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15th IHSS Meeting- Vol. 3
ADVANCES IN
NATURAL ORGANIC MATTER
AND
HUMIC SUBSTANCES RESEARCH
2008-2010
Vol. 3
POSTER PRESENTATIONS
Proceedings Book of the Communications presented to the
15th Meeting of the International Humic Substances Society
Tenerife - Canary Islands. June 27- July 2, 2010
Editors:
J.A. González-Pérez, F.J. González-Vila & G. Almendros
Vol. 3 Page - 1 -
15th IHSS Meeting- Vol. 3
Maquetación: Carlos Marfil Daza
Published on-line in: Digital.CSIC (http://digital.csic.es/), the Institutional
Repository of “Consejo Superior de Investigaciones Científicas” (CSIC).
© 15th Meeting of the IHSS, Tenerife, Canary Islands. 2010
URI:
Vol. 3 Page - 2 -
15th IHSS Meeting- Vol. 3
CONTENT Vol. 3
POSTER PRESENTATIONS SESSION B
Natural Organic Matter and Humic Substances in
Aquatic Systems and Sediments
WAT1 (1) Characterization of humic acid from the river bottom sediments of
Burigonga: complexation studies of metals with humic acid. M.A. Rahman, A. Hasan, A.
Rahim, A.M. Shafiqul Alam
WAT2 (5) Adsorption of fulvic acids by activated carbon. O. Samsoni-Todorova, L.
Savchyna, N. Klymenko
WAT3 (19) Examining the effect of humic acid on gag pipe corrosion in sea water.
A.R.Sardashti, R.Kafian
WAT4 (33) Color removal by coagulation from water containing aquatic humic
substances with different apparent molecular size. E. Sloboda, C. Tolledo Santos, A. Di
Bernardo Dantas, L. Di Bernardo, E.M. Vieira
WAT5 (57) pH effect in aquatic fulvic acid from Brazilian river. S. da Costa Saab, E.R.
Carvalho, R.B. Filho, M. R. de Moura Aouada, L. Martin-Neto, L.H.C. Mattoso
WAT6 (75) The role of organic matter in the transport of suspended minerals in the
estuarine zone. E.V. Lasareva, A.M. Parfenova, E.A. Romankevich
WAT7 (86) Organic material of uneven-age anthropogenic origin lakes. S. Zalmanova
WAT8 (99) Comparative measurement of hydrophobic organic matter dissolved in
water by the XAD resin method and the polarity rapid assessment method (PRAM). M.
Philibert, A. Revchuk, D. Quiros, A. Roh, M. Suffet
WAT9 (122) Microbial changes in the spectroscopic characteristics and molecular
weight of dissolved organic matters extracted from diverse source materials. J. Hur, B.M. Lee, T.-H. Lee, K.-Y. Jung
WAT10 (165) Dynamics of humic matters in fen bog water in conditions of climate
change. E.S. Ivanova, E.S. Voistinova, J.A. Kharanzhevskaya
WAT11 (168) Structural characteristics of deep groundwater humic substances in
Horonobe area, Hokkaido, Japan. M. Terashima, S. Nagao, T. Iwatsuki, Y. Sasaki, Y.
Seida, H. Yoshikawa
WAT12 (197) Browning of stream water during hydrological events. D.O. Andersen
WAT13 (199) Fluxes of natural and combustion-derived organic matter into the coastal
ocean off Southern Brazil. D.C. Podgorski, J.Y. Paeng, T. Dittmar, M.S.M.B. Salomao, C.E.
Rezende, M.C. Bernardes, B. Cooper
WAT14 (203) The changes of water organic contamination under the influence of
ultrasounds. L. Stepniak, E. Stanczyk-Mazanek, U. Kepa
WAT15 (238) Unexpected uniformity of humic substances in thermal waters. K. Kovács,
C. Sajgó, A. Brukner-Wein, Z. Kárpáti, A. Gáspár, E. Tombácz, P. Schmitt-Kopplin
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WAT16 (240) Basic by-products formation during chlorination of water containing
humic substances. E.V. Trukhanova, M.Y. Vozhdaeva, L.I. Kantor, E.A. Kantor
WAT17 (261) Research of the physics and chemical properties on sediments of the
Lobelia lakes in West Pomeranian region of Poland. L. Mielnik, J. Czekała
WAT18 (282) Ratio of color to chemical oxygen demand as an indicator of quality of
dissolved organic matter in surface waters. A.I. Konstantinov, N.S. Latyshev, P.A. Ivkin,
I.V. Perminova
WAT19 (313) UV-Vis spectrometry and size-exclusion chromatography study of
seasonal dynamics of quality of dissolved organic matter. A.I. Konstantinov, E.V.
Trukhanova, M.Y. Vozhdaeva, L.I. Kantor, I.V. Perminova
WAT20 (324) Humin contribution to sedimentary organic matter the Adriatic Sea. F.
Rampazzo, D. Berto, M. Giani, L. Langone
WAT21 (342) Influence of pre-ozonation of solutions of fulvic acid on equilibrium
adsorption on activated carbon. I. Kozyatnyk, L. Savchyna, N. Klymenko
WAT22 (358) Study of estuarine sediments in Galway Bay. R. Mylotte, M.H.B. Hayes, C.
Dalton
WAT23 (364) Effect of river floods on marine organic matter fluorescence. E. Parlanti, S.
Relexans, F. Ibalot, S. Huclier-Markai, R. Nicolau, S. Mounier, Y. Lucas
WAT24 (365) Study of colloidal organic matter transformation processes at superficial
sediment interfaces. E. Parlanti, S. Relexans, D. Amouroux, R. Bridou, S. Bouchet, G. Abril,
H. Etcheber
WAT25 (369) Resolving ahthropogenic and natural organic matter using hypy. X.
Zhang, C.E. Snape, W. Meredith, Y. Sun
WAT26 (370) Organomineral association patterns of humic substances in different
Venezuelan estuarine mangroves. A. Méndez, Z. Hernández, G. Almendros, X.L. Otero, F.
Macías, W. Meléndez
Natural Organic Matter and Humic Substances Interactions
INT1 (7) Relationship between organic carbon forms and selected trace elements in
grassland soils. L. Pospíšilová, P. Škarpa, V. Petrášová, M. Konečná"
INT2 (9) Investigation of humic substances by particle size distribution of soils and by
determination of zeta potential. S. Joó, J. Tóth, G. Samu, R. Földényi
INT8 (41) Interactions of organic compound with NOM need water: strong waterinduced enhancement of carbamazepine sorption on peat. M. Borisover, M. Sela, B.
Chefetz
INT9 (44) Adsorption of metal ions on humic acid derived from Turkish lignite. B.Z.
Uysal, D. Öztan, U.G. Zafer, Ö.M. Doğan, S. Anaç, M. Özdingiş, Z. Olgun
INT10 (48) Characteristics of humic acids isolated from heavy metals contaminated soils
at the copper-smelter “Legnica” (S-W Poland). A. Maciejewska, J. Kwiatkowska-Malina
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INT11 (50) The interaction of Cu 2+ with humic acids of different soils. G.V. Motuzova,
H. Dergam, A.A. Stepanov
INT12 (53) Influences of humic acids on the pattern of oxidation products of
tetrabromobisphenol A derived from a catalytic system using Iron(III)-tetrakis(psulfophenyl)porphyrin and KHSO5. M. Fukushima, Y. Ishida, S. Shigematsu
INT13 (54) Adsorption of trihalomethanes by humin: batch and fixed bed column
studies. G. da Costa Cunha, L.P. Cruz Romão, M. Cardoso Santos, B.R. Araújo, S.
Navickiene, V.L. de Pádua
INT14 (56) Combined effects of humic matter and surfactants on PAH solubility: Is
there a mixed micellization? H. Lippold
INT16 (76) Complexation of Copper(II) ions with humic acids and EDTA studied by
high resolution ultrasonic spectrometry. M. Klucakova, M. Pekar
INT17 (80) CE-ICP-MS as speciation technique to analyze the complexation behavior of
Europium, Gadolinium and Terbium with humic acid. C. Möser, R. Kautenburger, H.P.
Beck
INT19 (92) Determinations of ability of extracted HSs coordinated with metal ions. K.-F.
Ding, Q.-H. Fan, Y.-Y. Zhang, W.-S. Wu
INT22 (102) Characterization of zinc binding ability of dissolved hydrophilic organic
matter from the Seine River and major wastewater effluents. Y. Louis, B. PernetCoudrier, G. Varrault
INT23 (108) Molecular size distribution of metal complexes with pore water dissolved
organic matter determined by HPSEC and ICP-MS. N. Makarõtševa, V. Lepane, T.
Alliksaar
INT24 (160) Influence of Pb(II) ions on semiquinone radicals of humic acids and modle
compounds. J.M. Jerzykiewicz, M. Witwicki
INT25 (164) The effect of natural organic matter on the formation and solubility of
M(OH)4 solid phases (Th(OH)4, Zr(OH)4 Ce(OH)4). S. Antoniou, I. Pahalidis
INT27 (172) Adsorption of polycyclic aromatic hydrocarbons (PAHs) onto engineered
and natural nanoparticles. L. Marino, D. Mondelli, N. Senesi
INT28 (174) The challenge of building a humic-metal binding constants database. M.
Filella, W. Hummel, P.M. May, J. Puy, F. Quentel
INT29 (175) Heavy metal compounds with organic substance and methods of their
definition. T.M. Minkina, G.V. Motuzova, O.G. Nazarenko, S.S. Mandzhieva
INT30 (183) Concentrations of iron in the interactions of some acid ones organic with
minerals. C.F.D. Bassan; A.A. Paccola; P. de Magalhães Padilha
INT31 (215) Fluorescence study of adsorption mechanisms of flubendiamide onto humic
acids. I. Cavoski, V. D’Orazio, T. Miano
INT33 (243) Size exclusion characterization of dissolved organo-mineral complexes in
soils of the Southern Far East. T.N. Lutsenko, A.S. Volk
INT35 (251) Sorption of pharmaceuticals to humic substances. H. Mori, T. Ohtani, I.
Fukuda, H. Ashida, N. Fujitake
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INT36 (258) Influence of aromaticity degree on the aggregation of Humic Substances.
M. Drastík, J. Kučerík, O. Zmeškal, A. Čtvrtníčková, F. Novák
INT37 (283) Aggregation of humic acids in solution. Vapor pressure osmometry,
conductivity and mass spectrometric study. E.M. Peña-Méndez, D. Fetsch, J. Havel
INT38 (290) Sorption of silanol-modified humic acids onto different solid supports
including silica gel, clay and sand. I.V. Dubinenkov, A.B. Volikov, V.A. Kholodov, E.M.
Garanin, I.V. Perminova
INT39 (294) Imprinted humics-based sorbents as selective trap for metal Ions. E.
Kasymova, R.P. Koroleva, E.M. Khudaibergenova, N. Hertkorn, S.J. Jorobekova, A.D.
Pomogailo, K. Kydralieva
INT41 (307) Flow injection analysis (FIA) for fast monitoring of gold nanoparticles
formation from various precursors and theirs separation by using humic acids. E.M.
Peña-Méndez, A.I. Jiménez Abizanda, J.J. Arias León, J. Havel
INT42 (312) Spectrofluorimetric study of the interaction of Gold (III) and humic acids
under the formation of gold nano-particles. E.M. Peña-Méndez, F. Jiménez Moreno, J.E.
Conde González, J. Havel
INT43 (329) Metal binding by humic acids extracted from recent sediments from the
SW Iberian coastal area. J.M. de la Rosa, M. Santos, F.J. González Vila, H. Knicker, J.A.
González Pérez, M.F. Araújo
INT44 (363) Polycyclic aromatic hydrocarbons (PAHs) - dissolved organic matter
(DOM) interactions studied by solid phase microextraction (SPME). C. De Perre, K. Le
Menach, A.-M Dorthe, C. Béchemin, H. Budzinski, E. Parlanti
Environmental Applications of Natural Organic Matter
and Humic Substances
ENV1 (32) Use of humin for removal of phosphorus from sewage treatment station
effluents: influences of time and pH. L. Camargo de Oliveira, W.G. Botero, A.G. Ribeiro
Mendonça, J.C. Rocha, A. dos Santos, A.H. Rosa
ENV2 (37) Phytoremediation of a soil polluted with multiple heavy metals using MSW
compost as organic carbon source. K. Farrag, G. Brunetti, P. Soler, F. Nigro
ENV3 (55) Hydrogels filled with humic-rich lignite for various environmental
applications. M. Pekař
ENV4 (125) Mitigation of GHGs emission from soils by a catalyzed in situ photooxidative polymerization of soil humic molecules. A. Piccolo, R. Spaccini
ENV6 (155) Sorption of endocrine disruptors by humic substances from sediment
samples collected on Guarapiranga reservoir, São Paulo state-Brazil. B. Barboza Cunha,
W.G. Botero, L. Camargo de Oliveira, G. Carvalho Leite, D. Goveia, V. Moschini Carlos,
M.L. Martins Pompêo, L. Cardoso de Morais, L. Fernandes Fraceto, A. Henrique Rosa
ENV7 (180) Dual effect of humic acid on the degradation of pentachlorophenol by
Iron(II) and H2O2. K.C. Christoforidis, M. Louloudi, Y. Deligiannakis
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ENV8 (191) Radiotracer method in the investigation of humic substances sorption on
carbon-based nanomaterials. M.G. Chernysheva, G.A. Badun
ENV9 (195) Study of flow-through sample preparation methods for group of pesticides
determination in soil by reversed-phase high-performance liquid chromatography. M.
Chalányová, M. Hutta, I. Procházková
ENV11 (227) Humus substance role in technogenic soil formation at Priokhotie mining
industry developments. A.F. Makhinova, A.N. Makhinov
Industrial Production and Commercial Applications
IND2 (49) Study of copper extraction efficiency by humic acid/polypyrrole on paraffinimpregnated graphite electrode. M. Antilén, M. Araus, M. Pérez, F. Armijo, R. del Río,
M.A. del Valle
IND3 (71) Sorption of silanized humic derivatives onto montmorillonite clay. V.A.
Kholodov, V.M. Zelikman, K. Hatfield, I.V. Perminova
IND4 (87) Peat humic acids as the redox mediators for textile technologies. I.Y.
Vashurina, Y.A. Kalinnikov, I.V. Perminova
IND5 (124) Peat humic acids as surfactants. O. Purmalis, M. Klavins
IND6 (128) Efficiency and application prospect of humatized mineral fertilizers.
O.Gladkov, R. Poloskin. G.O. Andreevich, P.R. Borisovich
IND7 (139) Physical-chemical properties and application potential of humates prepared
from regenerated lignites. J. David, J. Kučerík
IND9 (178) Removal of tributyltin biocide by using black carbon. L. Fang, O.K.
Borggaard, H. Marcussen, P.E. Holm, H.C.B. Hansen
IND10 (221) Evaluation of the efficiency of fulvic and humic acids (Agrolmin Bravo and
Cerrado) in soybean production in the Brazilian savanna. L.T. Dias Cappelini, D.
Cordeiro, L.A. Artimonte Vaz, L.F. Artimonte Vaz, E.B. Azevedo, E.M. Vieira
IND11 (274) Pyrolisis parameters evaluation in the biochar preparation process. E.I.P.
de Rezende, A.P. Mangoni, I. Messerschmidt, A.S. Mangrich, E.H. Novotny, M.H. R.
Velloso
IND12 (277) Extraction of high-value lipids from Irish peats. R. McInerney, D.J. Hayes,
J.J. Leahy, M.H.B. Hayes
IND13 (278) Fluorescence of aqueous solutions of commercially produced humic
substances. O. Yakimenko, A. Izosimov, D. Shubina, V. Yuzhakov, S. Patsaeva
IND17 (326) Assessment of the oil shale byproducts use as soil conditioner: study of
sorption and biodegradation of phenol models with soil. R. Garrett Dolatto, G. Abate, I.
Messerschmidt, B. Fraga Pereira, A.S. Mangrich, C. Posser Silveira, C.N. Pillon
IND18 (330) Humic acids from fines of residual coal type material: preparation and
characterization. G.M. Maurício, A.C.S. Wimmer, E.A. Brocchi, A.C. Vidal, R.A. Nunes
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IND19 (350) Assessing the effect of a bio-accelerated composting process using
analytical pyrolysis (Py-GC/MS). F. Pérez-Barrera, K. Akdi, F.J. González-Vila, J.A.
González-Pérez, T. Verdejo
Natural Organic Matter and Humic Substances Biological
and Physiological Effects
PHY1 (10) A turning point of wheat breeding and humic substances. R. Shahryari
PHY2 (17) Response of maize genotypes to changes in chlorophyll content at presence of
two types humic substances. R. Shahryari, B.S. Moghanlou, A.M. Pour Khaneghah
PHY4 (31) Bioactivity of chemically transformed humic matter on plant root growth.
L.P. Canellas, L.B. Dobbss, F.L. Olivares, N.O. Aguiar, L.E.P. Peres, R. Spaccini, A. Piccolo,
A.R. Façanha
PHY5 (35) Effect of two humic substances as bifertilizers on germination and seedling
growth of maize genotypes. R. Shahryari, N. Bahari, M. Khayatnejad
PHY6 (107) Comparative evaluation of the inhibitory action of compost humic fractions
on two soil-borne phytopathogenic fungi. A. Traversa, E. Loffredo, N. Senesi
PHY9 (147) Effect of liquid humic compounds extracted from plant based-compost to
soil microorganisms. F. Suárez-Estrella, M.C. Vargas-García, G. Guisado, M.J. López, J.
Moreno
PHY11 (235) Analysis of the sorption properties soils after the application of sewage
sludges and conventional organic fertilizers. E. Stańczyk-Mazanek, L. Stępniak, U. Kępa
PHY13 (273) Interactions between plant-root exudates and soils in extracting humic-like
substances. D. Pizzeghello, A. Muscolo, A. Ertani, S. Nardi
PHY14 (276) Bioactivity of humic acids from vermicompost at increasing maturity
stages. N.O. Aguiar, L.P. Canellas, F.L. Olivares, J.G. Busato, LG.JR.S. Silva; E.H. Novotny,
A.R. Façanha
PHY15 (281) Root Growth promotion by humic acids from urban organic residues. K.
Jindo, C. García-Izquierdo, L.P. Canellas
PHY16 (284) Direct and indirect effects of humic substances of different origin on the
green algae Monoraphidium braunii. C.E. Gattullo, H. Bährs, J. Qianru, C.E.W. Steinberg,
E. Loffredo
PHY17 (318) Effects of compost water-extracts on the germination and growth of
slickspot peppergrass (Lepidium papilliferum). E. Loffredo, A.J. Palazzo, A. Traversa, T.L.
Bashore, N. Senesi
PHY18 (328) The action of humic acids promoting plant shoot development are
associated with nitrate-related changes on the plant hormonal balance. V. Mora, E.
Bacaicoa, E. Aguirre, R. Baigorri, M. Garnica, M. Fuentes, A.M. Zamarreño, J.C. Yvin, J.M.
García-Mina
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Health and Medical Applications of Natural Organic Matter
and Humic Substances
MED1 (2) Inclusion complexes of aspirin in fulvic acid offer enhanced dissolution,
permeability, stability and better pharmacodynamics. K. Anwer, A. Mirza, S.P. Agarwal,
A. Ali, Y. Sultana
MED3 (84) The effect of fulvic and humic acid supplementation on the intensity of the
immune response in rats. A.V. Vucskits, I. Hullár, E. Andrásofszky, N. Hetényi, J. Csicsor,
A. Móré, J. Szabó
MED4 (85) Evaluating potential nephrotoxicity of compost derived humic acid to
African mud catfish (Clarias gariepinus) grown in static water culture. I.M. Adekunle,
O.R. Ajuwon
MED5 (145) Treatment of pilonidal sinus by humic acid salts. M. Dizman, A. Tutar
MED6 (194) Stabilization of iron oxide magnetic nanoparticles with different
morphology in aqueous suspensions using humic substances. A.Y. Polyakov, A.E. Goldt,
T.A. Sorkina, E.A. Goodilin, I.V. Perminova
MED7 (241) Neutralisation of the anticoagulant effect of naturally occurring humic
acids and synthetic humic acid-like polymers by protamine sulphate. H.P. Klöcking, N.
Mahr, S. Kunze, R. Klöcking
MED8 (289) Protolytic properties of alkoxysilylated versus natural humic materials
aimed at use as stabilizers for magnetic fluids. T. Sorkina, A. Goldt, A. Polyakov, A.
Dubov, I. Toth, A. Hajdu, E. Goodilin, E. Tombacz, I. Perminova
MED9 (321) Halogen-free preparation and preliminary characterization of humic
substances from different substrates. C. Kleiner, C. Barthel, R. Junek, R. Schubert, J.I.
Schoenherr, R. Klöcking
Young Researchers in Humic Substances and
Natural Organic Matter IHSS Travel Award)
TA1 (4) Influence of surface chemistry and structure of activated carbon on adsorption
of fulvic acids from water solution. T.V. Poliakova, L.A. Savchynaand, N.A. Klymenko
TA2 (16) Studies by chimiometric methods of the interaction between Pb(II) and humic
acids. S. Orsetti, E. Andrade; F. Molina
TA3 (193) Seasonal dynamics of biomass and copper concentrations in ectohumus of
forest soils impacted by copper industry in South-West Poland. A. Medyńska, C. Kabala
TA4 (207) Limitation for study of humic substances or NOM using high resolution and
accuracy mass-spectrometry. G. Vladimirov, E. Nikolaev
TA5 (220) A study of interaction between pharmaceuticals and humic substances. L.
Ansone, M. Klavins
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TA6 (222) Characterization of soil organic matter of treated sewage effluent irrigated
areas. B.H. Martins, L. Macedo dos Santos, D.M.B.P. Milori, L. Martin-Neto, C.R. Montes
TA7 (234) Modelling differential absorbance spectra of SRFA during complexation with
copper and lead. D.J. Dryer, G.V. Korshin, M.F. Benedetti
TA8 (255) Behavior of soil carbon in amended areas with sewage sludge. B.H. Martins,
T.L. de Almeida, S. Gaiad, D.M.B.P. Milori, L. Martin-Neto
TA9 (257) Gold(III) and nanogold interaction with humic acids: spectrophotometry,
capillary electrophoresis and mass spectrometric study. N.R. Panyala, E.M. Peña-Méndez,
J. Havel
TA10 (259) Abiotic treatment of rice bran using an accelerator including organo-iron
compound. H. Kanno, N. Tachibana, M. Fukushima
TA11 (266) Effect of humic substances on uranium removing by bacterium Bacillus
polymyxa IMV 8910 from aqueous solution. I. Shevchuk, N. Klymenko
TA12 (360) Humic acids inspired hybrid materials as heavy-metal adsorbents. P. Stathi,
Y. Deligiannakis
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Natural Organic Matter and Humic Substances in Aquatic Systems
and Sediments
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Characterization of Humic Acid From the River Bottom Sediments of
Burigonga: Complexation Studies of Metals with Humic Acid
Mohammad Arifur Rahman*, Abu Hasan, Abdur Rahim and A. M. Shafiqul Alam
Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh
E-mail: marahman76@yahoo.com
1. Introduction
Humic substances are ubiquitous in waters, being found wherever organic matter is decomposed or
has been transported. Their importance in agriculture and soil sciences has been acknowledged for
over 150 years. Aquatic scientists have been slower in appreciating their importance, but now realize
that they may constitute as much as 95% of the total dissolved organic matter in aquatic systems and
often are equal to or greater than the concentrations of inorganic ions present. Moreover, the
structures, molar masses and functional groups of humic acid vary depending on origin and age [1, 2].
The river Buriganga, which runs by the side of the Dhaka city, is at present one of the most polluted
rivers in Bangladesh. The amount of untreated wastes, both domestic and industrial, being released in
the Buriganga is tremendous and increasing day by day. So, the structure of humic acid in the river
Buriganga would be different. Therefore, it is required to determine the structure of humic acid of the
Buriganga River in Bangladesh.
Iron is a transition metal. High concentration of iron in the river water made scaling in the boiler of
water purification system. Cadmium is not only heavy metal but also toxic in nature. It is one of the
most harmful elemental pollutants and is of particular concern because of its toxicities to humans.
Pollutant cadmium in water may arise from industrial discharges.
Humic acid represent the dominant part of dissolved organic matter in freshwater supplies [3, 4]. The
elimination of metals and humic acid upon drinking water treatment is mainly performed coagulation
with hydrolyzed metal species. Considerable attention has then been focused on this removal step as
uncoagulated humic materials lead to severe in the following treatment stages. Indeed. Membrane
fouling, trihalomethanes formation during chlorine disinfection, or biological regrowth in the
distribution network, have all been linked to the presence of residual humic substance in the clarified
water [5]. Three main mechanisms are generally invoked to explain the removal of humic acid and
metals by coagulation: charge neutralization/complexation preferentially applies at acidic pH and finds
experimental support from stoichiometric relationships between coagulant demand and dissolved
organic matter concentration, and from suspension restablization upon overdosing [6]. On the other
hand, under conditions favouring metal hydroxide precipitation, physical enmeshment and/ or
adsorption onto the freshly formed precipitate are assumed to play a major role in humic substance
elimination [7]. So, complexation of iron and cadmium with humic acid would be helpful to reduce
iron and cadmium concentration from the surface water of the river.
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There are many investigations of complexation of metals with humic acid were carried out [1, 8, 9].
Moreover, many characterization studies also carried out in different countries. However, molar
masses and functional groups of humic acid varied with origin and its surrounding environment. Since
the Buriganga is very much polluted river in Bangladesh its humic acid structure might be different.
So, the structure of humic acid and its interaction with metals would be different. No information is
available regarding the structure of humic acid and complexation study of humic acid with metals of
the Buriganga River. Therefore, it is urgently required to determine the structure of humic acid and
study of the metals and humic acid complexation of the Buriganga River. This work involves with the
characterization of humic acid of the Buriganga River and its complexation study with iron (III) and
cadmium (II).
2. Materials and Methods
Sampling: The river bottom sediments in the Burigonga River were collected in 0.15m depth from the
surface of river bottom sludge at 24th July (rainy season) in 2008 at 30oC atmospheric temperature
from five sampling stations. The sampling areas(23o42’N 90o24’E to 23o92’N 90o53’E) are as follows:
a. Bibi Shaheb’s Ghat, b. Forashgonj Ghat, c. Shahid Shaheb’s Ghat, d. Shagorer Dock and e.
Talukder Ghat. The sampling sites are located near about to Dhaka (Capital city of Bangladesh).
Methods: In order to characterize and study of the complexation with metal ions, sediment samples
were collected from five different places in Burigonga River. The Humic Acids were extracted with
the following standard procedure [10]. The extracted Humic Acids were characterized with the help of
IR, FTIR, SEM, EDX and CHNS analyzer. A complexation study of the Humic Acid with iron and
cadmium was carried out by using SEM, EDX, UV-Visible spectrophotometer and AAS.
Characterization of Humic Acid and Study of the Metal Complexes: Fourier Transform Infrared
(FTIR) Spectroscopy (SHIMADZU KN S72-120, Japan), UV-Visible Spectroscopy ((UV-160A,
SHIMADZU, Japan), Scanning Electron Microscopy (SEM, HITACHI S-3400N) combined with
Energy Dispersive X-ray (EDX, Princeton Gamma Tech Imix-PC with ultra-thin window detector)
and CHNS Analyzer (‘Elementar’ Germany) were used to evaluate the structure of humic acid and its
complexation with metals. Moreover, pH meter (Hanna 210), Conductometer (EYLA) and Atomic
Absorption Spectrophotometer (AA Analyst 800, Perkin Elmer, USA) were also used to measure pH,
conductance and the metal concentration in the river Buriganga.
Complexation Study of Humic Acid and Metals: In order to evaluate the complexation efficiency of
humic acid with metals accurately 25mg standard humic acid (BDH, England) was taken in 1L
deionized water for preparing 25 mg/L humic acid. Then 0.5 M NaHCO3 (E Merck, Germany) was
also added into the suspension to provide a carbonate alkalinity similar to that of natural waters [1].
Before the coagulant injection, the pH of synthetic waters was adjusted to pH 6 by drop wise addition
of 0.1 M HCl [1]. The coagulant, commercial Ferric Chloride (FeCl3) (BDH, England) and Cadmium
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Sulfate (3CdSO4. 7H2O) (BDH (England) of various concentrations 3x10-4 M, 4x10-4 M, 5x10-4 M,
6x10-4 M, 7x10-4 M were injected into 20.0 mL humic acid solution. A rapid mix period for 5 minutes
at 200 rpm followed by slow stirring 50 rpm for 40 min was done for the mixture. At the end of the
mixing, the coagulated suspension was allowed to settle in graduated conic plastic holder for 24 hours.
About 10 mL of supernatant was withdrawn with graduated pipette from upper of solution retained
after complex formation. pH, Conductivity, Concentration and absorbance were monitored of the
supernatant with the pH meter, Conductometer, AAS and UV- visible spectrometer with a resonance
of 254.0 and 436.0 nm respectively [12]. For the comparison study, pH and molar conductance of
3×10-4 M, 4×10-4 M, 5×10-4 M, 6×10-4 M, 7×10-4 M of ferric chloride and cadmium sulfate solution
were monitored before and after complexation. Finally, the complex of humic acid and metals were
dried with oven at 50–60o C. Then the complexes were characterized with SEM and EDX analyzer.
3. Results and Discussion
From the characterization study of extracted humic acid with FTIR, it was revealed that humic acid of
the Buriganga River contain aromatic hydrocarbons, enolic aldehyde/ ketone, carboxylate anions,
carboxylic acids, intermolecular hydrozen bond (polymeric form) and aryl carboxylic acids etc. Humic
acid analysis by CHNS showed that the higher C/N ration. These higher C/N values in sediment
containing higher organic carbon may be produced due to the degradation of organic matter (waste
materials) present in the Buriganga River. A complexation study of the humic acid with iron and
cadmium was carried out. The Fe(III):humic acid and Cd(II):humic acid complex were analyzed and
characterized with AAS, UV-Visible spectrophotometer, SEM, EDX respectively. The complexation
reaction is mainly responsible for charge neutralization of humic acid entities thus reducing HA
colloid stability [13]. From this study, it was found that iron and cadmium could make a complex at
pH 6.0 which was confirmed by EDX spectrum. Since iron and cadmium can make a complex with
humic acid of the Buriganga River, this complexation or coagulation process can be used to remove
the high concentration of iron and cadmium from the surface water of this river. We simply assume
that humic acid can be existed in a low-density network of hydrophobic and hydrophilic moieties.
Such nanocolloids change conformation according to their ionization state, can be built either from
amphiphilic polymers or assemblies of small molecules, and accordance with previous models of the
secondary structure of humic substances [14, 15, 16]. The formation of humic acid aggregates is then
controlled by the number of coagulant species and two dynamic aspects: (i) the reconformation of
humic network, and (ii) the collision rate of destabilized particles. An overall shrinkage of
anionic
humic network is indeed expected upon binding cationic coagulant species, which promotes the
formation of intra- and inter-particle hydrophobic moieties particle hydrophobic domains according to
the extent of neutralization. This suggests that, in addition to coagulant species, hydrophobic moieties
participate in the floc build up. The classical aggregation mechanisms proposed in the literature to
explain the coagulation of humic acid include charge neutralization/precipitation at acidic pH, and
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adsorption and /or sweep-flocculation in a hydroxide precipitate at alkaline pH. From our work, it can
assumed that a similar charge neutralization/complexation with hydrolyzed iron and cadmium species
occurs at the acidic pH, thus increasing the number of carboxylic groups available for complexation.
It should be noted that hydrolyzed iron and cadmium species are known to strongly interact with NOM
carboxylic groups, and that the humic network remain unaffected even in the overdosages range at
acid pH with highly charged. As most of features of Buriganga River humic acid are similar to
freshwater humic acid. The complexation/aggreagation model described above should be general.
Acknowledge
The authors acknowledge the International Foundation for Science (IFS) for their financial support to
carry out this research work.
References
1. J.M. Siéliéchi, B.S. Lartiges, G.J.Kyem, S. Hupont, C. Frochot, J.Theime, J.Ghanbaja, J.B. d’Espinose de la
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Caillerie, O.Barres, R.Kamga, P.Levitz,L.J.Michot ;Changes in humic acid conformation during coagulation
with ferric chloride: Implication for drinking water treatment; Water Res. 42,2111–2123, 2008.
G.Davies and E.A Ghabbor, Humic Substances: Structures, Properties and uses, Royal Society of
Chemistry, Cambridge, 1998.
E. Tripping, Cation Binding by humic substances: Cabridge University Press: Cambridge, UK, 2002.
J. Zumstein, J. Buffle, Circulation of pedogenic and aquagenic organic matter in a eutrophic lake. Water
Res. 23 (2), 229–239.
N. Narkis, M. Rebhun, Stoichioche,istry relationship between humic and fulvic acids and flocculants. J.
AWWA, 325–328, 1977.
C.F. Lin, T.Y., O.J. Hao, Effects of humic substances characterization on UF performance. Water Res. 34,
1097–1106, 2000.
P. Bose, D. A. Reckhow, Adsorption of natural organic matter on preformed aluminum hydroxide flocs, J.
Envirn. Eng. 124, 803–811, 1998.
J. Dries, L. Bastiaens, D. Springael, S. Kuypers, S. N. Agathos, L. Diels, Effect of humic acids on heavy
metal removal by zero-valent iron in batch and continuous flow column systems, Water Res.39, 3531–3540,
2005.
S. Suteerapataranon, M. Bouby, H. Geckeis, Interaction of trace elements in acid mine drainage solution
with humic acid, Water Res. 40, 2044–2054, 2006.
R.S Swift, Organic matter characterization. In: D. L. (1996); D.L. Sparks et al. (Ed.), Methods of Soil
Analysis, Part 3: Chemical methods. SSSA Book, Series No. 5. SSSA and ASA, Madison.
Regina M.B.O. Duarte, Eduarda B.H. Santos, Armando C.Duarte, Spectroscopic characteristics of ultra
filtration fractions of fulvic and humic acids isolated from an eucalyptus bleached Kraft pulp mill effluent,
Water Res.37, 4073–4080, 2003.
M. A. Rahman., S. Kaneco, T. Suzuki, H. Katsumata, K. Ohta, A. M. Shafiqul Alam, Development of
sintering materials by sea sediments and TiO2 for the cleaning technology, Pak. J. Anal. Environ. Chem. 8
(1 & 2), 2007.
V. Jung, V. Chanudet, J. Ghanbajo, B. S. Lartiges, J. –L. bersillon, Coagulation of humic substances and
dissolved organic matter with ferric salt: an electron energy loss spectroscopy investigation, Water Res. 39,
3849–3862, 2005.
K. Ghosh, M. Schnitzer, Macromolecular structures of of humic substances, Soil Sci.129, 266–276, 1980.
Piccolo, The supramolecular structure of humic substances, Soil Sci. 166, 810–832, 2001
J. F. L. Duval, K. J. Wilkinson, H. P. Van Leeuwen, J. Buffle, Humic substances are soft and permeable:
evidence from their electrophoretic mobilities, Eniviron. Sci. Technol. 39, 2005.
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Adsorption of Fulvic Acids by Activated Carbon
Olena Samsoni-Todorovaa*, Liudmyla Savchynab, Natalia Klymenko b
a*
National Technical University of Ukraine “ Kyiv Polytechnic Institute”, 37 Peremogy Pr.,
Kyiv 03056, Ukraine; bInstitute of Colloid Chemistry and Chemistry of Water, Ukrainian
National Academy of Sciences, 42 Vernadsky Avenue, Kyiv 03680, Ukraine
E-mail: samsoni@online.ua
1. Introduction
It is known that the presence of humic substances in nature water complicates of its treatment
for drinking purposes and it is reason for generation a number of toxic, mutagenic, or
carcinogenic substances [1, 2]. Unfortunately a little information is available today on the
molecular structure of fulvic acids, their associativity in the presence of salt ions and their
molecular weight than on humic acids. This substantially hampers generalization of the
various experimental results and theoretical substantiation of the most effective operating
procedure for adsorption units, whether in facilities used for the production of drinking water or
in technological systems designed to prepare deionized water.
Our purpose was to obtain the model conceptions of fulvic acids molecules and degree of their
ionization on the basis of adsorption measurements. The final object is to improve the
activated carbon use in the water treatment plant.
2. Materials and Methods
We used activated carbon of Akant-meso trade mark that had made by high-temperature water
steam activation of anthracite modification of the Donetsk Basin deposit. The surface and the
porous structure of Akant-meso were determined by nitrogen adsorption at 77 K with
Quantachrom device.
A fulvic acid preparation was obtained from peat by the Forsyth method [3]. Solutions of
sodium fulvates at pH 7 and 12 were obtained by adding corresponding quantities of sodium
hydroxide. Concentrations of fulvic acid solutions were established by the organic carbon
content which was determined by Shіmadzu TOC-V CSN device.
3. Results and Discussion
It is known that the model conceptions of the chemical structure of fulvic acid molecules can
be obtained by adsorption measurements. This is achieved by comparing of the limiting
equilibrium adsorption of fulvic acid (а∞) on activated carbon with a known surface and pore
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size (rp) with the value а∞ calculated by model. It is obvious, that the pore size of activated
carbon must be equal to the minimum or greater than effective size of researched fulvic acid
molecules (rmin).
Sizes of fulvic acid molecules depend on their aggregation and wettability. In fresh water (pH
5–10, salt content <0.01 M), fulvic acids (concentration <100 mg/dm3) are unassociated or
slightly associated, and their molecules are unfolded. At concentrations between 0.1–0.4 g/dm3
there occurs a "medium-degree" association, and at >0.4 g/dm3 fulvic acid associates forms
dense aggregates of quite large sizes [4]. The carbon concentration in soil fulvic acids changes
from 43.7 to 53.1%, and in water fulvic acids from 41.6 to 51.1%. The molecular weight of
fulvic acids, as given by the various sources and also determined by different methods, varies
over a rather broad range. In very dilute solutions it probably ranges from 600 to 1300 Da.
Most of investigators estimate the volume of fulvic acid molecules in interval of 1.2–2.1 nm3.
Structural-adsorptive characteristics of activated carbon Akant-meso are listed in Table 1.
Table 1: Structural-adsorptive characteristics of activated carbon Akant-meso
Adsorbent
SBET, m2/g
Sme, m2/g
Va, сm3/g
Vmi, сm3/g
Vme, сm3/g
Vma, сm3/g
Akant-meso
950
345
0.55
0.37
0.14
0.04
The isotherms of the adsorption of fulvic acids on the activated carbons were satisfactorily
described by a relationship analogous to the Langmuir equation.
At pH 2 the ionization of both phenol and carboxyl groups was suppressed, which made
possible the densest packing of fulvic acid molecules in the adsorption layer.
The relatively flat structure of nonassociated molecules of fulvic acids agrees with a flat
configuration of adsorbed molecules, because aromatic structures are oriented with the
surface of the benzene rings parallel to the plane of the carbon surface [5]. In this arrangement
the length of the molecule axis perpendicular to the adsorbent surface, or the "thickness" of the
molecule, must be 0.4 nm (the “thickness” of the benzene ring). The projection area of the
benzene ring itself must be ~ 0.63 nm2. Therefore the short axis of a "flat" molecule cannot be
shorter than 0.75–0.8 nm. The Van der Waals volume of the fulvic acid molecule is in the
interval from 1.2 to 2.1 nm3, corresponding, with a molecule "thickness" of 0.4 nm, to a
molecule projection area of 3.0 to 5.0 nm2.
As can be seen from Table 1, practically the entire surface area of the activated carbon Akantmeso is that of pores with the radius rp > 0.8 nm, meaning that all this surface must be
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accessible to such fulvic acid molecules. The limiting specific adsorption of fulvic acids at pH
2, which we found from the adsorption isotherm for Akant-meso, was 207.7 mgC/g. The
quantity of adsorbed fulvic acids corresponding to this value depends on the carbon content in
the molecule (Table 2).
Table 2: Relationship between limiting value of fulvic acid adsorption on Akant-meso at pH 2 and
carbon concentration in molecule and molecular weight
а∞ at (Мw)n, mmol/g
Carbon concentration
а∞,
in molecule, %
mg FA/g
800
900
950
980
1000
1100
48.0
432.7
0.54
0.48
0.45
0.44
0.43
0.39
49.0
423.8
0.53
0.47
0.45
0.43
0.42
0.38
50.0
414.5
0.52
0.46
0.44
0.42
0.41
0.38
Based on the data in Table 2, we calculated, for an Akant-meso surface area of 950m2/g, areas
screened by a fulvic acid molecule ωFA ("landing surface" of a molecule):
ωFA = SBEТ(r ≥ 0,8 nm) ⋅ 1018 / а∞ ⋅ NА.
The results obtained are listed in Table 3.
Table 3: Areas of "landing surfaces" of fulvic acid molecules in compact adsorption layer
ωFA at (Мw)n, nm2
Carbon concentration in
fulvic acid molecule, %
800
900
950
980
1000
1100
48.0
2.92
3.28
3.46
3.57
3.65
4.00
49.0
2.98
3.35
3.54
3.65
3.72
4.09
50.0
3.04
3.42
3.62
3.73
3.81
4.19
As can be seen from the data in Table 3, the area of the "landing surface" varies from 2.92 to
4.19 nm2 corresponding to a Van der Waals volume of the molecule of 1.16 to 1.68 nm3. These
values well conform with those reported in the literature.
At pH 7 and 12 respectively, the carboxyl and the phenol groups of the fulvic acid molecules
were ionized resulting in an electrostatic repulsion of the anions and an increase in their
hydration, which reduced the anion packing density and, consequently, the а∞ value. The а∞
values for fulvic acids adsorbed on the activated carbon at pH 2, 7, and 12 and their
proportions are shown in Table 4.
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Table 4: Values of а∞ of fulvic acids adsorbed on activated carbons
Adsorbent
Akant-meso
а∞, mg C/g at pH
2
7
12
207.7
136.6
91.1
The largest value of fulvic acid adsorption was registered at pH 2 (see Table 4). Such
conditions are unacceptable for the production of drinking water but can be realized at thermal
power plants if the adsorptive water purification of natural organic compounds is carried out
after the H-cationization stage. As can be seen from Table 4, this would increase the
efficiency of Akant-meso by 50%.
4. Conclusions
It was shown that the adsorption measurements of values of the limiting equilibrium specific
adsorption of fulvic acids on activated carbon can get conceptions of the fulvic acids
molecules size in the aqueous solution. It was found that the most efficient activated carbons
for a thorough removal of fulvic acids from natural waters will be those similar to Akant-meso
in porous structure, i.e., having an average effective mesopore radius >0.8 nm.
References
1.
2.
3.
4.
5.
V. V. Goncharuk, N. A. Klimenko, L. A. Savchina, T. L. Vrubel and I. P. Kozyatnik, Journal of
Water Chemistry and Technology, 28 (2006) 2–49.
G. Garnier, S. Mouner and J. Y. Benaim, Water Res., 38 (2004) 3685–3692.
L. N. Aleksandrova, Organic Soil Matter and Processes of its Transformation [in Russian],
Nauka, Leningrad, 1980, p. 288.
D. Buffle, Complexation Reactions in Aquatic Systems: An Analytical Approach, Ellis Horwood
Limited, New York, 1988, p. 692.
A. M. Koganovskii, T. M. Levchenko and V. F. Kirichenko, Adsorption of Dissolved Substances
[in Russian], Nauk. Dumka, Kiev, 1977, p. 223.
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Examining the Effect of Humic Acid on Gag Pipe Corrosion in Sea Water
A.R.Sardashti*, R.Kafiana
Department of Chemistry,Faculty of Science, University of Sistan and Baluchestan, P.O.Box
98135-674, zahedan, IRAN
E-mail: sardasht@hamoon.usb.ac.ir
1. Introduction
Corrosion, is an electrochemical process in which a potential –difference happens either
between two metals or two different parts of a same metal. the created potential-difference
can be measured in relation to standard electrod.electrical potential of this metal may be more
or less than standard limit [1].organic acids dissolved in water, such as Humic acid, exist
mostly with high concentration in water of marsh as well as, in waters which were not taken
from such places. The lands, covered with plants mainly, and surface-fleeing waters, partly,
possess such material [2]. One of new projects of gas-pipe lines passage, cross countries
because of security reasons, may be performed through the bottom(depth)ce, of seas and
ociens.Since the Humic substances of certain water of humic acid have the properties of
Rodox and ionic exchange ,corrosion ukelihood by humic substances is very great [3].
Because, pH of sea water, in most areas varies, ranging from 7.2 to 7.6, therefor, in usual
temperature degrees and small changes pH has no effect of usual rate of steel corroding. This
can be an economic loss. So, in this project along with examining the rate of corrosion, for
preventing purposes tests of electrochemical polarization and SEM on water of Oman Sea as
well as a few preventers, were conducted
2. Materials and Methods
To conduct the examination of electrochemical polarization and cycle voltamer, providing a
surface-having electrode which plays the role of work-procedure electrode, is necessary. At
first, we prepare an electrode sample which is made of the materials involved in gas pipe,
with surface of 1 cm2 and set it with (twins) glue, then smooth it sand paper, wash it with
distilled water and dry it, finally. Then we sink the electrode-sample in sea-water or other
mixture with stable pH, or having inhibtator, so that the corrosion takes place.Now, we put
the electrode sample along with reference electrode (Ag/Agcl) and opposite electrode in 50 ml
water of sea or other mixtures that form our electrochemical cell. At this time, we provide the
cell with gas for 15 minutes. Now draw the polarization curve and record the voltamgram CV
of corroded piece. in experiments of weight-reduction, we do as the same and then, get out the
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15th IHSS Meeting- Vol. 3
sample from solution and after drying, we will weigh it depositions have brown colour).in
element decomposition of electrode sample, by solving 0.02g of it, in concentrated
clorohydric acid inside a joujet balloon and in water of two-times distillation, we reach it to
desired volume. Content elements of prepared solution are defined by technique of Flame
atomic absorption
3. Results and Discussion
The results of elemental Analysis of steel sample (forming substances of gas pipe) are showed
in Table 1. In measuring the weight reduction in sea –water, it is 10 mg. In mixture of seawater and 5mg extracted humic acid, the reduction reaches to 20mg. Average potential of
corrosion, resulted from some electrochemical polarization curves, in sea-water is E= 0.98
volts and I=51 µA (time of half-hour) (Fig 1) .By adding Benzoic acid(02.g) as a inhibitor
potential of corrosion in a time of half-hour, is E=- 0.92volts(Fig2) and I=32µA and in
period of one-hour, becomes E=-0.84 volts I=1.9µA. Brown-colour deposits would
disappear(Fig3).By adding 10 mg humic acid to 50 ml sea water, corrosion potential becomes
E=-1.04volts I=90 µA (Fig 4).in other word, by increasing humic acid, corrosion increases.
This result can be approved by experiments of weight-reduction. average potential of
corrosion, resulted from few curves of polarization of electrode sample, in Buffer pH=7, will
be 0.817 volts .By putting 5 mg humic acid in Buffered solution ,corrosion potential becomes
0.805 volts.pH shows the effect of humic acid on corrosion of electrode sample[4, 5].
Table1: Elemental Analysis of steel sample
Element Al
Cr
Fe
Ni
Cu
Zn
Cd
Pb
%(w/w)
19
63.92
0.005
0.76
0.52
0
0
19.8
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Figure1: Electrochemical polarization of electrode sample in sea water (in period of half hour)
Figure2: Electrochemical polarization of electrode sample in sea water content 0.20 g of Benzoic acid
(in period of half-hour)
Figure 3: Electrochemical polarization of electrode sample in sea water content 0.20 g of Benzoic acid
(in period of one-hour)
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Figure4: Electrochemical polarization of electrode sample in sea water content 10mg of humic acid
(in period of half-hour)
Acknowledgments
The author’s is grateful to Dr. Hussein from university of Kerman (Faculty of Science) for
corporation in this work
References
1.
2.
3.
4.
5.
L.FP.dick and L.M.Rodrigues, corrosion science journal, 62, 1 (2006) 35
A.Mshams El Din, Desalination, 238(2009)166-173
S.M.A.Hossenini, A.Azimi, corrosion science, 51(2009)728-732
G.A.Zhang, Y.F.cheng, corrosion science (2009)
Z.Y.Lit, X.G.Li, C.W.Du, G.L.Zhaj, Y.F.cherg, corrosion science 50(2008)2251-2257
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Color Removal by Coagulation from Water Containing Aquatic Humic
Substances with Different Apparent Molecular Size
Eliane Slobodaa*, Camila Tolledo Santosb, Angela Di Bernardo Dantasb, Luiz Di Bernardob,
Eny Maria Vieiraa
a
b
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil;
Escola de Engenharia de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil
E-mail: elisloboda@hotmail.com
1. Introduction
Humic substances originate from soil humic material and aquatic plants. In surface waters
humic substances generally account for 50 to 70% of the dissolved organic matter (DOM) [1].
When aquatic humic substances (AHS) are not efficiently removed during the water treatment
steps, they can cause several problems such as serving as substrate for the growth of
microorganisms, complexing with metals such as Fe, Mn, Pb and others and making their
removal difficult, causing corrosion in piping, and producing substances with unpleasant
tastes and odors, some of which are toxic and potentially carcinogenic when preoxidation is
performed using free chlorine [2].
The coagulation of humic acid seems to be brought about two major mechanisms, depending
on the pH conditions: adsorption of the humic acid on the precipitate of Al(OH)3(s) at pH
above 6; and precipitation of the humic acid by the neutralization of charge through a soluble
or incipient solid-phase aluminum hydrolysis species in the range of pH 4.0 to 5.5 [3, 4]. This
study aimed to verify the influence of the apparent molecular size of AHS on the effectiveness
of coagulation with aluminum sulfate and characterize the DOM in different fractions de
AHS.
2. Materials and Methods
The water samples were collected from the Itapanhaú river (true color intensity in the order of
300 Hazen units), Bertioga, São Paulo State, Brazil. Extraction of humic substances was made
according to the procedure adopted by the International Society of Humic Substances (IHSS)
[5]. The extracted humic substances were filtered through a membrane with 0.45 μm pores
(Millipore) and this fraction was denoted as F1. Fraction F1 was then separated by
ultrafiltration using polyethersulfone membranes (Vivalflow50) into 3 apparent molecular
size fractions: F2: from 100 kDa to 0.45 μm, F3: from 30 to 100 kDa and F4: < 30 kDa.
Water from an artesian well was used to prepare 4 experimental samples: water sample I (F1),
water sample II (F2): water sample III: (F3) and water sample IV (F4). Water sample color
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intensity was 100 ± 5 Hazen units, turbidity 5.0 ± 0.5 NTU with added kaolinite, temperature
20 ± 1°C. Solutions of NaOH and HCl were used to adjust the coagulation pH.
Jar test equipment (ETICA) and direct filtration, was used to carry out the tests. The following
parameters were adopted to perform the tests: rapid mixing time = 30 s; velocity gradient =
1000 s-1; filtration time = 20 min; filtration rate = 60 m/d; each bench-scale sand filters
consisted of a 15 cm layer of sand with grain sizes of 0.30 to 0.59mm. The coagulant
employed was a commercial liquid aluminum sulfate with 7.28% (w/w) of Al2O3. Color
measurements were based on the procedure recommended by the Standard Methods for the
Examination of Water and Wastewater (1998) [6].
The characterization of DOM in Itapanhaú river was made as outlined in Figure 1 and
described in the sequence.
2 L of river water
filtered through
0.45μ m at pH 2
Elution with
NaOH 0.1 M
HPON
XAD-8
HPOA
Elution with
NaOH 0.1 M
Extraction with
acetonitrile/water
for 48 h, 50ºC in
Soxhlet extraction
TPHN
XAD-4
TPHA
Extraction with
acetonitrile/water
for 48 h, 50ºC in
Soxhlet extraction
HPI
Figure 1: Fractionation of DOM
The hydrophobic fraction (HPOA) and transphilic (TPHA), respectively adsorbed on XAD-8
and XAD-4 resins were eluted according to the methodology proposed by Malcolm and
MacCarthy (1992) [7]. After the water passed through the two resins, samples were collected
and were identified as hydrophilic fraction (HPI). The acetonitrile was removed from extracts
in rotaevaporador at 90 °C. The extracts were identified as hydrophobic neutral fractions
(HPON) and neutral transphilic (TPHN). Measurements of total organic carbon (TOC) in the
fractions were made by the spectrophotometric method (TOC Analyzer 5000ª).
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15th IHSS Meeting- Vol. 3
3. Results and Discussion
Considering only the relationship of the mass of each of the fractions isolated in the MOD
XAD-8 and XAD-4, it was found that the largest proportion of the DOM Itapanhaú River
water is represented by the fraction HPOA (46%), and smaller quantities HPI fractions (18%),
TPHA (15%), TPHN (11%) and HPON (10%). It justifies the use of the methodology of
Thurman and Malcolm (1981) [8] to obtain the SHA River water Itapanhaú.
The results of the coagulation filtration tests on water samples I to IV using aluminum sulfate
as a coagulant are shown in Figures 2a to 2d.
a) Results of Water Sample I
b) Results of Water Sample II
c) Results of Water Sample III
d) Results of Water Sample IV
Figure 2: Diagram of aluminium coagulation and color removal domains for water samples I to IV
In water sample I (Figure 2a), Zone 1 for ≥ 95% removal is defined by the pH ranging from
4.7 to 5.3 and aluminum dosages between 1.9 and 2.7mg L-1, Zone 1.1 for ≥ 95% removal is
defined by the pH ranging from 5.5 to 6.0 with aluminum dosages of > 3.5mg L-1 Al. Zone 2
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15th IHSS Meeting- Vol. 3
(color removal ≥ 90%) is defined by the pH ranging from 4.4 to 6.2 and aluminum dosages of
> 1.9mg L-1 Al.
Figures 2b and 2c show similar results, but for water sample IV (Figure 2d), indicating only
one zone for ≥ 95% removal efficiency, which was defined by the pH ranging from 4.9 to 5.5
and aluminum dosages of > 4.2mg L-1 Al; Zone 2 (≥ 90% removal efficiency) was defined by
the pH ranging from 4.5 to 5.6 and aluminum dosages of > 2.7mg L-1 Al.
4. Conclusions
To achieve the same degree of color removal, the water samples with lower apparent
molecular sizes required higher doses of aluminum sulfate. The coagulation probably
occurred as a result of charge neutralization through a positively charged hydrolyzed
aluminum species at lower pH values, while at higher pH values it probably occurred due to
the adsorption of humic and fulvic acids on the Al(OH)3(s) precipitate.
References
1. G.R. Aiken, in G.R. Aiken et al. (Eds.), John Wiley and Sons, New York, 1985, p. 363–385.
2. L.D. Bernardo and A.D.B. Dantas (2005). Methods and Techniques for Water Treatment, RiMa,
São Carlos, 2005.
3. P.N. Johnson and A. Amirtharajah, Res. and Techn. J. AWWA., 75 (1983), 239.
4. G. A. Edwards and A. Amirtharajah, J. AWWA., 77 (1985), 57.
5. http://www.ihss.gatech.edu, acessed in jun 2008.
6. Standards Methods for the Examination of Water and Wastewater 1998 20th Edition, APHA,
AWWA, AWPCF, Washington, DC, USA.
7. R.L. Malcolm and P. MacCarthy, Environ. Intern., 18(1992), 607.
8. E.M. Thurman and R.L. Malcolm, Environ. Sci. Technol., 15 (1981), 466.
Vol. 3 Page - 28 -
15th IHSS Meeting- Vol. 3
pH Effect in Aquatic Fulvic Acid From Brazilian River
Sérgio da Costa Saaba*, Eduarda Regina Carvalhob, Rubens Bernardes Filhob, Márcia Regina
de Moura Aouadab, Ladislau Martin-Netob, Luiz Henrique C. Mattosob
a
Departamento de Física, UEPG, Av. Carlos Cavalcanti 4748, CEP 84030-999, Ponta Grossa
PR Brazil; b Embrapa Instrumentação Agropecuária Rua XV de novembro 1452, CEP 13560970, São Carlos,SP, Brazil
E-mail: scsaab@uepg.br
1. Introduction
Presence of humic substances (HS) in a water supply is undesirable for several reasons, for
instance: it produces esthetical problems as color in the water; stabilizes dispersed and
colloidal particles during coagulation processes; leads to formation of biodegradable organic
compounds during ozonation and thereby enhances regrowth of microorganisms within the
water-distribution systems [1].
Atomic Force Microscopy (AFM) technique can image surfaces with atomic resolution by
scanning a sharp tip across the surface at forces smaller than the forces between atoms [2].
AFM is a technique which has been employed to study the morphologies of humic and fulvic
acid [3–5]. It is a powerful tool to characterize small colloids, as well as colloid
agglomeration, adsorption onto surfaces, or modification in morphologies affected by changes
in the physical-chemical properties.
The objective of this work was to get AFM images of aquatic acid fulvic (AFA) Brazilian
river, and zeta potential with pH change to verify the structural and morphologic change of
the AFA.
2. Materials and Methods
The aquatic HS were isolated from a sample collected from a tributary stream of River
Itapanhaú within of the State Park called "Serra do Mar". This is an environmental protection
area located in the seaboard, 7th UGRHI of 11th group of UGRHI from São Paulo State,
Brazil.
The extraction of fulvic acids from the river samples was made followng the methodology
suggested by International Humic Substances Society (IHSS) [6]. The imaging of AFM of
AFA samples was carried out at two pH values 3.0 and 9.0. These were used to identify
structural changes of FA when the pH varies. Images were obtained using the AFM
microscope Didimension V, Veeco. Tapping mode was used and Silicon SPM.
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15th IHSS Meeting- Vol. 3
For the study of zeta potential in function of the pH, AFA samples in a suspension of 100mg
sample in 1L distilled water milliQ were used. The suspension was sonificated for 30 min in a
60W bath ultrasound in 20 mL parts. pH was adjusted with the addition of 0.1M HCl or
NaOH at 20 oC and after 24 h the pH was readjusted. The equipment used was Malvern
Instruments, Zeta sizer nano ZS model Zen 3600.
3. Results and Discussion
Figure 1a (up) shows de AFA AFM images at pH 3.0 and in figure 1a (down) the height and
diameter of AFA particles on the mica sheet measured from the two straight lines indicated in
figure 1a (up). Agglomerates in the shape of pyramids can with diameter around 150–300 nm
and 10–55 nm high are observed.
4
60
1
2
1
2
50
height / nm
40
height / nm
1
2
3
3
30
2
20
1
10
0
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8 0 .9
1 .0
1 .1
1 .2
1 .3
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
X / μm
X / μm
a) pH 3.0
b) pH 9.0
Figure 1: AFA AFM image at pH a) 3.0 and b) 9.0Figure 1b (up) shows the AFA AFM images at pH
9.0. Figure 1b image indicates a more open distribution of AFA on the mica sheet when compared
with figure 1a image at pH 3.0. Dimensions can be observed in figure 1b (below) where the AFA
height on the mica at pH 9.0 was between 2.5–4.0 nm and diameter between 100–300 nm. With pH
increase, AFA particles expand and repel one another electrostratically. Strengths become weaker, due
to H bonding, van der Waals interactions and interactions of π electrons from adjacent molecules, with
dissociation of carboxylic and phenolic groups, generating negative charges [7] as shown in figure 2
(zeta potential)
Figure 2 shows the zeta potential variation with the pH of the river AFA sample. Zeta
potential becomes more negative with the increase in pH. There is a sharp increase in the
negative charge from pH 7.0, this fact coincides with the beginning of phenolic acid groups
ionization, with a gradual increase of these groups from pH 7.0. Thus, the contribution of
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15th IHSS Meeting- Vol. 3
phenolic acids for the formation of AFA negative charges in solution is more important than
the carboxylic groups.
0
ZP / mV
-30
-60
-90
2
4
6
8
10
12
Mean
Figure 2: Zeta potential variation with the AFA sample pH
4. Conclusions
This work showed that through atomic force microscopy techniques, structural change in
fulvic acid of a Brazilian river was identified when the solution pH varied. Results suggest
that in acid pH weak electrostatic interactions and hydrogen bonding are responsible for
aggregates formation while in alkaline pH electrostatic interactions are strong due to increase
in the phenolic groups ionization and low hydrogen interaction forming more open structures.
References
1.
2.
3.
4.
5.
6.
7.
E. R. Carvalho; L. Martin-Neto; D.M.B.P. Milori; J. C. Rocha; A. H. Rosa, J. Braz. Chem. Soc.,
15 (2004) 421.
F. L. Leite; P. S. P. Herrmann; J. Adhesion, Sci. Technol., 19 (2005) 365.
K. Namjesnik-Dejanovic and P. A. Maurice, Colloids and Surf. A: Physicochem. Eng. Aspects,
120 (1997) 77.
M. Plaschke; J. Rothe; T. Schäfer; M. A. Denecke; K. S. Dardenne; K. Pompe; H. Heise, Colloids
Surf. A: Physicochem. Eng. Aspects, 197 (2002) 245.
J. M. Gorham; J. D. Wnuk; M. Shin; H. Fairbrother, Environ. Sci. Technol., 41 (2007) 1238.
R. S. Swift; in Sparks (Ed), Methods of Soil Analysis Part 5, Soil Sci.Soc.Am.:, Madison, 1996,
p.1018.
R. A Alvarez-Puebla. and J .J. Garrido, Chemosphere, 59 (2005) 659.
Vol. 3 Page - 31 -
15th IHSS Meeting- Vol. 3
The Role of Organic Matter in the Transport of Suspended Minerals in the
Estuarine Zone
Lasareva E.V.a, Parfenova A.M.a, Romankevich E.A.b
a
Chemistry Department Moscow State University, 1 Leninskie gory, 119991 Moscow, Russia;
b
Shirshov Institute of Oceanology, 36 Nachimovski prospect, 117997 Moscow, Russia
E-mail: elasareva@ya.ru
1. Introduction
The idea of V.I. Vernadsky, concerning the important role of boundary zones in the ocean, as
zones of considerable biogeochemical activity, especially takes place in the marginal filter —
zone of river and seawater mixing in river mouth. Large-scale processes of flocculation and
coagulation of dissolved (colloidal) and suspended matter, sorption on newly formed surfaces
take place in this zone. All these processes result in that 93–95% of suspended matter and 20–
40% of dissolved matter of river discharge (pollution included) is deposited in the zone [1].
Our observations of the seasonal distribution of particulate and dissolved organic matter (OM)
concentrations in the Severnaya Dvina river and the Amazon river estuaries showed a
tendency to inverse patterns of distribution of particulate (POC) and dissolved (DOC) organic
carbon. It was shown that the decrease in DOC concentrations is accompanied by the growth
of POC concentrations. This pattern is observed at salinity 1–8 ‰ and may be considered as a
result of coagulation and flocculation processes [2].
2. Material and methods
To confirm the nonconservative behavior of DOM and to understand the role of OM in the
coagulation and flocculation processes in estuarine zone we conducted experiment, using
model systems – suspensions of different clay and carbonate minerals under increasing
salinity from 1 up to 35‰. Montmorillonite and kaolinite (1 g/L) were studied as clay
minerals, carbonate minerals —calcite and aragonite were prepared in laboratory [3]. The
addition of model organic substances —chitosan (as flocculent and fulvic type OM) and
humic acids (HA) under varying salinity values allows us to study the role of OM (humic and
fulvic types) on the processes of coagulation and flocculation. In thre study 1mL of 0.05 %
chitosan solution (Mw=300000) and 0.1 mL of 1 g/L HA (Humintech Ltd., Germany)
solution were added to the mineral suspensions. The optical density of suspensions after
mixing of minerals with salt (NaCl) and OM was used as an indicator of stability of the model
systems.
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15th IHSS Meeting- Vol. 3
3. Results
The laboratory experiments showed that kaolinite suspensions are more stable under different
salinity conditions than montmorillonite ones. The addition of chitosan solution to clay
suspensions leads to flocculation of montmorillonite suspensions just at small salinity (1 ‰)
and does not effect kaolinite flocculation under studied conditions. The addition of HA
solution to clay and carbonate suspensions does not change greatly the behaviors of
suspensions at different salinity. It is interesting to mention that the joint action of HA and
chitosan solutions leads to the flocculation of kaolinite at 7‰ (Fig. 1).
1
2
3
4
1,2
1,0
D
0,8
0,6
0,4
0,2
0
2
4
6
8
10
12
14
16
18
20
22
S, ‰
Figure 1: Dependence between the optical density of the kaolinite suspension and the salinity (1), the
same 3 hours after of НА addition (2), the same 20 hours after of НА addition, (3), the same with
addition of НА and chitosan (4)
2. Conclusions
The laboratory data allow to explain in-situ investigations concerning the difference in clay
and carbonate behavior in estuarine systems and show that the joint action of OM of humic
and fulvic type may lead to removal of clay mineral in estuarine zone, while the presence only
humic or fulvic type OM may stabilize suspensions and allow them migrate to long distance.
The mechanisms of flocculation and coagulation of clay and carbonate minerals under
increasing salinity in estuarine zone are considered. The further investigation may clarify a
number of related problems, as the transport of mineral, nutrients and pollutants from river to
ocean, the explanation of sedimentation rates, and the role of colloidal fraction in the
mechanisms of dissolved and suspended matter removal.
References
1. A.P. Lisitsyn. The marginal filter of the ocean. Oceanology. 1995, V. 34, № 5. P.671.
2. A. Vetrov, E. Romankevich. Carbon Cycle in the Russian Arctic Seas. 2004, Springer, 331p.
3. J.L. Wray, F. Daniels. Precipitation of calcite and aragonite. J. Am. Chem. Soc. 1957, V. 79, № 9.
P.2031.
Vol. 3 Page - 33 -
15th IHSS Meeting- Vol. 3
Organic Material of Uneven-Age Anthropogenic Origin Lakes
Sofia Zalmanova
St. Petersburg State Agrarian University, Faculty of Soil Science and Agroecology, Soil
Science and Soil Ecology Department name of Pr. L. N. Aleksandrova, Petersburg’s road, 2,
Pushkin, St. Petersburg, Russian Federation 196601
E-mail: lisofang@yandex.ru
1. Introduction
Global stability of biosphere depends on preservation and maintenance of water ecosystem
functioning. In this case artificial lakes should possess ability to self-restoration and dynamic
adaptation to external influences, and also not to cause basic changes in developed
ecosystems, surrounding lakes. In this connection the questions linked to studying water
ecosystems are actual. The accent of the research work has been made on a problem of
organic material (basically submitted by HS) and fresh lake bottom sediments, as to the most
important characteristic of an ecological condition lake ecosystem.
For the description of a lake ecosystem ecological condition the following researches have
been carried out: the chemical estimation of water in investigated reservoirs (definition of
oxidation-reduction potential, pH, quantities of humic organic substances, the contents of
heavy metals); the chemical estimation of lake sediments (definition of oxidation-reduction
potential, pH, losses of ignition, hygroscopic humidity, chemodestruction fractionating of
organic material); the estimation of an ecological condition of reservoirs on biological
parameters (the maximum(supreme) water vegetation and macrozoobenthos).
2. Materials and Methods
In this research work the problem of artificial lakes is considered by the example of the park
Suoranda (Leningrad region). Investigated lakes have appeared on a place of extraction of
sand. It is the most widespread reason of occurrence of lakes of an anthropogenic origin in
our region. As a result of biological succession the career landscape to the external attributes
becomes similar to typical for Northwest of Russia moraine landscapes with a characteristic
combination of lakes and woody hills. The given territory functions as technogenic
disturbance of natural environment landscape and represents interest for research as allows
expanding representations about restoration of natural ecosystem after anthropogenic
intervention [2].
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Humic substances (HS) are extracted from lake water by the sorption method. For this aim the
lake water with HS was acidified by a sulfuric acid up to рН = 1-2, then it was filtrated
through special gel sorbent, on which humic substances were sorbed. Desorption of HS from
a sorbent have been made by using an alkaline solution (0.1 M NaOH).
For an estimation of qualitative composition of lake sediment organic matter the method of
chemodestruction fractionating (CDF) was used. Various components of bottom sediments
organic matter (BSOM) or various parts of organic macromolecules (including HS) have a
different resistance to biota enzymes. Relatively difficult (stable) and easy degraded (labile)
organic compounds (or fragments of macromolecules) have different role in BSOM system. A
ratio of stable and labile BSOM parts is an important characteristic of ecosystem [1].
The method of CDF concerns the physic-chemical analysis of soils. It is based on different
susceptibility of SOM components to an oxidizing agent. That the higher a rate of organic
matter decomposition in soil, the higher an ability of its destruction by mineral oxidizers. The
stronger the oxidizing solutions, the more the oxidation of BSOM components. On this basis,
we can divide the SOM into labile and stable parts. The resistance of BSOM components to
oxidation was connected with both the chemical composition and the spatial three-dimension
structure of macromolecules, especially so far as concerns the native humic substances. The
easily oxidized organic compounds and/or fragments of macromolecules actively take part in
trophical cycle of the biota of lake sediment and higher plants, as main source of substances
and energy. This fraction stipulates the biochemical properties. In turn, the difficult oxidized
organic material influence on physical and physic-chemical properties. The biota of lake
sediments can decays the difficult degraded material to the easily degraded organic
compounds [1].
For an establishment of red-ox potential of lakes the contents of iron in sediments and water
has been determined: the ratio of ferrous and ferric iron presented aerobic and anaerobic
conditions and character of oxidation-reduction processes. Iron was determined by
colorimetric method as the painted complex with С7Н6О6·2Н2О.
Measurements of concentration of metals in samples of water have been made by a method
atomic absorption spectrometry [3].
The ecological status of artificial lakes of various age was estimated by O.N. Mandryka and
L.V. Kulangievoj in indissoluble connection with character of a landscape and inherent in its
plant associations various type [2].
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15th IHSS Meeting- Vol. 3
3. Results and Discussion
In the table 1 the integrated ecological estimation of the investigated reservoirs based on the
characteristic of properties lake sediments and water, and also on biological parameters of
reservoirs is resulted are presented.
Table 1: The comparative characteristic of Marsh lake and Career - 1
The characteristic of research objects
Lake Marsh
Age of a reservoir
About 50 years
рН of waters
5,95 (poorly sour)
рН of lake bottom sediments
6,85 (neutral)
The contents humic substances in water of
~1
reservoirs, mg TOС/l
Distribution of organic substance in lake
Non-uniform between
bottom sediments
fractions, are present by the
semidecomposed plant
remains
The contents of organic substance in lake
8,7
sediments, %
Geochemical conditions
Regenerative, there are
attributes of bogging
Prevailing processes of transformation of
organic substance in lake sediments
Type of a reservoir
Saprobity [2]
destructive
Dystrophic
1.504 (between oligo-and βmesosaprobic)
Career-1.
About 6-7 years
6,09 (poorly sour)
6,67 (neutral)
No
Uniform, than 0,25 mm
are dated for fraction less
8,4
Regenerative, but with
the big enrichment by
oxygen
Synthetic
Oligotrophic
1.805 (between
oligosaprobic and βmesosaprobic)
In organic matter of Career - 1 sediments synthetic processes dominate above destructive, and
over lake Marsh on the contrary — prevalence of destructive processes.
On the data obtained from CDF of organic substance of lake adjournment is received, that as a
result of the processes of transformation of organic substance the unstable and unbalanced
system of components of organic substance lake sediments of both lakes is formed kinetically.
In sediments of lake Marsh the quantity ferrous iron was more, than in lake sediments of
Career - 1, in this lake is more expressed development marsh process. In lake sediments of
Career - 1 prevails quantity ferric iron that apparently, specifies enrichment of oxygen in this
reservoir.
Concentration of copper, lead, cadmium, iron, zinc in the samples of water of both lakes
corresponded to the established ecological norms. In the samples of water of both reservoirs
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15th IHSS Meeting- Vol. 3
concentration of manganese and iron exceeds maximum concentration limit, therefore under
the contents of manganese and iron in waters of the investigated objects the given lakes are
suitable only for the recreational purposes.
Less than 0.25 mm Career - 1 contained sediments of fraction of more fine-material an
organic material, than lake Marsh, except for that in sediments of lake Marsh there was a
significant amount of large fossils that corresponds to the type of these lakes: oligotrophic –
Career -1 and dystrophic - Marsh lake.
4. Conclusions
On the basis of the carried out researches, it is possible to tell, that organic matter of water
and bottom sediments of lakes is in interaction with other characteristics of lakes and together
they form representation about ecological conditions of lakes. The lake Marsh in which
contents of organic matter prevails can be attributed to lakes of the dystrophic type, Career-1
— oligotrophic type. The ecological condition Career-1 and lakes Marsh is characterized as
poorly polluted.
References
1. A. I. Popov, V. P. Tsiplenkov, M. A. Nadporozhskaya., G. T. Frumin An estimate of qualitative
humus composition by the chemodestruction fractioning// Humic Substances in the Global
Environment and Implications in Human Health / Abstracts, 6th Int. Meeting IHSS. Italy.
Monopoli (Bari), 1992, p. 240.
2. O.N. Mandryka., L.V. Kulangieva, The Concept of transformation technogenic reservoirs in
ecologically steady complexes for recreation the population near of megapolis // The Theory and
practice of restoration of internal reservoirs. Works of the international scientific practical
conference. (St.-Petersburg, October, 15-18, 2007). — SPb., 2007, p. 248-253.
3. I. Havesov, D. Tsalev The Method of atomic absorption spectrometry/ Translation. from
Bulgarian. — Leningrad, 1983.
4. I. V. Baranov The Organic materials in lake bottom sediments and water basins and its
bioproductive value, Theses of reports of IV congress All-Union Geobiotic Association. In 3
parts, Ch. 1.-Kiev, 1981, p. 98-99.
Vol. 3 Page - 37 -
15th IHSS Meeting- Vol. 3
Comparative Measurement of Hydrophobic Organic Matter Dissolved in
Water by the XAD Resin Method and the Polarity Rapid Assessment
Method (PRAM)
Marc Philibert, Alex Revchuk, David Quiros, Arthur Roh, Mel Suffet*
Environmental Science and Engineering Program - UCLA, School of Public Health, Room
46-081-CHS, Charles E. Young Drive South Los Angeles, CA 90095-1772, USA
E-mail: msuffet@ucla.edu
1. Introduction
Dissolved organic matter (DOM) is a mixture of thousands of molecules prevalent in water
bodies. Dissolved organic matter is responsible for multiple water treatment issues including
trihalomethanes formation and membrane fouling. A better understanding of the physical and
chemical characteristics of DOM (e.g. polarity and size fractions) is necessary to find ways of
controlling these water treatment problems. Multiple measurements over time are needed at
ambient pH to define how these problems occur. Therefore, a quick probing polarity method
is needed for the evaluation. The polarity rapid assessment method (PRAM)1.2 was chosen to
study these problems at ambient pH and ionic strength with the PRAMs solid phase extraction
cartridges of different polarity in parallel. The classical methods of evaluating the polarity of
DOM is the XAD resin series column adsorption method3. The resin method has been used
for over two decades. The objective of this study was a comparison of Resin3 and PRAM1,2
methods to try to relate data in the literature using the XAD resin method and the PRAM
method. During the evaluation, the PRAM method was run in series as the XAD resin method
is completed to develop a more consistent evaluation of comparative polarity. Also, the pH
was run at ambient pH and adjusted to <2 as used in the XAD resin method.
2. Materials and Methods
The water samples used in this study include seven samples from the City of Fort Collins, and
one humic standard (Suwannee River, IHSS). Two sets of influent and effluent samples from
the city’s water treatment plant taken in separate months, and three samples from the city’s
main water resources were studied. These samples were all analyzed using the XAD resin
fractionation method1 and PRAM2,3. Ultrafiltration4 and fluorescence spectroscopy5 of the
fractions studied from these methods was used to further evaluate the similarities and
differences between the methods. The basis for the fluorescent regions described by the
fluorescence method relies on isolates from the XAD methods. The fluorescent regions
obtained are still used to analyze PRAM fractions as no PRAM study has been completed to
interpret fluorescence regions.
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3. Results and Discussion
Figure 1: represents the comparison between C18 and the XAD-8 retention for DOC at the Fort
Collins Water Utility at pH <2. The results from the two methods are poorly correlated pH <2
indicating that the two methods do not sorb the same material and one cannot be substitued for the
other. This hinders the possibility of using historical XAD-8 data alongside recent PRAM results.
y = 0.3985x + 18.283
R2 = 0.2498
corre lation C18 (pH 2) and XAD-8 for DOC
70.00
60.00
C18 (pH 2)
50.00
40.00
30.00
20.00
10.00
0.00
25.00
35.00
45.00
55.00
65.00
75.00
XAD-8
Figure 1: Correlation of C18 and XAD-8 for DOC for Fort Collins
Figure 2 shows the fluorescence analysis of the Fort Collins Water Treatment Facility’s raw
water for, PRAM (pH neutral and pH< 2) and XAD fractions (pH < 2). It is observed that the
lowering the pH impacted the fluorescence response. It is clear from this figure that the
characteristics of every sample analyzed are different. The main peaks for all the samples are
from the II, III and V regions according to the paper by Chen et al., (2003) based upon the
resin method. However the comparison between the C18 and XAD resins at pH 2 shows that
the Fluorescent region I is more important for the C18 cartridge while the XAD resins have a
higher peak in the Fluorescent region IV. This leads to the conclusion that the two methods
while retaining a so-called similar fraction, of ‘hydrophobic compounds’ as defined in the
methods have enough differences that their direct comparison is not straightforward and
confirms the poor correlation observed on Figure 1.
Percentage intensity FCWTF influent June 2008
60
Raw ambient pH
C18 pH2
C18 ambien pH
XAD-8
Raw pH2
XA1D-8/XAD-4
% intensity
50
40
30
20
10
0
I
II
region III
IV
V
Figure 2: Fluorescence percentage intensity for FCWTF influent in June 2008 at ambient pH and pH 2
for raw, C18 and pH =2 only for XAD-8 and XAD-8/XAD-4
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15th IHSS Meeting- Vol. 3
Figure 3 shows the fluorescence results of UF fractions and of their respective C18 cartridge
effluents. Once again we see that the C18 cartridge increases the importance of the region I.
This means that that region is the least hydrophobic (most hydrophilic) as it completely goes
through the cartridge. The other regions all decrease in importance, with regions III and V
decreasing the most. This highlights that these two regions are the most hydrophobic as they
are retained by the C18. The effect of the UF is different. The peaks retain their importance
through the different membranes though they don’t react in the same way. The main peak of
region III increases through the three membranes. Region III representing molecules of fulvic
acid-like functionality. This increase of region III is counterbalanced by decreases in region I
and IV. This shows that regionsI and IV are responsible for the production of larger
molecules. Molecules in these regions are representative of microbial by-products and
aromatic proteins that are indeed likely to be larger.
FCWTF influent
Raw
1k
1k C18
5k
5k C18
10k
10k C18
% intensity
80
60
40
20
0
I
II region III
IV
V
Figure 3. Fluorescence characteristics of UF fractions and their C18 fraction
4. Conclusions
This study shows that the two methods used here: the XAD resin adsorption and PRAM
though used for similar purposes are not directly comparable to define hydrophobicity. The
comparison of the two methods focused mainly on the C18 cartridge and the XAD-8 and
XAD-8/XAD-4 resins. All three of these are used in the literature to evaluate the hydrophobic
character of organic matter. We show here that these two methods are not equivalent and
retain different fractions of the organic matter. This emphasizes the difficulty of defining the
hydrophobic fraction of a sample. Both methods assume that the material they are retaining is
hydrophobic as this fraction is retained by a hydrophobic adsorbing material though there is
no correlation between the retention of these materials. The difference between the retention
of these two methods is seen more clearly in the fluorescence intensity measurements. We
notice that the C18 cartridge’s effluent prominently lets region I (aromatic protein) material
through whereas the XAD method removes that region. This fluorescent region is defined by
Vol. 3 Page - 40 -
15th IHSS Meeting- Vol. 3
aromatic protein and is consistent for interpretation by both methods. Also, XAD resins are
more partial to the region type IV molecules. This could be due to the interpretation of
fluorescence data from a point of view of XAD isolation for region IV. Can the differences
and potential complementary use of the two methods be utilized for evaluation of polarity is
an open question?
Finally this study shows that the use of the PRAM method coupled with fluorescence and/or
ultrafiltration can be used to gather more insight into the hydrophobic fraction of organic
matter. The ultrafiltration method described here allows one to interpret the size distribution
of the samples. Once the UF method is run, the collected effluent is run by PRAM and finally
the PRAM effluent material is run on fluorescence. This allows a comparison of the
hydrophobic fractions of each size fraction as well as their fluorescence signature. This
method can be used to evaluate correlations between these fractions and different parameters
of concern such as THM formation or membrane fouling.
Acknowledgements
The authors would like to thank Dr. Judy Billica of the City of Fort Collins Utilities for her
help in obtaining all the samples. We thank the partial support of research funding from the
City of Fort Collins, City of Greeley, Tri-Districts, Northern Colorado Water Conservancy
District and the Metropolitan Water District of Southern California. Many thanks also to Dr.
Eric M. V. Hoek of the Civil and Env. Eng. Dept. at UCLA for use of his XAD Resin set-up.
References
1. Rosario-Ortiz, F. L., Snyder, S. and Suffet, I. H. (2007a). Characterization of dissolved organic
matter in drinking water sources impacted by multiple tributaries. Water Res. 41: 4115–4128.
2. Rosario-Ortiz, F. L., Snyder, S. and Suffet, I. H. (2007b). Characterization of the polarity of
natural organic matter under ambient conditions by the polarity rapid assessment method (PRAM).
Environ. Sci. Technol. 41: 4895–4900.
3. Singer, P.C, Schneider, M., Brandt, J.E. and Budd, G.C. (2007). "MIEX for removal of DBP
precursors: pilot-plant findings". J. Amer. Water Works Assoc. 99: 128–139.
4. Revchuk, A. D. and I. H. Suffet, 2009. Quality Assurance of Ultrafiltration Separation for Humic
Substances by Chemical Probes. Water Res., In press.
5. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K. Fluorescence Excitation-Emission Matrix
Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci, Technol.
2003, 37 (24).
Vol. 3 Page - 41 -
15th IHSS Meeting- Vol. 3
Microbial Changes in the Spectroscopic Characteristics and Molecular
Weight of Dissolved Organic Matters Extracted from
Diverse Source Materials
Jin Hur*, Bo-Mi Lee, Tae-Hwan Lee, Ka-Young Jung
Dept. of Earth and Environmental Sciences, Sejong University, Seoul, 143- 747, Korea
E-mail: jinhur@sejong.ac.kr
1. Introduction
Dissolved organic matter (DOM) plays important roles in aquatic ecosystems by supplying
nutrients and energy to heterotrophic organisms, by functioning as a carbon carrier in a carbon
cycle, and by altering light environments. Many environmentally related DOM reactivities are
known to be correlated with the physico-chemical properties and the composition of DOM,
which highly depend on the precursor materials (i.e., sources). The temporal and the spatial
variations in the apparent characteristics of DOM in rivers and lakes are likely to be attributed
to the respective mixing of different DOM sources and the combination of several natural
fractionation and/or transformation processes. In general, the degree of the variations
observed in watersheds tends to be much smaller than those among the DOM freshly
produced from diverse source materials [1]. Microbial transformation has been suggested as a
crucial factor to explain the relatively smaller variations. Despite a number of the studies that
investigated microbial transformation on DOM characteristics, little effort has gone to explore
potential microbial changes in correlations among DOM characteristics. The objectives of this
study were as follows: (1) investigate microbial changes in the selected characteristics of the
DOM prepared from diverse source materials and (2) compare the correlations among some
selected DOM characteristics before versus after microbial incubation.
2. Materials and Methods
The DOM sources for this study included diverse organic materials that may serve as the
precursor materials for organic constituents in watersheds (i.e., treated sewage, suspended
algae, attached algae, paddy field soil, field soil, sediment, reed, leaf, and weed). A constant
volume (250 mL) of DOM samples for microbial incubation was prepared in sterile 300-mL
Erlenmeyer flasks by diluting the DOM extract solutions to 30 mg C/L. Aliquots (4 mL) of
the prepared inoculum solution were added to each incubation flask. The flasks were sealed,
incubated in the dark at 20 °C for 28 days, and gently shaken every day. Concentrations of
dissolved organic carbon (DOC) were determined using a Shimadzu V-CPH analyzer.
Absorption spectra were measured at 1-nm increments over the wavelength range 200–600
Vol. 3 Page - 42 -
15th IHSS Meeting- Vol. 3
nm with a spectrophotometer (Evolution 60, Thermo Scientific). Synchronous fluorescence
spectra of the samples were recorded with a luminescence spectrometer (Perkin-Elmer LS50B). Excitation and emission slits were adjusted to 10 nm and 10 nm, respectively. The
excitation wavelengths ranging from 250 to 600 nm were used with constant offsets (Δλ = 30
nm). Size exclusion chromatography (SEC) was used to determine apparent weight- and
number-average molecular weight (MWw and MWn) values of DOM samples following the
methodology reported by Hur and Schlautman [2].
3. Results and Discussion
Microbial changes in synchronous fluorescence spectra of DOM: Synchronous fluorescence
spectra of the DOM after incubation are presented in Fig. 1.
6.0
(a) Effluent
5.0
DOC-normalized QSE
-1
(QSE-L mg C )
DOC-normalized QSE
(QSE-L mg C-1)
6.0
(before incubation)
4.0
3.0
2.0
1.0
0.0
(b) Effluent
(after incubation)
5.0
4.0
3.0
2.0
1.0
0.0
250
300
350
400
450
500
550
600
250
300
350
Wavelength (nm)
6.0
(c) Algal-derived
5.0
DOC-normalized QSE
-1
(QSE-L mg C )
DOC-normalized QSE
-1
(QSE-L mg C )
6.0
(before incubation)
4.0
Lake algae
3.0
Attached algae
2.0
1.0
0.0
500
550
600
(d) Algal-derived
5.0
(after incubation)
4.0
Lake algae
3.0
Attached algae
2.0
1.0
300
350
400
450
500
550
600
250
300
350
Wavelength (nm)
400
450
500
550
600
Wavelength (nm)
6.0
6.0
DOC-normalized QSE
-1
(QSE-L mg C )
(e) Terrestrial
5.0
(before incubation)
4.0
Field soil
3.0
Lake sediment
2.0
Paddy soil
1.0
(f) Terrestrial
5.0
(after incubation)
4.0
Field soil
3.0
Lake sediment
2.0
Paddy soil
1.0
0.0
0.0
250
300
350
400
450
500
550
250
600
300
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
6.0
6.0
(g) Plant-derived
5.0
DOC-normalized QSE
-1
(QSE-L mg C )
DOC-normalized QSE
-1
(QSE-L mg C )
450
0.0
250
DOC-normalized QSE
(QSE-L mg C-1 )
400
Wavelength (nm)
(before incubation)
4.0
Leaf litter
3.0
2.0
Weed
Reed
1.0
(h) Plant-derived
5.0
(after incubation)
4.0
Reed
3.0
Weed
2.0
Leaf litter
1.0
0.0
0.0
250
300
350
400
450
500
550
600
250
300
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Figure 1: Comparison of DOC-normalized synchronous fluorescence spectra for dissolved organic
matters (DOM) from diverse sources prior to versus after incubation
Vol. 3 Page - 43 -
15th IHSS Meeting- Vol. 3
Substantial changes were observed for the algal- and the plant-derived DOM groups. For both
DOM groups, the major changes were the decrease of protein-like fluorescence (PLF) region
and the enhancement of other HS-associated fluorescence regions. In contrast, relatively small
changes were observed for the spectra of the other DOM samples. Irrespective of the DOM
investigated, the fluorescence shifted to longer wavelengths after incubation. The trend of the
fluorescence shift may be associated with enrichment of more condensed aromatic structures
with electron-withdrawing substituents and/or more conjugation of aliphatic chains [3]. The
simultaneous occurrence of the PLF disappearance and the enhancement of HS-like
fluorescence characteristics imply the possibility of the mutual relationship between the two
fluorescence regions.
Changes in the correlations among DOM characteristics by microbial incubation: For this
study, no correlation was found between SUVA and fluorescence index (FI) values before
incubation (Fig. 2a). The result was not consistent with the previous report of a negative
correlation between aromatic carbon content and FI values for aquatic fulvic acids. After
incubation, however, the correlation became slightly negative although it was not statistically
significant (P = 0.117) (Fig. 2b). This comparison suggests that the typical trend of decreasing
FI with higher SUVA values may not be applied to such freshly produced DOM samples and
also that the typical trend may be limited to the DOM collected in natural waters, in which
various natural transformation processes are occurring.
2.50
(a)
2.0
1.5
Treated sewage
Algal-derived
Terrestrial
Plant-derived
1.0
Before incubation
Fluorescence Index (FI)
Fluorescence Index (FI)
2.5
0.5
(b)
2.00
1.50
Treated sewage
Algal-derived
Terrestrial
Plant-derived
1.00
0.50
0.00
0.50
1.00
1.50
2.00
2.50
0
1
SUVA280 (L mg C-1m -1)
2
3
4
5
SUVA280 (L mg C-1m -1 )
1600
1600
1200
Treated sewage
Algal-derived
Terrestrial
Plant-derived
800
Before incubation
400
r = 0.848, p = 0.004
(d)
MWn (g/mol as PSS)
(c)
MWn (g/mol as PSS)
r = 0.561, p = 0.117
After incubation
1200
Treated sewage
Algal-derived
Terrestrial
Plant-derived
800
After incubation
400
r = 0.483, p = 0.188
0
0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.00
Spectral slope (S275-295 )
0.01
0.02
0.03
0.04
0.05
0.06
Spectral slope (S275-290)
Figure 2: Correlations between SUVA280 values and fluorescence index for diverse sources of DOM
prior to incubation (a) and after incubation (b). Correlations between spectral slope (275–290 nm) and
MWn values for diverse sources of DOM prior to incubation (c) and after incubation (d).
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15th IHSS Meeting- Vol. 3
Several previous studies have shown that spectral slope is negatively correlated with MW of
DOM [4]. For this study, however, the opposed trends were observed between spectral slope
and MW differed for the DOM samples before and after incubation (i.e., freshly produced
versus biodegraded DOM). Before incubation, a significant negative correlation was observed
between the two DOM descriptors whereas the correlation became weakly positive after
incubation (Figs. 2c and 2d). The results suggest that although the typical trend between
spectral slope and DOM MW may be applied to diverse sources of freshly produced DOM, it
is impossible to generalize the trend for aquatic environments with microbial degradation
occurring.
4. Conclusions
In general, the extent of microbial changes in the selected DOM characteristics including
SUVA, fluorescence, and MW values was more pronounced for the DOM types containing
higher biodegradable carbon content (i.e., algal-derived and plant-derived DOM). Irrespective
of the DOM sources, microbial changes resulted in the enhancement of HS-like structures and
the associated characteristics. Microbial utilization of biodegradable organic substances
appears to be prerequisite for the enhancement. This study provided significant evidences that
microbial transformation affected the correlation among several DOM descriptors. The results
imply that the correlations established based on freshly produced DOM may not be applied to
aquatic environments where microbial degradation is occurring.
Acknowledgements
This work was supported by National Research Foundation of Korea Grant funded by the
Korean Government (No. 2009-0058569).
References
1.
2.
3.
4.
K.P. Wickland, J.C. Neff, G.R. Aiken, Ecosystems 10 (2007) 1323–1340.
J. Hur, J. and M.A. Schlautman, Environ. Sci. Technol. 37 (2003) 880–887.
J. Hur, J., M.-H. Park, M.A. Schlautman, Environ. Sci. Tech. 43 (2009) 2315–2321.
J.R. Helms, A. Stubbins, J.D. Ritchie, E.D. Minor, D.J. Kieber, K. Mopper, Limnol. Oceanogr. 53
(2008) 955–969.
Vol. 3 Page - 45 -
15th IHSS Meeting- Vol. 3
Dynamics of Humic Matters in Fen Bog Water in Conditions of Climate
Change
Ivanova E.S.*, Voistinova E.S., Kharanzhevskaya J.A.
Siberian Research Institute of Agriculture and Peat, Gagarina st. 3, Tomsk, 634050, Russia
E-mail: Ivanova_e_s@bk.ru
1. Introduction
Processes of organic substance transformation are one of the main phases of the biological
cycle. They ensure general stability of the biosphere. Main processes of organic substance
transformation are decomposition with liberation of СО2 and СН4 and humification, process
of formation of a special class of compounds —humic acids. Despite the fact that there is a
wide range of materials dedicated to this subject, there are still some not yet fully investigated
issues. This work presents the study of correlation dependencies of humic substance content
in swamp waters on main hydrometeorological parameters: levels of swamp waters,
temperature of peat beds, oxidation-reduction potential and humidity of peat beds. Analysis
was carried out using the data for vegetative periods of seven years.
2. Materials and Methods
A native fen situated on a low river terrace of Bakchar river in Bakhchar district of Tomsk
region, Russia, has been chosen as a model for study of humic substances content in bog
waters. Average thickness of peat deposit in the central open part of the fen is 3–4 m,
maximal depth is marked in near-slope part of the terrace basin – 5,5 m. The peat deposit of
the central part of the fen mass to the depth of 2–2,5 meters in the top is composed of swamp
hypnum and sedge-hypnum types of peat. Fraction of sedge peat in composition of the peat
deposit of fen increases from the flood-plain part to near-terrace slope as well as diversity of
peat types it’s composed of.
In terms of their composition waters of the studied fen pertain to geochemical type of
hydrocarbonated calcium-magnesium waters. Values of bog water mineralization range from
32.67 to 110.81 mg/L. Average content of macro-components in bog waters: HCO3 (87.82
mg/L). Ca (20.98 mg/L). Mg (8.88 mg/L). Fe (5.17mg/L). Medium reaction is weakly acidic.
close to neutral (рН 5.28–7.07).
Research methods included the study of organic substance content depending on main factors
of natural processes that occur in swamps: peat humidity, peat bed temperature, level of
swamp waters and oxidation-reduction potential. Analysis of humic substances was carried
Vol. 3 Page - 46 -
15th IHSS Meeting- Vol. 3
out according to [1]. Samples for chemical analysis, determination of humic substances,
measurement of bog water levels were taken from stilling wells according to [2].
Observations of oxidation-reduction potential (ORP) and temperature regimes were conducted
per layers of 10 centimeters down to mineral ground be means of fixed measuring elements
[3, 4]. Peat samples for humidity measurement were taken from all depth levels according to
[5]. Observations of ORP processes, peat deposit temperature, its humidity, and taking of
samples for chemical analysis and determination of humic substances were conducted once a
month from May to September 2002 to 2008.
3. Results and Discussion
Bog waters are characterized by high concentrations of fulvic acids (from 33.8–137.9 mg/L)
and low concentration of humic acids (from 1.2 to 19.81 mg/L). Monthly average
concentration of humic acids in bog water for a year over a period of study didn’t change and
amounted to 7.5±1.3mg/L in contrast to fulvic acids whose content was more variable –
70.3±20 mg/L. Changes of average concentration of humic acids over the period of
observation were cyclic. Maximum values of humic acids were registered in 2004, in the
conditions of the highest bog water levels (up to +12 in August), the highest amount of
precipitation (190 mm in July) over the period of study and high air temperatures. In 2006 the
content of humic acids in the bog waters was the lowest, in the conditions of the lowest bog
water levels over the period of seven years (average level for vegetative period -28 cm).
Concentration of fulvic acids from 2004 to 2007 was gradually decreasing. In 2004 content of
fulvic acids was at its maximum. Decrease of concentration over the following years of
observations was registered at the time when the bog water level decreased. Fulvic acids are
very soluble in comparison to humic acids and easily migrate both in forms of free acids and
as parts of compounds.
The conducted analysis indicated the necessity of investigation of correlation and regression
dependencies of humic substance content in bog waters on main hydrometeorological
parameters. Within limits of one parameter, coefficients of correlation not only differ in
magnitude but also are of different signs. Differences in dependencies of humic substances on
peat deposit temperature and ORP are, first of all, connected with non-uniformity of water
regime, consequently, with differences in limiting factors (water level, air temperature, etc.).
It’s hard to distinguish parameters that are crucial in determining the concentration of humic
substances of bog water. High correlation of fulvic acids with levels of bog waters is observed
during high-water years, whereas during other years either the coefficient of correlation
Vol. 3 Page - 47 -
15th IHSS Meeting- Vol. 3
changes its sign or this relationship becomes weaker. No stable positive relationship was
revealed for any of the introduced parameters. Dependencies between the amount of summer
precipitation and humic acids are weaker and not so unambiguous: some of correlations are
positive while some are negative. At the same time a close interrelation between precipitation
and fulvic acids was registered over a period of several years.
4. Conclusions
A peat deposit is a multi-component system where reaction of oxidation and reaction of
different nature and of different speed proceed simultaneously. We have registered close
dependency between the amount of humic acids and ORP of peat deposit: quantitative
characteristic of humic acids increases (R2=0,85) as conditions change from oxidizing to
reducing ones. Close dependency was also recorded with hydrothermal regime and humidity
of peat deposit. Consequently, indirect interrelation was observed between humic acids and
the above mentioned parameters. Besides, we registered close dependency between
concentration of fulvic acids and levels of bog waters.
References
1. Y.Y. Lurje, Unified Methods of Water Analysis, 1973, p.376.
2. А.А. Reznikov, Е.P. Mulikovskaya, Y.I. Sokolov, Methods of Natural Water Analysis, Nedra,
Moscow, 1970, p. 488.
3. L.I. Inisheva, V.I. Yukhlin, F.F. Zelinger, Determination of ORP by means of ESK-1 device,
Tomsk CTIC, Тоmsk, 1975, № 35-75, p. 2.
4. L.I. Inisheva, N.G. Inishev, F.F. Zelinger, V.I. Yukhlin, Determination of soil and peat
temperature by means of ММТ-4, Tomsk CTIC, Тоmsk, 1975, № 36-75, p. 4.
5. GOST 11305-83. Peat. Moisture Determination Methods, Standards Publishing House, Moscow,
1983, p. 7.
Vol. 3 Page - 48 -
15th IHSS Meeting- Vol. 3
Structural Characteristics of Deep Groundwater Humic Substances
in Horonobe Area, Hokkaido, Japan
Motoki Terashimaa*, Seiya Nagaob, Teruki Iwatsukic, Yoshito Sasakia, Yoshimi Seidaa,
Hideki Yoshikawaa
a
Geological Isolation Research and Development Directorate, Japan Atomic Energy Agency (JAEA),
4-33 Muramatsu, Tokai-mura, Naka-gun, Ibaraki 319-1194, Japan; bInstitute of Natural and
Environmental Technology, Kanazawa University, Wake, Nomi, Ishikawa 923-1224, Japan;
c
Horonobe Underground Research Center, Japan Atomic Energy Agency (JAEA), 432-2 Hokushin,
Horonobe-cho, Teshio-gun, Hokkaido 098-3224, Japan
E-mail: terashima.motoki@jaea.go.jp
1. Introduction
In geological disposal system of high level radioactive waste, carrier effects of humic substances
(HSs) on migration of radionuclides are one of concerns, because HSs have a binding ability to metal
ions and a possibility to be mobile in geological medium. In general, the metal-ion binding ability and
mobility of HSs can strongly depend on their structural characteristics. For example, the metal-ion
binding ability of HSs is related to the amount and types of acid functional groups of HSs. In addition
the mobility (i.e., sorption and diffusion) of HSs in geological mediums can be dominated by their
size. On the other hand, the characteristics of HSs depend on their origin. In Japan, the geological
disposal system has been planned to be constructed at deep underground below 300 m depth. Thus,
information of the structural characteristics of HSs in deep groundwaters is required for a better
understanding of the effects of HSs on migration of radionuclide in geological disposal system.
However, information on the characteristics of deep groundwater HSs in Japan is lacking. Nagao et al.
showed the characteristics of HSs from the saline groundwater in argillaceous rock layer (790 – 1200
m depth) in the Mobara area [1,2], and the groundwaters in sedimentary rock layer (ca. 160 m depth)
and granitic rock layer (c.a. 180 m depth) in the Tono area [3]. Ueda and Sakamoto characterized the
HSs isolated from the shallow groundwater in sandy soil layer (ca. 50 m depth) [4]. Among these, only
characteristics of Mobara groundwater HSs are provided as the information on the deep groundwater
HSs below 300 m depth.
In this study, dissolved HSs, i.e., fulvic (FA) and humic acids (HA), were isolated from deep
groundwater at a depth of ca. 500 m in the Horonobe area, Hokkaido, Japan. The isolated groundwater
HSs were characterized by elemental analyses, spectroscopic analyses (UV-Vis, Fluorescence, FT-IR,
NMR), and size fractionation analysis. The structural characteristics evaluated were discussed on the
basis of the comparisons with HSs from surface waters and other groundwaters.
2. Materials and Methods
The groundwater in sedimentary rock (495 – 550 m depth) was collected from the borehole (HDB-10)
near the Horonobe Underground Research Center by using the packer type groundwater sampling
Vol. 3 Page - 49 -
15th IHSS Meeting- Vol. 3
system. The chemical components of the Horonobe groundwater are shown in Table 1. To extract
dissolved HSs, the groundwater was pumped up and passed through DAX-8 column after filtration
and acidification. After the extraction, the HSs were purified in our laboratory, according to the IHSS
method. Their weights that were finally obtained as a powder were as follows: 300 mg FA and 100
mg HA for total treated water of 2929 L in October 2007, 615 mg FA and 12 mg HA for total treated
water of 1743 L in October 2008.
Elemental compositions of C, H and N were determined with an elemental analyzer (Yanagimoto,
MT-6), and S was analyzed by ion chromatography after transformation to SO42-. Ash contents were
also determined by means of combustion at 550 ºC. 1H and
13
C NMR spectra were recorded by a
Bruker AVANCE K500 spectrometer. A pre-saturation method and an inverse gated decoupling
method were applied for the 1H and
13
C NMR measurements, respectively. UV-Vis spectra were
measured by Hitachi U-3300 spectrophotometer using 1-cm quartz cell. Three-dimensional excitation
emission matrix (3-D EEM) spectra were measured by a Hitachi F-4500 fluorescence
spectrophotometer. Relative fluorescence intensity is expressed in terms of standard quinine unit
(QSU). A QSU corresponds to fluorescence intensity of standard quinine sulfate (10 μg L-1 in 0.05 M
H2SO4) at an excitation / emission wavelength of 345 / 450 nm. Molecular size distribution was
determined by using ultrafiltration method. The ultrafiltration was sequentially conducted using the
series of ultrafilters with molecular weight cut-off of 100k, 30k, 10k, and 5k Daltons.
Aquifers
Horonobe
Mobara1
Tono2
Table 1: Chemical components of Horonobe groundwater, Mobara and Tono
Na+
ClSO42- HCO3K+
Sampling date
Depth / m
pH
-1
Oct, 2007
Oct, 2008
495.89~550.00
495.89~550.00
792~1202
160
6.9
6.9
7.9
9.6
100
129
3020
---
5300
4930
10700
---
mg L
7100
< 1.0
7300
< 2.0
18800
22
-----
2230
2290
903
---
TOC
47.3
22.0
55.7
0.11
3. Results and Discussion
The elemental compositions of the Horonobe HSs are given in Table 3. The CHN compositions are
almost same compared with those of Mobara and Tono HSs. However, remarkable differences were
observed for O. In the Horonobe HSs, the values of O are evaluated to be 25 – 32%, while the values
of 38 – 46% are observed for the Mobara and Tono HSs. This relatively lower percentage of O implies
that the contents of acid functional groups (e.g., carboxyl and phenolic hydroxyl) in the Horonobe
groundwater HSs are lower than the others.
1
H and 13C NMR spectra of the Horonobe FA are shown in Figure 1. Both spectra of Horonobe FA
exhibited large broad peaks assigned to aliphatic proton and carbon species. In addition,
13
C NMR
spectrum showed large sharp signal originating from carboxyl carbon at 165 – 190 ppm and
considerably small broad signal assigned to phenol carbon at 145 – 165 ppm. These results indicate
that the Horonobe FA is mainly composed of aliphatic carbon and most of the acid functional groups
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15th IHSS Meeting- Vol. 3
are carboxyl groups. These results were also supported by the UV-Vis and FT-IR measurements. The
composition of carbon species estimated from 13C NMR spectrum in Horonobe FA is summarized in
Table 4. When the composition was compared with that of the Mobara FA originated from deep
groundwater, it was found that the Horonobe FA has relatively low carboxyl content and aromaticity.
Based on the results of elemental analyses and 13C NMR, the carboxyl contents were calculated. In the
Horonobe FA, the carboxyl content was evaluated to be 7.08 meq g-1. This value was comparable to
that of Suwannee River fulvic acid (6.47 meq g-1), but was slightly lower than that of Mobara FA (8.15
meq g-1). These results suggest that the Horonobe FA has a capability of binding to radionuclide.
Table 3: Elemental composition (ash-free basis, %) in Horonobe groundwater HSs and those in other
groundwater HSs from literatures
Aquifers
Depth / m
HSs
C%
H%
N%
O%
S%
Ash%
Horonobe 07
495.89~550.00
Horonobe 08
495.89~550.00
Mobara 1
792~1202
Tono 2
160
FA
59.54
6.85
1.77
29.68
2.16
＜0.01
HA
FA
HA
FA
HA
FA
HA
64.40
60.33
56.43
53.50
50.80
47.8
46.4
6.79
6.84
6.11
5.65
4.67
5.6
6.9
2.36
1.81
5.51
1.37
2.48
0.6
8.7
25.00
29.73
31.94
39.55
42.02
46.0
38.0
1.45
1.29
n.d.*
n.m.**
n.m.
n.m.
n.m.
＜0.01
0.10
1.09
2.38
1.06
n.m.
n.m.
*n.d. : not detected, **n.m. : not measured, 1Nagao et al., 2000, 2009 (ref.2), 2Nagao et al., 2009 (ref.3)
Figure 1: 1H and 13C NMR spectra of Horonobe groundwater fulvic acid
Table 4: 13C NMR estimates of carbon distribution in Horonobe groundwater fulvic acid samples and Mobara
groundwater fulvic acid from literature
Fulvic acids
Aliphatic-C
(5-48)
Carbon species % δ, ppm
Methoxyl and
Aromatic-C
Carboxyl-C
carbohydrate-C
(48-110)
(110-165)
(165-190)
Carbonyl-C
Aromaticity*
(190-220)
Horonobe 07
40.0
19.7
21.0
13.2
4.3
0.26
Horonobe 08
41.2
21.3
16.1
14.1
7.3
0.20
Mobara 1)
38.0
19.0
21.7
18.3
3.0
0.28
*Aromaticity = (% of 110-165 ppm) / (% of 5-165 ppm), 1Nagao et al., 2000, 2009 (ref.2)
Measurements of 3D-EEM spectra showed the special character of Horonobe HSs. In general, it is
recognized that relative fluorescence intensity (RFI) of fulvic acid is higher than that of humic acid. In
the Horonobe HSs, however, the RFI of humic acid at the excitation / emission wavelength of 320 /
385 nm was about 5 times higher than that of fulvic acid. This is the first case of inversion
Vol. 3 Page - 51 -
15th IHSS Meeting- Vol. 3
phenomenon in RFI trend of HSs. Thus this indicates that the Horonobe HSs may have a unique
structural characteristic.
The size distributions of Horonobe HSs and those of
Mobara and Tono HSs are shown in Figure 2.
Regardless of their origin, more than 80% of FA was
distributed into size fraction below 10 kDa. In
addition, 50 – 60% of FAs was found in size fraction
below 5 kDa. In contrast, the distributions of HAs
depended on the origin. The 80% fraction in the
Horonobe HA was distributed below 10 kDa, while
about 35% fractions in the Mobara and Tono HAs
were found in same size range, indicating that the
size of Horonobe HA is smaller than those of
Mobara and Tono HAs. In previous study, it is
proposed that Mobara FA was distributed into
smaller size fraction, compared to Nordic Lake FA
Figure 2: Size distribution of Horonobe
groundwater HSs. Data for the Mobara and Tono
HSs are referred from Nagao et al., 2009
[2]. Thus, this indicates that, in same manner as Mobara and Tono groundwater FAs, the deep
groundwater FA in Horonobe area is relatively smaller than surface water FA.
4. Conclusions
The dissolved HSs in deep groundwater at depth of 495 – 550 m were extracted, and their structural
characteristics were investigated with regard to acid functional groups and molecular size. Based on
the series of analysis, following findings were obtained, (i) FA is dominant fraction of HSs in
Horonobe deep groundwater, (ii) the deep groundwater FA has carboxyl groups of which content is
the comparable amount as that of surface water FA, and (iii) more than 50% of the deep groundwater
FA is found in the size fraction below 5kDa. These findings can be useful for understanding the metalion binding ability and mobility of deep groundwater HSs in geological disposal systems.
Acknowledgements
We thank Prof. N. Fujitake at Kobe University for the measurement of NMR spectra. This study was
partly funded by the Ministry of Economy, Trade and Industry of Japan.
References
1. S. Nagao, Y. Nakaguchi, N. Fujitake and H. Ogawa, Entering the Third Millennium with a Common
Approach to Humic Substances and Organic Matter in Waters, Soil and Sediments. IHSS, Toulouse, 2000,
p.1143.
2. S. Nagao, Y. Sakamoto, R.R. Rao and N. Fujitake, Humic Substances Research, 5/6 (2009) 9.
3. S. Nagao, T. Iwatsuki and K. Hama, Journal of Nuclear Fuel Cycle and Environment, 15 (2009) 77.
4. M. Ueda and Y. Sakamoto, Journal of Nuclear Fuel Cycle and Environment, 12 (2006) 31.
Vol. 3 Page - 52 -
15th IHSS Meeting- Vol. 3
Browning of Stream Water During Hydrological Events
Dag Olav Andersen
University of Agder, Department of Natural Sciences, Service box 422, 4604 Kristiansand,
Norway
E-mail: dag.o.andersen@uia.no
1. Introduction
Long-term data series show increased water colour and dissolved organic carbon (DOC)
concentrations in Norwegian surface waters [1, 2] and more generally in northern Europe and
North America [3]. Several mechanisms have been proposed to explain these observations but
isolating single factors is notoriously difficult [4]. In this context seasonal variations in water
colour, DOC and molecular size were studied in the two inlet streams of Lake Terjevann,
southernmost Norway.
2. Materials and Methods
Lake Terjevann is about 2.5 km from the coastline, about 8 km west of Kristiansand in the
southernmost part of Norway. The catchment consists of four sub-catchments A, B and C+D
of 0.50, 0.24 and 0.35 km2, respectively. The altitude ranges from 22 to 128 m above sea
level. The soil cover is thin (0 – 70 cm) with abundant outcrops of bedrock, mainly felsic
augen-gneiss. The vegetation is dominated by conifers, mainly Scots pine, with some oak and
birch. About 50 % of sub-catchment A was forested with Norway spruce about 50 years ago.
The climate is maritime with monthly mean temperatures commonly just below 0 oC in
December, January and February and just above 14 oC in June, July and August. The annual
mean deposition is about 1300 mm with maxima during late autumn and winter. Sea-salts as
well as long-range transported acidic sulphur and nitrogen compounds are common.
Samples of drainage water from sub-catchments A and B were collected almost weekly in
clean glass bottles and transported to the laboratory where conductivity, pH, absorbance (200700 nm) and HPSEC fractions were measured a few hours after sampling. Conductivity and
pH were measured with standard Radiometer equipment calibrated against KCl and
Radiometer buffers of 4.01 and 7.00 respectively. The UV-VIS spectra were obtained on
filtered (0.45 µm) samples by a Shimadzu Multispec photodiode array spectrophotometer and
a 10 mm quartz cuvette. The same instrument with a 10 mm quartz flow-cell was used as a
detector (254 nm) in the HPSEC system in combination with a Perkin Elmer Series 200 pump
and a Tosoh TSK-G3000SW column (7.5 x 600 mm with a 7.5 x 75 mm guard). A sodium
Vol. 3 Page - 53 -
15th IHSS Meeting- Vol. 3
acetate buffer with pH 7.0 was used as mobile phase. DOC was measured on filtered (0.45
µm) and acidified (pH 2) samples by a Shimadzu TOC-V total organic carbon analyzer with
an ASI-V sampler unit.
3. Results
During the autumn and winter of 2004 the weekly depths of rainfall varied between 0 and
about 45 mm (Fig. 1a) while the deposited amounts of sea-salts were relatively low. During
this period, the stream water quality fluctuated slightly in conductivity and pH (Fig. 1b) while
water colour and DOC (Fig. 2a) co-varied with rainfall and stream flow. During higher flow,
higher molecular weight (HMW) materials dominated the DOC (Fig. 2b).
50
45
0,40
a)
0,35
40
25
0,20
20
0,15
15
3
0,25
30
Discharge (m /s)
Rainfall (mm)
0,30
35
0,10
10
0,05
5
0
0,00
01.09.2004
29.09.2004
27.10.2004
24.11.2004
22.12.2004
19.01.2005
16.02.2005
16.03.2005
13.04.2005
11.05.2005
08.06.2005
0,40
Cond. (mS/m)
pH
m3/s
b)
Conductivity; pH
11,0
0,35
0,30
10,0
0,25
9,0
0,20
8,0
0,15
7,0
0,10
6,0
0,05
5,0
0,00
Discharge
13,0
12,0
06.07.2005
-0,05
4,0
01.09. 29.09. 27.10. 24.11. 22.12. 19.01. 16.02. 16.03. 13.04. 11.05. 08.06. 06.07.
04
04
04
04
04
05
05
05
05
05
05
05
Figure 1: a) Rainfall (mm) measured at Kjevik airport (bar chart) and discharge (m3/s) and b)
conductivity (mS/m), pH and discharge in stream water from sub-catchment B.
Vol. 3 Page - 54 -
15th IHSS Meeting- Vol. 3
20,0
a)
DOC (mg/L)
mg Pt/L
DOC; Colour
16,0
12,0
8,0
4,0
0,0
01.09.04
29.09.04
27.10.04
24.11.04
22.12.04
19.01.05
16.02.05
16.03.05
13.04.05
11.05.05
08.06.05
06.07.05
80
b)
HMW-fractions (%)
70
60
50
40
30
20
10
01.09.04
29.09.04
27.10.04
24.11.04
22.12.04
19.01.05
16.02.05
16.03.05
13.04.05
11.05.05
08.06.05
06.07.05
0,050
240-280
280-320
320-400
400-700
c)
Absorbance / DOC
0,045
0,040
0,035
0,030
0,025
0,020
0,015
0,010
01.09. 29.09. 27.10. 24.11. 22.12. 19.01. 16.02. 16.03. 13.04. 11.05. 08.06. 06.07.
04
04
04
04
04
05
05
05
05
05
05
05
Figure 2: a) DOC (mgC/L), water colour (mgPt/L), b) part HMW fractions (%) and c) normalized
specific absorbance values for UV and VIS light in stream water from sub-catchment B.
Vol. 3 Page - 55 -
15th IHSS Meeting- Vol. 3
In early January 2005 strong south-westerly winds entrained increased sea-salts into the
atmosphere. Combined with relatively large amounts of rainfall, the catchments were heavily
loaded. As a result the conductivity of the stream water increased from about 7 to 11 mS/m
without a significant decrease in pH and the high conductivity persisted for several months
(Fig. 1b). A concomitant decrease in water colour and DOC that also lasted for several
months occurred in spite of the heavy rain and increased flow, e.g. January 11 (Fig. 1b).
During this period, lower molecular weight materials dominated the DOC in the stream water
(Fig. 2b).
Rain low in sea-salts in May and June diluted the salt concentration in the soil indicated by
the decrease in stream water conductivity that returned to about pre-event levels (Fig. 1b) as
well as increased water colour and DOC (Fig. 2a), with higher proportions of HMW materials
(Fig. 2b).
4. Discussion
The results indicate an increase in coloured HMW stream water DOC absorbing at longer
wavelengths during hydrological events. Shifts in peak maxima to longer wavelengths are
generally ascribed to conjugation and increasing numbers of chromophores [5], i.e.
unsaturated HMW materials. Plant and soil organic matter are the two major sources for DOC
in streams and rainwater percolating plant organic matter has been shown to obtain higher
concentrations of coloured organic acids than from soil organic matter [6]. Flushing of the
upper soil horizons seems to increase the export of coloured DOC from the catchment.
5. Conclusions
Rainfall amounts define the hydrological flow paths in soils. Increased DOC and more
coloured HMW DOC characterise the drainage from the upper soil horizons. Generally,
browning of downstream lake waters may be explained, at least partly, by this mechanism.
References
1. H. Liltved, R. Wright and E. Gjessing, Vann, 1 (2001) 70.
2. D. Hongve, G. Riise and J.F. Kristiansen, Aquat. Sci. 66 (2004) 231.
3. B.L. Skjelkvåle et al., Envir. Pollut., 137 (2005) 165.
4. N. Roulet and T.R. Moore, Nature, 444 (2006) 283.
5. D.A. Skoog, D.M. West, F.J. Holler and S.R. Crouch. Fundamentals of analytical chemistry,
Thomson Brooks/Cole, USA, 2004, Chapter 26, p. 784.
6. E.M. Thurman, Organic geochemistry of natural waters, Martinus Nijhoff/Dr W. Junk Publishers,
Dordrecht, 1985, Chapter 2, p. 67.
Vol. 3 Page - 56 -
15th IHSS Meeting- Vol. 3
Fluxes of natural and combustion-derived organic matter into the coastal
ocean off Southern Brazil
David C. Podgorskia, JiYoung Paengb, Thorsten Dittmarc, Marcos S.M.B. Salomaod, Carlos E.
Rezended, Marcelo C. Bernardese, Bill Cooper a*
a
Florida State University, Department of Chemistry and Biochemistry, Tallahassee, Florida
USA; bFlorida State University, Department of Oceanography, Tallahassee, Florida, USA;
c
Max Planck Research Group for Marine Geochemistry, University of Oldenburg (ICBM),
Oldenburg, Germany; dUniversidade Estadual do Norte Fluminense, Laboratório de Ciências
Ambientais, Campos dos Goytacazes, Brazil; eUniversidade Federal Fluminense,
Departamento de Geoquímica, Niterói, Brazil
E-mail: cooper.chem.fsu.edu
1. Introduction
Combustion-derived black carbon is a potentially significant source of terrestrial organic
matter in the ocean, especially in South Brazilian catchments where the burning of sugar cane
is common practice. In this presentation we describe our efforts to identify and quantitate
black carbon in riverine DOM fluxes and the fate of these fluxes in the estuaries and coastal
zone of the North Fluminense region of Brazil. We utilized a variety of analytical tools to
identify the major source terms of dissolved organic matter (DOM), including ultrahigh
resolution mass spectrometry.
2. Materials and Methods
Riverine and estuarine water samples were obtained during a series of cruises in May, 2008
along the North coast of Rio de Janeiro State. The DOM was extracted using solid-phaseextraction (SPE) methods described previously [1]. The salt free extracts were oxidized with
nitric acid in a microwave digestion system and the benzenepolycarboxylic acids (BPCAs)
formed from condensed aromatic structures were separated and quantified with highperformance liquid chromatography and diode-array detection [2].
Ultrahigh resolution mass spectra were acquired on the home-built 9.4 T FT-ICR mass
spectrometer located at the National High Magnetic Field Laboratory, Tallahassee, Florida
[3]. Negative-Ions were produced by an external electrospray ionization source.
3. Results and Discussion
The first analysis of ultrafiltered water samples from Rio de Janeiro state showed that the
residues of pasture and mainly sugar cane burning are transported in the river system to a
large degree as low-molecular weight solutes (<1000 Da) and then distributed in the coastal
Vol. 3 Page - 57 -
15th IHSS Meeting- Vol. 3
zone. BCPA analyses confirmed the presence of condensed aromatic structures (i.e.
combustion-derived organic matter) in waters all along the coast of Rio de Janeiro.
Bulk compositional information on this same DOM was obtained by FT-ICR mass
spectrometry. We identify combustion-derived organic matter by assigning each molecular
formula obtained from the mass spectrum a number to be used in a modified aromaticity
index (A.I.mod) proposed by Koch and Dittmar [4]. The formulas, and those in the same
homologous series, are considered to be aromatic if AImod > 0.5 and condensed aromatic if
A.I.mod ≥ 0.67 [4]. The data is also projected on traditional van Krevelen Diagrams for further
visual interpretation.
4. Conclusions
Analyses of benzenepolycarboxylic acids (BPCAs) formed from the oxidation of condensed
aromatic structures identified significant amounts of black carbon being transported down
rivers and into the coastal zones of Southeastern Brazil. Molecular compositional information
obtained by ultrahigh resolution mass spectrometry confirmed the presence of both aromatic
and condensed aromatic compounds in these same samples.
Acknowledgements
Financial support for sampling and analyses was provided by the National Science
Foundation (NSF-OISE-0710744). Mass spectra were obtained at the National High Field FTICR Facility (NSF-DMR-06-54118). The Brazilian researchers are fellows of the National
Institute for Science and Technology (TMCOcean, CNPq 573.601/2008-9) and Bilateral
Cooperation supported by CNPq (490658/2006-7) and NSF.
References
1. T. Dittmar, B. Koch, N. Hertkorn, G. Kattner, Limnol. Oceanogr. Meth. 6, 230 (Jun, 2008).
2. M. P. W. Schneider, R. H. Smittenberg, T. Dittmar, M. W. I. Schmidt, Geochim. Cosmochim. Acta
73, A1181 (Jun, 2009).
3. M.W. Senko, C. L. Hendrickson, L. PassaTolic, J. A. Marto, F. M White, S. H. Guan, A. G.
Marshall, Rap. Comm. Mass Specrom. 10, 1824-1828 (1996)
4. B. P. Koch, T. Dittmar, Rap. Comm. Mass Spectrom. 20, 926 (2006).
Vol. 3 Page - 58 -
15th IHSS Meeting- Vol. 3
The Changes of Water Organic Contamination under the
Influence of Ultrasounds
L. Stepniak*, E. Stanczyk-Mazanek, U. Kepa
Częstochowa University of Technology, Institute of Environmental Engineering
Brzeźnicka 60a, 42-200 Czestochowa, Poland
E-mail: stepniak@is.pcz.czest.pl
1. Introduction
Humic substances (HS) constitute of about 60–80% of the total content of the organic matter
which is present in natural water. The indirect share of HS within the accumulation of heavy
metals, the creation of stable suspensions and toxic substances (DBPs) determine the
necessity of their removal. The type and the sequence of the processes in the contemporary
water treatment systems are often selected in order to optimize the removal of natural organic
matter. In this context, the unconventional ultrasonic method is considered as the removal
mode of HS from water [1]. The literature-based research indicates that this method was
described on the basis of the effects obtained with the use of prepared water, most frequently
with the commercial humic acids (HA) preparation. The effectiveness of the ultrasonic impact
on HA model solutions is evaluated on the basis of the TOC index decrease. The results
showed that the effectiveness of reducing the TOC increased together with the intensity of the
ultrasound and exposure time (35% for example at the intensity of 42 W/cm2 and the time
of 20 min.) [2]. The best effect was observed at pH 3 in comparison with the effect obtained
at pH 5 and pH 11 [3]. It is believed that the sonochemical effects are associated with radical
oxidation reactions (chemical degradation of HA) as well as with the impact forces generated
during the annihilation of cavitation bubbles (mechanical fragmentation of HA) [2]. Due to
the composition of HS in surface water (where fulvic acid are predominant), the verification
of these effects in natural water environment is justified.
2. Materials and Methods
The substrate for the research was surface and prepared water. The analysis of the natural
surface water showed that the increased organic contamination indices: colour, oxygen
consumption, TOC (DOC), UV254. High intensity of water colour occured mainly because
of the presence of soluble HSs which predominated in the content of water samples.As the
prepared water we used a solution of commercial HA (Fluka) in deionized water. These
samples had the HA concentration of 30 mg/l, the content of which, determined by the TOC
index, was similar to one given for the natural water (Tab.1).
Vol. 3 Page - 59 -
15th IHSS Meeting- Vol. 3
Table .1: The physico-chemical analysis of the investigated water samples
Index
Unit
Prepared
Natural
pH
–
5.12
6.55–7.36
colour
mg/l(Pt)
80
40–45
turbidity
NTU
10.3
6.02–14.09
oxygen consumption mgO2/l
12.14
7.07–10.04
mg/l
–
total iron
0.75–1.05
absorbance UV254
1/cm
0.68
0.17–0.43
TOC
mgC/l
12.2
10.69–14.20
DOC
mgC/l
9.66
7.91–11.97
The samples of both waters underwent sonification at the natural pH solution, as well as at pH
3 and pH 9. The water samples (500 ml volume) were sonificated with the use of the highpower ultrasonic generator of Sonics&Materials VC-750 (20 kHz) equipped with a sonotrode
of 1.9 cm diameter (2.83 cm2 surface). As a variable process parameter the vibration
amplitude (12–60 μm) was taken, influencing the intensity of ultrasonic field. The maximum
intensity was high–37.8 W/cm2 (the density of power–0.2 W/ml). Changes in water organic
impurity were controlled mainly by the TOC index analyses. The TOC were determined with
the use of Analyzer Multi N/C 2100S (according to PN-EN 1484:1999). The selected results
of the research presenting the effects of the investigated process throughout the changes of the
TOC index depending on the amplitude, pH and the type of water were evaluated.
3. Results and Discussion
Studying the prepared water pH influence on the HA removal with the ultrasound method, the
highest effect was obtained at pH 3 (Fig. 1a). The decrease in the process effect at higher pH
level (natural pH 5.12) is related to the increase of the HA dissociation level. In strongly
alkaline medium, HA make proper solutions. In the case of natural water the effect of the
process at the acidic and non-corrected reading was similar. At the alkaline habitat the
decrease of the TOC has not been observed almost either. The effect of ultrasonic parameters
visible in the examination results indicates that the vibration amplitude has a major
importance for the process effects. Increasing the vibration amplitude enables an increase in
ultrasonic field intensity to be obtained, which for A=60 μm amounted to approx. 40 W/cm2.
For the greatest amplitude value and a sonification time of 10 minutes, the most favourable
reduction of the TOC index (by 4 mgC/dm3) was noted. The effect of removing water organic
contaminants in the sonochemical oxidation processes was also indicated by a reduction in the
oxygen consumption index.
Vol. 3 Page - 60 -
15th IHSS Meeting- Vol. 3
b) natural water
TOC, mgC/dm3
TOC, mgC/dm3
a) prepared water
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
acid
alkaline
natural
0
10
20
30
40
50
60
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
70
acid
alkaline
natural
0
10
Amplitude A, μm
20
30
40
50
60
70
Amplitude A, μm
Figure 1: The influence of the vibration amplitude (t=10 min.) and pH on the TOC index changes in
the water
In order to verify his dependence the further tests were made for the higher mount of natural
water samples. The samples were taken from the same source and were slightly varied
in terms of the initial TOC value. The most efficient maximum amplitude of vibrations 60
μm, was assumed. The average effectiveness of the process obtained in these tests was
compared with the effectiveness for the prepared water from the previous series (Fig.1b),
which is presented in Fig.2.
A=60 μm, t=10 min.
35
Effectiveness, %
30
acid
natural
alkaline
25
20
15
10
5
0
prepared w ater
natural w ater
Figure 2: The effectiveness of the TOC reduction for the prepared water and the mean effectiveness
for the natural water (SD= 2.1–3.1) in relation to the pH
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15th IHSS Meeting- Vol. 3
The obtained results reveal that the effectiveness of the process for the researched natural
water was the highest at the natural pH (6.55–7.36) and amounted to over 25%. These
incompatibilities need to be explained in the research conducted on fulvic acids (FA)
solutions, dominating in natural surface types of water. As it was stated in other research, that
as smaller-sized and more dissociated molecules, are removed in ultrasound field mainly with
the involvement of radical mechanism. Simultaneously, the results of this research indicate
the lower ultrasonic effect for FA in comparison with HA [2, 3]. Regardless of the kind
of water, the disadvantageous influence of the alkalinity, which inhibits the radical processes
of oxidation, was proved.
4. Conclusions
The effect of ultrasonic field intensity, as defined by the vibration amplitude, on process
effectiveness was confirmed for water investigated. The maximum effectiveness of the
ultrasonic method, i.e. 32% (60 μm, 10 min), was achieved for prepared water at the acid
reaction. The effect of pH on the process effectiveness for natural water may be connected
with the share of KH and KF in the composition of HSs. The sort of the fraction of the acids
in
water
depending
on
the
pH
influences
the
intensity
of
the
effectiveness
of ultrasounds. The intensity depends on the share of mechanical and chemical degradation
of the researched humic compounds. What influences the ultimate effect is the content
of different organic compounds in natural water, revealed by the amount of organic carbon.
As a result of this, the effectiveness of the sonochemical influence, at the non-corrected
natural water reaction was higher than for the prepared water.
Acknowledgements
This investigation was supported by the statutory research fund No. BW/401/203/07 of the
Częstochowa University of Technology.
References
1. T. J. Mason, E. Joyce, S.S. Phull, J.P. Lorimer, Ultrasonics Sonochemistry, 10 (2003) 319–323.
2. V. Naddeo, V. Belgiorno, R. Napoli, Desalination, 210 (2007) 175–182.
3. F. Chemat, P.G. Teunissen, S. Chemat, P.V. Bartels, Ultrasonics Sonochemistry, 8 (2001) 247–
250.
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Unexpected Uniformity of Humic Substances in Thermal Waters
Krisztina Kovácsa*, Csanád Sajgób, Alice Brukner-Weinb, Zoltán Kárpátid, András Gáspárc,
Etelka Tombácza, Philippe Schmitt-Kopplinc
a
University of Szeged, Department of Physical Chemistry and Material Science, 6720 Szeged,
Aradi Vt. 1., Hungary; bInstitute for Geochemical Research, Hungarian Academy of Sciences,
1112 Budapest, Budaörsi St. 45., Hungary; cHelmholtz Zentrum München, German Research
Center for Environmental Health, Institute of Ecological Chemistry, Department of
BioGeoChemistry and Analytics, 85764 Neuherberg, Ingolstädter Landstr. 1., Germany;
d
Budapest Sewage Works Ltd., North-Pest Wastewater Treatment Plant; 1044 Budapest,
Timár St. 1, Hungary
E-mail: kkriszta@chem.u-szeged.hu
1. Introduction
Thermal water is warm, hot groundwater tapped from deeper aquifers. According to the local
geothermal gradient value (50 ºC km-1 in Pannonian Basin) the water temperature increases in
a function of aquifer depth. Traditionally groundwater from natural springs and wells has
supplied drinking water and fed artificial spas for a long time. Humic substances are proven to
have biologic effects (e.g. anti-inflammatory and anti-viral activity), so the balneological use
of thermal waters is of great importance. In addition, heat of thermal waters provides local,
import independent, renewable energy source, which is free from pollution emission and
unaffected by weather conditions. Low temperature (<150 ºC) geothermal resources can be
used directly for heating (district heating, greenhouse heating, fish farming and industrial
drying processes). To preserve the sustainability of geothermal energy resource it is necessary
to inject the cooled thermal water back into the aquifer. The quality of the recycled water is
essential for the reliability and long-term use of the injection well.
The quantity and quality of organic matter present in thermal waters definitely influence their
uses and recycling efficiency (e.g. the presence of beneficial organic compounds in
therapeutic application, fouling in filters used in recycling process). We investigated the effect
of the changes in conditions such as temperature, pressure, presence of oxygen on the
properties of humic and fulvic acids after the groundwater outcrops.
2. Materials and Methods
Two groundwater samples of 46 and 83 ºC were taken from wells tapped from 993 m and
2103 m from the region of Makó (Southeast Hungary, Pannonian Basin) in February 2006
and January 2007, respectively. The water samples were preserved by pH adjusting next day
when they have already cooled down. Groundwater samples were collected from the same
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15th IHSS Meeting- Vol. 3
wells in February 2008. However, the acidification was performed immediately after the
sampling in the field. The acidified samples were stored below 5 ºC until processing.
Isolation of humic substances was according to the procedure of International Humic
Substance Society “Method for Preparation of IHSS Aquatic Humic and Fulvic Acids” [1].
The isolated humic and fulvic acids were characterized by potentiometric acid-base titration
(equilibrium titration, CO2-free condition, 0.01M NaCl as background electrolyte), FTIR
spectroscopy (KBr technique, Perkin-Elmer 1600 Series) and ESI-FT-ICR mass spectrometry
(Bruker APEX Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR/MS)
equipped with a 12 Tesla superconducting magnet and an APOLLO II ESI source, negative
ionization mode). The samples were dissolved in mixture of methanol and water (in the
volume ratios 99:1 and 99.5:0.5 MeOH:H2O, total volume 2 mL) directly before the analysis
and analyzed at 10 mg L-1 concentration. Humic acid solutions were prepared using trace
amount of NH4OH for complete dissolution (40 μL 28w% NH4OH). Additional details on
calibration, molecular formulae determination can be found in a recent paper [2].
3. Results and Discussion
Isolation method permits the gravimetric determination of humic and fulvic acid
concentration in water on the basis of the amounts of isolated material in the reference to the
volume of the water sample. The results of the same sample in each year show only a small
difference in the concentration of humic fractions (Table 1).
Table 1: Humic and fulvic acid concentration in thermal waters determined by gravimetry.
Sampling year
Humic acid
(mg L-1)
Fulvic acid
(mg L-1)
2006
1.9
1.2
2007
1.9
0.9
2006
6.7
2.7
2008
7.0
2.6
Depth (m)
993
2103
The acid-base properties of the isolated organic acids were investigated by potentiometric
acid-base titration. Some pH-dependent ionization curves are illustrated in Fig. 1. The amount
of charged groups is equivalent to the dissociated acidic groups of the acids. The amount of
charged groups across the pH range and the total acidity value at pH 10 are very similar for
both humic and fulvic acids from different samplings. Before the titration NaOH was not
added to the solutions of humic acids, so the complete dissolution of the humic acids took
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place in the course of titration. Due to the hindered dissolution in acidic region, the measured
points of humic acids did not run smoothly in succession until pH 7–8 as those of fulvic acids.
Figure 1: The pH-dependent dissociation of acidic groups on humic and fulvic acids isolated from
different samplings of thermal waters tapped from 2103 and 993 m
The infrared (IR) absorbance values belonging to different wave numbers, i.e., 2925, 1710
and 1620 cm-1 can be used for representing the aliphatic, carbonyl and aromatic content of the
humic substances (Fig. 2). Ratios of these absorbance values are suited to illustrate the
differences and changes in aliphatic, carbonyl and aromatic content [3]. The differences
between the ratios of humic and fulvic acids from different samplings are negligible.
Figure 2: Ratios of IR absorbance values for humic and fulvic acids from different samplings (e.g. Aal
/ Acar expresses the ratio of absorbance at 2925 cm-1 to tt at 1710 cm-1)
The ultrahigh-resolution FT-ICR mass spectrometric data was converted to molecular
formulae. The elemental compositions for these peaks can be calculated from mass spectra,
allowing their H/C and O/C atomic ratios to be calculated and plotted on the van Krevelen
diagram [4]. The positions of the patterns occupied by points differ slightly in case of humic
acids obtained from 993 m depth thermal water. Additional detailed investigation on
molecular composition is needed.
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15th IHSS Meeting- Vol. 3
Figure 3: van Krevelen diagrams of humic acids from different samplings of thermal waters tapped
from 993 and 2103 m
4. Conclusions
The characteristics of humic and fulvic acids isolated from thermal waters of various depths
are different. However, these properties do not show considerable changes depending on the
sampling method, i.e. the date of acidification of the water sample. It can be supposed that the
changes in conditions such as temperature, pressure, presence of oxygen do not affect the
properties of humic and fulvic acids after outcropping groundwater. This observation may
confirm the recalcitrant feature of humic substances in groundwater, too and facilitate the
design and operation of geothermal works from these materials of point of view.
Acknowledgements
The research was supported by the Hungarian National Science Foundation (OTKA) through
grant T-48829 and IHSS Training Award 2007.
References
1. E.M. Thurman and R.L. Malcolm, Environ. Sci. Technol., 15 (1981) 463.
2. A. Gaspar, E.V. Kunenkov, R. Lock, M. Desor, I. Perminova, Ph. Schmitt-Kopplin, Rapid
Commun. Mass Sp., 23 (2009) 683.
3. G.P. Lis, M. Mastalerz, A. Schimmelmann, M.D. Lewan, B.A. Stankiewicz, Org. Geochem., 36
(2005) 1533.
4. S. Kim, R.W. Kramer, P.G. Hatcher, Anal. Chem., 75 (2003) 5336.
Vol. 3 Page - 66 -
15th IHSS Meeting- Vol. 3
Basic By-Products Formation During Chlorination of Water Containing
Humic Substances
Ekaterina V. Trukhanovaa*, Margarita Yu. Vozhdaevaa, Lev I. Kantora, Evgeniy A. Kantorb
a
Municipal enterprise “Ufavodokanal”, Rossiyskaya str., 157/2, 450098 Bashkortostan,
Russia; b Ufa State Oil Technical University, Komarova str., 1, 450089, Bashkortostan,
Russia
E-mail: e.truhanova@mail.ru
1. Introduction
Organic composition of natural water is formed by soil and peat humus, plankton, higher
aquatic vegetation, animal organisms, as well as by organic compounds introduced into water
reservoirs by urban settlements being developed, by industrial and agricultural facilities [1].
Organic materials forming water composition may be both of natural and anthropogenic
origin. Humic compounds are natural materials that have the most significant effect on water
quality (by amount and by composition) being precursors of formation of numerous water
treatment by-products, particularly after chlorine disinfection of water.
Chlorination of natural water containing humic materials is followed by formation of
chlorine-bearing toxic, mutagenic and cancerogenic substances [2]. Trihalomethanes (THMs)
and haloacetic acids (HAAs) [3] are main by-products of chlorine disinfection of water.
Therefore, problem of HAAs and THMs formation during water treatment (Fig. 1) and
problem of control of their content are topical, especially for regions with a high humic
content in water.
Figure 1: Scheme of HAAs and THMs formation during natural waters chlorination [4]
No data is currently available in Russia on pattern of HAAs distribution in regional drinking
water, as well as no approved control procedures. In this connection, this research is focused
on studying dynamics of THMs and HAAs formation in drinking water of the city of Ufa.
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15th IHSS Meeting- Vol. 3
2. Materials and Methods
Determination of HAAs in water: A procedure of HAAs determination in water has been
developed by us on the basis of EPA 552.1 and 552.2 methods. This procedure is developed
in order to determinate monochloroacetic acid (MCAA) (within the range of 0.005–0.04
mg/dm3), dichloroacetic acid (DCAA), thichloroacetic acid (TCAA), monobromoacetic acid
(MBAA), chlorobromoacetic acid (CBAA) (0.001–0.04 mg/dm3), dibromochloroacetic acid
(DBCAA), dichlorobromoacetic acid (DCBAA) (0.001–0.02 mg/dm3). This procedure is
based on a twofold liquid-liquid extraction of HAAs with methyl-tert-butyl ether (MTBE),
followed by conversion of HAAs into methyl ethers with acid methanol and gas
chromatography with electron-capture detector (GC-ECD).
Determination of THMs in water: Determination of THMs in water was carried out with a
static mode sample preparation, followed by a Headspace Analysis. This procedure allows to
identify: chloroform (within the range of 0.0006–0.007 mg/dm3), bromodichloromethane and
dibromochloromethane (0.0003–0.03 mg/dm3), bromoform (0.001–0.1 mg/dm3).
3. Results and Discussion
Water samples of two water supply systems – river water intake system (RWIS) and
infiltration water intake system (IWIS) – were used to study HAAs and THMs content in
drinking water monthly from October 2006 through October 2009.
Diverse composition of HAAs forming in drinking water of RWIS and IWIS depends mainly
on source water quality, as well as on composition of organic and inorganic impurities in
water.
As Fig. 2 shows, THMs and HAAs content in RWIS water is approximately equal, while in
IWIS water the amount of HAAs formation predominates over the amount of THMs
formation. Total THMs content in RWIS drinking water within the entire period of the study
exceeds approximately 4 times total THMs content in IWIS water – 0.022 and 0.0056 mg/dm3
respectively. In the case of HAAs this difference amounts to 2.5 times – 0.024 and 0.0095
mg/dm3.
Average seasonal THMs concentrations indicated that during autumn drinking water sampling
THMs content is twice higher in water systems under study than during winter-spring period.
This can be explained by diverse humic composition in source water during these periods.
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15th IHSS Meeting- Vol. 3
0,06
С, mg/dm 3
С, mg/dm
3
0,06
0,05
0,04
0,03
0,02
0,05
0,04
0,03
0,02
0,01
0,01
N
O
ct
ob
ov e r
em
be
D
ec
r
em
be
Ja
r
nu
a
ry
Fe
br
ua
ry
M
ar
ch
Ap
ril
M
ay
a)
0
Ap
ril
M
ay
O
ct
o
N
ov ber
em
be
D
ec
em r
b
Ja e r
nu
ar
Fe
y
br
ua
ry
M
ar
ch
0
Total THMs
b)
Total HAAs
Figure 2: Total THMs and HAAs from October 2008 through May 2009: a) in RWIS drinking water, b)
in IWIS drinking water
4. Conclusions
Dynamics of formation of water chlorination by-products (haloacetic acids and
trihalomethanes) which occur during chlorination of water containing humus, is different,
depending on season and on water treatment procedure. Total content of by-products under
study occurring in RWIS water after a complex treatment exceeds several times similar
content in water from an infiltration water intake. This fact is explained by a high content of
humic substances, which are precursors of THMs and HAAs formation, in river water,
comparing to water from under-river wells that has passed filtering gravel beds. It is possible
to note a clearly remarkable seasonal prevalence in forming of chlorination by-products.
Therefore, study of pattern of THMs and HAAs distribution and their content in drinking
water is important while organizing drinking water quality analytical control.
References
1. A.V. Slipchenko, L.A. Kulskiy, E.S. Matskevich. Modern condition of water impurities oxidation
methods and chlorination prospects. Water Chemistry and Technique, 1990, vol. 12, №4, p. 334–
336.
2. L. Ts. Bonter., L. P. Alekseyeva, Ya. L. Khromchenko. Effect of organic impurities in natural
water on formation of toxic volatile halogen-alkanes during chlorination. Water Chemistry and
Technique, 1986, vol. 8, №1, p.37–41.
3. V.V. Goncharuk, N.A. Klimenko, L.A. Savchina, et al. Modern problems of drinking water
treatment technique. Water Chemistry and Technique, 2006, №1, vol. 28.
4. Rook J.J., Formation of galoforms during chlorination of natural waters. J. AWWA. 1976, V.68,
№3, p. 168
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15th IHSS Meeting- Vol. 3
Research of the Physics and Chemical Properties on Sediments of the
Lobelia Lakes in West Pomeranian Region of Poland
Lilla Mielnika*, Jacek Czekałab
a
West Pomeranian University of Technology in Szczecin, Department of Physics and
Agrophysics, ul. Papieża Pawła VI 3, 71-459 Szczecin, Poland; bDepartment of Soil Science
and Land Protection, University of Life Sciences in Poznań, ul. Szydłowska 50, 60-656
Poznań, Poland
E-mail: lilla.mielnik@zut.edu.pl
1. Introduction
Bottom sediments are the important element of all lakes. These sediments are considered to be
a valuable and unique source of processes occurring in the aquatic environment. The quality
of organic matter is an important feature with respect to the physicochemical conditions on
the lake bottom. The approximate value of the amount and quality of the organic matter, as
well as its decomposition, can be determined by the organic carbon to total nitrogen ratio
Corg/Ntot [Kamaleldin et al., 1997, Meyers, 2003, Twichell et al., 2002, Mielnik, 2005,
Mielnik et all 2009]. The Corg/Ntot ratio in lake sediments is often used as an indicator of
time changes in the organic matter cycles in aquatic ecosystems [Kamaleldin et al. 1997].
The work presents and discusses the results of research on the physics-chemical composition
of bottom sediments of Lobelia lakes located in West Pomeranian Region in Poland.
2. Materials and Methods
The research material was the bottom sediments of the Lobelia lakes. To do the investigations
one chose reservoirs, which differ in morfometric parameters and drainage basin development
The lake sediments were sampled from the superficial layer (at the depth of up to 25 cm) in
duration of the summer stagnation. The samples were collected in two fields: (i) in the costal
area in the littoral zone (samples designated L), (ii) at the maximum depth of the basin in the
profundal zone (samples designated P). In the air dried sediments there was determined the
content of organic carbon Corg by Orlov and Grindel method and the content of total nitrogen
Ntot by Kiejdahl method. The Corg/Ntot values were determined based on the results obtained.
3. Results and Discussion
Values of analysed parameters Corg and Ntot were various and changed in terms of lake
character, as well as in terms of the field where the sediments were collected in the lake basin
(Table 1). Due to the distinct distribution of depth, sedimentation takes place in a different
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15th IHSS Meeting- Vol. 3
way in each lake in the shallow zone as well as in the deeper one. In the bottom sediments of
the investigated lakes the Corg contents ranged from 0.4 to 29.5 %. This indicates there is a
different contribution of organic and mineral substances to the examined sediments.
This showing at different participation of organic and mineral substances in examined
sediments.
Examining the total nitrogen Ntot content in sediments provides important information about
the quality of sedimentary organic matter. The concentration of Ntot in the investigated
sediments is significantly high from 0.05 to 3.13 %. Bottom sediments from littoral zone in
two lakes: Morskie Oko i Ciemino were exception (the concentration of Ntot in this sediments
0.03 and 0.05 %). High nitrogen content in lake sediments results from high protein content in
the organic matter. This is a product of the life activity of aquatic organisms, as well as the
decomposition of plant and animal residues.
Table 1. Elemental composition of the sediments of the studied lakes
Lake
Jelonek
Zone
L
Maximum depth[m]
3.5
pH
5.9
P
Kociołek
L
16.3
5.7
P
Wielkie
Oczko
L
Morskie Oko
L
10.0
7.6
P
19.2
7.0
P
Ciemino
L
12,6
P
8.1
Corg
Ntot
Ptot
Corg/ Ntot
[%]
[%]
[g/kg]
21.0
1.76
1.82
11.9
20.1
1.78
1.80
11.3
2.4
0.10
0.12
21.1
22.0
1.61
1.94
13.7
20.8
1.50
0.55
13.8
20.3
1.60
2.18
12.7
0.4
0.03
0.05
13.4
29.5
3.13
2.55
9.4
0.7
0.05
0.18
13.3
13.0
1.59
1.52
8.2
L – litoral zone, P – profundal zone.
Calculated values of the Corg/Ntot ratio are be reflected of differentation Corg and Ntot contents
in the examined sediments too. The Corg/Ntot ratio for examined sediments was practically the
same for all the lakes at the range of 8.2 to 13.8. This indicates the predominant contribution
of plankton to organic matter production, and the rather low contribution of higher plants
containing lignin and cellulose. The sediments in littoral zone of Kociołek lake are the
exception. In this sediments the Corg/Ntot ratio were 21.1 and suggests the big participation of
the macrophytes and higher plants rich in lignin and cellulose and poor in protein [Meyers and
Ishiwatari 1993, Meyers 1997, Mielnik et all 2009].
When analyzing the Corg/Ntot ratio, it should be taken into consideration that the selective
decomposition of the organic matter by microorganisms during its slow diagenesis may be
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15th IHSS Meeting- Vol. 3
modified by its elemental composition as well as by the Corg/Ntot ratio of the organic matter in
sediments [Kamaleldin et al. 1997 Routh et al. 1999].
The biological mineralization of the phosphorus depends on biochemical transformations of
carbon and nitrogen. The tendency of changes in Ptot content in the examined sediments is
similar to that observed for Corg and Ntot content.
4. Conclusions
The differences in sediments in respect of their chemical properties result from the individual
features of each lake, including the morphometric differences, and the character of the
drainage basin for which the lake is a sedimentation tank. The flora growing over the water
body is a significant factor differentiating the properties of the sediments.
Acknowledgements
This work was supported by the Polish Ministerial Research Project: No.0547/P01/2008/34.
References
1. M. H. Kamaleldin, J.B. Swinehart, R.F. Spalding, Journal of Paleolimnology, 18, (1997), 121–
130.
2. P.A. Meyers, R. Ishiwatari, Organic Geochemistry, 20 (1993,) 867-90
3. P.A. Meyers. Organic Geochemistry 34, (2003) 261–289.
4. L. Mielnik, (in polish), Inżynieria Rolnicza 4(64), (2005) 31-36.
5. L. Mielnik, R. Piotrowicz, P. Klimaszyk, Oceanological and Hydrobiological Studies, XXXVIII,
3, (2009), 69-76.
6. J. Routh, T.J. McDonald., E.L. Grossman, Organic Geochemistry, 30, (1999), 1437-53.
7. S.C. Twichell, P.A. Meyers, Organic Geochemistry 33, (2002) 715–722.
Vol. 3 Page - 72 -
15th IHSS Meeting- Vol. 3
Ratio of Color to Chemical Oxygen Demand as an Indicator of Quality of
Dissolved Organic Matter in Surface Waters
Andrey I. Konstantinova*, Nikolay S. Latyshevb, Petr A. Ivkinb, Irina V. Perminovaa
a
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-3,
119991 Moscow, Russia; bOJSC «NII VODGEO», Komsomolskii prospect, 42-2, 119048
Moscow, Russia
E-mail: konstant@org.chem.msu.ru
1. Introduction
The majority of surface waters contain significant amounts of dissolved organic matter
(DOM). To assess the content of DOM, different parameters are used, such as color, chemical
oxygen demand and total organic carbon (TOC). Those parameters give direct or indirect of
the content of DOC, but they do not characterize its quality. The important parameter of DOM
which defines its hydrophobic-hydrophilic balance is the content of aromatic carbon. It has
been numerously shown [1] that the absorbance value at 254 nm normalized to mass
concentration of DOM expressed on organic carbon (OC) basis (known as SUVA parameter)
gives a good indirect estimate of aromaticity of DOM. In this study, we have hypothesized
that a ratio of two bulk parameters traditionally used to characterize water quality such as
color and chemical oxygen demand (COD) may serve as an analogue of SUVA in
characterizing quality of DOM, nominally, of its aromaticity as well as of the contribution of
humic matter into the total pool of DOM To prove this hypothesis, the existence of the
relationship was tested between direct aromaticity estimates provided by
13
C NMR and
values of color to COD parameters for a set of well characterized humic materials. The
obtained relationship was further extended to aromaticity assessment of DOM present in the
samples of medium and high colored surface waters.
2. Materials and Methods
Two samples of medium and highly colored surface water (the Volkhov River (Russia) and
Orsha River (Belarus) were used in this study sampled according to standard procedure [2].
Seven samples of humic substances (HS) isolated from different sources according to IHSS
technique [3] were studied: fulvic acids (FA) and humic acids (HA) of sod-podzolic soil
(Moscow region, Russia) – SFA and SHA; HA and non-fractionated humic substances (HS)
of high-land sphagnum peat (Tver region, Russia) – PHA and PHF; coal HA of leonardite
(Humintech Ltd, Germany) – CHA; HS of the Istra River (Moscow region, Russia) and the
Suwannee River (IHSS standard, USA) – AHF and SRDOM, respectively.
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15th IHSS Meeting- Vol. 3
Color of water was determined using chromate-cobalt units according to standard procedure
[4]. Chemical oxygen demand (COD) was determined by the Kubel method [5]. UV-Vis
analysis was conducted using spectrophotometer Cary-50 (“Varian”, USA) using 1 cm quartz
cuvettes. Organic carbon content in the model humic materials was determined using Carlo
Erba Strumentazione elemental analyzer (Italy). Quantitative 13C solution state NMR spectra
were acquired using Avance 400 spectrometer (Bruker, Germany) operating at 100 MHz
carbon-13 frequency. The spectra were recorded on the samples dissolved in 0,3 M
NaOD/D2O at concentration of 80 mg/mL. Carbon-13 NMR spectra were acquired with a 5
mm broadband probe, using CPMG pulse program with 7,8 s relaxation delay and acquisition
time about 0,2 s and INVGATE procedure. Aromatic carbon content (CAR) was determined as
integral intensity in the spectral region from 110 to 165 ppm according to [6].
3. Results and Discussion
To characterize SUVA values of model humic materials used, UV absorbance was registered
at 254 nm and normalized to the concentration of solution expressed on organic carbon basis.
To explore if the color to COD ratio can be used as a parameter of aromaticity of DOM, the
model set of humic materials was analyzed for color and COD values as well. The obtained
results are given in Table 1. The corresponding correlation plots are shown in Figure 1.
Table 1: Obtained physicochemical characteristics of the model HS samples
DOC
(mg/L)
Abs254
SUVA254
(L/[mgС*cm])
CAR
(%)
SFA
7,00
0,476
0,043
31
Color
(Cr-Co
units)
55,2
SHA
8,02
0,406
0,042
34
128,0
10,2
12,6
PHF
8,97
0,493
0,044
34
135,4
12,2
11,1
PHA
6,69
0,742
0,085
38
202,9
18,0
11,2
CHA
5,07
0,682
0,100
56
185,1
9,8
18,8
IRDOM
11,65
0,472
0,032
26
39,9
9,0
4,4
SRDOM
10,48
0,493
0,038
30
99,8
13,1
7,6
Samples
Color/COD
COD
(mgO2/L) (Cr-Co
units*L/mgO2)
7,6
7,3
As follows from the obtained results, there are statistically significant relationships observed
between the values of color to COD ratio and Car as well as between color to COD ratio and
SUVA254.
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15th IHSS Meeting- Vol. 3
Figure 1: Correlation plots of CAR vs Color/COD (left) and SUVA254 vs Color/COD (right)
At the next stage of this study Color/COD values were used to estimate quality of DOM after
water treatment. For this purpose, the medium colored water samples from the Volkhov River
and highly colored samples from the Orsha River were treated with the coagulants and the
floculant. Water treatment efficiency coefficients (η) were calculated according to the
equation (1).
.
η (%) = 100% (Xorigin – X)/Xorigin,
(1)
where: X – Color, COD and Color/COD values; index “original” means non-treated water sample
Characteristics of water quality measured and η values calculated are shown in Table 2. As it
can be seen, water treatment efficiency is slightly less for medium colored water samples as
compared to the highly colored ones. This corroborates well the lower value of Color/COD
parameter indicating less aromaticity of the medium colored water sample and its lesser
susceptibility to the action of coagulants and flocculants.
Hence, the parameter of Color/COD can be used for prediction of efficiency of water
treatment. The higher values of this parameter may be indicative of better suitability of
traditional coagulants-flocculants technologies to treatment of the corresponding water
samples. This can be the case because the higher Color/COD values can be provided by the
considerable content of high-molecular weight HS that have a high negative charge and are
able to form stable water insoluble compounds with coagulant molecules. Low values of
Color/COD can indicate high concentration of low-molecular fractions of DOM. In this case,
water treatment with coagulants is less efficient.
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15th IHSS Meeting- Vol. 3
Table 2: The quality assessment of the original and treated samples of the river waters
Sample
№
1.0
(original)
1.1
1.2
Source
The
Volkhov
River
1.3
2.0
(original)
2.1
2.2
The
Orsha
River
2.3
Coagulant
amounts, mg/l
AOC
PS
Color
COD
Color/COD
Cr-Co
units
η, %
mgO2/L
η, %
Cr-Co
units
L/mgО2
η,
%
-
-
-
93.4
-
19,9
-
4,7
8
-
16,6
82
9,7
51,4
1,7
-
8
14,0
85
11,3
43,3
1,2
4
4
27,2
71
15,3
23,1
1,8
-
-
288,7
-
38,5
-
7,5
30
-
19,5
93
10,3
73,2
1,9
-
18
27,6
90
13,2
65,5
2,1
5
15
34,8
88
16,2
57,9
2,2
63,
6
73,
6
62,
1
74,
8
72,
3
71,
3
4. Conclusions
1. Color/COD parameter is developed to characterize the content of aromatic carbon in DOM.
Its applicability for this purpose was proven using a set of well characterized HS.
2. The introduced parameter was used to asses efficiency of water treatment technology. High
values of the parameter indicate a high efficiency of water treatment based on coagulantflocculant approach, low values indicate that alternative approach (for example, oxidation)
should be used.
References
1. S.J. Traina, J. Novak, N.E. Smeck, J. Environ. Qual., 41 (1990) 151–153.
2. GOST R 51592-2000 Water. The general sampling requirements (in Russian).
3. R.S. Swift, Organic Matter Characterization, in D.L. Sparks (Ed.), Methods of Soil Analysis, Part
3. Chemical Methods, Soil Sci. Soc. of America, Madison, WI, 1996, p. 1018–1020.
4. GOST 3351-74 Drinking water. Techniques to determine taste, odor, chromaticity and turbidity
(in Russian).
5. Yu.V. Novikov, K.O. Lastochkina, Z.N. Boldina, Techniques to research water quality of water
reservoirs, Meditsina, Moscow, 1990 (in Russian).
6. D.V. Kovalevskii, A.B. Permin, I.V. Perminova, V.S. Petrosyan, Moscow State University
Bulletin, Series 2 (Chemistry), 41 (2000) 39–42 (in Russian).
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UV-Vis Spectrometry and Size-Exclusion Chromatography Study of
Seasonal Dynamics of Quality of Dissolved Organic Matter
Andrey I. Konstantinova*, Ekaterina V. Trukhanovab, Margarita Yu. Vozhdaevab,
Lev I. Kantorb, Irina V. Perminovaa
a
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3,
119991 Moscow, Russia; bMunicipal enterprise “Ufavodokanal”, Rossiyskaya str. 157/2,
450098 Ufa, Bashkortostan Republic, Russia
E-mail: konstant@org.chem.msu.ru
1. Introduction
The necessity of upgrading natural water treatment techniques which would improve
performance of conventional techniques is urged by new achievements in understanding
nature of dissolved organic matter (DOM) as well as by development of new analytical
instrumentation [1, 2]. The important issue for research is elucidating the feature of DOM
which impacts the most efficiency of water treatment technology used for drinking water
production. This feature might be connected to the source of natural water, as well as to
seasonal conditions. The objective of this research was to study seasonal dynamics of the
quality of DOM in surface and infiltration waters using UV-Vis spectrometry and sizeexclusion chromatography (SEC).
2. Materials and Methods
Nineteen raw and treated water samples were studied. The samples were taken from the
surface water intake system (SWIS): four samples of surface water (SW) were taken from the
River Ufa in July and November 2008, and in February and April 2009, four samples of
filtered water (FW) were taken after the surface water passed several treatment steps (primary
chlorination with low doses of chlorine, chemical treatment and high-rate filters with burnt
rock filtering medium), and four samples of drinking water (FWCl) (filtered water undergone
secondary chlorination). The water samples from infiltration water supply system (IW) were
also included into the experimental data set represented by four samples of non-chlorinated
water samples taken in July and November 2008. and in February and April 2009, and three
samples of drinking water taken from the infiltration water supply system that passed
chlorination in November 2008, and February and April 2009.
UV-Vis spectrometry analysis was conducted using spectrometer Cary-50 (Varian, USA)
equipped with quartz cuvettes with optical path length of 1 cm.
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15th IHSS Meeting- Vol. 3
SEC system consisted of a solvent pump, a packed column and a UV-detector with variable
wavelength as described elsewhere [3]. The UV-absorbance was measured at 254 nm. The
SEC column was 15x250 mm packed with Toyopearl HW-55S (“Toso-Haas”, Japan). 0,03M
phosphate buffer with pH 6,8 was used as a mobile phase at a flow rate of 1 ml/min. The
column was calibrated using sodium polystyrenesulfonates (PSS) (Da): 4480, 14000, 20700,
45100, and 80840 (Polymer Standard Service, Mainz, Germany). Blue dextran (2000 kDa)
served as a void volume probe, acetone – as a permeation volume probe.
Dissolved organic carbon (DOC) content was measured photometrically with Skalar SAN plus
Segmented Flow Analyzer («Skalar», Netherlands). The method is essentially based on
decomposition of dissolved organic matter using ultraviolet irradiation and potassium
persulphate oxidation to carbon dioxide. Carbon dioxide diffuses through special gaspermeable silicone membrane and is absorbed by phenolphthalein solution. Optical density of
the phenolphthalein solution is measured at a wavelength of 550 nm.
3. Results and Discussion
UV-Vis absorbance spectra of the samples studied are given in Figure 1. They are typical for
natural DOM. To characterize UV absorptivity of the water samples, specific UV absorbance
(SUVA) was calculated at 254 and 280 nm which correspond to wavelengths characteristic of
absorbance of aromatic groups. SUVA is defined as the UV absorbance of a water sample at a
given wavelength normalized to DOC concentration. The results obtained are given in Table
1.
0,25
0,2
IW-nov
0,15
IWCl-nov
SW-nov
FW-nov
0,1
FWCl-nov
0,05
0
200
300
400
500
wavelength, nm
Figure 1: Typical UV-Vis absorbance spectra of the samples studied
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15th IHSS Meeting- Vol. 3
As it can be seen from Table 1, the filtered river water sample (FW) has the highest SUVA
value at both wavelengths, though, at the same time, its DOC concentration decreased 1,7
times compared to water from surface water supply (the River Ufa) (SW).
Table 1: UV absorbance (А) and SUVA of the autumn samples studied (at 254 and 280 nm)
Sample
IW-nov
IWCl-nov
SW-nov
FW-nov
FWCl-nov
Dissolved organic carbon
(DOC), mg/L
1,7
1,5
4,2
2,5
2,7
A254
0,0293
0,0232
0,0622
0,0511
0,0429
SUVA254,
L/(mgС*cm)
0,0173
0,0154
0,0148
0,0204
0,0159
A280
0,0215
0,0165
0,045
0,038
0,030
SUVA280,
L/(mgС*cm)
0,0126
0,0110
0,0107
0,0153
0,0111
The results obtained indicate that water treatment steps (filtration of water samples) mostly
cause a decrease in aliphatic matter and increase in aromatic one. Secondary chlorination
causes partial decomposition of aromatics-rich organics, which is reflected as a decrease in
SUVA values for chlorinated water samples (FWCl and IWCl) as compared to less
chlorinated (FW) and non-chlorinated (IW) samples. The similarity of DOC values for the
SW samples might indicate an increase in low-molecular chloroorganic compounds –
chlorination by-products.
Typical SEC chromatograms in Kd scale are given in Figure 2. Molecular weight
characteristics calculated from the chromatograms obtained are given in Table 2.
Figure 2: Typical chromatograms of the samples studied: surface (left) and infiltrated water
IW-samples taken in autumn are characterized with the lowest Mw and Mp values, summer
samples have the highest ones, and winter and spring values – medium values. Maximum
molecular weight (MW) of spring samples is caused by the high rate of humus formation in
the spring. Increased microbiologic activity in summer is accompanied by degradation of
organic matter and a decrease in average MW of DOM of IW. In autumn and winter, when
the rate of humus formation is the lowest. Mw and Mp are the lowest.
Spring samples have maximum Mw and Mp for SW which is caused by floods. Provided that
DOM being highly concentrated in river waters is more susceptible to decomposition during
water treatment, it is reflected in maximum decrease in Mw and Mp of spring samples after
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15th IHSS Meeting- Vol. 3
filtration and chlorination. Decreasing Mw and Mn after chlorination in autumn, winter and
spring water samples might indicate their enrichment with low-molecular weight chlorination
byproducts. This is in sync with the results of UV-Vis study of autumn water samples. For a
summer sample (FW-jul) after chlorination (FWCl-jul) an increase in Mw and Mn was
observed that might be indicative of more complete decomposition of low-molecular weight
components of DOM in summer samples as compared to those of other seasons.
Table 2: Molecular-weight characteristics of the samples studied
Sample
IW-jul
IW-nov
IW-feb
IW-apr
IWCl-nov
IWCl-feb
IWCl-apr
SW-jul
SW-nov
SW-feb
SW-apr
FW-jul
FW-nov
FW-feb
FW-apr
FWCl-jul
FWCl-nov
FWCl-feb
FWCl-apr
Molecular weights, Da
Number-average (Mn)
Weight-average (Mw)
6050
4050
5070
4130
5750
4290
5860
4460
4740
3500
5110
3430
4990
2590
5780
3590
5830
4340
5960
3510
6730
4350
6070
4150
5480
3970
5620
3690
5270
3140
6510
5150
5160
3560
4160
2120
4010
2410
Peak (Mp)
6700
5820
6260
6230
5260
5950
6110
6440
6560
6870
7310
6770
6280
6420
6190
6890
5820
5210
4730
Polydispersity
(Mw/Mn)
1,5
1,2
1,3
1,3
1,4
1,5
1,9
1,6
1,3
1,7
1,5
1,5
1,4
1,5
1,7
1,3
1,5
2,0
1,7
4. Conclusions
The seasonal dynamics of structural and molecular weight characteristic of dissolved organic
matter is determined by seasonal changes in microbiological activity and in the rate of humus
formation including seasonal floods for surface water. In accordance with the results of UVVis and SEC studies, efficiency of surface water treatment during spring season drops
substantially.
References
1. E.M. Perdue, in G.E. Likens (Ed.), Encyclopedia of Inland Waters, Academic Press, NY, 2009, p.
806-819.
2. J. Peuravuori and K. Pihlaja, in L.M.L. Nollet (Ed.), Handbook of Water Analysis, CRC Press,
NY, 2nd edn., 2007, p. 435-447.
3. I.V. Perminova, F.H. Frimmel, A.V. Kudryavtsev, N.A. Kulikova, G. Abbt-Braun, S. Hesse, V.S.
Petrosyan, Environ. Sci. Technol., 37 (2003) 2477-2485.
Vol. 3 Page - 80 -
15th IHSS Meeting- Vol. 3
Humin Contribution to Sedimentary Organic Matter of The Adriatic Sea
F. Rampazzoa*, D. Bertoa, M. Gianib, C. Baldina, L. Langonec
a
Istituto Superiore per la Protezione e la Ricerca Ambientale, Brondolo Chioggia (Ve), Italy;
b
Istituto Nazionale di Oceanografia e di Geofisica Sperimentale, Trieste, Italy; cIstituto di
Scienze Marine, ISMAR-CNR, Bologna
E-mail: f.rampazzo@icram.org
1. Introduction
A substantial proportion of the so-called refractory organic matter (OM) in waters and
sediments is constituted by humic substances (HS) that are structurally complex
polyelectrolytic, dark coloured organic acids which are formed from the decomposition of
plant, animal and microbial tissues and tend to be more recalcitrant than their precursors [1].
They are defined according to fractionation schemes, based on solubility, in humic acids
which are soluble in dilute alkaline solution and precipitate under acid condition, fulvic acids
which are soluble in both base and acids and humin (HM) insoluble at all pH conditions.
Due to its insolubility in alkali, HM is difficult to study and few investigations were
performed in marine environments.
The aim of this study is to quantify the contribution of HM to sedimentary organic matter and
to investigate its origin (autochthonous or allochthonous sources) and biogeochemical
features in order to investigate the formation pathway of HS in the Adriatic sediments.
2. Materials and Methods
Sediments cores were sampled in three sites located respectively in a coastal area of central
Adriatic sea (0-25 cm), in the middle Adriatic pit (0-25 cm) and in the southern Adriatic pit
(0-35 cm) during the SIT-1 cruise carried out in the framework SESAME project during
February 2008. The extraction of HS from sediments was performed following the method of
the International Humic Substance Society modified following Moreda-Pineiro [2].
Organic carbon (Corg) and total nitrogen were determined in sediments and residual HM by a
CHN Elemental Analyzer. Corg was determined after removal of carbonates with HCl. Weight
percentages of nitrogen were determined following the same procedure. The content of HM in
the sediments was expressed as mg C/g sediment (dry weight) and as % of HM-carbon on the
total Corg in sediment. Stable isotopic ratio of organic carbon (13C/12C) was determined by a
CHNS-O analyzer coupled with an Isotope Ratio Mass Spectrometer. The results were
expressed in parts per mil from the international standard VPDB (Vienna Pee Dee Belemnite).
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15th IHSS Meeting- Vol. 3
3. Results and Discussion
Corg content in sediments was lower in the southern Adriatic pit (0.35 ± 0.20 %), while higher
values were observed in the coastal central Adriatic (0.65 ± 0.04%) and in the meso Adriatic
pit (0.61 ± 0.08 %).
HM contribution to the sedimentary organic matter was relevant (Table 1) in all stations,
increasing with the depth along the core, in particular in the southern Adriatic pit.
HS can constitute up to 40-68% of the organic carbon in other coastal and oceanic sediments
[3] or up to 64-100% of organic matter in some estuaries [4] where the humic acids are 4-28%
of the total HS.
Table 1: Corg/N ratios and humin concentration in the Adriatic sediments. Coastal Central Adriatic Sea
(CCA), Middle Adriatic Pit (MAP) and Southern Adriatic Pit (SAP) sediments
Coastal central
Adriatic CCA
Parameters
Middle Adriatic Pit
MAP
Southern Adriatic
Pit SAP
N Mean Dev.Std. N Mean Dev.Std. N Mean Dev.Std.
Corg/N (mol/mol)
4
8.9
0.4
4
7.4
0.6
6
6.7
0.9
Chumin in sediment (mg/g)
4
4.7
0.7
4
4.2
0.3
6
2.8
1.4
Chumin/Corg %
4
72.6
13.6
4
69.2
9.0
6
86.1
11.1
Corg/N ratios in the sediments of the southern Adriatic pit were in the range typical of
phytoplankton. These values are comparable with respect to those of the residual HM (Fig.1)
suggesting a common origin of the sedimentary organic matter. This hypothesis is also
confirmed by the isotopic ratio (δ13C) of sediments.
In the station located in the coastal zone of the middle Adriatic, more subjected to continental
organic matter input, the highest Corg/N ratios of HM fractions were observed (Fig.1).
The increase of Corg/N ratio in the HM of the coastal station with respect to the values
reported for the bulk sedimentary organic matter could indicate possible higher degradation
processes of the HS during sedimentation in the water column and burial in the sediments. It
cannot be excluded that a part of this HM could derive from allocthonous material refractory
to degradation like vascular plants compounds rich in lignin that may be preserved or only
partially altered by oxidation.
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15th IHSS Meeting- Vol. 3
10.0
9.0
Corg/N molar ratio
8.0
7.0
6.0
5.0
4.0
SAP
MAP
CCA
Corg/N sediments (mol/mol)
Corg/N humin (mol/mol)
Stations
Figure 1: Corg/N molar ratio in sediments and sedimentary humin of the. Coastal Central Adriatic Sea
(CCA), Middle Adriatic Pit (MAP) and Southern Adriatic Pit (SAP). Mean values of all the core
standard deviation and standard error are represented
4. Conclusions
A large amount of sedimentary organic matter (up to 80%) in Adriatic Sea is due to the HM
fraction pointing out the possible relevance both of the different origin of the organic matter
depending on the zone and of the humification processes.
Acknowledgements
We thank the captain and the crew of the R/V Urania of CNR for the assistance during the
VECSES1 cruise. The research activity has been possible thanks to the financial support of
the VECTOR Italian national project and the SESAME EU-FP6 project (contract n. 36949).
References
1. J.D. Coates, K.D.Cole, R., Chakraborty, S.M.O’Connor and L.A. Achenbach, Appl. Environ.
Microbiol. 68 (2002) 2445.
2. A. Moreda-Piñeiro, A.B. Barrera and P.B, Barrera, Anal. Chim. Acta 524 (2004) 97.
3. A. Nissenbaum and I.R.Kaplan, Limnol. Oceanogr. 17 (1972) 570.
4. L. Tremblay and J.P. Gagné, Org. Geochem. 38 (2007) 682.
Vol. 3 Page - 83 -
15th IHSS Meeting- Vol. 3
Influence of Pre-Ozonation of Solutions of Fulvic Acid on Equilibrium
Adsorption on Activated Carbon
I. Kozyatnyk*, L. Savchyna, N. Klymenko
a
Institute of Colloid Chemistry and Chemistry of Water, National Academy of Sciences of
Ukraine, 42 Vernadsky Avenue, Kyiv 03680
E-mail: koziatnik@ukr.net
1. Introduction
Adsorption on activated carbon (AC) is one of the best methods for removing of natural organic
matters (NOM) in the technology of high quality drinking water preparation. Activated carbon is used
in the technological scheme for the final removal of the NOM before chlorination to prevent the
formation of chlorinated disinfection by products in most water supply [1].
In [2] it was proposed to use the constants of adsorption equilibrium applying to the concentration of
the mixture of organic substances “conditional component” for quantify the absorbability of a
multicomponent mixture of organic substances from aqueous solutions. Using “conditional
component” approach [2] is most successfully accomplished through the application of ideal
adsorption solution theory [3–5] for estimation of equilibrium adsorption parameter of
multicomponent NOM.
Different ability to the adsorption for factions of FA on the AC is stipulated primarily by the value of
their hydrophilic-lipophilic balance and the availability of the porous space of the AC for the
penetration of molecules of FA with different molecular weight. It is shown the higher the degree of
hydrophilicity of the adsorbate, the lower the change of its Gibbs free energy of adsorption [2].
The effectiveness of adsorption of NOM on the biologically active carbon is determined by the value
of biodegradable organic carbon (BDOC) in the content of total organic carbon in solution. It is known
that the biologically degradable organic matters more hydrophilic compared with bioresistant. So
perhaps there should be a correlation between the BDOC content in the solution of FA and the
magnitude of the free energy of adsorption of FA and products of their destruction on the BAC.
In [6] it was proposed an approach for assessing the rational degree of organic matter oxidation in
water before to subsequent stages of treatment. It is known that the effectiveness of biofiltration of
organic matter on the AC depends on the change of Gibbs free energy of adsorption (-ΔG0а): the
higher the rate, the lower the contribution of the biodegradable component in the overall efficiency of
the process of biofiltration and vice versa [7]. Ozonation converts dissolved organic carbon from
hydrophobic to hydrophilic organic carbon without significant removal of dissolved organic carbon.
The positive role of this transformation is particularly effective when filtering water through BAC [8].
Thus, the purpose of this study was to evaluate the parameters of equilibrium adsorption of NOM (for
example, FA) from aqueous solutions and their changes after ozonation solutions before adsorption.
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15th IHSS Meeting- Vol. 3
2. Materials and Methods
As the object of study were used fulvic acids (FA), obtained by the Forsyth method [9] from highmoor peat. The object of investigation was KAU carbon, which is obtained by treatment of crushed
fruit stones with concentrated alkali and hot hydrochloric acid (after washing with water), washing,
carbonization and activation with steam. The major adsorptive characteristics of that carbon were as
follows: the total specific surface of 1036.4 m2/g, the limiting adsorptive pore volume of 0.51 cm3/g,
the microporous space volume of 0.16 cm3/g. The investigations were conducted using the AC fraction
of 0.5–1.0 mm.
The ozonation of the FA solutions has been performed as follows: 5 L of the FA solution have been
treated with the ozone/airy mixture. The ozone concentration in that mixture was 5 mg/L. The delivery
rate of that mixture was 2.5 L/min. The solutions were ozonized for 2, 4 and 6 min, and the
corresponding ozone dosages were 6, 12 and 18 mg/L.
The BDOC part in the TOC has been determined using technique represented in [10–12]. It is pointed
out in these papers that, at the BDOC determining, it is the best thing to fix the microorganisms at the
inorganic carriers (quartz sand) and to use the microorganisms from the potable water treatment
stations and the distributing systems.
We investigated the adsorption equilibrium in systems with non-ozonized and ozonized solutions of
FA at pH 6. For ozonation were used solutions of FA with different initial concentration of dissolved
substances: a)1 series of experiments - the content of TOC 12.7–14.7 mg C/L; b) 2 series of
experiments - the content of TOC 31.5–33.7 mg C/L. Assessment of adsorption characteristics of AC
samples at adsorption from aqueous solutions of FA was carried out by the Freundlich model, using
the approaches given in [13,14], and the method of “conditional component” [2].
3. Results and Discussion
At the first it is necessary to evaluate the equilibrium adsorption capacity of the AC for qualitative and
quantitative assessment of the effectiveness of activated carbon for water purification from NOM.
Removing of the NOM leads to a decrease in colour and content of BDOC, and removal of precursor
of chlorine disinfection by-products formation. Adsorption isotherms can be used to determine the
minimum dose of adsorbent and the ratio of the liquid-sorbent in the dynamic experiments.
At the different content of TOC in the initial solution, the change of the adsorption is different in
ozonated solutions at the same equilibrium concentration. The dependence of the equilibrium
adsorption on the concentration of the initial solution is a feature of adsorption from multi-component
NOM solutions, as noted in several studies [15, 16]. The change in ozone dose has different effects on
reducing the adsorption capacity of the AC. The smallest decrease in the magnitude of adsorption is
observed at a dose of 12 mg O3/L, the largest – with 6 mg O3/L. This shows the different chemical
nature and molecular size of the products of the NOM oxidation.
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15th IHSS Meeting- Vol. 3
Freundlich equation constants was defined in order to
evaluate the influence of ozonation on the parameters
of
equilibrium adsorption.
Furthermore,
it
а, mg С/g
1000
was
100
estimated the change in free energy of adsorption of
10
FA ozonation products using the method of conditional
component. Figure shows the adsorption isotherms of
FA from aqueous solutions in the coordinates of the
0,1
modified Freundlich equation [2–4] when the initial
concentration of TOC in solution was 31.5–33.7 mg/L.
Isotherms at initial concentration of TOC 12.7–14.7
mg/L have the same shape.
○–1
■–2
▲– 3
♦–4
1
1
10
100
Сeq , mg С/l
Isotherms of FA adsorption from ozonated and nonozonated solutions:
1 – non-ozonated; 2 – 6 mg O3/l;
3 – 12 mg O3/l; 4 – 18 mg O3/l
As it is seen from Fig. the adsorption isotherm of non-ozonized FA solution at initial TOC
concentrations 31.5–33.7 mg C/L in logarithmic coordinates of the Freundlich equation has two
distinct areas that are characterized the adsorption capacity of different FA factions in solution. As a
result of ozonation two distinct areas disappear on the FA adsorption isotherms in the Freundlich
equation coordinates, and adsorption isotherms are described by a straight line. That is, the
composition of the FA becomes more uniform with respect to the adsorption capacity.
Table 1: Changing in the Freundlich equation constants, BDOC and (-ΔGa0) values at the ozonation of
FA solutions
TOC, mg
C/L
14.7
12.7
13.7
13.6
31.6
31.5
32.1
33.7
Ozone dose,
mg C/L
0
6
12
18
0
6
12
18
BDOC (part of the TOC),%
48.2
56.6
51.0
49.8
52.6
47.9
45.8
47.2
Freundlich equation constants а
КF
1/n
0.33
1.73
0.69
0.94
0.64
1.27
1.52
0.77
0.54
1.52
0.91
1.28
1.56
1.02
0.84
1.40
(-∆Gа0),
kJ / mol
19.08
17.37
18.08
19.05
18.85
19.05
19.76
19.76
As seen from Table 1, ozonation leads to an increase in the BDOC content with lower initial
concentrations of TOC in solution. Change of free energy of adsorption (-ΔGа0) also decrease that
confirms our assumption about the correlation of these values. Change the dose of ozone has
practically no effect on the change in the percentage BDOC in ozonated solutions when we ozonated
of solutions with higher initial content of the BDOC. Biodegradable organic carbon value decreases
from 52.6% in non-ozonated solutions to 47.2–47.9% in ozonated solutions. According to these the
value of (-ΔGа0) changes from 18.85 kJ/mol up to 19.05 – 19.76 kJ/mol.
Increasing of the ozone dose leads to an increase in the value of the coefficient KF and reduce 1/n, due
to a change in the chemical properties of ozonation products compared to initial multi-component
solution of FA. The constants KF and 1/n for non-ozonized solution were calculated for the first plot,
which characterizes the adsorption of low-adsorbed faction of FA. Averaging properties of FA
ozonation products in relation to the adsorption capacity generally leads to an increase in the constant
Vol. 3 Page - 86 -
15th IHSS Meeting- Vol. 3
KF and decrease the constant 1/n. Ozonation of FA solutions by dose 12 mg/L probably leads to the
formation of by-products with properties different from those in other ozonated solutions. We can
conclude that it is difficult to judge unambiguously about the influence of ozonation on the adsorption
of FA from aqueous solutions on the AC on the basis of analysis of the Freundlich equation constants
changes. Therefore it is more appropriate to use a another approach, associated with the definition of
the correlation between the percentage of BDOC in solutions of FA and the change in Gibbs free
energy of adsorption of products of FA ozonation.
It is evident from present data that ozonation of the FA solutions with high relative BDOC content
leads to a reduction of its value in total organic carbon. This fact can have positive effects in the
subsequent process of coagulation because of hydrophobization of FA ozonation products. However, it
is expedient to increase the value BDOC in the content of TOC at the sequential combination of
ozonation and biofiltration through activated carbon to improve the process.
4. Conclusions
Thus, it can be concluded that ozonation of FA solutions with high initial BDOC content by ozone
doses that are economically and technologically acceptable, leads to a decrease in the BDOC value
compared with non-ozonized solution. Ozonation of FA solutions leads to equalization of the
adsorption ability of FA factions compared with non-ozonized solution. Ozonation of FA solutions
increases the adsorption energy of FA in the most of the investigated systems with a high initial
BDOC content. This may worsen the conditions for biofiltration through biologically active carbon,
but also improve the conditions of coagulation treatment of water. Prediction of the effectiveness of
ozonation NOM solutions before filtration through BAC is more appropriate by determination the
value of the free energy of adsorption.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
S.D Faust and Aly O.M. Chemistry of Water Treatment, Lewis Publishers, Boca Raton, 1999, p.581.
A.M. Koganovskij, N.A. Klymenko, T.M. Lievchenko and I.G. Roda, Adsorption of Organic Matters from
Water, Khimia, Leningrad, 1990, p. 256. In Russian.
G.W. Harrington and F.A. DiGiano, J. Amer. Water Works Assoc., 81 (1989) 93.
E.H. Smith and W.J.Jr. Weber, Water Air Soil Pollut., 53 (1990) 279.
H. Sontheimer, J.C. Crittenden and R.S. Summers, Activated Carbon for Water Treatment, DVGW–
Forschungstelle, Karlsruhe, 1988, p. 690.
N.A. Klymenko, L.V. Nevynna, Yu.V. Sydorenko, O.G. Shvidenko, and Yu. O. Shvadshina, J. Water
Chem. Technol., 29, 1 (2007), 15.
N.A. Klimenko, M. Winther-Nielsen, S. Smolin, L. Nevynna and Y. Sydorenko Water Res., 36 (2002) 5132
W. Nishijima, W.H. Kim, E. Shoto and M. Okada, Water Sci. Technol., 38, 6 (1998) 163.
L.N. Alexandrova, Soil Organic Matter and Processes of its Transformation, Nauka, Leningrad, 1980. In
Russian.
J. Joret, Y. Levi, T. Dupin and M. Gilbert, Proc. AWWA Annual Conference, Orlando, FL., 1998, 1715.
S. Trulleyova and M. Rulik, Science of the Total Environment, 332 (2004) 253.
U. Raczyk-Stanislawiak, J. Swietlik, A. Dabrowska and J. Nawrocki, Water Res., 38 (2004) 1044.
E.H. Smith, Water Res, 28 (1994) 1693.
E.H. Smith and W.I. Weber, Water Air Soil Pollut., 53 (1990), 279.
S. Qi and L. Schideman, Water Res., 42 (2008), 3353.
F.S.Li, A. Yuasa, H. Chiharada and Y. Matsui, J. Colloid Interface Sci., 265, 2 (2003), 265.
Vol. 3 Page - 87 -
15th IHSS Meeting- Vol. 3
Study of Estuarine Sediments in Galway Bay
R. Mylottea*, M. H. B. Hayesa, C. Daltonb
a
Chemical and Environmental Science Dept., University of Limerick, Ireland; bDept. of
Geography, Mary Immaculate College, Limerick, Ireland
E-mail: rosaleen.mylotte@ul.ie
1. Introduction
Oceans are the largest global carbon pool and are estimated to hold approximately 38,000
PgC (petagrams of carbon) (Mahli, 2002). The oceanic sediments contain 150 Pg of organic
matter (OM) (Ridgewell and Edwards, 2007). The oceans potentially could absorb and store
vast quantities of anthropogenic carbon dioxide (CO2), a potent greenhouse gas. Cold
turbulent waters dissolve CO2 while warm waters dissolve less CO2 and can even release
CO2, switching the oceans to a source of CO2. Global warming has focused attention on
oceans in an effort to reduce atmospheric CO2 and to help prevent acidification of the ocean
surface.
Core samples are being studied from the transitional waters in Galway Bay. A main focus of
the study is the effect that the estuary is having on the bay especially, with regards to the
organic matter (OM) present. OM is washed into the Bay from the River Corrib and its
tributary streams. OM is a reservoir of carbon (in sediments) and an important sink. Studying
the organic and inorganic colloidal components contained within the estuarine sediments can
give indications of changes that have occurred over time to the composition of the matter
transported to the estuary and will provide an insight into the composition of carbon
sequestered in the sediments.
The humic substances will be extracted from each of the four cores at different depths. Humic
substances (HS) are the refractory end products from the degradation of plant and microbial
organic material (Lepane, 1999). HS is composed of humic acid (HA), fulvic acid (FA) and
humin. HS were traditionally thought to be high molecular weight molecules (Laird, 2008).
Work by Piccolo (2001) as elaborated Sutton and Sposito (2005) has suggested that HS are a
supramolecular association of many relatively small and chemically diverse organic
molecules that form clusters linked together by hydrogen bonds and by hydrophobic
interactions. The project is studying in detail the compositions of the HS at different depths
and their associations with the sediments.
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2. Location
Galway Bay is located in the west of Ireland between Co. Galway and Co. Clare. The River
Corrib flows from Lough Corrib into Galway Bay. Core samples were taken at increasing
distances from the River Corrib estuary (see Fig. 1). Four cores were taken using a vibrocorer
(Geo-corer 6000) supplied by the Geological Survey of Ireland aboard the Marine Institute’s
ship the Celtic Explorer (Cruise No. CE09-04). Core samples are up to 6m in depth. Surface
grab samples were also taken using a Day grab.
Figure 1: Map of Coring Positions in Galway Bay
3. Materials and Methods
The sediment samples are pretreated with HCl to remove any CaCO3 present (H+ exchanged).
This involves adding 1M HCl to the sediment and leaving it to stand until the reaction is
complete. The OM is extracted and analysed using methods developed by the Carbolea
Research Group. The methods employed involved exhaustive extraction using 0.1M NaOH
(at pH 7, 10.5 and 12.6), 0.1M NaOH + 6M Urea, and DMSO + 6% H2SO4. All extractions are
carried out under nitrogen. The humic substances (HS) are isolated using the XAD-8 and
XAD-4 resin in tandem procedure (Hayes et al. 2008). Solid and liquid state NMR will be
employed to investigate the compositions of the organic fractions (Song et al., 2008). The
sediment (core 1 0-25cm) has being fractionated into sand, silt and clay. The isolated clay
will be analyzed using X-ray diffraction to determine the minerals present. The Walkley
Black procedure (Allison, 1965) will be used to determine the total organic matter in the
sediments at different depths and different fraction. Elemental analysis and XRF core
scanning will determine what elements are present in the sediment. The Itrax XRF can also
take high resolution images allowing visualization of the layers present in the sediment. Dr.
Catherine Dalton will investigate the palaeontology of the sediments at different depths.
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Changes in chemical composition can be related to palaeontology differences observed. A
master core will be dated radiometrically. Microfossils will be dated using
14
C Accelerator
Mass Spectrometry. Diatoms and foraminifera will be examined to investigate biological
diversity. The quality of the intact core, sedimentation rates, permeability and chemistry will
be analysed using the Geotek multi sensor core logger. Sediment core lithology will be
examined to determine the rock present and the associated minerals.
4. Results/Discussion/Future Work
From the extractions (Grab samples A1001, D1004 and core 1 0-25 cm), pH 7 and pH 10.5
(adjusted using 0.1M NaOH) yielded no OM in the supernatent. Extractions at pH 12.6 and
6M urea + 0.1M NaOH resulted in breaking of the hydrogen bonds and OM was released.
Therefore the sediment has been exhaustively extracted at this pH. The particulate matter was
removed by centrifugation and filteration. Further extractings with DMSO + 6% H2SO4 are
ongoing.The humic fractions will be isolated using resins. Core’s 1 to 4 have been scanned,
ga
m
m
a
(S
M
ag
ne
tic
S
us
ce
pt
ib
ili
ty
(g
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de
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ity
V
el
(m
/s
)
W
av
e
P
P
C
or
e
W
av
e
A
m
p
T
hi
ck
ne
ss
(c
m
)
the results are being analysed.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
10.0
10.5
11.0
11.5
12.00.0
0.2
0.4
0.6
0.8
1.0 0
400 800 1200160020000.0
1.0
2.0
3.0 0. 0
2.0
4.0
6.0
Figure 1: GeoTek MSCL Plot for Core 1
The preliminary results from core 1 (Fig. 1) have been compiled. Further analysis of the
results is necessary. Pictures were taken with a digital camera of the cores (see Fig. 2) as they
were split using the GeoTek core splitter.
Bottom
Top
Figure 2: Picture of a split core 1 (2-3 metres) showing visable layers
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Cores 1 to 4 have been scanned with the Itrax XRF scanning instrument. This generates a vast
amount of information which needs to be analysed. The clay has been found to be aggregrated
in the sediment. Currently there are experiments underway to disaggregrate the clay. The
efficacies of sonication and the addition of a deflocculant (sodium hexametaphosphate), or a
combination of both, are being determined. Clay will be analysed by XRD to identify the
minerals present that are associated with carbon sequestration. Soil minerology can help to
protect HS from degradation. This highlightes the importance of investigating the clay
minerology. This work will examine the composition of the carbon sequestered in oceanic
sediments.
Acknowledgements
This work is funded by the HEA through the PRTLI IV scheme. Thanks to my supervisors,
Prof. Michael Hayes and Dr. Catherine Dalton. Thanks to the Marine Institute, Dr. D. Toal,
Dr. C. Dalton and F. Melligan, all of whom were involved in collecting the core samples. I
also thank Dr. S. McCarron and Dr. J. Turner who are technicians in NUI Maynooth and
UCD.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Allison, L.E., (1965) ‘Organic carbon’ In: Method of Soil Analysis, Part 2, Chemical and
Microbiological Properties (eds. Black, C.A., et al.), pp. 1367-1378. American Society of
Agronomy, Inc., Madison, WI.
Hayes, T. M., Hayes, M. H. B., Skjemstad, J. O., Swift. R.S., (2008), 'Studies of compositional
relationships between organic matter in a grassland soil and its drainage waters', European
Journal of Soil Science. 59: pp. 603-616.
Laird, D. A., Chappell, M. A., Martens, D. A., Wershaw, R. L. and Thompson, M, (2008),
'Distinguishing black carbon from biogenic humic substances in soil clay fractions', Geoderma,
143 : page 115-122
Lepane, V., (1999), 'Comparison of XAD resins for the isolation of humic substances from
seawater', Journal of Chromatography A, 845: pp. 329-335
Mahli, Y., (2002), ‘Carbon in the atmosphere and terrestrial biosphere in the 21st century’,
Philosophical Transactions of The Royal Society, 360: pp. 2925-2945
Piccolo, A., (2001), 'The Supramolecular Structure of Humic', Soil Science, 166: pp.810-832
Ridgewell, A. and Edwards, U., (2007), 'Geological Carbon Sinks', p.76, in Raey, D., Hewitt, C.
N., Smith, K and Grace, J., (eds.), Greenhouse Gas Sinks, UK, CABI Publishing
Song, G., Novotny, E. H., Simpson, A. J., Clapp, C. E., Hayes, M. H. B., (2008), ‘Sequential
exhaustive extractions, and characterisations using solid and solution state NMR, of the humic,
including humin, components in a Mollisol soil’, European Journal of Soil Science, 59: pp. 505516.
Sutton, R. and Sposito, G, (2005), 'Molecular Structure in Soil Humic Substances: The New
View', Environmental Science and Technology, 39 : pp. 9009-9015
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Effect of River Floods on Marine Organic Matter Fluorescence
Edith Parlantia,b*, Stéphane Relexansa,b, Fabienne Ibalota,b, Sandrine Huclier-Markai c,d, Rudy
Nicolauc, Stéphane Mounierc, Yves Lucasc,
a
Université de Bordeaux, UMR 5255, ISM, Groupe LPTC, 351 Cours de la Libération
Talence, F-33405 France; b CNRS, UMR 5255, ISM, Groupe LPTC Talence, F-33405
France; c Université du Sud Toulon-Var ; Laboratoire PROTEE-CAPTE, B.P. 132, La Garde
Cedex, F-83597 France; d Université de Nantes, Laboratoire Subatech, Ecole des Mines de
Nantes, CNRS/IN2P3, 4 Rue A. Kastler, BP 20722, 44307 Nantes Cedex 3, France.
E-mail: e.parlanti@ism.u-bordeaux1.fr
1. Introduction
Dissolved organic matter (DOM) consists of a mixture of macromolecular compounds with
wide ranging chemical properties and diverse origins. Fluorescence spectroscopy has been
applied for characterizing fluorescence properties of coloured dissolved organic matter
(CDOM) for several decades. This technique yields important information on the dynamics
and chemical nature of bulk CDOM as a function of its fluorescence intensity and fluorescent
functional groups. The monitoring of the fluorescent DOM has often been used to distinguish
between water masses from various sources [1, 2], to follow the distribution of water masses
[3] or, equally to study the mixing processes in coastal and estuarine waters [4, 5]. In the
1990s, three-dimensional excitation emission matrix (EEM) spectroscopy came into more
common use for characterizing fluorescence properties of CDOM. EEM spectroscopy
provides highly detailed information and the data can be analysed as excitation spectra,
emission spectra or synchronous scan spectra. This technique reveals the complete
photophysical system of the complex multi-chromophore macromolecular CDOM and is now
largely used for the characterization of fluorescent organic material in aquatic environments.
2. Materials and Methods
The fluorescence spectra were recorded with a Fluorolog SPEX FL3-22 Jobin Yvon
Fluorometer. The fluorescence EEM spectroscopy involved scanning and recording of 17
individual emission spectra (260-700 nm) at sequential 10 nm increments of excitation
wavelength between 250 and 410 nm.
In order to discuss the results of the fluorescence analysis of the different samples, we
considered on the one hand the ratios of the intensities of the main fluorescence bands. On the
other hand, we applied to aquatic environments the humification index (HIX) [6] in order to
estimate the maturation of DOM in soils. Referring to this humification index HIX, we also
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15th IHSS Meeting- Vol. 3
built another parameter BIX (biological index) to characterize the autochthonous inputs
(biological origin) to DOM.
The goal of this study was to characterise the organic matter inputs from a small river
(Eygoutier River) into the Mediterranean Sea (Toulon Bay). We studied the organic matter
inputs during the lowest water levels of the river and the flood events for two years (2004 and
2005). Raw water samples were filtered through a 0.7 µm GF/F glass fibre filter. Samples
were treated against bacterial growth by adding 100 µL of 98 % sodium azide.
3. Results and Discussion
The increase of terrestrial DOM inputs in seawater was very well correlated with the increase
of the river flow. Samples were collected every hour from the very beginning of the first rain
for each flood. The influence of floods on Mediterranean coastal water CDOM was mainly
detected on surface water samples. Major modifications of the quality and quantity of DOM
were observed. DOM was then in particular characterized by higher HIX values. This index
appeared to be a good indicator of the impact of the floods in seawater. Even if the effects
were less significant for the deep water samples collected, we could observe some
modifications of DOM due to the flood inputs. The terrestrial inputs did not reach however
the most remote sites studied.
4. Conclusions
This work showed that the fluorescence intensity ratios as well as the HIX and BIX indexes
were particularly well adapted to the characterization and classification of CDOM in marine
and coastal environments. The HIX index appeared to be a good indicator of the impact of the
floods in seawater. Even though some CDOM modifications due to the flood events were
observed for the deep water samples, they were really less impacted than surface waters.
Acknowledgements
This work was supported by the French national program ECODYN.
References
B.J.H. Matthews, A.C. Jones, N.K. Theodorou, A.W. Tudhope, Mar. Chem., 55 (1996) 312- 317.
P.G. Coble, Mar. Chem., 51 (1996) 325-346.
R.F.Chen, J.L.Bada, Mar. Chem., 37 (1992) 191-221.
M.M. De Souza-Sierra, O.F.X. Donard, M. Lamotte, Mar. Chem., 58 (1997) 51-58.
C.E. Del Castillo, P.G. Coble, J.M. Morell, J.M. Lopez, J.E. Corredor, Mar. Chem., 66 (1999) 35–
51.
6. A. Zsolnay, E. Baigar, M. Jimenez, B; Steinweg, F. Saccomandi, Chemosphere, 38-1 (1999) 4550.
1.
2.
3.
4.
5.
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Study of Colloidal Organic Matter Transformation Processes at Superficial
Sediment Interfaces
E. Parlantia,b*, S Relexansa,b, D Amourouxc, R Bridouc, S Bouchetc, G Abrild, H Etcheberd
a
Université de Bordeaux, UMR 5255, ISM, Groupe LPTC, 351 Cours de la Libération
Talence, F-33405 France; b CNRS, UMR 5255, ISM, Groupe LPTC Talence, F-33405
France; c Université de Pau et des Pays de l’Adour, CNRS, UMR 5034, LCABIE, av P.
Angot, Pau, F-64053 France; d Université de Bordeaux, CNRS, UMR 5805, EPOC, Avenue
des Facultés, Talence, F-33405 France
E-mail: e.parlanti@ism.u-bordeaux1.fr
1. Introduction
The maximum turbidity zone (MTZ) of an estuary is especially characterized by intense
cycles of settling and resuspension of anoxic mud fluid. Moreover dissolved organic matter
(DOM) accumulates in the MTZ where it has a longer residence time and is then submitted to
flocculation and sedimentation processes that modify the size distribution of the
macromolecules during the transit of organic material to the marine medium [1]. Due to
intense diurnal, tidal and seasonal cycles as well as to high organic matter amounts,
superficial sediments are submitted to a lot of redox oscillations and show a great reactivity.
The role played by the colloidal fractions is of great interest to understand the variability of
sediment reactivity during these oxic/anoxic cycles. The aim of this study was to simulate in
vitro series of oxic/anoxic cycles in coastal and estuarine superficial sediments. A detailed
study of DOM fluorescence behaviour during oscillating oxic/anoxic conditions is reported.
2. Materials and Methods
The incubator used for this work (Fig.1) was developed by Commarieux and Abril [2].
Figure 1: Description of the incubator used to simulate oxic/anoxic oscillations in suspended sediment
samples
The forcing parameters studied in the experiments were mainly aerobic and anaerobic
conditions. Gas allowed moving suspended sediment from oxic to anoxic conditions, and vice
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versa. Continuous measures were possible in both gaseous and dissolved phases. Fine
superficial sediments from the Arcachon Bay and from the Adour Estuary (South western
France) were collected as marine and estuarine samples respectively. As DOM is a mixture of
organic macromolecules with a broad range of molecular size and weight it was fractionated
according to molecular size by using tangential-flow ultrafiltration using a molecular size cutoff membrane of 500 Da. Each isolated fraction (filtrate and retentate) was then characterized
using EEM spectroscopy.
3. Results and Discussion
This study showed significant modifications of DOM during oxic/anoxic oscillations. During
anoxic/oxic transitions we observed a decrease of DOM fluorescence intensity and a relative
increase of the proportion of components of molecular size >500Da. A global decrease in
fluorescence intensity of filtrates was observed all along the experiments indicating a decrease
of the relative proportion of small molecules (< 500 Da). A light increase of this fraction
proportion was however observed at the anoxic/oxic transition. Weak variations of the
fluorescence intensity ratios were observed: increase of Iγ/Iα and Iα'/Iα during the
anoxic/oxic transition and in oxic phase. Similar trends were observed for the two sediments
with an increase of DOM fluorescence intensity in oxic phases. The fluorescence indices HIX
and BIX variations highlighted rapid modifications of fluorescent DOM in response to the
redox oscillations.
4. Conclusions
A great reactivity of sediment organic matter was observed during redox oscillations. The
results show global changes of the intensity of fluorescent dissolved organic matter during the
transition phases, as well as rapid modifications of its quality. Tangential ultrafiltration allows
to observe differences in the size of molecules between the redox phases. The proportion of
molecules greater than 500 Da notably increases during the transition to oxic phases.
Acknowledgements
This work was supported by the French national program ECODYN
References
G. Abril, H. Etcheber, P. Le Hir, P. Bassoullet, B. Boutier, M. Frankignoulle, Limnology and
Oceanography, 44 (1999) 1304-1315.
2. M.V. Commarieux, G. Abril European Geoscience Union 1st meeting. Nice, April 25-30 Abstract
book (2004)
1.
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Resolving Ahthropogenic and Natural Organic Matter Using “hypy”
Xiaoyu Zhanga*, Colin. E. Snapeb, Will Meredithb, Yongge Suna
a
Earth Science Department, Zhejiang University, 38, Zheda Road, Hangzhou, China, 310027;
b
Department of Chemical and Environmental Engineering, University of Nottingham,
University Park, Nottingham NG7 2RD, UK
E-mail: xiaoyu.zhang@nottingham.ac.uk
1. Introduction
Completed in the Sui dynasty (581-618BC), the 1800 kilometre-long Beijing–Hangzhou
Great Canal is under consideration for designation as world cultural heritage site. Due to the
industrial output from the 18 major cities along the canal, representing one fifth of China's
industrial output and heavy use for transportation, parts of canal have been subject to severe
industrial pollution. The severe ecological deterioration endangers its potential for world
cultural heritage. Systematic study of roles of highly heterogeneous of natural organic matter
(NOM) in sediments play on the mixed combustion residues (black carbon) derived both
naturally from biomass and anthropogenic sources, together with coal and oil-derived
contaminants is key to better understand not only the biogeochemistry behavior of man-made
pollution in natural environment, but also the effects on the pollutant transportation and
transformation.
To help resolve and apportion the various possible inputs of macromolecular material and
black carbon (BC) in sediments collected from Great Canal, extraction and liquid
chromatography are being used. Temperature staged hydropyrolysis (hypy), which has great
power to convert biomass and labile OM completely into volatile products while providing
extremely good preservation of hydrocarbon moieties in kerogens is also utilized. The residue
of hypy comprising black carbon, together with extremely thermally mature kerogen are
important fraction of the structure of sediment and play significant role to the absorption and
transfer of man-made pollutants. GC-MS was used to help get information of products from
sequential steps.
2. Methods
The total organic carbon (TOC) of the samples in sequential stages were measured with
Elemental Analyser to get the organic carbon loss during sequential processing. The samples
were extracted using Soxhlet apparatus for 72 hours with a mixture of dichloromethane and
methanol (70:30). The resulting extracts were fractionated using open column liquid
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chromatography (silica: alumina) into aliphatic, aromatic and polar fractions by sequential
elution with 15 ml hexane, 15 ml hexane:DCM (60:40), 15 ml DCM: methanol (50:50).
The saturated and aromatic hydrocarbon fractions were then analyzed by gas
chromatography-mass spectrometry (GC-MS), with components were identified on the basis
of their mass spectra, GC retention times and comparison with literature mass spectra.
The extraction residue is then subjected to hypy to 550 oC in order to release the hydrocarbons
bound within solvent insoluble organic matter which can then be characterized by GC-MS to
resolve the NOM and anthropogenic inputs. The carbon residue remaining after hypy is
defined as the BC content of the sediment.
3. Data and discussion
The Canal Sediment sample contains total carbon (TC) of 3.35%, total inorganic carbon (TIC)
of 0.28%. The relatively high proportion of BC in the sediment may due to the extensive
distribution of coal combustion residue.
Table 1: Carbon content in sequential stages
Samples Starting carbon
EOM carbon
Hypy released carbon
Residual carbon (BC)
TOC (%)
3.07
0.70
1.16
1.21
Proportion of original TOC (%)
22.8
37.8
39.4
The extractable organic matter (EOM) was then fractionated by liquid column chromatograph
and analyzed with GC-MS. The Carbon Preference Index (CPI) value of aliphatic was
calculated as the following formula (Zheng et al., 2007):
CPI = 1/ 2 ⎡⎣( ∑ Ci + Ci + 2 + L Ci +8 ) / ( ∑ Ci −1 + Ci +1 + L Ci + 7 ) + ( ∑ Ci + Ci + 2 + L Ci +8 ) / ( ∑ Ci +1 + Ci +3 + L Ci +9 ) ⎤⎦
In which, i=25
Usually n-alkanes derived from the cuticular waxes of higher plants have strong odd/even
predominance and give CPI values >5. In contrast, n-alkanes from bacteria and algae give low
CPI values, around 1 (Cranwell et al., 1987). The CPI for the Canal Sediment is 1.08,
suggesting that the pollutants in the Canal Sediment are largely derived from crude oil. The
distribution of n-alkanes in the the aliphatic hydrocarbon fraction extracted from the sediment
is shown in Figure 1 and suggests that the oil input has undergone significant biodegradation,
as evidenced by high Unresolved Complete Mixture (UCM) below the chromatogram
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baseline and Pristane/C17 ratio of 2.74. The slight predominance of odd n-alkanes over even
combined with the apparent elevated abundance of C25, C27 and C29 which usually derived
from cuticular waxes are illustrated with terrestrial plant sources. (Powell, 1988; Murray and
Boreham, 1992; Sarmiento and Rangel, 2004; Basant et al., 2005).
Figure 1: Total ion chromatogram (TIC) of the aliphatic hydrocarbon fraction extracted from sediment
from the Great Canal, China
Figure 2: Total ion chromatogram (TIC) of the aromatic hydrocarbon fraction extracted from sediment
from the Great Canal, China
The polycyclic aromatic hydrocarbon (PAH) distribution shown in figure 2 display a
dominance of “parent” PAHs such as phenanthrene and pyrene, with relatively low abundance
of alkyl substituted PAH as methyl phenanthrene and methyl pyrene. Such a distribution is
typical of biodegradation oil (Volkman, 1984) or high temperature coal tar (Sun et al., 2003).
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Further work on the characterizing the hypy generated fraction, together with soils and
sediments samples collected from eastern China as references will be carried out to get more
information on the structure of NOM to the fate of man-made pollutants.
4. Summary
The Beijing-Hangzhou Great Canal sediment is found to be highly heterogeneous comprising
various complex macromolecules. Organic matter in the Canal sediment were extracted
sequentially according to their distinctive properties. The measurement for the organic matters
suggested that the Canal sediment is characterized by the highly man-made pollution. The
high BC content in the sample suggests combustion residues derived mainly from
anthropogenic combustion residues. Significant biodegradation of the crude oil derived input
is evidenced by the UCM distribution and hydrocarbon ratios. CPI and elevated C25, C27 and
C29 n-alkanes suggest the input of plant derived organic matter. Such systematic study of the
structural composition of the above organic matter continuum in the Canal sediments is
significant to better understand not only the biogeochemistry behavior in natural environment
but also the effects on the pollutant transportation and transformation and could give
instruction to the treatment.
References
1.
2.
3.
4.
5.
6.
Basant et al., 2005 G.G. Basant, S.B. Rajendra, K.B. Ashok, K. Dinesh, L.P. Kusum, K.M.
Adarsh, P.G. Jagdish, C.D. Gaur and J.T. Nizhat, Geochemical characterization and source
investigation of oils discovered in Khoraghat–Nambar structures of the Assam–Arakan Basin,
India, Organic Geochemistry 36 (2005), pp. 161–181.
Murray and Boreham, 1992 A.P. Murray and C.J. Boreham, Organic Geochemistry in Petroleum
Exploration, Australian Geological Survey Organization, Canberra (1992) 230 p.
Powell, 1988 T.G. Powell, Pristane/phytane ratio as environmental indicator, Nature 333 (1988),
p. 604.
Sarmiento and Rangel, 2004 L.F. Sarmiento and A. Rangel, Petroleum systems of the Upper
Magdalena Valley, Colombia, Marine and Petroleum Geology 21 (2004), pp. 373–391.
Volkman, J.K., Alexander, R., Kagi, R.I., Rowland, J. & Sheppard, P.N., 1984. Biodegradation of
aromatic hydrocarbons in crude oil from the Barrow Sub-basin of Western Australia. Organic
Geochemistry, 6, 619-632.
Zheng, Y., Zhou, W., Meyers, P.A., Xie, S., 2007. Lipid biomarkers in the Zoige-Hongyuan peat
deposit: indicators of Holocene climate changes in West China. Organic Geochemistry 38, 19271940.
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Organomineral Association Patterns of Humic Substances in Different
Venezuelan Estuarine Mangroves
Adriana Méndez a*, Zulimar Hernández b, Gonzalo Almendros b,
Xosé Luis Otero a, Felipe Macías a, Williams Meléndez c
a
Dpto. Edafología y Química Agrícola, Universidad de Santiago de Compostela (USC) 15782,
Santiago de Compostela (Spain); bCentro de Ciencias Medioambientales (CSIC), Serrano 115B,
28006-Madrid (Spain); cInstituto de Ciencias de la Tierra (ICT), Universidad Central de Venezuela
(UCV), Caracas 1010 A (Venezuela)
E-mail: adriana.mendez.moreno@rai.usc.es
1. Introduction
Mangroves have been the subject of interest by several researchers concerned with humic
substances. The physicochemical conditions of these hydromorphic soils (low redox potential,
anaerobic conditions…) together with a large variety of high intensity seasonal environmental
factors may have a large impact on the geochemical processes determining its dynamics. In
general, these ecosystems store a high amount of organic matter (OM) as a consequence of
inputs from fast-growing tropical vegetation and the constant contribution by riverine
sediments. This represents a suitable scenario to study not only factors related to the
performance of soil C sequestration, but also the nature, origin, dynamics, evolution and
organomineral interactions of its OM. Assuming the criterion that the extent to which plant
and microbial biomass are transformed into humic substances ought to be considered as
objective measurement of the performance of the mangrove as an active C sink, this study
focuses on assessing the quality of the soil OM in three estuarine mangroves in Venezuela,
aiming to establish its potential contribution to terrestrial C balance.
2. Materials and Methods
A total of eight mangrove soil samples were collected at different depths within the
Venezuelan insular territory (Margarita Island, Nueva Esparta State) and coastal region
(Falcón State) [1]. The sampling was carried out in three estuarine systems: i) plant
communities of Rhizophora (soil profile labelled as P09) in the Laguna de La Restinga, ii)
plant communities of Thalassia (P05) in the Laguna de Las Marites and iii) plant
communities of Avicennia and Rhizophora (P10 and P11) in the Golfete de Cuare.
Standard procedures were employed for the sequential isolation of the different humus
fractions [2]. A previous physical separation of the partially decomposed organic particles
was carried out by flotation in CHBr3-ethanol mixture (free organic matter, FOM (ρ< 1.8
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g·mL-3). The isolation of the extractable humic fractions was carried out by treating the soil
with 0.1M Na4P2O7 and 0.1M NaOH, five times each. The total humic extract was then
precipitated by the addition of 6M HCl to separate the humic acids (HA) from the fulvic acids
(FA). The alkali-insoluble soil residue contains the humin. The content of total organic carbon
(TOC), HA and FA were determined by wet partial oxidation with K2Cr2O7. The HA was
purified for its further chemical characterization.
The optical densities of the HAs were measured at 465 (E4) and 665 (E6) nm. The visible
spectra were acquired in a Hewlett-Packard model 8452A VIS-UV spectrophotometer. The E4
is often taken as an index for progressive humification [3] and the E4/E6 ratio was used as
index mainly of HA molecular size [4]. Infrared spectra were determined by a Shimadzu
FTIR-8400 PC and KBr pellets with 2 mg sample.
3. Results and discussion
The content of TOC varies over a wide range, between 4 and 280 g · kg-1, depending on soil
profile depth and the type of vegetation. Upper horizons, under vegetation of Rhizophora
(samples P11, P09 and to a lesser extent, P05) showed high TOC values, whereas deep
horizons, under Avicennia vegetation (sample P10), showed comparatively lower TOC values.
The mangroves with comparatively high OM concentration (soils which a priori could be
considered as behaving as potential C sinks) were also associated to high amounts of
particulate organic fractions (Fig. 1), i.e., the FOM, which is the case of mangrove plant
communities of Rhizophora (P11 and P09), whereas soils under plant communities of
Avicennia or Thalassia displayed a low content of FOM. The distribution of total C along to
soil depth evidenced an abrupt decrease of the particulate organic fraction and a significant
increase in the concentration of humin (strongly linked to mineral fraction) in all soil profiles.
The HA/FA ratio also changed with depth, depending on the area and type of plant
community. In mangroves developed from Avicennia (P10) the increase the concentration of
HA-type substances suggested active soil OM accumulation processes attributed to anaerobic
conditions in this zone, whereas in mangroves of Rhizophora (P11 and P09) the content of
HAs decreased in comparison with the amount of FA-type substances. In the latter case
approximately 20% of TOC consisted of colloidal fractions (HA+FA), with a low molecular
weight and low HA/FA ratio (~ 0.4).
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15th IHSS Meeting- Vol. 3
Figure 1: Visible and infrared spectra of the HAs, and distribution of the TOC in the different organic
matter fractions in soil from Venezuelan mangroves. Bars height (and the numbers above) corresponds
to total soil C in samples from different soil depths (in cm). P11 and P09: Rhizophora, P05: Thalassia,
P10: Avicennia. HA: humic acid, FA: fulvic acid, FOM: free organic matter, Humin
In general, the values of E4 in HAs from mangrove soil (Fig. 2) were relatively low (< 1 AU),
which could indicate weakly condensed humic substances, derived mainly from aquatic
biomass. The lack of a gradient of progressive aromatization with depth could be related with
the continuous contribution of sediments and plant debris of different origin. In this sense, the
E4 could be used as an indicator of OM quality, with independence of its value as source
indicator (allochthonous or autochthonous biomass) and evolution into the mangrove soil.
Some differences in the condensation processes of the HA are suggested by the E4/E6 ratio.
The high values of E4/E6 are observed in Rhizophora profile (P09), indicate small molecular
weight of HAs as regards the other samples, coinciding with the concentration of HA
observed in the Fig 1.
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15th IHSS Meeting- Vol. 3
Infrared spectra (not shown) suggested significant differences between HAs mainly as regards
the intensity of the peak centred at 1720 cm-1, indicating oxidative processes and/or terrestrial
origin. To lesser extent, the spectra differ in intensity of alkyl structures (2920 and 1460 cm-1)
inversely paralleling the E4 values and indicating less degree of humification in HAs with
more marked aquatic signature also reflected in the intensity of the amide (1660, 1540 cm-1
bands) revealed in the resolution-enhanced spectra.
4. Conclusions
In Venezuelan mangroves, the OM show high amount of particulate organic fractions in the
uppermost horizon, whereas the amount of humin (or C strongly linked to minerals) increase
with the depth of the soil profile. Nevertheless, the soil OM quality (which could be
considered as a surrogate indicator of the time of residence) is not correlated with depth or
vegetal community. In fact, the E4 and the carboxyl content of the HAs seem to be suitable
indicators to evaluate the local soil C quality. Mangroves with noticeable amount of soil C (in
depth) and high OM quality could be considered as the most active C sinks, as may be the
case of Golfete de Cuare.
Acknowledgements
We acknowledge with thanks the technical staff of the laboratories of Soil Science of the USC
and the UCV their help in the analysis and preparation of the samples.
References
1. M.B. Barreto, Acta Biológica Venezuelica, 4, (2004), 24.
2. P. Duchaufour and Jacquin, Bulletin de l'Association Française pour l' Étude du Sol, 1, (1975) 29–
36.
3. S.J. Traina, J. Novak and N.E. Smeck, Journal of Environmental Quality, 19 (1990) 151–153.
4. J.S. Chen and C.Y. Chiu, Geoderma, 117 (2003) 129–141.
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Natural Organic Matter and Humic Substances Interactions
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15th IHSS Meeting- Vol. 3
Relationship Between Organic Carbon Forms and Selected Trace Elements
in Grassland Soils
Pospíšilová Lubica*, Škarpa Petr, Petrášová Veronika, Konečná Marie
Mendel University of Agriculture and Forestry in Brno, Department of Agrochemistry, Soil
Science, Microbiology and Plant Nutrition, Zemědelská 1, 613 00 Brno, Czech Republic
E-mail: lposp@mendelu.cz
1. Introduction
Type of management is main factor influencing organic carbon content and its sequestration in soils.
Every type of farming should fulfil some basic conditions: it should be optimal with respect to yields,
considerate with respect to natural environment and should unified new and traditional forms of
farming with respect to regions [1]. Labile carbon content is supposed to be one of important factor for
anthropogenic effect evaluation. In our contribution content of stabile carbon forms (total organic
carbon, humic substances carbon, humic acids and fulvic acids carbon) and labile carbon form (hot
water extractable carbon) in different grassland soils will be discussed. Further we would like to
follow correlation between different carbon forms and selected trace elements.
2. Materials and Methods
Objects of our study were following grassland soils: Haplic Cambisol (locality Tři Kameny), Haplic
Cambisol (locality Rapotin), Haplic Stagnosol (locality Sluneční)), and Haplic Stagnosol (locality
Bílčice). Total organic carbon content was determined by oxidimetric titration method according to
[2]. Humic substances carbon, humic acids carbon and fulvic acids carbon were detected by short
fractionation method [3]. Labile organic carbon was determined by hot water extraction method [4].
Total and labile trace elements content was determined by flame or electrothermal atomic absorption
spectrometry after extraction of the soil samples in the aqua regia (total content) and in the solution of
0.01M CaCl2 (labile form). Description of the methods applied, and of the materials studied including
references [5].
3. Results and Discussion
Average values of different carbon forms determined in spring 2008 and spring 2009 are showed in
Table 1. Results showed that the highest total organic carbon was in Haplic Cambisol (Tři Kameny).
The highest amount of HA was determined in Haplic Stagnosol (Bílčice). FA amount was higher to
compare with HA content. Quality of humic substances was low. Ratio HA/FA was less than 1. Labile
carbon content was the highest in Haplic Stagnosol (Sluneční)) and decreased in order: Haplic
Stagnosol (Sluneční) > Haplic Cambisol (Tři Kameny) > Haplic Stagnosol (Bílčice) > Haplic
Cambisol (Rapotín). Determined trace element content is given in Table 2. Correlations between labile
carbon form and labile Zn was found. Labile Zn and Cd well correlated with humic substances carbon
(stabile form).
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15th IHSS Meeting- Vol. 3
Table 1: Stabile and labile carbon forms in studied soils
TOC
C labile
HS
HA
FA
%
mg/kg
mg/kg
mg/kg
mg/kg
Sluneční
Tři Kameny
Rapotín
Bílčice
2.2
2.9
1.34
1.96
2, 200
1,720
240
690
7.0
9.0
6.0
10.75
2.5
3.1
2.4
4.1
6.5
5.9
3.6
6.6
0.4
0.5
0.6
0.6
Sluneční
Tři Kameny
Rapotín
Bílčice
1.8
2.2
1.30
2
2,300
1,750
270
750
9.0
13.0
5.5
9.0
3.0
4.0
2.1
4.0
6.0
9.0
3.4
5.0
0.5
0.45
0.6
0.8
Soil types
Spring 2008
H. Stagnosol
H. Cambisol
H. Cambisol
H. Stagnosol
Locality
HA/FA
Spring 2009
H. Stagnosol
H. Cambisol
H. Cambisol
H. Stagnosol
Table 2: Trace elements content in studied soils (spring 2008)
Soil types
Locality
Total
content
mg/kg
H. Stagnosol
H. Cambisol
H. Cambisol
Sluneční
Přemyslov
Rapotín
Zn
72.33
81.18
59.69
H. Stagnosol
Bílčice
94.93
Labile
forms
Mo
Cd
Pb
Cu
Co
5.99 8.28 29.78 0.153 0.230
5.61 7.92 22.04 0.133 0.186
13.87 20.26 7.55 0.212 0.366
Zn
0.039
0.535
0.127
Cd
Cu
Co
0.012 0.034 0.028
0.051 0.028 0.049
0.081 0.040 0.024
13.97 20.75 27.22 0.205 0.252
0.519
0.098 0.060 0.055
4. Conclusions
We can conclude that grassland soils contained low amount of humic acids. Sorption and mobility of
trace elements was mainly influenced by fulvic acids. Correlation between labile Zn and Cd and
carbon content was detected.
Acknowledgements
This work was supported by the project MŠMT No. 2B08039.
References
1. J. Bouma, Implementing soil quality knowledge in land-use planning. In: P. Schjonning, S.
Elmholt, B.T. Christensen (eds.), CABI Publ., 2004, 283–295.
2. D. W. Nelson and L. E. Sommers, Total carbon, organic carbon, and organic matter. Page A. L.,
Miller R. H., Keeny D. R. (Eds.). SSSA Publ., Wisconsin, 1982, 539–579.
3. D. S. Orlov, Soil Chemistry, MGU, Moscow, 1985, p. 376.
4. M. Körschens, A. Wiegel, E. Schulz, J. Plant Nutr. Soil Sci. 4/98 (1998), 409–424.
5. F. J. Stevenson, Humus Chemistry – Genesis, Composition, Reactions. New York: Wiley & Sons,
1982, p. 443.
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Investigation of Humic Substances by Particle Size Distribution of Soils and
by Determination of Zeta Potential
Szilvia Joóa, Judit Tóthb, Gyöngyi Samua, Rita Földényia*
a
University of Pannonia, Department of Earth and Environmental Sciences, 8200 Veszprém,
Egyetem u. 10., Hungary; bInstitute of Materials and Environmental Chemistry, Chemical
Research Center, Hungarian Academy of Sciences, Hungary
E-mail: foldenyi@almos.uni-pannon.hu
1. Introduction
Soils are characterized by their particle size distribution basically. Particle size distribution
(PSD) influences the specific surface of materials hereby the adsorption capacity of the
adsorbent [1]. Adsorption processes have special role in the fate of pollutants in the
environment. The composition of the aqueous media can influence the particle size
distribution of soil and the transport processes of chemicals [2–3].
The humic substances (HS) which are very important components of the soils can adsorb
pollutants. Their solubility depends on the pH of the soil solution while the type of the
dissolved HS is determined by the type of the soil [4–5].
Surfactants are used everywhere and form one group of the main water and soil pollutants.
Since their role is to dissolve the non- or hardly water soluble materials as well as to form
stable emulsion, suspension etc. these compounds seem to be very harmful to the
environment.
The electrocinetic (zeta) potential let us conclude the stability of solution or suspension [6]
which affects the fate of the suspension in natural waters. Stability of solution affects
sedimentation, adsorption, aggregation, transportation of contaminants in soils. Better
stability means further transport.
The aim of the present work is to compare the PSD of the soils (determined in suspensions),
the composition as well as the stability of the soil solution, and to find relation among them.
Our investigations were focused on the role of anionic and cationic surfactants in the soil
solution. The model compounds chosen for investigations are used most frequently as
detergent in cosmetics (sodium dodecylsulphate: SDS), as forming agents in pesticide
formulations (Supragil WP) or in industry (alkyltrimethylammonium bromide: Cetrimide).
SDS and Supragil WP represent the anionic, while Cetrimide the cationic type of surfactants.
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2. Materials and Methods
The particle size distribution of different soils being typical in Carpathian Basin was
investigated by laser diffraction meaning a quite new method in PSD measurements [7]. In
this case suspensions made from the appropriate soil (hernozem, brown forest and sandy soil)
and alginite were analyzed by Master Sizer 2000 equipment. Every sample was studied in
distilled water. Furthermore sandy soil suspensions were made not only in pure water but
even in different aqueous solutions as follows:
HA. 0.1 mol/dm3 NaCl; HB. 0.1 mol/dm3 phosphate buffer (pH=6.6); HC. 0.01 mol/dm3
phosphate buffer (pH=6.84); HD. 0.1 mol/dm3 NaCl and 0.01 mol/dm3 phosphate buffer
(pH=6.6); HE. 0.1 g/dm3 Supragil WP; HF. 0.1 g/dm3 Supragil WP and 0.1 g/dm3 NaCl; HG.
0.1 g/dm3 Cetrimide; HH. 0.1 g/dm3 Cetrimide in 0.1 mol/dm3 NaCl solution; HI. 0.01
mol/dm3 CaCl2; HJ. 0.1 g/dm3 SDS; HK. 0.1 g/dm3 SDS in 0.01 mol/dm3 CaCl2 solution; HL.
0.1 g/dm3 SDS in 0.1 mol/dm3 NaCl solution; HV. distilled water.
The sandy soil suspensions were measured in different media after removing organic matter
by H2O2, too.
Critical micelle concentration (CMC) of surfactants was determined by measuring surface
tension by Traube stalagmometer in 0.01 mol/dm3 phosphate buffer (pH=7).
Adsorption of surfactants was investigated on sandy soil by static equilibrium experiments.
The analysis of equilibrated solutions was carried out by the so-called two-phased titration
[8]. Solutions of fulvic acid (FA, Organit Ltd.), sodium humate (HANa, Roth+Co, Karlsruhe)
with 35 mg C/dm3 concentration and suspensions of sandy soil were examined at different pH
values (5; 6; 7) by Zeta Sizer equipment (Malvern) using dynamic light scattering. Solution is
instable if zeta potential (ζ) <30 mV and it is stable if ζ >30 mV.
3. Results and Discussion
Different soils in pure water have different particle size distribution (Fig. 1). Surfactants
strongly influenced the PSD (Fig. 2.), while the salt solutions have less effect.
In Supragil WP (HE) and Cetrimide (HG) containing suspensions the size of soil particles
decreased slightly while SDS (HJ) had similar but more significant effect. In the presence of
surfactants the smaller, colloidal particles - like the humic substances - got into solution by
means of dispergation named as solubilization effect [5,9] which can enhance the transport of
contaminants. In salty media (HK, HL) SDS promoted formation of aggregates, which cannot
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15th IHSS Meeting- Vol. 3
be observed at the other investigated tensides. The proportion of bigger particles in the
suspension increased with higher SDS concentration. It can be explained by formation of
aggregates which were arisen either from the HS molecules or from HS and SDS molecules.
CaCl2 also assisted aggregate formation.
Figure 1: Particle size distribution of different soil suspensions obtained in pure water
Figure 2: Particle size distribution of sandy soil in surfactant containing suspensions
After removing organic matter (mostly FA, [5]) of the sandy soil by H2O2, surfactants in
themselves had no effect on smaller inorganic particles, however, in the presence of salts
aggregates could be observed.
According to the results of static equilibrium experiments surfactants adsorbed on sandy soil
in more layers caused by hydrophobic interaction between the solute and the mostly
hydrophobic surface (organic matter, quartz) of the adsorbent.
The CMC of surfactants as well as the pH influenced the zeta potential and the stability of the
suspension. If the tenside concentration was higher than the CMC the suspension proved to be
stable in the case of anionic surfactants (Fig. 3.a), if their concentration was lower, the
suspension was instable.
Cetrimide as a cationic surfactant could result in positive zeta potential of the system (Fig.
3.b). ζ=0 was observed at different tenside concentrations depending significantly on the type
of the dissolved HS.
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15th IHSS Meeting- Vol. 3
0
CMC (Supragil WP at pH=7): 646 mg/l
-20
CMC (Cetrimide at pH=7): 1820 mg/l
sandy soil, pH=7
FA, 35 mg C/l, pH=7
HANa, 35 mg C/l, pH=7
boundary of stability
40
30
-30
Zeta potential (mV)
Zeta potential (mV)
-10
sandy soil, pH=7
FA, 35 mg C/l, pH=7
HANa, 35 mg C/l, pH=7
boundary of stability
-40
-50
-60
-70
20
10
0
-10 0
500
1000
1500
2000
2500
3000
-20
-30
-40
-80
10
100
1000
10000
-50
Supragil WP concentration (mg/l)
Cetrimide concentration (mg/l)
Fig 3.a
Fig 3.b
Figure 3: Investigation of zeta potencial in different surfactant containing solutions
4. Conclusions
The investigated surfactants influenced the particle size distribution. Especially significant
was the solubilization effect of SDS used widely as a detergent. The removal of organic
matter content of the sandy soil did not resulted in similar process proving the special
interaction between the HS and surfactant molecules.
Adsorption of surfactants on sandy soil was mostly governed by hydrophobic interaction.
If the tenside concentration was higher than its CMC, the suspension became stable with
Supragil WP and SDS. The cationic surfactant Cetrimide behaved as a counter ion of HS
polyanions and was able to form even stable colloid system with positive zeta potential while
the stability caused by anionic tensides (SDS and Supragil WP) could be observed due to the
repulsive power of the negative charges.
According to these results the surfactants can influence material transport in the soil,
including hardly dissolving contaminants that may be adsorbed on soil particles, too.
References
1. D. Baenninger, P. Lehmann, H. Flühler, Eur. J. Soil. Sci. 57 (2006) 906.
2. R. Haque, V. H. Freed (Eds.), Environmental Dynamics of Pesticides. Plenum Press, New York
and London, (1975) 115.
3. M. S. Wilson (Ed.), Advances in Soil Organic Matter Research: The Impact on Agriculture and
the Environment. Redwood Press Ltd., Wiltshire (1991) 121.
4. E. Illés, E. Tombácz, Colloids Surf., A: Physicochem. Eng. Asp., 230 (2004) 99.
5. T. Ertli, A. Marton, R. Földényi, Chemosphere, 57 (2004) 771.
6. Malvern Instruments: Zetasizer Nano Series User Manual, MANO 317, 3.1, 2007.
7. G. Eshel, G. J. Levy, U. Mingelgrin, M. J. Singer, Soil Sci. Soc. Am. J. 68 (2004) 736.
8. Á. Patzkó, Laboratory Practices in Colloid Chemistry (in Hungarian), JATEPress, Szeged (1996)
54.
9. J .P.Gao, J. Maguhn, P. Spitzauer, A. Kettrup, Water Res. 32 (1998) 2089.
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Interactions of Organic Compound with NOM Need Water: Strong WaterInduced Enhancement of Carbamazepine Sorption on Peat
Mikhail Borisovera*, Maggie Selaa,b, Benny Chefetza
a
Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization,
The Volcani Center, Bet Dagan, Israel; bThe Department of Soil and Water Sciences, The
Hebrew University of Jerusalem, Rehovot, Israel.
E-mail: vwmichel@volcani.agri.gov.il
1. Introduction
Natural organic matter (NOM) controls distributions of multiple organic compounds between
different environmental compartments. The role of NOM-associated water in sorbate-NOM
interactions is not well understood, in contrast to solute-bulk water interactions. Recently, a
concept was suggested to explain the role of NOM-bound water in sorption of organic
compounds which included consideration of cooperative water-induced disruption of intraNOM interactions upon a penetration of organic sorbate [1, 2]. Yet, there is no clarity how a
type a NOM hydration effect (i.e. enhancement or suppression of sorbate interactions) and its
magnitude are related to the structure of organic sorbates. In this research, we examined the
effect of a hydration of a model NOM sorbent, Pahokee peat, on interactions of a probe
organic compound, carbamazepine (CBZ; 5H-dibenzo[b,f]azepine-5-carboxamide). The CBZ
characterized by relatively large molar volume (186.5 cm3/mol) was compared, in terms of
interactions with NOM, with phenanthrene (PHEN) having similar molar volume (178.2
cm3/mol), and, in terms of NOM hydration effect, with smaller specifically interacting
sorbate, phenol (87.8 cm3/mol). This comparison provided an insight in a relation between an
involvement of water in sorbate-NOM interactions and a size of organic sorbate.
2. Materials and Methods
Pahokee peat (supplied by IHSS) was freeze-dried and used as a model (dehydrated) NOM
sorbent. Sorption of CBZ by peat was examined in batch experiments from water and, at
different extents of peat hydration, from inert solvent, n-hexadecane (HD), according to the
protocols similar to that described in Refs. [2, 3].
3. Results and Discussion
Sorption of CBZ from water on fully hydrated NOM is stronger than its sorption on
dehydrated NOM from HD (Fig. 1A). To eliminate the differences in CBZ-solvent
interactions, solution concentrations were normalized by the CBZ solubility in a specific
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solvent. Thus, interactions of CBZ with NOM are enhanced at the presence of NOM-bound
water by factor of 20-40 (Fig. 1B). This profound effect of NOM hydration on CBZ-NOM
interactions is associated with specific CBZ-NOM interactions (elucidated by comparing the
distribution coefficients of CBZ and PHEN between fully hydrated NOM sorbent and the
inert reference state, [3]). Water-induced CBZ sorption enhancement appears also in HD
environment when the partial NOM hydration is increased. The CBZ sorption enhancement
starts at significantly higher water activities as compared with earlier observations of NOM
hydration effect on phenol sorption [2]. This enhancement was modeled, using Link Solvation
Model [1, 2], to provide an estimate for a number of water molecules involved in a
Sorbed concentration, mg/kg
penetration of a large-size organic sorbate into the NOM interior.
A.
B.
1000
1000
X 20-40 times
100
100
0.1
1
10
1E-3
Solution concentration, mg/L
Sorption from water
0.01
0.1
1
Solution concentration/solubility (~activity)
Sorption from n-C16H34
Figure 1: Sorption isotherms of carbamazepine on differently solvated model NOM
4. Conclusions
A strong water-induced enhancement of sorbate-NOM interactions is associated with greater
hydration of NOM moieties upon penetration of CBZ molecules.
Acknowledgements
This research was supported by Research Grant No. IS-3322-06 from BARD, The United
States-Israeli Binational Agricultural Research and Development Fund. Help from Nadezhda
Bukhanovsky (The Volcani Center, Israel) is greatly appreciated.
References
1. M. Borisover and E.R. Graber, Langmuir, 18 (2002) 4775.
2. E.R. Graber, L. Tsechansky and M. Borisover, Environ. Sci. Technol. 41 (2007) 547.
3. M. Borisover and E.R. Graber, Environ. Sci. Technol., 37 (2003) 5657.
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Adsorption of Metal Ions on Humic Acid Derived from Turkish Lignite
Bekir Zühtü Uysala*, Duygu Öztana, Ufuk Gündüz Zafera,
Özkan Murat Doğana, Selahaddin Anaçb, Mustafa Özdingişb, Zeki Olgunb
a
Chemical Engineering Department, Faculty of Engineering and Clean Energy Research and
Application Center (CERAC) , Gazi University, Maltepe, 06570 Ankara, Turkey; bTurkish
Coal Enterprises, Hipodrom Cad., No:12, 06330 Ankara, Turkey
E-mail: bzuysal@gazi.edu.tr
Abstract
Adsorption of some metal ions (Pb+2, Cu+2, Zn+2, Cr+6, Ni+2) on humic acid produced in a
pilot plant from Turkish Ilgın lignite was investigated in this work. The effects of adsorption
time, solution pH, metal concentration and temperature on humic acid adsorption capacity
were investigated. The humic acid adsorption capacity was found to follow an order as Pb+2 >
Cu+2 > Zn+2 > Cr+6 > Ni+2 .
1. Introduction
Environmental contamination with heavy metals represents a potential threat to humans,
animals and plants. Many of them are soluble in water, therefore become more available for
living systems and accumulate in the environment. Removal of heavy metals from waste
streams employs various technologies, which are often expensive. Use of inexpensive natural
sorbents such as zeolites, fly ash, coal has been considered as a promising alternative for this
purpose [1]. A large number of organic humic substances are also increasingly being applied
worldwide especially in agricultural applications. Turkish Coal Enterprises (TKI) of Turkey
has been producing humic acid and other humic containing substances in a pilot plant since
2008. The potential of using the humic acid produced by TKI for removal of heavy metal ions
from liquid waste streams was aimed to be assessed in this research.
2. Materials and Methods
Humic acid, produced from Ilgın lignites, was provided from the pilot plant of Turkish Coal
Enterprises (TKİ). The kits used for the analyses of Pb+2, Cu+2 , Zn+2 , Cr+6 , Ni+2 were
purchased from Hach-Lange. A Hach DR/4000 spectrophotometer and quartz cuvettes were
used for all absorbance measurements. Calibration curves for all the metal ions were first
developed employing Lambert-Beer Law and used in the analyses later.
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3. Results and Discussion
Optimum temperature and pH intervals, adsorption time and maximum adsorption capacities
(g metal / kg Humic Acid) were determined for each metal ion as shown in Table 1 and Fig 1.
Table 1: Optimum operating conditions and humic acid adsorption capacity
Metal Ion
Temperaure
(oC)
20
22
20
15
15
Nickel
Chrome
Zinc
Copper
Lead
pH
6
2.8
4.5
3, 4, 6
3
Adsorption time to
reach equilibrium, min
15
240
120
15
5
Adsorption Capacity
(g metal / kg Humic Acid)
12.64
37.93
97.50
154.33
196.90
Adsorption capacity
(g metal/kg HA)
Nickel
200
Chrome
150
Zinc
Copper
100
Lead
50
0
Amount of HA (kg)
Figure 1: Humic acid adsorption capacities for different heavy metals
It can be seen in Figure 1, the maximum adsorption capacity of humic acid was observed for
lead. For the others an order in the sequence of Pb+2 > Cu+2 > Zn+2 > Cr+6 > Ni+2 was obtained.
A similar order (Pb > Fe > Cu > Zn > Ni ) and similar magnitudes of adsorption capacities
were reported by Arctech Inc for their Humasorb-cs product [2].
4. Conclusions
In this work, the adsorption of heavy metal cations, Pb+2, Cu+2 , Zn+2 , Cr+6 and Ni+2 from
aqueous solutions on humic acid derived from Turkish Ilgın lignite was studied. The results
show that humic acid adsorption capacity followed the order of Pb+2 > Cu+2 > Zn+2 >, Cr+6 >
Ni+2. Optimum pH values were determined to be different for each metal as reported earlier
[3] and found to be between approximately 3 and 6.
References
1. M. Havelcova, J. Mizera, Sorption of metal ions on lignite and the derived humic substances,
Journal of Hazardous Materials, 161 (2009) 559.
2. www.arctech.com visited in December 2009.
3. P. A. Brown, S. A. Gill, Metal Removal from wastewater using peat,Wat. Res., 34
a. (2000) 3907.
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15th IHSS Meeting- Vol. 3
Characteristics of Humic Acids Isolated from Heavy Metals Contaminated
Soils at the Copper-Smelter “Legnica” (S-W Poland)
Alina Maciejewska, Jolanta Kwiatkowska-Malina*
Department of Spatial Planning and Environmental Sciences, Warsaw University of
Technology, 1 Politechniki Sq., 00-661Warsaw, Poland
E-mail: J.Kwiatkowska@gik.pw.edu.pl
1. Introduction
The presence of organic matter in soil is important not only with respect to plant nutrition but
to environmental pollution, as well. Heavy metals, such as: Cu, Pb, Zn and Cd can accumulate
in top soils. Humic substances (HS) and humic acids (HAs) of soil organic matter are
important in trapping and subsequent transport of heavy metals in the environment, due to the
presence of functional groups in their structures. The S-W region of Poland, particularly the
Legnicko-Głogowski district, is heavily contaminated with heavy metals [1], with the
maximum contents of Cu and Pb measured in the vicinity of the Copper-Smelter “Legnica”,
that amount to several grams per kilogram of soil. Despite considerable improvements of the
air quality in the last 2 decades, soil contamination with heavy metals in this region have not
considerably decreased , and it is likely to pose a long-term hazard to the food chain, ground
and surface waters and soil microorganisms [2]. Phytoextraction is commonly used to remove
heavy metals from soil by concentrating them in the harvestable parts of plants [4]. However,
in some cases it may be more efficient to limit the biological activity of heavy metals in soils
by transferring heavy metals into forms not available for plants. Organic matter present in soil
can form organometallic compounds, the so-called chelates, with heavy metals lowering their
accessibility for plants and soil micro-organisms, as well as their potential leaching to the
adjacent ground and surface waters.
The aim of this study was to compare the influence of soil contamination level on the quantity
and selected qualitative parameters of HAs extracted from soils from the area of the Copper–
Smelter “Legnica” heavily contaminated with heavy metals (mostly Cu and Pb).
2. Material and Methods
The research was made on the soil developed from silt loam on clay (WRB – Deluvial
Brown) from zone I and II (0.1 km and 1.0 km from the main emitter, respectively)
contaminated with heavy metals: zone I - 4985 (Cu), 1236 (Pb), 294.6 (Zn), 2.82 (Cd) [mg
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15th IHSS Meeting- Vol. 3
kg−1] and zone II - 1008 (Cu), 413.0 (Pb), 194.5 (Zn), 1.51 (Cd) [mg kg−1]. The soil from zone
I showed neutral, while from zone II - alkaline reaction (Table 1).
Soil samples were taken from the 0-30 cm horizon, air-dried, mixed and sieved (φ = 1-mm)
prior to analyses. The total organic carbon (TOC) was determined by the TOC analyzer
(Shimadzu 5000), the total nitrogen (Nt) - using the standard Kjeldahl method, pH potentiometrically in H2O and 1 M KCl, and the hydrolytic acidity (Hh) - using the Kappen's
method.
Humic acids were extracted from soil samples according to the IHSS standard method [6].
The values of absorbance at wavelength: 280 (A280), 400 (A400), 465 (A465), 665 nm (A665)
were determined for separated HAs. VIS spectra were performed for 0.02% HAs solutions in
0.1 M NaOH, and UV-spectra were determined after fivefold dilution by the Lambda 20
Perkin-Elmer Analyzer. Based on the determined absorbance values at wavelengths of: 280
(A280), 465 (A465), 600 (A600), and 665 nm (A665), the following absorbance ratios were
calculated: A2/4 - at wavelengths of 280 and 465 nm, A2/6 - at wavelengths of 280 and 665 nm,
A4/6 - at wavelengths of 465 and 665 nm. Simultaneously, the values of the coefficient Δlog K
= log A400 - log A600 were calculated for HAs. The FT-IR spectra were obtained in the 4000 to
400 cm-1 wavelength range by the Nicolet 5PC FTIR spectrophotometer on KBr pellets
obtained by pressing, under reduced pressure, uniformly prepared mixtures of 1 mg sample
and 400 mg KBr, spectrometry grade, with precaution taken to avoid moisture uptake. The
elemental composition of HAs was determined using the CHN 2400 Perkin-Elmer Analyser.
The oxygen content was calculated from the difference [100% - (%C+%H+%N)], in relation
to the ashless sample weight. Based on the HAs elemental composition, the values of atomic
ratios (H:C, O:C, N:C, O:H) were calculated, and the internal oxidation degree was
determined referring to the formula [3]: ω = (2O+3N-H):C (where: C, H, O, N - represent
contents of carbon, hydrogen, oxygen and nitrogen in atomic percentage, respectively).
3. Results and Discussions
The properties of examined soils (Table 1), particularly: reaction, hydrolytic acidity and the
organic carbon content, may cause a decrease of solubility (mobility) of heavy metals in top
soil, and consequently of their phytoavailability. The contamination levels of soil had no
influence on contents of organic carbon and nitrogen. The ratios of organic carbon to nitrogen
for both zones were similar and characteristic for polish arable soils.
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15th IHSS Meeting- Vol. 3
Table 1: Physicochemical and chemical properties of soils at the Copper-Smelter “Legnica”
pH
Sample
TOC
1 M KCl cmol(+) kg−1 of soil
7.05
0.44
7.30
0.39
H2O
7.30
7.55
zone I
zone II
Hh
Nt
g.kg-1 of soil
0.94 0.091
0.88 0.091
TOC:Nt
10.33
9.67
The absorbance values at wavelengths of 280, 465 and 665 nm of the HAs extracts are
presented in Table 2. Extracts from the soil samples (zone I) had higher absorbance values as
compared to zone II indicating higher carbon contents. However, it is not clearly reflected in
the elemental composition of HAs (Table 3). Although the TOC contents in soil samples from
zone I were higher compared to zone II, however, the absorbance values do not reflect it. The
values of A2/4 in both cases were similar, but the A4/6 and A2/6 absorbance ratios of HAs
extracts from zone II were lower than for zone I. HAs extracted from soil from zone II
compared with these from zone I had higher oxygen content, which indicates evolution
towards oxidation, was supported by an increase in O:C ratio. Humic acids extracted from
soils from both zones had similar carbon contents, as well as H:C and N:C ratios. HAs from
zone II were richer in nitrogen and oxygen, and were characterised by lower value of the ω
parameter, as compared to these from zone I. The data show that HAs in both zones have the
elemental composition similar to these from arable soils [5].
Table 2: Spectral properties of soil alkali extracts - the Copper-Smelter “Legnica”
Sample
zone I
zone II
A280
5.3
4.3
A465
0.94
0.76
A600
0.304
0.261
A665
0.161
0.142
A2/4
5.62
5.66
A2/6
32.9
30.0
A4/6
5.86
5.34
ΔlogK
0.763
0.728
Table 3: The eelemental composition of humic acids (in atomic percentage) extracted from soils at the
Copper Smelter “Legnica”
Sample
zone I
zone II
C
H
N
34.54 43.52 2.81
34.55 43.98 2.84
O
18.14
18.24
H:C
O:C
1.26
1.27
0.525
0.528
Ο:Η
N:C
ω
0.417 0.081 0.032
0.415 0.082 0.029
The FT-IR spectra of HAs from soils from both zones feature the following common bands
(Fig. 1): 3380 cm-1 (O–H stretching of various functional groups); 2925 and 2850 cm-1
(aliphatic C–H group stretching); 1720 cm-1 (C=O stretching of carboxyl groups); 1650 cm-1
(aromatic C=C stretching and COO- symmetric stretching); 1508 cm-1 (amide II band); 1424
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15th IHSS Meeting- Vol. 3
cm-1 (amide III band); 1380 cm-1 (O–H deformation and C-O stretching of phenolic OH
and/or antisymetric stretching of COO- groups); a broad band at 1225 cm-1 (C-O stretching
and O-H deformation of carboxyl and C-O stretching of aryl ethers and phenols); 1040 cm-1
(C-O stretching of polysaccharides). The major differences are in the relative intensity of
peaks assigned to carboxyl groups, which are higher in the case of HAs from zone II than
from I.
I zone
%T
II zone
4000
3500
3000
2500
2000
W avenumbers
1500
1000
500
cm
-1
Figure 1: FT-IR-spectra of HAs extracted from soils at the Copper–Smelter “Legnica”
4. Conclusions
The lower absorbance ratios of HAs from the soil samples from zone II as compared with
zone I indicate higher carbon contents. However, it is not clearly reflected in the elemental
composition of HAs. Higher oxygen content in HAs from zone II indicates evolution towards
oxidation. Examination of FT-IR spectra showed that metal ions react mainly with carboxylic
groups of the HAs. The contamination level of heavy metals (mostly Cu and Pb) has not the
influence on the quantity and selected qualitative parameters of HAs extracted from soils.
References.
1.
2.
3.
4.
5.
A. Karczewska, Zesz. Naukowe AR Wrocław, 432 (2002).
B.J. Alloway, Heavy Metals in Soils (2nd ed.), Blackie Academic and Professional, 1995.
J.A. Zdanov, Biochimija, 30, 6, (1965) 1257.
M.W.H. Evangelou, M. Ebel and A. Schaeffer, Chemosphere, 63 (2006), 996
N. Szombathova, B. Debska, M. Lacko-Bartosova, A. Zaujec, S.S. Gonet, Acta Sci.Pol.,
Agricultura 3(2), 37 (2004).
6. S.S. Gonet, B. Debska, Environ. Int., 24 (1998) 603.
Vol. 3 Page - 119 -
15th IHSS Meeting- Vol. 3
The Interaction of Cu 2+ with Humic Acids of Different Soils
Motuzova G.V., Dergam H., Stepanov A.A.
Moscow State University, Faculty of Soil Science, 119991, Moscow, Leninskie Gory,
Moscow University, Faculty of Soil Science
E-mail: motuzova@mail.ru
1. Introduction
The growth of productive human activity leads to the increase of the environmental pollution
by heavy metals. Heavy metals are accumulated in the soils, interacting with soil components.
The most important are the interactions of heavy metals with humic substances. Recent
studies demonstrated the ability of humic substances to bind the ions of heavy metals strongly
and loosely, namely in the exchangeable state, in the form of outer-and inner-sphere
complexes. But insufficient attention is paid to the changes that occur with humic acids (HA)
themselves under the influence of heavy metals. These processes deserve the special attention,
because of their danger for ecosystem.
The objects of the investigation were the samples of upper humus horizons of soils, that have
been developed in different conditions: 1) soddy-podzolic (S-P) heavy loamy soil, developed
on the moraine deposits (fallow, Leningradskij region); 2) chernozem (CH) heavy loamy on
the loess deposits (Voronezh region), 3) meadow serozem (MS) light loamy on the loess
deposits (Syria,Mesopotamia).
2. Materials and Methods
The HA samples were received throw extractions by 0, 1 N. NaOH solution. There were
determined the elemental composition of HA, their molecular mass distribution (by gel
filtration); IR spectra, hydrophilic-hydrophobic properties, 1H NMR spectra, acid-base
properties (by the reverse titration). There were determined some parameters of complexes
formation of the HA with Cu 2+ ions: a) the content of Cu in fractions <10 kDa and> 10 kDa;
b) the distribution of Cu between fractions HA + Cu with different properties (molecular
masses, amphiphilic properties; c) the properties of the HA in the fractions- products of their
interaction with Cu
2+
(C content in the fractions, molecular masses distribution, amphiphilic
properties, 1H NMR spectra).
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15th IHSS Meeting- Vol. 3
3. Results and discussion
Humic acids of the investigated soils are characterized as by the general, so by some specific
properties. They are united by the elemental composition, dominance of aliphatic structures,
consisting of components, that exhibit the hydrophobic properties, by the predominance of
molecules with average masses (18000-25000 a.m.u.), the uniform set of the structures of
aliphatic and aromatic parts of the HA. The greatest differences are noticed between the HA
of chernozem and HA of serozem. Humic acids of chernozem are richer with carbon, they
content less nitrogen, molecular-mass distribution is more homogeneous, their aliphatic part is
less developed (the number of methyl groups is less, the number of carboxyl groups is more
than in HA of serozem). Humic acids of soddy-podzolic soils occupy an intermediate position
between the HA of chernozem and serozem.
At the next stage the complexes of HA-Cu were obtained by the interaction of HA with the
solution of Cu (NO3) 2 at pH 7,0. The properties of HA-Cu complexes of three soils have also
both the general and specific features. There were revealed two types of ligands in the
structure of HA, that are able for complexation with Cu2+. The complexation capacity of the
centers of the first type of HA ligands of three soils is low and close in magnitude (24 -26
mmol/100g HA), but the stability of complexes, formed by them is relatively high (log K1
4,8-5,9) and changes in order CH> S-P> MS. The complexation capacity of the second type
of HA ligands of three soils is 2,3-3 times higher (75-59 mmol/100g HA ) and changes in
order: CH> S-P> MS. But the sustainability of these complexes is not large (lg K2 3,2-2.8).
The difference of complexation ability of three soils is in accordance with the content of the
functional groups in HA and their degree of dissociation (HA of CH> HA of S-P> HA of
SER).By the adding of copper salts to the solution of HA the most part of Cu2+ in the soddypodzolic soil (79%) and chernozem (59%) is bound in complexes with HA. In the serozem the
free forms of Cu 2+, their complexes with inorganic ligands and with organic ligands with low
molecular masses are dominated (63%). The highest specific activity in the complexes
formation with Cu
2+
show the HA species with low molecular masses (especially in HA of
serozem), the least active show the species of HA with high molecular masses. But due to the
domination of the species with average molecular masses in the composition of HA of all
three soils just namely these species are predominantly involved in the complexation with
Cu2+ (for soddy-podzolic soil and chernozem 81-88%, for serozem, -65% from HA total).
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15th IHSS Meeting- Vol. 3
The highest specific activity in the complexes with Cu
2+
show the species of HA from three
soils that exhibit the hydrophylic properties, especially in HA of chernozem. The share of
these complexes constitutes 65 % and 55% in chernozem and soddy-podzolic soils. HA of
serozem occupy the intermediate positions; the portion of the complexes consists of 72%.
As a result some properties of HA have changed after their interaction with Cu
2+
. It is
attested by the changes of 1HNR spectra, molecular mass distribution of HA, their
amphiphilicity, the change of metal concentration in the dialysate. It is assumed that the
restructuring of the initial metal-humus complexes under the influence of the ions Cu 2+ takes
place, accompanied by the separation of the long aliphatic chains from the aromatic rings of
the HA, the appearance in their composition of the unsubstituted structures, the changing in
the molecular-mass distribution of HA and increasing of their hydrophobicity.
4. Conclusion
Interaction of humic acids with Cu2+ leads to changes of the state as the metal, so the
properties of humic acids. The last can be classified as the sign of humus degradation.
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Influences of Humic Acids on the Pattern of Oxidation Products of
Tetrabromobisphenol a Derived from a Catalytic System using Iron(III)tetrakis(p-sulfophenyl)porphyrin and KHSO5
Masami Fukushima*, Yosuke Ishida, Satoko Shigematsu
Laboratory of Chemical Resources, Division of Sustainable Resources Engineering, Graduate
School of Engineering, Hokkaido University, Sapporo 060-8628, Japan
E-mail: m-fukush@eng.hokudai.ac.jp
1. Introduction
Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant. The
leaching of the TBBPA from landfills is facilitated in the presence of humic acids (HAs),
which can enhance the water solubility of hydrophobic organic pollutants [1]. TBBPA is
known to be able to act as an endocrine disruptor [2]. Thus, studies on the transformation of
TBBPA are important to elucidate its fate in environments. While oxidation may be major
degradation processes in environments, there have been a few reports on the oxidation of
TBBPA [3, 4]. In addition, influences of HAs on the oxidation of TBBPA have not been
investigated. On the other hand, it had been reported that an iron(III)-tetrakis(psulfonatophenyl)porphyrin (FeTPPS) can oxidize chlorophenols [5]. This suggests a
possibility for applying the FeTPPS/KHSO5 catalytic system to the oxidation of TBBPA. In
the present study, we studied the influences of HAs on the pattern of byproduct of TBBPA,
derived from the catalytic oxidation in the FeTPPS/KHSO5 system.
2. Materials and Methods.
Humic acids. An HA was extracted from a Shinshinotsu peat soil (Hokkaido, Japan) and
purified according to a method, recommended by the International Humic Substances Society
[6]. To alter the amounts of phenolic moieties, the HA was treated with hydroquinone (HQHA) according to the method, as reported by Perminova and coworkers [7]. The results of
elemental and acidic functional group analyses for the HAs are summarized in Table 1.
Table 1: The results of elemental and acidic functional group analyses for HAs
HA
HQ-HA
Functional Groups
(meq g-1 C)
Elemental Composition (%)
Samples
%C
54.5
59.2
%H
5.35
5.01
%N
2.17
2.24
%O
35.1
30.7
%S
0.66
0.87
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%ash
2.22
2.01
COOH
3.2 ± 0.1
4.1 ± 0.4
Phenol-OH
7.3 ± 0.3
11 ± 0.1
15th IHSS Meeting- Vol. 3
Oxidation tests for TBBPA. A 25 mL aliquot of 0.02 M NaH2PO4/Na2HPO4/citrate buffer at
pH 4 – 8, which contained 0 or 50 mg L-1 of HAs, was placed in a 100-mL Erlenmeyer flask.
Stock solutions of TBBPA (0.01 M in acetonitrile) and FeTPPS (0.2 mM) were added to set
the final concentrations at 50 μM and 5 μM, respectively. After mixing vigorously, aqueous
0.01 M KHSO5 was added to set the final concentration at 125 μM, and the flask was then
shaken in a thermostatic shaking water bath at 25±0.1. After a 1, 5, 15 or 30 min reaction
period, 1 mL of 1 M ascorbic acid aqueous was added, and pH of the solution was adjusted to
11 – 11.5 by the addition of aqueous K2CO3. Subsequently, 5 mL of acetic anhydride was
added dropwise to the solution, and 0.5 mL of a 1 mM anthracene hexane solution was added
as an internal standard for the GC/MS analyses. This mixture was doubly extracted with 15
mL of n-hexane, and the extract dehydrated with Na2SO4 anhydride. After filtration, the
extract was evaporated under a stream of dry N2, and the residue was then dissolved in 0.25
mL of n-hexane. A 1 μL aliquot of the extract was introduced into a GC-17A/QP5050 GC/MS
system (Shimadzu). A Quadrex methyl silicon capillary column (0.25 mm i.d. × 25 m) was
employed in the separation. The temperature ramp was as follows: 65 for 1.5 min, 65 – 120 at
35 min-1, 120 – 300 at 4 min-1 and a 300 hold for 10 min. Concentrations of TBBPA before
and after the oxidation were analyzed by an HPLC, as described in a previous report [5].
3. Results and Discussion
Influence of solution pH of TBBPA degradation. Figure 1 shows the influence of solution pH
on percentages of the degradation of TBBPA. The percent of TBBPA degradation increased
with an increase in solution pH. In particular, more than 90% of TBBPA was degraded at pH
8 in the absence and presence of HAs. Because the pKa value of TBBPA is reported to be 7.4
[4], one of reasons for this trend can be attributed to the fact that water solubility of TBBPA is
enhanced in weak-alkaline condition. The range of pH for leachates from landfills is reported
to be 7 – 12 [1]. Thus, oxidation products from TBBPA were investigated at pH 8. Although
Br- and BrO3- were analyzed by ion chromatography, these were not detected in all reaction
mixtures. Thus, debromination is not considered in the catalytic oxidation of TBBPA via the
FeTPPS/KHSO5 system. This suggests that further polymerized brominated compounds are
produced as a result of catalytic oxidation.
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TBBPA degradation (%)
100
80
60
40
FeTPPS + KHSO5
FeTPPS + HA + KHSO5
FeTPPS + HQ-HA +KHSO5
20
0
4
5
6
7
8
pH
Figure 1: Influence of solution pH on percentages of the TBBPA degradation
Influences of HAs on the oxidation products of TBBPA. To identify the oxidation products, nhexane extracts from the reaction mixtures were analyzed by means of GC/MS. Figure 2
shows the chromatograms in the absence and presence of HA. In the absence of HA
(FeTPPS+KHSO5), no clear peaks for the oxidation products were observed. However, in
FeTPPS + HA + KHSO5 (and FeTPPS + HQ-HA+ KHSO5, data not shown), clear peaks for
4-(2-hydroxyisopropyl)-2,6-dibromophenol acetate (2HIP-2,6DBP, m/z 352) and trimer (m/z
836) of 2,6DBP appeared. Because debromination cannot occur in the catalytic oxidation of
TBBPA, more polymerized byproducts of brominated phenol may be formed in FeTPPS +
KHSO5. However, in FeTPPS + HA+ KHSO5, the [2HIP-2,6DBP] were much smaller than
those of degraded TBBPA.
O
C CH3
O
Br
Br
Br
Br
CH3
H3C
C
Br
O
O
O
Br
H3C
C
CH3
Br
OH
C
Br
m/z 836
CH3
OH
m/z 352
FeTPPS+HA+KHSO5
TBBPA
FeTPPS + KHSO5
15
20
25
30
35
40
45
50
55
Retention time (min)
Figure 2: GC/MS chromatograms of n-hexane extract from the reaction mixtures (pH 8)
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15th IHSS Meeting- Vol. 3
To estimate the mass balance of bromine in the FeTPPS/HAs/KHSO5 catalytic system, the
HA fractions in the reaction mixtures were separated, as described in a previous report [5].
The contents of bromine in the separated HA and HQ-HA were 7.6% and 9.6%, respectively.
Considering the amounts of remained TBBPA in the reaction mixture, it was estimated that
64% and 91% of the oxidized TBBPA were incorporated into HA and HQ-HA, respectively.
From the pyrolysis-GC/MS analysis of HA fractions in reaction mixtures, the bound
brominated species in HAs were identified as bromophenols that can be derived from the
oxidation intermediates of TBBPA.
4. Conclusions
Oxidation of TBBPA via the FeTPPS/KHSO5 catalytic system was not significantly enhanced
or inhibited in the presence of HAs. However, more polymerized byproducts may be formed
in the absence of HA. In the presence of HA, low-molecular-weight byproduct, such as 2HIP2,6DBP, was produced and approximately 64 – 91% of oxidized TBBPA were bound to
polymeric structures of HAs. Although more polymerized compounds from TBBPA only are
difficult to soluble in water, low-molecular-weigh byproducts and HA-bound byproducts are
relatively soluble in water. Thus, transportability of brominated byproducts from TBBPA is
decelerated in the absence of HA. However, the transportability of brominated byproducts
may be enhanced in the presence of HAs. These results suggest that the HAs can serve as
carrier of brominated byproducts, derived from catalytic oxidation of TBBPA, to aquatic
environments.
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research from the JSPS (21310048).
1. References.
2. M. Osako, Y.-J. Kim and S. Sakai, Chemosphere, 57 (2004) 1571.
3. S. Kitamura, T. Suzuki, S. Sanoh, R. Kohta, N. Jinno, K. Sugihara, S. Yoshihara, N. Fujimoto, H.
Watanabe and S. Ohta, Toxicol. Sci., 84 (2005) 249.
4. K. Lin, W. Liu and J. Gay, Environ. Sci. Technol., 43 (2009) 4480.
5. S.-K. Han, P. Bilski, B. Karriker, R.H. Sik and C.F. Chignell, Environ. Sci. Technol., 42 (2008)
166.
6. M. Fukushima, H. Ichikawa, M. Kawasaki, A. Sawada, K. Morimoto, K. Tatsumi, Environ. Sci.
Technol., 37 (2003) 386.
7. R.S. Swift, In, Methods of Soil Analysis Part 3, Soil Science Society of America, Madison, 1996,
p. 1018.
8. I.V. Perminova, A.N. Kovalenko, P. Schmitt-Kopplin, K. Hatfield, N. Hertkorn, E.Y. Belyaeva,
V.S. Petrosyan, Environ. Sci. Technol., 39 (2005) 8518.
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Adsorption of Trihalomethanes by Humin: Batch and Fixed Bed Column
Studies
Graziele da Costa Cunhaa, Luciane Pimenta Cruz Romãoa*, Mônica Cardoso Santosa,
Bruno Rafael Araújoa, Sandro Navickienea and Válter Lucio de Páduab
a
b
Department of Chemistry, Federal University of Sergipe, 49100-000 Aracaju, SE, Brazil;
Department of Sanitary and Environmental Engineering, Federal University of Minas Gerais,
30110-001 Belo Horizonte, MG, Brazil
E-mail: luciane@ufs.br
1. Introduction
During the end of the 19th century and beginning of the 20th century, diseases caused by pathogenic
microorganisms, such as typhoid fever, dysentery and cholera, increasingly became a focus of
attention, especially in relation to potable water quality. To address this problem, in 1908 chlorine was
first used as a disinfectant in New Jersey, USA, and has continued to be widely used in water
treatment stations (WTSs) worldwide. Its various compounds destroy and/or inactivate the organisms
responsible for many illnesses hence greatly reducing human mortality caused by diseases
disseminated via hydric systems [1]. Despite the benefits provided by disinfection, use of chlorine and
other compounds has attracted the attention of the scientific community, due to reactions with natural
organic matter (NOM) that can generate subproducts that may be undesirable from the human health
perspective. The trihalomethanes are frequently found in water treatment systems, with chloroform
(CHCl3), dichlorobromomethane (CHBrCl2), dibromochloromethane (CHBr2Cl) and bromoform
(CHBr3) being the most common. Various studies have reported on the high carcinogenic and
mutagenic potentials of these compounds. Commercial activated carbon is the material most widely
used for adsorption of THMs in water treatment stations (WTSs) worldwide, due to its high removal
capacity. Nevertheless, its efficiency is dependent on the need for large dosages in short time periods,
and its use is limited by the associated cost. To reduce costs of treatment, alternative adsorbents are
sought that are more economically viable, easily disposed of, and above all may be readily regenerated
without losing their properties. Bioadsorbents have merited special attention, due to their availability
and abundance, and significantly lower cost compared to synthetic adsorbents. Amongst these
adsorbents is peat, an organic soil formed continuously by a complex process of decomposition and
humification of plant residues by microbiological oxidation in flooded environments. Humin is the soil
organic matter that remains after removal of humic and fulvic acids, defined as the fraction that is
insoluble in aqueous solution at any pH, and possesses higher molecular weight and carbon content
compared with other peat humic fractions. High porosity and surface area are indicative of humin’s
potential as an adsorbent [2]. The objective of the present work was therefore to assess the
performance, in batch and fixed bed column systems, of humin used either in natura or immobilized
on sodium silicate, respectively, for adsorption of the main trihalomethanes found in water supply
systems.
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2. Materials and Methods
The peat sample was collected from a peat bog in the vicinity of Santo Amaro das Brotas, Sergipe
State, Brazil. The sample was air-dried, ground using a pestle and mortar, and sieved first through a 9mesh grid to remove roots and twigs, and then through a 48-mesh grid to obtain a uniform particle
size. The in natura peat was not used as an adsorbent, because of the dissolution of humic and fulvic
acids at the pH range of 6.5-9.0 typically found for water from treatment stations. The procedure for
immobilization of the humin biomass on sodium silicate was similar to that described by [3]. Batch
tests to investigate the influence of initial concentration, the kinetics and the adsorption isotherm were
conducted individually for each of the THMs studied, with agitation (150 rpm, 25 ± 0.2 ºC) in amber
glass flasks. All tests were performed using 10 mL of THM solution and 0.1 g of humin. A Millex-HV
0.45 μm syringe-driven filter unit was used for removal of the supernatant for subsequent analysis.
After filtration, the THM concentrations were quantified using a gas chromatograph (Varian, USA)
fitted with an electron capture detector (GC-ECD), coupled to a purge and trap (PT) system. Internal
standard was added at the time of injection, in order to avoid any losses. A blank solution was
prepared without the humic material, and all experiments were performed in triplicate. Humin
immobilized on sodium silicate was packed into a glass column (20 cm × 2.0 cm i.d.), through which
the THM solution was percolated, in descending flow, using a peristaltic pump. The influences of
height and flow rate on THM adsorption were monitored. Breakthrough curves (C/C0 vs time, where C
is the concentration exiting the column, and C0 the initial concentration) were obtained by collection
of aliquots in amber flasks after different time intervals (up to 360 min). The concentrations of THMs
remaining after exiting the column, and in the solution at the end of the experiment (to identify
possible losses by volatilization) were determined by GC/ECD-PT.
3. Results and Discussion
The effect of contact time on the adsorption of the THMs was studied using the concentration (250 μg
L-1) that showed highest percentage removal. There was rapid removal in the first few minutes, with
equilibrium achieved at around 240 min, indicative of fairly fast kinetics. The percentage removals of
bromoform, dibromochloromethane, dichlorobromomethane and chloroform were 83.2 ± 0.1%, 78.0 ±
0.1%, 77.0 ± 0.1% and 74.6 ± 0.1%, respectively. The selectivity of adsorption followed the order
CHBr3 > CHBr2Cl > CHBrCl2 > CHCl3, however without any significant differences (P > 0.05),
suggesting that the active sites on the humin did not exhibit any preference in adsorption of the THMs
studied. In addition, significant correlation (P < 0.05) was obtained between the adsorption
percentages of the THMs by humin, as a function of time (bromoform, r = 0.98;
dibromochloromethane, r = 0.96; chloroform, r = 0.99; dichlorobromomethane, r = 1.0). The validity
of the pseudo first and second order kinetics, and intra-particle diffusion models was used to analyze
the linear equations (t/qt) vs. t, log (qe – qt) vs. t, and qt vs. t0.5, respectively. The experimental data
obeyed pseudo second order kinetics, as can be seen in Table 1. The values of k22 were greater than
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15th IHSS Meeting- Vol. 3
those of k12 and ki2, as well as those of R22. Intra-particle diffusion was also a contributing factor.
However, the lines do not pass through the origin, indicating that this mechanism did not determine
the rate of the overall process, and the existence of a complex mechanism consisting of adsorption and
intra-particle transport. The values of qe (experimental) and qt (theoretical, obtained from the angular
coefficient of the straight line) show good agreement.
Table 1: Pseudo first order, pseudo second order and intra-particle diffusion kinetic parameters for
adsorption of THMs on humin
THMs
Bromoform
Chlorodibromomethane
Bromodichloromethane
Chloroform
qt
qe
-1
µg·mg
19.60 19.51
18.81 17.73
18.05 17.89
21.01 20.45
k12 · 10-5
R1 2
min-1
6.67
1.53
8.90
3.18
k22·10-4
R2 2
(µg·g-1·min)
0.477
0.389
0.804
0.508
3.38
7.34
1.67
1.39
ki2 · 10-4
Ri2
µg·g-1·min0.5
0.999
0.999
0.999
0.997
2.60
3.90
1.57
1.00
0.984
0.989
0.817
0.963
The experimental data fitted the Freundlich model, since in addition to giving the highest correlation
coefficient (R2) value (Table 2), the separation factor (RL), an essential characteristic of the Langmuir
isotherm, was not favorable (RL > 1). The constants Kf and n are related to the maximum adsorption
capacity and the adsorption intensity, respectively. The Kf value showed the following order of
selectivity of humin for the THMs: CHBr3 > CHBr2Cl > CHBrCl2 > CHCl3. This behavior was also
confirmed by the experimental values, with the constant, n, showing favorable adsorption of the
THMs. Humin was efficient for THM removal, with concentrations reduced by at least 83.0%
following treatment.
The influence of flow rate on adsorption of THMs by humin immobilized on sodium silicate was
investigated using two different flows (2 and 3 mL min-1). From the results presented in Figures 1a
and 1b, increased flow caused a significant (P < 0.05) reduction in the adsorption capacity of the
humin for the THMs. This was because increased flow reduced the residence time of the solute in the
adsorbent bed, which then reduced solute diffusion into the pores of the adsorbent. Figures 1c and 1d
show the breakthrough curves obtained for different bed heights (2 and 4 cm), at a constant flow rate
of 2 mL min-1.
4. Conclusions
The humin has been shown to be effective for adsorption of the main THMs found in water supply
systems. Adsorption was relatively fast, with a maximum adsorption of 83.2% in batch experiments.
Using a fixed bed column, adsorption results demonstrated the efficacy of humin as an adsorbent,
extracting 99.7% under optimized conditions of TTHMs in the systems studied. The adsorption
capacity of a fixed bed employing humin immobilized on sodium silicate showed the same selectivity
as batch adsorption.
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Figure 1: Breakthrough curves for adsorption of THMs by humin immobilized on sodium silicate in
column experiments. Conditions: Bed height 2 cm, flow rate a) 3 and b) 2 ml min-1; flow rate 2 mL
min-1, bed height c) 2 and d) 4 cm; e) adsorption of TTHMs; T = 25 ± 0.1°C
Acknowledgements
The authors thank MCT/CNPq (proc. n° 550062/2007-2) for financial support of this study.
References
1. A.D. Nikolaou, S.K. Golfinopoulos, G.B. Arhonditsis, V. Kolovoyiannis, T.D. Lekkas,
Chemosphere. 55 (2004) 409.
2. G. De La Rosa, J.R. Peralta-Videa, J.L. Gardea-Torresdey, J. Hazard. Mater. 97 (2003) 207.
3. A.P.S. Batista, L.P.C. Romão, M.L.P.M. Arguelho, C.A.B. Garcia, J.P.H. Alves, E.A. Passos,
A.H. Rosa, J. Hazard. Mater. 163 (2009) 517.
4. O. Ozdemira, M. Turana, A.Z. Turanb, A. Fakia, A.B. Enginc, J. Hazard. Mater. 166 (2009) 647.
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Combined Effects of Humic Matter and Surfactants on PAH Solubility:
Is There a Mixed Micellization?
Holger Lippolda*
a
Forschungszentrum Dresden-Rossendorf, Institut für Radiochemie
E-mail: h.lippold@fzd.de
1. Introduction
It has been recognized that solid-liquid distribution and transport of hydrophobic
contaminants such as PAH (polycyclic aromatic hydrocarbons) are governed by their
interaction with humic matter, which is present on sediment surfaces as well as in solution,
acting as a sink or a mobilizing agent, respectively [1, 2]. As surface-active compounds,
humic substances are often compared to surfactants. Emerging environmental technologies
involve a deliberate application of surfactants to enhance the sorption capacity of soils and
aquifer materials [3, 4], or to increase the efficiency of soil washing procedures and pumpand-treat operations for groundwater decontamination [5]. Whereas contaminant binding to
humics as well as to surfactants has been extensively studied, there is a notable lack of
literature on their combined action in mixed systems. This topic is, however, important
because environmental influences of surfactants are inevitably associated with the effects of
the ubiquitous natural organics. Since both are amphiphilic, it seems conceivable that mixed
micelles can be formed, involving synergistic or antagonistic effects in the solubilization of
organic compounds.
In this study, we have examined the joint influence of humic acid (HA) and surfactants
(cationic, anionic) on the water solubility of pyrene as a representative of PAH, at surfactant
concentrations below and above the critical micelle concentration (CMC). In order to detect
and characterize interaction processes, we have investigated the octanol-water partitioning of
HA in the presence of various surfactants, using radiolabelled humic material. In particular,
the hypothesis of a micellar nature of dissolved humic substances has been addressed.
2. Materials and Methods
All chemicals and HA were purchased from Sigma-Aldrich (Germany). The HA was purified
by acid-base treatment, followed by dialysis. For experiments on octanol-water partitioning, it
was radiolabelled with
131
I (Amersham, Germany), adopting the Iodogen method [6].
Surfactants and 14C-labelled pyrene were used as received.
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Pyrene solubilities were determined by liquid scintillation counting (Beckman LS 6000, GMI,
USA) after shaking an excess amount with surfactant / HA solutions in glass vials for 7 days
and passing the supernatants through membrane syringe filters. The results were corrected for
adsorption losses and colour quenching.
For determining octanol-water partition ratios, solutions of
131
I-labelled HA and surfactants
(pre-equilibrated for 15 h) were overlaid with octanol (2 mL / 2 mL in PP test tubes) and then
gently shaken for 24 h. Samples of both phases were analyzed by gamma counting, using a
1480 Wallac Wizard 3” (Perkin Elmer, USA).
Size exclusion chromatography was performed by means of an HPLC equipment HP 1100
(Hewlett-Packard, USA) with a TSKgel G3000 PWXL column (Phenomenex, USA), run with
phosphate buffer as eluent.
3. Results and Discussion
The water solubility of pyrene is increased in the presence of HA, which acts as a carrier due
to hydrophobic interaction of both components. When adding the cationic surfactant
dodecyltrimethylammonium bromide (DTAB), this solubility enhancement was found to be
cancelled; the humic colloids were precipitated as a consequence of charge compensation by
the organo-cations.
Interestingly, an antagonistic effect was also observed on addition of an anionic surfactant,
sodium dodecylsulfate (SDS). While no precipitation was induced in this case, the solubility
of pyrene was reduced by half and remained constant on further addition. Only at surfactant
concentrations above the CMC, the solubility increased sharply owing to micellar
incorporation. The presence of HA did not cause any change in the CMC of SDS, as is
normally observed on addition of a second amphiphilic compound. Furthermore, the effects of
HA and micellar SDS on pyrene solubility turned out to be strictly additive. Consequently,
they are based on distinct processes, occurring independently of each other, i.e., there is no
mixed micellization with humic molecules acting as a co-surfactant.
The octanol-water partition ratios of HA changed significantly in the presence surfactants.
The partitioning equilibrium was shifted towards the organic phase on addition of cationic
surfactants, and towards the aqueous phase on addition of anionic surfactants. Based on these
findings, different modes of interaction could be identified, as shown in Fig. 1.
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15th IHSS Meeting- Vol. 3
(a)
(b)
Figure 1: Schematic representation of association modes between humic colloids and cationic (a) and
anionic surfactants (b) as derived from octanol-water partitioning experiments
The binding mechanism shown in Fig. 1(b) provides an explanation for the decline in pyrene
solubilization in systems of HA and SDS. Obviously, a competitive situation arises in the
hydrophobic binding of the PAH and the surfactant tail groups. The fact that the pyrene
molecules cannot be displaced completely supports the proposition that different binding sites
exist in humic colloids: weak near-surface sites and strong inner sites [7].
The size distribution of the colloids was found to be unaffected by the association with
anionic as well as with cationic surfactants. A general micellar character (as originally
suggested by Wershaw [8]) is thus unlikely since a co-aggregation should then entail
substantial disruptions and rearrangement processes.
4. Conclusions
A humic-bound mobilization of hydrophobic pollutants is not facilitated by surfactants that
may be present at contaminated sites, and surfactant-aided flushing procedures are neither
impaired nor enhanced by dissolved organic matter. Both cationic and anionic surfactants
associate with humic colloids, which could be demonstrated in octanol-water partitioning
experiments with radiolabelled HA, but our studies did not provide any indication of a
formation of mixed micelles. It is thus questionable whether dissolved humic substances are
organized in micelle-like aggregates.
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Acknowledgement
This research has been supported by funding from the German Federal Ministry of Education
and Research (BMBF), contract number 0330537.
References
1.
2.
3.
4.
5.
6.
7.
8.
S.W. Karickhoff, Chemosphere, 10 (1981) 833.
C.T. Chiou, P.E. Porter, D.W. Schmedding, Environ. Sci. Technol., 17 (1983) 227.
S.A. Boyd, J.F. Lee, M.M. Mortland, Nature, 333 (1988) 345.
J. Wagner, H. Chen, B.J. Brownawell, J.C. Westall, Environ. Sci. Technol., 28 (1994) 231.
C.C. West and J.H. Harwell, Environ. Sci. Technol., 26 (1992) 2324.
P.J. Fraker and J.C. Speck, Biochem. Biophys. Res. Commun., 80 (1978) 849.
J.J. Pignatello and B. Xing, Environ. Sci. Technol., 30 (1996) 1.
R.L. Wershaw, J. Contam. Hydrol., 1 (1986) 29.
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Complexation of Copper(II) Ions With Humic Acids and EDTA Studied by
High Resolution Ultrasonic Spectrometry
Martina Klucakova*, Miloslav Pekar
Brno University of Technology, Faculty of Chemistry, Purkynova 118, 612 00 Brno, Czech
Republic
E-mail: klucakova@fch.vutbr.cz
1. Introduction
Ultrasound spectroscopy is based on observing interactions of ultrasound wave with studied
system. Two principal characteristics are measured – ultrasound velocity and attenuation
(decrease in amplitude). Velocity reflects local elasticity and density of material, which are
determined by molecular arrangements, conformation and solvation shell. Attenuation reflects
in homogeneous samples fast relaxation (chemical) processes and in heterogeneous samples
scattering of ultrasound wave, which can be used e.g. for particle sizing. Considering the
measured parameters are strongly sensitive to molecular conformation and inter- and intramolecular interactions ultrasound spectroscopy is used as method for investigation of
chemical reactions [1–3].
Metal binding of humic acids (HA) is the subject of many studies. In our previous works [4–
7] complexation of humic acids and by metal ions has been studied by conductometry,
potentiometry, UV/VIS and FT-IR spectrometry. This study utilizes High Resolution
Ultrasound Spectrometry (HRUS) for analysis of interactions between copper(II) ions and HA
and EDTA as a humic-like model.
2. Materials and Methods
HA were isolated from South Moravia lignite by standard alkaline extraction as described
elsewhere [4, 8]. More details on the chemical structure of the initial lignite matrix, as well as
that of the isolated HA, can be found in previous papers [8-10]. The basic characteristics of
used humic sample are listed in Tab. 1.
Table 1: Characterization of used humic sample (normalized on dry ash-free HA)
C
H
N
S
O
(at. %) (at. %) (at. %) (at. %) (at. %)
43.9
40.2
0.7
0.2
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15.0
total acidity
(mmol/g)
4.94
15th IHSS Meeting- Vol. 3
Humic sol was prepared by dissolution in concentrated NaOH and mixing with 15% (wt,)
solution of HEPES (Aldrich) up to pH = 7, The content of HA in final solution was 14.23 g/L
EDTA was dissolved in the solution of HEPES, Final concentration of EDTA in HEPES
buffer was 5.72 mmol/L (pH = 7).
Ultrasonic spectrometer with high resolution HR-US 102 (Ultrasonic Scientific, Ireland), was
utilized for measurement of basic ultrasonic parameters, The device consists of two
independent cells tempered at 25 °C, Reference cell was filled by HEPES buffer (EDTA
measurement) or HEPES with NaOH (HA measurement), Measuring cell was filled by EDTA
or HA solution prepared by ways described above and titrated by CuCl2 up to saturation of
their acidic groups, Velocity (U) and attenuation (N) in both cells was measured, the resulting
differences ΔU and ΔN between reference and sample cell we can see at graphs, Blank
experiments with NaOH in HEPES, EDTA and HA versus HEPES were carried out to
correction of data obtained from titration experiments, All HRUS measurements were done at
four different frequencies.
3. Results and Discussion
The dependencies of measured ultrasonic velocity and attenuation on degree of saturation of
acidic groups in HA and EDTA by CuCl2 are shown in Figs, 1 and 2, According the results
both compounds are able to bind the bivalent Cu2+ ions by two functional groups, In case of
EDTA all binding sites can be occupied and saturation is represented by ratio Cu2+/H+ = 0.5.
Complexation of HA is more complicated, HA contains many various binding sites with
different strength which can be also gradually occupied, On the other hand, each Cu2+ ion
may not be able to find two suitable acidic groups for its binding for structure and
conformation of HA particles, The steric effects are the reason, why some acidic groups can
remain vacant, A part of Cu2+ ions can be bonded only by one acidic group but our quantumchemical computing showed that such arrangement is hardly likely [11], The maximum of
measured ultrasonic parameters then quadrates with ratio Cu2+/H+ = 0.4.
The differences between complexation behaviour of HA and EDTA are clearly evident also
from slopes of measured dependencies, The slope of decrease of ΔU with degree of saturation
of binding sites is -1.31 ± 0,04 m/s for HA and -0.58 ± 0,01 m/s for EDTA, It shows on more
rigid structure of Cu-HA complexes, which can be caused by formation of inter- and intramolecular bridges.
Vol. 3 Page - 136 -
15th IHSS Meeting- Vol. 3
2+
+
Cu /H (mol/mol)
0
0,2
0,4
0,6
0,8
1
1,2
0
-0,1
Δ U (m/s)
-0,2
-0,3
-0,4
-0,5
-0,6
Figure 2: The dependence of ΔU on saturation of acidic groups in HA (circles) and EDTA (triangles)
in their titration by CuCl2
2+
+
Cu /H (mol/mol)
0
0,2
0,4
0,6
0,8
1
1,2
10
8
Δ N (1/m)
6
4
2
0
-2
Figure 3: The dependence of ΔN on saturation of acidic groups in HA (circles) and EDTA (triangles)
in their titration by CuCl2
The stronger influence of complexation on HA is probably caused by its aggregation and
formation of much bigger particles in comparison with EDTA, which corresponds with data
obtained for titration of the saturated organic compounds, because Cu-HA aggregates can
sediment in contrast to Cu complexes with EDTA, The marked HA aggregation was also
confirmed by strong increase of ultrasonic attenuation for Cu2+/H+ > 0,4.
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15th IHSS Meeting- Vol. 3
4. Conclusions
Measurement of ultrasonic velocity and attenuation in titration of HA and EDTA as “humiclike” model by CuCl2 showed that HRUS is very sensitive method for detection and analysis
of complexation of organic compounds, It was confirmed that saturation of acidic groups in
HA is not 100 % probably due to their structure and steric effects (in contrary to EDTA), The
formation of relatively big Cu-HA particles (able to sediment) was observed, On the other
hand, aggregation of EDTA caused only indistinctive increase of attenuation.
Acknowledgements
This work was supported by government funding – Czech Science Foundation, project, Nr,
104/08/0990.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
B,I, Kankia, T, Funck, H, Uedaira and V,A, Buckin, J, Solution Chem,, 26 (1997) 877,
E,D, Kudryashov, C, Smyth, B, O’Driscoll and V,A, Buckin, Spectroscopy, 18 (2003) 26,
W,-B, Ko and V,A, Buckin, Elastomer, 41 (2006) 57,
M, Klucakova, M, Kalab, M, Pekar and L, Lapcik, J, Polym, Mater,, 19 (2002) 287,
M, Klucakova and M, Pekar, J, Polym, Mater,, 20 (2003) 145,
M, Klucakova and M, Pekar, in E,A, Ghabbour and G, Davies (Eds,), Humic Substances:
Molecular Details and Applications in Land and Water Conservation, Taylor & Francis, New
York 2005, Chapter 11, p,167,
M, Klucakova and M, Pekar, Colloid, Surface, A, 286 (2006) 126,
M, Klucakova and M, Pekar, Colloid, Surface, A, 252 (2005) 157,
J, Kucerik, M, Klucakova and M, Pekar, Petrol, Coal, 45 (2003) 58,
P, Peuravuori, P, Zbankova and K, Pihlaja, Fuel Proc, Technol,, 87 (2006) 829,
M, Klucakova, P, Pelikan, L, Lapcik, B, Lapcikova, J, Kucerik and M, Kalab, J, Polym, Mater,, 17
(2000) 337.
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CE-ICP-MS as Speciation Technique to Analyze the Complexation
Behavior of Europium, Gadolinium and Terbium with Humic Acid
Christina Möser*, Ralf Kautenburger, Horst Philipp Beck
Institute of Inorganic and Analytical Chemistry and Radiochemistry, Saarland University,
Campus C4.1, 66123 Saarbrücken, Germany
E-mail: c.moeser@mx.uni-saarland.de
1. Introduction
For the long-term disposal of radioactive waste, detailed information about geochemical
behavior of radioactive and toxic metal ions under environmental conditions is necessary. The
work with the radioactive actinides needs specialized high security standards. In order to
avoid these standards the lanthanides europium, gadolinium and terbium were used as
homologues of the actinides americium, curium and berkelium. Their similar chemical and
physical properties permit a conferment to the behavior of the actinides.
In the spotlight of the investigations stand the sorption and desorption behavior of the
lanthanides onto Opalinus clay. Natural organic matter can play an important role in the
immobilization or mobilization of metal ions due to complexation and colloid formation. This
complexation behavior could interfere the sorption of metal ions onto Opalinus clay. In
addition to humic acid (HA), in Opalinus clay natural appear organics like lactate, formiate or
propionate were used. As a selected technique, capillary electrophoresis (CE) was hyphenated
with inductively coupled plasma mass spectrometry (ICP-MS). With this method, both the
uncomplexed metal ions and metal organic complexes can be simultaneous detected in one
analysis step. As medium for these experiments additionally to 10mM sodium perchlorate
solution synthetic prepared pore water was used. The influence of the high concentration of
cations like Magnesium and Calcium in the pore water is very important and needs specific
notice.
2. Materials and Methods
For preparation of all used solutions MilliQ deionised water (18,2 MΩ) was used. The humic
acid is commercially available from Aldrich (St. Louis, USA; HA sodium salt) and was
purified through some precipitation and dissolution steps with 0.1 M HCl and 0.1 M NaOH /
0.01 M NaF. For detection in the ICP-MS the humic acid became iodinated with iodine, iodic
acid and sodium iodide (ultrapure, Merck) [1]. The single element standards of Eu, Gd, and
Tb were of ultrapure quality (nitrates) and receivable from Merck. As electropheric buffer a
mixture of 100 mM acetic acid / 10 mM Na-acetate was used. Before starting experiments the
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buffer was filtered and degassed in an ultrasonic bath. The complexation experiments were
carried out with a lanthanide concentration of 500 ppb, 25 mg/L HA and 10mM NaClO4 /
synthetic pore water. The used salts for pore water preparation were of p. a. quality or better.
The NaClO4 was p. a. quality. The samples were equilibrated in a horizontal shaker for 72 h at
25 °C.
As speciation technique a CE was hyphenated with ICP-MS. Beside excellent
separation performance we obtained a high sensitivity for the determination of rare earth
metals like europium (representative for lanthanides) and its organic complexes. The
connecting piece between CE and ICP-MS was a homemade interface. The fused-silica
capillary of the CE was flexibly fitted into a MicroMist 50 µl nebulizer with a Cinnabar
cyclonic spray chamber. The chamber can be chilled to a temperature of 4 °C for best
sensitivity. 200 ppb of cesium (ultrapure, Merck) was added to the CE separation buffer to
observe the capillary flow. A make-up fluid including 4 ppb Ho (ultrapure, Merck) as an
internal standard was combined with the flow from the capillary within the interface to obtain
a fluid throughput high enough to maintain a continuous nebulization [2]. The conditions for a
optimal separation were 1.5 psi for sample injection and 3 psi with 30 kV for the separation
process. A description of the analytic conditions and operating parameter can be found in [3].
3. Results and Discussion
In these report we show the influence of the high cation concentration in the synthetic pore
water on the complexation behavior of humic acid. Fig. 1 shows a typical electropherogram
with three Eu species (red line). The green line pictured the run of the Cs curve. The first
decrease of the curve corresponds to the start of the sample migration. The second minimum
of the curve shows the point of neutrality (EOF). With these minimum neutral species
migrate; behind this point migrate the negative species. The first Eu signal (peak1) represents
the uncomplexed metal before CE separation. The aquatic Eu3+ ion was complexed with the
acetate in the electrolyte buffer and migrates during the separation as EuAc2+ towards the CE
cathode. Peak 2 and peak 3 pictured the HA complexed metal ions. Peak 3 shows the strong
bound Eu, peak 2 the weak bound Eu. During the separation the weak bound Eu3+ dissociates
due to the attached voltage out of the HA complex and migrates as peak 2 towards the
cathode. The strong bound Eu is stabile during the separation and migrates as negative
charged complex behind the other peaks.
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153
133
Eu
9000
Peak 2
Peak 3
Cs
60000
8000
50000
7000
40000
[counts]
6000
Peak 1
5000
30000
4000
20000
3000
2000
10000
1000
0
0
50
100
150
0
200
Migration tim e [sec]
Figure 1: Typical electropherogram of uncomplexed and HA-compexed Eu species and Cs as CE flow
marker in 10 mM NaClO4 solution
The electropherogram with synthetic pore water (Fig. 2) shows a completely different run of
the Eu and Cs curve. In this case peak 2 represents the free Eu3+ ions which are complexed
with the acetate in the electrolyte buffer and migrate during the separation as EuAc2+ towards
the CE cathode. Peak 1 and 3 pictured the HA complexed Eu-ions. The blue curve represents
the migration of the iodated HA. This curve shows the correlation between the Eu- and the
HA-migration.
133
Peak 3
12000
153
10000
Eu
I
120000
127
100000
8000
[counts]
Cs
140000
Peak 2
80000
6000
60000
Peak 1
4000
40000
2000
20000
0
0
50
100
0
150
Migraton tim e [sec]
Figure 2: Typical electropherogram of uncomplexed and HA-compexed Eu species, Cs as CE flow
marker and iodide as HA migration marker in synthetic pore water
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The first Eu-humate complex migrates with the sample front (Cs signal, green line) and
presents the most positive charged complex. The Eu-humate complex in peak 3 migrates with
the EOF. Due to the high amount of competing cations in the pore water the Eu-humate
complex is uncharged.
4. Conclusion
In summary, the results show that the competing cations in the synthetic pore water have a
great influence of the complexation behavior of the HA. From this it follows that
investigations under natural conditions are very important for the comprehension of the
complex system.
Acknowledgements
The author thanks the BMWi for financial support (grant no. 02 E 9683 and 02 E 10196).
References.
1. D. Braun, H. O. Wirth, Patentschrift 1 088 227, 1961, Deutsches Patentamt.
2. R. Kautenburger, H.P. Beck, J. Chromatogr. A, 1159, 2007, 75.
3. R. Kautenburger, H. Anal. At. Spectrom., 24, 2009, 934.
Vol. 3 Page - 142 -
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Determinations of Ability of Extracted HSs Coordinated with Metal Ions
Ke-fei Ding, Qiao-hui Fan, Yu-ying Zhang, Wang-suo Wu*
Radiochemistry Laboratory, School of Nuclear Science and Technology, Lanzhou University,
Lanzhou, Gansu P.R. China
E-mail: wuws@lzu.edu.cn
Humic substances (HSs) are the major fraction of dissolved organic compounds present in
water and soils, which could be separated into humic acids (HAs), fulvic acids (FAs) and
humin according to their solubility in acid and alkaline solution [1]. HSs are considered
natural polyelectrolytic organic compound of complex structure involving a proportion of
aromatic rings with a larger number of attached –OH and –COOH groups, which result in the
interaction with metal ions to form complexes easily [2]. Since the strong complex ability,
HAs and FAs play important roles as ligands in the mobilization of metal ions in soils and
aqueous of environment, and they also affect the bioavailability and toxicity of these metal
ions. In most cases, the free metal ion, rather than all species of metal, is correlated to
toxicity, so complexes with humic substances as ligands generally reduces metal toxicity [3].
The objectives of this research are to model and predict the complexation behavior of HSs
with metal ions and radionuclides. Ion exchange method was used to determine the
conditional stability constants of complexes (i.e., β) between HA or FA with selective cations,
i.e., Ni2+, Cu2+, UO22+ and Th4+ [4].
HA and FA were extracted from the soil of Huajia county (Gansu province, China) according
to a references standard of the International Humic Substances Society (IHSS), which were
used in following all the experimental studies. Cross-polarization magic angle spinning
(CPMAS) 13C NMR spectra of humic substance was divided into six chemical shift regions,
0-60, 61-80, 81-110, 111-160, 161-180 and 181-210 ppm (Figure 1). These regions were
referred to as aliphatic, heteroaliphatic, acetal, aromatic, carboxyl, and carbonyl regions. In
addition, predictive weight-average molecular (Mw) to the method of Moriguchi et al. [5], in
which absorbance coefficients (ε) of humic substance at 280 nm on UV-vis spectroscopy are
applied to the empirical equation:
M w = 3.99ε + 490 (1)
The result shows that the molecular weights of HA and FA were ~3132.18 and ~1691.15,
respectively.
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15th IHSS Meeting- Vol. 3
Figure 1: Cross-polarization magic angle spinning (CPMAS) 13C NMR spectra of HA and FA
In this study, the Schubert method was used to determine the metal complexes of HS. The
fundamental assumption was made, namely, the metal ion was the central group, thus the
complexes ML1, ML2, ML3….were formed in the aqueous solution and the conditional
stability constants are defined by:
βi =
[ MLi ]
[ M ][ L]i
i=1, 2, 3 ……
(2)
Where M is the metal ion, L is the free ligand and [ ] stands for the concentration. The
distribution coefficient (λ0) of metal between resin and aqueous solution in the absence of
ligand could be defined by:
λ0 =
[ M ]ads
[M ]
(3)
In the presence of hydrolyzed species (M(OH)i), buffer-complexed species (MBj), bicarbonate
and carbonate species M(HCO3)m and M(CO3)n in the aqueous phase, Eq(3) convert to Eq.(4):
λ1 =
[M ] +
∑ [M (OH ) ] + ∑
i
i
[ M ] ads
b
[ MBb] +
∑
m
[ M (CO3 ) n ] +
∑
n
[ M ( HCO3 ) n ]
(4)
Eq(4) was also further modified to include the effect of complexation by HSs to form ML1,
ML2, ML3,. . .
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15th IHSS Meeting- Vol. 3
λ2 =
[M ]ads
[M ] +
∑ [M(OH) ] + ∑ [MBb] + ∑
i
i
b
m [M (CO3 ) n ] +
∑
n [M (HCO3 ) n ] +
∑
k [MLk ]
(5)
The concentration of HSs complexes can be substituted by conditional stability constants ߚ1;
ߚ 2; ߚ3. At a constant pH, a constant buffer concentration, and a constant bicarbonate or
carbonate concentration, combination of Eqs. (3)–(5) leads to the following equation:
1
λ2
The plots of
1
λ2
−
1
λ1
−
1
λ1
=
1
λ0
( β 1 [ L ] + β 2 [ L ] 2 + β 3 [ L ]3 + ⋅ ⋅ ⋅ ⋅ ⋅ ⋅
(6)
vs. [L] were achieved and the conditional stability constants would be
obtained by using quadratic polynomial equation fitting method. The results have been listed
in Table 1. It is clear that the complexes of metal ions with HSs mainly formed 1:1 and 1:2,
and the mono-molecular formation is predominant. The value of ߚ of HA-M was larger than
that of FA-M, since the larger macular weight of HA results in the larger volume and more
coordinate groups than FA. In addition, the complex ability of HSs with metal ions was
obviously influenced by the kind of metal ions and reaction temperature.
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Table1: Conditional stability constants of metal ions-HA and FA complexes
Metal
Ni2+
Cu2+
UO22+
Th4+
Ligand
HA
FA
HA
FA
HA
FA
HA
FA
r
pH
4.70
4.80
4.80
4.77
4.60
4.60
2.69
2.64
Logߚ1
Logߚ2
5.932
3.537
5.555
4.084
5.223
4.540
6.290
4.218
5.451
4.819
1.315
3.711
3.464
5.631
3.733
0.99356
0.99373
0.99870
0.99932
0.99874
0.97537
0.98384
0.99216
References
1. C.L. Chen, X.K. Wang, H. Jiang, W.P. Hu, Colloid. Surf. A. 302 (2007) 121.
2. F.J. Stevenson, Humus Chemistry: Genesis, Composition, Reactions, 2nd ed. Wiley, New York
1994.
3. D. Gondar, R. López, S. Fiol, J.M. Antelo, F. Arce, Geoderma 135(2006) 196.
4. W.M. Dong, H.X. Zhang, M.D. Huang, Z.Y. Tao, Appl. Radiat. Isot. 56(2002)959.
5. T. Moriguchi, M. Tahara, K. Yaguchi, J. Colloid Interf. Sci. 297(2006) 678.
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Characterization of Zinc Binding Ability of Dissolved Hydrophilic Organic
Matter from The Seine River and Major Wastewater Effluents
Louis Yoanna, Pernet-Coudrier Benoîta,b, Varrault Gillesa*
a
Laboratoire Eau Environnement et Systèmes Urbain (LEESU), Université Paris-Est, 61
Avenue du General de Gaulle, F 94010 Créteil Cedex, France ; b Eawag, department water
resources and drinking water, Überlandstrasse 133, 8600 Dübendorf, Switzerland
E-mail: varrault@univ-paris12.fr; yoann.louis@leesu.enpc.fr
1. Introduction
Complexation of trace metals in aquatic ecosystems by natural organic matter (NOM) and
inorganic ligands, i.e. their chemical speciation, determines their mobility, bioavailability and
toxicity [1, 2]. Natural organic matter, which is ubiquitous in the environment, is known to
play important roles in the fate of many contaminants due to its complexing properties. A
better determination of NOM structural and functional properties can greatly improve our
understanding of the underlying mechanisms responsible for heavy metals complexation.
Over the past few decades, many studies have been published regarding the capacity of
dissolved organic matters (DOM) to complex metals and especially copper. It is interesting to
note however that the published data pertain mainly to the so called “humic substances” (HS)
and demonstrate the ability of these substances to complex metals. HS are derived from
oxidative and hydrolytic biodegradation of plants and animals [3] and they make up 40–60%
of DOC in natural surface water [4].
In urbanized aquatic system, the hydrophobic characteristic of DOM is weaker as a result of
various urban DOM discharges and the strong primary productivity induced by these
discharges. Previous study has shown that non-hydrophobic organic matter represents more
than 50% of DOC in urbanized aquatic system [5]. However, because of the difficulty in
isolating the hydrophilic fraction of DOM, no information is available regarding hydrophilic
DOM in urbanized aquatic system and its influence on Zn complexation.
As a matter of fact, this work focus on zinc interactions with 3 isolated DOM fractions
fractionated according to polarity criteria, i.e. hydrophilic (HPI), hydrophobic (HPO) and
transphilic (TPH) fractions from four sampling sites located on the urbanized basin of the
Seine River (France) upstream and downstream Paris city. Suwannee River Fulvic Acid
(SRFA) obtained from the International Humic Substances Society (IHSS) has also been
characterised in order to compare properties of DOM from urban aquatic system with natural
humic substances
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15th IHSS Meeting- Vol. 3
2. Materials and Methods
Among the four sites studied on the Seine River basin, the first one located upstream of Paris
city (called Méry sur Marne) will be considered as our natural reference because of little
anthropic impact and those downstream of Paris (called Andrésy and Méricourt) as the most
impacted by urbanization as well as the treated effluent of the biggest wastewater treatment
plant (WWTP) of Parisian conurbation (Seine-Aval WWTP). For each sample, from 250 L to
1000 L were collected during a dry weather period (Fig. 1).
Figure 1: Sampling site on the Seine River basin (France) with on site filtration, softening and
concentration by reverse osmosis
Samples were filtered onsite through subsequent 10 µm and 0.45 µm polypropylene cartridge.
The samples were then softened on sodium cation-exchange and concentrated by reverse
osmosis (RO) in order to reduce the volume. Sample filtration, softening and concentration
were carried out in line and onsite so as to limit process duration and potential DOM
biodegradation (Fig. 1). The RO concentrate was then acidified and filtered back at the
laboratory on non-ionic macroporous Amberlite® DAX-8 resins (acrylate ester) and
Supelite® XAD-4 (divynil benzene) combined with one another. This allows us to fractionate
DOM into three fractions according to polarity criteria: hydrophobic (HPO), transphilic (TPI)
and hydrophilic (HPI) fraction [5].
Among the techniques allowing trace metals speciation in natural waters, voltammetry, and in
the case of this study differential pulse anodic stripping voltammetry (DPASV), is really
appropriate because it offers high sensitivity and enough selectivity for zinc measurement.
Obtained voltammetric data are related to the physico-chemical characteristics of the
electroactive metal species, and information on speciation parameters of the investigated
metal could be obtained [6].
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The pseudopolarography [7] technique, based on successive DPASV measurements at
different deposition potentials, was also employed in this study. This technique allows
discrimination/separation of different fractions of metals, from the electrochemically labile to
the inert ones, regarding the analytical window of the method. It is successfully applied for
the speciation of metals in model solutions and in natural waters, both for the labile and for
the inert metal complexes. According to the theory and to experimental evidences [7 and ref
therein], one or more reduction waves could be obtained (similarly like in classical
polarography) which correspond to one labile and/or to one or more inert metal complexes.
In order to work on a wider analytical window, zinc additions were performed in a
logarithmic mode [8] (from nano-molar to about 14 micro-molar) which also allows a better
accuracy of the complexing parameters determination.
3. Results and Discussion
Depending on the studied DOM fraction, pseudopolarography experiments point out different
behaviour with one (Figure 2 on the right) or two wave (on the left) showing the specificity of
each extracted fraction.
Figure 2: Pseudopolarography experiments representative of a HPO (on the left) and a HPI (on the
right) DOM fraction. x axis: different deposition potentials with step of 0.05V from -0.8 (labelled 1)
to -1.6V (labelled 17), y axis: scanning potential for stripping, z axis: Intensity for [ZnT]=13.8µM
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However for all the studied fractions, a deposition potential of -1.05 V give a good sensitivity
to analyse the labile zinc. Experiments carried out with logarithmic zinc additions give new
data (stability constant and ligands concentration) concerning the zinc-NOM interactions in an
urbanized area especially with HPI fraction. From the analysis, it is emerging a tendency
showing that the HPI fraction has a higher affinity for zinc than respectively the TPH and the
HPO fractions. This tendency was also observed on the same samples for copper and mercury
on previous study carried out in our lab.
4. Conclusions
Thanks to this study and the news determined complexing parameters usable in
modelization/prediction study, the behavior of zinc (which is a metal always present in
urbanized aquatic system mainly due to its use in roof and gutter) will be better understood in
such complex ecosystem than the urbanized rivers with especially the hydrophilic DOM
fraction.
Acknowledgements
We would like to thanks the French Ministry of agriculture for the post doctoral grant of Y.
Louis, and B. Pernet-Coudrier for the DOM fractionation made during his PhD with the
financial support of the French Ministry of research and higher education.
References
1. J. Buffle, Complexation Reactions in Aquatic Systems: An Analytical Approach, M. Masson and J.
F. Tyson, Ellis Horwood, New York, 1988, p.692.
2. A. Tessier and D. R. Turner, Metal Speciation and Bioavailability in Aquatic Systems, J. Buffle
and H. P. Van Leeuwen, John Wiley & sons, Chichester, 1995, p. 696.
3. F.J. Stevenson, Humus Chemistry – Genesis, Composition, Reactions, John Wiley & Sons, New
York, 1994.
4. B. Martin-Mousset, J.P. Croué, E. Lefebvre, B. Legube, Distribution et caractérisation de la
matière organique dissoute d’eaux naturelles de surface, Water Res., 31 (1997), 541.
5. B. Pernet-Coudrier et al., Chemosphere, 73 (2008) 593.
6. Y. Louis et al., Mar. Env. Res., 67 (2009) 100.
7. R. Nicolau et al., Anal. Chim. Acta., 618 (2008) 35.
8. C. Garnier et al., Anal. Chim. Acta., 505 (2004) 263.
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Molecular Size Distribution of Metal Complexes with Pore Water Dissolved
Organic Matter Determined by HPSEC and ICP-MS
Natalja Makarõtševaa*, Viia Lepanea, Tiiu Alliksaarb
a
Institute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn,
Estonia; bInstitute of Geology, Tallinn University of Technology, Ehitajate tee 5, Tallinn,
Estonia
E-mail: natalja.makarotseva@ttu.ee
1. Introduction
It is widely known that natural organic matter is influencing the fate and behavior of
contaminants in aquatic environment; however, the interaction mechanisms are relatively
poorly understood. The goal of the present study was to investigate which organic fractions
bind to specific metals.
2. Materials and Methods
In the present study pore water extracted from the sediments from Lake Peipsi, Estonia, was
investigated using high-performance size exclusion liquid chromatography (HPSEC) with
fluorescence detection and inductively-coupled plasma mass-spectrometry (ICP-MS).
Sediment core was collected on ice using Russian type peat corer from the central part of
Lake Peipsi (58°47.213´; 27°19.299´) in March, 2007. The core was sliced into 1 cm thick
sub-samples. Before the analysis, pore water was extracted from the solid phase by
centrifugation and filtration.
Pore water dissolved organic matter (DOM) from different depths was separated and
fractionated using HPSEC. 100 mM phosphate buffer was used as a mobile phase at the flow
rate 0.5 mL/min. Injected volume was 20 µL. Fluorescence signal was detected at 340/420 nm
(humic-like fluorescence). HPSEC column was calibrated using polystyrene sulphonate
standards (PSS) with molecular weights of 17, 13, 6.8, and 4.3 kDa dissolved in the mobile
phase. Collected HPSEC fractions were analyzed with ICP-MS for selected metals, such as
Al, Ca, Mn, Ni, Cu, Zn, Ag, Cd, and Pb, in order to reveal which molecular weight fractions
complexed them. Mentioned metals were also measured directly in not fractionated pore
water samples by ICP-MS.
3. Results and Discussion
Common chromatogram of pore water DOM with marked collected fractions is shown on Fig.
1. Phosphate buffer is widely used for DOM separations but its usage in case of ICP-MS
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15th IHSS Meeting- Vol. 3
analysis could be problematic, because phosphates itself may contain metal impurities. In the
present study the attempt to minimize buffer contamination influence was done by injecting
the buffer as a blank sample to the HPSEC column and fractionating it at the same retention
times as the collected pore water samples.
Results showed that Ca was mostly complexed with DOM eluted at 19.5-23.0 min (3rd
fraction). The molecular weights of that fraction corresponded to number average molecular
weight Mn of 600 Da, and weight average molecular weight Mw of 700 Da. The presence of
other investigated metals was detected in all collected fractions and their contents were not
differing much. Thus, under the used conditions it was not possible to distinguish which
fraction contains more metals. The possible reason for it was that the content of metals was
very low and it was close to ICP-MS LOD.
1
(a)
1000
(b)
800
0,6
Ca, ug/L
Detect or signal, mV
0,8
0,4
0,2
2
1
4
0
5
10
15
20
400
200
3
0
600
0
25
30
0
-0,2
1
2
3
4
Fraction
Retention time, min
Figure 1: (a) HPSEC example chromatogram of pore water DOM, with marked fractions; (b) Ca
content in the separated fractions by ICP-MS
In this study Ca was determined in fractionated pore water samples from different depths.
Other metals distributions in depth profile were also constructed.
4. Conclusions
The obtained information is valuable for understanding the binding and transport of metals in
aquatic water bodies. Also, this knowledge enables to draw conclusions about the
bioavailability of metals towards the organisms living in lake ecosystems.
Acknowledgements.
The authors thank the Department of Analytical Chemistry of Masaryk University, Brno,
Czech Republic, for kind possibility to use ICP-MS.
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Influence of Pb(II) Ions on Semiquinone Radicals of Humic Acids and
Model Compounds
Maria Jerzykiewicz*, Maciej Witwicki
Faculty of chemistry, Wroclaw University, F. Joliot-Curie 14 St., Wroclaw, Poland
E-mail: mariaj@wchuwr.pl
1. Introduction
The complexation of Pb2+ with humic acids macromolecules leads to the formation of a new
kind of radical species characterized by unusually low giso values (~ 2.001) [1,2]. Noteworthy,
these new radicals appears to be very stable.
In this paper we decided to investigate the interaction of Pb(II) ions with humic acids and
their model compounds in details. We hoped to obtain an insight into the components of the
g-tensor and how they change upon the interactions of the parent radicals with the Pb(II) ions.
On the other hand, the systematic theoretical DFT study of the model complexes between
Pb(II) ions and semiquinone radicals was preformed, examining different possible
deprotonation forms of the ligands and different possible binding schemes with the metal
cation. We expected that the correlation between theoretically predicted and experimentally
obtained g-tensors would reveal the structural types responsible for the low giso values
observed by the EPR spectroscopy, allowing for characterization of the best model mimicking
the Pb(II) coordination to HA which is an important problem for environmental chemistry [3].
2. Materials and Methods
3,4-dihydroxybenzoic acid (34dhb) and gallic acid (3,4,5-trihydroxybenzoic acid, 345thb)
were purchased from Aldrich. Humic acids were extracted from peat (HAP) of Odra river
lowland near Wroclaw (Lower Silesia, Poland) and from compost (HAC) derived from the
Municipal Composting Plant in Zabrze (Upper Silesia, Poland). The isolation was carried out
using standard IHSS procedure [4]. To obtain the complexes of Pb(II) ions with HAC, HAP,
34dhb, and 345thb in powder form, a constant amount of a solid ligand L (1 mmol of 34dhb
and 345thb or 50 mg of HAC and HAP) was treated with a fixed volume of lead acetate
solution (20 mL) of concentration appropriate to obtain various initial Pb(II):L molar ratios
from 1:1 to 5:1. The solid product was filtered and dried at room temperature, and the EPR
spectra of powder samples were recorded.
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X-band EPR (9.6 GHz) spectra were recorded at the room temperature on a Bruker ESP 300E
spectrometer equipped with a Bruker NMR gauss meter ER 035 M and a Hewlett-Packard
microwave frequency counter HP 5350B. As investigated systems contain lead, all singlepoint calculations of the g-tensor were preformed within the two-component approach using
spin-orbit ZORA relativistic formalism, as implemented by van Lenthe in Amsterdam Density
Functional (ADF) version 2007.01. Three different functionals were used: (a) local density
approximation of Vosko, Wilk and Nusair (VWN) with the exchange gradient correction
proposed by Becke (B) and the correlation term developed by Perdew (P86); (b) VWN local
density approximation with the exchange gradient correction and the correlation term by
Perdew and Wang (PW91); (c) B exchange gradient correction and the correlation term by Lee,
Yang and Parr (LYP). More about computational approach in [5].
3. Results and Discussion
Formation of low-g radicals upon the Pb(II) interaction. Treatment of solid HAC and HAP
with lead acetate solution results in a characteristic modification of the EPR signal. Total
signal intensity increases as compared to that recorded for the pure HAs. The native radical
concentration in the HAC was found to be about 0.3 × 1018 spins per gram. After formation of
Pb(II)-HAC complex the concentration increases even to 1.7 × 1018 spins per gram.
Subtraction of the HAs signal from the signal of Pb-treated HAs reveals a new radical line, as
reported previously [2]. The g values for new radicals in both Pb-HAs systems are
significantly smaller than those for native radicals that occur in HAs: geff = 2.0012 for PbHAC compared to geff = 2.0033 for HAC and 2.0013 for Pb-HAP compared to 2.0036 for
HAP. Nevertheless, no significant dependence of EPR parameters on concentration of Pb(II)
was observed. It needs to be noticed that the presence of indigenous signal due to HAs in the
spectra of Pb(II)-treated HAs proves that the native radicals, which are probably located in the
humic acid macromolecular matrix, are not affected by the Pb(II) ions. Pb(II) seems to be
bonded mainly to the outer macromolecular fragments. Furthermore, if the Pb(II) ions were
involved in the interaction with the native radical sites in HAs giving the g ~ 2.001 signals,
the complexes could not be generated from model dihydroxybenzoic acids (since
dihydroxybenzoic acids do not contain the native radicals). Therefore, it can be assumed that
the radical complexes formation is preceded by the Pb(II) ions coordination to the
diamagnetic ligand. In contrast to HAs, the initial concentration of Pb(II) ions appeared to be
a major factor determining the EPR parameters observed for radical complexes with 34dhb
and 345thb ligands. Two types of spectra were observed depending on Pb(II) concentration
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15th IHSS Meeting- Vol. 3
(see Figure 1). For the system prepared using molar ratio Pb(II):L = 3:1, the powder EPR
spectra indicating formation of complex 1 are similar to those reported previously (geff ~
2.001) [1]. However, the spectra of the Pb(II):L= 2:1 system differ distinctly from those
observed when molar ratio Pb(II):L=3:1 was used. They are stronger and slightly anisotropic,
with larger geff, but still below the giso values for the radical anions derived from the parent
hydroxybenzoic acids. In addition, the powder EPR spectra for the 2:1 system show satellite
splitting due to the anisotropic hyperfine interaction of an unpaired electron with the
207
Pb
nucleus (I= 1/2, 22.1% abundance)- figure 1. Therefore, the second complex (complex 2) has
been identified. The parameters of hyperfine couplings with the
207
Pb nucleus strongly
suggest a covalent interaction between the Pb(II) ion and a radical ligand in complex 2.
Figure 1: EPR spectra of radicals in Pb(II)-34dhb powders with different metal ion to ligand ratio
DFT Computations for Model Complexes of Pb(II) with Semiquinone Radical Species. Three
different forms of ligand were considered: L2-•, HL-•, and H2L•. The radical derived from
34dhb is an ambidentate ligand, and many possible isomers exist for every form. Two binding
sites (carboxylic or phenolic oxygens) were taken into account for the ligand acting as either
mono- or bidentate. First, the geometry of the Pb(II):L 1:1 model complexes and their g
parameters were calculated. Next, analogous calculations were performed for model 1:2
complexes that were derived from those 1:1 complexes in which a reasonable agreement
between the theoretical and experimental g-tensors was achieved.
Systematic theoretical studies for the Pb(II)-34dhb system has shown that the structures with
a significant accumulation of the spin population on the Pb atom cannot explain the shifts of
experimentally observed g-tensor components. DFT investigations show, that decrease of the
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spin population on all oxygen atoms in favour of the carbon atoms of a benzoic ring, observed
for the complexation via carboxyl oxygen atoms while both hydroxyl oxygen atoms are
protonated, can reproduces the experimental results and therefore can be responsible for the
observed shift of the g-tensor. The binding mode and the form of ligands present in Pbsemiquinone complexes have been determined and the reaction mechanism, which may lead
to the formation of such complexes, has been proposed.
4. Conclusions
X-band and high field EPR spectroscopy has been applied to investigate the complexes of
Pb(II) ions with the semiquinone radicals of HA and their model compounds (34dhb and
345thb). They are characterized by unusually low g-parameters in comparison to the parent
radicals. For the model compounds, a formation of two complexes (complex 1 and complex
2) has been revealed by two different EPR spectra. For complex 2, a splitting of the spectrum
due to the anisotropic hyperfine interaction with the
207
Pb nucleus (I=1/2, 22.1%) has been
observed.
References
1. E.Giannakopoulos, K. C. Christoforidis, A Tsipis, M. Jerzykiewicz,Y. J. Deligiannakis, Phys.
Chem. A 2005, 109, 2223
2. M. Jerzykiewicz, Geoderma 2004, 122, 305
3. E. Tipping, E. Cation Binding by Humic Substances, 1st ed.; Cambridge University Press:
Cambridge, U.K., 2002
4. R.S. Swift. Organic matter characterization, In Methods of Soil Analysis. Part 3 Chemical
Methods - SSSA Book Series no. 5, Madison, WI, 1996, pp.1011–1069,
5. M. Witwicki, M. Jerzykiewicz, A.R. Jaszewski, J. Jezierska, A. Ozarowski, J. Phys. Chem. A
2009, 113, 14115–14122.
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The Effect of Natural Organic Matter on the Formation and Solubility of
M(OH)4 Solid Phases (Th(OH)4, Zr(OH)4 Ce(OH)4)
Stella Antoniou, Ioannis Pashalidis*
Chemistry Department, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus
E-mail: pspasch@ucy.ac.cy
1. Introduction
The chemical behavior and mobility of metal ions in aquatic environments depends strongly
on the solubility of the solid phases/minerals present. (Oxo)hydroxide solid phases are usually
the predominant secondary phases of tetravalent metal ions in natural aquatic systems, and are
also important sinks for (radio)toxic metals (e.g. Th(IV), Pu(IV), Zr(IV)) in the near field of
nuclear waste repositories and heavily contaminated water systems. Therefore, in order to
understand the chemistry and mobility of (radio)toxic metals, knowledge of the solubility and
stability of relevant solid phases/minerals is of fundamental importance. In natural water
systems where humic acid is present, the complexation with humic acid plays a significant
role in the geochemical behavior and migration of thorium and other actinide ions in the
geosphere [1 - 4]. Hence, the impact of humic acid on the stability of M(IV) solid phases and
species predominantly formed is of particular interest.
To ascertain the effect of humic acid complexation on the solid phase formation and
subsequently the chemical behavior of M(IV) (e.g. Th(IV) and Zr(IV)) in aqueous solutions,
the stability of M(OH)4 has been studied as a function of the humic acid concentration in 0.1
M NaClO4, in the pH range from 3 to 5. The solid phase formation of redox sensitive metal
ions has been investigated for Ce(IV) hydroxides formed by alkaline precipitation from
aqueous solution containing various amounts of humic acid.
2. Materials and Methods
M(IV) and humic acid stock solutions were prepared by dissolution of Th(NO3)4 5H2O
(Merck Co), ZrO(NO3)2 xH2O (Aldrich) and Ce(NO3)4 (Kristallhandel Kelpin) and humic acid
sodium salt (Aldrich) in de-ionized water, respectively. M(OH)4 solid phases were prepared
by alkaline precipitation of M(IV) from pure aqueous solutions or solutions containing
different amounts of NOM (0, 0.1, 0.3 and 0.5 g l-1 humic acid), under normal atmospheric
conditions at 25 oC. For solubility studies Th(OH)4 and Zr(OH)4 solids were conducted with
40 ml of pure 0.1 M NaClO4 solutions or 0.1 M NaClO4 solutions containing 0.1 g l-1 humic
acid under normal atmospheric conditions at 25 oC. pH was adjusted by 0.1 M NaOH or 0.1
M HClO4 and was measured using a glass electrode (Hanna Instruments pH 211). The
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analytical M(IV) concentration in solution was determined by spectrophotometry (UV 2401
PC Shimadzu) using arsenazo III according to a previously described method [5].
For physicochemical characterization, the precipitates were separated from the solutions by
centrifugation, washed (2 times) with de-ionized water, air-dried and characterized by
Thermogravimetry (TGA-50, Shimadzu) and x-ray diffraction (XRD 6000 Shimadzu). FTIRATR spectroscopy (IR Prestige-21 Shimadzu) and XPS measurements were performed on
solid samples after separation and thermal treatment of the samples at 100 oC under vacuum
conditions. The XPS measurements were carried out at the ICE/HT-FORTH Labs in Patras,
Greece.
3. Results and Discussion
Investigation of pure Th(OH)4 and Zr(OH)4 solid phases and the same solids in equilibrium
with aquatic solutions of 0.1 M NaClO4 containing 0.1 g/L of humic acid by ATR-FTIR
spectroscopy shows similar features independent of the presence of humic acid. In the
spectrum predominate two intensive absorption bands at 3610 cm-1 and 1629 cm-1 in the FTIR
spectrum, which correspond to O-H stretching and to H-O-H bending, respectively. The
absorption band at 1081 cm-1 and 1074 cm-1 correspond to the Th-OH and Zr-OH stretching,
respectively. Thermogravimetric analysis of the Th(OH)4 and Zr(OH)4 solids prior contacting
with humic acid solutions, show mass losses (about 32%) with temperature, which according
to stoichiometric calculation data correspond to M(OH)4 solid phases. After equilibration with
humic acid solutions, the thermogravimetric data indicate only on a higher mass loss (about
5%), which is attributed to excess physisorbed water on the humic acid treated solids [4]. Xray diffraction analysis of pure Th(OH)4 and Zr(OH)4 solid phases and the same solids in
equilibrium with aquatic solutions of 0.1 M NaClO4 containing 0.1– 0.5 g/l of humic acid
indicates also only the presence of M(OH)4 solid phases. Based on the Scherrer equation [6]
the particle/crystallite sizes have been evaluated and are summarized in Table 1.
Table 1: Particle size of Zr(OH)4 and Th(OH)4 solid phases prior and after contact with aqueous
solutions of 0.1M NaClO4 containing 0.1-0.5 g/l of humic acid
[HA], mg/l
Particle size, nm
Th(OH)4
Zr(OH)4
0
8
9.4
0.1
7.5
9.0
0.3
7.6
9.3
0.5
7.7
9.2
According to data summarized in Table 1, the presence of humic acid in solution does not
affect the particle size of the Th(OH)4 solid phase. The absence of any effect on the crystallite
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size of Th(OH)4, which in contrast to the effect of humic acid on the crystallite size of U(VI)
solid phases (e.g. UO2(OH)2 [3] and UO2CO3 [4]), can be attributed to the higher stability of
the M(IV) solid phases compared to the U(VI) solid phases.
Solubility measurements were performed with Th(OH)4 under normal atmospheric conditions
in aquatic solutions of 0.1 M NaClO4 containing 0 and 0.1 g/L of humic acid. According to
solubility measurements corresponding to the systems with or without humic acid, the
Τh(OH)4 solid phase determines solubility. Fluctuations which are observed in the thorium
concentration were attributed to the formation of Th(IV) colloids in the pH range 3-5 [2]. In
this study the formation of Th(IV) colloids has been confirmed by ultrafiltration experiments
using membrane filters of different pore size (0.45μm, 0.22μm, 30kD, 100kD). According to
the ultrafiltration experiments, decreasing pore size of the ultrafiltration mebranes results in a
significant decrease of the total Th(IV) concentration in solution. Furthermore, lower Th(IV)
concentration in humic acid - containing solution after filtration indicates the formation of
larger particles generated upon humic acid - Th(IV) colloid interaction.
To investigate the effect of NOM to the solid formation of redox-sensitive (oxidizing)
tetravalent ions, Ce(IV) ions have been precipitated upon addition of alkaline aqueous
solutions containing different amounts (0 - 0.5 g/l) of humic acid. The resulting solid phases
have been separated, vacuum dried and characterized by means of XPS analysis regarding the
relative amount of Ce(IV) in the solid phase as a function of the humic acid (NOM)
concentration in solution. The evaluation of the XPS spectra (Figure 1) resulted in a linear
correlation between the humic acid concentration in the test solution and the reduced amount
of Ce(IV) in the precipitated solid phase, indicating that in the presence of NOM oxidizing
ions may be reduced affecting solid phase composition. The latter is important for the
chemical behavior and migration of actinide (e.g. Pu(IV) ions) in the geosphere.
4. Conclusions
The results obtained from this study lead to the conclusions that (a) M(OH)4 is stable and
remains the solubility limiting solid phase even in the presence of increased NOM (humic
acid) concentrations in solution, (b) the presence of NOM (humic acid) results in increased
hydrophylicity of the solids but doesn’t affect the crystallite size and the solubility product of
M(OH)4, (c) M(OH)4 solubility is basically pH depended and governed by the presence of
colloidal species and (d) NOM (humic acid) may reduce redox-sensitive (oxidizing) metal
ions and result in increased amount of the reduced species in the solid phase affecting its
solubility behavior.
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Figure 1: Relative amount of Ce(IV) in the solid phase as a function of the humic acid (NOM)
concentration in the test solution
Acknowledgements
The research leading to these results has received funding from the Cyprus Research
Promotion Foundation (Grant agreement No. ΠΕΝΕΚ/ΕΝΙΣΧ/0308/05). The authors thank
Dr Elina Siokou for the XPS measurements.
References
1. J.I. Kim, Mat. Res. Soc. Symp. Proc., 294 (1993) 3.
2. V. Neck and J.I. Kim, Radiochim. Acta, 89 (2001) 1.
3. C. Kolokassidou and I. Pashalidis, J. Radioanal. Nuclear Chem., 279 (2009) 523.
4. S. Antoniou, C. Kolokassidou, K. Polychronopoulou and I. Pashalidis, I., J. Radioanal.
Nuclear Chem., 279 (2009) 863.
5. S.B. Savvin, Talanta, 8 (1961) 673.
6. Jenkins, R., Snyder, R.L., Introduction to x-Ray Powder Diffractometry, J. Wiley & Sons,
N. York, 1996.
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Adsorption of Polycyclic Aromatic Hydrocarbons (PAHs) onto Engineered
and Natural Nanoparticles
Lina Marino, Donato Mondelli, Nicola Senesi
University of Bari, Dip. Biologia e Chimica Agrof. Amb., Via G. Amendola 165/A, 70126,
Bari, Italy
E-mail: senesi@agr.uniba.it
1. Introduction
Nanoparticles have been demonstrated to have a very high sorption capacity for a variety of
organic contaminants from water and soil (soil remediation) [1]. However, there are
unanswered questions about the impact of engineered nanomaterials and nanoproducts on
human health and the environment [2]. Among nanoparticles, fullerene C60 has received
considerable attention due to its unique characteristics and numerous potential applications
[3]. Fullerene C60 is virtually insoluble in water [4], where it forms clusters that present closed
interstitial spaces within the aggregates into which organic compounds can diffuse and remain
trapped [5]. Adsorption of organic compounds by fullerene have been shown to depend to a
great extent on its dispersion state in water [6]. Fullerenes are reported as weak sorbents for
organic compounds including PAHs, but are very efficient for the removal of organometallic
compounds [7].
The specific objective of this study is to evaluate the use of fullerene C60 as an adsorbant of
organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), from aqueous
solutions, compared with the adsorbent effect of different types of soil with various organic
carbon (OC) and clay contents, texture, pH and cation exchange capacity (CEC), as such and
with the addition of suitable amounts of fullerene (F), compost (C) and humic acid from
compost (HAC).
2. Materials and Methods
Soils. ES (Elliott Soil), a clay soil of the IHSS (International Humic Substances Society)
collection with 2.9% OC; S2, a silt loam soil with 1.1% OC; S3, a loamy sand soil with 0.1%
OC (Table 1).
The soil samples were homogenized in a ball mill (Retsch MM 200) and used both as such
and added with 5% F or 1% C or 1% HAC.
Nanoparticles. Fullerene C60 (F) with purity>99,5% was obtained from Sigma-Aldrich and
used as received.
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Compost (C) from vegetable wastes with 38.5% OC.
Humic acid from compost (HAC) with about 50% OC.
Chemicals. Pyrene (Sw: 135 µg/L at 25 °C, purity>99,0%) was purchased from Fluka
Analytical and used as received. Its stock solutions were prepared in methanol (HPLC grade).
Adsorption Experiments. The adsorption kinetics and adsorption isotherms of pyrene onto the
various soil substrates were determined using a Batch Equilibrium Method (OECD, 2000) and
the high-performance liquid chromatography (HPLC) technique with a fluorescence-detector
operating at 337 nm in excitation and 377 nm in emission.
Table 1. Some physical and chemical properties of soils examined
Soil
ES
S2
S3
Textural
Class
(USDA)
Clay
Silt Loam
Loamy sand
pH
(1:2.5
H2O)
6.8
8.1
8.5
Organic
carbon
(%)
2.9
1.1
0.1
Total
nitrogen
(%)
0.25
0.12
0.01
Total
carbonate
(%)
< 0.1
5.9
8.9
Adsorption kinetics. 50 mg of soil were suspended in 40 mL of 0.01 M CaCl2 aqueous
solutions containing pyrene at a concentration of 100 µg/L in glass flasks. A blank sample
(without soil) at the same concentration of pyrene was prepared. The mixtures were then
mechanically shaken for five different time periods: 12, 24, 48, 72 e 96 h. At the end of each
time period, the suspensions were centrifuged at 12000 rpm for 20 min, and the supernatants
were analyzed by HPLC to determine the residual concentration of pyrene in solution. All
experiments were conducted in triplicate at a temperature of 25 °C.
Adsorption isotherms. Aliquots of 50 mg of the various soil substrates were added in glass
flasks with 40 mL of 0.01 M CaCl2 aqueous solutions containing pyrene at concentrations of
40, 60, 80, 100, and 120 µg/L. Equilibration was achieved by mechanical shaking of mixtures
for 72 h at 25 °C in the dark. All experiments were conducted in five replicates. Suspensions
were then centrifuged at 12000 rpm for 20 min, and the supernatants were analyzed for the
equilibrium concentrations, Ce, of pyrene by HPLC. Three standard samples (without soil) at
concentrations of 30, 60 and 120 µg/L were also analyzed in each set of sorption isotherm
experiments. The amount of pyrene adsorbed onto substrate (x/m, in µg g-1) was calculated as
the difference between the initial concentration and the equilibrium concentration (Ce, in µg
mL-1) of pyrene in solution. Experimental data were fitted to both a linear and non linear
Freundlich and Langmuir equations.
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3. Results and Discussion
Adsorption kinetics. Adsorption of pyrene onto the examined soils occurs in two phases, a
rapid one in less than 48 hours of contact amounting to more than 90% of total adsorption,
and a slow one that may need several hours until attainment of equilibrium. The rapid
adsorption phase would occur on the most reactive and/or accessible sites of substrate,
whereas sites less reactive and/or less sterically accessible would be involved subsequently in
the slow phase.
Adsorption isotherms. Generally, experimental adsorption data for pyrene onto the various
substrates fit better in Langmuir isotherms. The adsorption isotherms indicate that pyrene has
a moderately high affinity for the substrate in the initial stages of adsorption, whereas it has
increasing difficulty in finding vacant sites, until reaching a maximum of adsorption.
The KD values (Table 2) indicate that addition of compost and HAC to soils is more efficient
than fullerene for pyrene adsorption.
Further, the KOC values (Table 2) differ for the samples examined, thus indicating that the
structural and chemical properties of the organic matter in the samples, and not only its
amount, affect markelly the extent of adsorption.
Table 2. Distribution coefficients (Kd) and organic carbon partition
coefficient (Koc), for Langmuir adsorption isotherms of pyrene onto
substrates studied
Koc (x103)
Substrate
KD
ES
1324
46
ES+5% F
1459
53
ES+1% C
2226
69
ES+1% HAC
2175
66
S2
275
25
S2+5% F
304
29
S2+1% C
473
43
S2+1% HAC
411
27
S3
0.2
0.2
S3+5% F
6.2
6.5
S3+1% C
141
29
S3+1% HAC
112
19
C
9721
25
HAC
3995
8
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References
1. L. Dai (Ed.), Carbon Nanotechnology: Recent Developments in Chemistry, Physics, Materials
Science and Applications, Elsevier, Boston, 2006.
2. V. L. Colvin, The Potential Environmental Impact of Engineered Nanomaterials, Nat. Biotechnol.
21, pp. 1166-1170, 2003.
3. H. W. Kroto, J. R. Heath, S. C. Obrien, R. F. Curl, R. E. Smalley, C60-Buckminsterfullerene,
Nature 318, pp. 162–163, 1985.
4. D. Heymann, Fullerene, Sci. Technol., 4, p. 509, 1996.
5. X. Cheng, A. T. Kan, M. B. Tomson, J. Nanopart. Res., 7, p. 555, 2005.
6. X. Cheng, A. T. Kan, M. B. Tomson, J. Chem. Eng. Data, 49, p. 675, 2004.
7. E. Ballesteros, M. Gallego, M. Valcarcel, J. Chromatogr. A 2000, p. 869, 101.
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The Challenge of Building a Humic-Metal Binding Constants Database
Montserrat Filellaa*, Wolfgang Hummelb, Peter M. Mayc, Jaume Puyd, François Quentele
a
Institute F.-A. Forel, University of Geneva, 10 route de Suisse, CH-1290 Versoix,
Switzerland; bPaul Scherrer Institut, Laboratory for Waste Management, CH-5232 Villigen,
Switzerland; cSchool of Mathematical and Physical Sciences, Murdoch University, Murdoch,
WA 6150 Australia; dDepartament de Química, Universitat de Lleida, Rovira Roure 191, E25198 Lleida, Spain; eUniversité de Bretagne Occidentale, CNRS UMR 6521, 6 avenue V. Le
Gorgeu, F-29238 Brest Cedex, France
E-mail: montserrat.filella@unige.ch
The fraction of natural organic matter (NOM) more refractory to degradation, often known as
fulvic and humic compounds, plays a decisive role in trace element chemistry in
environmental and engineering systems. Accordingly, a significant amount of research has
been devoted to its characterization as well as to the determination of binding constants to
quantify its interaction with trace elements. However, in spite of the effort deployed, the
difficulties encountered when trying to compare complexation constants reported in the
literature or to find constant values for less studied elements remain well-known problems.
The reason is that our ability to measure and interpret the complexation equilibria of humic
substances is severely constrained by their ill-defined nature that, together with certain of
their characteristics, hinder the application of the experimental and interpretation methods
usually applied in the field of stability constant determination. This has led to the
development of a wide range of interpretation models for the representation and quantification
of the binding properties of humics, which adds a further difficulty for the practical
application of existing data.
Since no systematic compilation of published data exists, we have undertaken the gathering of
all data published on the complexation of trace elements with humic substances over the past
50 years. Our ultimate goal is the critical analysis and the interpretation of all existing data
with the objective of providing a robust framework for further research as well as a useful tool
for practical applications.
The first step in this endeavour is the object of an IUPAC-sponsored project: the development
of a comprehensive database of published values of humic-metal binding constants. The
building of such database represents a considerable challenge. On the one hand, it has to face
problems common with ‘classical’ thermodynamic equilibrium constant databases such as the
need of gathering information published in journals and reports not always readily available
or the adoption of criteria that minimize the inherent degree of subjectivity associated with
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any evaluation of data quality. On the other hand, new aspects need to be carefully considered
before deciding which type of information needs to be extracted and included in the database.
These further requirements are a direct consequence of the above-mentioned ill-defined nature
of the humic substances that has led to the use of a wide variety of experimental methods and
interpretation approaches.
The IUPAC project has started in 2009 with the collection of publications in electronic form
for subsequent information extraction by the members of this project. Currently this collection
comprises some 600 files and is expected to increase further. The rate of ’hard-to-find‘ journal
articles and reports until now stayed below 3% but may also increase when we will screen our
collected papers for further references with the goal of an exhaustive compilation.
Interesting and immediate spin-offs of the project are: (i) the exhaustive compilation of all the
studies ever published, irrespective of the quality or applicability of the data contained; (ii) the
identification of the elements for which little or no information exists; (iii) the elaboration of
recommendations for authors and editors concerning the information that needs to be included
in any publication for the data to be meaningful.
Acknowledgements
The work described is sponsored by IUPAC (project 2008-025-1-500).
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Heavy Metal Compounds with Organic Substance and Methods for their
Definition
Tatiana M. Minkinaa, Galina V. Motuzovab, Olga G. Nazarenkoc, Saglara S. Mandzhievaa*
a
Department of Biology and Soil Science, Southern federal University, ul. Bolshaya Sadovaya
105, Rostov-on-Don, 344006 Russia; bFaculty of Soil Science, Moscow State University,
Vorob'evy gory, Moscow, 119992 Russia; cAgroecological Faculty, Don State Agrarian
University, Persianovskii, Rostov-on-Don oblast, 346493 Russia
E-mail: msaglara@mail.ru
1. Introduction
Heavy metals (HMs) are related to the chemical properties of soils in various ways. The
uptake of heavy metals by soils was found to be dependent on the soil composition and
properties [1]. Special attention is paid to the participation of organic matter in these
processes. Heavy metals interacted with the soil organic matter forming various compounds.
The aim of this work was to study the interaction of heavy metals with the soil organic matter
2. Materials and Methods
A pot experiment was conducted with the upper (0- to 20-cm) layer of an arable calcareous
clay loamy ordinary chernozem with the following properties: pH water, 7.2; particles <0.01
m, 59%; CaCO3, 1.1%; Corg, 2.3%; exchangeable cations (meq/100 g): Ca2+ 29; Mg2+ 3; Na+
0.1. A draining layer (a 3-cm thick layer of claydite and a 3-cm thick layer of washed river
sand) was placed in polyethylene vessels of 4 L. The layer was covered with 4 kg of triturated
(to <5 mm) and thoroughly homogenized soil, to which Pb, Cu, and Zn were applied as dry
acetates separately at rates of 100 and 300 mg/kg. The samples were wetted to the total
moisture capacity.
We analyze the methods of the metal compounds definition and to develop the scheme of
metals compounds distribution bound to organic matter. The total contents of the metals in the
soil were determined after the decomposition of the soil with HF + HClO4. Both sequential
and parallel extractions were used for determining the metal compounds bound with organic
matter. The sequential extraction of HMs was conducted according to the Tessier method [2].
From a single sample, the following HM compounds were sequentially extracted: the
exchangeable HM compounds (with a 1 M MgCl2 solution with pH 7.0), the HM compounds
bound to carbonates (with a 1 M CH3COONa solution with pH 5.0), the HM compounds
bound to iron and manganese oxides (with a 0.04 M NH2OH HCl solution in 25%
CH3COOH), and the HM compounds bound to organic matter (with a 3.2 M CH3COONH4
solution in 20% HNO3 after oxidation of the organic substances with 30% H2O2 in the
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presence of 2 M HNO3 and heating to 85 ºC). From separate samples, HM compounds were
extracted with an ammonium acetate buffer (AAB) with pH 4.8 [3] and a mixture of AAB and
1% EDTA with pH 4.8 [4]. In both cases, the extraction was performed at a soil to solution
ratio of 1 : 5; the time of the extraction was 18 h. The content of the metals in all extracts was
determined by atomic absorption spectrophotometry.
We suppose that the content of metal in the 3.2 M CH3COONH4 extract after oxidation with
30% H2O2 in the presence of 2 M HNO3 and heating to 85oC characterizes the metal
compounds strongly bound to organic components of the soil. Hydrogen peroxide in an acid
environment is an active oxidant of organic substances in the soil. According to different
authors, this treatment oxidizes 80–95% of the organic substances in the soil [5, 6]. Isolation
of these forms was preceded by the displacement of the equilibrium-exchangeable metal ions
bound to carbonates and nonsilicate compounds of iron, aluminum, and manganese [2].
The AAB with pH 4.8 presumably solubilizes all the potentially exchangeable ions, among
which are those retained by organic substances as the main carriers of exchangeable positions
in the soil exchange complex [7]. Metal compounds extractable by an AAB + EDTA solution
are classified among the potentially mobile forms. Along with the exchangeable ions, these
compounds include the organomineral complexes of metals. Solov'ev [4] and McLaren and
Crawford [8] also believe that the treatment of soils with this mixed reagent predominantly
solubilizes the metals loosely bound to organic substances.
We calculated the contents of the following groups of compounds [9]:
(1) metal compounds strongly bound to organic and mineral soil components (from the
difference between the total metals in the soil and their potentially mobile compounds), and
(2) metal compounds bound in complexes with organic matter (from the difference
between the contents of metals in the AAB + EDTA and AAB extracts).
The relationships between the analyzed metal compounds are shown in Fig. 1.
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Figure 1: The scheme of metal compounds distribution bound to organic matter
3. Results and Discussion
Lead, copper, and zinc interacted with the soil organic matter when applied to an ordinary
chernozem in a pot experiment. Two years after the treatment, an appreciable part of the
metals applied was found in the organic substances, predominantly in a loosely bound state.
These organic substances were supposed to be organomineral complexes, the formation of
which resulted in the partial destruction of humic acid molecules. In the contaminated soils,
the organomineral compounds made up about half the total content of metals in the organic
matter; in the uncontaminated soils, their share was no more than 10% (Table 2).
Table 2. Contents of Pb, Cu, and Zn in the organic substances and the potentially mobile compounds
(mg/kg, above the line; % of the total content in the soil, under the line)
Experimental
Pb
Cu
Zn
treatment
1
2
1
2
1
2
Control
6.8/29
0.8/4
4.4/10
0.5/1
1.3/2
0.7/1
100 mg/kg
57.4/49
13.0/10
55.7/40
13.9/10
6.4/4
8.0/5
300 mg/kg
156.0/49
89.0/18
131.0/38
76/17
21.6/6
156/12
Note: (1) content of metal strongly bound to organic matter; (2) potentially mobile metal compounds
The series of metals arranged in accordance with their content in the potentially mobile
organomineral compounds is similar to that found for the metals strongly bound to organic
matter: Pb > Cu > Zn. humus substances. This series also corresponds to that of the metal ion
radii values (Pb > Cu > Zn), which suggests that the ion size of the metals affects their
retention by the organic substances.
It is notable that, in the contaminated soils, the content of zinc in the potentially mobile form
was even higher than that in the compounds strongly bound to organic matter. This could be
an artifact, in part, caused by the transition of metal ions retained by the amorphous Al and
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Mn compounds (if they were not completely removed at the pretreatment stage) into the AAB
+ EDTA extract, along with the organomineral zinc compounds. In fact, zinc predominantly
accumulated in the sesquioxide-bound fractions of the contaminated soils.
In the control treatment, the content of metals in the AAB + EDTA extract varied from 1% of
the total content for Cu and Zn to 4% for Pb. The increase in the content of potentially mobile
compounds in the contaminated soils depended not only on the nature of the metal and the
rate applied, but also on its application mode. At the separate application, the share of
potentially mobile Pb and Cu, which are more active complexing agents, increased from 3–
6% of the total content at a rate of 25 mg/kg to 17–18% at 300 mg/kg; the share of Zn
increased from 3–12%.
Acknowledgments
This work was supported in part by the Ministry of education and science of Russian
Federation (project nos. № 2.1.1/3819).
References
1. T.M. Minkina, G.V. Motuzova and O.G. Nazarenko, Interaction of heavy metals with the organic
matter of an ordinary chernozem, Eurasian Soil Sci., 2006, № 7, P. 702–710. [in Russian].
2. A. Tessier, P. G. C. Campbell and M. Bisson, Sequential Extraction Procedure for the Speciation
of Particulate Trace Metals, Anal. Chem., 1979, 51, 844–850.
3. N.K. Krupskii and A.M. Aleksandrova, Determination of Mobile Forms of Trace Elements, in
Trace Elements in the Life of Plants, Animals, and Humans, Kiev, 1964, pp. 34-36 [in Russian].
4. Laboratory Manual for Agricultural Chemistry, Ed. by V.G. Mineev, Mosk. Gos. Univ., Moscow,
1989, [in Russian].
5. N.G. Zyrin, G.V. Motuzova, V.D. Simonov and A.I. Obukhov, Trace Elements in Soils of
Western Georgia, in Forms of Trace Elements in Soils, Moscow, 1979, pp. 3–160 [in Russian].
6. J.A. Omuite, Sodium Hypochlorite Treatment for Organic Matter Destruction in Tropical Soils of
Nigeria, Soil. Soc. Am. J., 1983, 44, 878–888.
7. G.V. Motuzova and N.Yu. Barsova, Reserve of Mobile Metal Compounds in Carbonate-Free Soils
and Its Determination, in Proceedings of the II International Conference "Heavy Metals,
Radionuclides, and Biophilic Elements in the Environment", Semipalatinsk, Kazakhstan, 2002,
Vol. 1, pp. 173–178.
8. R.G. McLaren and D.W. Crawford, Studies on Soil Copper: 1. The fractionation of copper in
soils, J. Soil Sci., 1973, 4, p. 172.
9. D.L. Ladonin and S.E. Margolina, Interaction between Humic Acids and Heavy Metals,
Pochvovedenie, 1997, No. 7, 806–811 [Eur. Soil Sci. 30 (7), 710–715 (1997)].
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Concentrations of Iron in the Interactions of Some Acid Ones
Organic with Minerals
Cassia Fernanda Domingues Bassana, Ademércio Antonio Paccolab, Pedro de Magalhães
Padilhab
a
University of Marília, Department of Agrarian Sciences; bDepartment of Natural resources
UNESP-Botucatu; cDepartment of Chemistry UNESP-Botucatu. Brazil
E-mail: cfbassan@unimar.br
1. Introduction
The external and superficial portion of the terrestrial crust is formed by several types of rocky
bodies, subjects to conditions that alter its physical form and chemical composition, that is to
say, the disintegration and decomposition. In certain cases, organic acids formed by the
decomposition of the organic matter, aid in the chemical processes of meteriorization,
together with other chemical factors and with the alteration of original minerals in other
secondary ones due to the temperature variation, oxide-reduction reactions and chemical
substances secreted by micro-organism and unicellular alga.
The iron meets in the soil in appreciable amounts in the primary minerals, as in the
ferromagnesians and in the biotite; in the accessories as ilmenite (FeTiO3), magnetite (Fe3O4)
e pyrite (FeS2); in the secondary minerals, as in the hydrated oxides (limonite – Fe2O3.nH2O;
turgite – 2Fe2O3.H2O e goethite – Fe2O3.H2O). It also happens in having composed organic
(humate of Fe), in salts, as phosphate and in small amounts, as exchangeable ion and in the
solution. The Fe it is one of the most abundant elements of the terrestrial crust (about 5,1%),
being just overcome by the oxygen, for the siliceous and for the aluminum. In the soils,
however, it can suffer extreme variations. The iron is absorbed in the ferrous or ferric forms.
Deficiencies of iron have been associated the soils of high pH, being common in calcareous or
alkaline soils. "Results of the conversion of the Fe2+ to Fe3+ and subsequent precipitation as
Fe(OH)3 ". The solubility of the iron in the soil is, largely, controlled by the solubility of the
hydrated oxides of Fe3+. The Fe3+ inorganic in solution it varies with the pH and it reaches a
minimum between 6,5 and 8,0. The species hydrolytic constitutes most of the ions Fe3+ in
solution. Above pH 8,0 Fe(OH)4- it is the predominant ion. The activity of the ions of Fe3+ in
the solution of the soil is difficult of being calculated by virtue of the great amount of
colloidal iron. It is increased that the presence of the natural quelates in the soils. The activity
of the Fe3+ in solution decreases 1000 times for each increase of an unit of pH. Where the
drainage and the aeration are good the ferric compositions they prevail and where is
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15th IHSS Meeting- Vol. 3
inadequate they are formed more soluble ferrous compositions. In addition to the effects of
the oxygen on the state of oxidation of the Iron, it should be increased that certain bacterium
are capable to accomplish the transformation of the Fe2+ in Fe3+. Organic acids produced by
microorganisms and the CO2 liberated by the roosts of the plants, they can contribute to the
solution of the precipitate iron.
The present work studies the behavior of minerals of iron that composes part of the soils of
the humid tropical areas and its interaction with some organic acids.
2. Material and Methods
The used minerals were: hematite (Fe2O3), goethite (HFeO2) e magnetite (Fe3O4) that
commonly they happen in areas humid intertropical, being coming of the following Brazilian
areas: Itabira-MG, Botucatu-SP and Registration-SP, respectively.
Prepare of the samples: The minerals were triturated and sifted separately in mesh of
0,05mm. It leaves of the mineral samples they were used in I prepare it of sheets for
mineralogical determinations for diffratometryc of ray-X and other part, 1 g for sample, was
conditioned to react with organic acids, separately. For a collection of glass flasks with cover,
properly sterilized, 1g of mineral were transferred, 0,5g of inoculate * and 250 mL of solution
of organic acid 0,02 mol.L-1.
The experiment was driven under weekly soft agitations for 53 days.
Experimental determinations: It leaves of the samples of minerals they were examined with
relationship its composition for Diffractometryc of Ray-X (XRD), seeking the confirmation of
the studied mineral. It leaves of the solutions they suffered digestion with acid nitric and
peroxide of hydrogen after 53 days, and they were analyzed in ICP – Inductively Coupled
Plasma.
3. Results and Discussions
The largest extracted concentration of iron was of the oxide of iron goethite for the oxalic
acid, followed by the mineral magnetite for the citric acid and the hematite for the oxalic acid.
*inoculate: Solution microbiological inoculate with extract of soil of earth purple (TE) under forest vegetation, according to
NISHIGUCHI (1999), presenting a relationship respectively C:N:P:K 250:1:0,2:0,2, for the microorganisms development.
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Table 1: Concentrations (mg.Kg-1) of iron (Fe) presents in the extracts of organic acids and water,
determined by ICP
MINERALS
Hematite
Goethite
Magnetite
Acetic
2,15
17,90
31,60
ÁCIDS
e WATER
Butiric
Citric
Fenic
Latic
Malic
Oxalic
Propionic
1,48
80,53
5,18
56,00
65,73
2335,36
1,73
57,85
659,93
451,18
177,88
171,65
12076,88
29,73
28,20
4613,00
61,70
1017,20
1229,20
1798,28
116,33
-, below or very close of the limit of detection of the technique (0,004 mg.Kg-1)
Tanic
116,02
1487,52
2955,00
Water
23,18
101,78
11,10
Figure 1 – Concentrations of Fe (mg.Kg-1) extracted or solubilized for the organic acids and
water, determined by ICP
4. Conclusions
The results suggest that, in most of the systems, it happened the quelatization of the metallic
ions for the organic molecules, proposing the formation of a salt of the respective minerals;
the particularity of acid rights in the solubilization of the iron, if showing more efficient those
with more than a carboxyl a and that they present hydroxyls, that is to say, with larger amount
of hydrogen you ionized and larger carbonic chain.
References
1. CHAPMAN, P. M. et al. Evoluation of bioaccumulation factors in regulating metals.
Environmental Science & Technology News, v. 30, p. 448-452, 1996.
2. DANA, J. D. Manual de mineralogia. Rio de Janeiro: Ao Livro Técnico, 1969, p. 271.
3. EIRA, P. A.; CARVALHO, P. C. T. A decomposição da matéria orgânica pelos microrganismos
do solo e sua influência nas variações de pH. Revista de Agricultura, v. 45, p. 15-21, 1970.
4. HART, B. Trace metal complexing capacity of natural Waters: a review. Environmental
Technology Letters, v. 2, p. 95-110, 1981.
5. MALCOM, R. The uniqueness of humic substances in each of loll, stream and marine
environments. Analytical Chemical Acta, v. 232, p. 19-30, 1990.
6. NISHIGUCHI, I; PACCOLA, A. A. Estudo da degradação de resíduos de endosulfan em palha
de café (Coffea arábica L.. 1999. 39f. Dissertação (Mestrado em Agronomia) – Faculdade de
Ciências Agronômicas da Universidade Estadual Paulista de Botucatu, Botucatu, 1999.
7. ROCHA, J. C. et al. Relative liability of trace metals complexed in aquatic humic substances
using ion-exchanger cellulose-htphan. J. Brazilian Chem. Soc., v. 8, p. 239-243, 1997.
Vol. 3 Page - 173 -
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A Fluorescence Study of Adsorption Mechanisms of Flubendiamide onto
Humic Acids
Ivana Cavoskia, Valeria D’Oraziob*, Teodoro Mianob
a
CIHEAM-IAMB, Ist. Agron. Mediterr. di Bari,Via Ceglie 9, 70010, Valenzano, Bari, Italy;
b
Univ. of Bari, Dip. Biol. Chim. Agrof. Amb., Via G. Amendola 165/A, 70126, Bari, Italy
E-mail: dorazio@agr.uniba.it
1. Introduction
Flubendiamide, N0-[1,1-dimethyl-2-(methylsulfonyl)ethyl]-3-iodo-N-{4-[2,2,2 tetrafluoro-1(trifluoromethyl)ethyl]-0-tolyl} phthalimide is a powerful insecticide belonging to a new
chemical class (the phthalic acid diamides), widely used against lepidopteran pests on a large
variety of annual and perennial crops. Its residues and metabolite, the desiodo flubendiamide,
were determined in a number of crops. Flubendiamide is almost insoluble in water, and since
soils exhibit a marked affinity for hydrophobic organic compounds, they exert an essential
role in controlling the environmental fate of these chemicals. In addition, soil sorption of most
hydrophobic organic compounds is directly related to soil organic matter (SOM) content and
especially to its humic fractions (HA). In facts, they show a large reactivity towards these
compounds, mainly as a function of their functional groups as well as their molecular and
structural arrangements [1, 2]. In this study, fluorescence spectroscopy was utilized to
investigate on the physico-chemical mechanisms involved in flubendiamide adsorption onto
HAs of different origin.
2. Materials and Methods
Flubendiamide (FLU) (CAS N°: 272451-65-7), purity 98%, was purchased from Sigma
Aldrich (Steinheim, Germany) and used to prepare the initial working solution (1 mg/mL in
ACN) was prepared. The two humic acids used in this study, were isolated from a) a graybrown podzolic soil (Normandy, France) according to
the International Humic Substances Society (IHSS)
procedures (HA S); and purchased from b) the IHSS,
(lot 1S101H), Suwannee River (HA SR). A stock
solution of 5 mg HA in 50 mL 0.05M NaOH at pH 8
was prepared for both HAs; 4 mL of each HA solution
Figure 1: Flubendiamide structure
were added with 0.05, 0.1 and 0.2 mL of FLU solution,
respectively FLU1, FLU2 and FLU3, were mechanically shaken for 24 hours at room T. A
blank solution for each HA was prepared as control at the same conditions. Both controls and
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15th IHSS Meeting- Vol. 3
interaction solutions were directly analyzed by fluorescence spectroscopy, using a PerkinElmer (Norwalk, CT) LS 55 luminescence spectrophotometer equipped with the WinLab
4.00.02 software for data processing. Total luminescence spectra, in the form of excitationemission matrices (EEMs, contour maps), were recorded over the emission wavelength range
from 300 to 600 nm, increasing sequentially by 5 mm steps the excitation wavelength from
250 to 500 nm. The EEM plots were generated as contour maps from spectral data by using
Surfer 8.0 software (Golden Software, Inc., 2002, Golden, CO).
3. Results and Discussion
Data reported on Table 1 indicate significant differences between the two HA. On the whole,
the higher O content measured for the HA SR is mainly due to the occurrence of acidic
functional groups, whereas in the HA S the
Table 1: Elemental composition (g kg−1),
atomic ratios, acidic functional group
content (cmol kg−1) and E4/E6 ratios of the
examined HAs
oxygen-containing groups seem to be related to
non acidic groups, such as hydroxyl, methoxyl,
ethers, ketones, and quinones. Further, HA S
sample appears to be characterized by a greater
aromaticity degree and lower polarity with
respect to the first one, with higher H/O and
lower (O+N) /C ratio. In general, chemical data
suggest a more aromatic character for the HA S
and a mainly aliphatic and acidic character for the
HA SR, characteristics confirmed by E4/E6
values. Such properties are expected to result in a
well different chemical reactivity and residual
adsorbing capacity of these HAs toward the nonpolar flubendiamide molecule. Figures 2 and 3
show the contour maps (EEMs) of HA S and HA
SR, respectively, and their interaction products with flubendiamide at different
concentrations. The EEMs spectra of both HAs show two common peaks, 1 and 2, identified
by the excitation/emission wavelenght pairs (EEWPs) 430-440ex/510-522em and 390400ex/476-500em, respectively, and characterized by greater relative fluorescence intensity
(RFI) values in the HA S sample with respect to HA SR one.
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15th IHSS Meeting- Vol. 3
The
excitation
wavelengths
of
the
fluorophore 1 are in the range of those
generally
ascribed
to
extensively
conjugated quinones and phenols with an
elevated
polycondensation
degree,
whereas those of the peak 2 can be
correlated to components like esculetin
and/or scopoletin structures [3]. The
slightly higher values of the HA S EEWP
can be likely related to a greater
condensation degree of these aromatic
Figure 2: EEMs of HA S and its interaction products
groups. Finally, a third additional peak (3)
appears in the HA SR sample, centered in
the shorter wavelength region (EEWP
345-355ex/450-464em)
and
generally
ascribed to flavones and isoflavones like
structures [3].
The changes occurring in the EEMs
spectra of the HA-FLU products suggest,
for both HAs, the involvement of all
fluorophores in the interaction mechanism
at various extent, as a function of FLU
Figure 3: EEMs of HA SR and its interaction products
concentration
and
HA
sample.
In
particular, the peak 1 seems not involved in the interaction with the lowest FLU
concentration, especially in the HA S sample, whereas the RFI value decrease and the EEWPs
shift indicate its involvement with FLU both at intermediate and at highest concentration. On
the contrary, the peak 2 appears involved in the interaction regardless of FLU concentration in
the HA S, and only marginally in the HA SR sample. Finally, the peak 3 (HA SR) strongly
appears to interact with FLU for each concentration tested, as remarked by the RFI and
EEWPs values changes. In order to better evaluate the changes occurring in the EEMs spectra
of interaction products, the EEMs spectra of FLU molecule dissolved in three solvents with
different polarity indexes were recorded, and the RFI values changes were evaluated as a
function of the medium (Fig. 4). As shown in Fig. 4, the RFI value of both FLU peaks
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15th IHSS Meeting- Vol. 3
increases markedly with decreasing medium polarity, and this effect is due to the presence of
CF3 substituent, that lead to an efficient quenching
Figure 4: EEMs of FLU in acetonitrile (ACN), methanol (MeOH) and ethyl acetate (Ethyl Acet)
because of the turning of the aromatic amide moiety of FLU (Fig. 1); this effect results more
evident in the HA SR sample because of the polar properties of this latter. Additionally, the
greatest RFI value measured in the sample HA SR FLU 3 can be likely ascribed to decreased
rotation of the macromolecular aggregate occurring at the highest FLU concentration. In the
case of HA S FLU no significant changes were observed in the RFI values. The FLU
substituents were not effective, FLU molecule may result entrapped inside the HA.
4. Conclusions
On the whole, fluorescence data seem to suggest that flubendiamide adsorption can occur
onto HA as a function both of the aromatic degree by means of hydrophobic bonds, as it
happened for HA S, and of acidic functional groups availability by means of hydrogen bonds,
as it happened for HA SR. Further studies may help in revealing further insights on the
suggested mechanisms.
References
1. R.P. Schwarzenbach, P.M. Gschwend, D.M. Imboden, Environmental Organic Chemistry, Wiley,
New York, 1993.
2. R.R. Engebretson, T. Amos and R.V. Wandruszka, Environ. Sci. Technol., 30(1996) 990.
3. O.S. Wolfbeis, in S.G. Schulman SG (Ed.), Molecular Luminescence Spectroscopy, Part I, Wiley,
New York, 1985, p 167.
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Size Exclusion Characterization of Dissolved Organo-mineral Complexes in
Soils of the Southern Far East
Tatiana N. Lutsenkoa*, Alexandra S. Volkb
a
Pacific Institute of Geography, Far Eastern Branch of the Russian Academy of Sciences,
Radio St. 7, 690041 Vladivostok, Russia; bInstitute of Chemistry, Far Eastern Branch of the
Russian Academy of Sciences, 100-letiya Vladivostoka Av. 159, 690022 Vladivostok, Russia
E-mail: luts@tig.dvo.ru
1. Introduction
The illuviation processes of the dissolved organo-mineral species are quite expressed in soils
of the Southern Far East. In our research we aim to study DOC relationship with Fe and Al in
mountain-taiga brown soils of the Sikhote-Aline applying a method of size exclusion
chromatography (SEC). On the basis of these efforts we expected to determine differences in
binding properties of DOC exposed to different ecological conditions of synthesis.
2. Materials and Methods
The study area is one of the west slopes of the Sikhote-Aline mountain ridge (South of the
Russian Far East). Lysimetric waters were collected in zero-tension lysimeters (for the period
from July to September) below organic and mineral horizons of the main types of mountaintaiga brown soils: humic Cambisol (750 and 950 m a.s.l.); and dystric Cambisol (1250 and
1400 m a.s.l.). The lysimetric waters were filtered through 0,23 μm filters.
Table 1: Characterization of the lyzimetric waters of mountain-taiga brown soils [1]
Soil, elevation
Humic Сambisol, 750 m
Humic Сambisol, 950 m
Dystric Сambisol, 1250 m
Dystric Сambisol, 1450 m
Horizon
рН
DOC, mM
Fe, mM
Al, mM
А1
5.63
3.45
0.0091
0.0126
B2hf
5.25
1.47
0.0034
0.0119
А1
4.71
4.10
0.0127
0.0167
В1hf
4.70
2.65
0.0118
0.0210
А2
4.19
4.86
0.0127
0.0141
В4hf
5.16
1.28
0.0118
0.0211
А1А2
4.50
1.92
0.0095
0.0200
BhfC
5.17
1.06
0.0039
0.0222
The filtrates were concentrated by freezing and rotary evaporation by a factor of 5-10.
Fractionation of DOС and study of its binding with Fe and Al were performed on Sephadex
G-25. A set of standard organic substances was taken for column calibration. Absorbance of
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DOС fractions was measured at 665 and 465 nm to determine E4/E6 ratios. DOC
concentrations were determined by the dichromate technique followed by colorimetry. The
content of Fe was analyzed by AAS-method. Aluminum was determined by a
spectrophotometry with the organic reagent anthrazochrom after decomposition of organic
matter. Potentiometric titrations of DOC fractions were performed in a PC-controlled system
under constant argon flow. DOC fractions, accumulated in 7 runs of SEC, after rotary
evaporation were dissolved in 10 ml of 0.1M KCl adjusted to pH=10 by addition of 0,1M
KOH for complete solubilization. Prior to titration the solution was readjusted to pH=2,5 with
0,1M HCl to attain complete protonation of functional groups. Results of potentiometric
titration were processed using the density function method which yields pK-spectra of DOC.
3. Results and Discussion
During fractionation on the Sephadex G-25, the DOC of soil waters is separated into three
major molecular weight fractions. The first fraction has a high molecular weight (HMW) and
is characterized by a limit of MM > 5000 Da. MW for the middle molecular weight (MMW)
Conductivity
fraction spans 650-1500 Dа; the low-molecular weight (LMW) fraction spans 200-400 Dа.
Ratio D4/D6 testifies that the HMW fraction
200
C, μS/cm
contains more aromatic molecules (D4/D6
100
5,2-6,2), but MMW fraction (D4/D6 6,3-
DOC, mM
0
10
8
6
4
2
0
100
200
250
DOC
are more aliphatic. Investigation of the
of
dissociation
and
the
concentrations of functional groups has also
100
150
200
250
shown that the fractions of DOC differ in
the chemical nature. Potentiometric titration
Fe
0,04
data indicated that the content of the most
0
100
0,2
Al, mM
10,5) and LMW fraction (D4/D6 10,3-15,0)
degree
0,08
Fe, mM
150
200
150
250
fraction is almost three times higher than in
Al
0,1
acidic groups (pK 2-4) in the HMM
the MMW fraction. For the LMM fraction,
0
100
150
200
250
Elution volume, ml
Figure1: SEC of the lyzimetric waters
the concentration of acidic groups is lower
than the detection limit of the method
because of insufficient amount of the
fraction organic matter.
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15th IHSS Meeting- Vol. 3
SEC of the natural water samples is complicated by the fact that the peaks of MMW and
LMW fractions overlap the elution peaks of dissolved salts, which leads to some uncertainty
in interpretation. However, we can estimate the complexation ability of the HMW fraction of
DOC since its elution peak does not coincide with the elution of dissolved salts.
A portion of DOC, Fe and Al is sorbed by Sephadex. We take this fourth fraction into account
as labile compounds possessing affinity for gel. A share of fraction 4 (Figure 2) is calculated
by difference between introduced and eluted amounts of DOC, Fe and Al.
There is a tendency of decreasing of the amount of HMM fraction in the distribution of DOC
organic horizons with the increase of elevation. This tendency can be also tracked in the
distribution of dissolved Fe and Al in the organo-mineral forms of its migration. In the
warmest conditions (elevation 750 m), DOC consisting of more than 65 % of HMW
substances migrates. More than 98 % of dissolved Fe and 68 % of Al are associated with this
fraction. In the colder conditions (elevation 950 m), the amount of HMW fraction goes down
to 60 %, the quantity of Fe and Al presented by this form simultaneously reaches to 71,3 %
and 72 %, respectively. The smallest amount of the HMW fraction (53,8 %) is characteristic
for the most acidic solutions (pH 4,19) of the soil at elevation of 1250 m. The solution pH
also limits the binding of the metals: about 60 % of Fe and 40 % of Al are associated in HMW
complexes. In connection with the pH of waters and, correspondingly, reduction in the
stability of DOC and Fe complexes, a share of labile fraction (4) of the latter exceeds 25 %.
Humic cambisol (750 m)
Humic cambisol (950 m)
Dystric cambisol (1250 m) Dystric cambisol (1400 m)
100
A1
A1
80
A1
A1A2
DOC
Fe
Al
60
DOC, Fe, Al, %
40
20
0
80
1
2
60
3
4
1
2
B2hf
3
4
1
2
3
4
1
2
B4hf
B1hf
3
4
Bhfc
40
20
0
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
Fractions
Figure 2: Distribution of DOC, Fe, Al (% of total) in SEC fractions of soil waters: 1–HMW fraction,
2–MMW fraction, 3–LMW, 4-fraction of DOC and metals absorbed by Sephadex
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15th IHSS Meeting- Vol. 3
From studying elevation dynamics of Al speciation, it is possible to see that it, to a lesser
degree, reflects dynamics of the speciation of DOC than does dissolved Fe, although, on the
whole, the tendency is similar. Aluminium forms weaker complexes with DOC than Fe, thus
the dependence of Al behaviour on changes in DOС speciation is not so obvious.
A stoichiometric ratio Мe:С for the HMW complexes is 3-4 atoms of Fe and 6-10 atoms of Al
per a chain of thousand carbon atoms. In the course of migration in the soil, the fractions of
DOC are saturated by Fe and Al and coagulate as well as are sorbed at the surface of minerals.
In the organo-mineral complexes of the lower horizons, absolute shares of metals increase up
to 4-9 atoms of Fe and 13-45 atoms of Al per a chain of thousand carbon atoms, respectively.
In the mineral horizons the multiformity of DOC, Fe and Al speciation is more expressed.
The capacity of DOC to bind Fe and Al becomes less pronounced, their migration is
supported by fractions of DOС with МW 200-400 Dа as well as fraction of labile, maybe,
hydrolytic forms.
4. Conclusions
From the results of lysimetric waters SEC in the present study, DOC was separated into three
major fractions. Differences between the DOC production and transformation conditions are
expressed in different ratios of its fractions and binding Fe and Al. As shown by us earlier, a
share of humic acids reduces and of fulvic acids grows in composition of DOC from the lower
to upper parts of the slope. In the elevation series of soils, an acidity of produced acids
increases which results in increasing the acidity of solutions. As a result of action of these
factors, basic identified elevation tendency is a reduction in formation of the high-molecularweight fractions and intensity of precipitation processes of organo-mineral complexes in the
soil profile and intensification of processes of their illuviation as the low-molecular weight
and labile forms.
Acknowledgements
The author wish to thank Dr. V.S. Arzhanova, senior researcher of Pacific Institute of
Geography of FEB RAS for the samples of lysimetric waters and discussion of the data
obtained. The author thanks Dr. S. Yu. Bratskaya, senior researcher of Institute of Chemistry
of FEB RAS for the potentiometric titrations of the concentrated SEC fractions of DOC.
References
1. V.S. Arzhanova and P.V. Yelpatievsky, Mountain geosystems: geochemistry, functioning and
dynamics (Sikhote-Aline Mountains, Southern Russian Far East), Dalnauka, Vladivostok, 2005,
p.68-69 (in Russian).
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Sorption of Pharmaceuticals to Humic Substances
Hisayo Mori, Tomoya Ohtani, Itsuko Fukuda, Hitoshi Ashida, Nobuhide Fujitake
Graduate School of Agricultural Science, Kobe Univ., Rokkodai 1, Kobe 657-8501, Japan
E-mail: fujitake@kobe-u.ac.jp
1. Introduction
Pharmaceuticals and personal care products (PPCPs) in the environment have received much
attention during the last decade. Approximately 100 compounds have been detected in
wastewaters, streams, ground-waters, and drinking waters in several countries (e.g.[1]). These
substances enter the aquatic environment via the effluent of sewage-treatment plants as a
result of their incomplete removal. Not only in aquatic environment, but some medicines from
the application of sludge or manure were persistent in agricultural soils [2] and they were
absorbed by vegetables [3]. Therefore, these compounds may act as allergens for human
beings, if not actually below the acute toxicity level. In addition, it is concerned that PPCPs,
especially antibiotics, could induce drug-resistant bacteria in aquatic or soil ecosystems.
In this study, we focus the interactions between pharmaceuticals and humic substances.
Humic substances are ubiquitous in virtually all terrestrial, and coexist with pharmaceuticals
in environments. Little is known, however, of the behavior of these compounds in the
presence of humic substances. There is a possibility that the bioactivity of pharmaceuticals is
changed if they interact with humic substances. So we examined the sorption experiments
with 4 pharmaceuticals and 10 humic substances isolated from different environmental
conditions.
2. Materials and Methods
atenolol
ciprofloxacin
ibuprofen
fluoxetine
Figure 1: Structural formulae of 4 pharmaceuticals
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The pharmaceuticals, such as an antihypertensive (atenolol), a nonsteroid anti-inflammatory
drug (ibuprofen), an antidepressant (fluoxetine hydrochloride), and an antibiotic
(ciprofloxacin) were selected. Humic materials were purchased or isolated from freshwater,
sediment, peat, and soils using the quasi-IHSS methods, and 1 aquatic fulvic acid, 7 soil
humic acids, and 2 soil fulvic acids were obtained. All humic samples were characterized by
liquid-state 13C NMR spectroscopy.
The organic carbon normalized sorption coefficient (Koc) was determined according to the
fluorescence quenching method [4]. The experimental aqueous phase was prepared with fixed
pharmaceutical concentrations and varied concentrations of humic substances (1–5 mg C L-1)
in phosphate buffer, pH 7.2, and 0.01 ionic strength. The final concentrations of
pharmaceuticals used were as follows: atenolol, 500 μg L-1; ibuprofen, 700 μg L-1; fluoxetine,
1500 μg L-1; ciprofloxacin, 150 μg L-1. The solutions were shaken for 24 hours at 25 ºC in the
dark and the fluorescence intensity was measured by Jasco FP-6200 spectrofluorometer.
The excitation / emission wavelengths used were 230/302 nm for atenolol, 230/293 nm for
ibuprofen, 230/294 nm for fluoxetine, and 270/410 nm for ciprofloxacin with slit widths of 5
nm. The correction factor for inner filter effects and the concentration quenching of humic
materials were calculated with absorbance values of excitation and emission wavelengths for
each solutions [4]. The corrected fluorescence intensity of pharmaceuticals in the presence of
humic substances (F) and absence (F0) was used in the Stern-Volmer equation, which
described the decrease in fluorescence in the presence of quencher (humic substances). Values
of F0/F formed linear plots against concentration of humic substances, with the Koc values
Fo/F
calculated from the slopes.
Stern-Volmer equation: F0/F = 1 + K [Humic substance]
3. Results and Discussion
The Stern-Volmer plots for fluorescence
quenching of atenolol with 10 humic substances are
Concentration of humic substances (mgC L-1)
given in Fig. 2. It shows linear relationships for
any combinations, and similar results were
obtained on all the other target pharmaceuticals
Figure 2: Stern-Volmr plot for
quenching atenolol with 10
humic substances
(not all shown in Figure). The differences of slopes between humic materials indicate that
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15th IHSS Meeting- Vol. 3
they have different binding affinities to pharmaceuticals respectively.
Table 1: Log Koc between 10 humic substances and 4 pharmaceuticals
LogKoc
Soil humic acids
Soil fulvic acids
Aquatic fulvic acid
Suwannee NOM a
a
atenolol
ibuprofen
Reference [5]
fluoxetine ciprofloxacin
Figure 3: Log Koc between 10 humic substances and 4 pharmaceuticals
The LogKoc values between humic materials and pharmaceuticals shows in Table 1. They
ranged widely from 4.42 to 6.82 for ciprofloxacin, while ranging from 4.8 to 5.86 for the
other three drugs. These results suggest the different ranges of values should depend on the
chemical properties of pharmaceuticals used. Then, the most of values were plotted in the
range from 5 to 6 (Fig. 3) and comparable binding affinities to PAHs (LogKoc＝3.7- 5.4) (e.g.
Vol. 3 Page - 184 -
15th IHSS Meeting- Vol. 3
[6]), whereas some exceeded these values. In addition, our data were higher than the values of
Suwannee river NOM with atenolol, ibuprofen, and fluoxetine (shown in Fig. 3 as the
asterisk) [5].
All humic samples were characterized by liquid-state 13C NMR spectroscopy (Fig. 4), and the
correlation coefficients between LogKoc and the distribution of carbon species in humic
substances were calculated. The result indicated the LogKoc values correlated with the Aryl,
O-Aryl, and alkyl carbons (e.g. Aryl carbon: r2 = 0.86, O-Aryl carbon: r2 =0.78, and alkyl
carbon: r2 = -0.81, for ciprofloxacine).
Aquatic fulvic acid
Soil fulvic acids
Soil humic acids
(%)
Figure 4: The distribution percentage of carbon species by 13C NMR spectroscopy
4. Conclusions
Ten humic substances were interacted with 4 pharmaceuticals such as atenolol, ibuprofen,
fluoxetine, and ciprofloxacin. The LogKoc values between humic materials and
pharmaceuticals were a comparable level of interactions with PAHs, whereas some
combinations led to exceed these values. The result indicated the Koc values correlated with
the Aryl, O-Aryl, and alkyl carbon moieties of humic substances.
References
1. B. Halling-Sorensen, S.N. Nielsen, P.F. Lanzky, F. Ingerslev, H.C. Holten Lutzhoft, and S.E.
Jorgensen, Chemosphere., 36(1998) 357.
2. M. Rabolle and N.H. Spliid, Chemosphere, 40 (2000) 715.
3. K. Kumar, S.C. Gupta, S.K. Baidoo, Y. Chamder, and C.J. Rosen, J. Environ. Qual., 34 (2005)
2082.
4. T.D. Gauthier, E.C. Shane, W.F. Guerin, W.R. Seitz, and C.L. Grant, Environ. Sci. Technol., 20
(1986) 1162.
5. H. Yamamoto, A. Hayashi, Y. Nakamura, and J. Sekizawa, Environ. Sci., 12 (2005) 347.
6. I.V. Perminova, N.Y. Grechishcheva, and V.S. Petrosyan, Environ. Sci. Technol., 33 (1999) 3781.
Vol. 3 Page - 185 -
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Influence of Aromaticity Degree on the Aggregation of Humic Substances
Martin Drastíka, Jiří Kučeríka, Oldřich Zmeškala, Anna Čtvrtníčkováa, František Novákb
a
Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00, Brno, Czech
Republic; bBiology Centre AS CR, v. v. i., Institute of Soil Biology, Na Sádkách 7, 370 05
České Budějovice, Czech Republic
E-mail: xcdrastik@fch.vutbr.cz; kucerik@fch.vutbr.cz
1. Introduction
Humic substances are for a long period classified according to the isolation procedure (which
reflects also the solubility in water according to pH) into three groups – humic acids (HA),
fulvic acids (FA) and humin. Distinguishing between HA and FA is basically just formal
because as shown several times, there is no strict line which separates these two groups. As
published by Kučerík et al. [1] even the aggregation patterns are very similar and can be
described by the same mathematical apparatus.
Aggregation of HAs in solutions was studied intensively by various techniques such as smallangle neutron scattering, small-angle X-ray scattering, turbidimetry or scanning electron
microscopy and consequently utilizing fractal analysis [2, 3]. However, those techniques have
undisputable limitations which are associated mainly with the limited concentration range and
inconvenient possibility of changing the concentration or composition of measured solution
during running experiment. Recently, it has been demonstrated that this problem can be
overcame by the coupling of ultrasonic spectroscopy and consequent fractal analysis [1]. The
purpose of this work is to extend the recent results which suggested the correlation between
aggregation behavior represented by fractal dimension of sodium humates and fulvates and
their chemical composition.
2. Materials and Methods
Humic and fulvic acids were isolated from individual soil horizons of long-term research stands
by standard procedure and recommendations published in [4] were taken into account. Freezedried samples were converted to Na+ salts by titration to pH 7.2 using 0.1 M NaOH.
To monitor ultrasonic velocity High Resolution Ultrasonic Spectroscopy HRUS 102 device
(Ultrasonic-Scientific, Dublin, Ireland) was employed. All measurements were carried out the
same way as published in [1, 5 ,6]. The ultrasonic velocity (U) was measured in the
concentration range from 0.001 to 3.5 g/L. For easier observation of potential interactions, the
concentration increment of ultrasonic velocity (I) was determined using the relation published
in [5], i.e. I=(U–U0)/(U0mρ0).
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15th IHSS Meeting- Vol. 3
3. Results and Discussion
Measured data were processed as published elsewhere [1] utilizing non-linear fitting.
Obtained parameters will be correlated with primary characteristics (elemental composition,
aromaticity, carboxyl carbon) and treated by fractal dimension analysis developed by Drastík
et al. [6]. Figure1 represents the dependence of increment of ultrasonic velocity I (non-linear
fitting included) and fractal dimension D on concentration. It was already demonstrated that
fractal dimension D can reveal the differences in the way of aggregates formation for various
samples. The validity of observations done in references [1] and [6], where samples of IHSS
were analyzed, will be examined.
Figure 1: Dependence of fractal dimension D and increment of ultrasonic velocity I on concentration
Acknowledgements
The financial support of Ministry of Education of the Czech Republic, project MSM
0021630501.
References
1. J. Kučerík, M. Drastík, O. Zmeškal, A. Čtvrtníčková, WSEAS Transactions on Environment and
Development, 5 (2009) 705.
2. J. Rice, J.S. Lin, Environ. Sci. Technol. 27 (1993) 413.
3. N. Senesi, F.R. Rizzi, P. Dellino, P. Acquafredda, Colloid Surface A, 127 (1997) 57.
4. R.L. Malcolm in B. Allard, H. Borén and A. Grimvall (Ed.), Humic substances in the aquatic and
terrestrial environment, Springer Berlin/Heidelberg, 1991, Chapter 16.
5. J. Kučerík , D. Šmejkalová, H. Čechovská, M. Pekař, Org. Geochem., 38 (2007) 2098.
6. M. Drastík, A. Čtvrtníčková, O. Zmeškal, J. Kučerík, Energy and Environmental Engineering
Series, WSEAS Press, 2009, 163–168.
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Aggregation of Humic Acids in Solution. Vapor Pressure Osmometry,
Conductivity and Mass Spectrometric Study
E. M. Peña-Méndeza*, D. Fetschb, J. Havelb, c
a
Department of Analytical Chemistry, Nutrition and Food Chemistry, Faculty of Chemistry,
University of La Laguna, Campus de Anchieta, 38071 – La Laguna, Tenerife, Spain;
b
Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 61137
Brno, Czech Republic; cDepartment of Physical Electronics, Faculty of Science, Masaryk
University, Kotlarska 2, 61137 Brno, Czech Republic
E-mail: empena@ull.es
1. Introduction
During last two decades there was a considerable change in view of the structure and
molecular weight of humic acids (HA). HA were found to be supramolecular
associations/aggregates of relatively low molecular weight components [1,2]. The knowledge
of aggregation is quite fundamental because its important role in the processes taking place in
the environment.
The aim of this work was to evaluate the possibilities to apply non invasive methods such as
vapour
pressure
osmometry
(VPO),
conductivity
and
matrix
assisted
laser
desorption/ionization time of flight mass spectrometry (MALDI) for studying the aggregation
of HA in solution and to elucidate the process.
2. Materials and Methods
The Vapor Pressure Osmometer from Knauer (Berlin, Germany) was used to measure vapor
pressure of solutions. Conductometry experiments were performed on the conductometer OK104 of Radelkis (Budapest, Hungary). Mass spectra were measured via Laser Desorption
Ionization (LDI), i.e. without the use of any matrix. Time of Flight (TOF) MS measurements
were carried out on the AXIMA CFR from Kratos Analytical (Manchester, UK) mass
spectrometer equipped with the nitrogen laser (wavelength 337 nm) and
spectra were
measured either in the linear or positive ion modes.
3. Results and Discussion
Vapour pressure osmometry measurements of HA aqueous solution prove aggregation of HA
when increasing their concentration (Fig. 1); after the diluting of the solutions the aggregation
was shown as reversible. It is also evident from Fig. 1 that several aggregates are formed.
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15th IHSS Meeting- Vol. 3
Aggregation, due to hydrophobic interactions of alkyl groups, π–π interactions, hydrogen
bonds, and supramolecular interactions, etc. explains for decades suggested high molecular
VPO Re
Ressistance Difference
weights of HA.
Fig. 1 Vapour pressure osmometry
measurements of HA aqueous solution
as a function of HA concentration.
Humic acid
aggregation was also
studied by MALDI TOF MS. The
aggregates, due to weak interactions
HA (mgl-1)
through
the
different
functional
groups present in their structure (hydrophobic and/or π–π, etc.), are broken down by the
action of the laser. The mass spectra are complex, showing many components with low m/z
values in the range ~200–1000 Da while mostly no peaks are observed for m/z values greater
than 1000 Da.
4. Conclusions
The results obtained by VPO, conductivity and confirmed by MALDI TOF MS show that
studied HA consist of a mixture of numerous number of compounds with much lower
molecular weight than still often suggested in the literature. Also the direct soil analysis by
MALDI yield almost the same mass spectra like those for isolated HAs.
Acknowledgements
This work was supported by the Grant Agency of the Czech Republic (project KAN
101630651 and the Ministry of Education, Youth and Sports of the Czech Republic (projects
MSM0021622411 and LC06035). E.M. Peña Méndez thanks for the partial support from the
University of La Laguna.
References
1. J. Havel, D. Fetsch, E.M. Peña-Méndez, P. Lubal, P. and J. Havliš. In Understanding and
Managing Organic Matter in Soils, Sediments and Waters (Swift R.S. and Spark K.M., Eds.).
IHSS, Australia. 1991.
2. E.B. Kujawinski, M.A. Freitas, X. Zang, P.G. Hatcher, K.B. Green-Church, and R.B. Jones. Org.
Chem. 33 (2002) 171.
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Sorption of Silanol-Modified Humic Acids onto Different Solid Supports
Including Silica Gel, Clay and Sand
Ivan V. Dubinenkova, Alexander B. Volikova, Vladimir A. Kholodovb, Evgeny M. Garanina,
Sergey A. Ponomarenkoc, Irina V. Perminovaa*
a
Department of Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russia;
b
Dokuchaev Soil Institute, Pyzhevskiy per. 1, 119017 Moscow, Russia
c
Institute of Synthetic Polymeric Materials of the Russian Academy of Sciences, 117393
Profsoyuznaya street 70, Moscow, Russia
E-mail: iperm@org.chem.msu.ru
1. Introduction
Restoration of the polluted and degraded soil belongs to crucial environmental problems of
the 21st century. It is well-known that humic substances (HS) play a key role in soil fertility
by forming stable microaggregates which, retain water and structurize soil [1]. In addition, HS
regulate geochemical fluxes of metals due to high chelating ability and play protective role in
the polluted environments by binding heavy metals and organic pollutants into nonbioavaileble complexes.
In our previous research, we have demonstrated that the directed modification of HS is a
powerful tool for manufacturing humic materials with controlled properties [2]. In this study
we demonstrate that incorporation of silanol groups into the structure of HS allows for
production of the derivatives that show considerable promise as soil conditioners. This is due
to intense formation of stable soil aggregates stimulated by an increased content of soil
organic matter. The soil conditioners of the art will bring about formation of the stable
organic coating on the soil mineral surfaces.
The goal of this work was to estimate sorption of silanol-modified HS derivatives on the
different inorganic supports including silica gel and soil minerals – quartz sand and bentonite
and kaolinite clay.
2. Materials and Methods
Leonardite humic acids (CHP) isolated from the commercial potassium humate (Powhumus,
Humintech Ltd., Germany) were used for all modifications. 3-amino-propyltriethoxy-silane
(APTES) was used for treatment of CHP. The choice of APTES was provided by the presence
of reactive amino groups in its structure which can yield amide bonds upon reaction with
carboxyl and carbonyl groups [3, 4]. In addition, APTES is commercially available
organosilane suitable for preparative production of the corresponding derivatives.
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15th IHSS Meeting- Vol. 3
Silanol derivatives of CHP were obtained by its condensation with APTES. The reaction was
run at three different APTES-to-humics ratios, nominally: 0.2, 0.5, and 1.0 g of APTES per g
of CHP. These ratios corresponded to different extents of modification associated with
carboxyl groups available within the humic backbone. The corresponding amounts of APTES
were calculated in accordance with 1:1 stoichiometry of modification reaction on the basis of
the amount of COOH groups present in the CHP sample (3.8 mmol per g). Depending on
modification degree, the corresponding samples were designated CHP-APTES-20, CHPAPTES-50, and CHP-APTES-100. One sample was synthesized with tetraethoxysilane (TES)
substituting 10% of APTES out of the amount needed for 100% modification. TES was added
for increasing the number of silanol groups in the sample. The corresponding sample was
designated CHP-APTES-TES. The reaction was carried out at 130-140 °C for 5 hours.
Sorption of the modified HA was carried out on silica gel, quartz sand, bentonite and kaolin
clays. Quartz sand and silica gel were used as obtained from the manufacturer. Clays were
thoroughly soaked in 0.001 M CaCl2 prior to use in the experiments for saturation with Ca2+.
A weight of sorbent accounted 50 mg, 100 mg and 500 mg for silica, clays and sand,
respectively. The total volume of experimental solution was 10 mL, concentration of humic
samples was set in the range from 0.1 to 4 g/L. All sorption experiments were conducted in
phosphate buffer (0,03 M, pH 6.0). Equilibrium time was 24 hours.
3. Results and Discussion
To achieve maximum sorption affinity of the silanol modified humic derivatives for the
different Si-containing supports carrying hydroxyl groups, the sorption experiments were run
at pH 6. This pH was shown to provide for the maximum binding of silanol-HS to silica gel.
This finding corroborates well the reported data on the optimum conditions for formation of
siloxane bonds between silanol groups [5].
Figure 1 shows sorption isotherms for the silanol derivatives samples with different
modification degree on silica gel. As it was expected, the maximum sorption was observed for
the sample with maximum modification degree (CHP-APTS-100), which displayed the
maximum affinity for silica gel surface.
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15th IHSS Meeting- Vol. 3
q, mg/g
350
300
250
200
150
100
50
0
0
0,5
1
1,5
2
2,5
3
3,5
Ceq, g/l
.
Figure 1: Sorption isotherms of the silanol derivatives of leonardite HA onto silica gel.
▲ - CHP-APTES-100; ● - CHP-APTES-TES; ■ - CHP-APTES-50; ♦ - CHP-APTES-20
The CHP-APTES-100 isotherm fits to Lengmuir isotherm model. The sample showed high
affinity and sorption activity within a wide range of concentrations. At the same time, the
other samples tested: CHP-APTES-50, CHP-APTES-20 and CHP-APTES-TES displayed Sshaped isotherms. This might indicate differences in filling in the silica surface with the
humic derivatives. It should be noted, however, that 100% modified sample showed a rather
low water solubility. The optimal ratio between sorption affinity and solubility was observed
for 50%-modified sample – CHP-APTES-50.
Given these properties of CHP-APTES-50, it was selected for conducting comparative
sorption studies on different solid matrices. Figure 2 shows sorption isotherms for CHPAPTES-50 onto different sorbents. It can be seen that it displays the maximum sorption
affinity for silica gel and activated bentonite clay. The sorption isotherms on clays were of
Langmuir shape, while the sorption isotherms on silica gel were of S-shape. It might be
indicative of different sorption mechanisms. The sorption values at maximum concentration
of the silanol-derivative tested – 4 g/L - reached about 100 mg/g for silica gel and activated
bentonite. For non-activated bentonite, it was much less and accounted for 50 mg/g. For
kaolinite it did not exceed a value of 30 mg/g. There was no considerable sorption of the
tested derivative observed on quartz sand. This was true for all other samples.
Vol. 3 Page - 192 -
15th IHSS Meeting- Vol. 3
q, mg/g
160
140
120
100
80
60
40
20
0
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Ceq, g/l
Figure 2. Sorption isotherms of CHP-APTES-50 derivative onto different solid supports.
▲ - activated bentonite; + - silica gel;* - kaolinite; ● - quartz sand; ♦ - non activated bentonite
4. Conclusions
The sorption of silanol-modified HS on different solid supports including silica gel, quartz
sand and clays was studied. It was shown that the silanolized humic derivatives had high
affinity for sorption on silica gel and clays which are characterized with highly developed
surface area. At the same time, very limited sorption was observed on quartz sand with low
surface area. Maximum sorption achieved was 300 mg per gram of silica gel for CHPAPTES-100 derivative at 4 g/L concentration and a weight of silica gel of 50 mg.
The conclusion could be made that the silanolized HS can be used as soil conditioners for
increasing the pool of stable soil organic carbon due to sorption on clay particles.
Acknowledgements
This research was supported by the grant of RFBR 10-03-00803 and of the NATO CLG
983197.
References
1. Martin J.P. and Waksman S.A., Soil Sci., 52 (1941) p. 381.
2. Perminova I.V., Ponomarenko S.A., Karpiouk L.A., and Hatfield K. PCT application №
/RU2006/000102.
3. Prado, A. G. S., Sales, J. A. A., Airoldi, C. J., Therm. Anal. Calorim. 70 (2002) p.191.
4. Koopal, L. K., Yang, Y., Minnaard., A. J. Coll. Surf. 141 (1998) p.385
5. Shabanova N.A., Sarkisov P.D, Fundamentals of sol-gel technologies, 2004
Vol. 3 Page - 193 -
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Imprinted Humics-Based Sorbents as Selective Trap for Metal Ions
Elvira Kasymovaa, Rozalina P.Korolevaa, Elnura M.Khudaibergenovaa, Norbert Hertkornc,
Sharipa J. Jorobekovaa, Anatolii D.Pomogailob, Kamila Kydralievaa
a
Institute of Chemistry and Chemical Technology, National Academy of Sciences, Chui ave.
267, Bishkek 720071, Kyrgyz Republic; bInstitute of Problems of Chemical Physics, RAS,
Ac. Semenov ave, 1, Chernogolovka, Moscow region, 142432; cHelmholtz Zentrum
München, German Research Center for Environmental Health, Institute of Ecological
Chemistry, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
E-mail: k_kamila@mail.ru
1. Introduction
Selective binding of the traced metals with humic acids (HA) has very important value for the
removal of heavy metals and radio-nuclides from migration cycles. Additional
functionalization of HA causes the increase of their sorption capacity and the selectivity of
binding of metal atoms.
2. Materials and Methods
To increase the efficacy of the proposed sorbents we have been used the technique of
“adjustment” of polymeric complexes to the template on the stage of their synthesis or
formation of three-dimensional structure. The essence of this technique is the use of
"imprinted polymers," molecular chains imprinted with empty binding sites that match the
size and shape of specific kinds of metal ions. These polymers can be used to selectively trap
and contain a desired species of ion for removal from solution. Previously such approach was
used for the selective binding of strontium ions in the regions contaminated as a consequence
of Chernobyl disaster, adjusted polymeric sorbents on the basis of co-polymers of diacrylate
strontium with sterol, methylmetacrylate, acrylic acid and cross-linking agent – dimethacryl
ether ethyleneglycol, etc. [1].
3. Results and Discussion
To create imprinted sorbents, a target metal ion such as zinc, cobalt, nickel or copper was
sandwiched between a pair of ligands as humic acids and m-aminophenol (shortly – HAA).
After these sandwich complexes are cross-linked into a polymer, the metal ions are washed
away with HCl, leaving empty sites of the right size to fit similar ions. The hollow spaces are
able to encase and rebind the target molecule if encountered with for example a water sample
containing the molecule. The rebinding has a specificity connected to both the geometrical
structure of the hollow site and the chemically functional groups within the site.
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15th IHSS Meeting- Vol. 3
A set of imprinted sorbents were synthesized and characterized in terms of elemental and
functional groups composition and molecular weight distribution, FTIR and NMR
spectroscopy. Batch equilibrium studies using imprinted and non-imprinted polymer solutions
were conducted to determine metal-binding capacities.
M1
Cross-linking
M1
M1
Removing of M 1
M1
M1
M1
M1
M 1 + M 2 + M 3 ...
M1
M1
"Template" polymer
Figure 1: Scheme for template synthesis (“template” arrangement of polymer sorbent)
Imprinted sorbents possess significant speed of sorption (the balance is set up in several
minutes), sorption capacity (0.5–3.0 mg-eq M/g) and selectivity of sorption onto the
“adjusted” sorbents.
In a solution with equal concentrations of two different ion species including both copper and
zinc, the polymer captured 23 copper ions for every ion of zinc. The HAA imprinted with zinc
much prefers the zinc ions over the other metal ions, grabbing zinc over nickel, the nearest
competitor, in a ratio of five to one.
4. Conclusions
This research has demonstrated that molecular imprinting of metals using humics-based
sandwiches can be used to selectively induce binding of target metals based on shape
differences of their topologies.
Acknowledgements
This work supported by grant of the International Science and Technology Centre (#ISTC
KR-1316).
References
1. A.D. Pomogailo, et al. Doklady Akademii nauk, 1994, 335, 749–752.
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Flow Injection Analysis (FIA) for Fast Monitoring of Gold Nanoparticles
Formation from Various Precursors and Theirs Separation by Using Humic
Acids
Eladia María Peña-Méndeza, Ana I. Jiménez Abizandaa*, Juan José Arias Leóna and Josef
Havelb,c
a
Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry,
University of La Laguna, 38071 La Laguna, Tenerife, Spain; bDepartment of Chemistry,
Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic;
c
Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlarska 2,
61137 Brno, Czech Republic
E-mail: aijimene@ull.es
1. Introduction
Unique properties of nanomaterials accelerate the development in biotechnology, industry,
medicine, etc. The delocalized electrons in the metal clusters can undergo a collective
excitation called surface plasmon resonance. Gold nanoparticles (GNP) represent one of the
most widely studied nanoparticles systems [1, 2]. Many different chemical and physical
methods to produce nanogold particles with different size and shape are described. Thus,
GNP can be obtained reducing gold (I, III) salts with reducing agents like AlBH4, citric acid
or sodium citrate, hydroxylamine, tin (II) chloride and organic compounds such as amines,
hydroquinone and/or natural organic compounds. Unfortunately, nanoparticles tend to form
aggregates in solution as a consequence of their small size. One of the most effective
strategies to avoid this fact is to protect the colloids obtained with protecting agents. In
addition, high molecular weight polymers improve function of the particle surface for their
application in bioanalytical methods.
In this work auric acid (HAuCl4) and reducing agents like gallic or H2O2, etc. were used to
produce GNP in aqueous solution. The HA can also react with Au(III) in solution, generating
different size GNP. The aim was to study possibility of FIA for the separation of GNP and
the influence of HA in the formation and separation of nanoparticles with different size.
2. Materials and Methods
Hewlett–Packard HP 8453A diode array spectrophotometer equipped with quartz cuvettes of
1 cm light path and 4 ml inner volume. Gilson Minipuls-2 peristaltic pump fitted with PVC
tubes. All other tubing and connectors were made of Teflon. Crison digital pH-meter
furnished with a glass–saturated calomel double electrode.
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15th IHSS Meeting- Vol. 3
The Suwannee river standard HA (1S101H). The 200 mg·l-1 stock solution was prepared by
dissolving the corresponding weight in 36 mM NaOH. Auric acid, HAuCl4 trihydrate was
from Sigma-Aldrich (Steinheim, Germany). Sodium hydroxide was from Merck (Darmstadt,
Germany). All other reagents were of analytical grade purity. De-ionized water used to
prepare all solutions was double distilled from MilliPore system.
For FIA studies, 10–50 μL of solutions was injected in the FIA system.
3. Results and Discussion
Classical FIA was used e.g. for rapid, highly sensitive and routine automatic determination
of ionic forms of gold in different samples [4] and to study the catalysis of GNP in some
reactions [5]. In this work, FIA with Diode Array Detector (DAD) was used to follow the
nanoparticles formation and separation of various size GNP.
Instrumental set up as the length of the capillary, temperature, flow rate, additives, etc. were
optimized. Even if only partial separation of the GNP was obtained, it enables fast
characterization of the particles produced. Specifically, separation enables to get UV/Vis
spectra of individual GNP plasmons and monitoring the kinetics of GNP formation.
0.16
B
Absorbance
A
0.12
GNP
HA
GNP+HA
2GNP+2HA
GNP+4HA
0.08
0.04
0
0
200
600
1000
1400
time (s)
Figure 1: Flow Injection Analysis set up (A) and an example of GNP separation and effect of HA (B)
Suwannee river standard (SW), +HA, +2HA, +4 HA: different additions of SW to GNP
As can be seen from Fig. 1, the addition of HA to GNP improves the separation of different
size nanoparticles. An increase in the HA concentration produces a displacement in the FIA
peak to early times. Thus, HA not only act as a reducing agent but also contribute to modify
the rate of displacement of GNP formed.
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15th IHSS Meeting- Vol. 3
4. Conclusions
It was found that FIA with long path capillary can be used to separate GNP. The separation
mechanisms is suggested to be similar to turbulent chromatography. Additions of HA to
GNP improve the separation. Humic acids are probably adsorbed on the GNP surface
stabilizing them and/or GNP are simultaneously encapsulated with HA forming
supramolecular complexes. Flow Injection Analysis technique developed enable fast
monitoring of GNP formation.
Acknowledgements
Grant Agency of the Czech Republic, projects no. 525/06/0663 and 202/07/1669, Academy of
Sciences of the Czech Republic (project KAN 101630651) and the Ministry of Education,
Youth and Sports of the Czech Republic (projects MSM0021622411 and LC 06035) are
acknowledged. Canary Autonomic Government by research project PI 2007/011 is
acknowledged. E.M.P., A.I.J. and J.J.A. thank the partial support of the University of La
Laguna (Spain).
References
1. Y. Sun, Y. Xia, Science 298, (2002), 2176.
2. B.K. Jena, C.R. Raj, Biosens. Bioelectron. 23, (2008),1285.
3. E.M. Peña-Méndez, J.R. Hernández-Fernaud, R. Nagender, J. Houška and J. Havel, Chem. Listy
102,(2008), 1394.
4. D.G. Themelis, A.V. Trellopoulos, P.D.Tzanavaras, M. Sofoniou, Talanta 72, (2007), 277.
5. L. Wang, P. Yang, Y. Li, H. Chen, L. Maoguo, L. Fabao, Talanta 72 , (2007), 1066.
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Spectrofluorimetric Study of the Interaction of Gold (III) and Humic Acids
under the Formation of Gold Nano-Particles
Eladia María Peña-Méndeza, Francisco Jiménez Morenoa, Jose Elías Conde González,
Josef Havelb,c
a
Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry,
University of La Laguna, 38071-La Laguna, Tenerife, Spain; bDepartment of Chemistry,
Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic;
c
Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlarska 2,
61137 Brno, Czech Republic
E-mail: fjimenez@ull.es
1. Introduction
Humic acids are a complex mixture of partially "decomposed" and otherwise transformed
organic materials from different sources. The chemistry of their formation is quite complex
and still it is not completely understood. There are several subclasses of humic acids (tannins,
lignins, fulvic acids, etc.). A substantial fraction of the mass of the humic acids is containing
carboxylic acid functional groups which endow these molecules with the ability to chelate
positively charged multivalent ions (Mg2+, Ca2+, Fe2+, Fe3+, Al3+ and most other "trace
elements") [1]. The fluorescent structures are minor components in humic substances.
Fluorescence spectroscopy is fast, relatively easy and powerful method to follow such
fluorescence structures but also method for providing knowledge about the chemistry and
nature of the interactions between gold (III) and HA. The reduction of Au(III) may be due to
functional groups such as e.g. amino, hydroquinones and phenolics groups normally present
on humic acids; functional groups which are recognized to be efficient reducing agents for
gold cations [2].
The aim of the work is to investigate the interaction taking place between gold (III) and soil
humic acids (HA).
2. Materials and Methods
Perkin-Elmer (Beaconsfield, Buckinghamshire, UK) spectrofluorimeter equipped with a
xenon lamp and quartz cuvettes of 1 cm path length and 4 mL inner volume. Crison
(Barcelona, Spain) digital pH-meter furnished with a combined glass–saturated calomel
double electrode. Lauda (Königshofen, Germany) MS6 thermostat. Ultrasonic cleaner
(Selecta, Seville, Spain) was also used.
The HA soil IHSS standard stock solution (200 mg·L-1) was prepared by dissolving the
corresponding weight in 36 mM NaOH. Auric acid, HAuCl4·3H2O, was purchased from
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Sigma-Aldrich (Steinheim, Germany). Sodium hydroxide was from Merck (Darmstadt,
Germany). All reagents were of analytical grade purity. All aqueous solutions were made
using ultrahigh purity water purified using a Mill-Q Plus system (Millipore Co).
3. Results and Discussion
The current study employed fluorescence spectroscopy to investigate the interactions of
humic acid (HA) with gold (III). The results of kinetics show that gold (III) is in the first stage
bound to HA, most probably complexed to-
160
SH and/or amino groups present in the humic
pH = 5.05, HA + Au(III)
pH = 3.02, HA
120
Fluorescence
from the HA are reducing Au(III) to Au (0)
of. The redox reaction is pH dependent (cf.
pH = 8.58, HA + Au(III)
140
structures and later on the reducing groups
generating gold nano-particles of various size
pH = 3.02, HA + Au(III)
pH = 3.02, HA + AuNP
pH = 3.02, AuNp
100
pH = 3.35, Au(III)
80
60
Fig. 1).
The nano-particles were also characterized by
scanning electron microscopy (SEM). Because
fluorescence of GNP is also influenced by HA,
40
20
0
250
the interaction GNP-HA can also be suggested.
350
450
550
Wavelength (nm)
650
4. Conclusions
Humic acids are interacting with Au(III) under some kind of complexation. Finally, humic
acids are reducing Au (III) to gold nano-particles in a reaction which consists of several steps.
Also, the interaction between humic acid and gold nano-particles was proved. Highly
homogeneous in size gold nano-particles were prepared but at different conditions GNP of
varied size are formed.
Acknowledgements
Grant Agency of the Czech Republic, projects no. 525/06/0663 and 202/07/1669, Academy of
Sciences of the Czech Republic (project KAN 101630651) and the Ministry of Education, Youth and
Sports of the Czech Republic (projects MSM0021622411 and LC 06035) are acknowledged. Canary
Autonomic Government by research project PI 2007/011 is acknowledged. E.M.P-M., J.E.C. and F.J.
thank the partial support of the University of La Laguna (Spain).
References
1. V.L. Pallem, H.A. Stretz and M.J.M. Wells, Environ. Sci. Technol., 43 (2009) 7531.
2. R.A. Alvarez-Puebla, D.S. dos Santos, and R.F. Aroca, Analyst, 132 (2007) 1210.
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Metal Binding by Humic Acids Extracted from Recent Sediments from the
SW Iberian Coastal Area
De la Rosa, J.M.a*, Santos, M.a, González Vila, F.J.b, Knicker, H.b, González Pérez, J. A.b,
Araújo, M.F.a
a
Instituto Tecnológico e Nuclear, Estrada Nacional 10, 2686-953, Sacavém, Portugal;
b
IRNAS-CSIC, Av. Reina Mercedes, 10, 41012-Sevilla, Spain
E-mail: jmrosa@itn.pt
1. Introduction
It is well known that humic substances (HS) play a key role in a range of environmental
issues, such as soil and water acidification, nutrient control, weathering, mobility and
distribution of heavy metals, ecosystem buffering, etc [1]. In almost all of those issues, cation
binding is recognized to be an important factor, which has been studied in many papers [1–4].
These macromolecules are also interesting because of their structural features, which includes
binding sites with different complexing strength, able to form inert and labile complexes with
inorganic cations (metals) [5] and organic compounds. The information on the compositions
and functional groups of HAs is critical for understanding their reactivity with organic and
inorganic contaminants. However, due to their complexity and heterogeneity, it is difficult to
determine the HAs structures. Bearing in mind the fact that cation-humic interactions depend
on the presence of reactive acidic functional groups, such as carboxylic and phenolic groups
[6], their characterization and quantification may enlighten the metal-humic interactions. The
major aim of this study is to report information about the amounts of metals bound in “not
labile” forms to sedimentary humic acids from estuarine and coastal sediments taken in the
Guadiana estuary, Tinto and Odiel River mouth areas, and in the adjacent continental shelf,
within the northern Gulf of Cadiz (Spain). The analytical strategies followed included the
total concentration of organic carbon (TOC), inorganic carbon (TIC), nitrogen, sulphur and a
series of fundamental heavy metals (Fe, As, Cr, Cu, Hg, Ni, Pb and Zn) in both, bulk
sediments and humic acids (HA). In order to check the relationship between the presence of
carboxylic and phenolic groups and the amount of metals “captured” by HA, they were
characterized by using analytical Pyrolysis-GC/MS and solid state 13C-NMR spectroscopy.
2. Materials and Methods
Area of study and sediment sampling. Surface sediment samples (0 to 20 cm depth) were
collected on northern of the Gulf of Cadiz under the auspices of the Spanish BACH-Project
[7]. It is a site of geological and environmental interest, highly influenced by water run-off
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and sediment load derived from the Guadiana River, which is a major river of the Iberian
Peninsula (742 km of length). The flow of the Guadiana River has been extensively modified
during last century due to a range of anthropogenic activities, including damming, mining,
urbanisation, deforestation and dredging. A set of representative samples was selected for this
study, they consist of three samples from the Guadiana estuary (GE209; GE220; GE226), one
sample from the Tinto estuary (TE25), one sample from the Odiel estuary (OE39), and two
samples from the adjacent continental shelf (S131; S155). All samples were stored frozen in
glass containers to avoid microbial growth. Before analysis, samples were freeze-dried,
thoroughly ground in a mortar mill and homogenized.
Elemental Analysis. Carbon, nitrogen and sulphur contents were determined in triplicates by
using an elemental analyzer. TOC was measured on decarbonised samples (HCl) and TIC was
calculated from the difference between TC and TOC [8].
Humic acids extraction. HAs were extracted with a mixture 0.1 M Na4P2O7 and 0.1 M NaOH
and the dark brown supernatant (total humic extract) was precipitated by adjusting the pH to
2, de-ashed, redissolved in 0.1 M NaOH and centrifuged. The residue was discarded, and the
brown of sodium humate supernatant was reprecipitated with HCl and dialysed in to remove
the salts introduced during the extraction procedure. The HA was then freeze dried and kept
for further chemical characterization.
13
C NMR spectroscopy. Solid-State 13C-NMR spectroscopy analyses of HAs were performed
on a Bruker DSX 200 spectrometer, operating at a 13C resonance frequency of 50.3 MHz. A
commercial Bruker double bearing probe and phase-stabilized zirconium dioxide rotor was
used. The cross-polarisation (CP) technique was applied during magic angle spinning (MAS)
of the rotor. For quantification, the spectra were divided into different chemical shift regions
according to [9].
Pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). Pyrolysis of HAs was
performed in a double shot Frontier Laboratories pyrolyzer (model 2020) directly connected
to a GC-MS system Agilent 6890 equipped with a fused silica capillary column HP 5MS (30
m × 250 μm × 0.25 μm inner diameter). The detector consisted of an Agilent 5973 mass
selective detector (EI at 70 eV). Standard chromatographic conditions and identification of
individual compounds procedure were carried out according to [10].
Metal analysis. The analytical determination of major and minor metals (Fe, As, Cr, Cu, Hg,
Ni, Pb and Zn) in both bulk sediments and HA samples were performed by digesting 10 mg of
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samples in a microwave oven (5 min 300W) with a mixture of H2O2 and HNO3. All the
samples were diluted to 50 mL with ultra pure water (18MΏ) and keep refrigerated before
analysis. The resulting solutions were analyzed by ICP-MS (Perkin Elmer ELAN DRCe) and
the metals quantified by external calibration. For correcting any instrument drifts Rh 10 ppb
was added as an internal standard to all the samples and standard reference materials.
3. Results and Discussion.
The TC values ranged from 12.8 to 31.1 g kg-1, which are typical values for coastal and
marine sediments [8]. Greater TOC values were found in riverine and in the Guadiana
estuarine samples. The TN values were found to be very similar and ranged between 0.9 and
2.0 g kg-1. The C/N ratios in the sediments studied ranged between 7 and 12. Larger C/N
ratios (≥ 12) were observed for the estuarine samples, indicative of a large contribution of
terrestrial detritus. Sulphur (TS) contents in the samples ranged from 1.8 to 13.5 g kg-1.
Larger values were observed in the Odiel estuary sample (OE39), and the smallest value was
in the marine sample S131.
Total contents of Fe, Cu, Zn, Pb and Cr metals in bulk sediments were significantly increased
in samples located in the Tinto and Odiel river
Figure 1: Contents of selected metals in Humic acids
mouths (TE25; OE39), which is explained by
3750
the fact that those rivers drain a catchment area
3500
that traverses the pyritic belt in the Southwest
3000
3250
1750
compared to the samples located in the
continental shelf, pointing to a transport of
ppm
of the Iberian Peninsula. Guadiana estuarine
sediments presented lower metal contents
Cu
Zn
Cr
Ni
Pb
1500
1250
1000
750
500
250
sedimentary material due to the influence of
strong river discharges.
Figure 1 shows the contents of several metals
0
TE 25 OE 39 S 131 S 155 GE209 GE220 GE226
in HAs. Retention capacity of particular metals on HAs may be ordered in the following
sequence Fe>>>Cu>>Zn>Cr. This preferential binding order of metal-HA doesn’t correlate
with the metal content order in bulk sediments.
The CP-MAS
13
C NMR spectrum of the seven HAs all exhibited major peaks at 30 ppm
(alkyl C), 55 ppm and 71 ppm (O-alkyl carbons), 130 ppm and 152 ppm (aromatic carbons),
173 ppm (carboxylic carbon), and 196 ppm (carbonyl carbon). Aromaticity was significantly
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higher in the Tinto estuarine sample (TE25; 40.9 %) and Guadiana estuary (GE209; 34.4 %)
HAs, contrasting with the aliphaticity of the marine HAs (S131 and S155; 70.2 and 74.3 %
respectively), in which aquatic plants are the dominant contributors to the HS. The carboxylic
and phenolic C parameter accounts the % of reactive acidic functional groups in the HAs [6],
an increase was shown for HAs isolated from estuarine samples, so they might have further
binding sites able to form complexes with metals.
Pyrolysis-GC/MS experiments revealed similar compositions of pyrolysates generated from
all samples. These were characterized by the presence of a complex mixture of phenols,
benzenes, naphthalenes, indanes, pyrroles, alkylated homologues with a low degree of
alkylation and a scarce contribution of fatty acids and low relative abundances of n-alkanes
and n-alkenes. A slight increase in the aromatic and carboxylic compounds released from
riverine HAs was observed confirming results obtained by 13C NMR spectroscopy.
4. Conclusions
Overall, the results of this study demonstrate that the combination of techniques used yield
valuable information concerning the nature of HAs in recent sediments. There was a positive
correlation between metal content and functionality (acidic carboxylic and phenolic groups).
Nevertheless this study in still in progress, so hopeful results are expected with the aim of
increase the knowledge in the metal-humic interactions.
Acknowledgements. We thank Ms. Trinidad Verdejo (IRNAS-CSIC) for assisting us in the
Py-GC/MS spectrometry.
References
1. E. Tipping, Cation Binding by Humic Substances, Cambridge University Press, Cambridge, 2002.
2. J.A. Marinsky and J. Ephraim, Environ. Sci. Technol., 20 (1986) 349.
3. F.J. Stevenson, Humus Chemistry: Genesis, Composition, Reactions, John Willey & Sons, New
York, 1994.
4. A.E. Martell and R.D. Hancock, Metal Complexes in Aqueous Solutions, Kluwer, New York,
1996.
5. R. Yamamoto and S. Ishiwatary, Sci. Total Environ., 279 (1992) 117.
6. J.C. Masini, G. Abate, E.C. Lima, L.C., Hahn, M.S. Nakurama, J. Lichtig, and H.R. Nagatomy,
Anal. Chim. Acta, 364 (1998) 223.
7. BACH project, REN 2002-04602-C02-01. Environmental Geochemistry of Sediments from the
Huelva Coast.
8. J. Nieuwenhuize, Y.E. Maas, J. Middelburgh, Mar. Chem., 45 (1994) 217.
9. H. Knicker, H.-D. Lüdemann, Org. Geochem., 23 (1995) 329.
10. J.A.González Pérez, C.D. Arbelo, F.J. González Vila, A. Rodríguez, G. Almendros, C.M. Armas,
O. Polvillo. J. Anal. Appl. Pyrolysis, 80 (2007) 369.
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Polycyclic Aromatic Hydrocarbons (PAHs) - Dissolved Organic Matter
(DOM) Interactions Studied by Solid Phase Microextraction (SPME)
Chloé De Perrea,b, Karyn Le Menacha,b, Anne-Marie Dorthec,d, Christian Béchemine,
Hélène Budzinskia,b*, Edith Parlantia,b*
a
Université de Bordeaux, UMR 5255, ISM, Groupe LPTC, 351 Cours de la Libération
Talence, F-33405 France; b CNRS, UMR 5255, ISM, Groupe LPTC Talence, F-33405
France; c Université de Bordeaux, UMR 5255, ISM, Groupe NSysA, ENSCPB, 16 Avenue
Pey Berland Pessac, F-33607 France; d CNRS, UMR 5255, ISM, Groupe NSysA Pessac, F33607 France; e IFREMER, LER/PC, BP 7, L’Houmeau, F-17137 France
E-mail: e.parlanti@ism.u-bordeaux1.fr; h.budzinski@ism.u-bordeaux1.fr
1. Introduction
Polycyclic Aromatic Hydrocarbons (PAHs) are highly toxic pollutants, with carcinogenic
properties for some of them (US-EPA priority substances). Dissolved organic matter (DOM)
in aquatic environments is well known to play an important role in the fate of organic
pollutants. Indeed, DOM should bind these compounds modifying their distribution,
bioavailability, biodegradation and subsequently their toxicity towards aquatic organisms. In
order to calculate the partitioning coefficient (KDOC) of each pollutant to DOM, it is necessary
to measure concentrations of free and DOM bound pollutant fraction. Few analytical
techniques allow the measurement of only free compound concentration but most of them
may modify the interactions during the analysis, making the study of these interactions a real
challenge. The goal of this study was therefore to develop a reliable technique that permits to
quantify rapidly total and free organic pollutant concentrations: solid-phase microextraction
coupled to gas chromatography-mass spectrometry (SPME-GC-MS). We aimed also at
highlighting environmental parameters which influence PAH/DOM interactions.
2. Materials and Methods
SPME-GC-MS was chosen for the analysis of PAHs, especially since it allows the
quantification of both freely dissolved and DOM-bound PAH concentrations simultaneously,
with a negligible disturbance of the equilibrium DOM-PAH [1, 2]. SPME technology is
constituted of a polymeric fiber that is introduced into the water samples in order to
adsorb/absorb, according to their affinity with the fiber coating, the only free PAHs that are
then desorbed by heating in the GC injector. KDOC values were calculated for individual
compounds and for a mixture of 4 and 16 PAHs, with variations of DOC and PAH
concentrations. DOM samples were characterized by their optical properties by means of
excitation-emission matrix (EEM) spectroscopy and UV-visible absorption. Moreover, full
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and fractional factorial designs were performed to investigate the effect of environmental
parameters like salinity, pH, DOC and PAH concentrations on the interactions.
3. Results and Discussion
A series of tests was performed to optimize SPME parameters for the analysis of PAHs.
Results showed that fiber coating in polydimethylsiloxane (PDMS) seems to provide the best
efficiency with a time of analysis of only one hour, low limits of detection and quite good
reproducibilities. It was shown that the strength of interactions highly depends on DOM
origin and structure and on type of PAHs. Humification and aromaticity of DOM were not the
driving factors of interactions and fresh material of aquatic DOM could have stronger
interactions with PAHs than humified aquatic fulvic acids. Salinity was not an important
factor since it modified significantly neither KDOC values nor optical properties of DOM. On
the contrary, pH and DOC concentrations could strongly affect interactions and had appeared
to be interrelated for some samples. PAH concentration was shown to affect interactions:
KDOC values tended to decrease at high concentrations of PAHs.
4. Conclusions
SPME is an appropriate tool to study interactions of many PAHs simultaneously at low
concentrations with DOM. This study showed that interactions of PAHs with DOM are very
complex and depend on many factors. Some of them have no effect on interactions, such as
salinity, while some others highly affect KDOC values such as pH and DOC concentrations.
Nevertheless, interactions and environmental factor effects have been shown to depend on
PAHs and DOM origin and structure and appear to be difficult to apprehend. Indeed, in this
study three different DOM were studied and three different complex behaviors were pointed
out, so the phenomena that occur in natural waters should be a challenge to model.
Acknowledgements
"Region Aquitaine", FEDER, ORQUE program (CPER) and INSU (EC2CO programIMOTOX project) are acknowledged for financial support and the French Ministry of
Research for the PhD grant of C. de Perre. We also gratefully acknowledge C. Vérité
(IFREMER LER/PC) for the dissolved organic carbon analyses, R. Brizard and J.C. Billy
(IFREMER LGP, La Tremblade) for the algae cultures.
References
1. J. Poerschmann, Z. Zhang, F.D. Kopinke, J. Pawliszyn, Analytical Chemistry, 69 (1997) 597-600.
2. M.B. Heringa, J.L.M. Hermens, TrAC Trends in Analytical Chemistry, 22 (2003) 575-587.
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Environmental Applications of Natural Organic Matter and Humic
Substances
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Use of Humin for Removal of Phosphorus from Sewage Treatment Station
Effluents: Influences of Time and pH
Luciana Camargo de Oliveira*a, Wander Gustavo Boteroa,b, André Gustavo Ribeiro
Mendonçaa, Julio Cesar Rochaa, Ademir dos Santosa, André Henrique Rosac
a
Institute of Chemistry of Araraquara – UNESP, Araraquara, SP, Brazil; bFederal University
of Alagoas, Arapiraca, AL, Brazil; c Department of Environmental Engineering – UNESP,
Sorocaba, SP, Brazil
E-mail: lcamargo@iq.unesp.br
1. Introduction
Phosphorus-containing substances have widespread applications in industry, agriculture and
in the home. These chemicals are ultimately transported to waterways where, despite
treatment of effluents, they can cause serious pollution problems. Although phosphate is not
considered to be toxic, it is a nutrient that in excess can cause algal overgrowth and
eutrophication. Under conditions of low oxygen content it is anaerobic bacteria that are
mainly responsible for decomposition of organic matter, so that instead of oxidation,
reduction takes place, producing noxious compounds such as hydrogen sulphide,
methanethiol and ammonia. In addition, phosphates can produce dense layers of foam,
reducing the surface tension of water, to the detriment of aquatic fauna. Removal of
phosphorus from freshwaters is therefore of environmental interest [1, 2].
For many years, control of phosphorus in surface waters has focused on point sources, such as
detergents in domestic effluents, however more recently attention has shifted to diffuse
sources. In Europe, it is estimated that 50 % of the phosphorus present in surface waters
derives from diffuse sources, due to its use in agriculture. Large annual inputs of phosphate
fertilizer, some of which leaches to waterways, are responsible for substantial environmental
impacts. Hence, there is a need for substances that could assist in retention of phosphate, and
that could ideally be subsequently used as natural fertilizers [1].
Humic substances (HS) are promising agents that might be able to perform this dual function.
Enriched with nutrients, they have already been marketed as inorganic fertilizers. Humin, the
fraction of HS that is insoluble throughout a wide range of pH, has received less scientific
attention than other HS fractions [3]. Nonetheless, there have been reports in the literature
concerning its interaction with organic compounds (herbicides, insecticides, fungicides, PAHs
and PCBs) [3, 4], as well as a small number of studies involving inorganic substances [5].
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The present work examines humin-phosphorus interactions from the perspective of the possible
use of humin to remove phosphorus from sewage treatment station effluents, for later application
in agriculture as a natural fertilizer.
2. Materials and methods
Extraction of humin. Humin was isolated from peat samples collected in the vicinity of the Mogi
River, in the municipality of Rincão, São Paulo State, Brazil, using alkaline extraction procedure
recommended by the International Humic Substances Society (IHSS) [4].
Preparation of standard solutions.A 1000 mg L-1 phosphorus (as phosphate-P) stock standard
solution was prepared, and dilutions made as required immediately prior to the experiments
concerning pH and temperature, as well as for construction of calibration curves.
Influence of pH on humin-phosphorus interactions. One hundred mg quantities of humin were
weighed out into polypropylene flasks, and 50 mL of 10 mg L-1 phosphorus standard solution
added, at pHs varying from 2 to 8. After pH adjustment, the solutions were left under agitation for
24 hours, and then filtered and the phosphorus contents quantified by inductively coupled plasma
atomic emission spectrometry (ICP-OES).
Influence of time on humin-phosphorus interactions. Mixtures of humin and phosphorus were
prepared as above, and the pH adjusted to the value at which greatest adsorption of phosphorus
occurred. After pH adjustment, the solutions were agitated for periods of 5, 10, 30, 180, 1440 and
4320 minutes, then filtered and the phosphorus contents measured.
Use of humin with sewage treatment station effluents. Adsorption of phosphorus was investigated
by adding 10 mg of humin to 50 mL portions of effluent, either as received or after adjustment of
pH to 3.0. The labile phosphorus contents of the samples were measured before and after humin
addition. The mixtures were filtered after the maximum adsorption time, and the phosphorus
contents quantified.
3. Results and discussion
The maximum adsorption of phosphorus by humin occurred at pH 3.0 (Fig. 1). At pH 8.0, which
is typical of sewage treatment station effluents, adsorption was 24.2 %. Experiments were then
performed at these two pHs (3.0 and 8.0), to determine the effect of time on the adsorption
process (Fig. 2a). At pH 3.0, the humin adsorbed ~84 % of the added phosphorus after 72 hours,
after which no further changes in adsorption were observed. At pH 8.0, the humin adsorbed ~31
% of the phosphorus after 72 hours. The adsorption process was more efficient at pH 3.0.
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Adsorbed phosphorus (mg L-1)
6
5
4
3
2
1
0
3
4
5
6
7
8
Figure 1: Influence of pH on phosphorus adsorption
by humin. Initial phosphorus concentration:
pH
10 mg L-1
90
pH 3.0
pH 8.0
0,9
70
Concentration ratio (Cl-C)/Cl
Phosphorus adsorption (%)
80
a)
)
60
50
40
30
20
b)
pH 3.0
pH 8.0
0,6
0,3
0,0
10
0
0
1000
2000
3000
4000
5000
0
Time (min)
2000
4000
Time (min)
Figure 2: a) Influence of time on phosphorus adsorption by humin; b) Kinetic study of the interaction
between humin and phosphorus
The data obtained were analysed from the perspective of chemical kinetics [6, 7]. Results are
illustrated in Figure 2b, where Cl is the limit concentration of phosphorus, and C the
concentration after different time intervals. From this, it was possible to estimate the times
required to reach equilibrium between the phosphorus ions and humin. At pH 3.0, the time
required for equilibrium to be achieved was 180 minutes, while at pH 8.0 it was 160 minutes,
indicating that the kinetic behaviour of phosphorus-humin interactions is similar at different
pHs.
When humin was added to samples of sewage treatment station effluent, adsorptions varied
from 22 to 32 % at pH 8.0, and from 89 to 92 % at pH 3.0 (Table 1), in agreement with the
values previously obtained using phosphorus standard solutions (Fig. 2a).
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Table 1: Adsorption by humin of phosphorus in sewage treatment station effluent samples
Effluent
sample
pH
1
2
3.0
8.0
Initial phosphorus content,
prior to humin addition
(mg L–1)
5.54 ± 0.07
3.16 ± 0.08
Final phosphorus
content, after humin
addition (mg L–1)
0.51 ± 0.09
2.29 ± 0.10
Removal (%)
89.3–92.3
22.4–32.4
4. Conclusions
Preliminary studies of phosphorus-humin interactions indicate that humin is able to adsorb
phosphorus across a range of pHs. Humin is therefore a promising material for removal of
phosphorus from effluents, with an additional potential benefit being the ability to use the
products as sources of nutrients in agriculture. This work is in development and as soon as
possible the authors will present major data as full paper.
References
1. J.C. Rocha, A.H. Rosa, A.A. Cardoso, Introdução à Química Ambiental, Bookman, Porto Alegre,
2009.
2. I.R.S. Chao, T.H. Ferraz, H2O Água, (2007) 40.
3. J. Rice, Humin. Soil Science, 166 (2001) 848.
4. J. Zhang, M. He, Y. Shi, J. Hazard. Mat., 166 (2009) 802.
5. G. De La Rosa, J.L. Gardea-Torresdey, J.R. Peralta-Videa, I. Herrera, C. Contreras, Bioresour.
Technol., 90 (2003) 11.
6. J.C. Rocha, É. Sargentini Júnior, L.F. Zara, A. H. Rosa, A. Santos, P. Burba, Talanta, 53 (2001)
551.
7. P. Burba, Van Den Bergh, Anal. Bioanal. Chem., 378 (2004) 1637.
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15th IHSS Meeting- Vol. 3
Phytoremediation of a Soil Polluted with Multiple Heavy Metals Using
MSW Compost as Organic Carbon Source
Karam Farraga, Gennaro Brunettib*, Pedro Soler-Rovirac, Franco Nigrod
a
Central Lab for Environmental Quality Monitoring (CLEQM), National Water Research
Center, Ministry of Water Resources and Irrigation, Egypt; bDip. Biologia e Chimica AgroForestale ed Ambientale, University of Bari, Via Amendola 165/A, Bari, Italy; cCentro de
Ciencias Medioambientales (C.S.I.C.), Serrano 115 bis, Madrid, Spain; dDip. Protezione delle
Piante e Microbiologia Applicata, University of Bari, Italy
E-mail: brunetti@agr.uniba.it
1. Introduction
Phytoremediation can be defined as the combined use of plants, soil amendments and
agronomic practices to remove pollutants from the environment or to decrease their toxicity
(1). Finding the optimum plant species for remediation of a specific soil is a key point
affecting the achievement of the objective, as well as the selection of appropriate soil
amendments which would improve soil conditions, allowing plant survival and growth (2).
2. Materials and Methods
Analytical methods: pH and EC were measured in 1:2.5 and 1:2 sample:water ratio,
respectively. Organic carbon (OC) was determined by dichromate oxidation and titration with
ferrous ammonium sulphate; total nitrogen (N) was assessed by Kjeldahl method. Soil
available phosphorus (P) was quantified by Olsen method. Heavy metals contents were
determined by ICP in extracts from samples digested in HNO3: H2O2 (7 mL+1 mL).
Chromium oxidation test (Bartlett test) was performed by addition of CrCl3 and UV-VIS
spectrophotometer measure (540 nm) of coloured complex between Cr (VI) and
diphenylcarbazide (3).
Soil samples were collected from a polluted area located at Altamura area (Apulia, Southern
Italy), where industrial wastes increased soil heavy metals content up to following levels (mg
kg-1): Cd 2, Cr 1277, Cu 89, Ni 132, Pb 166, and Zn 497. Main characteristics of soil were:
silty loamy texture; pH (H2O) = 8.5; EC = 0.32 dS m-1; OC = 66.1 g kg-1; N (total) = 7.2 g kg1
; C/N ratio = 9.1; P = 83.5 g kg-1.
A greenhouse experiment was performed using Brassica napus as accumulator plant and
testing the addition to the soil of organic amendment (MSW compost) and a bacterial strain
(Bacillus licheniformis BLMB1). Compost analysis showed the following main values: pH
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(H2O) = 8.7; EC = 1.25 dS m-1; OC = 232 g kg-1; N = 14.8 g kg-1. Treatments were as follows:
T1 (soil), T2 (soil + compost 10%), T3 (soil + B. licheniformis BLMB1 10%), T4 (soil +
Compost 10% + B. licheniformis BLMB1 10%). Bacterial strain was applied to the soil as
aqueous suspension (10%, w/v) containing 108 cells ml-1. Data statistical analysis, regression
models and correlations matrixes were performed using Statgraphics Plus 5.1 software.
3. Results and Discussion
The values of Cr3+ oxidation test resulted lower in the treatments with highest amounts of OC,
and a significant (P<0.001) negative correlation (-0.806***) between the variables was found.
Linear regression analysis (Fig. 1) also evidenced a significantly high relationship between
the two parameters.
0.6
y = -0.0089x + 0.7939
r2 = 0.6505 (p<0.001)
mol Cr(VI)
0.5
T1
0.4
0.3
T4
T3
T2
0.2
0.1
0.0
38
43
48
53
58
-1
g OC kg soil
Figure 1: Lineal regression of OC content and Cr(III) oxidation capability of soil
These results indicate that higher OC soil contents prevent Cr3+ oxidation to Cr6+, which is
environmentally more dangerous than the former. As reported in literature, this behaviour
could rely on two already recognised evidences, i) organic matter act as an electron donor,
thus preventing Cr oxidation,
and ii) bacteria enhance soil processes for Cr reduction.
However, also the humic acids fraction from compost would immobilize and therefore reduce
Cr3+ tendency to be oxidized (5) by forming Cr3+ stable complexes with donor groups (4).
This hypothesis is in accordance with other works (6) where the addition of composted
materials decreased the soluble and exchangeable soil Cr, increasing the organic-bound
fraction. To be successful, phytoremediation needs appropriate soil conditions for the plant
growth. Statistical analysis of B. napus vegetative parameters showed a significantly high
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correlation between soil OC content and plant height (0.904***), foliar area (0.902***), and
dry weight (0.955***). Therefore, treatments with OC inputs (T1 and T3) improved plant
biomass (Fig 2) due to the direct and indirect benefits of organic matter fractions on soil and
plants.
3,3
3,2
60
3,1
40
3
20
g (dry weight)
g OC kg-1 soil
80
2,9
0
2,8
T1
T2
T3
TOC
T4
Plant DM
Figure 2: Results for soil OC content and Brassica napus biomass production
Our data also indicate that supplying soil with organic C and microorganisms Bacillus
licheniformis BLMB1 enhance the extraction of some heavy metals and their accumulation in
B. napus. This effect was more evident for copper and lead (Fig. 3).
150
mg kg-1
120
90
60
30
0
T1
T2
T3
Pb
Cu
T4
Figure 3: Copper and lead contents in shoots of Brassica napus
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Significantly high correlations were found between soil OC and metal content in shoots
(0.660**, and 0.658*, for Cu and Pb, respectively), as compared to the untreated control.
Similar results were reported by other (2) for Cu, in a site polluted by several heavy metals. In
the case of copper, compost organic fractions, such as humic acids, would allow the formation
of surface complexes (7) thus facilitating the release to soil solution and maintaining Cu
available for plant extraction. These aspects have particular relevance in the phytoremediation
practices, considering that not only metals extraction by plant but also vigorous growth and
higher biomass production need to be pursued in order to obtain the highest metal
phytoextraction.
4. Conclusions
The preliminary results from our experiment confirm the advantages of using appropriate
amendment in combination with an optimum plant species for phytoremediation purposes.
Addition of exogenous organic carbon and B. licheniformis BLMB1 reduced the tendency of
Cr3+ to be oxidized, preventing the accumulation and the environmental damage of the Cr6+
form. Compost organic matter increased plant biomass and enhanced the phytoextraction of
some heavy metals. The combination of both effects (soil immobilization and plant
extraction) will be the target for our next research activities on phytoremediation, in order to
obtain deepest knowledge about these processes
Acknowledgements
This work was founded by Regione Puglia (Italy) through the research project POR Puglia
2000-2006, Misura 1.8 – Azione 4: “Monitoraggio siti inquinati”. Supporto scientifico alle
attività di recupero funzionale ed il ripristino ambientale del sito inquinato dell’Alta Murgia.
References
1. D.E. Salt, R.D. Smith, L. Raskin, Phytoremediation Annual Review of Plant Physiology and Plant
Molecular Biology 49, (1998) 643-668.
2. R. Clemente, D.J. Walker, M.P. Bernal, Environmental Pollution 138, (2005) 46-58.
3. Metodi ufficiali di analisi chimica del suolo. Decreto Ministeriale del 13 Settembre 1999.
4. H. Kerndorff and M Schnitzer, Geochim. Cosmochim. Acta, 44 (1980) 1701-1708.
5. R.J. Bartlett, Environmental Health Perspectives 92, (1991) 17-24.
6. N.S. Bolan, D.C. Adriano, R. Natesan, B.J. Koo, J. Environ. Qual. 32, (2003) 120-128.
7. N. Senesi, G. Sposito, J.P. Martin, Sci. Tot. Environ. 55, (1986) 351-362.
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Hydrogels Filled with Humic-Rich Lignite for Various Environmental
Applications
Miloslav Pekař
Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University of
Technology, Purkyňova 118, 612 00 Brno, Czech Republic
E-mail: pekar@fch.vutbr.cz
1. Introduction
Lignite is a young coal with a high content of organic matter, especially humic acids. It could
not be therefore viewed as a fuel but rather as a valuable natural product and chemical raw
material [1–3]. Lignite can be used also in its natural state which represents the most costeffective way, e.g. as a sorbent, in-situ remediation agent or soil conditioner. Direct
application of lignite in its natural state is not “user-friendly” and, further, very small lignite
particles (dust) can suffer from stability problems, e.g. in soil. This work aimed at
incorporating lignite into poly(vinyl alcohol) (PVA) hydrogels to obtain easy usable material
capable of uptake of sufficient amount of water or aqueous solutions.
2. Materials and Methods
Lignite mined in the South Moravia (Czech Republic; for details see [1,2,4]) was used in its
natural state after the removal of water (105 °C) to the equilibrium value in ambient
laboratory atmosphere, i.e. to about 7%.
Poly(vinyl alcohol hydrogels were prepared by the freeze-thaw procedure [5]. PVA (Mowiol
28–99, Fluka) was prepared as 5% aqueous solution. Part of the solution was used directly to
prepare control (lignite-free) samples. Part of the solution was thoroughly mixed with defined
amount of milled lignite (particle size below 0.2 mm). Solution and suspensions were poured
into microtitration plates and placed to freezer (about –18 °C) for 16 h. They were then left to
thaw at laboratory temperature for 8 h. The freeze-thaw cycle was repeated four times.
Resulting hydrogels were left to dry in the air at laboratory temperature. In this way, pellets of
diameter of about 4 mm were prepared containing up to 90% (by weight) of lignite in the dry
state (xerogel).
Xerogels swelling was tested using deionized water until the constant mass was attained.
Swelling degree was calculated as the weight difference between the swelled gel and initial
xerogel relatively to the weight of the xerogel.
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Sorption capabilities were tested by two simple batch tests. First one used methylene blue in
the concentration of 100 mg/L, second one applied cupric ions in the concentration of 0.025
mol/L. Ratio hydrogel:solution was 0.1 g per 10 mL of the solution in the case of methylene
blue and 0.5 g per 10 mL in the case of copper. Concentrations of the dye and copper ions
were determined spectrophotometrically.
3. Results and Discussion
Filling with lignite lowered the swelling degree as can be seen in Fig. 1 but even the gel
containing about 90% of lignite was able to swell about a half of its weight. Lignite in the
freshly mined state contains about 50% of water, consequently, hydrogel swelling can be
attributed both to PVA network and lignite.
Sorption tests were made on hydrogel containing 33, 83, and 89% of lignite (in xerogel).
Sorption on the natural lignite gave final concentration of methylene blue of 6 mg/L and
cupric ions of 0.0055 mol/L in these tests. Control hydrogel and hydrogel with the lowest
amount of lignite showed no measurable sorption affinity for methylene blue. Hydrogels
containing 83 and 89% of lignite decreased the methylene blue concentration to 7 and 2 mg/L,
respectively. This indicates that at sufficiently high lignite filling in hydrogels the lignite
active sites for methylene blue are well accessible to the methylene blue solution penetrating
the hydrogel structure and hydrogel sorption capability is close to that of natural lignite.
Example of results obtained for copper sorption test is given in Fig. 2. Control hydrogel
showed only weak sorption capacity for copper which then increased with increasing filling
by lignite but still was appreciable lower than for the natural lignite. Active sites for copper
sorption are probably partially blocked by interactions of lignite functional groups with
hydroxyl groups of PVA, i.e. participate in forming the hydrogel network.
4. Conclusions
In summary, physical PVA hydrogels can be successfully filled by natural lignite particles
while still possessing acceptable swelling properties and sorption capabilities, especially for
organic sorptives. The optimum lignite filling is about 80% (by weight in the dry gel form).
Lignite can be thus immobilized and prepared in the more user-friendly form that combines
water retention and sorption capacities as well as contents of humic substances for various
sorption, remediation or agricultural applications.
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swelling degree (%)
200
150
100
50
0
33
37
43
83
89
lignite content (% in xerogel)
Fig..1. Influence of lignite on hydrogel swelling
0.02
Cu
2+
concentration (mol/l)
0.025
0.015
0
33
83
89
lignite contents (% in xerogel)
Fig. 2. Influence of lignite on copper concentration in equilibrium after the sorption from
the 0.025 mol/l copper solution
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Acknowledgements
Assistance of M.Dvořáková in experimental work is gratefully acknowledged. This work was
supported by government funding – Czech Science Foundation, project. Nr. 105/05/0404.
References
1. M. Klučáková, L. Doskočil, P. Bušinová and M. Pekař, in International Conference on Coal
Science & Technology (ICCS & T), Conference Proceedings CD. 2009, P12, p. 1–13.
2. M. Pekař, I. Sýkorová and I. Koutník, in Twenty-Fourth Annual International Pittsburgh Coal
Conference, CD-ROM Proceedings. PCC: Pittsburgh, 2007, P3–4, 13 pp.
3. M. Pekař, M. Klučáková, L. Omelka and P. Zedníčková, in Humic Substances – Linking Structure
to Functions, Proceedings of the 13th Meeting of the International Humic Substances Society.
F.H. Frimmel, G. Abbt-Braun, Eds., Schriftenreihe Bereich Wasserchemie Engler-Bunte-Institut
der Universität Karlsruhe, 2006, vol. 45-II, p. 1029–1032.
4. I. Sýkorová and O. Michna, Zesz. Nauk. Polit. Slas., Gornictwo, 249 (2001) 177.
5. I. Galeska, T.K. Kim, S.D. Patil, U. Bharwaj, D. Chattopadhyay, F. Papadimitrakopoulos and D.J.
Burgess, AAPS Journal, 7 (2005) E231.
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Mitigation of GHGs Emission from Soils by a Catalyzed in situ Photooxidative Polymerization of Soil Humic Molecules
Alessandro Piccolo*, Riccardo Spaccini
Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali,
Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy
E-mail: alessandro.piccolo@unina.it
1. Introduction
In 2005, agriculture accounted for an estimated emission of 5.1 to 6.1 GtCO2-eq/yr (10– 12%
of total global anthropogenic emissions of greenhouse gases (GHGs)). However, measures to
mitigate GHGs emission from agricultural soils are limited to improved cropland practices
such as crop rotation, nutrient management, tillage/residue management, agroforestry, and
return to natural vegetation. These practices are not only far from substantially reducing
GHGs emissions from soils or permanently stabilizing soil organic matter, but are also
predicted to hardly match more than a maximum of 25% of the GHGs reductions required by
the Kyoto Protocol within 2050. Despite the knowledge that GHGs release from soil largely
derives from biochemical transformations of plant litter and soil humus (SH), no new and
much wished biotechnological measures are adopted so far to augment mitigation. Here we
propose an innovative approach to mitigate GHGs emissions from soils based on the in situ
photo-polymerization of SH under biomimetic catalysis. Three Mediterranean soils of
different physical and chemical properties were added with a synthetic watersoluble ironporphyrin, irradiated by solar light, and subjected to 15, and 30 wetting and drying cycles.
2. Materials and Methods
Soil Samples and Characterization. Soil samples were collected from the surface layers (0-30 cm)
of three agricultural plots from south-central Italy: 1. Porrara (Avellino), 2, Colombaia (Caserta),
3. Itri (Latina). Samples were air dried, sieved through a 4.75 mm sieve, and used for
characterization and incubation experiments.
Photo-polymerization experiments. For each replicate (n = 3), 60 g of air dried soil sample was
placed on a Petri dish (12 cm diameter) and soil moisture was kept at 40% of water holding
capacity (WHC) by adding X mL of water (X = 20, 17, 10 mL, for Porrara, Colombaia and Itri
soil, respectively) in order to obtain a control series. The polymerized series were similarly
prepared (n = 3) and added with 0.24 μmol of synthetic water-soluble iron-porphyrin (mesotetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate
of
iron(III)
chloride,
Fe-(TDCPPS)Cl)
dissolved in the X mL of water pertaining to WHC of each soil. After preparation, both control
and polymerized series were left under natural solar radiation throughout the following
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treatments: (i) covered with a Petri dish and incubated for 5 d; (ii) submitted to 15 wetting/drying
cycles; (iii) submitted to 30 wetting/drying cycles. During wetting/drying cycles, samples were
uncovered after 5 d incubation and distilled water was added, whenever samples became dry
(approximately once a week), to reestablish WHC.
Aggregate stability. An air–dried sub–sample (30 g) was placed on the top sieve of a set of three
nested sieves (1.0, 0.50, and 0.25 mm) and submerged into 2 cm of distilled water for 30 min.
After this time, the sieves were manually oscillated (up and down 4 cm) for 30 times during 1
min. Recovered aggregate fractions were oven–dried at 60°C, weighed, and stored at room
conditions. The mean weight diameter index in water (MWDw) used for the determination of
aggregate stability was calculated according to the equation:
n
MWDw = ∑ X iWi
i =1
where Xi is the mean diameter of each aggregate fraction and Wi is the proportion of the total
sample weight occurring in the i–th fraction. The amount of OC (%) in each aggregate fraction
was normalized to the weight of each fraction: OC content in fraction (g kg–1) × mass of
recovered fraction (g kg–1) / total OC recovered (g kg–1).
Soil respiration. Soil respiration was evaluated by a dynamic absorption method. Briefly, 9 g of
air dried and rewetted soil sample (< 2 mm) were placed on a air-tight soil respiration flask in
which a CO2-free air was continuously fluxed by a peristaltic pump. The CO2 emitted from soil
was then captured in a trap containing a 0.01 M NaOH solution. The amount of CO2 absorbed in
this solution was determined after 27 days by back titration with 0.01 M HCl after addition of 7
mL of 0.5 M BaCl2. The ambient CO2 concentration was determined by inserting blank samples
(i.e.: no soil) into the respiration system.
Statistical analysis. A Student’s t-test was used to compare values obtained for control and
treatments, and difference was considered to be significant at the level of P ≤ 0.05.
3. Results and Discussion
The humified organic matter in soil (70–80% of SOM) represents the principal potential C
sink in the biosphere, whose advanced comprehension may help to mitigate CO2 emissions
from soil. Soil humus is composed by the hydrophobic and heterogeneous aliphatic and
aromatic molecules progressively surviving the microbial transformation of dead biological
tissues [1]. Recent scientific evidence shed new light on the chemical nature of humus by
describing humic molecules as heterogeneous but relatively small in mass (≤ 1000 Da) [2],
rather than the previously assumed macropolymers [3]. Humic molecules were shown to be
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tightly associated in supramolecular structures, which are prevalently stabilized by
noncovalent hydrophobic bonds [1]. Based on this, it is reasonable that small aromatic humic
molecules could be covalently linked to each other by oxidative coupling reactions under
appropriate catalysis, thereby enhancing their molecular size and complexity. It is already
shown that larger and more chemically-stable humic molecules were obtained by treating
humic solutions in oxidative (H2O2) conditions, with a phenoloxidase enzyme, such as
peroxidase [1], or a biomimetic catalyst, such as an iron-porphyrin [4]. Both the enzymatic
and biomimetic catalysts accelerate the oxidative coupling of phenols via a free-radical
mechanism. Moreover, the biomimetic catalysis increased the molecular dimension of humic
matter in solution simply by photo-oxidation under solar radiation, without the need of an
additional oxidant [5]. By applying photo-oxidative catalysed conditions on humic phenolic
monomers, a rapid formation of new intermolecular C-C and C-O-C bonds led to several
identified (up to tetramers) and unidentified oligomers [6–8].
The photo-polimerization technology can be applied in situ on soils. The catalyzed photooxidative formation of covalent bonds among soil phenolic molecules would chemically
stabilize SH by increasing the content of chemical energy in humic structures and
consequently reducing the extent of SH biomineralization. Moreover, increasing the mass of
humus molecules would result in linking together soil particles to larger soil aggregates and
thus improving soil physical quality.
Figure 1: Mean Weight Diameter in water (MWDw) for the three soils, Porrara, Colombaia, Itri,
before and after photo-polymerization treatment for 5 d incubation, and 15 and 30 wetting and drying
cycles. Error bars indicate standard error (n = 3). The asterisks denote a significant difference between
control and treatment at the level of P ≤ 0.05
The occurrence of SOM photo-polymerization was suggested by the mean-weight diameter of soil
aggregates in water (MWDw), an index of aggregate stability, that significantly increased over control
for all the three soils after the 5 d incubation (Fig. 1), though to a different extent depending on the
intrinsic composition of each soil. This indicates that the photo-polymerization treatment, by
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increasing the molecular mass and cross-linking of humic molecules, promoted a tighter association
among soil particles and formation of larger water-stable aggregates. An enhanced soil aggregate
stability in the photo-polymerized samples was still kept after 15 w/d cycles, but it was lost for all
soils after 30 w/d cycles.
Figure 2: Soil respiration (mg CO2.g-1 of soil) from soils, Porrara, Colombaia, Itri, before and after
photo-polymerization treatment for 5 d incubation, and 15 and 30 wetting and drying cycles. Error
bars indicate standard error (n = 3). The asterisks denote a significant difference between control and
treatment at the level of P ≤ 0.05
A stronger chemical and physical stabilization of OC after the catalytic photo-polymerization of soils
can be inferred by the amount of respired CO2 (Fig. 2). Microbial mineralization of SH was
significantly inhibited in the photo-polymerized samples, as compared to control, for all soils after 5 d
incubation and even after 15 w/d cycles. While the reduction of CO2 emission was significant for
Porrara and Itri soils also after 30 w/d cycles, this was no longer true for the Colombaia soil. The CO2
respiration behaviour of Porrara and Itri soils confirmed that the catalytic photo-polymerization
strongly stabilized their SH. The different result for the Colombaia soil after 30 w/d cycles may be
attributed to its larger amount of OC, thus limiting the treatment effect. The OC stabilization obtained
in Porrara, Colombaia, and Itri soils mitigated the CO2 emission by, respectively, 12.8, 7.4, and 29.4 %
after 5 d incubation, 8.3, 5.6, 18.7 % after 15 w/d cycles, and 7.0, -2.0, 15.9 % after 30 w/d cycles.
Such mitigation corresponded to 0.34, 0.16 and 0.20 Mg of CO2.ha-1 for Porrara, Colombaia, and Itri
soils, respectively, after 5 d incubation, and still to 0.18 and 0.20 Mg of CO2.ha-1 for Porrara and Itri
soils, respectively, even after severe disaggregation of 30 w/d cycles. The results confirm the
effectiveness of the proposed technology.
References
1. A. Piccolo, Adv. Agron. 75 (2002) 57.
2. A. Piccolo and M. Spiteller, Anal. Bioanal. Chem. 377 (2003) 1047.
3. A. Piccolo, P. Conte and A. Cozzolino. Soil Sci. 166 (2001) 174.
4. A. Piccolo, P. Conte and P. Tagliatesta, Biomacromolecules 6 (2005) 351.
5. D. Smejkalova and A. Piccolo, Biomacromolecules 6 (2005) 2120.
6. D. Smejkalova and A. Piccolo, Environ. Sci. Technol., 40 (2006) 1644.
7. D. Smejkalova, A. Piccolo and M. Spiteller, Environ. Sci. Technol., 40 (2006) 6955.
8. D.Smejkalova, P. Conte and A. Piccolo, Biomacromolecules, 8 (2007) 737.
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Sorption of Endocrine Disruptors by Humic Substances from Sediment
Samples Collected on Guarapiranga Reservoir, São Paulo State-Brazil
Bruno Barboza Cunhaa,b, Wander Gustavo Boteroa, Luciana Camargo de Oliveiraa,
Guilherme Carvalho Leiteb, Danielle Goveiaa,b, Viviane Moschini Carlosb, Marcelo Luiz
Martins Pompêoc, Leandro Cardoso de Moraisb, Leonardo Fernandes Fracetob, André
Henrique Rosa*b
a
Institute of Chemistry – UNESP, Araraquara-SP, Brazil; b Department of Environmental
Engineering – UNESP, Sorocaba-SP, Brazil; Institute of Biosciences – USP, São Paulo,
Brazil
E-mail: ahrosa@sorocaba.unesp.br
1. Introduction
Endocrine disrupting chemicals (EDCs) are defined as exogenous substances that alter the
functions of the endocrine system and consequently cause adverse health effects in an intact
organism, or its progeny. Researches about EDCs have been increasing due to their
widespread occurrence, persistence, bioaccumulation and potential adverse effects on
ecosystem functioning and human health [1–3].
The EDCs are considering an emergent and dangerous class of contaminants in aquatic
systems because already are present in high concentrations natural waters and the actual
wastewater treatment plants can’t remove them. In aquatic systems there are many factors that
can control the transport, availability and reactivity of EDCs like: pH, redox potential and
specially content/characteristics of humic substances present in the water and sediment.
However, there are only a few papers in the literature associated with the interactions between
Humic Substances (HS) from sediments and EDCs [1, 3, 5]. Then, the primary objective of
this study was to characterize the interactions beetwen Bisphenol A (BA), Estrone (E), 17β–
Estradiol (E2) and 17α–Ethinylestradiol (EE) in two different sediments collected on
Guarapiranga Reservoir, São Paulo City-Brazil. The adsorption process was evaluated and
Langmuir/Freudlich parameters were obtained from sorption experiments.
2. Materials and Methods
Sorption experiments. The sorption experiments were done with two different sediments
collected at Guarapiranga Reservoir, São Paulo State, Brazil. All equilibrium batch
experiments were conducted in triplicates in 100-mL glass vials. Between 0.1 and 1 g of
sorbent were weighed into the vials, which were filled with 50 mL of milli-Q water with 2 mg
L-1 of each endocrine disruptor (BA, E, E2 and EE). Vials were shaken on a horizontal plate
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shaker at 180 rpm for 1 day at a constant temperature of 25 oC. Blanks were set up using the
same solid-to-water ratios as the samples but without adding BA, E, E2 and EE. Similar
experiments were done to check the equilibrium sorption with the same conditions, except
that 1 g of sorbent were weighed into the vials and were taken off aliquots of 1 mL at 5, 10,
15, 30, 60, 120, 240,360, 480 and 1440 min.
Chromatography analysis. Optimization of SPE extraction of analytes using Nexus cartridges
(Varian) was performed according to the Nexus applications recommendations for endocrine
disruptors. The derivatization of standard solutions and samples was performed in a test tube,
according to the procedure proposed by Jeannot [4] and the Varian GC-MS system was
employed for analysis.
3. Results and Discussion
Sorption experiments. Adsorption kinetics curves measured for BA, E, E2 and EE in two
sediments with different contents or organic matter are shown in Figure 1. In all cases,
adsorption of EDC onto sediments examined appears to occur in two steps, a rapid one
occurring in the first few hours of contact (in this case less than 2 h), which generally
corresponds to more than 80% of total adsorption, and a slow one that may need several hours
until the attainment of the equilibrium.
100
100
90
90
80
70
40
60
-1
-1
50
q (μg g )
BA
E
E2
EE
60
q (μ g g )
BA
E
E2
EE
80
70
50
40
30
30
20
20
10
10
0
0
0
a)
200
400
600
800
1000
1200
1400
1600
0
200
b)
Time (min)
400
600
800
1000
1200
1400
1600
Time (min)
Figure 1: Adsorption kinetics curves of EDCs in a) sediment with less organic matter content and b)
sediment with more organic matter content
EE appears to be the most adsorbed EDC onto all substrates. The rapid adsorption phase
would occur on the most reactive and/or accessible sites of the sediment, whereas the slower
adsorption may reflect the involvement of less reactive and/or more sterically hindered sites.
In all cases, an equilibration time of 24 h has been considered adequate and has been used
for the measurements of adsorption isotherms.
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15th IHSS Meeting- Vol. 3
Sorption isotherms. In this work were tested Langmuir and Freudlich sorption models. The
Langmuir parameter were calculated by regression analysis using the equation
qe =
bQ o Ce
, where qe is the concentration of a chemical adsorbed in the solid phase (µg
(1 + bCe )
g1); b is affinity; Qo is the Langmuir coefficient; Ce is the equilibrium solution concentration
(µg L-1) Freundlich parameters were calculated by regression analyses using the equation
qe = k e + C e
(1 / n )
, where qe and Ce has the same meaning of Langmuir equation; kF is the
Freundlich coefficient((µg g-1)/(µg L-1)1/n) and 1/n the Freundlich exponent, and are
summarized in Table 1.
Table 1: Langmuir and Freudlich parameters obtained from sorption experiments
Isotherm
Sediment
Compound
P1
BA
P1
E
P1
E2
P1
EE
P2
BA
P2
E
P2
E2
P2
EE
Langmuir parameters
b
r
Qo
0.125
-0.344
0.7926
0.200
-1.174
0.5528
0.399
-0.418
0.7561
0.510
-0.429
0.9936
0.193
-0.370
0.9432
0.243
-0.371
0.9601
0.255
-0.388
0.9824
0.668
-0.466
0.9966
Freudlich parameters
kF
n
r
11
4.29x10
-0.033
0.7416
9.17
-0.482
0.2935
57.00
-0.239
0.5537
7.42
-0.593
0.9631
5.63x10-7
0.123
0.9700
2.98x10-6
0.140
0.9826
8.09x10-6
0.148
0.9920
5.58x10-4
0.225
0.9999
Table 2 – Pseudo second order kinetics parameters obtained from sorption experiments
Sediment
P1
P1
P1
P1
P2
P2
P2
P2
Kinetic
Compound
BA
E
E2
EE
BA
E
E2
EE
h
0.525
3.866
0.952
4.359
17.844
2.748
4.697
23.952
qe
30.93
24.21
26.30
73.86
27.89
36.44
59.21
86.80
Parameters
K
5.49×10-4
6.59×10-3
1.38×10-3
7.99×10-4
2.30×10-2
2.07×10-3
1.34×10-3
3.18×10-3
r
0.9953
0.9992
0.9968
0.9978
0.9986
0.9993
0.9998
0.9999
Data presented in Table 1 shows that the sorption performed with the sample containing higher humic
substances content is more suited to the Freudlich model. All kinetics experiments have a good fit of
pseudo second order kinetics model, with correlation coefficients grater than 0.9953. It is possible to
see in Table 2 that there is a higher rate of adsorption for the samples with higher content of HS,
except for estrone that can interact preferentially with organominerals present in the sediment samples.
Vol. 3 Page - 227 -
15th IHSS Meeting- Vol. 3
4. Conclusions
These results demonstrate that the availability of endocrine disruptors could be directly
related to the presence of humic substances in aquatic systems. Consequently, studies of the
interactions between AHS and endocrine disruptors are vital for a better understanding of the
transport and/or reactivity of this type of contaminant in the environment.
Acknowledgements
The authors gratefully acknowledge the funding of their work by FAPESP and CNPq
(Brazilians Agencies).
References
1. R. Liu, J.L. Zhou, A. Wilding, Journal of Chromatography A, 1038 (2004) 19.
2. G.R. Aiken, R.L. Wershaw, P. MaCcarthy, Humic Substances in Soil, Sediment and Water—
Geochemistry, Isolation and Characterization, Wiley, New York, 1985.
3. J. Lintelmann, A. Katayama, N. kurihara, L. Shore, A. Wenzel, Pure Appl. Chem., 75, 5, (2003)
631.
4. R. Jeannot, H. Sabik, E. Sauvard, T. Dagnac, K. Dohrendorf, J. Chromatogr., 974 (2002) 143.
5. E. Loffredo, N. Senesi, Developments in Soil Science, 28A (2002), 143.
6. A. Navarro, S. Endo, T. Gocht, J. A. C. Barth, S. Lacorte, D. Barceló, P. Grathwohl,
Environmental Pollution, 157 (2009) 698.
7. L.P.C. Romão, G.R. Castro, A.H. Rosa, J.C. Rocha, P.M. Padilha, H.C. Silva, Anal. Bioanal.
Chem., 375 (2003) 1097.
8. D.J. Hawke, K.J. Powell, J.E. Gregor, Marine Fresh. Res.,47 (1996) 11.
9. M.L. Pacheco, E.M. Pena-Méndes, J. Havel, Chemosphere, 51 (2003) 95.
10. S. Chen, W.P. Inskeep, S.A. Williams, P.R. Callis, Soil Sci. Soc. Am. J., 56 (1992) 67.
11. R.S. Swift, in: D.L. Sparks, (Ed), Methods of Soil Analysis: Chemical Methods, SSSA, Maddison,
1996, p.1011.
Vol. 3 Page - 228 -
15th IHSS Meeting- Vol. 3
Dual Effect of Humic Acid on the Degradation of Pentachlorophenol by
Iron(II) and H2O2
Konstantinos C. Christoforidis, Maria Louloudi, Yiannis Deligiannakis
Laboratory of Physical Chemistry, Department of Environmental and Natural Resources
Management, University of Ioannina, Seferi 2, 30100 Agrinio, Greece
E-mail: kchristofo@gmail.com; mlouloud@cc.uoi.gr; ideligia@cc.uoi.gr
1. Introduction
Pentachlorophenol (PCP) is a well known organochlorine compound used extensively as
wood preservative. Chlorophenols are highly toxic and are poorly biodegradable [1] therefore
efficient abiotic methods are needed for their removal from the environment [2].
Fenton reaction [3] has been extensively used for the degradation of a broad range of organic
pollutants. The HO· radicals generated from the Fenton reaction are highly reactive radicals
[4]. Since HO· radicals are nonselective, they can react with non-pollutant substances such as
natural organic mater. This postulates a pervasive effect of humic acid (HA) on a Fenton
system which can be crucial for the detoxification of natural waters. However the effect of
HA on the catalytic efficiency of the Fenton reaction still remains controversial, since the
degradation of organic pollutants has been reported to be either inhibited [5,6] or enhanced
[6,7] in the presence of humic materials. For example, recently has been reported that the
initial catalytic conditions may either increase or decrease HO· production [8].
In the present study, the efficiency of the Fenton reaction on the decomposition of PCP in the
presence of HA was studied by catalytic and EPR methods. The data reveal that ratio [HA/Fe]
is the determining factor which can result in either enhancement or inhibition of PCP
decomposition. An EPR-based method is proposed for the estimate of the optimal [HA/Fe]
ratio in order to enhance the catalytic performance.
2. Materials and Methods
HA used was a lignite sample extracted from a mining site of Greece according to the
protocols of International Humic Substances Society (IHSS) [9]. PCP was purchased from
Aldrich and H2O2 was obtained as a 30% solution from Fluka. FeSO4·7H2O (Merck) was used
in the Fenton reactions. For the EPR study of Fe-HA complexation Fe2(SO4)3·xΗ2Ο (Riedelde Haën) was used. 2-isopropanol was obtained from Merck.
Vol. 3 Page - 229 -
15th IHSS Meeting- Vol. 3
All aquatic stock solutions were prepared in ultra-pure Milli-Q water. An 8g/L HA stock
solution was incubated for 24 h at pH 12 adjusted with NaOH. Afterwards, the solution was
brought to pH 3 by adding H2SO4 and stored at 4oC. Aquatic stock solutions of FeSO4·7H2O
(100 mg/L, at pH 1 adjusted with H2SO4) and of H2O2 (3330 mg/L) were prepared. A stock
solution of 2000 mg/L PCP was prepared in acetonitrile. Catalytic mixtures were prepared at
pH 3.5, in the absence and presence of 20 mg/L HA, containing 13 mg/L PCP and varied
concentration of H2O2 (4.35–54 mg/L) and FeSO4·7H2O (0.87–32.5 mg/L). All reactions
were kept in dark during the reaction time. Quantification of PCP was performed with HPLC.
For the EPR experiments 0.4mM of Fe2(SO4)3·xΗ2Ο were incubated with 230, 1710 and 5110
mg/L HA for 2 h at pH 3.5.
3. Results and Discussion
PCP decomposition. Control experiments have shown that [H2O2] does not affect
significantly the degradation of PCP in either the classic or the HA-modified Fenon reaction.
On the other hand, [FeSO4·7H2O] appeared to have a decisive effect on PCP degradation.
Specifically, depending on the concentration of FeII, 60–100% and 35–100% of PCP was
removed by the classic and HA-modified Fenton reaction. Control experiments in the
presence of 2ml isopropanol in all HA-modified Fenton reactions resulted to no conversion of
PCP within 9 days (data not shown). This suggests that the degradation of PCP in the
presence of HA is due to the formation of OH· like in the classic Fenton reaction. However,
for [FeSO4·7H2O] below 19.5 mg/L in the presence of HA the kinetics is severely lower
compared to the unmodified Fenton reaction.
100
A
100
B
% [PCP] Remained
Decrease Increase
80
80
Decrease Increase
60
60
40
40
20
20
0
0
0
5
10
15
20
25
30
0
5
[FeSO4Ž7H2O] (ppm)
10
15
20
25
30
Figure 1: Effect of [FeSO4·7H2O] on PCP removal in the absence („) and in the presence of 20 mg/L
HA ({). Reaction time (A) 3 days and (B) 9 days. Catalytic conditions: 13 mg/L PCP, 52 mg/L H2O2
Vol. 3 Page - 230 -
15th IHSS Meeting- Vol. 3
Reaction Yield. The effect of varying [FeSO4·7H2O] on the decomposition of PCP at 3 or 9
days in the presence and absence of HA is compared in Fig. 1. The data show that there is a
crossing in the reaction yield vs. [FeSO4·7H2O] which occurs at [FeSO4·7H2O] = 6.5 mg/L.
This crossing implies the existence two –or more- interfering mechanisma. In particular, in
the presence of 0.87 and 2.6 mg/L FeSO4·7H2O the presence of 20 mg/L HA inhibited PCP
removal -at any reaction time- under the conditions of our experiment (Fig. 1). On the other
hand, at higher FeSO4·7H2O concentration (i.e. >6.5 mg/L), the decomposition of PCP
increased dramatically compared to the reaction without HA. At 6.5 mg/L of FeSO4·7H2O a
characteristic point appeared where the performance of the catalytic reaction was the same in
the presence or absence of HA. These results show that HA has a rather complex effect on the
removal of PCP, i.e. it can act either as an enhancer or as an inhibitor of the Fenton reaction
depending on the FeSO4·7H2O concentration.
Overall, under the conditions of our experiments, HA appears to play a significant dual role
on the decomposition of PCP by Fenton reaction. The data revealed that HA can act either as
an inhibitor or as an enhancer. A key observation is that the function of HA is correlated with
the iron concentration. The beneficial role of HA in total PCP conversion is observed only
when [FeSO4·7H2O]>6.5 mg/L. Based on the results presented in Figure 1, the total
conversion of PCP is determined by the ratio HA/Fe. In particular when [HA
(mg)]/[Fe(μmol)]<0.85 the total PCP conversion is increased otherwise it remains constant or
decreases with respect to the non modified Fenton reaction. The same trend was observed for
the initial rates as well.
EPR Spectroscopy. Figure 2A shows EPR spectra of Fe2(SO4)3·xH2O at various HA
concentrations. Three different HA concentrations were selected, i.e. 5110, 1710 and 230
mg/L corresponding to HA/Fe ratios similar to reactions with [FeSO4·7H2O] 0.87 mg/L, 2.6
mg/L and 19.5 mg/L respectively.
At pH 3.5 in the absence of HA no EPR signal was observed. This is due to the fact that in
aqueous solution at pH>2.7 FeIII forms hydroxylated species Fek(OH)m which are EPR silent.
However, at low pH values (pH=1) a signal with g=4.3 and E/D=0.33 -characteristic of
mononuclear high spin FeIII - was observed. On the other hand, in the presence of HA at pH
3.5 a high spin FeIII with g=4.3 was detected. This shows that HA mentains mononuclear FeIII
species, acting as chelator of Fe.
Vol. 3 Page - 231 -
Signal Intensity
A
dX''/dH (au)
(e)
(d)
(c)
400
800
1200
40
B
Increase Decrease
of PCP
of PCP
oxidation oxidation
30
1600
2000
2400
[FeSO4•7H2O]=0.87 ppm
20
[FeSO4•7H2O]=2.6 ppm
No HA
10
III
(b)
(a)
% [Fe Adsorbed + (EPR silent species)]
15th IHSS Meeting- Vol. 3
[FeSO4•7H2O]=19.5 ppm
0
0
1500
3000
4500
[HA] (ppm)
Magnetic Field (G)
Figure 2: A) EPR spectra of 0.4mM Fe2(SO4)3·xH2O incubated in the absence of HA, spectrum (a)
and (b) (spectrum (b) has 30 % v/v glycerol) and in the presence of (c) 230, (d) 1710 and (e) 5110
mg/L HA for 2 h at pH 3.5. B) [FeIII-HA+ Fe-SL] % vs. [HA] used for each sample. Each case is
labelled with the [FeSO4·7H2O] of the corresponding Fenton reaction
Figure 2B shows the percent of the sum of FeIII that has been adsorbed by HA (Fe-HA) along
with the EPR silent Fe species (Fe-SL), i.e. FeIII reduced to FeII by HA [10] or dimers of iron.
It is observed that increase of the HA concentration resulted to an increase of [Fe-HA + FeSL]. Based on the data in Figure 2B, the % of [Fe-HA + Fe-SL] where the oxidation of PCP
is increased in the presence of HA is estimated to be less than 7.5%, otherwise it is decreased
(areas indicated by the arrows).
Comparing the catalytic with the spectroscopic EPR data we observe that (a) in cases where
the presence of HA inhibits the decomposition of PCP, the [Fe-HA + Fe-SL] ratio is 40% and
25% ([FeSO4·7H2O]=0.87 and 2.6 mg/L) respectively, while it is only 1% for the case where
HA improves the modified Fenton reaction ([FeSO4·7H2O]=19.5 mg/L). Furthermore, HA
could act as a sink of HO·. In this way HA inhibits the oxidation of PCP by the Fenton
reaction, competing in this way with PCP and resulting to a significant degrease of the
reaction’s efficiency. Reaction of HA with OH· has been reported in the literature in a photoFenton system [11].
Overall, our results indicate that FeIII EPR intensity, along with high [Fe-HA + Fe-SL] ratio
determines the catalytic efficiency depending on the HA/Fe ratio.
4. Conclusions
Based on the catalytic results, the only way that HA may affect catalytic removal of PCP is:
(I) to chelate strongly Fe without allowing it to react with H2O2 and
(II) to promote the Fenton redox cycle of Fe [12].
Specifically, case (I) would hold when [HA(mg)]/[Fe(μmol)]>0.85 while case (II) when
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15th IHSS Meeting- Vol. 3
[HA(mg)]/[Fe(μmol)]<0.85. This could be explained by the presence of two different sites in
HA able to chelate Fe. This is supported by the observation of Senesi et al. who demonstrated
the presence of at least two different binding sites of Fe in humic substances using EPR
spectroscopy [13]. HA may also compete with the substrate and react with the produced OH·
but our results can be explained only if cases (I) and (II) are occuring.
While other works have demonstrated that the catalytic decomposition of pollutants by the
Fenton reaction can be either increased or decreased in the presence of humic substances, the
present work reports that both functions can occur depending on the initial catalytic
conditions. Special care should be given on the ratio [HA/Fe]. However, given the complexity
and diversity of HA samples [14], this ratio may vary from sample to sample. In this aspect,
EPR spectroscopy can act as a powerful tool in order to extract the appropriate HA/Fe ratio
where the ratio [Fe-HA + Fe-SL] relative to the initial [Fe] is not high (<7.5%) in order to
enhance the catalytic performance in the presence of HA.
References
1. K.I. Abe and K. Tanaka, Chemosphere, 35 (1997) 2837.
2. M. Pera-Titus, V. Garcia-Molina, M.A. Banos, J. Gimenez, S. Esplugas, Appl. Catal. B: Environ.,
47 (2004) 219.
3. C. Walling, Acc. Chem. Res., 8 (1975) 125.
4. G.V. Buxton, C.L. Greenstock, W.P. Helman, A.B. Ross, W. Tsang, J. Phys. Chem. Ref. Data, 17
(1988) 513.
5. M. Lindsey, M.A. Tarr, Environ. Sci. Technol., 34 (2000) 444.
6. M.Z. Li, P.J. Shea, S.D. Comfort, Chemosphere, 36 (1998) 1849.
7. M. Fukushima, K. Tatsumi, Environ. Sci. Technol., 35 (2001) 1771.
8. C. Ciotti, R. Baciocchi, T. Tuhkanen, J. Hazar. Mater., 161 (2009) 402.
9. M. Drosos, M. Jerzykiewicz, Y. Deligiannakis J. Coll & Intrf. Sci. 332 (2009)78–84.
10. M.D. Paciolla, S. Kolla, S.A. Jansen, Advan. Environm. Res., 7 (2002) 169.
11. M. Fukushima, K. Tatsumi, Environ. Sci. Technol., 35 (2001) 3683.
12. Y. Sun, J.J. Pignatello, J. Agric. Food Chem., 40 (1992) 322.
13. N. Senesi, S.M. Griffith, M. Schnitzer, Geochim. Cosmochim. Acta, 41 (1977) 969.
14. F.J. Stevenson, in Humus Chemistry: Genesis, Composition, Reactions; John Wiley & Sons, Inc.,
New York, 1994.
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Radiotracer Method in the Investigation of Humic Substances Sorption on
Carbon-Based Nanomaterials
Maria G. Chernysheva, Gennadii A. Badun
Lomonosov Moscow State University, Leninskie Gory, 119992, Moscow, Russia
E-mail: masha.chernysheva@gmail.com
1. Introduction
Nanomaterials of carbon are widely use in different fields. The physical, chemical, and
electronic properties of carbonaceous nanomaterials are strongly coupled to carbon’s
structural conformation and, thus, its hybridization state. Mutable hybridization states of
carbon account for the diversity of organic compounds as well as the considerable differences
among carbon’s bulk configurations, which changes from sp3 for nanodiamond to sp2+π for
graphene. Studying the properties of carbonaceous nanomaterials is the important field of
modern science of material. Now days carbon nanomaterials particularly carbon nanotubes
(CNT) are tested as a unique substrate for sorption of biomolecules including peptides [1] and
proteins [2, 3]. In review [4] single-walls carbon nanotubes were also tested for removal of
contaminants in drinking water. It is appear to be perspective to study the binding of humic
substances with different types of nanocarbon. The goal of this research was to compare the
sorption ability of nanodiamonds and graphene unto humic materials by means of
radiochemical approach [5] using tritium labeled humic materials and liquid scintillation
spectrometry.
2. Materials and methods
Brown coal humic acids (CHA) (commercially available preparation Powhumus (Humintech,
Germany)) was used. Tritium label was introduced in CHA by means of thermal activation
method. The labeling technique and purification procedures were previously described in Ref.
[6].
Sorption experiments with carbon nanomaterials were conducted at room temperature in
0.028 M phosphate buffer (pH 6.8) for the initial concentrations from 1 to 360 mg/L. Sample
of nanomaterial was placed in the test-tube followed by the addition of [3H]-CHA solution in
phosphate buffer. Mixture was sonicated during 15 min. Then a bit of suspension was picked
out, centrifuged and its counting rate was measured by liquid scintillation counter RackBeta
1215 (Finland). Equilibrium concentration of CHA solution was calculated from the specific
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15th IHSS Meeting- Vol. 3
radioactivity of CHA and radioactivity of the solution. Adsorption amount of CHA was
calculated as a difference between initial and equilibrium amount of CHA in the solution.
3. Results and Discussion
Equilibrium CHA concentrations were from 0.2 to 150 mg/L. Comparative analysis of
adsorption isotherms obtained shows that on both nanodiamonds and graphene CHA
adsorption increase with concentration growth until 100±10 for nanodiamonds and 30±5
mg/L for graphene. Then it archive plateau and does not change for nanodiamonds, but start
increase again in case of graphene at 120±10 mg/L. The value of adsorption per gram of
nanocarbon in plateau region was 20±5 mg/g for both materials. It has to be noted that
desorption under the same conditions was ca. 30 % for HS on nanodiamonds and less then
10 % in case of graphene. It was also found that graphene with adsorbed CHA forms stable
suspension in aqueous solution while graphene itself is to hydrophobic to be placed in water.
4. Conclusions
Radiochemical assay for the first time was applied for studying sorption of HS on carbonbased nanomaterials. This approach allow conduction of sorption experiment with HS in wide
range of concentrations from ultra low (less then 1 mg/L) to hundreds mg/L. The difference in
the adsorption isotherms results in the influence of carbon structure.
Acknowledgements
This work was supported by Russian Federal Agency of Education (project # 2351P).
References.
1.
2.
3.
4.
5.
6.
G.R. Dieckmann et al., J. Am. Chem. Soc. 125 (2003) 1770.
L. Song et al., Colloids and Surfaces B. 49 (2006) 66.
L.E. Valenti et al., J. Colloid Interface Sci. 307 (2007) 349.
V.K.K. Upadhyayula et al., Sci. Total Environment. 408 (2009) 1.
A.V. Severin, G.A. Badun, Z.A. Tyasto. Radiochemistry, 51 (2009) 55.
G.A. Badun et al, Radiochimica Acta, (2010) (in press).
Vol. 3 Page - 235 -
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Study of Flow-Through Sample Preparation Methods for Group of
Pesticides Determination in Soil by Reversed-Phase High-Performance
Liquid Chromatography
Mária Chalányová*, Milan Hutta, Ivana Procházková
Department of Analytical Chemistry, Faculty of Natural Sciences,
Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovakia
E-mail: chalanyova@fns.uniba.sk
1. Introduction
Soil is a heterogeneous system containing organic and inorganic matter. It contains solid
particles, soil colloids and gases among which equilibrium is established [1]. Soil from this
point-of-view is a complex matrix and the isolation of analytes from soil is a complex
analytical problem [2]. From literature survey clearly follows that the soil sample pretreatment is usually a critical and time consuming step also due to presence of humic
substances. The basis of successful analytical method for determination of pollutant residues
in complex matrices at trace concentration levels is the use of selective isolation techniques
[3, 4] enabling isolation of analytes with high recovery and with minimum quantity of matrix
co-extracts. Flow-through methods, namely matrix solid phase dispersion (MSPD) and offline flow-through solid-liquid extraction (FSSLE) for isolation eight common pesticides
(atrazine, propazine, simazine, terbutrine, metoxuron, cloquintocet-mexyl, cypermethrin
and permethrin) from soils before their RP-HPLC analysis were studied.
2. Materials and Methods
Procedure of MSPD pre-treatment of soil sample:The bottom of plastic column was filled by
0.05 g silica or florisil and mixture of 0.50g of contaminated and uncontaminated soil sample
homogenized with around 0.50 g of sorbent. The mixture of 8 pesticides was eluted with 2.00;
2.50 and 3.00 ml of 100% of methanol, resp. Collected extract volume was reduced to 0.20 or
0.50 ml and reconstituted in 0.50 or 1.00 mL volume in the mobile phase methanol:water 1:1
(v/v).
Procedure of off-line flow-through solid-liquid extraction (FTSLE) pretreatment of soil
sample: bottom part of CGC column (150x3 mm) was filled with mass of Silica L 40/100
ranging from 0.05 g to 0.20 g. This layer was topped by fill and tap method by 1.00 g of soil
sample (alternatively, original soil or soil fortified by pesticides at various concentration
levels). The rest of the column volume was filled up by glass beads (0.5 mm diameter).
Studied analytes were extracted by various volumes of methanol by flow rates alternatively
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15th IHSS Meeting- Vol. 3
switched between 0.5 and 0.3 mL/min. Further processing of the extract was the same as is
described above.
3. Results and Discussion
Influence of mobile phase pH to the pesticides retention was thoroughly studied for definition
of optimal separation conditions. Methanolic mobile phase pH was adjusted by aqueous
phosphate buffer in the pH range 2.5-5.5. Dependence of retention times of individual
investigated pesticides on pH is shown in Fig. 1.
35 30 metoxuron
25 simazine
atrazine
20 propazine
15 terbutrine
cloq
10 cypermethrin
permethrin
5 0 2
2,5
3
3,5
4
4,5
5
5,5
pH
Figure1: Dependence of retention time of selected pesticides on methanolic mobile phase pH
The optimal chromatographic conditions chosen for the separation of 8 pesticides were:
Purospher Star RP 18e (50x4mm) analytical colum, separation of analytes was achieved by
linear gradient elution from methanol:aqueous pH buffer 50:50 (v/v) to 100% methanol in 35
minutes. The injection volume was 0.02 mL. UV spectrophotometric detection was done at
235 nm wavelength. The mobile phase flow rate was 0.5 ml/min.
The work is presenting results of the study of several parameters affecting the extraction
efficiency of 8 pesticides at concentration levels around 1-2.5 mg / kg dry soil.
MSPD: For MSPD pretreatment of soil sample optimal conditions for given pesticides were
achieved by use of silica L 40/100. Experimental conditions for isolation of pesticides are as
follows: desorption by 3.00 mL of 100% methanol, reduction of the extract volume to 0.50
mL by evaporation under dry air stream, make-up the volume to 1.00 ml. Extraction recovery
for the mixture of 8 studied pesticides achieved 2.5 mg/g dry soil, within the range 56-94%.
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Table 1: Extraction recovery of selected pesticides from soil contaminated by pesticides in
comparison to their standard addition into the extract of the same soil MP/20 at 2
concentration levels
Recovery (%)
1.0 µg/g
2.5 µg/g
Extract Std.
Analytes Extract Std.
Contam. Soil
Contam. Soil
dd
dd
metoxuron
96,1 ± 3,9
62,6 ± 1,8
107,2 ± 2,9
67,5 ± 0,3
simazine
108,4 ± 3,3
78,3 ± 1,2
109,5 ± 2,6
94,3 ± 4,2
atrazine
102,1 ± 6,4
71,3 ± 0,7
107,3 ± 8,3
77,8 ± 1,1
propazine
82,0 ± 2,9
64,5 ± 0,7
102,7 ± 2,5
71,3 ± 4,4
terbutrine
79,6 ± 0,1
65,6 ± 0,1
90,1 ± 2,9
69,1 ± 1,4
cloq
67,7 ± 0,5
48,3 ± 1,4
89,5 ± 0,6
56,4 ± 1,2
cypermethrin
68,5 ± 5,7
57,7 ± 1,1
89,4 ± 2,1
69,2 ± 1,6
permethrin
69,8 ± 8,3
63,5 ± 1,2
81,7 ± 0,5
60,9 ± 3,2
FTSLE: For FTSLE soil pretreatment the optimal mass of silica bottomed in the column is
0.50 g. Experimental conditions for the isolation of pesticides are as follows: desorption by
3.00 mL of 100% methanol, flow rate 0.30 ml/min, reduction of the extract volume to 0.50
mL by evaporation under dry air stream, make-up the volume to 1.00 mL. Extraction recovery
for the mixture of studied pesticides achieved 2.5 mg/g dry soil was within the range 61-88%.
Table 2: Recovery of pesticides using FTSLE at 0.3 mL/min from soil contaminated by
pesticides in comparison to their standard addition into the extract of the same soil MP/20 at 2
concentration levels
Recovery (%)
2.5 µg/g
1.0 µg/g
Extract
Std.
Extract
Std.
analytes
Contam. Soil
Contam. Soil
dd
dd
metoxuron
105,1 ± 1,8
73,3 ± 2,4
106,4 ± 5,7
67,3 ± 1,0
simazine
105,5 ± 4,2
81,6 ± 2,4
109,3 ± 3,0
78,2 ± 2,5
atrazine
102,4 ± 3,9
81,1 ± 0,7
104,5 ± 4,8
72,8 ± 6,2
propazine
103,9 ± 2,5
81,8 ± 1,4
106,1 ± 4,2
73,3 ± 4,0
terbutrine
98,6 ± 0,9
83,5 ± 0,2
101,1 ± 3,2
75,4 ± 6,1
cloq
91,6 ± 0,2
88,3 ± 0,5
84,8 ± 2,2
68,4 ± 5,0
cypermethrin
62,6 ± 2,4
61,1 ± 3,0
65,0 ± 2,8
56,5 ± 1,7
permethrin
62,8 ± 3,3
63,2 ± 2,1
62,1 ± 0,4
54,6 ± 5,6
Volumes: Vfraction=3.0 mL; Vafter evaporation=0.5 mL; Vfinal= 1.0 mL.
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15th IHSS Meeting- Vol. 3
4. Conclusions
Under the defined conditions both studied sample pre-treatment methods gave comparable
recoveries of pesticides investigated by RP-HPLC.
Acknowledgements
The work was supported by grants VEGA 1/0870/09, APVV-0595-07 and VVCE-0070-07.
References
1. C.Sánchez-Brunette, B. Albero, J. L. Tadeo, Determination of Pesticides in Soil, in J. L. Tadeo,
(Ed.,) Analysis of Pesticides in Food and Environmental Samples, Chapter 8., CRC Press, Boca
Raton, 2008, pp. 207-230.
2. T. A. Howell, in: D. Hillel, (Ed.), Encyclopaedia of Soils in the Environment, Oxford, UK,
Elsevier Press, 2004, pp. 379–386.
3. M. Chalányová, M. Paulechová, M. Hutta, J. Sep. Sci., 29 (2006) 2149.
4. I. Rybár, R. Góra, M. Hutta, J. Sep. Sci., 30 (2007) 3164.
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Humus Substance Role in Technogenic Soil Formation at Priokhotie Mining
Industry Developments
Makhinova А.F., Makhinov А.N.
Institute of Water and Ecology Problems FEB RAS, 65, Kim Yu Chen st., Khabarovsk,
Russia
E-mail: mahinova@ivep.as.khb.ru
1. Introduction
Open mining and intensification of mineral extraction in Priokhotie cause destruction of
enormous forest massifs. Forest soil resources being a deficit, the problem of soil reclamation
and cultural technogenic landscapes formation becomes most urgent. Technologies of mine
reclamation and technogenic soil formation first of all involve intensive reclamation of a
humus-organogenic horizon and in particular covering the planned surface of the dump with
humus-accumulative horizons of mountain-taiga soils removed at the initial stage of field
development [1]. Reclamation of technogenic landscapes still remains a single-action activity.
At present actually no ecological monitoring is implemented [5]. However, a technogenic soil
profile shows significant changes in physical and chemical characteristics and soil-formation
processes, which cause soil degradation and which mechanisms are weakly-studied.
The work was focused on the studies of physical and chemical specifics of recultivated soils
and the role of humus substances in soil profile degradation.
2. Materials and Methods
The objects of study were technogenic soils formed at reclaimed dumps in the Taryng-Lata
River basin. Observation sites were chosen on the 12-8о gradient mountain slope of NE
position in Ayano-Maisky District of Priokhotie. Dump reclamation with an organogenic 20
cm-layer was performed immediately after the stripping operations were completed. Two
observation sites were made on the reclaimed land down the slope 50 meters apart. The
distance between the site profiles was 6 meters. An alluvium-diluvium layer was 48 cm-think
at the upper site and 56 cm-thick at the lower site. To prevent slipping down of filled-up and
organogenic layers the section wall, perpendicular the slope lengths, was enforced with metal
mesh pieces 3 meters long and up to 45 cm deep. The thickness of upper layers was 20 cm.
When a stable grass cover appeared three years after reclamation, two types of technogenic
soils at different slope gradients were studied: 1) with a filled-up mineral-organogenic layer
on a metal mesh with D-3 cm cells (Profiles 1 and 2); 2) with a filled-up organogenic weakly-
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decomposed layer (Profiles 3 and 4). Standard methods were used to estimate technogenic
soil density, chemical and physical characteristics, granulometric composition and carbon
concentrations [3].
Observation site 1 (P-1 and P-3). Analytical studies showed no differentiation of organic
carbon content in the technogenic soil profile. In Profile 1, characterized with a rich mineralorganogenic layer, inner-ped pores were colored with a dark-grey humus cutan. In P-3 (20-30
cm layer) brown films were found on the sides of ped structures and water filtration rate was
increased.
Some experimental studies were undertaken to describe pore space and prevailing mode of
soil water migration. 100 cm-long glass pieces were fixed of the metal mesh on the profile
walls along the slope. Water was poured on the profile surface till a 2 cm-water column was
formed on the frozen rock layer. It was found out that compared to technogenic soil with a
filled up mineral-organogenic layer (P-1), in technogenic soil with a filled up organogenic
layer (P-3) the descending water flow rate was 1.21 times higher and the inner-lateral
discharge of water solution was 1.35 times more rapid (Table 1). All the measurements were
repeated three times.
Table 1: ome Physical and Chemical Characteristics of Technogenic Soils
№
РР
Layer
thickness,
cm
Р1
0-20
20-35
35-45
0-20
20-35
35-45
Р3
Р2
Р4
0-20
20-35
35-45
0-20
20-35
35-45
Den- %Po (V)
Sum of
-res filtr. fractions , %
sity
water salt
g/сm in
cm/
>0,01 <0,0
3
vol- sec
01
ume
Site 1, NE slope 12о, no soil frost in 50 cm-layer
5.21 4.35 1.92
0,8
34
3.6
77.09 4.95
5.21 4.41 2.72
2.17 41
3.6
83.09 5.34
5.32 4.53 2.85
2.38 48
4.2
82.79 3.81
4.71 3.91 1.62
0.8
69
5
--5.2
4.19 2.62
2.17 38
3.9
83.01 5.48
5.32 4.29 2.85
2.38 51
4.2
82.15 3.81
Site 2, СВ slope 8о, soil frost is observed deeper 50 cm
5.01 4.09 1.92
0.8
33
3.6
81.02 6.12
5.26 4.28 2.84
2.17 40
3.1
86.01 5.48
5.32 4.52 2.98
2.38 48
3.7
89.15 4.81
4.72 3.91 1.63
0.8
68
4.4
--5.21 4.19 2.79
2.17 36
3.7
86.01 5.48
5.32 4.29 2.85
2.38 49
4.1
89.15 4.81
РН
Unit
weight
g/сm3
С
Total
%
CHA
CFA
6.82
1.95
0.36
89,0
2.01
0.31
0.76
0.19
0.11
0.22
--
7.81
1.93
0.36
89,0
2.01
0.31
0.75
0.19
0.11
0.22
--
Observation site 1 (P-2 and P-4). The mountain slope is gentle; no evident differences
between the profiles are observed. However, compared to the profiles of Site 1, both profiles
revealed weighting of mechanical composition by layers; profile thickness increased by 5-7
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15th IHSS Meeting- Vol. 3
cm. Field studies also revealed humus-ferrous cutans in the mineral layer (20-30 cm) of P1
under the organo-mineral layer; brown films on the sides of ped structures were rarely
observed.
3. Results and Discussion
Morphological studies at the observation sites revealed that a) thickness of upper filled up
mineral organogenic and organogenic layers decreased; b) all mineral layers of technogenic
soils became more dense; c) mechanical washing out of filled up layers changed their color
from brown into dark-brown; structural fragments became more solid and clayey fractions in
them increased. Land reclamation with definite boundaries between the soil layers caused the
formation of technogenic soils different from original forest soils. Soil layer transformation
degree in the whole profile primarily depends on the character of humus-organogenic layers,
used for soil recultivation, as well as on the degree and duration of their watering and the
slope gradient. Warm-time rains accelerate washing out of filled up organogenic-mineral soil
into the supporting layers [2]. High concentrations of washed out humus increase the
formation of peds, different in size and surface specifics, and cause their “glueing” and layer
packing.
Morphological studies of ped surfaces show that in P-3 a brown film on the aggregate surface
is ferriferous, that indicates the presence of more aggressive fractions of fulvic acids, which
form an organogenic layer, while being decomposed. Ferric hydroxide prevalence in the
upper 20-30 cm layer restrains their mobility and excludes alluvial genesis of cutans
(penetrated from the upper layer). The main formation mechanism of differences in
technogenic soils may be of a microbiological nature on the background of heterogeneity of
organic matter in the filled up layers. Increased concentrations of Fe ions that form ion
bounds of a crystallization type result in the formation of solid aggregates, and thus in a wellmarked inter-aggregate porosity in the technogenic soil layers. That is why, the rate of
filtration through inter-ped cracks increases (Table 1). Similar changes of soil filtration
characteristics were also described by several other authors [4].
There are no big differences between profiles 2 and 4 (site 2). Still, the following differences
from the first (upper) site should be noted: a) increase of technogenic profile thickness and
density of a 20-40 cm layer; b) water capillary disperse indicates weaker permeability of the
layers; c) permafrost melting goes much slower in the lower layers due to weak inner-lateral
water discharge; lower layers are temporary over wet; d) sod pedogenesis prevails in P-2;
e)mycelium appears at the bottom of the upper matted and organogenic layers.
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4. Conclusions
Technogenic landscape reclamation in Priokhotie causes irreversible physical and chemical
changes and technogenic soil formation. Dump reclamation with organogenic (organomineral and other) soil layers accelerates sod pedogenesis and technogenic soil formation, in
most cases characterized with formation processes, different from those of disturbed soils.
Due to the absence of sod pedogenesis on the steep slopes (>10о) at the initial stage of
technogenic soils formation, the filled up layer thinners to 7 cm: a) in P-3 an organogenic
layer is rapidly decomposed; b) in P-1 an organo-mineral layer is rapidly washed out through
a large pore space into the supporting layers. Direction of soil forming processes depends on
the specifics of a layers used for recultivation. The use of coarse organogenic layers
intensifies the formation of aggressive humus fulvic acids, processes of fersiallitization and
the formation of ferriferous films on the sides on peds. Organo-mineral complexes in humous
soil layers used for land reclamation foster the processes of alluvial and humus discharge and
formation of drip humus-ferriferous inter-aggregate films. Thickness of diluvia deposits plays
a significant role in technogenic soil formation on the gentle slopes and impacts the length of
permafrost processed and the formation of a zone of capillary-suspended moisture. Appearing
anaerobic processes most often cause gleization inside the peds and misbalanced Fe secretion
on the ped sides.
References
1. Е.V. Abakumov, E.I. Gagarina. Land reclamation in post-technogenic landscapes and physical
characteristics of dump ground // Proc. All-Russia Conf. “Fundamental Physical Studies in Soil
Science and Melioration” М., 2003. P. 262-264.
2. J.L. Anderson, J. Bouma. Relation between hydraulic conductivity and morphometric data of an
argillic horizon //Soil Sci. Soc, Am. Proc. 1973. V.37. P. 408-413.
3. А.F. Vadunina, Z.А. Korchagina. Methods for studying physical characteristics of soil. М.:
Agropomizdat, 1986. 241p.
4. J. Bouma. Hydropedology as a powerful tool for environmental policy research // Geoderma. V.
131. #3-4. P. 275-286.
5. A.F. Makhinova, A.N. Makhinov. Risk assessment of soil degradation and possible soil
recultivation in mining in Priokhotie region //V.1. From Molecular Understanding to Innovative
Applications of Humic Substances. Proceeding of the 14th Meeting of Internati-onal Humic
Substance Society. September 14-19, 2008, Moscow-Saint Petersburg, Russia, Editors: I.V.
Perminova, N. A. Kulikova, Vol.P, Sapiens, Moscow, 2008, P.273-278.
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Industrial Production and Commercial Applications
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Study of Copper Extraction Efficiency by Humic Acid/Polypyrrole on
Paraffin-Impregnated Graphite Electrode
Mónica Antilén*, Miguel Araus, Mauricio Pérez-Ponce, Francisco Armijo, Rodrigo del Río,
M.A. del Valle
Pontificia Universidad Católica de Chile, Vicuna Mackenna 4860, 7820436, Chile
E-mail: mantilen@puc.cl
1. Introduction
Due to its polyfunctionality, humic acid, HA, is one of the most powerful chelating agents
among the existing natural organic substances: they are able to complex heavy metals [1],
inorganic anions and halogens [2], organic acids [3], aromatic compounds [4], pesticides and
herbicides [5]. This allows establishing that HA may alter the availability, transport, fixation,
and toxicity of environmental contaminants. On the other hand, it is well known that the
electrochemical activity of conducting polymers, such as polypirrole (Ppy) is accompanied by
the insertion and ejection of anions from the electrolytic solution according to the reaction:
Ppy
+
xA−
⎯oxidation
⎯⎯→
reduction
←⎯⎯⎯
[(Ppy)x+ (A−)x]
+
xē
where A is a dopant anion to compensate the positive charges generated during the oxidation
process and x is the (level) dopant [6]. Because in this case polypyrrole is the type of polymer
that can be p doped/undoped, it has been previously utilized to remove contaminants such as
arsenic species [7], and results showed that the matrix has a preference for AsO43− species.
Moreover, the natural properties of PPy as anion exchanger explain why it has been widely
employed for film preparation in solid phase micro-extraction coupled to chromatographic
techniques [8], an advanced technique to obtain analytical samples of volatile and
semivolatile organic compounds. The formation of composite by the insertion of polyethylene
glycol (PEG) has been previously reported to improve Ppy properties. The results
demonstrated that at low PEG concentration an open structure of the film is obtained and
consequently the insertion and ejection of ions to the electrolyte is accelerated [6]. From the
abovementioned background about the different approaches by which an electrode can be
modified to incorporate HA in a reproducible way, obtaining devices with improved
sensitivity and within the spectrum allowed by this kind of matrix, a novel alternative for the
preparation of electrodes in a simple, reproducible and using chelating properties of humic
substances, along with the of p-doping of conducting polymers properties, predicting a
synergistic effect, is proposes in this article.
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15th IHSS Meeting- Vol. 3
2. Materials and Methods
Apparatus: All electrochemical experiments were performed on a VoltaLab PGZ100
potentiostat system using a three-compartment/three-electrode glass cell under an argon
atmosphere. Paraffin-impregnated graphite (0.28cm2) was used as working electrode (PIGE).
A Pt wire (20 cm2) was the counter electrode. All potentials quoted in this article were
measured versus a Ag|AgCl reference electrode. PIGE was prepared by impregnating
spectroscopic graphite rods with melted paraffin wax under vacuum [9].
Chemicals and solutions: HA (Sigma–Aldrich) was purified by precipitation as previously
described [9]. Stock solutions containing Cu(II) in 20 mM HCl + 100 mM KCl (solution B)
were prepared by dissolution of the appropriate amount of CuCl2 in twice-distilled water
according to standard procedures. Pyrrole (Sigma–Aldrich) was purified by distillation, stored
at 4ºC, and protected from light. All other reagents used were analytical grade (Merck). All
solutions were deaerated with pure Ar for at least 20 min
Preparation of modified PIGE: As for PIGE, four electrodic surfaces were characterized by
cyclic voltammetry: (1) PIGE, (2) PIGE electrochemically modified by pyrrole (PIGE/PPy),
(3) PIGE modified with HA transferred and immobilized onto the electrode surface using the
abrasive transfer technique [10] and then electropolymerized with pyrrole (PIGE/HA/PPy),
and (4) PIGE modified with HA and then electropolymerized with pyrrole-polyethylene
glycol (PIGE/HA/PPy-PEG). Electrochemical growth of PPy or Ppy-PEG films was carried
out on PIGE or PIGE/HA from a 1.4 mM pyrrole + solution A (50 mM Na2SO4 + 30 mM
H2SO4) by potentiodynamic methods with the application of five successive cycles between −
0.2 and 1.0 V [9], while PPy-PEG films were prepared with 1.0 g L−1 PEG (M.W. 1000).
Extractions: Three electrode surfaces, PIGE/PPy, PIGE/HA/PPy, and PIGE/HA/PPy-PEG,
were studied in solutions containing 22.32 mM CuCl2 + 20 mM HCl + 100 mM KCl (solution
C , cell A) and 20 mM HCl + 100 mM KCl (solution B, cell B) to select the doping–undoping
potentials to perform the extractions. The total volume of electrolyte in each cell was always
10 mL. Each extraction was carried out as follows: the modified PIGE was immersed in
solution C and potentiostatically perturbed at 0.4 V for 5 min, after which it was dipped into
solution B, wherein it was potentiostated at -0.6 V for 5 min. Initially and after several
extractions, the copper concentration in solutions A and B was determined by inductively
coupled plasma/optical emission spectrometry (ICP–OES) on a Varian Liberty series II
instrument.
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3. Results and Discussion
Figure 1 depicts the stable voltammetric profile of PIGE without modification, PIGE
modified with Ppy, PIGE modified with HA-Ppy, and the same PIGE modified with HAPEG-Ppy. The comparative response of each electrode clearly shows that all modifications
were successfully accomplished, causing an increase in the capacitive response. However, the
incorporation of HA led to a current drop (not showed) indicating clearly that it is a nonconducting solid, where no redox processes occur, demonstrating also that adsorption of HA
on the PIGE surface takes place [9]. Consequently, PIGE-HA-PPy and PIGE-HA-Ppy-PEG
modifications produce the highest current values. This has been ascribed to the type of
porosity and morphology of the obtained polymer as a result of HA and PEG incorporation [6,
9].
Figure 1: Potentiodynamic response of
PIGE; PIGE/Ppy; PIGE/HA/Ppy and
PIGE/HA/Ppy-PEG in 20 mM HCl and
100 mM KCl between -0.6 V and 0.4 V
at 0.1 V s−1 (I is current intensity and E is
the voltage)
Table 1 summarize the results obtained with the modified electrodes tested for copper
extraction, where the electrode possessing HA and in addition a composite of PPY-PEG is the
most efficient to extract Cu(II) species. This high efficiency could be explained due to a HA−
Cu(II) species interaction as 1:1 complexes, which would be boosted with the help of the
doping-undoping process of the conductive composite Ppy-PEG. The chemical speciation
obtained by Geochem [11], indicated that about 95% of Cu(II) is as CuCl42- which is essential
in order that Ppy-PEG exchanges these anions from its film. Because both species (HA and
CuCl42-) bear negative charge, weak interactions are expected. However, the results indicate
that this is not the case, because it was electrochemically demonstrated that a drop of the
Cu(II) signal (data not shown) occurs in the presence of HA. Since copper chloride ion has a
formal charge of 2+, a phenolate addition to the electrophilic center followed by protonation
and water release might take place in a similar manner to other anions [9]. Therefore,
adsorption mechanisms other than electrostatic ones (charge) may occur, since the possibility
of forming cross-bridges between acid molecules has been suggested [12].
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Table 1: Copper (II) extracted as a function of modified PIGE electrode
Modified
electrode
Extraction
number
Initial
Cu(II)
(g L-1)
1.267 ± 0.013
Extracted
Cu(II)
(g L-1)
0.00160 ± 0.00002
0.00177 ± 0.00004
PIGE/PPy
10
PIGE/HA/PPy
10
1.398 ± 0.014
1.262 ± 0.013
0.00281 ± 0.00003
0.00290 ± 0.00003
PIGE/HA/PPy-PEG
10
1.530 ± 0.015
1.532 ± 0.015
0.00810 ± 0.00003
0.00830 ± 0.00003
On the other hand, average charge results for all modified electrodes indicated the existence
of a reversible doping/undoping process in acid medium, which means that the stability of the
electrode would remain unchanged even after 10 extractions.
4. Conclusions
Finally, the use of a composite (PPy-PEG) has enabled improving the porosity of the polymer
which in turn, in the presence of HA, has proved to be the most efficient Cu(II) extractor.
Acknowledgements
The authors thank to DIPOG, PUC-Chile and Financiamiento Basal para Centros Científicos
y Tecnológicos de Excelencia, under grant FB-0807 for funding this research project.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
P.Lubal, D. Široky, D. Fetsch, J. Havel, Talanta, 47 (1998) 401.
S.C. Myneni, Science ,295 (2002) 1039.
A. Cozzolino, P. Conte, A. Piccolo, Soil Biol.& Biochem., 33 (2001) 563.
K. Nam and J.Y. Kim, Environ. Pollution, 118 (2002) 427.
U. Klaus, T. Pfeifer, M. Spiteller , Environ. Sci. Technol., 34 (2000) 3514.
R. Schrebler, P. Cury, H. Gómez, R. Córdova, L.M. Gassa, Bol. Soc. Chil. Quím., 47 (2002) 537.
M.A. del Valle, G.M. Soto, L. Guerra, J. Vélez, F.R. Díaz, Polymer Bull., 51 (2004) 1436.
J.Wu and J. Pawliszyn, J. Chromatogr A, 909 (2001) 37.
M. Antilén and F. Armijo, J. Appl. Polym. Sci., 113 (2009) 3619.
F. Scholz and B. Meyer, Electroanalytical Chemistry, A series of Advances, Marcel Dekker: New
York, 1998, p 8.
11. D. R., Parker, W.A. Norvell, and R. L. Chaney. Chemical equilibrium and reaction models, SSSA
Spec. Publ. 42, ASA and SSSA, Madison,1995, p 253.
12. K.M. Sparks, J.D. Wells, B.B. Johnson, Aust. J.Soil Res., 35 (1997) 89.
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Sorption of Silanized Humic Derivatives onto Montmorillonite Clay
Vladimir A. Kholodova*, Vladimir M. Zelikmanb, Kirk Hatfieldc, Irina V. Perminovab
a
Dokuchaev Soil Science Institute, Pyzhevskiy per. 7, 119017 Moscow, Russia; bDepartment
of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, 119991, Moscow,
Russia, c University of Florida, Gainesville, Florida, USA
E-mail: vkholod@mail.ru
1. Introduction
Composite materials on the basis of humic substances (HS) and alumosilicate might be used
in the field of environmentally sound technologies. However, relative low sorption capability
of HS toward alumosilicate confines application of these materials. Affinity of HS to
alumosilicate could be increased by treatment of HS with organosilanes [1]. The goal of this
work was to compare sorption capability toward montmorillonite clay of silanized and native
HS.
2. Materials and Methods
The montmorillonite clay from the deposit “10 khutor”, Khakassia, Russia was used. Clay
was dispersed by ultrasonic treatment several times with separation of coarse fraction by
decantation. After that, montmorillonite was Ca2+ saturated by fivefold treatment with 0.001
M CaCl2.
The silanized humic materials were derived by treatment of HS from leonardite (the
preparation “Powhumus”, Humintech Ltd., Germany) by aminopropyltroethoxysilane as
described in [2].
Sorption capability of HS was estimated in batch adsorption experiments. Stock solution of
HS (silanized or native) containing 0.001 Ca2+ at pH 5.5 was added to 50 mg of
montmorillonite to the final concentrations of 25-4000 ppm. Solutions were shaken during
48 h. Equilibrium concentrations of humic substances ([HS], ppm) were determined by
optical density at 465 nm. HS amount adsorbed onto montmorillonite (Cads, mg×g-1) was
calculated as a difference between initial and equilibrium HS concentrations.
3. Results and Discussion
General adsorption isotherms curves are shown on Fig. 1.
Initial parts of both isotherms had L-form. Sorption of native HS on montmorillonite reached
plateau near 20 mg×g-1. At the same time, sorption of silanized HS linearly increased up to 80
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mg×g-1. After equilibrium concentration of 1500 ppm, the inflection point was observed on
the sorption isotherm of the silanized HS. It might be explained by the beginning of
multilayer sorption of HS.
Cads, mg×g-1
180
160
Alkoxysilylated HS
140
Native HS
120
100
80
60
40
20
[HS], ppm
0
0
500
1000
1500
2000
2500
Figure 1: Adsorption isotherm silanized and native HS onto montmorillonite.
Thus sorption capacity of silanized HS was much higher as compared to the native HS. The
obtained data suggest considerable increase in mineral affinity of HS as a result of
introduction of silanolic groups.
4. Conclusions
The higher sorption capacity toward montmorillonite clay of alkoxysilylated HS as compared
to the native HS was demonstrated. Synthesis of silanized humic materials is a powerful tool
for manufacturing new hybrid materials.
Acknowledgements
The research was supported by NATO CLG #983197.
References
Karpiouk L.A., Perminova I.V., Ponomarenko S.A., Muzafarov A.M., Hatfield K. In: From
Molecular Understanding to Innovative Applications of Humic Substances; Proceedings of the
14th International Meeting of the International Humic Substances Society, I.V. Perminova, N.A.
Kulikova, (Ed.), Vol. II, Humus Sapiens, Moscow, 2008. p. 521-524.
2. Perminova I.V., Ponomarenko S.A., Karpiouk L.A., Hatfield K. Humic derivatives, methods of
preparation and use. PCT application. Pub. No.: #WO/2007/102750.
1.
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Peat Humic Acids as the Redox Mediators for Textile Technologies
Irina Yu. Vashurinaa*, Yuri A. Kalinnikova, Irina V. Perminovab
a
Ivanovo State University of Chemistry and Technology, Engels Prosp., 7, 153000 Ivanovo,
Russia; bDepartment of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 13, 119991, Moscow, Russia
E-mail: irina-new@bk.ru,
I. Introduction
One of the essential qualities of humic acids (HA) is their high catalytic activity in diverse
redox transformations that occur in soils, peats, coals etc. It is attributable to the quinonoid
redox-active units of HA as well as to transition metals that form stable complexes with
functional groups of HA and participate in electron transfer reactions. Scientific interest to
humic substances as redox mediators is steadily growing due to the progress in the
understanding of their role in abiotic and biotic redox transformations of ecotoxicants in
contaminated environments. At the same time, there is almost no information available on the
application of humic substances in industrial technologies that rely on redox reactions.
In the present study, the possibility is assessed to use peat HA as redox mediators for textile
technologies, namely, vat printing of cellulose fabrics and preparation of native starch gels for
cellulose yarn sizing.
2. Materials and methods
HA were extracted from lowland peat mined in the centre of European Russia (Kostroma
region) as described elsewhere [1]. UV-VIS spectroscopy was used to study the reduction
kinetics of HA and dye in aqueous solutions. Viscometry and rheology was used to
characterize starch gels. Potentiometric titration, other analytical and specific textile protocols
were also used in our studies.
3. Results and discussion
The kinetic and potentiometric data obtained have revealed that the reduction of HA by
sodium hydroxymethanesulfinate and their follow up oxidation by benzene chlorosulfamide
(the reagents used in the tested textile technology) in aqueous alkaline media is reversible
from 88 to 92%. The activation energy of redox transformations in HA is about tenfold less as
compared to that of vat dyes reduction and native starch oxidation.
Using 1-4-diaminoderivatives of anthraquinone (the dyes Acid Green 27 and Acid Blue 80) as
the water soluble models of vat dyes, catalytic activity of peat HA in vat dye reduction by
sodium hydroxymethanesulfinate and sodium dithionite was shown. The effect is comparable
to that of conventional redox catalysts (such as β-anthraquinone sulfonic or 1,2-dihydroxy
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anthraquinone-3-sulfonic acids), but achieved using concentrations an order of magnitude
lower. The addition of 0.05-0.10 gOC·kg-1 of HA causes 2-20-fold increase in the dye
reduction rate constant and an increase in reaction output up to 97-98%.
It was revealed that at high pH values (10-13) peat HA effectively dismutated supeoxide ionradical and peroxide ions formed in vat dyeing and printing compositions because of
interactions of reducing agents with oxygen, thus precluding the accumulation of superoxide
and peroxide and favoring dye reaction with the reducing agent. This allowed for a twofold
reduction in the amount of sodium hydroxymethanesulfinate in printing pastes.
HA were recognized as chemical and structural modifiers of starch hydrogels used for cotton
yarn sizing. Chemical modification consists in deeper starch oxidation (the content of
oxidized groups in corn starch increased by 50%), full consumption of oxidizing agents and
less than half the gelation period. Structural modification reveals in essential decrease in
starch gel viscosity and a transformation to a Newtonian flow regime.
Due to their enhanced fluidity and adhesion to cellulose together with better elasticity of
formed films, HA modified starch gels provide complex improvement of the properties of
sized yarn and reduce yarn break while spinning.
4. Conclusions
This investigation produced new HA containing compositions for textile materials vat dyeing
and printing and for cotton yarn sizing that markedly differ from the existing ones by less
consumption of aggressive chemicals (reducing and oxidizing substances, alkali), full
utilization of vat dyes, starch, reducing and oxidizing agents, replacement of some toxic
textile auxiliaries by biocompatible HA, shortening the duration of thermal treatments. This
makes the HA-based technologies more efficient and it introduces elements of green
chemistry into textile industry.
Acknowledgements
This research was supported by the Russian Foundation for Basic Research (grants 05-0496405 and 06-04-08048) and by the Foundation for the Assistance to Scientific Innovative
Enterprises (contract No 4302 р/6530).
References
1. Lowe L.E. Studies on the nature of sulphur in peat humic acids from the Fraser River Delta,
British Columbia // Sci. Total Environ., 1992, 113 (1-2), P. 133-145.
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Peat Humic Acids as Surfactants
Oskars Purmalis, Maris Klavins
University of Latvia, Latvia
E-mail: oskars.purmalis@lu.lv; maris.klavins@lu.lv
1. Introduction
Humic substances are a general category of naturally occurring, biogenic, heterogeneous
organic substances that can be characterised as being yellow to black in colour, of high
molecular weight and refractory. They form most of the organic component of soil, peat and
natural waters, they influence the process of formation of fossil fuels, and they play a major
role in the global carbon geochemical cycle. Structure of humic substances is characterised by
presence of numerous aromatic, carboxylic and phenolic functionalities, linked together with
alkylmoieties, imparting a measure of flexibility to the polymer chains [1]. Surface tension
measurement defined humic substances as surface active substances [2, 3]. Unless micellar
structural model of humic substances has been suggested, in the same time there are only a
few studies about the factors that affect the surface tension of humic solutions.
The ability to influence surface tension of solutions of humic substances depends on
decomposition degree and botanical composition of the peat. The surface tension-pH curves
of humic substances featured a minimum for all solutions, declining steeply from higher and
lower pH values. Surface tension of solutions of humic substances decreased with increasing
concentration. The objective of this study was to study changes in surface activity variation in
peat profile of humic substances.
2. Materials and Methods
The peat samples were collected in the Kronu Dzelves Bog (0 – 350 cm) and Ploce bog (0 130 cm) (Latvia), both bogs botanical origin – fuscum peat. Humic acids were extracted and
purified using procedures recommended by the International Humic Substances Society
(IHSS) [4].
Surface tension measurements were taken with tensiometer Krüss K6 (Krüss GmbH), fitted
with a 19 mm diameter platinum ring. Samples were prepared for measurement diluting till
the appropriate concentration standard solution at humic substances (1000 mg/l) and
equilibrating for 24 hours. Solutions were placed in a shallow glass dish of 50 mm diameter
and the platinum ring was inserted in the middle of the container to avoid edge effects and
equilibrated for 90 min. The ring was raised through manual operation of the torsion
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mechanism and the tension readings at the instant of surface detachment were noted. All
measurements were taken in triplicate at a temperature of 22 0C and mean results are used in
the figures with standard deviation not more than ±2 mN/m.
Hydrophobicity of humic substances has been characterized by their distribution between
water and polyethylene (PEG) phases (PEG 20000, Fluka) [5] as distribution coefficient
KPEGW (analogous to octanol water distribution coefficient – Kow).
Elemental analysis (C, H, N, S, and O) was carried out using an Elemental Analyzer Model
EA-1108 (Carlo Erba Instruments).
3. Results and Discussion
To study the changes of ability of humic substances to influence surface tension, we have
used well characterised humic substances (Table 1), isolated from bogs in Latvia.
Studied humic substances demonstrate ability to influence surface tension of their solutions
(Figure 1, 2). In concentration intervals from 50 mg/l to 1000 mg/l γ dropped from 57,6 – 62,9
mN/m to 53 – 55,2 mN/m. The surface tension of solution of humic substances at fixed
concentration so can be used to describe their surfactant properties and among studied humic
substances comparatively high variability could be observed.
Surface tension, mN/m
.
64
62
60
58
56
54
52
0
200
400
600
800
1000
HA concentration, mg/l
Figure 1: Variation of surface tension of humic acid (isolated from Ploce bog) solutions
depending on the location depth (cm):
0-30 cm (▲); 31-60 cm (●); 61-85 cm (○); 86-110 cm (■); 111-130 cm (♦)
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There exist significant differences in the ability of humic acids to influence surface tension of
their solutions. Peat humic substances isolated from Ploce bog (Latvia) from consecutively
increasing depths do have major differences in their ability to modify tension of aquatic
solutions (Figure 1). The surface tension of solutions of humic substances is decreasing with
increasing depth of their location in peat column (age of the peat and their humification
Surface tension, mN/m
degree).
63
62
61
60
59
58
57
56
55
54
0
0
0
0
0
0
0
0
0
0
0
0
5
0
.30 0.6 0 .8 1.1 1.3 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0
.0 .30 .6 0 .85 .10 0.0 .10 .20 .30 .40 .50 .60 .70 .80 .90
0
0
0
0
0
0
0
0
0
0
1
0
0
0
e
A A
A
A
A
A
A
A
A
A
H
ce
ce
ce
ce
oc
Pl Plo Plo Plo Plo lve e H e H e H e H e H e H e H e H e H
v
v
v
v
v
v
v
v
v
e
l
l
l
l
l
l
l
l
l
z
D Dze Dze Dze Dze Dze Dze Dze Dze Dze
Figure 2: Variation of surface tension of humic acid (isolated from Ploce and Kronu Dzelves
bog) solutions: concentration – 100 mg/l
Table 1: Elemental and functional composition of humic substances used in the study
Humic Acid
Ploce 0.0 – 0.30
Ploce 0.30 – 0.60
Ploce 0.60 – 0.85
Ploce 0.85 – 1.10
Ploce 1.10 – 1.30
Dzelve 0.0 - 0.10
Dzelve 0.10 - 0.20
Dzelve 0.20 - 0.30
Dzelve 0.30 - 0.40
Dzelve 0.40 - 0.50
Dzelve 0.50 - 0.60
Dzelve 0.60 - 0.70
Dzelve 0.70 - 0.80
Dzelve 0.80 - 0.90
Dzelve 0.90 - 1.00
C, %
51.51
51.13
51.12
52.11
57.50
42.36
50.62
51.62
51.92
53.16
50.88
48.05
53.95
52.55
53.34
H, %
4.80
4.81
4.87
4.61
4.94
4.19
4.56
4.85
4,48
4.84
4.56
4.05
4.94
4.82
4.87
N, %
2.12
2.06
2.05
1.60
1.92
2.30
2.61
2.77
2.57
2.24
2.25
2.04
2.32
2.17
2.39
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mEq COOH/g
5.66
5.80
6.01
5.91
5.10
3.90
5.00
4.70
4.60
4.30
4.50
4.70
4.50
4.30
4.30
KPEG/W
6.35
6.23
5.77
10.75
8.98
4.98
7.37
9.30
8.63
7.15
6.65
7.04
7.19
2.78
2.71
15th IHSS Meeting- Vol. 3
Humic acids isolated from Kronu Dzelve bog, demonstrates higher variability, particulary in
first 50 cm of peat column. It depends on hydrological regime fluctuations and peat
humification effect irregularity on the top layer of peat bog. In the deeper layers Kronu
Dzelve bog peat humic acids demonstrates the same results as Ploce bog peat humic acids.
The effect of humic substances on surface tension depends on their amphiphilic character and
tendency to accumulate at the water-air interface. It is known that the behaviour of humic
substances in aquatic solutions depends on their concentration, pH, metal ion concentrations
[6, 7] and the same factors determines the influence of humic substances on the surface
tension and the formation of pseudomicelles, since both are manifestations of the same
solution properties.
4. Conclusions
The surface tension-pH curves of humic substances featured a minimum for all solutions,
declining steeply from higher and lower pH values. The decrease in surface tension with
decreasing pH reflects the gradual neutralization of acidic sites, which created amphiphilic
species that migrated to the surface. Surface tension of solutions of humic substances
decreased with increasing concentration. Thus there exist direct links between peat
decomposition degree, botanical composition, structure of humic substances and their ability
to influence surface tension of aquatic solutions.
References
1. Engebretson, R.R., von Wandruszka, R. (1994) Microorganization in dissolved humic acids.
Environ. Sci. Technol., 28, 1934 – 1941
2. Engebretson, R.R., von Wandruszka, R. (1996) Quantitative approach to humic acid associations.
Environ. Sci. Technol., 30, 990 – 997
3. Gašparovic, B., Cosovic, B. (2003) Surface-active properties of organic matter in the North
Adriatic Sea. Estuarine, Estuar. Coast. Shelf. S., 58, 555 – 566
4. Guetzloff, T.F., Rice, J.A. (1994) Does humic acid form a micelle? Sci. Total Environ., 152, 31 –
35
5. Klavins, M. (1998) Aquatic humic substances. University of Latvia, Riga, 234 pp.
6. Tan, K. H. (2005) Soil sampling, preparation, and analysis - second edition. N.Y.: Taylor &
Francis group, 623 pp.
7. Zavarzina, A.G., Demin, V.V., Nifanteva, T.I., Shkinev, V.M., Danilova, T.V., Spivakov, B.Ya.
(2002) Extraction of humic acids and theit fractions in poly(ethylene glycol)-based aqueous
biphasic systems. Anal.Chim.Acta, 452, 95-103.
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Efficiency and Application Prospects of Humatized Mineral Fertilizers
Oleg Gladkov*, Rodion Poloskin
Open Company "SPA" “AET” Ltd (RET Ltd Company), Saint-Petersburg, Russia, 195112
E-mail: gladkov@humate.spb.ru
1. Introduction
Development of a new market niche of the fertilizers combining for end users the tempting
properties of organic and mineral fertilizers draws attention of scientists and consumers for a
long time. Using of such fertilizers allows to raise plant uptake of nutrients on 20-50 % and,
accordingly, to decrease the application rates, or it is essential to raise productivity at the same
application rates. Thus the general expenses for application of fertilizers essentially decrease,
and the yield of non-polluted agricultural production and the maintenance of soil fertility at
the minimum impact on the environment are provided.
2. Materials and Methods
The Company "SPA" "AET" Ltd (RET Ltd Company) is engaged in the development of
"know-how" and researches of the efficiency of application of industrial humates and
humatized organic-mineral fertilizers (HOMF), including the phosphoric and complex
fertilizers, more than 15 years. In 1999 after the long-term researches the company let out on
the market the concentrated humic product under the trade mark "LignohumateR". The
product is made on the unique patented technology of accelerated humification of commodity
ligno-sulfonates. The product is a 20% water solution or completely soluble powder. The
comparison of "Lignohumate" with the products of other leading world manufacturers has
shown that their physical and chemical characteristics do not concede to the analogues.
At the same time Lignohumates have a number of positive features such as increased content
of fulvic and other low-molecular acids, and also of macro- and microelements.
3. Results and Discussions
The basic marks of Lignohumate have been examined and registered in different countries,
they are widely used in the agriculture of Russia, the CIS countries, EU, the North America,
China. The constructed typical industrial module allows to let out more than 1500 tons/year of
humic product in recalculation on a solid. It has already made the company one of the largest
manufacturers of humates in Russia.
In the present report we give the information only about one of the perspective directions of
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application of Lignohumate - in a composition with the mineral fertilizers. For the realization
of this target our company cooperates with the variety of the Russian scientists and experts of
the industrial companies, manufacturers of mineral fertilizers, together with the foreign
companies among which are CSC "ARVI" (the Lithuanian Republic), "Amagro" (Czechia),
«RЕTRIVALL» Inc. (Canada), "Radostim" (Germany), etc. At the factories were fulfilled the
technologies of humatizing fertilizers with the humic additive component directly “in melt”
as well as covering of ready granules with humic additive “on the surface”
The tests of physical and chemical properties of such fertilizers have shown that the addition
of Lignohumate to mineral fertilizers considerably improves the basic functional properties of
fertilizers (dusting and blocking) in comparison with the standard mineral analogues. The
entering of humic organic component gives new properties to the mineral fertilizers. First of
all, their structure changes that causes the changes of physical and chemical properties, such
as dusting, durability of granules, blocking, for example, drawing on ammophos of 0,5-1 % of
Lignohumate in the form of a cover reduces the dusting on 37 %. The drawing of
Lignohumate on a carbamide granule reduces by 30 % its blocking and by 30-35 % increases
the durability of granules (1,3 kg/g, at the durability of standard fertilizer of 0,95 kg/g). Some
information on the most important and indicative researches of agricultural efficiency of
application of such fertilizers is resulted
The Efficiency of Application of Humatized Carbamide:For carrying out of tests two parties of
humatized carbamide with the various maintenance of Lignohumate have been made. The
first trial party was with drawing of humic additive on the surface and the industrial party
with placing of Lignohumate “in melt” before granulation was made on the Open Society
"Akron". Both parties have been directed to the different organizations for the tests on grain
crops. The results received by the experts are well correlated that guarantees their objectivity.
Some results of these tests are shown here.
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The conducted tests have allowed to conclude the following:
- The use of both parties of humatized carbamide leads to the increase of productivity, quality
of production and nitrogen operating ratio;
- The optimum concentration of Lignohumate as a part of fertilizer for the efficiency and
profitability is within 0,36-0,5 %,
- The bigger efficiency is received using the fertilizer with the put humic cover;
- The technological tests of industrial party of humatized carbamide have shown that the use
of Lignohumate “in melt” improves the durability and water resistance of granules, reduces
the washing out of easily soluble nutrients of fertilizers.
Other model experiments of application of humatized fertilizers have been spent on the basis
of three types of complex NPK fertilizers of the Companies CSC "ARVI" (Lithuania). The
manufacture of the trial parties was carried out in a real production cycle with the minimum
technological completions. Some results of agricultural tests of three types of humatized NPK
fertilizers at the cultivation of rape are resulted on the figures.
Fig.3, Fig.4, Fig.5
4. Conclusions
The carried out researches have allowed to draw the following conclusions:
- At the manufacture of fertilizers the improvement of durability of granules and the quality of
the technological processes of granulation is noted;
- On all tested cultures the trial fertilizers provide the reduction of the vegetative period, the
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increase in productivity and quality of production in relation to the standard analogue;
- The application of humatized NPK fertilizers allows to recommend the decrease of norms of
their entering in relation to the standard NPK not less than for 25-30 % without productivity
decrease;
- The major result of researches is the acknowledgement of the possibility of reception of high
effect at rather small concentration of Lignohumate in the fertilizer within 0,1-0,3 %.
The last circumstance does not so considerably change the fertilizer cost price in comparison
with the base analogue (for 15-20 EURO/TON) that is considerably below the economic
efficiency of application of such fertilizers. The received results have allowed the company
CSC "ARVI" & CO to register the new kind of NPK fertilizers with the additive of
Lignohumate under the mark “ARVI Extra plus” on EU market. The first industrial party of
these fertilizers was put to Germany in the end of 2008. Now long-term contracts are being
coordinated.
The carried out researches and the reached technological experience allow to develop the
new, much more capacious market of application of humic products in different countries. At
the same time the necessity of development of joint scientific researches with the
manufacturers of mineral fertilizers for the expansion of this direction of application of
humates amplifies.
References
1. Oleg Gladkov, Iren Sokolova “Lignohumate - Newly Developed Humic Preparation for
Recultivation and Restoration of Humic soil”. NATO Advanced Research Workshop “Use of
Humates to Remediate Polluted Environments: from Theory to Practice” Zvenigorod, Russia,
September 23-29 2002. Soil Science Department, Moscow State University, 119992, Moscow,
Russia
2. Olga Yakimenko, Oleg Gladkov, Rodion Poloskin, Vera Кing « LIGNOHUMATE – A NEW
MEMBER OF HUMATES FAMILY». Humic Science & Technology Conference IX. March 22 to
24, 2006, Northeastern University, Boston, MA, USA
3. Oleg Gladkov, Rodion Poloskin, Vera King, Olga Yakimenko “Commercial Humates from
Lignosulfonate: Production, Properties and Use”. Humic Science & Technology Conference IX.
March 22 to 24, 2006, Northeastern University, Boston, MA, USA
4. Wolfgang Novik, Uwe Bёm, Oleg Gladkov, Virginius Streimikis, “The Pilot Project on
Manufacture of Humatized Fertilizer “ARVI Extra” and “ARVI Extra plus” and the First Results
of its Application in Germany”. Fifth International Conference “Humic Preparations and Phytohormones in Agriculture”, February, 16-18th, 2010, Dnepropetrovsk, Ukraine.
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Physical-Chemical Properties and Application Potential of Humates
Prepared from Regenerated Lignites
Jan David, Jiří Kučerík
Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00, Brno, Czech
Republic
E-mail: kucerik@fch.vutbr.cz; xcdavid@fch.vutbr.cz
1. Introduction
At the beginning of the 21st century, our world is facing a set of challenges which are overall
related to the state of soils and to the old environmental loads. The search of new resources
for the chemical industry and agriculture is also of a great interest. Humic substances,
primarily humic acids (HAs) and their salts (i.e. humates) are thought to be helpful in
designing of the particular solutions of those problems.
HAs are heterogeneous mixture of organic compounds, the dark and refractory alkali-soluble
fraction of soil organic matter [1]. Recently, the scientific discussion was brought up on the
structure of HAs, resulting in the recognition, that HAs represent the collections of diverse
low-molecular-mass molecules held together by hydrophobic bonds and hydrogen-bonding
interactions [2, 3]. Using various techniques, it was also proved, that from very low
concentration HAs in aqueous solutions form aggregates of various dimensions, which may
be crucial for the future HAs’ industrial and agricultural applications [4, 5].
The aim of this research is focused on the production and characterization of HAs originating
from the pre-treated South-Moravian lignite (Czech Republic). Lignite is the youngest type of
coal, usually called as brown coal. It is the first product of coalification and the intermediate
between peat and subbituminous coal [6]. The most frequent lignite pretreatment described in
literature is wet regeneration/oxidation with solutions of strong oxidizers; it has been used
both for coal [7] and lignite [8]. This regeneration may increase the yield of extractable HAs
and modify their structure (in particular by increasing the ratio of aromatic and semiquinone
structures and content of polar elements such as O and N). That modification is indisputably a
way how to extend the application potential of the HAs. Following research is the
continuation of our previous pilot study [9] with the aim to characterize regenerated HAs via
various methods as thoroughly as possible and to design the particular applications of
regenerated HAs (especially in agriculture, environmental protection and applied chemistry).
Even though recently a progress has been done in the research of the physical chemistry of
HAs in diluted solutions by means of Dynamic Light Scattering (DLS) and High Resolution
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Ultrasonic Spectrometry [10, 11], the knowledge about behavior of diluted humates is still
limited. The combination of results obtained from those techniques with other methods and
approaches (see Discussion) would provide knowledge on the relations between physical
properties such as hydration and conformation, chemical structure (elemental analysis) and
their biological activity and sorption capacity. Such approach should bring the deeper insight
into the chemistry of lignite humic substance as well as to promote their technological and
agricultural applications.
2. Materials and Methods.
Regeneration of lignite and extraction of HAs. South-Moravian lignite (Mine “Mír”,
Mikulčice, Lignit Hodonín, Czech Republic) was regenerated in the similar way as in
previously published research [7, 8] (50 g of lignite, 500 mL of agent, 40 °C, stirring for 20
minutes) by (10; 20; 30; 40; 50; 65 wt%) nitric acid and by (5; 10; 20; 30 wt%) hydrogen
peroxide agents. The HAs were extracted using the modified IHSS alkali extraction (0.5
mol·L-1 sodium hydroxide and 0.1 mol·L-1 Na4P2O7) method. The HAs were purified by 5
wt% hydrofluoric acid (and then dialyzed against deionized water until no Cl– anions were
present. Finally, the samples were freeze-dried using Labconco Freezone 4.5 system, with the
Vacuubrand RZ6 pump. When needed from the point of water solubility, obtained HAs were
transferred to the water soluble potassium salt via titration with 0.1 mol·L-1 potassium
hydroxide on the Schott TitroLine Alpha plus automatic titrator to static pH 7.2 and
ammonium salt (titrated with an excess of diluted ammonium hydroxide to pH 7–9).
Samples were characterized by techniques described in the Discussion.
3. Results and Discussion
As the first step, results obtained by elemental analysis (Perkin Elmer 2400 elemental
analyzer) showed that regeneration of lignite has significant influence on the elemental
composition of the resulting HA. Regeneration with nitric acid increased the H/C, N/C and
O/C ratio, while regeneration with hydrogen peroxide increased the H/C ratio.
The research continues in combining the data from techniques such as High Resolution
Ultrasonic Spectrometry and densitometry (Ultrasonic Scientific HR-US 102 and Anton Paar
DMA 4500 Density meter) determining the number of water molecules hydrating a humic
molecule in the humate solution at specific concentration. The results were correlated with
aggregate dimensions obtained from DLS (Coulter N4 Plus Particle sizer). Considerations are
supported by Diode Array Detector (DAD) detected High Performance Size Exclusion
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Chromatography (HPSEC) (Gilson UltiMate 3000 Chromatography station). Final
conclusions will be done with respect to the chemical characteristics gained from not only
from elemental analysis, but also from solid state
13
C Nuclear Magnetic Resonance and
Excitation-Emission Fluorescence Spectrometry, i.e. the efficiency of the pre-treatment of
parental lignite with respect to the concentration and character of oxidant and physicochemical properties of obtained HAs.
The extraction and regeneration of supra mentioned HAs was done with the purpose of further
practical application. From this point of view, two main purposes for HAs are strongly
emerging. The first one lies in agriculture exploiting the humics “hormone-like” biological
activity or just simply the stimulating effects on the plant growth [12–14], the second one is in
the possibility to adsorb or bind metals and adsorb or solubilize organic pollutants [15, 16].
The environmental applicability (interactions with selected contaminants) of south-Moravian
lignite and HAs derived from it are being tested according to Conte et al. [17] and von
Wandruszka and Newell [16].
Finally, the root growth enhancement possibilities are being tested on the germination and
root growth of maize (Zea mays) in the defined light and temperature conditions [9].
For all these applications, the surface activity of HAs seems to be one of important
parameters, therefore before the application tests, surface tension of samples of various
concentrations were measured via KSV Sigma 700 Surface tension meter with du Noüy ring.
Selected results will be presented and discussed on the conference.
Acknowledgements
The funding of this research by the Ministry of Education, Youth and Physical Training of the
Czech Republic in terms of the MSM0021630501 research project is acknowledged.
Presenting author thanks for all the support for his travelling costs.
References
1.
2.
3.
4.
5.
6.
7.
F.J. Stevenson, Humus Chemistry, John Wiley & Sons, New York, 1994. p. 33.
R.L. Wershaw, Environ. Sci. Technol., 27 (1993), 814–816.
A. Piccolo, Adv. Agron., 75 (2002) 57–134.
R.R. Engebretson, R. von Wandruszka, Org. Geochem., 26 (1997), 759–767.
R. von Wandruszka, Geochem. Trans., 2 (2000), DOI: 10.1039/b001869o.
B. Mikulášková, et al., Chem. Listy, 91 (1997), 160–168.
R. Rausa et al., in N. Senesi, T. M. Miano (Ed.), Humic Substances in the Global Environment
and Implication on Human Health, Amsterdam: Elsevier, 1994, 1225–1244.
8. Kučerík et al., Petroleum and Coal, 45 (2003), 58–62.
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9.
10.
11.
12.
13.
14.
Z. Vlčková et al., Soil Biol. Biochem., 41 (2009), 1894–1901.
N.E. Palmer, R. von Wandruszka, Fresenius J. Anal. Chem. 371 (2001), 951-954.
J. Kučerík et al., Org. Geochem., 38 (2007), 2098–2110.
A. Muscolo et al., Soil Sci. Soc. Am. J., 71 (2006), 75–85.
L.P. Canellas et al., Ann. Appl. Biol., 153 (2008), 157–166.
B. Antošová et al., in M. I. Barroso (Ed.), Reactive and Functional Polymers Research Advances,
NovaScience Publishers, 2007, 191–203.
15. M. Havelcová, et al., J. Haz. Mat., 161 (2009), 559–564.
16. R. von Wandruszka, J.D. Newell, Environ. Prog., 21 (2002), 209–214.
17. P. Conte et al., Environ. Pollut., 135 (2005), 515–522.
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Removal of Tributyltin Biocide by Using Black Carbon
Liping Fang*, Ole K. Borggaard, Helle Marcussen, Peter E. Holm and Hans Chr. B. Hansen
Department of Basic Sciences and Environment, Faculty of Life Sciences, University of
Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C., Denmark
E-mail: fang@life.ku.dk
1. Introduction
Black carbon (BC) is the product of incomplete combustion of fossil fuel and biomass, which
is ubiquitously in environment [1]. Charcoal and soot have been distinguished as two main
forms of BC [2]. BC exhibits strong sorption with hydrophobic organic compounds (HOCs),
highly contributing to total sorption of HOCs to soils and sediments [3]. Therefore, the
application of BC as sorbent becomes a promising and low-cost option for purifying industrial
wastewater and groundwater.
In this study, we i) characterized two BCs, e.g., soot and charcoal; ii) surveyed the sorption
capacity of tributyltin biocide by BCs (soot and charcoal) at different pH values.
2. Materials and Methods
Tributyltin chloride ((C4H9)3SnCl) (96%) was purchased from Sigma Aldrich (Denmark). All
other reagents used were pro analysis. A 1000 mg L-1 TBT stock solution was prepared by
dissolving the corresponding amounts of TBT in methanol.
Wheat charcoal was produced as follows: finely ground wheat straw was combusted by in the
muffle furnace at 300 °C for approximate 24 h. Soot as carbon black was donated by
TIMCAL (Switzerland). Wheat charcoal was ground with ball mill to fine powder. The
resulting powder was washed with acids in order to remove silica and salts.
The element composition of the three BCs was determined on a CHN elemental analyzer
(Flash EA 1112, Thermo Scientific). The contents of carboxylic acid groups and phenolic
groups on the BCs were determined by titration of BC suspensions.
Sorption experiments were determined by adding appropriate amounts of tributyltin (TBT)
stock solution into suspensions of the charcoal (50-250 mg/L) and soot ( 60- 400 mg/L) in pH
4, 6 and 8 buffers to obtained initial TBT concentrations in the range 0.42 to 8.42 µmol L-1.
The suspensions were shaken for 24 h in the dark at room temperature (22±1 ºC). Finally,
each suspension was filtrated into10 mL volumetric flask with a mixed cellulose ester syringe
filter, and added 30 µL 69-70% HNO3. The loss of TBT during the whole sorption and
pretreatment processes was estimated by performing both types of experiments without
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adding BC in triplicate. Less than 5 % and 10 % were lost during sorption and filtration,
respectively. The acidified filtrates were analyzed by GFAAS (Perkin Elmer, Denmark).
3. Results and discussion
The selected characteristics of two BCs are listed in Table 1, it shows wheat charcoal contains
relatively high oxygen comparing with that of soot (Table 1), which also could be reflected
from their substantial contents of oxygen - contained functional groups (-COOH, Ar-OH). In
addition, the ash is blow <1% for wheat charcoal and soot.
Table1: Characterization of two Black Carbons
Charcoal
Soot
a
Functional group (mmol g-1)
Carboxylic
Phenolic
0.7
0.75
0
0
Atomic ratio
H/C
O/C
0.62
0.27
-a
0.01
H content is lower than detection limit
Sorption of TBT to soot is negligible at pH < 4 and then increases dramatically reaching a
constant plateau of about 95 % at pH > 6 (Figure 1). Soot possesses no charged functional
groups (Table 1), and in aqueous solution TBT exists as TBT+ ((C4H9)3Sn+) and TBTOH
((C4H9)3Sn OH) depending on pH (Eq. 1, [4]). Therefore, hydrophobic sorption of uncharged
TBT (TBTOH) is considered the only sorption mechanism.
pKa =6.3
(1)
The sorption edge for wheat charcoal also increase with increasing pH from limited sorption
at pH < 3 to maximum sorption around pH 7 but the raising curves are less steep than the soot
sorption edge and the sorption maxima are followed by slight declines (Fig. 1). The sorption
edge resembles those reported for TBT sorption by minerals and humic substances [5, 6], but
higher relative sorption (Cs, pH/Cs, pH 3) at high pH. Since the charcoal possess charged surface
sites, both TBT+ and TBTOH may be sorbed by charcoal. The shape of the sorption edges
may therefore reflect a combination of two kinds of sorption including hydrophobic sorption
of TBTOH on uncharged sites and electrostatic sorption of TBT+ on negative surface sites.
Figure: 1. Sorption edge of wheat charcoal, and
soot versus pH, respectively (long dash, solid
line); TBTOH proportion versus pH (dotted line)
simulated by Visual Minteq (50 μg L-1 TBTCl,
I= 0.02 M NaNO3).
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The empirical nonlinear Langmuir model was applied to fit the observed data from the
sorption experiments (Fig. 2). In agreement with sorption edge results (Fig. 1), the Langmuir
sorption maximum (Cs,max) increases from about 5×103 µmol kg-1 at pH 4 to approx. 1×105
µmol kg-1 at pH 8 with soot.
The Langmuir binding constant, KL may indicate trends in the affinity between sorbents and
sorbate. It can be seen that KL of the charcoal is considerably higher than that of soot,
especially at pH 4. At this pH, sorption is almost restricted to reaction with TBT+, as this form
constitutes >99% of the TBT at pH 4 (Figure 1). The larger KL for wheat charcoal than for
soot may therefore indicates stronger bonding of the electrostatic than hydrophobic sorption,
since soot is limited to hydrophobic sorption, which is very little at pH 4. With increasing the
pH value, the KL of wheat charcoal decreases showing the decline of bonding between
charcoal and TBT. Nevertheless, the Cs, max of charcoal increases owing to the increase of
charged sites on surface. In contrast, soot shows higher KL at pH 8 than that at pH 4 and 6,
which elucidates that stronger bonding between soot and TBT at pH 8. It could be ascribed to
the hydrophobic form TBTOH dominates at high pH values (>99% at pH 8). Consequently, it
could strongly bind with the hydrophobic surface of soot, resulting in higher sorption
capacity.
Figure 2: Langmuir sorption isotherms of soot and wheat charcoal at three constant pH values (filled
circles: pH 4, empty circles: pH 6, filled triangles: pH 8). All the curves were fitted with all raw data
obtained in triplicates. Vertical bar: standard error
4. Conclusion
This work has been to survey the sorption of TBT to BCs at different pHs, showing the uptake
ability of TBT by BCs much relays on pH factor. At pH 4, both of hydrophobic and
electrostatic sorption are relatively weak, whereas, the sorption reaches maxima at pH 6 for
both soot and charcoal. Moreover, due to the decreasing of TBT+, the TBT sorption to
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15th IHSS Meeting- Vol. 3
charcoal appears to be slight attenuated. As an important component in environment, BCs
therefore could influence the bioavailability, transformation, etc of TBT. However, by taking
the advantage of those properties, the application of BCs on purification of wastewater shows
to be an alternative choice.
References
1. M.W.I. Schmidt and A.G. Noack, Global Biogeochemical Cycles, 14 (2000) 777.
2. M. Elmquist., G. Cornelissen, Z. Kukulska, and O. Gustafsson, Global Biogeochemical Cycles 20
(2006) GB2009.
3. T.D. Bucheli and O. Gustafsson, Chemosphere 53 (2003) 515.
4. C.G. Arnold, A. Weidenhaupt, M.M. David, S.R. Muller, S. B. Haderlein and R. P.
Schwarzenbach, Environmental Science & Technology 31 (1997) 2596.
5. M. Hoch, J. Onso-Azcarate and M. Lischick, Environmental Toxicology and Chemistry 21(2002)
1390A.
6. Weidenhaupt, C. G. Arnold, S.R. Muller, S.R. Haderlein and R.P. Schwarzenbach, Environmental
Science & Technology 31(1997) 2603.
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Evaluation of the Efficiency of Fulvic and Humic Acids (Agrolmin Bravo
and Cerrado) in Soybean Production in the Brazilian Savanna
L.T. Dias Cappelinia, D. Cordeiroa, L.A. Artimonte Vazb, L.F. Artimonte Vazb, E. Bessa
Azevedob, E.M. Vieirab*
a
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil;
b
Agrolatino Ltda, Rua João Pessoa, 996 – Matão – São Paulo
E-mail: eny@iqsc.usp.br
Introduction
The average yield for the soya culture in Brazil is 2,629 kg/hectare or 43.80 bag/hectare [1], a
very low value when compared to the national agricultural productivity potential, in real
conditions of cultivation, approx. 4,000 kg of grain per hectare or 66.70 bag/hectare [2].
Therefore, new alternatives and technologies that promote agricultural productivity have been
developed respecting the concepts of environmental sustainability and enabling high annual
production without promoting deforestation. There is a growing interest in the technology of
applying humic substances by using products made from humic and/or fulvic acids in
agriculture.
This study has the scope of stimulating research in this sector and evaluating the existent new
resources, focusing on products made from humic and fulvic acids, with the aim of increasing
productivity of the soya culture in tropical climate, complying with the new environmental
requirements.
2. Materials and Methods
The present experiment was conducted in Oxisol III on October 21st, 2008, and the harvest
occurred on February 23rd, 2009. The treatments were: Agrolmin with Bravo Cerrado and
conventional fertilization application to the soil during the planting operation and Bravo
Cerrado with conventional fertilization application, applied to the leaves, which were
compared with standard treatments adopted in the property.
A randomized block design was used with 3 treatments and 11 repetitions. In the experiment,
each parcel consisted of six lines, spaced by 0.45 m, with a length of 2 m, and a total area of
5.4 m2 for each parcel. Since the total number of parcels equals to 33, the area occupied by the
soya experiment was equal to 178.2 m2.
The description of treatments, products, doses and time of application on the soya crop are
listed in Table 1.
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V4, V8 e R4
Table 1: Description of treatments, products, doses and timing of application for soya
Dose
Applicatio
Treatments
Foliar
Dose
(kg/hectare)
Period
(T1) (02-20-20) + 0,2%
Bravo
350
3 L/hectare
B + Agrolmin
Cerrado
+150 kg KCl/hectare
(T2) (02-20-20) + 0,2%
Bravo
350
3 L/hectare
B
Cerrado
+ 150 kg KCl/hectare
Zn
90 g/hectare
Mn
150 g/hectare
(T3) (02-20-20) + 0,2%
Cu
1 g/hectare
350
B
B
1 g/hectare
+ 150 kg KCl/hectare
S
135 g/hectare
N
150 g/hectare
Soil sampling was performed in the whole area, collecting the samples in the depth of 0 to 20
cm. Leaf samples were collected at the R1 stage – beginning of flowering – to assess the
nutritional status of the culture.
The application of Agrolmin with Bravo Cerrado and conventional fertilization application
was performed together with the operation of planting in spray jet directed at seed precoverage. However, the application of Bravo Cerrado with conventional fertilization was
made on the leaves.
3. Results and Discussion
The results for roots dry mass (RDM) and shoot dry mass (SDM) production of the plants and
the yield of soybeans are shown in Figures 1 to 3. Comparing the treatments, it can be
observed that the conventional fertilization with 300 kg/hectare of the 02-20-20 formulation
plus 150 kg/hectare of KCl (T1) presented the lowest RDM. The application of conventional
fertilizer plus 3 L/hectare of Bravo Cerrado (T2) increased the RDM in 7.4%. However, this
result is not statistically different from the conventional fertilization. On the other hand, the
combination of the conventional fertilization plus Bravo Cerrado and 20 L/hectare of
Agrolmin increased significantly (P ≤ 0.05) the production of RDM. This increase was 0.5
g/plant, corresponding to 16.6%. Considering that Agrolmin is a product made from humic
substances containing significant amounts of humic and fulvic acids that play an important
role in the induction of root growth [3,4], this result was expected. It was also observed a
significant increase in the amount, size, and persistence of nodules, indicating higher rates of
biological nitrogen fixation biological [5].
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The analysis of the leaves indicated that increment, showing nitrogen average levels of 57.1,
48.8, and 45.3 g/kg for Agrolmin with Bravo Cerrado and conventional fertilization (T1),
Bravo Cerrado with conventional fertilization (T2), and conventional (T3) treatments,
respectively.
As occurred with the RDM production of soya plants, the addition of 3 L/hectare of Bravo
Cerrado with conventional fertilization (T2) and 20 L/hectare of Agrolmin with Bravo
Cerrado and conventional fertilization (T1) increased the SDM production. This increase was
statistically significant and corresponds to 7.7% and 21.0% for Bravo Cerrado with
conventional fertilization and Agrolmin with Bravo Cerrado and conventional fertilization,
respectively, when compared to conventional fertilizer (T3). Given that there was an increase
in root growth, the volume of soil explored by the roots was greater, increasing the absorption
of water and nutrients, fact that is reflected on SDA production of soya plants. Furthermore,
the combination of mineral sources of nutrients with Bravo Cerrado and Agrolmin increases
the usage efficiency of those nutrients, which can be seen by gains in the SDM production.
The soybeans production significantly increased [6] (P ≤ 0.05) with the application of 20
L/hectare of Agrolmin with Bravo Cerrado and conventional fertilization (T1), when
compared to the conventional treatment (T3). This increase was of 1,133 kg/hectare from
soybeans, accounting for 24.5%. The leaf analysis indicated that 5.5 g/kg of phosphorus in the
treatment that received the application Agrolmin with Bravo Cerrado and conventional
fertilization (T1) against 2.7 g/kg for treatment with Bravo Cerrado with conventional
fertilization (T2) and 2.87 g/kg for conventional treatment (T3). These effects indicate higher
rates of recovery of the applied mineral fertilizers, resulting in greater agricultural
productivity.
The application of Bravo Cerrado with conventional fertilization promoted a better foliar
nutrition compared to the conventional treatment. This fact can be explained by the chelating
effect promoted by humic and fulvic acids added to the nutrient sources of Bravo Cerrado. A
better nutritional balance was also reflected in the grains productivity of parcels treated with
Bravo Cerrado with conventional fertilization (T2), which produced 8.7% more (335 kg/ha)
than the control area (T3). Although this production increase was not significant, it clearly
shows the effect of a better nutrition on production.
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4. Conclusions
The application of Bravo Cerrado with conventional fertilization promoted an increase of
7.4% in the production of RDM, 7.7% in the production of SDM, and 8.7% in soybean
production. On the other hand, the application of the commercial product Agrolmin with
Bravo Cerrado increased the roots dry mass production of soya plants by 16.6%, the shoots
dry mass by 6.7%, and consequently the grain yield by 24.5%.
The productivity increase with the application of Bravo Cerrado and Agrolmin with the same
doses of mineral fertilizers is a viable alternative to reducing production costs, as well as
reducing environmental impacts due to maintaining the extent of the cultivated area.
Acknowledgements
The authors thank CAPES, FAPESP, CNPq, for financial support and Agrolatino Ltda.
References
1. Ministério da Agricultura, Pecuária e Abastecimento. Companhia Nacional de Abastecimento
Superintendência. Regional do Paraná. Gerência de Desenvolvimento e Suporte Estratégico / Setor
de
Apoio
à
Logística
e
Gestão
da
Oferta.
Soja,
(2009).
http://www.conab.gov.br/conabweb/download/sureg/PR/Soja%20Junho%202009.pdf (12/01/10).
2. III Simpósio de Plantas Oleaginosas. Realidades e Potencialidades Brasileiras, (2009).
ESALQ/USP - Piracicaba/SP. http://www.pecege.esalq.usp.br/soja/ (12/01/10).
3. CHEN, Y; AVIAD, T. (1990), Effects of humic substances on plant growth. In: McCARTHY P.;
CLAPP, C.E.; MALCOLM, R.L. & BLOOM, P.R., (Eds.) Humic substances in soil and crop
sciences: selected readings. Madison: SSSA. 161-186.
4. VAUGHAN, D.; ORD, B.G. (1976), Uptake and incorporation of C14-labelled soil organic matter
by roots of Pisum sativum L. Journal of Experimental Botany, Oxford, 32, 679-687.
5. LEE, Y. S.; BARTLETT, R. J. (1976), Stimulation of plant growth by humic substances. Soil
Science Society of America Journal, Madison, 40, 876-879.
6. BENITES, V. de M.; POLIDORO, J.C.; MENEZES, C.C.; BETTA, M. (2006). Aplicação foliar
de fertilizante organo-mineral e soluções de ácido húmico em soja sob plantio direto. Embrapa,
Rio de Janeiro. 6p. (Circular Técnica, 35).
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Pyrolisis Parameters Evaluation in the Biochar Preparation Process
E. Inayve P. de Rezendea, A.P. Mangonia, I. Messerschmidta, A.S. Mangricha*,
E.H. Novotnyb, M.H.R. Vellosob
a
Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas, C.P. 19081,
81531-990, Curitiba, PR, Brazil; bEMBRAPA Solos, R. Jardim Botânico, 1024, 22460-000,
Rio de Janeiro, RJ, Brazil
E-mail: mangrich@quimica.ufpr.br
1. Introduction
Considerable efforts are being proposed to mitigate the environmental problems caused by the
significant increase of CO2 concentration in the atmosphere. The methodology of using
biochar, obtained by pyrolysis of biomass, as a organic soil conditioner to mitigate this
problem has gained support of a considerable number of components of the scientific
community. The use of biochar in soil to produce positive effects on soil fertility was applied
in the Amazon region by pre-Columbian indigenous community1. David R. Montgomery in
its 2020 visions about care with world soil´s sad: “Over the next few decades, approaches
such as low-till and organic methods could restore native soil fertility and store enough soil
organic matter to offset global fossil-fuel emissions by 5–15%. Offsets, and soil fertility,
could be further increased through adding biochar — charcoal made by heating organic
wastes”2. Biochar presents as structural features characteristic condensed aromatic
compounds, hydrogen-deficient, highly resistant to oxidation, and therefore the action of soil
microorganisms, thereby contributing to carbon sequestration. Yet it may be partially oxidized
in their peripherals aromatic groups, producing carboxylic and phenolic groups that contribute
to soil CEC, buffering the acidity, complexing ions and inorganic structures, retaining water
via hydrogen bonds and, consequently, increasing security (stabilization) and fertility of soil
3,4
. In the pyrolysis of biomass study in our laboratory, methods have been developed not only
to produce biochar, but also aim to produce bio-fuels. Towards the development of scientific
knowledge, technology and innovation in the use of organic by-products, especially derived
from the biofuel industries, this work has been carried out to prepare "biochar" from the
castor oil cake, through the pyrolysis at low temperatures ( 300-350 ° C) and deficiency of air.
2. Material and Methods
The castor oil cake, gridding in ball mills to a particle size of 80 meshes was placed in
porcelain boats in the inner glass tube furnace EDG FT-40 microprocessor-controlled. The
factors assessed by a 23 factorial design were: heating rate (V), final temperature (T) and
warm-up period (P), at the levels of 5 and 10 ° C min-1, 300 and 350 ° C and 30 and 60 min,
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respectively. The solid material obtained was characterized by EPR, FTIR and NMR
spectroscopy.
EPR spectroscopy. The EPR spectra were obtained at room temperature (~ 300 K) on a
Bruker EMX spectrometer operating at X-band (~ 9.5 GHz) using 100 kHz modulation
frequency and 0.05 mT amplitude modulation. The parameters values were obtained by the
treatment of experimental spectra with the aid of WinEPR software and weak pitch standard
from Bruker Company.
FTIR spectroscopy. The FTIR analyses were performed on a spectrophotometer model FTIR
Biorad Excalibur Series (FTS-3500 GX) with the spectra resolution of 4 cm-1 in the region
from 4000 to 400 cm-1. The sample pellets for analyses were made using approximately 1 mg
of the biochar sample and 99 mg of KBr spectroscopic grade and submitting the homogenized
mixture to pressure. For each spectrum 32 scans were summed.
NMR spectra. Solid-state 13C NMR experiments were carried out using a Varian VNMRS 500
MHz spectrometer at
13
C and 1H frequencies of 125.7 and 500.0 MHz, respectively. The
technique used was variable amplitude cross-polarization (VACP).
3. Results and Discussion
EPR spectra. By EPR spectroscopy the g-factor, spin density and power saturation of the
signal values were obtained. The values of the g-factor of EPR found are around 2.003,
indicating the presence of free radicals in organic structures (OFR). The parameter values of
spin density and power saturation of the signal can be seen in Figure 1. For the spin density
parameter, the highest values are of those samples with longer periods of heating (P1, P2, P5,
P6). For the power saturation of the EPR signal, the sample P2 (V = 30 0C min-1, T = 350 0C,
P = 60 min and D = 11.81 x 1018 spins g-1) sustained the greatest power of EPR while the
sample P4 (V = 30 0C min-1, T = 350 0C, P = 30 min and D = 4.60 x 1018 spins g-1) showed a
lower saturation power. Thus, the sample P2, by EPR spectroscopy, is presented with more
integration spin arrangement of aromatic structures (better dissipates the energy resulting
from the relaxation of spins), supporting more power, suggesting that the heating time was the
most important factor for the formation of stable internal structure of the sample.
FITR spectra. The FTIR spectra of the samples were very similar, all featuring mainly a broad
band at 3680-3300 cm-1, associated with the O-H stretching from alcohol carboxylic acids and
water. Energy absorption in 1625 cm-1 attributed to the structural vibrations of C = C
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aromatics, asymmetric stretching of C = OO- and bending of O-H groups. Shoulder at 1706
cm-1 is assigned to C = O stretching of ketone, esther and carboxylic acid.
P (min)
Eixo y
P6
P5
60
12,16
2,04
P2
13,88
2,68
P1
11,81
5,66
9,31
4,11
Eixo z
P7
P8
30
8,72
1,93
300
P4
4,60
1,74
350
4,03
1,76
P3
v (\ C min‐1)
7,70
4,48
5
10
Eixo x
Figure 1: Diagram for the effects in planning 23 interpretation (Bold values inside the circles
correspond to spin density (x1016 spins g-1) and those below without bold are power (x10-4 W))
NMR spectra. To aid in the analyses of the results, the Multivariate Curve Resolution (MCR)
procedure was carried out using the software ‘The Unscrambler® v9.7’ (CAMO Software
AS). The basic goals of MCR are: the determination of the number of components co-existing
in the chemical system; the extraction of the pure spectra of the components (qualitative
analysis); and extracting the concentration profiles of the components (quantitative analysis).
The results of this analysis indicate that the set of analysed samples can be modelled by
means of a two component mixture – binary, one of a partially carbonised material
(Component 1, Fig. 2a), with aromatic groups presenting poor ring condensation (129 ppm)
and alkyl groups. The other estimated compound is still less carbonised, presenting features of
the precursors, like O-alkyl (72 ppm) and di-O-alkyl (shoulder at ~110 ppm) from cellulose;
O-aryl
(143
ppm)
from
lignin
and
240
210
180
150
120
90
60
30
0
Estimated Concentration (%)
Compound 1
Compound 2
aliphatic
carboxyl
(173
ppm).
Compound 1
Compound 2
100
80
60
40
20
0
-30
P1
P2
P3
P4
P5
P6
P7
P8
Samples
13
C Chemical Shift (ppm)
Figura 2 – Results of multivariate curve resolution (MCR) analysis. (a) Estimated spectra; (b)
Estimated concentrations
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The samples treated at lower temperature (300 °C - P3, P4, P7 and P8) were those that
showed signals from the precursor (castor oil cake). In other words, the cited oxygen
substituted groups present in the compound 2 are preserved from the castor oil cake due to the
low carbonisation temperature employed, since this temperature avoided that these
thermolabile compounds were decomposed or altered, unlike the samples treated at 350 °C.
Acknowledgements
CNPq, DQ/UFPR, Ao Professor Dr. Ronny R. Ribeiro.
References
1.
2.
3.
4.
S. Bruun and J. Luxhoi, Environ. Sci. & Technology, March 1 (2008);
D. R. Montgomery, Nature, 463, p. 26-32 (2010);
E. H. Novotny at al., J. Braz. Chem. Soc., 20 (6), p. 1003-1010 (2009);
T. Whitman and J. Lehmann, Environ. Sci. & Policy, p. 1024-1027 (2009).
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Extraction of high-value lipids from Irish Peats
Raymond McInerneya, Daniel John Hayesa, J.J. Leahya, Michael HB. Hayesa
a
University of Limerick, Limerick, Ireland
E-mail: Raymond.McInerney@ul.ie
1. Introduction
Lipids, which range from simple n-fatty acids or n-alcohols to more complex cyclic
terpenoids and steroids, are insoluble in water but extractable with non-polar solvents e.g.
hexane, chloroform, benzene or ether [1]. Lipids are generally not components of humic
substances but they occur in associations with humic molecules. They can, however, be
incorporated in humic structures through esterification with the carboxyl and hydroxyl
components in humic acids, especially [2].
For the characterization of lipids in complex mixtures it is important to isolate the
components freed from contamination, as evidenced by well resolved peaks in
chromatographic procedures [3].
Organic solvent extraction of peat can give wax yields ranging from 5 to 15%. The amounts
depend on the source of the peat, its pretreatment, and the solvent used [4].
The components of the waxes have relatively wide molecular weight profiles, and
functionalities that range from n-alkanes to a variety of structures with reactive functionalities.
2. Materials and Methods
Low density (LD), medium density (MD), and high density (HD) peat samples from raised
bogs were provided by Bord na Mona (the Irish Peat Board). Samples were air dried and
sieved.
Solvent Extraction of Peat Samples. Each of the peat samples was extracted under reflux for
4.5 hours in a 1:1 toluene/ethanol (1:1 v/v) mixture, then filtered under reduced pressure. The
filtrate was rotary evaporated to remove the bulk of the solvent, and the remaining solvent
was evaporated in a fume cupboard. The residual wax was then dried in a vacuum oven at 40
o
C for at least 3 hours.
Derivatization of Sample for Gas Chromatography-Mass Spectrometry Analyses. The
procedure described by Jansen et al. [5] was used for the clean-up and derivatization of the
wax samples. The temperature programme for the GC-MS analysis was: 50 °C, 2 min; heating
at 40 °C/min to 80 °C; holding at 80 °C for 2 min; heating at 20 °C/min to 130 °C;
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15th IHSS Meeting- Vol. 3
immediately followed by heating at 4 °C/min to 300°C; and finally holding at 300 °C for 10
min. Elemental analysis of the peat waxes used an Elemental Analyzer.
3. Results and Discussion
The elemental analyses of the peat waxes are given in Table 1. Hydrogen to carbon atomic
ratio of 1.7 and higher indicates that these components are long chain aliphatic compounds;
i.e. mainly fatty acids, alcohols and sterols.
Figure 1 shows the chromatogram of the wax from the HD peat sample. The compositions of
the different peaks were identified using the GC/MS library and their relevant yields were
quantified by the introduction to the filtered extracts of internal standards of the deuterated
lipids, n-eicosane-d42 and eicosanoic-d39 acid.
a:
HD Wax
MD Wax
LD Wax
C
75.02
73.91
72.76
Table 1: Elemental composition of the peat wax samples
H
O
N
S
H/Ca
11.56
11.0
0.45
1.98
1.85
10.90
12.86
0.38
1.94
1.77
10.33
14.16
0.51
2.24
1.70
O/Ca
0.11
0.13
0.15
Atomic ratio
Figure 1: GC Analysis of wax extracted from the high density peat using ethanol/toluene
A list of the possible components that are to be found in the wax extracted from the HD peat
samples are given in Table 2. The waxes extracted from the MD and LD peat samples
contained a similar list of components (not shown here). The major products identified, and
their potential uses were:
Docosanoic acid, also known as behenic acid, is an n-carboxylic acid, a fatty acid, formula
C21H43COOH. In appearance. It is a white to cream coloured crystalline or powder material,
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15th IHSS Meeting- Vol. 3
MP 74–78 °C, BP 306°C. Commercially, it is often used to give smoothing properties to hair
conditioners and moisturizers.
Table 2: Identifications of the different peaks in the wax of the high density peat
Retention
Time (min)
11.00
23.56
27.69
33.69
35.23
37.18
38.72
42.13
45.46
Name of Compound
% mass (dry mass) wax
n-Butanoic acid
Hexadecanoic acid
Decanoic acid
1-Docosanol
Docosanoic acid
Tetracosan-1-ol
Tetracosanoic acid
Hexacosanoic acid
7,8, D-hydro-1-biopterin
0.02
0.26
0.14
0.12
0.20
0.12
1.08
1.59
0.13
1-Docosanol is a long chain 22-carbon primary alcohol. It is also known as behenyl alcohol.
It is used as an emollient in skin care. It has a major use as an antiviral agent, specifically for
treatment of "cold sores" caused by the herpes simplex virus. It acts by inhibiting fusion
between the human plasma cell membrane and the viral envelope.
β-sitosterol is a common phytosterol, MP 139 oC. It is either used alone or in combination
with similar phytosterols in reducing blood levels of cholesterol, and it is sometimes used in
treating hypercholesterolemia. It has a positive effect on androgenetic alopecia (causing hair
loss in males), and is also used in the treatment of prostatic carcinoma and breast cancer.
Decanoic acid or capric acid, a saturated fatty acid, CH3(CH2)8COOH. It is used in organic
synthesis and industrially in the manufacture of perfumes, lubricants, greases, rubber, dyes,
plastics, food additives and pharmaceuticals.
Tetracosanoic acid (or lignoceric acid), is the saturated fatty acid, C23H47COOH. It is a
byproduct of lignin production. It is the most abundant fatty acid in skin ceramides [6].
Tetracosanol is a constituent of polycosanol, a mixture of alcohols. Tetracosanol and other
long chain fatty alcohols, and their esters are known to improve the physical performance of
athletes. Components comprising such alcohols and esters are contained in vegetable oil.
4. Conclusion
Long-chain aliphatic alcohols (polycosanols) and fatty acids (with more than 20 carbon
atoms) are of considerable interest as healthcare and personal care products. Distillation and
fractionated by short-path distillation would provide fractions that are rich in docosanol (C22)
and tetracosanol (C24) and other long chain fatty alcohols that may be found in different peats.
Acknowledgements
We acknowledge the financial and sample contributions of Bord na Mona.
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References
1. Dinel, H., M. Schnitzer and G.R. Mehuys, 1990. Soil lipids: origin, nature, content, decomposition
and effect on soil physical properties. In: Bolag, J.M., Stotzky, G. (Eds.), Soil Biochemistry, Vol.
6. Marcel Dekker, New York, pp. 397–429.
2. Mathur, S.P. and R.S. Farnham, 1985. Geochemistry of humic substances in natural and cultivated
peatlands. In: Aiken, G.R., McKnight, D.M., Wershaw, R.L. and MacCarthy, P., Editors, 1985.
Humic Substances in Soil, Sediment and Water, Wiley, New York, pp. 53–85.
3. Wiesenberg, G.L.B., L. Schwark and M.W.I. Schmidt, 2004. Improved automated extraction and
separation procedure for soil lipid analyses. Eur. J. Soil Sci. 55, pp. 349–356.
4. Howard A.J., and D. Hame, 1962. The extraction and constitution of peat wax. Chromatographic
fractionation of wax, Journal of the American Oil Chemists' Society, 39, 5
5. Jansen, B., K.G.J. Nierop, M.C. Kotte, P. de Voogt and J.M. Verstraten, 2006. The applicability of
accelerated solvent extraction (ASE) to extract lipid biomarkers from soils. Appl. Geochem., 21:
1006–1015.
6. Vávrová, K., J. Zbytovská, K. Palát, T. Holas, J. Klimentová, A. Hrabálek and P. Dole al, 2004.
Ceramide analogue 14S24 ((S)-2-tetracosanoylamino-3-hydroxypropionic acid tetradecyl ester) is
effective in skin barrier repair in vitro, Eur. J. Pharm. Sciences 21(5), 581–587
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Fluorescence of Aqueous Solutions of Commercially Produced Humic
Substances
Olga Yakimenkoa*, Aleksei Izosimova, Daria Shubinab, Viktor Yuzhakovb, Svetlana Patsaevab
a
Soil Science Department, Moscow State University, Moscow 119991, Russia;
b
Department of Physics, Moscow State University, Moscow 119991, Russia
E-mail: ola-yak@mail.ru
1. Introduction
Humic substances (HS) and especially their water-soluble fraction play a very important role
in environmental biogeochemistry [1-2]. Usage of commercially produced HSs in agriculture
and soil remediation is very perspective nowadays. However diversity of commercial HS
sources and technological know-how of their production cause the problem of their
characterization and classification.
Fluorescence spectroscopy is a powerful tool for rapid characterization of organic substances,
in particular dissolved organic matter (DOM) in natural waters of different origin [3-4].
Fluorescence emission spectra of humic substances naturally occurring in water are generally
characterized by a unique broad band, so called “humic-type fluorescence”, showing a
maximum wavelength (λem) around 420-460 nm depending on both the sample origin and
excitation wavelength (λex) [5-7]. Typically fluorescence of humic acids isolated from various
soils and soil-related materials is shifted to longer wavelength region (500-520 nm) [8-9]
comparably to humic substances of natural waters. Fluorescence spectra of commercial HS
differ depending on genesis of source material: coal humates show the emission maximum at
470 nm whether humates originated from peat, lake sediments and lignin derivatives
demonstrate maximum emission shifted towards shorter wavelengths at excitation
wavelengths 355 nm [10].
This objective of this work was to study the fluorescence properties of commercially available
HSs produced from various organic resources, and to find spectral parameters useful for their
rapid characterization and discrimination.
2. Materials and Methods
Fluorescence spectra were examined for commercially available sodium and potassium
humates manufactured in Russian Federation, USA, Canada and China from a number of
source materials differ in their origin and humification conditions: coalified materials (brown
coal, lignite, leonardite and humalite), peat, lake bottom sediment (sapropel) and organic
waste material (lignosulphonate).
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Fluorescence emission spectra were measured by luminescence spectrometer Solar CM 2203
under excitation at 270, 310 and 355 nm for the samples diluted in 10 times. The choice of
excitation wavelengths was based on our previous reports on DOM fluorescence [5-7]. Both
absorption and fluorescence measurements were made under room temperature for aqueous
solutions of humates at concentration 0.02 g l-1 and pH 6.0, placed in quartz cuvettes with 1
cm optical path length and 5 ml volume. Fluorescence quantum yield was estimated using
quinine sulphate solution as a reference.
3. Results
Fluorescence emission excited at different wavelength. Fig.1 illustrates that fluorescence
emission maximum position differs for commercial HS of different origin. Most of HS from
coals show the maximum near 500 nm, whether humates from young caustobioliths peat and
sapropel demonstrate emission maximum at shorter wavelength area (430-480 nm). Humic
product from lignosulphonate subjected to “artificial humification” has emission maximum in
UV-area (360 nm).
Sa-Plod from sapropel
BC-Hum from brown coal
Pe-IXP from peat
Pe-Eda from peat
Hu-Usa from humalite
Hu-Bsol from humalite
Le-Sah from leonardite
Le-Sp100 from leonardite
Li-Ion from lignite
Li-Sol80 from lignite
OW-LhNa from lignosulphonate
Intensity, rel. un.
2
1
0
300
400
500
600
700
Wavelength, nm
Figure 1: Fluorescence spectra excited at 310 nm for aqueous solutions of commercial HSs
In contrast to studied earlier samples of DOM in natural waters or soil extracts [5-7],
commercially available HSs did not exhibit noticeable shift of fluorescence maximum
position along with increasing excitation wavelength: emission wavelength keeps practically
constant for all commercial HS, except for HS produced from peat.
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Fluorescence quantum yield (QY). Fluorescence QY for commercial HSs (see Fig. 2) is
typically smaller than that of DOM in water (0.02-0.04), and is comparable for that of soil
water-extractable DOM (0.001-0.003). Fluorescence QY depends essentially on excitation
wavelength for natural HS, its value is increasing along with λex rising. In contrast, for
commercial HSs the QY value is either decreasing with rising of excitation wavelength, or
keeps constant. The former phenomenon is especially noticeable for coal humates, produced
from mature caustobioliths, whether the latter one was observed for HS from peat and
sapropel.
We explain this observation considering that natural HSs differed from commercial HSs in
bigger heterogeneity of fluorophores composition as evidenced by the behavior of their
emission maximum and QY changes along with increasing of excitation wavelength.
Fluorescence quantum yeild
0,04
ex 270
ex 310
ex 355
0,03
0,02
0,01
Le
-S
ah
Le
-H
PA
Le
-S
p1
00
Hu
-U
sa
Hu
-D
so
l
Hu
-B
so
l
O
W
-L
hN
a
Li
-B
G
Ha
Li
-S
ol
80
Li
-Io
n
Pe
-E
da
BC
-H
um
Sa
-P
lo
d
Pe
-IX
P
0,00
Figure 2: Fluorescence QY measured at excitation wavelengths 270, 310 and 355 nm for different
commercial HS samples (see Fig.1 for details of HSs)
4. Conclusions
UV-excited fluorescence spectra for aqueous solutions of commercially produced humic
substances were compared with that for earlier studied natural HSs of riverine and marine
origin. Two parameters such as fluorescence emission maximum (λem) and fluorescence
quantum yield were found very useful for rapid characterization of commercially available
HSs. Both parameters measured along with excitation wavelength rising from 270 to 355 nm
for solutions of commercially available HSs differs much from that for substances of natural
origin. In contrast to natural HS, commercially available humates do not exhibit noticeable
shift of fluorescence maximum position along with increasing excitation wavelength 270 to
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355 nm. For commercial HSs the fluorescence quantum yield value is either decreasing with
rising of λex or does not depend on it, demonstrating at opposite pattern comparably to natural
HS, for which QY is rising with increasing excitation wavelength. Distinct fluorescence
properties of humic substances observed along with variation of λex provide useful diagnostic
criteria for distinguishing between commercial and natural humic substances.
Acknowledgements
Financial supports of Russian Foundation of Basic Research (project 07-04-01510) and
Presidium of Russian Academy of Science (grant of Biodiversity Program) are deeply
appreciated.
References
1. D.S. Orlov, Humic Substances of Soils and General Theory of Humification. Balkema, Brookfield,
1995, p.266
2. I.V. Perminova, et al. in I. Twardowska, H.E Allen, M.H. Haggblom, S. Stefaniak, (Eds.), Viable
Methods of Soil and Water Pollution Monitoring, Protection and Remediation. Series IV: Earth
and Environmental Sciences, Springer, Netherlands, 69 (2005) 249-274.
3. M.М.D. Sierra , et al., Marine Chemistry, 47 (1994) 127-144.
4. P.G. Coble, et al., Marine chemistry, 51(1996) 325-346.
5. S.V. Patsaeva, EARSeL Advances in Remote Sensing, 3 (1995) 66-70.
6. A.S. Milyukov, et al., Moscow University Physics Bulletin, 6 (2007) 368-372.
7. O.M. Gorshkova, A.S. Milyukov, S.V. Patsaeva, V.I. Yuzhakov. Proc. SPIE 6263 (2006) 248255.
8. N. Senesi, et al., Soil Sci. 152 (1991) 259-271.
9. A. Zsolnay, et al., Chemosphere, 38 (1999) 45-50.
10. P. Volkov, O.Yakimenko. in Humic Substances – Linking Structure to Functions. Proc. of 13th
Meeting of the International Humic Substances Society (2006) 45-I: 261-265.
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Assessment of the Oil Shale Byproducts Use as Soil Conditioner: Study of
Sorption and Biodegradation of Phenol Models with Soil
Rafael Garrett Dolattoa, Gilberto Abateb, Iara Messerschmidtb, Betânia Fraga Pereiraa*,
Antonio Salvio Mangrichb, Carlos Posser Silveirac, Clenio Nailto Pillonc
a
FAPEG/Embrapa Clima Temperado, BR 392, km 78, CP 403, 96001-970, Pelotas-RS,
Brazil; bUniv. Federal do Paraná, Depto. de Química, CP 19081, 81531-990, Curitiba-PR,
Brazil; cEmbrapa Clima Temperado, BR 392, km 78, CP 403, 96001-970, Pelotas-RS, Brazil
E-mail: betaniapereira@yahoo.com.br
1. Introduction
A reasonable oil shale water volume is generated in the oil shale industrialization, arising
from the shale pyrolysis process. This oil shale water byproduct can be used as a soil
conditioner, due to be rich in important micronutrients to plants and crops. Nevertheless, a
high content of other not desirable compounds, such phenol and the derivatives o-cresol and
p-cresol, can be present in this oil shale water. Although the water would be diluted
previously to the application, it is very important to be aware of the phenolic compounds’
behavior in the presence of the soil sample in order to prevent and avoid the leaching of it to
the groundwater . In this way, in the first moment, the aim of this work was to study, the
interaction of phenol, o-cresol, and p-cresol models with a soil sample rich in clay fraction
that was collected in an experimental area near an oil shale industry. A comparison of
mercuric chloride and sodium azide methods was performed to investigate the existence of
phenolic compounds models biodegradation. The following studies will considerer the
interaction of the real oil shale water with soil samples.
2. Materials and Methods
Tests in Batch: One gram (± 0.1 mg) of soil was transfer to glass flask of with 30.00 mL of
CaCl2 0.01 mol L-1 [1]. The isotherms were obtained from 10 points in increasing
concentrations of each phenolic compound model in the range of 0.00 to 500.0 mg L-1. For
excitement, it was used an orbital bench, operating at 170 oscillations min-1 for 48 hours.
After this period the samples were centrifuged at 3000 rpm and concentrations remaining of
the phenolic compound models in the supernatants phases were determined by the method of
4-AAP [2].
Contact time: The time of apparent equilibrium between the phenol model and the soil sample
was studied using a mass of five grams (± 0.1 mg) of soil volume in 150.0 mL of phenol at a
concentration of 50.00 mg L-1 in an ionic medium of CaCl2 0.01 mol L-1. Similarly, blank
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evidence was prepared in the absence of phenol.
Microbial Inhibition: For this test similar conditions of the experiments performed at the
contact time were used. Some glass bottles were prepared only with soil samples (5.0000 g) in
150.0 mL of phenol at a concentration of 50.0 mg L-1 and in another group of bottles beyond
the soil it was also added HgCl2 100 mg L-1 acting as a microbial inhibitor [3]. The vials were
kept under agitation of 170 oscillations min-1 for 10 days. During this time aliquots of 5.00
mL of the suspensions were withdrawn, centrifuged at 3000 rpm for 5 minutes and the
concentration of phenol was determined by absorption spectrophotometry in the UV region λ fixed at 270 nm. Experiments were performed similarly to o-cresol and p-cresol. When the
phenol was not more detected in the supernatants, the samples were fortified with a new
injection of phenol (fortifications) from the stock solution, providing a concentration close to
50.0 mg L-1. All the inhibition experiments were performed in triplicate.
Also another study was conducted using a second type of microbial inhibitor. It was used the
solution of NaN3 drawing upon the same conditions of those studied with HgCl2, however, it
was used NaN3 solution with a concentration of 1% (w / v) in the middle of CaCl2 0.01 mol L1
as an inhibitor of microbial activity.
3. Results and Discussion
The soil sample (62.3% clay, 31.1% silt and 2.6% sand) was collected in the surface layer
between 0–18 cm, which is classified as clayey. The results of the sorption capacity of
phenolic compounds from the soil are shown in Fig. 1.
Figure 1 show that the isotherms for phenol and p-cresol are very similar, tending to linearity.
For phenol and p-cresol, the curves showed a very similar behavior and are almost linear, and
a removal between 10 and 4% was verified for phenol and one of between 25 and 4% for pcresol, for the first and the last point of the sorption isotherms, respectively. For o-cresol the
sorption was not significant, being near 3% for the first point of the curve and 0.7% for the
last point. The decrease of sorption for the three compounds suggests saturation of sorption
sites of soil, probably due to high concentration of phenols.
However, other processes can proceed in parallel to the sorption, such as, biological
degradation of phenol throughout the experiment.
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600
-1
Amount sorbed (μg g )
500
400
fenol
o-cresol
p-cresol
300
200
100
0
0
100
200
300
400
500
-1
Equilibrium concentration (mg L )
Figure 1: Sorption isotherms of phenolic compounds after 24 h of contact time with 1.0000 g of soil in
30.0 ml of CaCl2 0.01 mol L-1. Initial concentration of phenolic species between 5.00 and 500.0 mg L1
. The points shown the average result of three experiments
In this way, it was investigated the possibility of biodegradation of compounds in the soil
sample, solutions of HgCl2 100 mg L-1 and sodium azide NaN3 1% (m/v) were used as the
inhibitors microbial, and the results are shown in Fig. 2.
60
spike 1
spike 3
spike 2
55
50
-1
phenol (mg L )
45
40
35
30
25
20
(a)
(b)
(c)
15
10
5
L.O.Q.
0
0
1
2
3
4
5
6
7
8
9
10
contact time (days)
Figure 2: Monitoring of phenol concentration versus time in the absence of inhibitor (A), in the
presence of 100 mg L-1 HgCl2 solution (B), and in the presence of 10 g L-1 azide solution (C). Initial
phenol concentration and spike values, 50.0 mg L-1; soil mass, 5.0000 g; initial volume, 150.0 mL;
ionic medium, 0.01 mol L-1 CaCl2 solution. All points represent the medium result of three
experiments
Initially the suspension containing HgCl2 over 10 days of monitoring showed no reduction in
the concentration of phenol presentation that the effects of sorption and volatilization are not
very pronounced in these samples. Concomitantly, the suspensions containing phenol in NaN3
showed a similar behavior to the suspensions in HgCl2, but it was observed a decrease in the
concentration of phenol in relation to the concentration initially added. It suggests the
possibility of phenol adsorption by the soil around 20% when the sodium azide was used as
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an inhibitor. And it could be some influence of sodium in order to facilitate the process of
phenol adsorption in the soil. Also, the effect of sorption is not significant in the presence of
HgCl2, which could somehow block the adsorption sites of the soil, preventing the interaction
of phenol, through electrostatic interactions between phenol and soil. Moreover, in the
suspension with no addition of an inhibitor, the phenol is no longer detected in solution after
48 hours of contact in the experimental conditions studied. Although it was tested the ability
of soil in to degrade a new quantity of phenol in the solution (fortification 1), again, the
compound was not detected in the suspension after 72 hours. A second phenol fortification
was made in the same bottle (fortification 2) and it was again observed degradation. A new
fortification (fortification 3) was performed and after 48 hours, 50 % of the added phenol was
detected, indicating that possibly half of it was being degraded. The degradation of phenol
after new injections was considered a strong indication of the degradation of the compound
instead of the sorption process. Similar experiments were conducted using o-cresol and pcresol and similar results were observed in the microbial inhibition tests for the compound
phenol.
4. Conclusions
The study conducted with microbial inhibitors HgCl2 and NaN3 indicate the biodegradation of
phenol and o-cresol in the studied soil sample. In the following researches it will be studied
the sorption or biodegradation of phenolic compounds in Brazilian soil samples with different
characteristics (texture, organic matter, clay content, pH, CEC) where it was added solid and
liquid (oil shale retort water) by-products from the industrialization of oil shale in order to
verify the environmental safety these.
Acknowledgements
FAPEG/Embrapa Clima Temperado/PETROBRAS, DQ/UFPR, CNPq, Brazil.
References
1. OECD-Organization for Economic Co-Operation And Development. Guideline for the testing of
Chemicals. Adsorption-Desorption Using a Batch Equilibrium Method; Adopted: 21st January
2000, OECD/OCDE 106.
2. APHA – American Public Health Association. Standard Methods for the Examination of Water
and Wastewater. 1995. 19th Edition. Washington, DC, USA.
3. Viotti, P.; Papini, M., P.; Stracqualursi, N.; Gambá, C. Ecological Modelling, 182 (2005) 131-148.
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Humic Acids from Fines of Residual Coal Type Material: Preparation and
Characterization
G.M.Maurícioa, A.C.S. Wimmera, E.A.Brocchia*, A.C.Vidala, R.A.Nunesa
a
Catholic University of Rio de Janeiro, PUC-Rio, Brazil
E-mail: ebrocchi@puc-rio.br
1. Introduction
The United States have the biggest reserves of xystus in the world, followed by Brazil. The
major part of the Brazilian xystus is distributed along Irati, an area that contains one of the
largest amounts of xystus in the world. Irati comprises several Brazil states such as São Paulo,
Paraná, Santa Catarina, Rio Grande do Sul and Goiás.
In Goiás it has been developed by Petrobás a continuous xystus oil removing process which is
well recognized by both a low water consumption and a fine solid waste generation.
These fines are residual coal type and, as such, can be considered as a source for humic
substances (HS) production. In that context, several laboratorial scale methods were tested in
order to obtain a kind of HSs. The obtained material was characterized and has been tested in
environmental applications such as degraded soil recovery, production water treatment and
fertilization.
This work presents an initial evaluation of a selected extraction method applied on the
residual fines based on a preliminary infrared analysis of the obtained humic substance and its
comparison with the starting material original spectrum.
2. Materials and Methods
The material employed for this study was a fine coal type obtained by a xystus processing
industry. The residue pretreatment consisted of a brief granulometric adjustment, where the
material was triturated in a mill and screened to particles finer than 150 mesh (0.105 mm).
Then it was characterized and analyzed in order to determine C, H, O, N and S concentrations
as well as humidity and ashes content. The Humic Acid (HA) preparation was carried out
through different chemical treatment such as those using H2O2, formic acid, formic acid +
H2O2, KOH, NaOH and HNO3. Among the studied treatments, the oxidation method with
HNO3 was the one which showed the best result.
It has been carried out by placing 5.0 grams in a 1000 mL flask with 100 mL HNO3 25%. The
procedure was based on the method described by Trompowsy (2006) in which the whole
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sample is heated up to boiling but kept under reflux for 4 hours. After this the cold material
was placed in a number of a centrifuge tubes and left to rest for 12 hours. Then a 5000 rpm
centrifuge has been used for 20 minutes to produce both a solution and a precipitated. The
latter one has been put to react with 100 mL KOH 1M solution under mechanical agitation for
12 hours in a N2 atmosphere. The former (solution) has been again put to rest for 12 hours in a
set of centrifuge tubes before being centrifuged in the same previously conditions. The
generated solution was taken to pH about 1.0 by adding 6M HCl
and then placed in
centrifuge tubes. After 12 hours it has been centrifuged in the same condition (5000 rpm, 20
minutes) in order to obtain a precipitated which was to be the humic acid (HA). This material
has been left to dialysis before being freeze-dried.
The characterization of the obtained material was performed through conventional methods,
typically employed for the humic substances studies, such as visible UV, Infrared and ICP.
C, H, N e S analysis was implemented as described in ASTM-5291 method and the humidity
concentration was determined through drying procedure up to a constant weight in a stove at
105 ºC. The ashes were obtained in an oven at 550 ºC in order to evaluate its concentration.
The UV-Vis equipment has been employed to determine the E4/E6 ratio (Abs
465nm/665nm). The HA was dissolved at 20 mg/L in a NaHCO3 0.05 mol/L solution while
the pH solution was kept at 8.5. A spectrophotometer was used to perform the sample
scanning in the 190-800 nm range. An infrared spectrometer with Fourier transformed was
also employed (2500 to 25000 nm).
3. Results and Discussion
The starting material prior to the chemical treatments presented 2.61% and 86.20% humidity
and ashes level, respectively. The oxidation method with HNO3 has produced a HA weight
recovery, as compared with the initial sample mass, of about 6.5%. The sample
characterization results of the obtained material are presented below.
Table 1: Elementary analysis of the starting material (-150 mesh) and resulting HA
Species
C
H
N
S
% (m/m)
Fines
6.4
0.9
0.3
3.2
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HA
18.2
2.3
1.2
< 0.3
15th IHSS Meeting- Vol. 3
The humification number, given by the E4/E6 relation, was 0.3371. Figs. 1 and 2 show the
infrared spectra originated from the residual fine samples and from the obtained HA.
Figure 1 – Xystus fines infrared spectrum.
Figure 1. Infrared spectrum of the residual fine
Figure 2: Resulting infrared spectrum of HA obtained from the residual fines
Comparing the two infrared spectra one can notice that in the HA spectrum new bands
appeared in the 1400-3400 cm-1 range. These bands are related to the carboxyls and hidroxyls
bonds. According to literature data, the peaks indicated below have the following
relationships.
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15th IHSS Meeting- Vol. 3
• 3354.20 cm-1 → O-H and N-H
• 2287.35 cm-1 – 1838.11 cm-1 → C-H
• 1626.53 cm-1 – 1680.64 cm-1 → C=O
• 1510.80 cm-1, 1534.80 cm-1 and 1575.95 cm-1 → COO• 1285.29 cm-1 – 1424.55 cm-1 → deformation of O-H carboxyls
Conclusions
An alternative starting material has been used in order to extract humic acid from it. The
material presents some coal characteristics as it is consisted of fine particles generated as
residue in a xystus oil removing process.
Among several studied reactants and process it was clear that the HNO3 oxidation followed
by a number of separation steps was the one which gave best results. The obtained material,
having some HA features, was related to a weight recovery of about 6.5 % in respect to the
initial material mass.
In the infrared spectrum of the obtained material it was detected new band appearances in the
range between 1400 and 3400 cm-1 which can be related to typical HS existing bonds (O–H
and N–H).
These preliminary results allow us to conclude that the product obtained through chemical
oxidation with HNO3 has shown some compatible characteristics with those presented by a
typical HA. This fact is a great incentive to carry on the investigation on applying this residue
as an alternative starting material for extracting humic substances such as the humic acid. The
obtained HS also opens new possibilities of environmental interest since it has been tested in
the recovery of spoiled soils and in the production water treatment. Also, it has been planned
to apply some HS samples as agriculture fertilizer.
The whole idea is now being considered to be applied on materials related to the coal industry
as a mean of giving some use for the concentration process tailings.
References
1. GIEGUZYNSKA, E. et al. Compositional differences between soil humic acids extracted by
various methods as evidenced by photosensitizing and electrophoretic properties. Chemosphere 1
(2009) . Consulting: 12 /March/ 2009.
2. HAUMAIER, L.and ZECH, W. Black carbon – possible source of highly aromatic components of
soil humic acids. Org. Geochem. 23 (1995) 191–196.
3. RAJ, S. The nature and composition of coal humic acids. Ebasco services incorporated. Available
at: :http://www.anl.gov/PCS/acsfuel/preprint%20archive/Files/25_4_SAN%20FRANCISCO_0880_0058.pdf. Consulting: 23/Jan/2009.
4. TROMPOWSKY, P.M. Síntese e caracterização de substâncias húmicas semelhantes aos ácidos
húmicos de carvão de eucalipto, e sua interação com diclorofenol, cálcio, manganês e alumínio.
MSc Thesis.Viçosa University. MG. 2006. 107 p.
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Assessing the Effect of a Bio-accelerated Composting Process Using
Analytical Pyrolysis (Py-GC/MS)
F. Pérez-Barreraa*, K. Akdia, F.J. González-Vilab, J.A. González-Pérezb, T. Verdejob
a
A.M.C. Chemical/Trichodex S.A., P.I. La Isla, Avda. Rio Viejo, 44-45, 41700 Dos
Hermanas, Sevilla, Spain, bIRNAS, CSIC, P.O. Box 1052, 41080 Sevilla, Spain
E-mail: info@amcchemical.com
1. Introduction
The use of recycled materials in agriculture as fertiliser and as an organic amendment in
intensively cropped and organic matter-depleted soils is an important environmental strategy
[1]. There is a large variety of composted materials for which quality must be guaranteed to
ensure a safe agricultural use. Appropriate management of the composting process of urban
wastes is needed to avoid harmful effects caused by non-matured compost application [2]. At
the same time, excessive composting could lead to i) loss of N and polysaccharides with a role
in soil aggregation, and ii) immobilization of nutrients (mainly N and P), hence it has been
reported that such a “postmature” compost may be less favourable to plant nutrients uptake
than are less-matured composts [3].
Pyrolysis coupled to gas chromatography and mass spectrometry (Py-GC/MS) is a powerful
tool widely applicable in the characterization of complex organic mixtures with diverse origin
and is mainly used for the direct study of materials which, owing to their complexity, are
difficult to analyse by conventional methods like composted organic matter (OM) [4]. PyGC/MS. Pyrolysis of composted OM generates a wide range of products with diverse
chemical properties that can be related to their biochemical origin (aliphatic compounds and
methoxyphenols derived from lignin, cyclic ketones and furans from polysaccharides, Ncontaining molecules from proteins, organic acids...).
In this work Py-GC/MS has been applied to the study of the composting evolution of organic
a organic material composed of urban pruning residues and sewage sludge with and without
the addition of a microbial bio-accelerator (CBB).
2. Materials and Methods
The bio-accelerator CBB is a natural product developed by A.M.C. Chemical/Trichodex Co.
(www.amcchemical.com, Seville, Spain) and based in a mixture of organisms that, when
applied to fresh organic materials, is able to accelerate the composting process. Among
known catalytic activity of the selected microbial mix favoring the degradation of
lignocellulosic materials include xylanase and glucanase activities.
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15th IHSS Meeting- Vol. 3
The organic material used was composed of urban pruning residues and sewage sludge at a
ratio 30:70; 2) and was composted in the presence of CBB at the recommended dose and
without CBB using a “slow process” composting i.e, without using enforced aeration of the
windrow during the degradation phase. During the first 4 weeks of composting, the windrow
temperature was monitored periodically every 2–3 days.
Compost piles were sampled in different seasons and at regular time intervals during
composting. After sampling, fractions were sieved at a particle size < 5 mm, air-dried and
grinded before analysis.
Analytical pyrolysis (Py-GC/MS) was performed with a double-shot pyrolyzer (model 2020,
Frontier Laboratories) directly connected to a GC/MS system Agilent 6890 equipped with a
fused silica capillary column (J&W Scientific 5MS 30 m × 250 µm × 0.25 µm inner
diameter). The detector consisted of an Agilent 5973 mass selective detector (EI at 70 eV).
The analysis was performed at pyrolysis temperature 500 ºC with final temperature achieved
at a rate of 20 ºC min-1 and the end temperature was maintained for 1 min. The GC–MS
conditions were as follows: oven temperature was held at 50 ºC for 1 min and then increased
up to 100 ºC at 30 ºC min-1, from 100 to 300 ºC at 10 ºC min-1 and isothermal at 300 ºC for 10
min. The carrier gas used was He with a controlled flow of 1 ml min-1. Pyrolysis products
were identified using the Wiley and NIST computer libraries and attending to the relative
retention times and spectra reported in the literature. The chromatograms were then integrated
and the relative contents of the different products calculated on the basis of peak areas.
3. Results
The evolution of the temperature in compost piles amended with CBB (recommended BIO 1
and double recommended dose BIO 2) and
unamended (0) is shown in Figure 1. It is
apparent that untreated piles are unable to
sustain an adequate thermophilic stage to
appropriately
catalyze
the
degradation
processes. In the pile amended with CBB,
even at lower dose (BIO 1), a shoulder in
the curve is evident indicating the growth
Figure 1: Evolution of the temperature in piles of
compost SS amended and not with CBB with an
indication of the different composting stages
of fungi and lignin degradation.
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15th IHSS Meeting- Vol. 3
For practical purposes the chromatograms of
the products released by pyrolysis can be
analyzed at first sight when divided in three
main domains: Domain I (elution time c. 2–9
min): low molecular weight (MW) products
dominated by polysaccharides (Ps) + proteins
(Pr) derived products; Domain II (elution time
c. 9–19 min): where most lignin (Lg) derived
products
(methoxyphenols)
are
included;
Domain III (elution time >c. 19 min): high
MW compound where sterols and compounds
derived from lipids (Lip) elute [4].
When studying the pyrograms produced by the
un-composted wastes (starting material) and
composted material with and without addition
of CBB at thermophilic stage (Fig.2), the
effect of the bio-accelerator is apparent. The
non-treated
piles
show
a
very
similar
pyrogram to that of the starting material
indicating a low degree of transformation. This
is dominated by alkyl structures presumably
Figure 2: Pyrograms from un-composted wastes
(starting material) and composted material with
and without CBB at thermophylic stage
present as esters, or physically
entrapped in a matrix consisting of
carbohydrate and protein (Domain
I) which, after pyrolysis, yields only
small amount of furan derivatives,
cyclohexene, cyclopentadiene, Nbearing
aromatic
Figure 3: Evolution of the relative abundance of the
different families of compounds (% of total
chromatographic areas) as inferred by Py-GC/MS during
the composting process
and
products
some
(mainly
alkylbenzenes). The CBB treated
compost
showed
conspicuous
changes with respect to the starting
and
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fragments
un-treated
compost,
in
15th IHSS Meeting- Vol. 3
particular a decrease in fragments arising from polysaccharides and an increase in the relative
yields of aromatic products and methoxyphenols derived from lignin (lignin residues) is
observed. Finally, the yields of heterocyclic compounds (indole derivatives) were higher in
the starting materials decreasing progressively with composting time. The CBB bacterial
product effectively seems to favour composting also shortening composting times.
Analytical pyrolysis can be used as a semi-quantitative technique when working with relative
abundances of the different families of compounds as percentage of total chromatographic
areas. In Fig.3 the evolution of the main components of organic matter is depicted. At the
early stage of composting (after 3 weeks), in untreated piles only a decrease in the relative
abundance of polysaccharide constituents is observed whereas the effect of the bio-accelerator
is again apparent in the treated piles where a sharp decrease of polysaccharide, polypeptides
and lignin derived compounds is evident. This is in agreement with the degradation process of
complex lignocellulosic materials where hemicellulose and lignin are degraded and partly
transformed during thermophilic stage after easily degradable carbon sources have been
consumed [5 and references therein].
4. Conclusions
The pyrolysis technique used (double shot Py-GC/MS) is a valid tool to assess compost
evolution and was also informative in assessing to which extent compost transformation
reached an acceptable stabilization when final compost is sufficiently mature. In this respect,
Py-GC/MS was the efficacy of CBB compound in accelerating the composting process. The
technique also provides information about the main biogenic structures affected during
composting (polysaccharides, polypeptides, lipids and lignins). Furthermore, by calculating
percentual values this technique could also be developed into a semi-quantitative tool to study
composting.
References
1.
2.
3.
4.
C.García, T.Hernández, F.Costa, Waste Manage. Res. 10 (1992) 445.
I.Déportes, J.L.Benoit-Guyod, D.Zmirou, Sci. Total Environ. 172 (1995) 197.
M.J.Blanco, G.Almendros, Plant Soil 196 (1997) 15.
F.J.González-Vila, J.A.González-Pérez, K.Akdi, M.D.Gómis, F.Pérez-Barrera, T.Verdejo, Biores.
Technol. 100 (2009) 1304.
5. M.Tuomela, M.Vikman, A.Hatakka, M.Itävaara, Biores. Technol. 72 (2000) 169.
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Natural Organic Matter and Humic Substances Biological
and Physiological Effects
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A Turning Point of Wheat Breeding and Humic Substance
Reza Shahryari*
Islamic Azad University, Ardabil branch, IRAN
E-mail: shahryari@iauardabil.ac.ir
1. Introduction
Organic matter in the soil exists in three different forms: 1) Living plant and animal matter, 2) Dead
plant and animal matter and 3) decomposed plant and animal matter (humic substances)(4).
Differences in SOM and its turnover are related to changes in climate, parent rock, and vegetation and
to numerous complexes biological, chemical and physical soil processes (7). Humus is defined by
Stevenson (1994) as the total organic fraction in soils exclusive of non-decomposed plant and animal
material, their partial decomposition products, and the soil biomass. Thus humic substances (e.g.
humic acids and fulvic acids) make up the bulk of humus (7). The concentration of HS varies from
place to place; the values in seawater being normally from two to three mg/l (8). HS in soils and
sediments can be divided into three main fractions: humic acids (HA or HAs), fulvic acids (FA or
FAs) and humin (3). Humic acids are the fraction of humic substances that is not soluble in water
under acidic conditions (pH < 2) but is soluble at higher pH values. They can be extracted from soil by
various reagents and which is insoluble in dilute acid. Humic acids are the major extractable
component of soil humic substances. They are dark brown to black in colure. Fulvic acids are the
fraction of humic substances that is soluble in water under all pH conditions. They remain in solution
after removal of humic acid by acidification. Fulvic acids are light yellow to yellow-brown in color.
Humin is the fraction of humic substances that is not soluble in water at any pH value and in alkali.
Humins are black in color. Many investigators now believe that all dark colored humic substances are
part of a system of closely related, but not completely identical, high - molecular - weight polymers.
According to this concept, diferences between humic acids and fulvic acids can be explained by
variations in molecular weight, numbers of functional groups (carboxyl, phenolic OH) and extent of
polymerization (12).
In regard to the potential of the HA, continuous development has led to availability of various
commercial humic acid based products and they are widely marketed. The HA products are usually
available in the form of inexpensive soluble salts, referred to as potassium humate (2). Humates of
brown coal, peat and soils are mostly studied while sapropel humates (bottom organic sediments) are
much less known (6).
The following principal ways of HS action could be proposed: organism development, hormone-like
activity, nutrient carriers, catalysts of biochemical reactions and antioxidant activity (5). The
biological activity of HS encompasses all the activities of HS in regulating plant biochemical and
physiological processes, irrespective of their stimulatory or inhibitory roles. Mitigating activity of HS
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is observed under various stress conditions including both biotic and abiotic ones (7).
Nowadays, humic preparations are increasingly applied as stimulators in plant breeding (6 and 10).
Shahryari et al (11) believed that major aim of each wheat breeding program is to increase yield and
improve quality. Potassium Humate causes increase in crop quality and quantity (10).
Application of HA in agriculture as soil fertilizer and soil conditioner has been extensively discussed
in the literatures. To date, numerous researches have demonstrated conclusively that HS have
significant impacts on the soil structure and plant growth (2). Farmers use humates to accelerate seed
germination and improve rhizome growth. These materials are able to stimulate oxygen transport,
accelerate respiration and promote efficient utilization of nutrient by plants. These observations
prompted scientists to study the specific properties of humates and their possible benefits in improving
health and well being of humans and animals (4). Nevertheless, HA in proper concentrations can
enhance plant and root growth (1).
Seyedbagheri (9) evaluated commercial humic acid products derived from lignite and leonardite in
different cropping systems from 1990 to 2008. The results of those evaluations differed as a result of
the source, concentration, processing, quality, types of soils and cropping systems. Under their
research, crop yield increased from a minimum 9.4 percent to a maximum 35.8 percent. Also,
Shahryari et al (10) observed potassium humate increased average grain yield of bread wheat from
2.49 to 3.61 ton/ha in a well watered condition. This was 45% increase in yield.
In present study, we try answer to the question how humic substance increase wheat grains yield.
2. Materials and Methods
Six bread wheat genotypes (Gascogen, Sabalan, 4057, Ruzi-84, Qobustan and Saratovskaya-29)
planted in Agricultural Research Station of Islamic Azad University, Ardabil branch, Iran.
Experimental design was split plot on the basis of completely randomized block design with three
replications. Factor A was application of potassium humate or not; and factor B was genotypes.
Treatments by a sapropel derived potassium humate (1ml/l) were done at four stages: pre-planting on
seeds, tillering stage, stem elongation and after anthesis. Yield and some of yield related characters
measured. Those were grain yield, biomass per plant, spike number per square meter, seeds number
per spike, 1000 seed weight, Spike length, peduncle length and plant height.
Cause and effect of characters studied by use of the path coefficient analyses where grain yield was
kept as resultant variable and other contributing characters as causal variables. The most suit model
formed for grain yield, separately, in the both conditions of with potassium humate or without it.
Characters with non significant correlations removed and Path analysis were done for remained
characters.
3. Results and Discussion
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15th IHSS Meeting- Vol. 3
Increasing of grain yield by 45% under effect of potassium humate was unbelievable (10). Kulikova et
al. (5) expressed that in spite of numerous studies on the biological effects of HS, the mechanism of
their action remains unclear. But there must be a logical reason. A biometric procedure Such as Path
analysis leads us to understanding of the genetic association of traits and their contribution to yield.
Comparison of path coefficients in two different conditions of this study revealed there were more
complex relations between characters at presence of potassium humate (Figure 1). Cumulative effects
(significantly direct and indirect effects) of traits caused increase in yield.
In the first step of path analysis; peduncle length, plant height, 1000 seed weight and spike number per
square meter removed from model. Therefore these four traits had not effect on increasing of yield at
presence of potassium humate. In the other steps, traits with non significant regression removed from
regression model and continued path analysis. Resulted model revealed that seed weight per spike had
the most effect on yield increase with direct effect (r = 0.576) at presence of potassium humate. After
that spike length (r = 0.337), biomass (r = 0.254) and seed number per spike (r = 0.175) had total
correlation effects on increasing of grain yield.
Researchers must be tried to improve quality, while maintaining the yield level of standard cultivars in
breeding programs. Plant breeders focused on increasing of yield, pay attention to genetic of alleles
such as dominance or recessive; co dominance, incomplete dominance, polygenic, pleiotropic and
epistasis effects. On the other hand may be a linkage between a desirable and a non-desirable gene.
Such relations as these induce difficulties in breeding program. There are negative correlations
between crop yield and some of traits. For example in a breeding program, we want to increase protein
content of wheat flour but yield may decrease. And for successfully breeding for increasing of these
two characters together, we make an effort for a long time. But we can succeed to this important
subject by application of humic substance as a miracle natural biological material.
Figure 1: Diagrammatic representation of direct and indirect effects of variables on grain yield with
potassium humate treatment
4. Conclusions
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Potassium humate increased grain yield by the way of seed weight per spike. After that spike length,
biomass and seed number per spike were notable. These traits should be attending breeding programs
for extra increasing of yield at presence of potassium humate. This could be a turning point between
wheat breeding and humic substances application.
Acknowledgements
I would like appreciate from my Professors A. Gadimov (Institute of Botany at the Azerbaijan
National Academy of Sciences), E. Gurbanov (Faculty of Biology at the Baku State University,
Azerbaijan) and M. Valizadeh (Agriculture Faculty at the Tabriz University in IRAN) are greatly
acknowledged. The author also thanks R. Talai (Ardabil Agricultural and Natural Resources Research
Centre, IRAN) for assistance in statistical analysis.
References
1. M. Bacilio, P. Vazquez and Y. Bashan. Alleviation of noxious effects of cattle ranch composts on
wheat seed germination by inoculation with Azospirillum spp. Biol Fertil Soils. 2003. 38: 261–
266.
2. S.S. Fong, L. Seng and H. B. Mat. Reuse of Nitric Acid in the Oxidative Pretreatment Step for
Preparation of Humic Acids from Low Rank Coal of Mukah, Sarawak. J. Braz. Chem. Soc. 2007.
Vol: 18. No: 1. 41-46.
3. International Humic Substances Society. What are humic substances? Focus on form: Retrieved
May 27, 2009, from http://ihss.gatech.edu/ihss2/whatarehs.html
4. K.M.S. Islam, A. Schuhmacher and J.M. Gropp. Humic Acid Substances in Animal Agriculture.
Pakistan Journal of Nutrition. 2005. 4 (3): 126-134.
5. Kulikova N. A, E. V. Stepanova and O.V. Koroleva. Mitigating Activity of humic substances:
direct influence on biota. In: I.V. Perminova, et al. (ed.). Use of humic substances to remediate
polluted environments: from theory to practice. Springer Netherlands. 2005. Vol: 52. 285–309.
6. A.A. Perk and Popov A.A. Growth and development of plants under the action of different
fractions of sapropel humates. 2nd International symposium plant growth substances: intercellular
hormonal signaling and applying agriculture Abstracts. Kyiv, Ukraine. 2007. P: 149.
7. D. Pizzeghello, G. Nicolini and S. Nardi. Hormone-like activity of humic substances in Fagus
sylvaticae forests. New Phytologist. 2001. 151: 647–657.
8. A. Al-Rasheed Radwan. Water treatment by heterogeneous photocatalysis an overview. 4th SWCC
Acquired Experience Symposium held in Jeddah. 2005.
9. Mir-M. A. Seyedbagheri. Perspective on Over a Decade of On-Farm Research on the Influence of
Humates Products on Crop Production. Proceedings of the 14th meeting of International Humic
Substances Society. From molecular understanding to innovative applications of humic
substances. I.V. Perminova and N. A. Kulikova. (eds). 2008. 603- 604.
10. R. Shahryari, A. Gadimov, E. Gurbanov and M. Valizadeh. 2009. Application of potassium
humate in wheat for organic agriculture in Iran. Abstracts Book of Go Organic International
Symposium. The Approach of Organic Agriculture: New Market, Food Security and a Clean
Environment. Bangkok, Thailand. P: 59.
11. R. Shahryari, E. Gurbanov, A. Gadimov, M. Valizadeh, H. A. Hosseinpour, J. Bargiyan Khiyabani
and B. Teymuri. Wheat genotypes quality affected by potassium humate under terminal drought.
(In Persian). Proceedings of the 11th National Iranian Soil Sciences Congress. 2009. Gorgan, Iran.
12. Weber, J. 2009. Definition of soil organic matter. Focus on form: Retrieved Oct 10, 2009, from:
http://www.humintech.com/001/articles/article_definition_of_soil_organic_matter.html.
Vol. 3 Page - 303 -
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Response of Maize Genotypes to Changes in Chlorophyll Content at
Presence of Two Types Humic Substances
Reza Shahryaria*, Babak Shahmorad Moghanloub, Ali Mohammad Pour Khaneghahb
a
Islamic Azad University, Ardabil Branch, Iran; bYoung Researchers Club, Islamic Azad
University, Ardabil Branch, Iran
E-mail: rz_shahriari@yahoo.com
1. Introduction
Corn (Zea mays L.) grows in more countries than any other cultivated crops. It is a major
source of food for both Human and animals through the world [8]. Also, it is one of the major
crops in Iran.
According to the classical definition, Humic substances (HS) are "a general category of
naturally occurring heterogeneous organic substances that can generally be characterized as
being yellow to black color, of high molecular weight and refractory" [4]. The concentration
of HS varies from place to place. Their size, molecular weight, elemental composition,
structure, and the number and position of functional groups vary, depending on the origin and
age of the material [7]. HS are major components of the natural organic matter (NOM) in soil
and water as well as in geological organic deposits such as lake sediments, peats, brown coals
and shales. They make up much of the characteristic brown color of decaying plant debris and
contribute to the brown or black color in surface soils. HS in soils and sediments can be
divided into three main fractions: humic acids (HA or HAs), fulvic acids (FA or FAs) and
humin [3].
Nowadays, humic preparations are increasingly applied as stimulators in plant breeding [6 and
9]. Shahryari et al [10] believed that major aim of each wheat breeding program is to increase
yield and improve quality. Potassium humate causes increase in crop quality and quantity [9].
Research has confirmed that humic substances can indirectly and directly affect the
physiological processes of plant growth [11]. The following principal ways of HS action
could be proposed: organism development, hormone-like activity, nutrient carriers, catalysts
of biochemical reactions and antioxidant activity. In spite of numerous studies on the
biological effects of HS, the mechanism of their action remains unclear [4]. Yang et al [12]
expressed it is unknown how humic substances decrease chlorophyll accumulation. They may
inhibit the biosynthetic pathway of chlorophyll; stimulate the degradative pathway of
chlorophyll, or both [12]. This reduces chlorophyll accumulation and photosynthesis, which,
in turn, diminishes total plant growth [11].
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Liu et al. [5] evaluated the effect of a commercial preparation of humic acid on the
chlorophyll concentration of creeping bentgrass. Badsar et al [1] conducted an experiment for
determination effect of a sapropel derived HS on wheat genotypes under drought stress.
The aim of this investigation was to find-out the effects of two type humic substances derived
from different origins on chlorophyll content of maize genotypes.
2. Materials and Methods
An investigation was conducted for determination effect of two HS on chlorophyll content of
different maize genotypes. Twenty six grains of every seven maize genotypes cultured three
replicated in Petri dishes. Solution of 0.067 % w/v prepared from the both of liquid HS (Table
1). The volume of preparations was 1.5 L. Then, 15 ml solution was put in each Petri dish.
Petri dishes had put in the dark place within the laboratory temperature. Solution consumed
by seed, was to provide equal value during the germination.
Table 1: Compounds of liquid humic fertilizers based on peat and leonardite
Humic substances
Peat based
Leonardite based
Humic acids
(% w/v)
3.3
13.2
Fulvic acids
(% w/v)
0.9
3.3
Total humic extracts
(% w/v)
4.2
16.5
After completion of germination period (15 days later), three healthy germinated seeds from
every Petri dish transferred to small plastic pots into the greenhouse soil.
Factorial experiment used on the basis of completely randomized design in the three
replications. Factor A was solutions (peat and leonardite HS; and water as control). Factor B
was maize genotypes.
Chlorophyll content index (CCI) measured by a CCM-200 (made by Opti-Science Company)
for Fifteen-day seedlings. Measurements were done in the beginning, middle and bottom of
leaves. Mean of collected data for three seedlings per replication used for analysis of variance
by MSTATC software. Comparison of means was made by Duncan's Multiple Range Test.
3. Results and Discussion
ANOVA (table 2) revealed high significant differences between solutions, maize genotypes
and their interactions. Leonardite, water and peat produced 7.06, 6.31 and 5.45 CCI,
respectively. Comparison of means for genotypes was known that genotypes grouped to two
classes at probability level of 5 %. Genotypes with high CCI were ZP 434, OS 499 and 505.
Genotypes with low CCI were Golden West, Single Cross 704, ZP 677 and 500.
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Table 2: ANOVA for maize genotypes
CCIunder treatment of two types
humic substances
S.O.V
Solutions
Df
2
Comparison of CCI means for G ×S presented at
Table 3 and Figure 1. Genotypes Single Cross 704
and 505 had the highest CCI in leonardite HS
MS
**
13.611
treatment. After those genotypes OS 499 in water
**
Genotypes
6
8.743
S ×G
12
2.644 **
Error
42
0.794
in water were placed. Peat HS caused to decrease
CV (%)
-
14.20
CCI in 500, OS 499 and 505. This was similar to
**: significant at probability level of 1%
and leonardite, ZP 434 in water and leonardite, 505
result of Ferreti et al [2]. They reported that humic
substances apparently decreased the chlorophyll
content.
Leonardite HS increased CCI in Golden West and Single Cross 704 but Peat HS had not
effect on these two maize genotypes. Leonardite HS had not effect on 500, OS 499 and 505.
Genotypes ZP 677 and ZP 434 had not responses to application of both two types of HS. This
result was accordant with Liu et al [5] and Badsar et al [1]. They reported that chlorophyll
content was unaffected by HS. It is unknown how HS affect on chlorophyll content of leaves.
Table 3: Comparison of CCI means for G ×S
(W: water, P: Peat & L: Leonardite derived HS)
Genotype
W
P
L
ZP 677
5.67 CD
5.14 CD
5.51 CD
Golden West
5.49 CD
5.85 BCD
6.61 ABC
500
5.56 CD
4.29 D
5.38 CD
OS 499
8.01 A
5.78 BCD
7.96 A
ZP 434
7.71 A
7.35 AB
7.54 A
505
7.51 A
5.43 CD
8.20 A
Single Cross 704
4.21 D
4.32 D
8.22 A
Figure 1: Chlorophyll content index of maize
genotypes in humic substances derived from
peat (p) and leonardite (l); and water (w)
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4. Conclusions
Effects of HS on chlorophyll content of leaves are related to origin of HS and genetic
response of genotypes. Also, concentration of humic acids and fulvic acids in HS preparations
could be effective on chlorophyll contents. This should be studied.
References
1. M. Badsar, R. Shahryari and V. MollaSadegi. Effect of potassium humate on chlorophyll content
of wheat leaf under terminal drought condition. (In Persian). Proceedings of the 11th National
Iranian Soil Sciences Congress. 2009.
2. M. Ferretti, R. Ghisi, S. Nardi and C. Passera. Effect of humic substances on photosynthetic
sulphate assimilation in maize seedlings. Can. J. Soil Sci. 1991. 71:239–242.
3. International Humic Substances Society. What are humic substances? Focus on form: Retrieved
May 27, 2009, from http://ihss.gatech.edu/ihss2/whatarehs.html
4. N. A. Kulikova, E. V. Stepanova and O.V. Koroleva. Mitigating Activity of humic substances:
direct influence on biota. In: I.V. Perminova, et al. (ed.). Use of humic substances to remediate
polluted environments: from theory to practice. Springer Netherlands. 2005. Vol: 52. 285–309.
5. C. Liu, R. J. Cooper and D. C. Bowman. Humic acid application affects photosynthesis, root
development, and nutrient content of creeping bentgrass. Hort. Sci. 33: 1998. 1023–1025.
6. A. A. Perk and A. A. Popov Growth and development of plants under the action of different
fractions of sapropel humates. 2nd International symposium plant growth substances: intercellular
hormonal signaling and applying agriculture Abstracts. Kyiv, Ukraine. 2007. P: 149.
7. A. Al-Rasheed. Radwan. Water treatment by heterogeneous photocatalysis an overview. 4th
SWCC Acquired Experience Symposium held in Jeddah. 2005.
8. M. El-Khallal Samia, Tahani A. Hathout, Abd El Raheim A. Ashour and Abd-Almalik A. Kerrit.
Brassinolide and Salicylic Acid Induced Growth, Biochemical Activities and Productivity of
Maize Plants Grown under Salt Stress. Research Journal of Agriculture and Biological Sciences.
2009. 5(4): 380-390.
9. R. Shahryari, A. Gadimov, E. Gurbanov and M. Valizade. Application of potassium humate in
wheat for organic agriculture in Iran. Abstracts Book of Go Organic International Symposium.
The Approach of Organic Agriculture: New Market, Food Security and a Clean Environment.
Bangkok, Thailand. 2009. P: 59.
10. R. Shahryari, E. Gurbanov, A. Gadimov, M. Valizade, H. A. Hosseinpour, J. Bargiyan Khiyabani,
B. Teymuri. 2009. Wheat genotypes quality affected by potassium humate under terminal drought.
(In Persian). Proceedings of the 11th National Iranian Soil Sciences Congress. Gorgan, Iran.
11. C. M. Yang, M. H. Wang, Y. F. Lu, I. F. Chang and C. H. Chou. Humic substances affect the
activity of chlorophyllase. J. Chem. Ecol. 30(5): 2004. 1057-1065.
12. C. M. Yang, C. N. Lee and C. H. Chou. Effects of three allelopathic phenolics on the chlorophyll
accumulation of rice (Oryza sativa) seedling: II. Stimulation of consumption-orientation. Bot.
Bull. Acad. Sin. 45: 2002. 119-125.
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Bioactivity of Chemically Transformed Humic Matter
on Plant Root Growth
Luciano P. Canellasa*, Leonardo B. Dobbssa, Fábio L. Olivaresa, Natália O. Aguiara, Lázaro
E. P. Peresb, Riccardo Spaccinic, Alessandro Piccoloc, Arnoldo R. Façanhaa
a
UENF-NUDIBA Campos dos Goytacazes 28602-013, Rio de Janeiro, Brazil; bUSP-ESALQ,
Universidade de São Paulo (USP), Piracicaba, Brazil; cDipartimento di Scienze del Suolo,
della Pianta, dell’Ambiente e delle Produzioni Animali (R.S., A.P.) Università di Napoli
Federico II, Portici, Italy
E-mail:canellas@uenf.br
1. Introduction
The application of products derived from humic substances (HS) at low concentration on crop
plants and their potential to act as plant growth promoters have been creating increased
interest among farmers. However, there is little information about the mechanisms by which
HS influence biological activities in plants. Evidence of the physiological mechanism through
which HS exert their effects may depend on hormones and, in particular, on the presence of
auxin or auxin-like components in their structure. Chemical modification of HSs has been
widely used as a tool to understand their chemical structure [1]. Studies of chemical
transformation of HS followed by biomonitoring can provide new insights on humus
bioactivity. The aim of this studied was to evaluate the influence of chemical modifications of
humic structure on root stimulation. The HS were evaluated by elemental composition,
HPSEC, CP-MAS 13C NMR and DOSY H NMR spectroscopies, and their bioactivities were
monitored by following the morphological and biochemical traits of arabidopsis (Arabidopsis
thaliana eco col. 4), tomato (Licopersicum esculentum), and maize (Zea mays).
2. Materials and Methods
Humic substances were isolated from vermicompost produced with cattle manure and E.
foetida using 0.1M NaOH. The humic derivates were produced by acidic oxidation with
KMnO4 (D1); basic oxidation with KMnO4 (D2); Reduction with sodium borohydride (D3);
alkaline methanolic hydrolysis (D4); acid hydrolysis with H2SO4 (D5); acid hydrolysis by
dioxane in 2M HCl (D6); extraction of free lipids (D7); methylation (D8). The complete
description of all chemical reaction used can be found in the different chapters of reference
book edited by Hayes et al. [1].
composition, CP-MAS
13
Humic derivatives were characterized by elemental
C NMR, DOSY-H NMR, HPSEC and their effects on lateral root
emergence were evaluated using Arabidopsis, tomato and maize [2–3]. The activity of plasma
membrane H+-ATPase isolated from maize root seedlings was used as biochemical marker of
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15th IHSS Meeting- Vol. 3
humic bioactivity [2]. Four-day-old DR5::GUS transgenic Micro Tom tomato plants were
treated with HS and their chemical derivatives for easier detection of auxin-like activity.
3. Results and Discussion
The main results of HS characterization are showed in Table 1 and Fig. 2. All humic
derivatives promotes induction of lateral root emergence on Arabidopis, tomato and maize
(data not showed). The effect of HS and their derivatives on plasma membrane (PM) H+ATPase activity isolated from maize roots vesicles can be observed in Fig. 1B. The evidence
of auxin-like presence on HS structure are showed in Fig 2 as revealed by DR5::GUS gene
reporter. Table 2 shown the significance of hydrophobicity of HS on H+-ATPase induction.
These findings seem to indicate that, although the relationship between bioactivity and
molecular size varies considerably, other chemical features such hydrophobicity appears to
play a combined role with size in providing a bioactivity to the modified humic matter from
vermicompost. The root acidification mediated by plasma membrane H+-ATPase is important
for the regulation of cytoplasmic pH and the activation of cell wall-loosing enzymes and
proteins through acidification of apoplast [4]. This effect is closely related to the auxininduced cell growth as proposed by the acid-growth theory. It has been earlier postulated that
some HS may include compounds similar to indolacetic acid in their structure, and the
capacity of HS to promote root growth was attributed to these compounds [5–6]. In fact,
humic complex structures can be disrupted by simple organic acids exuded by plant roots and
microbes and small auxin-like molecules may then be released and act on the cell receptors in
plasma membrane [7]. Induction of the auxin responsive synthetic reporter DR5::GUS by
Micro Tom-type plants by all humic derivatives is a clear evidence that physiological
response of humic matter is due hormonal action (Fig. 2).
Table 1 – Elemental composition and area integration from CP-MAS 13C-NMR spectra
Elemental composition (%)
Sample
C
H
N
Bulk HS
D1
D2
D3
D4
D5
D6
D7
D8
25.24
28.14
31.25
25.00
21.64
31.60
47.84
27.60
33.23
2.38
2.80
3.02
2.24
1.99
2.94
3.08
2.38
2.98
2.74
4.32
4.07
3.69
2.04
3.37
2.71
2.69
4.40
a)
Chemical shift (CP-MAS 13C NMR) %
Aromaticityc
H/C C/N
0–40
40–110
110–160
160–200
HB/HI
1.30
1.84
1.56
1.77
1.13
1.28
0.68
1.17
1.59
23.20
25.40
24.70
22.00
21.20
25.70
20.80
20.30
25.00
42.90
38.70
39.90
43.90
42.40
35.10
42.70
43.30
41.20
23.90
26.20
25.10
24.20
25.80
30.70
27.30
25.60
23.90
10.00
9.80
10.20
9.90
10.60
8.50
9.30
10.80
9.90
0.89
4.18
1.06
0.99
0.86
0.89
1.29
0.93
0.85
0.96
3.82
3.98
4.13
3.87
3.25
3.66
3.90
4.18
12.37
11.71
12.09
13.01
12.69
12.54
18.12
13.53
13.02
C-alkyl + C-aromatic /C-polysaccharides + COOH
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15th IHSS Meeting- Vol. 3
Fig. 1A: Mw dimension calculated by DOSY H NMR data; B: Effect on maize root PM H+-Activity
Figure.2A: MicroTom DR5::GUS gene reporter seedlings treated with HS and their chemical derivatives
(D); Control plants=C
4. Conclusion
Here, we found that chemical modifications of a humic structure affect its root growth in
three plant species. The chemical modifications varied the composition, hydrophobicity, and
components molecular sizes in humic superstructures, and this was reflected in their capacity
to interact with plant cells. While a certain relation was shown between molecular size and
humic bioactivity, we found that this may not be the only criterion to evaluate humic effects
on root growth, and should be combined with the material hydrophobic character. It thus
appears that HS bioactivity on plants depend on sufficient hydrophobicity to allow
interactions with plant root cells, but the hydrophobic domains should concomitantly possess
a conformation sufficiently labile to release, possibly by the action of acidic root exudates,
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15th IHSS Meeting- Vol. 3
auxin-like molecules which exert a biological stimulation.
References
1. Hayes, M.H. et al. Humic Substances II: In Search of Structure. Chichester, Wiley, 1989.
2. Canellas, L. P.; Façanha, A. O.; Olivares, F. L.; Façanha, A. R.. Plant Physiol. 2002, 130, 1951–
1957
3. Dobbss, L., Medici, L.O., Peres, L.E.P., Pino-Nunes, L.E., Rumjanek, V.M., Façanha,
4. A.R., Canellas, L.P., Ann. Appl. Biol. 2008, 153, 157–166.
5. Sze, H.; Li, X.; Palmgren, M. G. Plant Cell. 1999, 11, 677–689.
6. Muscolo, A.; Cultrupi, S.; Nardi, S. Soil Biol. Biochem. 1998, 30, 1199–1201
7. Nardi, S., Pizzeghello, D., Muscolo, A., Vianello, A., Soil Biol. Biochem. 2002, 34, 1527–1536.
8. Canellas, L. P.; Teixeira Junior, L. R. L.; Dobbss, L. B.; Silva, C. A.; Medici, L. O.; Ann. Appl.
Biol. 2008, 153, 157–166.
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Effect of Two Humic Substances as Bifertilizers on Germination and
Seedling Growth of Maize Genotypes
Reza Shahryaria*, Niknam Baharia, Majid Khayatnejadb,
a
Islamic Azad University, Ardabil branch; Iran; bYoung Researchers Club, Islamic Azad
University, Ardabil Branch, Iran
E-mail: rz_shahriari@yahoo.com
1. Introduction
Humic substances (HS) are the result of organic decomposition and the natural organic
compounds comprising 50 to 90 % of the organic matter of peat, lignites, sapropels, as well as
of the no- living organic matter of soil and water ecosystems. According to the classical
definition, HS are "a general category of naturally occurring heterogeneous organic
substances that can generally be characterized as being yellow to black color, of high
molecular weight and refractory [1]. The biological activity of HS encompasses all the
activities of HS in regulating plant biochemical and physiological processes, irrespective of
their stimulatory or inhibitory roles. Humic matter from forest soils has a very complex
biological activity and depending on its origin, molecular size, and concentration exhibits
high or low stimulations of plant metabolic parameters. In forest soils, litter composition does
not influence the chemical characteristics of humic fractions greatly, as revealed by nuclear
magnetic resonance spectra, but it does influence biological activity and as a result forest
species are affected in different ways by their HS. HS are known to possess bioactivating
properties in relation to plants [2]. In spite of numerous studies on the biological effects of
HS, the mechanism of their action remains unclear [1]. However, farmers use humates to
accelerate seed germination and improve rhizome growth. These materials are able to
stimulate oxygen transport, accelerate respiration and promote efficient utilization of nutrient
by plants [3]. Nevertheless, humic acid in proper concentrations can enhance plant and root
growth [4]. Presence of HS is important during all stages of plants’ development but
particularly vital in the early stages. That is why the pre-planting treatment of seeds is very
important. Even before germination begins, vital forces are awakened, and the immune
system is stimulated [5]. To date, numerous researches have demonstrated conclusively that
HS have significant impacts on the soil structure and plant growth [6]. Gadimove et al [9]
concluded that humates are miraculous natural substances for increasing quantity and quality
of crop yields. They expressed that a practical- scientific perspective and programming need
to application of this technology in the world; especially in the developing countries. Also,
they expressed action rate of these materials are related to origin and quality of HS. Quality of
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15th IHSS Meeting- Vol. 3
commercial humates is related to procedure of extraction and percent of humic acids and
fulvic acids. We must note to these subjects for select and application of those for agricultural
applications.
In this investigation, effect of two types HS as biofertilizers were studied on germination and
seedling growth of different maize genotypes under cultivation in Moghan plain, Ardabil
province, Iran.
2. Materials and Methods
A laboratory study was conducted to evaluate the influence of HS on germination and early
growth of seven maize genotypes which were include of ZP 677, Golden West, 500, OS 499,
ZP 434, 505 and Single Cross 704. Solution of 0.067 % w/v prepared from the two type liquid
HS (Table 1).
Table 1. Compounds of liquid humic based on peat and leonardite
Compounds
Humics
Peat based
Leonardite based
Humic acids
(% w/v)
Fulvic acids
(% w/v)
Total humic extract
(% w/v)
3.3
13.2
0.9
3.3
4.2
16.5
Factorial experiment used on the basis of completely randomized design in the three
replications. Factor A was solutions (peat and leonardite HS; and water as control). Factor B
was maize genotypes. Twenty six seeds of maize genotypes were allowed to germinate in
Petri dishes in the dark room temperature. Solution consumed by seed, was to provide equal
value during the germination. Final germination percent measured after 15 days. Then three
germinated seeds of uniform appearance was transferred to separate small plastic pots into the
greenhouse soil and then grown in the room temperature. Watering only was made by water.
Fifteen-day seedlings were measured for seminal root lengths and coleoptiles length. Mean of
collected data for three seedlings per replication used for analysis of variance by MSTATC
software. Comparison of means was made by Duncan's Multiple Range Test. Linear
regression between seminal root lengths, coleoptiles length and germination percent
separately calculated for experimental solutions. Relative seminal root elongation percent and
relative shoot elongation percent calculated by following formula:
Relative root elongation % = {mean root length in test solution/ mean root length in control}
× 100.
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Relative shoot elongation % = {mean shoot length in test solution/ mean shoot length in
control} × 100.
3. Results and Discussion.
ANOVA (Table 2) showed that solutions produced significant differences for seminal root
lengths (p<0.01) and coleoptiles length (p<0.05); but it had not effect on final germination
percent. Peat HS by 3.76 cm and leonardite HS by 3.69 cm with each other had more seminal
root lengths than control (3.34 cm). Coleoptile length of water (4.41 cm) and leonardite HS
(4.40 cm) treatments was higher than treated by peat HS (3.95 cm).
Table 2: ANOVA for measured traits of maize genotypes affected by HS
MS
S.O.V
Df
Solutions
Seminal root
lengths
Coleoptiles
length
Germination
percent
2
1.074**
1.44*
228.93 ns
Genotypes
6
3.11**
0.48 ns
762.80**
S×G
12
1.62**
0.40 ns
256.66 ns
Error
42
0.18
0.45
156.04
CV (%)
-
11.90
15.88
16.35
ns: non significant differences; *: significant at p<0.05; **: significant at p<0.01
Genotypes had significantly differences (Table 2) for seminal root length (p<0.01) and
germination percent (p<0.01). The highest seminal root length was belonged to Single Cross
704 and OS 499, respectively with 4.44 and 4.41cm (Table 3). Also OS 499 had the highest
germination percent (87.62 %). Interactions between solutions and genotypes only were
significant for seminal root length (Table 2). Single Cross 704 and OS 499 had the highest
seminal root lengths respectively with 5.36 and 5.15 cm by use of leonardite HS (Table 4).
Considering the Figure 1 and Figure 2 can be stated that leonardite HS generally caused the
highest relative root and shoot elongation than peat HS. Relative root elongation of Single
Cross 704 and ZP 434 created by leonardite HS was very clearly higher than others, compared
with peat HS.
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Table 3. Mean comparisons of seminal root lengths and germination percent for under study maize
genotypes
Genotypes
Golde
ZP
677
Character
Single
n West
500
OS
ZP 434
Cross
505
704
499
seminal root lengths(cm)
Germination(%)
3.55 c
3.26 c
3.68 bc
4.41 ab
3.43 c
2.62 d
4.44 a
81.22 ab
63.68 c
84.20 a
87.62 a
79.06 abc
65.39 bc
73.51 abc
W
P
L
ZP 677
3.42 defgh
4.27 bcde
2.96 fgh
Golden West
3.77 cdef
3.03 fgh
3.18 fgh
500
3.03 fgh
4.47 abcd
3.56 cdefg
OS 499
3.06 fgh
4.24 bcde
5.15 ab
ZP 434
2.95 fgh
4.01 cdef
3.34 efgh
505
2.57 gh
2.94 fgh
2.35 h
Single Cross 704
4.59 abc
3.37 efgh
5.36 a
1
Potassium humate
based on peat
0.8
0.6
Potassium humate
based on leonardite
0.4
0.2
sin
gl
e
cr
os
s
70
4
50
5
49
9
zp
43
4
we
st
en
G
ol
d
zp
50
0
0
67
7
70
4
50
5
cr
os
s
gl
e
49
9
43
4
zp
O
S
we
st
1.2
s in
G
ol
d
en
zp
50
0
Potassium humate
based on leonardite
1.4
O
S
Potassium humate
based on peat
Relative shoot elongation %
Genotype
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
67
7
Relative root elongation %
Table 4: Mean comparisons of seminal root lengths for G ×S at probability level of 1%
(W: water, P: Peat & L: Leonardite HS)
Figure 1: Relative root elongation %
Figure 2: Relative shoot elongation %
Peat HS created significantly positive correlation (r = 0.82*) between seminal root length and
germination percent at probability level of 5%. There was similar positive correlation (r =
0.70ns) for leonardite HS. Control (water) treatment was not able to create significant
correlations between measured characters.
The results of this study showed that applied humic substances had effect on biological
characters of maize in early growth stage. There are similar reports about effect of HS on
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crops germination. Some of them expressed by Gadimove et al [7] and Sasaki et al [8]. They
were noted that treatment of tomato seeds with 0.01 % Potassium humate solution before
planting for 24 hours related to variety increased production by 20 – 25 %. Also, Pre- planting
seed treatment of cucumber with 0.01 % potassium humate solution for 24 hours increased
production by 38 %. And, time and percentage of germination investigated for hazel-nut seeds
in three concentrations of Potassium Humate (0.01, 0.02 and 0.03 ml/seed) and two times (12
and 24 hours). Related to variety, germination percentage was increased between 37.08 and
64.14 % and the highest germination percentage (53%) observed in concentration of 0.02
relative to control (43%).
4. Conclusions
Types of humic substances could play the same or a different role as a biofertilizer on maize.
In this study, applied Peat and leonardite HS with each other produced more seminal root
lengths than control. But coleoptiles length of control and leonardite HS treatments was
higher than treated by peat HS. These HS biofertilizers in small amounts have many effects
on plant growth. Thus need to continue researches by different amounts of desired HS
biofertilizres for information on how they impact.
References
1. N. A. Kulikova, E. V. Stepanova and O.V. Koroleva, Mitigating Activity of humic substances:
direct influence on biota. In: I.V. Perminova, et al. (ed.). Use of humic substances to remediate
polluted environments: from theory to practice. Springer Netherlands, 2005, Vol: 52. 285–309.
2. D. Pizzeghello, G. Nicolini and S. Nardi, Hormone-like activity of humic substances in Fagus
sylvaticae forests, New Phytologist, 2001, 151: 647–657.
3. K. M. S Islam, A. Schuhmacher and J.M. Gropp, Humic Acid Substances in Animal Agriculture.
Pakistan Journal of Nutrition, 2005, 4 (3): 126-134.
4. M. Bacilio, P. Vazquez and Y. Bashan, Alleviation of noxious effects of cattle ranch composts on
wheat seed germination by inoculation with Azospirillum spp, Biol Fertil Soils, 2003, 38:261–
266.
5. B. Levinsky, All about humates, Focus on form: Retrieved May 27, 2009, from
http://www.teravita.com/Humates/HumateIntro.htm
6. S. S. Fong, L. Seng and H. B. Mat, Reuse of Nitric Acid in the Oxidative Pretreatment Step for
Preparation of Humic Acids from Low Rank Coal of Mukah, Sarawak, J. Braz. Chem. Soc, 2007,
Vol: 18. No: 1. 41-46.
7. A. Gadimov, N. Ahmaedova and R. C. Alieva, Symbiosis nodules bacteria Rhizobium
leguminosarum with Peas (Pisum Sativum) nitrate reductase, salinification and potassium humus,
2007, Azerbaijan National Academy of Sciences.
8. O. Sasaki, I. Kanai, Y. Yazawa and T. Yamaguchi, Relationship between the chemical structure of
humic substances and their hygroscopic properties, Annals of Environmental Science, 2007, Vol:
1. 17-22.
9. A. G. Gadimov, R. Shahryari and A. G. Garayeva, A perspective on humic substances as natural
technological products with miraculous biological effect on crops, Transaction of the Institute of
Microbiology of Azerbaijan national Academy of Sciences, 2009, V. 7. p. 118-126.
Vol. 3 Page - 316 -
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Comparative Evaluation of the Inhibitory Action of Compost Humic
Fractions on Two Soil-Borne Phytopathogenic Fungi
Andreina Traversa, Elisabetta Loffredo*, Nicola Senesi
Dipartimento di Biologia e Chimica Agro-forestale e Ambientale, University of Bari,
Via G. Amendola 165/A 70126 Bari, Italy
E-mail: loffredo@agr.uniba.it
1. Introduction
The biological activity of a compost humic acid (C-HA) depends mainly on the type of
substrate used for composting and the type and duration of the process. Nowadays, the needs
of sustainable agriculture and the increasing tendency to adopt soil-less cultivation suggest
new management practices, such as an increasing use of compost as partial substitute of peat
in potting media for ornamental plants and in greenhouse nurseries of horticultural plants. The
recent literature supports the positive effects exerted by compost addition to growing media,
which especially originates from its humic fraction, that improves plant growth and protects
plants from different soil-borne phytopathogens such as fungi [1-5]. This latter evidence,
besides the ascertained organic matter benefits to soil and other growing media, will add value
to compost application and encourages deeper research in this aspect. The objective of this
study was to evaluate comparatively the capacity of different C-HAs to inhibit the growth and
activity of two widespread plant pathogenic fungi, Pythium ultimum and Sclerotinia
sclerotiorum, and to possibly relate this ability to some C-HA properties.
2. Materials and Methods
The HA samples were isolated from a mixed compost (MC-HA), a green compost (GC-HA)
and a coffee compost (CC-HA) by using the conventional procedure and characterized by
means of a series of chemical and spectroscopic analyses. Some properties of the HA samples
examined are referred in Table 1. Each HA sample suspended in PDA (potato dextrose agar)
substrate was tested at concentrations of 10, 50 e 200 mg/L on the growth in vitro in Petri
dishes of P. ultimum and S. sclerotiorum and, in the case of the latter fungus, the time of
appearance and the number of sclerotia (non active fungal structures) formed. All experiments
were performed in controlled conditions and replicated eight times. All data were statistically
analyzed by one-way analysis of variance (ANOVA) and the least significant differences test
(LSD). Further, in order to evaluate the possible relationships existing between C-HA activity
on the two fungi and C-HA chemical properties (Table 1), the correlation coefficients were
calculated between C-HA properties and the average of inhibition degree (percentage of
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decrease of radial mycelial growth with respect to the control) and, in the case of S.
sclerotiorum, of the number of sclerotia at 168 h.
Table 1: Some properties of C-HAs examined
a
HA sample
Ash a
%
MC-HA
3.2
GC-HA
CC-HA
COOH a
meq/g
Phenolic OH a
meq/g
Total acidity a
meq/g
E4/E6
ratio
3.38
2.94
6.32
8.08
3.4
3.34
1.80
5.14
8.24
4.9
3.08
2.55
5.63
8.54
on moisture free basis
3. Results and Discussion
With respect to the corresponding control (PDA alone), no morphological changes were
observed for P. ultimum and S. sclerotiorum mycelia as a function of the presence of any CHA examined at any concentration in the growing medium. These results are in agreement
with previous results observed in vitro tests for two formae speciales of Fusarium oxysporum
in the presence of soil and compost humic fractions [3]. Differently, since the first hours after
fungal inoculation and during the entire experimental time, the presence of any C-HAs in the
PDA medium produced in general an inhibition of the radial mycelial growth of P. ultimum.
Different growth decreases were measured with respect to the control as a function of the dose
**
***
***
45
40
35
Figure 1: Effects of MC-HA at
concentrations of 10 mg/L (light grey
bars), 50 mg/L (dark grey bars) and 200
mg/L (black bars) on the radial mycelial
growth of P. ultimum on PDA, with
respect to the control (white bars) as a
function of time
30
*
***
*
25
20
15
**
Radial mycelial growth (mm)
and the type of C-HA. The maximum inhibition was exerted by MC-HA (Fig. 1).
10
5
0
18
24
40
Hours
* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001 according to LSD test
In the case of S. sclerotiorum, none of the three C-HAs reduced significantly the growth of
the fungus during the experimental period (88 h). Conversely, sclerotial development
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appeared more sensitive than the radial growth to all C-HAs. Apparently, any C-HA sample at
any concentration stimulated markedly sclerotial initiation and greatly increased the number
of sclerotia in the plates (Fig. 2). After 168 h from the fungus inoculation, the number of
sclerotia resulted generally much higher in C-HA treatments, especially in CC-HA, with
respect to the control. These results indicated that the presence of C-HA determined the
occurrence of adverse nutritional conditions that arrest the fungal activity, with evident
benefit for plant health.
MC-HA
35
Number of sclerotia
Number of sclerotia
30
25
20
15
10
5
0
136
144
GC-HA
35
160
168
30
25
20
15
10
5
0
136
Hours
144
Hours
160
168
CC-HA
35
Number of sclerotia
30
25
20
Figure 2: Effects of C-HAs at concentrations of
10, 50 and 200 mg/L on the sclerotia formation
of S. sclerotiorum on PDA, with respect to the
control, as a function of time
15
10
5
0
136
C
10 mg/L
144
Hours
160
50 mg/L
168
200 mg/L
Some significant relationships were found between C-HA properties and fungal inhibition. In
the case of P. ultimum, a significant positive correlation occurred between phenolic OH
content and mycelial growth inhibition. The number of sclerotia formed were positively
correlated with ash and phenolic OH content, total acidity and E4/E6 ratio, and negatively
correlated with COOH group content. These results, which are in good agreement with
findings of a recent study performed in vitro on the fungus F. oxysporum [3], apparently
confirm what already hypothesized in that work, i.e., the inhibitory action of humic acids on
the growth of the fungi tested might be related more to hydrophobic than hydrophilic
properties of HA.
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4. Conclusions
This study confirmed what already demonstrated by the authors and other researchers with
different fungi and different humic fractions, that is the general depressing activity of humic
compounds on some soil-borne plant pathogenic fungi. The C-HA samples examined in our
work exerted a different action on the two phytopathogenic fungi considered. P. ultimum
appeared to be significantly inhibited by any C-HA at any concentration adopted, especially
by HA originated from the mixed compost. In the case of S. sclerotiorum, a marked
enhancement of sclerotial formations was observed in all C-HA treatments, whereas the
mycelial growth of this fungus was not significantly altered. The efficiency of C-HA studied
in controlling fungal growth was apparently related to the substrate of origin, i.e. the different
HA properties, the HA concentration and the type of fungus examined.
References
1. A. Bernal-Vicente, M. Ros, F. Tittarelli, F. Intrigliolo and J.A Pascual, Biores. Technol. 99 (2008)
8722.
2. A.M..Litterick, L. Harrier, P. Wallace, C.A. Watson and M. Wood, A review. Crit. Rev. Plant Sci.
23 (2004) 453.
3. E. Loffredo, M. Berloco, F. Casulli and N. Senesi, Biol. Fert. Soils 43 (2007) 759.
4. E. Loffredo, M. Berloco and N. Senesi, Ecotoxicol. Environ. Safe. 69 (2008) 350.
5. E. Loffredo and N. Senesi, Sci. Hortic. 122 (2009) 432.
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Effect of Liquid Humic Compounds Extracted from Plant Based-Compost
to Soil Microorganisms
F. Suárez-Estrella*, M.C. Vargas-García, G. Guisado, M.J. López, J. Moreno
Department of Applied Biology, Microbiology Division, CITE IIB, University of Almería, La
Cañada de San Urbano s/n, 04120 Almería, Spain
E-mail: fsuarez@ual
1. Introduction
The effects of humic compounds (HCs), the major component of soil organic matter, on plant
growth have been examined in recent works [1] (Nardi et al. 2002). Humic colloids also affect
the growth of soil microbial populations. Many soil microorganisms from different taxonomic
and functional groups respond favourably to the presence of HCs in in vivo or in vitro
experiments [2, 3].
The present study compares the effects of HCs extracted from compost based on horticultural
waste and those from a commercial liquid fertilizer on soil microbial populations using a pot
experiment carried out with tomato plants (Solanum lycopersicum L.).
2. Material and Methods
Humic-like substances. A concentrated humic extract from leonardite (LHs) provided from an
agricultural company were used to represent humates of fossil origin. On the other hand,
liquid HCs from compost based on horticultural waste (WCHs) were used.
Plants and substrates used in pot trials. Two varieties of tomato were used: certified cv.
‘Raf’, type Marmande and certified cv. ‘Durinta’. Both sandy soil (SS) and a semi-inert
substrate (IS) on the basis of mainly “vermiculite” were used separately to compare the effect
of HCs on microbial growth.
Plants were supplied weekly with LHs and WCHs in aqueous solutions at 0.7% but control
plants were not amended with HCs. Plants were located in a randomized block in the
greenhouse and grown for 60 days at constant temperature of 24 ± 1°C and relative humidity
of 75%.
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Estimation of microbial growth. After 7, 14, 28, 45 and 60 days, microbial counts were taken
on 3 separate soil samples from each experimental block. Total number of aerobic bacteria
(TB), actinomycetes (TA) and fungi (TF), cellulolytic population (CEL), hemicellulolytic
microorganisms (HEM), ligninolytic microorganisms (LIG) and nitrogen-fixing bacteria (NF)
were determined by plate counts. Ammonifiers (AM) and nitrifying bacteria (NIT) were
determined by the most probable number technique.
Statistical analysis. Data were subjected to one multifactorial analysis of variance (ANOVA)
in which counts of the different groups were compared for the different levels of humic
treatment (LHs, WCHs and Control), sampling time and pot substrate type (IS and SS). In
order to determine which means were significantly different (p < 0.05), multiple comparison
tests (Fisher’s least significant difference) were used. All experiments were carried out twice.
3. Results and Discussion
Microbial populations were affected when different HCs were added to all experimental
blocks. The effect observed depended on the origin of the amendment applied (LHs or
WCHs) as well as the tomato cultivar used (‘Raf’ or ‘Durinta’).
Table 1 shows the significant influence of the different factors on all microbial groups
analysed as well as the influence of the interactions between them. In general, several
differences were observed when humic extracts were added to different cultivars. In this
sense, the humic treatment significantly influenced on TB, TF, CEL, LIG and NF populations
when ‘Raf’ was used while this effect was observed on TB, TF, HEM, NF and NIT when
‘Durinta’ was used. On the other hand, in both cultivars, the interactions between the different
factors significantly influenced TA, TF, CEL, LIG and NF populations.
In the ‘Raf’-soil system, when HCs were added to the soil, populations of TA, HEM, AM and
NIT did not differ from those observed in the control treatment. On the other hand, counts of
TB, TF, NF and CEL populations in LHs and WCHs treatments were, in general, higher than
those obtained in control soils (data not shown). The population of nitrogen-fixing bacteria
(NF) was particularly higher in the WCHs treatment than in the LHs, and values from control
fell in between them. This effect was more evident from 28 days and when IS was used (data
not shown).
In the ‘Durinta’-soil system, microbial counts of TA, CEL, LIG and AM were not affected by
the addition of HCs to the soil. On the other hand, as observed in the case of ‘Raf’, counts of
TB, TF and NF populations were higher when LHs or WCHs were added. Contrary to the
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results obtained from the ‘Raf’-soil system, microbial counts of HEM and NIT were also
higher in amended substrates. NIT showed higher counts than those obtained in the case of
substrates added with LHs. These differences were higher during the second month and when
IS was used rather than SS (data not shown).
Table 1: Effect of Sampling time, substrate and humic treatment on microbial populations from
a plant-soil system. Significant differences are observed at 95% confidence level (p < 0.05).
Factors
Sampling time
Substrate kind
Humic treatment
Interactions
Sampling time X
Substrate kind
Sampling time X Humic
treatment
Humic treatment X
Substrate kind
Factors
TB1
p < 0.05
0.0000
0.0000
0.0087
p < 0.05
0.0000
TA2
p < 0.05
0.0000
0.0000
ns10
p < 0.05
0.0000
TF3
p < 0.05
0.0000
0.0000
0.0009
p < 0.05
0.0000
‘Raf’
CEL4
p < 0.05
0.0000
0.0000
0.0000
p < 0.05
0.0000
HEM5
p < 0.05
0.0000
0.0000
ns
p < 0.05
0.0000
LIG6
p < 0.05
0.0000
0.0000
0.0000
p < 0.05
0.0000
NF7
p < 0.05
0.0000
0.0000
0.0001
p < 0.05
0.0000
NIT8
p < 0.05
0.0001
0.0158
ns
p < 0.05
0.0001
AM9
p < 0.05
0.0000
0.0010
ns
p < 0.05
0.0000
ns
ns
0.0357
0.0000
0.0005
0.0000
0.0029
ns
0.0000
ns
ns
ns
0.0000
0.0000
0.0000
ns
0.0010
0.0029
TB1
p < 0.05
0.0000
0.0001
0.0005
p < 0.05
0.0000
TA2
p < 0.05
0.0000
0.0000
ns
p < 0.05
0.0000
‘Durinta’
TF3
CEL4
p < 0.05 p < 0.05
0.0000
0.0000
0.0000
0.0294
0.0060
ns
p < 0.05 p < 0.05
0.0000
0.0000
HEM5
p < 0.05
0.0000
0.0000
0.0000
p < 0.05
0.0000
LIG6
p < 0.05
0.0000
0.0000
ns
p < 0.05
0.0000
NF7
p < 0.05
0.0000
0.0000
0.0002
p < 0.05
0.0000
NIT8
p < 0.05
0.0000
0.0048
0.0000
p < 0.05
0.0000
AM9
p < 0.05
0.0000
0.0001
ns
p < 0.05
0.0000
ns
0.0036
0.0000
0.0197
0.0159
0.0025
0.0000
ns
ns
ns
0.0000
ns
0.0000
ns
0.0005
ns
Sampling time
Substrate kind
Humic treatment
Interactions
Sampling time X
Substrate kind
Sampling time X Humic
0.0393
treatment
Humic treatment X
ns
Substrate kind
1
Total Bacteria
2
Total Actinomycetes
3
Total Fungi
4
Cellulolytic Microorganisms
5
Hemicellulolytic Microorganisms
6
Ligninolytic Microorganisms
7
Nitrogen-Fixing Bacteria
8
Ammonifiers Microorganisms
9
Nitrifying Bacteria
10
non significant
The results obtained in this work have shown that the application of HCs has a significant
impact on several soil microbial groups. This response could be attributed to the nutritive
value of humates from WCHs and LHs. Also, Valdrighi et al. (1996) [4] suggest that
potassium added to WCHs has no stimulatory effects on microbial populations. Several
authors have confirmed that molecular characteristics of HCs may result in higher biological
activity due to enzymatic activation of nutrient uptake or modification of bacterial cell
permeability to nutrients [2, 5]. Counts of total aerobic bacteria, fungi and nitrogen-fixing
bacteria were higher in soils treated with HCs than in control soils (data not shown). This
effect was observed in both plant cultivars tested (‘Raf’ and ‘Durinta’). However, treatment
with WCHs promoted the highest counts of nitrogen-fixing (NF) bacteria with ‘Raf’, while
the highest counts of nitrifying bacteria were obtained with ‘Durinta’ (data not shown). It is
therefore possible that different plant-soil systems react differently to the presence of HCs [6].
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Soil microorganisms involved in nitrogen cycling have previously been studied as regards
their response to the application of HCs. Populations of autotrophic ammonia and nitrite
oxidizers increased in soil or axenic cultures amended with humates from composted
vegetable waste, especially at high rates [2, 4]. On the other hand, Acea et al. (2003) [7]
confirmed the importance of nitrogen-fixing bacteria to promote microbial crust formation,
enhancing C and N cycling microorganisms and increasing organic matter and nutrient
content in deteriorated soils.
4. Conclusions
Therefore, results derived from this preliminary work showed the potential for improving the
utilization of HCs extracted from compost based on plant waste (WCHs). The extraction of
these substances (WCHs) produced an extract which behaved as a stimulatory substance on
some microorganisms related to the plant roots.
References
1. S. Nardi, G. Concheri and G. Dell’Agnola G, in: Piccolo A (Ed) Humic Substances in Terrestrial
Ecosystems, Elsevier, Amsterdam, 1996, pp 361-406
2. M.M. Valdrighi, A. Pera, S. Scatena, M. Agnolucci and G. Vallini, Compost Science Utilization 3
(1) (1995) 30-38
3. G. Vallini, A. Pera, M. Agnolucci and M.M. Valdrighi, Biology and Fertility of Soils 24 (1997),
243-248
4. M.M. Valdrighi, A. Pera, M. Agnolucci, S. Frassinetti, D. Lunardi and G. Vallini, Agriculture
Ecosystem and Environment 58 (1996), 133-144
5. M. Tejada, C. García, J.L. González, M.T. Hernández, Soil Biology and Biochemistry 38 (2006)
1413-1421
6. D. Vaughan and R.E. Malcom, In: Vaughan D, Malcom RE (Eds) Soil Organic Matter and
Biological Activity, Martinus Nijhoff/Junk W, Dordrecht, 1985, pp 37-76
7. M.J. Acea, A. Prieto-Fernández and N. Diz-Cid, Soil Biology and Biochemistry 35 (2003) 513-524
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Analysis of the Sorption Properties Soils After the Application of Sewage
Sludges and Conventional Organic Fertilizers
Stańczyk-Mazanek Ewa*, Stępniak Longina, Kępa Urszula
Institute of Environmental Engineering, Czestochowa University of Technology,
ul. Brzeznicka 60a, 42-200 Częstochowa, Poland
E-mail: stanczykewa@wp.pl; stanczyk@is.pcz.czest.pl
1. Introduction
The sorption capacity of soil is regarded as one of the most important factors influencing the
fertility of the soil and the properties of plants grown. An extended sorption complex is an
element that retains and absorb various soil contaminants. The state of the sorption complex
is influenced, inter alia, by the following: organic matter content, humus compounds forming
during organic matter decomposition, silt minerals, pH, and hydrolytic acidity. The pH value
of soil very strongly determines its physical, chemical and biological properties. The reaction
of the soil influences the stability of the structure and associated air-water relationships. All
these factors provide optimal growing and yielding conditions for plants [1, 2]. The authors of
the present work undertake the analysis of the effect of application of sewage sludges and
selected organic fertilizers on the changes in the sorption properties of thus treated soils.
2. Materials and Methods
Sewage sludges and, for comparison purposes, also manure were introduced to sandy soils
(P). Tests were conducted under pot experiment conditions. The following doses of organic
fertilizers were applied: 0, 10, 50, 100 and 200 tonnes per hectare. Particular sewage sludges
and manure were mixed, respectively, with both soil types. The amounts of fertilizers were
calculated for 10 kg of soil (which was held in experimental pots) so that they corresponded
to the following doses: 10, 50, 100 and 200 tonnes of sewage sludge or manure (PO) per
hectare. Unfertilized sand constituted the control soil. So prepared soil samples were left for a
period of about 6 months, while maintaining constant humidity. After this period, test samples
were taken for analysis. The tests were carried out in 3 replications. The results represent the
mean of these replications. After 6 months from the fertilization, variations in active and
hydrolytic acidity in the grounds treated were analyzed. The content of organic matter; the
sum of bases, S, in the sorption complex (by the Kappen method); sorption capacity, T; and
the contents of humic acids in the soils treated were also determined.
Two soil types with the grain-size composition of loose sand were used for the fertilization
tests. The reaction of the first soil (which was designated as soil P1) was 8.31, while that of
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the other (soil P2) was 4.79. The sewage sludges used for the fertilization of selected soils
originated from 2 sewage treatment plants. These were designated by P and R, being the first
letters of the names of locations where the sewage treatment plants were situated. Sewage
sludge P was oxygen-stabilized. Sewage sludge R came from a biological sewage treatment
plant. Two sewage sludge types were taken from the sewage treatment plant. One of them was
dewatered by gravity without the addition of a polyelectrolyte. This was designated as sewage
sludge R (plot). The other sewage sludge was dewatered on a belt press with the use of a
polyelectrolyte, and was designated as sewage sludge R (press).
3. Results and Discussion
The results of the testing of the sandy soil (P1) treated with sewage sludges and manure,
respectively, are shown in Figures 1 and 2. Whereas, the results of the analysis of soil P2 are
illustrated in Figures 3 and 4.
pH
8,5
8
P1P(prasa)
P1R(prasa)
P1R(poletka)
7,5
P1O
7
6,5
Kontrola
10
50
100
200
hydrolytic acidity
9
2
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
P1P(prasa)
P1R(prasa)
P1R(poletka)
P1O
Kontrola
dose [t/ha]
10
50
100
200
dose [t/ha]
Fig. 1 Variations in the pH of sandy soil (P1)
Fig. 2 Variations in the Hh of sandy soil (P1)
after treatment
after treatment
10
pH
8
6
P2P(prasa)
P2R(prasa)
P2R(poletka)
4
P2O
2
0
hydrolytic acidity
2,5
2
10
50
100
200
P2R (pra sa)
P2R (pol etka)
1
P2O
0,5
0
Kontrola
P2P(p rasa )
1,5
Kontro la
10
50
100
20 0
dose [t/ha]
dose [t/ha]
Fig. 3 Variations in the pH of sandy soil (P2)
Fig. 4 Variations in the Hh of sandy soil (P2)
after treatment
after treatment
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15th IHSS Meeting- Vol. 3
The properties of the sorption complex of soils treated (soil P2 is shown) are summarized in
Table 1.
Table 1. Test results for the properties of the sorption complex of P2 soil mixes
Soil mix type
Determination
Organic matter
Sum of bases S
Sorption capacity
Humic acid
content [%]
[meq/100g soil]
T[meq/100g soil ]
content [%]
Control (P2)
0.6
2.55
3.63
0.025
PP 10
0.6
3.35
4.63
0.050
PP 50
1
6.86
8.4
0.165
PP 100
2.2
10.4
12.31
0.285
PP 200
4.4
20.2
22.53
0.495
PR 10 press
0.6
2.95
4.19
0.070
PR 50 press
0.8
11.25
12.6
0.155
PR 100 press
1.6
12.6
14.6
0.245
PR 200 press
2.8
25.3
27.63
0.515
PR 10 plot
0.6
5.5
6.59
0.065
PR 50 plot
1
8.55
9.83
0.195
PR 100 plot
1.8
12.5
13.93
0.250
PR 200 plot
3
27.9
29.81
0.620
PO 10
0.6
3.1
4.11
0.065
PO 50
0.8
6.1
6.93
0.135
PO 100
1.4
9.3
9.94
0.250
PO 200
3
15.7
16.19
0.435
4. Conclusion
In summing up the obtained investigation results it can be concluded that the application of
the organic fertilizers, both manure and sewage sludge, significantly influenced the sorption
properties of soil. As a result of organic fertilization, the physicochemical properties of the
soil are improved, the acidification is reduced, and the sum of exchange bases, humic
compounds, and sorption complex capacity increase [1,3,4]. Similar results have been
obtained in the present work. However, only manure used in the tests did always cause a
reduction in the acidity of soils treated with it. This phenomenon was observed for the
treatment of both basic and acidic sandy soils, alike. The application of manure reduced both
active and potential (hydrolytic) acidity. The acidity-reducing tendency of this fertilizer was
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visible for a long time after its application. Different responses of soil treated were observed
after the application of sewage sludge. A basic soil upon treatment with all sewage sludge
types underwent acidification, which was the greater, the higher was the sewage sludge dose.
It was observed that a higher increase in hydrolytic acidity in soils was caused by sewage
sludge with the addition of polyelectrolytes used for dewatering. Sewage sludges from plots
exhibited lower acidifying properties. This is probably dependent on the variable properties of
sewage sludges themselves. Navas et al. [3] observed a decrease in the pH of soils after the
application of sewage sludge at a rate of 0, 40, 80, 160 and 320 tonnes/hectare, respectively.
The reported increase in acidity (similarly as in the present work) was the higher, the larger
was the sewage sludge dose introduced to the soil. Similar results were obtained by Wong et
al. [5]. A drop in the pH of soil after the application of sewage sludge was also observed by
Forsberg and Ledin [6]. Other investigation results [7] indicate an increase in the pH of soil
after the introduction of sewage sludge. The differences are most likely to be due to the
properties of particular sewage sludges, which indicates very variable and often unpredictable
chemical and biological properties of sewage sludge.
References
1. Gorlach E., Mazur T.: Chemia rolna (Agricultural chemistry), Wydawnictwo Naukowe PWN,
Warsaw 2001.
2. Wang A.S., Scott Angle J., Chaney R.L., Delorme T.A., McIntosh M.: Changes in soil biological
activities under reduced soil pH during Thlaspi caerulescens phytoextraction, Soil Biology and
Biochemistry 38 (2006), 1451–1461.
3. Navas A., Bermudez F.,Machin J.: Influence of sewage sludge application on physical and
chemical properties of Gypsisols, Geoderma 87 (1998) 123–135.
4. Bieniek B., Różańska E., Bieniek A.: Wpływ ścieków przemysłu rolno-spożywczego na
właściwości sorpcyjne gleb mineralno-organicznych (The effect of agricultural & food industry
wastes on the sorption properties of mineral-organic soils), Zeszyty Problemowe Postępów Nauk
Rolniczych 2000, z. 472: 97–102.
5. Wong J.W.C., Fang L.M., Ma K.K.: Effects of sewage sludge amendment on soil microbial
activity and nutrient mineralization, Environment International, 24, No 8, 1998: 935-943.
6. Forsberg L.S., Ledin S.: Effects of sewage sludge on pH and plant availability of metals in
oxidising sulphide mine tailings, Science of the Total Environment 358 (2006) 21–35.
7. Bramryd T.: Effects of liquid and dewatered sewage sludge applied to a Scots pine Stand (Pinus
sylvestris L.) in Central Sweden, Forest Ecology and Management 147 (2001), 197–216.
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Interactions Between Plant-Root Exudates and Soils in Extracting Humiclike Substances
Diego Pizzeghelloa*, Adele Muscolob, Andrea Ertania, Serenella Nardia
a
Department of Agricultural Biotechnologies, University of Padova, Agripolis, Viale dell’
Universita` 16, 35020 Legnaro, Padova, Italy; bDepartment of Agricultural and Forest
Systems Management “Agriculture Faculty, Mediterranea” University of Reggio Calabria,
Feo di Vito 89126 Reggio Calabria, Italy
E-mail: diego.pizzeghello@unipd.it
1. Introduction
Plants have evolved with roots in close contact with the solid phase of the soil, therefore
organic acid root exudates, in plant–root–microbial interactions, have attracted much interest
not so much in terms of carbon source but as ‘signals’ for recognition or as precursors of
phytohormone production [1]. Recent studies have demonstrated that organic acids (i.e.
fumaric and succinic acids) occurring in the root exudates can dissociate humic substances
(HS) in low and high molecular weight structures. This interpretation may support the
hypothesis that the conformational behavior of dissolved humus in the rhizosphere, and
therefore also the interaction of humic components with plant-root cells, may be controlled by
the presence of root-exuded or microbe-released organic acids in the soil solution [2, 3].
Therefore, root exudates may be a better medium for extracting low molecular size (LMS)
organic fractions (humic-like substances) than currently used alkaline solutions (i.e. NaOH,
KOH). Our objective was to compare the chemical and biological activity of LMS organic
fractions extracted using maize (Zea mays L.) and two forest species (Picea abies and Pinus
sylvestris) root exudates to humic substances extracted by KOH [4]. The identification of
some organic acids species present in the LMS organic fractions have been investigated by
GC/MS technique and the biological activity of the humic-like substances extracted was
evaluated by determining their hormone-like activity.
2. Materials and Methods
Humic substances (HS) were extracted with common alkaline (KOH) extraction procedures
(4) from the A horizons of a Eutric Cambisol (EC) [8] and a Rendzic Leptosol (RL) [8]. The
EC developed under a field of Bermuda grass (Cynodon dactylon) located near the College of
the Faculty (Legnaro, Padova, Italy), and the RL was covered by a Scotch pine forest and
located near Cortina d'Ampezzo (Belluno, Italy).Two forest species (Picea abies Karst. and
Pinus sylvestris L.) and two commercial maize hybrids (Zea mays L. cultivars Mytos and
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15th IHSS Meeting- Vol. 3
Samantha; Dekalb, Italy) were used for the root exudates production. The root exudates were
collected from forest plants raised in sterile conditions, as described in a previous work [5],
and from maize seedlings treated with sterile Hoagland’s solution [6, 7]. The LMS
humic/organic substances were obtained by treating 2 g of soil (A horizon) with 20 ml of
water or 20 ml of root exudates and shaken them at room temperature for 16 h under a N2
atmosphere. The suspensions were centrifuged at 10 °C and 5000 g for 30 min and the
supernatants (extracts) were analyzed for total carbon. The GC/MS analysis was made on 2
ml freeze-dried exudate or extract by an HP 5890 gas chromatograph coupled with a
quadrupole HP 5971 A [9]. The separation and determination of the low molecular weight
organic and phenolic acids were made by HPLC following the procedures reported in
previous papers [9, 10]. The auxin-like and gibberellin-like activity of the LMS
organic/humic fractions was assessed by checking the reduction in the growth of watercress
(Lepidium sativum L.) roots and the increase in the length of lettuce (Lactuca sativa L.)
epicotyls [11].
3. Results and Discussion
Agrarian and forest seedlings released different types of exudates in the rhizosphere, and they
have higher extracting abilities towards carbon then water when in contact with poorer
substrates [7, 9]. The concentrations of oxalic and succinic carboxylic acids were always
higher in the P. abies and P. sylvestris than in the two commercial maize hybrid root
exudates. The two forest tree exudates differed in that the P. abies had a high content of
oxalic and L-malic acids, whereas the P. sylvestris contained citric acid. GC/MS spectra
revealed that the LMS organic/humic-like fractions had a greater variety of fatty acids than
the HS. Mytos extracted C15H31COOH and C16H23COOH from EC, whereas Sandek extracted
only C15H31COOH. The P. abies extracted C11H23COOH and C13H27COOH from both soils.
The fraction extracted from the EC by P. sylvestris revealed C13H27COOH and C17H35COOH.
All soil extracts by exudates exhibited a hormonal activity that was not present in either water
extracts or in the original exudates. Moreover, the extracts from the agrarian soil exhibited a
higher hormone-like activity with respect to the extracts from forest soil. The extracting
ability of the different species appear to be related to their different environmental conditions
[12]. Concerning the content in phenolics, benzoic acid and in minor extent phtalic acid,
resulted to be the more present in the LMS organic/humic-like components extracted by using
exudates. Analysis of the phenolic pool demonstrated specificity on the extracted molecules
not directly correlated with the phenolics present in the pure exudates. Studies have shown
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that phenolic compounds have been found to be highly effective in plant defense against
pathogens, nematodes, phytophagous insects [13]. In particular, benzoic acid is one of the
most active phenolic compound inhibitors of fungi [14].
4. Conclusions
This paper reconfirms that many different regulatory signals affect rhizosphere interactions.
Among these, the role of exudates in breaking out from the bulk humus the active humic
components endowed with hormone-like activity and pest inhibitors is evident. This suggest
that an appropriate combination of factors may be useful for the best use of humic substances
on plant's health and plant capacity to adapt to different environmental conditions.
References
1. H. Marschner, Mineral Nutrition of Higher Plants, Academic Press, London, 1995, p. 889.
2. S. Nardi, G. Concheri and G. Dell'Agnola, in A. Piccolo (Ed.), Humic Substances in Terrestrial
Ecosystems, Elsevier, Amsterdam, p. 361-406.
3. A. Piccolo, P. Conte, R. Spaccini and M. Chiarella, Biol. Fertil. Soils, 37 (2003) 255–259.
4. F.J. Stevenson, Humus Chemistry, Genesis, Composition, Reaction, second ed. Wiley, New York,
1994, p. 496.
5. S. Nardi, F. Reniero and G. Concheri, Chemosphere, 35 (1997) 2237-2244.
6. D.R. Hoagland and D.I. Arnon DI, Agricultural Experiment Station, Circular 347 (1950).
7. S. Nardi, E. Sessi, D. Pizzeghello, A. Sturaro, A. Rella and G. Parvoli, Chemosphere, 46 (2002)
1075–108.
8. FAO, Soil Map of the World: revised legend, FAO, Rome, 1990.
9. S. Nardi, M. Tosoni, D. Pizzeghello, M.R. Provenzano, A. Cilenti, A. Sturaro, R. Rella and A.
Vianello, Soil Sci. Soc. Am. J., 69 (2005) 2012-2019.
10. S. Nardi, D. Pizzeghello, L. Bragazza and R. Gerdol, J. Chem Ecol., 29 (2003) 1549-1564.
11. L.J. Audus, Plant Growth Substances, Leonard Hill, London, 1972.
12. T. Ingestad, Physiol. Plantarum, 13 (1960) 513–533.
13. F.D. Dakora and D.A. Phillips, Physiol. Mol. Plant Pathol., 49 (1996) 1-20.
14. M., Weidenborner, H. Hindorf, H.C. Jha and P. Tsotsonos, Phytochemistry, 29 (1990) 1103-1105.
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Bioactivity of Humic Acids from Vermicompost at Increasing Maturity
Stages
Natalia O. Aguiara*, Luciano P. Canellasa, Fabio L. Olivaresa, Jader G. Busatoa, Luis Gonzaga
JR. S. Silva.a, Etelvino H. Novotnyb, Arnoldo R. Façanhaa
a
Núcleo de Desenvolvimento de Insumos Biológicos para Agricultura - Universidade Estadual
do Norte Fluminense Darcy Ribeiro. Av. Alberto Lamego, 2000, Campos dos Goytacazes
28013-602, Rio de Janeiro, Brasil; bEmbrapa Centro Nacional de Pesquisa de Solos (CNPS),
R. Jardim Botânico, 1024, Rio de Janeiro, Brasil
E-mail: nattyaguiar@gmail.com
1. Introduction
The application of humic substances (HS)-derived products at low concentration and their
effects as plant growth promoters have been creating increased interest among farmers.
Despite of the technological potential, little information have been accumulated about the
mechanism by which HS influences biological activities in plants. It has been widely
demonstrated that humic acid (HA) can affect plant growth and metabolism, but scientific
efforts linking HA structure to biological activity have so far produced divergent results. The
relationship between different levels of bioactivity and variation at chemical structure of HS
persists as a challenge for scientific and technological purposes into the direction of
improvement of organic fertilizers based on humic matter. It was previously observed that HA
induced changes in the developmental program of root growth and in plant development by
proliferation of lateral emerging sites and induction of a plasma membrane (PM) H+-ATPases
in different plant species [1]. The aim of this work was to evaluate chemical changes and
plant growth-promoting effect of HA from different maturing stages isolated from
vermicompost, relating bioactivity and its chemical characteristics.
2. Material and methods
Vermicompost (VC) was prepared using cattle manure putted on concrete cylinder with 150 L
of capacity and humidity was maintained at 65–70%. After approximately one month, the
earthworm was introduced (Eisenia foetida) at a ratio of 5 kg worms per m3 of organic
residue. Two cylinders of each organic residue were used to sample at different time: 0, 30,
60, 90 and 120 days. Before sampling, the content of cylinder was vigorously mixed by
manual spade. The VC was chemically characterized (organic carbon, C-N ratio, CEC and
HA content). HA were isolated with 0.5M NaOH under N2 and precipitated with 6M HCl.
The diluted HF:HCl was used for 16 hours to decrease the ash content and after that HA was
washed with water, dialyzed (membrane cutoff 1000 Da) and freeze dried. The relative Mw
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index was obtained by the ratio of low: high and characterized by size exclusion (PolysepGFC-P 3000 (600 mm per 7.8 mm i.d.) column (Phenomenex) and reverse-phase (Supelco C18 column) high performance chromatography for hydrophobicity. Maize seedlings with
0.5 cm of root length was treated for 48 h with HA solution (20 mg C L-1 of AH and 2 mM
CaCl2 at pH 7.0). After this time, the seedlings were transferred to 40 mL of 2 mM CaCl2 at
pH 7.0. After 48 h the pH was evaluated using Thermo Orion pHmeter. A preliminary assay
was carry-out to verify a putative relationship between H+ extrusion and PM H+-ATPase
activity [2].
3. Results
Parameters related to organic matter evolution during vermicompost maturation stages. The
C-N ratio and lignin content, decreased and CEC and HA content increase (Fig. 1). The
relative Mw had shown no change and the hydrophobic content, determined by NMR, showed
small changes with the maturation process (Fig. 2). However the hydrophobic content,
determined by RP-HPLC showed changes with the maturation process (Fig. 3).
20
80
70
16
VC1
14
60
lignin (%)
CEC (Cmolc kg-1)
B
18
A
50
40
12
10
8
6
4
30
2
20
0
0
15.5
15
30
60
90
120
0
60
90
120
4
C
D
14.5
3.5
gCHA kg-1 VC
14
C/N ratio
30
13.5
13
12.5
12
3
2.5
2
11.5
11
1.5
0
30
60
90
120
0
vermicomposting time (days)
20
40
60
80
100
120
vermicomposting time (days)
Figure 1: Parameters related to organic matter stabilization according vermicompost maturity time
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Figure 2: 13C-CPMAS-NMR spectra from vermicompost at different maturing stages
Figure 3: HPSEC chromatograms - Mw distribution (A), RP-HPLC Hydrophobicity (B) index Mw (C)
and index hydrophobicity (D) of HA from vermicompost at different maturing stages (0, 30, 60, 90
and 120 days)
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Figure 4: A: Proton extrusion (mM H+ g-1 dry root) for 2 mM CaCl2 solution of maize root seedlings
treated with 48 hours of HA isolated from cattle manure vermicompost at different maturing stages (0,
30, 60, 90 and 120 days). B: relationship between H+ extrusion and PM H+-ATPase activity
At the end of vermicompost maturation it was observed a selective increase of C-aryl, O-aryl
and C-carboxyl species as a consequence of carbohydrates decrease (Fig. 2). The relative
molecular size/weight (Mw) revealed no changed with HA maturation (Fig. 3A and C), but
the hydrophobicity evaluated by RP-HPLC increase at 60-d and after this time decrease (Fig,
3 B and D). After 60-d of vermicomposting process the HA displayed bioactive effect
compared with control manifested by increaser of maize root seedlings H+ extrusion (Fig.
4A), which gave a good correlation with PM H+-ATPase activity (Fig. 4B).
We concluded that at the initial stages of vermicompost maturing process the HA had
diminished plant growth promoting effect. Increased bioactivity of HA evaluated by H+
extrusion at the solution and PM H+-ATPase activity by HA was achieved with enhanced
vermicompost maturing coupled with increase of hydrophobic domains on its structure,
mainly until 60 days. However the form of protection of bioactive molecules in the structure
of AH is not even well understood and needs to be more study. Furthermore the process of
humification by itself is not a sufficient to explain the promotion of plant growth promoted.
References.
1) Canellas, L.P., Façanha, A.O., Olivares, F.L., Façanha, A.R., Humic acids isolated from
earthworm compost enhance root elongation, lateral root emergence, and plasma
membrane H+-ATPase activity in maize roots. Plant Physiol. 130, 1951– 658 1957. 2002.
2) Canellas, L.P., Piccolo, A., Spaccini, R., Dobbss, L., Zandonadi, D., Olivares, F. and
Facanha, A.. Chemical composition and bioactivity properties of size-fractions separated
from
a
vermicompost
humic
acid.
Chemosphere
(2009),
doi:
10.1016/j.chemosphere.2009.10.018.
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Root Growth promotion by humic acids from urban organic residues
Keiji Jindo, Carlos García Izquierdo, Luciano Pasqualoto Canellas
CEBAS-CSIC. Campus Universitario de Espinardo. Apartado de correos 164. Murcia. E30100, Spain
E-mail: keijindo@cebas.csic.es
1. Introduction
The effect of HA on plant physiology is generally recognized to result in root growth
enhancement (Vaughan et al., 1985; Nardi et al., 2002) and enhance of nutrients uptake (Chen
et al., 2004; Pinton et al., 2007). Humic acids (HA) promotes plasma membrane (PM) H+
ATPase activity and it synthesis (Pinton et al., 1987; Canellas et al., 2002; Quaggiotti et al.,
2004). The major role of this enzyme on energetic metabolism PM H+-ATPase activity was
found to be a useful physiological indicator of HA bioactivities (Canellas et al., 2006).The
aim of this work was to evaluate the root seedling growth and PM H+-ATPase activity on
maize treated with HA isolated from sewage sludge and municipal solid wastes at initial and
final stage of composting process.
2. material and Methods
Organic materials from urban origin: Four different organic wastes were used in this study:
Sewage sludge (SS) from a municipal wastewater treatment plant in El Raal—Murcia,
compost produced from the abovementioned SS (SSC), organic fraction of MSW collected
from the treatment plant, and the compost produced from this organic material (MSWC).
HA extraction: The method of extraction of humic acid (HA) was followed by Stevenson. The
dialyzate was lyophilized and characterized chemically. Later, these 4 samples were
chemically characterized by solid-state nuclear magnetic resonance (13C CPMAS-NMR),
Maize: Maize seeds (var UENF 506) provided were surface-sterilized by soaking in 0.5%
NaCl for 30 min, followed by rinsing and then soaking in water for 6 h. The seeds were then
sown on wet filter paper and germinated in the dark at 28°C. After regression analysis, a new
experiment was carried out using 2mM C of each HA. Four-day-old maize seedlings with
roots approximately 0.5 cm long were transferred into a solution containing 2 mM CaCl2 (to
avoid any interference with nutrients contsittuents (Pinton et al.1999) and either 0 or 2 mM C
L-1 of HA . Roots were collected on the seventh day and scanned at 300 dpi to estimate their
length and area using Delta-T Scan image analysis software (Cambridge, UK) (Bouma et al.,
2000). Additional samples of root seedlings were collected for mitotic sites experiments and
lateral roots (all graphics of results are demonstrated with comparison with the control).
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Effect of H.A. on lateral roots growth
Roots Area
150
100
90
80
70
60
50
40
30
20
10
0
Relative roots area ratio (%)
Relative lateral roots ratio (%)
130
110
90
70
50
30
10
- 10
ss
css
msw
cmsw
SS
CSS
MSW
CMSW
mitosis
Relative ATPase activity
800
%
Relative mitosis (%)
600
200
180
160
140
120
100
80
60
40
20
0
400
200
0
SS
CSS
MWS
CMWS
ss
css
msw
cmsw
Biochemical assays: PM vesicles were isolated from maize roots grown with or without 20
mM C L-1 of bulk HS and each chemical derivative by a differential centrifugation method
(Canellas et al. 2002). The vesicles were either used immediately or frozen under liquid N2
and stored a t -70°C until use. Protein concentration was determined by the Lowry method.
ATPase activity in PM vesicles was determined by colorimetrically measuring the release of
Pi. Between 80 and 95% of the PM vesicle ATPase activity measured at pH 6.5 was inhibited
by vanadate (0.1 mmol L-1).
3. Results and discussion
All HA promoted root growth and proton pump activation in maize vesicles. Within a general
stimulation of root areas over control, HA more with hydrophobic character preferentially
increased root growth and proton pump induction. The conformational dynamics of humic
hydrophobic associations in the rhizosphere may release auxin-like plant growth promoters
and actively enhance plants biochemical activities.
4. Conclusion
The organic residues from urban source can be considered an appropriated source for HA
extraction and use as a root plant growth promoter. The HA differed in hydrophobicity but not
in molecular dimension with compost maturation. Their bioactivity enhance with the
composting time and were related with chemical molecular changes that happened mainly
with the increase of hydrophobic character.
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1. References
2. Canellas, L. P., D. B. Zandonadi, F. L. Olivares, and A. R. Façanha. 2006. In Nutrição Mineral de
Plantas. M. S. Fernandes (ed.). Sociedade Brasileira de Ciência do Solo, Viçosa, Brazil, pp. 175200.
3. Bouma, T. J., K. L. Nilsen, and B. Koutstaal. 2000. Plant Soil. 218:185-196.
4. Canellas, L. P., F. L. Olivares, A. L. Okorokova-Façanha, and A. R. Façanha. 2002. Plant Physiol.
130:1951-1957.
5. Vaughan, D., R. E. Malcolm, and B. G. Ord. 1985. Soil Organic Matter and Biological Activity.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 77-108.
6. Chen Y., Clapp C.E., Magen H. (2004) Soil Science & Plant Nutrition. 50, 1089
7. Pinton R., Varanini Z., Nannipieri P. (2007) The rhizosphere: biochemistry and organic
substances at the soil–plant interface, CRC Press, Madison, USA: pp. 447.
8. Quaggiotti, S., B. Ruperti, D. Pizzeghello, O. Francioso, V. Tugnoli, and S. Nardi. 2004. J. Exper.
Bot. 55:803-813.
9. Nardi, S., D. Pizzeghello, A. Muscolo, and A. Vianello. 2002. Soil Biol. Biochem. 34:1527
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Direct and Indirect Effects of Humic Substances of Different Origin on the
Green Algae Monoraphidium braunii
C. Eliana Gattulloa*, Hanno Bährsb, Ji Qianrub, Christian E.W. Steinbergb, Elisabetta
Loffredoa
a
Dipartimento di Biologia e Chimica Agro-forestale ed Ambientale, University of Bari,Via
Amendola 165/A, 70126 Bari, Italy; bInstitute of Biology, Freshwater and Stress Ecology,
Humboldt University at Berlin, Späthstr. 80/81, 12437 Berlin, Germany
E-mail: e.gattullo@agr.uniba.it
3. Introduction
Dissolved natural organic matter (NOM) is widespread and abundant in freshwater
ecosystems, exceeding the total living organic carbon by one order of magnitude [1, 2]. A
consistent part of NOM (50–80%) is made up by humic substances (HS) [1, 3]. It’s well
known that HS, because of their physical and chemical properties, modify the underwater
light climate, alter the bioavailability of inorganic micronutrients and xenobiotics and, after
their photodegradation, supply carbon and/or energy to heterotrophic organisms. New
ecophysiological studies, without disregarding the traditional conception that HS affect
indirectly the aquatic biocenosis, consider HS as natural environmental chemicals able to
interact also directly with organisms [4]. Due to the low molar mass of their building blocks,
HS can be taken up by organisms. Once internalized, they act like xenobiotics inducing
several specific or not specific stress responses. Specific reactions include reduction of
photosynthetic oxygen production, estrogenicity and chemical attraction. Non specific
reactions comprise physical and chemical membrane irritation, induction and modulation of
biotransformation enzymes, induction of stress defense proteins and internal oxidative stress
[5]. According to recent theories of aging and evolution, a mild stress may be benefit for
individuals, training their chemical defense system [5, 6]. Therefore, the study of interactions
between HS and organisms represents the first step to clarify the possible role of HS as mild
stressors able to modify the biochemical and physiological properties of individuals and, on a
larger scale, structure the biocenosis. Much information are reported in the literature on the
interactions between dissolved organic carbon and animals or aquatic plants. With regard to
phytoplankton, previous works [7, 8] revealed contrasting effects of HS on algal growth rate.
Moreover, HS lower the oxygen production in various green algae, but the mechanism of
action remains still obscure [9]. The present work is a part of a larger study on stress
responses of various green algae to different organic fractions. Here, the authors measured the
growth and some fluorescence parameters on the green algae Monoraphidium braunii (MB)
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exposed to two HS sources. A parallel experiment was carried out in the same conditions, but
using a nitrogen-free culture medium, to assess the attitude of HS to supply nitrogen to MB.
4. Materials and Methods
Monoraphidium braunii was obtained from the Algal Culture Collection, Göttingen, and
cultivated in axenic condition in FW04-medium [10]. The Suwannee river NOM, acquired
from the International Humic Substances Society, and the HuminFeed® (HF), a commercial
HS isolated from leonardite by alkaline extraction, were tested at the concentrations of 2, 5
and 20 ppm DOC. Algae in the logarithmic growth phase were used to inoculate FW04medium alone (control) or added with each concentration of NOM or HF, up to an initial
concentration of about 105 cells/mL. Algae solutions were cultivated in axenic conditions in
glass flasks, at 20 °C, under a cool white light set to 150 µM photons/m2s, using a
photoperiod of 12 hours. In each flask, a polyethylene bag filled by a solution of KHCO3 and
K2CO3 ensured the necessary CO2 supply to algae. After 2 and 4 days, algae cell number and
size were measured by a particle counter. Small aliquots of MB solutions were washed twice,
through centrifugation at 4000 rpm for 10 minutes and replacement of surnatant by fresh
culture medium, then adapted to the dark for 30 minutes and analyzed by the Phytoplankton
Analyzer (Walz Effeltrich, Germany) to determine the chlorophyll a content (chl) and various
fluorescence parameters like the maximum quantum yield of PSII in the dark adapted state
(Fv/Fm), the quantum yield of PSII in the light adapted state (ΦPSII), the photochemical (PQ)
and not-photochemical (NPQ) quenching. After 4 days, when algae reached the maximum
growth, the experiment was stopped and the antioxidative capacity of the water soluble
compounds (ACW) was measured by the Photochem (Analytik Jena, Germany), an
instrument which uses the method of photochemiluminescence. For this analysis, algae
samples were concentrated, then the pellet was suspended in a 0.1 M NaH2PO4 buffer, mixed
with glass beads (Ø 0.3 µm) and cleft by a speed mill. The protein content was measured
according to Bradford [11]. The ACW was not determined for algae treated with NOM. A
similar experiment was performed growing algae in FW04-medium nitrogen-free, alone
(control) or added with HF at the concentration of 1,5,10 and 20 ppm DOC. For both
experiments, treatments were replicated 4 times and data were statistically analyzed by oneway analysis of variance (ANOVA) and the SNK test.
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3. Results and Discussion
Neither NOM or HF modified algae cell number. HuminFeed determined a decreasing of cell
size, but only at the higher concentration. In previous works, Steinberg and other Authors
[7,8] assessed that HS usually modulate algae growth, but they also observed that algae
response pattern to HS exposure is species-specific. Therefore, it’s not surprising that MB
reaction to HS differed from what is reported in literature for other algae species. After two
days of exposure to NOM, fluorescence parameters like chl, Fv/Fm and NPQ increased at each
NOM concentration, with respect to the control. However, no significative differences were
revealed between the control and the different NOM treatments on the fourth day. The
interaction MB-HF did not affect any fluorescence parameters. These indexes are indicative
of the overall photosynthetic rate [12]. In particular, Fv/Fm refers to the maximum efficiency
of PSII, i.e. the efficiency if all PSII reaction centers are opened. NPQ express the efficiency
by which energy is dissipated in heat. Initially, MB reacted positively to each concentration of
NOM, showing an higher chlorophyll content and, consequently, a combination of both high
photosynthetic light use and heat dissipation. To the best of our knowledge, the only studies
in the literature to which compare our results refers to the aquatic plant Ceratophyllum
demersum [13]. Unlike MB, PSII electron transport chain of C. demersum was inhibited
significantly by much lower concentration of several NOM. The exposure to HF reduced the
Control
HF 2 ppm DOC
HF 5 ppm DOC
HF 20 ppm DOC
120
ACW (%)
100
80
60
*
**
**
ACW of MB (Fig.1). In particular, this
stress response increased with increasing
HF concentrations. Probably, HF is taken
up by algae and then metabolized with
40
oxidizing oxygen and nitrogen species as
20
byproducts, causing an internal oxidative
0
stress.
* P≤ 0.05; ** P ≤ 0.01; according to SNK test
Figure 1: ACW of algae exposed to different HF concentration
When a nitrogen-free medium was used for all treatments, included the control, algae
concentration rose in the presence of HF, especially at 5, 10 and 20 ppm DOC (Fig.2), while
cell size slowly decreased. This result represents the evidence that HS supply nitrogen and,
probably, also other nutrients to algae, accelerating their growth rate. With regard to
chlorophyll content and fluorescence parameters, all treatments were not significative
different to the control. In general, MB was not deeply stressed neither by NOM nor HF.
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Maybe, because of its origin from a DOC-rich environment (Grosse Fuchskuhle), this algae
species was well adapted to HS.
Cell concentration (%)
300
***
250
200
**
***
***
Control
HF 1 ppm DOC
HF 5 ppm DOC
HF 10 ppm DOC
HF 20 ppm DOC
150
100
50
0
** P≤ 0.01; *** P≤ 0.001; according to SNK test
Figure 2: Cell concentration after 5 days of exposure to differentconcentrations of HF
4. Conclusions
This study confirmed the recent theory that HS, upon their uptake, affect organisms directly.
HF provoked the internal oxidative stress, but did not interfere within the photosynthetic
electron chain. The experiment confirmed also that algae benefit of the exposure to HF if they
are cultivated in a nitrogen-poor medium. Hence, HS play an important role in the structuring
of water biocenosis, suppressing the most sensitive species and favoring the less sensitive.
References
1. E.M. Thurman, Organic Geochemistry of Natural Waters, Dr W. Junk Publishers, Dordrecht,
1985, p. 497.
2. R.G. Wetzel, Limnology. Lake and River Ecosystems, Academic Press, San Diego, 3rd edn., 2001,
p. 1006.
3. C.E.W. Steinberg and U. Münster, Humic Substances in Soil, Sediment and Water. Geochemistry,
Isolation and Characterisation, G.R. AikenD.M., McKnight, R.L. Wershaw and P. MacCarthy
(Eds.), Wiley, New York, 1985, 105–145.
4. C.E.W. Steinberg, A. Paul, S. Pflugmacher, T. Meinelt, R. Clöcking and C. Wiegand, Fresenius
Env. Bull., 12 (2003) 391–401.
5. C.E.W. Steinberg, T. Meinelt, M.A. Timofeyev, M. Bittner and R. Menzel, Env. Sci. Pollut. Res.,
15 (2008) 128–135.
6. E.J. Calabrese, Env. Pollut., 138 (2005) 379–412.
7. T.A. Karasyova, E.O. Klose, R. Menzel and C.E.W. Steinberg, Env. Sci. Pollut. Res., 14 (2)
(2007) 88-93.
8. V.Y. Prokhotskaya and C.E.W. Steinberg, Env. Sci. Pollut. Res., 14 (2007) 11–18.
9. C.E.W. Steinberg, S. Kamara, V. Prokhotskaya, et Al., Freshwater Biol., 51 (2006) 1189–1210.
10. A. Nicklisch, T. Shatwell and J. Köhler, J. Plankt. Res., 30 (2008) 75-91.
11. M.M. Bradford, Anal. Biochem., 72 (1976) 248-254.
12. K. Maxwell and G.N. Johnson, J. Experim. Botany, 51 (2000) 659-668.
13. S. Pflugmacher, C. Pietsch, W. Rieger and C.E.W. Steinberg, Sci. Tot. Env., 357 (2006) 169– 175.
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Effects of Compost Water-Extracts on the Germination and Growth of
Slickspot Peppergrass (Lepidium papilliferum)
Elisabetta Loffredoa*, Antonio J. Palazzob, Andreina Traversaa, Terry L. Bashorec,
Nicola Senesia
a
Dipartimento di Biologia e Chimica Agro-forestale e Ambientale, University of Bari,
Via G. Amendola 165/A 70126 Bari, Italy; bERDC-CRREL, Hanover, NH 03755-1290, USA;
c
HQ ACC/A3A, Airspace, Ranges, Airfield Operations Division, Langley AFB, VA 23665-2789,
USA
E-mail: loffredo@agr.uniba.it
1. Introduction
Compost addition to soil is a common practice, and amendment of soils low in organic
content can be used to improve soil fertility. Slickspot peppergrass (Lepidium papilliferum) is
a rare plant species of high conservation concern in southwestern Idaho, and grows in
depressed soils called slickspots. Slickspot soils are low in organic content and additions of
composts may be used to help establish new stands of this rare species. Compost waterextracts (C-WE) contain the portion of organic material that can pass through a 0.45 μm filter
membrane and is constituted of an heterogeneous mixture of molecules with different
molecular size and complexity, ranging from simple sugars and organic acids to relatively
high molecular weight humic colloids. The biological activity of C-WE depends mainly on
the type of substrate used for composting and the type and duration of the process. In some
cases, however, phytotoxic effects may originate from compost application depending on the
plant species type and age and environmental conditions. The aim of this work, which is part
of a more extensive research on the restoration of rare plant species, was to evaluate the
effects of C-WE from different composts on the germination and growth of slickspot
peppergrass, and possibly relate its biological activity to chemical and physico-chemical
characteristics of C-WE.
2. Materials and Methods
The three composts used in this work were a green compost (GC), a mixed compost (MC) and
a coffee compost (CC). The C-WE were obtained from each compost by extracting the matrix
with distilled water (1/10, w/v), centrifuging at 6000 rpm and filtering sequentially through
Whatman filters with particle size retention decreasing from 11 to 0.45 μm [1]. The C-WE
samples were characterized by means of chemical and physico-chemical methods, such as pH,
electrical conductivity (EC), total organic carbon (TOC) and E4/E6 ratio. The C-WE were
diluted with Nitch nutrient solution in the ratios of 1:2 (C-WE1:2), 1:5 (C-WE1:5) and 1:10 (C-
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WE1:10) (v/v) and tested on the germination and growth of slickspot peppergrass. In
germination experiments, the nutrient solution alone (control) and each diluted C-WE sample
were separately added in agar solution (1.5 % agar) to 60 seeds of slickspot peppergrass in
Petri dishes. Seed germination was achieved in a Phytotron growth chamber at 18 ± 1 °C
during the illumination period and at 10 ± 1°C in the dark with 8-h photoperiod. The
germination percentage was measured after 7 and 15 days, and primary shoot and root lengths
were measured on germinated seeds collected after 7 days. Subsequently, growth experiments
were performed on the 7-day germinated seeds, which were transplanted on new plates
containing the same C-WE samples and let to grow with a 12-h photoperiod and a
temperature of 23 ± 1 °C. After 15 and 30 days, live seedlings were counted and root and
shoot lengths were measured. Then, the 30-day grown seedlings were transplanted in plastic
pots containing peat, and irrigated periodically either with the nutrient solution alone or with
each diluted C-WE sample. All experiments were replicated three times and all data obtained
were analyzed statistically by one-way analysis of variance (ANOVA) and the least
significant difference test (LSD).
3. Results and Discussion
Some properties of the three C-WE samples examined are shown in Table 1. The pH and EC
values were similar for GC-WE and MC-WE whereas they resulted much higher or lower,
respectively, for CC-WE. The TOC content, the molar absorptivity at 280 nm (ε280) and the
E4/E6 ratio increased in the order: GC-WE < MC-WE < CC-WE. The relatively high values
measured for ε280 and E4/E6 ratio and other spectroscopic evidence would suggest, especially
for CC-WE, the occurrence of relatively low molecular weight aromatic molecules, such as
phenolic-like units, aniline-derived compounds, benzoic acid derivatives, polyenes, and polycyclic aromatic hydrocarbons, which are generally present in C-WE samples. A more detailed
presentation and discussion of the properties of the three C-WE can be found in Traversa et al.
[2].
Table 1: Some characteristics of the C-WE samples used
Sample
pH
EC
(dS/m)
TOC
ε280
-1
(mg L ) (L cm-1 mol-1)
GC-WE
7.4
3.14
140
26.8
3.6
MC-WE
7.0
3.28
186
32.5
4.3
CC-WE
8.3
1.66
375
38.9
6.9
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E4/E6 ratio
15th IHSS Meeting- Vol. 3
Results of germination experiments indicated that after 7 days, GC-WE and MC-WE at the
higher dilution and CC-WE at any dilution did not alter the germination percentage with
respect to the control, whereas a reduction of germination was observed in all the other cases.
After 15 days, a significant reduction of the germination percentage was measured only for
GC-WE and MC-WE at the lower dilution. Probably, the relatively high EC of the latter two
samples was responsible for the decrease of the germination percentage when a scarce
dilution was adopted. The effects of the three C-WE treatments at three dilutions on the shoot
and root length of seedlings after 7 and 30 days are shown in Figs. 1-2. No significant effect
was observed on primary shoot length for any C-WE treatment at the three dilutions, whereas
only in the case of GC-WE and MC-WE at the lower dilution a significant reduction of
primary root length was measured with respect to the control (Fig. 1). Also in this case, it can
be hypothesized that the higher EC of the two samples inhibited root elongation. After 15(data not shown) and 30-days growth, no differences of shoot and root lengths were observed
between the C-WE treatments and the control (Fig. 2). After 7-month growth, the plants
treated with the different C-WE showed better growth and health conditions with respect to
the control. At the lower dilution, the maximum benefit for plant growth was caused by GCWE treatment with respect to the other two samples (Fig. 3). The GC-WE produced the
highest plant stimulation at the lower dilution, whereas MC-WE and CC-WE showed the best
effects at the highest dilution (Fig. 3). Results obtained suggest that the molecular
composition of the C-WE plays an important role in their biological activity on plants.
4. Conclusions
The addition of C-WE to the germination and growth medium of slickspot peppergrass
generally did not alter the rate of germination and early growth when a low or moderate dose
was used. In particular, among the three different composts used, the coffee compost
presented a water soluble organic fraction completely tolerable by the seedlings also at a very
high dose. After a prolonged period of growth, favorable effects of any C-WE were observed
on slickspot peppergrass, and particularly by the WE from the green compost. This
information will be used, along with other environmental and genetic factors that affect seed
germination and early root and leaf growth, to better understand how restore this rare plant
species.
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140
SHOOT
ROOT
100
**
80
*
Length (%)
120
60
40
20
0
GC-WE MC-WE CC-WE
C
C-WE1:10
GC-WE MC-WE CC-WE
C
C-WE1:5
C-WE1:2
160
140
Length (%)
Figure 1: Effects of C-WE at
different dilutions on primary shoot
and root lengths of germinated
seeds after 7 days, expressed as
percentages of the control (100 %).
The vertical line on each bar
indicates the standard error for 3
replicates
ROOT
SHOOT
Figure 2: Effects of C-WE at
different dilutions on shoot and root
lengths of seedlings after 30 days,
expressed as percentages of the
control (100 %). The vertical line
on each bar indicates the standard
error for 3 replicates
120
100
80
60
40
20
0
C
GC-WE MC-WE CC-WE
C-WE1:10
C
GC-WE MC-WE CC-WE
C-WE1:5
C-WE1:2
Figure 3: Effects of MC-WE1:2,
GC-WE1:2 and CC-WE1:2 (from left
to right) on the growth of slickspot
peppergrass after about 7 months
Acknowledgements
This work was supported by the Research Contract n. W911NF-08-1-0076 of the US Army
RDECOM ACQ CTR – W911NF, Durham NC, USA. Project title: “Effects of Quality
Composts and Other Organic Amendments and Their Humic and Fulvic Acid fractions on the
Germination and Early Growth of Slickspot Peppergrass (Lepidium papilliferum) and
Switchgrass in Various Experimental Conditions”, funded by the Airspace, Ranges, and
Airfield Operations Division, HQ Air Combat Command, Langley AFB, VA.
References
1. A. Traversa, V. D’Orazio and N. Senesi, Forest Ecol. Manag., 256 (2008) 2018.
2. A. Traversa, E. Loffredo, C.E. Gattullo and N. Senesi, Geoderma (under revision) (2010).
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The Action of Humic Acids Promoting Plant Shoot Development are
Associated with Nitrate-Related Changes on the Plant Hormonal Balance
V. Mora, E. Bacaicoa, E. Aguirre, R. Baigorri, M. Garnica, M. Fuentes, A.M. Zamarreño, J.C.
Yvin, J.M. Garcia-Mina*
CIPAV- Roullier Group and Department of Chemistry and Soil Chemistry, Faculty of
Sciences, University of Navarra
E-mail: jgmina@timacagro.es
1. Introduction
A number of studies have shown the ability of humic substances (HS) to enhance the growth
of different plant species cultivated both in soils or inert substrate (1). However, the
mechanism or mechanisms responsible for these effects of HS are not well understood. Some
authors propose that this action of HS is principally indirect, by improving soil texture and the
availability of certain nutrients such as Fe and Zn (1). Other authors suggest that, besides
these HS effects on soil properties, there exist direct effects of HS on plant metabolism (2).
The question is whether these metabolic effects of HS are the consequence of previous effects
on nutrient uptake and translocation, or they are independent of them.
Several studies have demonstrated that the growth promoting effects of certain nutrients, such
as nitrate, are linked to effects on the distribution of specific plant hormones within the plant
(3). On the other hand, several studies describe the ability of HS to increase H+-ATPase
activity and nitrate root uptake (4). It is, thus, possible that the action of HS on plant growth is
related to nitrate root uptake and further root to shoot translocation and the effects of this
nitrate-related action on the hormonal balance. In turn, this HA effect might be related to a
previous action on root functionality (ATPase activity and root architecture).
In this presentation we analyze this working hypothesis. To this end we have investigated in
cucumber plants the time-course effect of the root application of a purified leonardite humic
acid (HA) on: (i) the growth of the root and the shoot; (ii) root H+-ATPase activity and nitrate
concentrations in roots and shoot; (ii) the concentrations of ethylene and IAA in the root; (iii)
the concentrations of polyamines (PAs), cytokinins (CKs) and ABA in root and shoot; (iv) the
root-shoot distribution of the main mineral nutrients.
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2. Material and Methods
The experimental design and the methodology used for measuring the main plant hormones
are described in (5). The analytical methods for analysing ethylene and polyamines are
described in (6).
The humic acid used in the experiments was extracted from a leonardite obtained from
Czechia. The extraction and characterization methods are described in (5). The analysis of the
main plant phytoregulators in HA revealed that their presence was negligible.
3. Results and Discussion
The application of HA caused a significant a significant increase in root growth and root
growth rates that was associated with significant increases in the root concentration of IAA
(after 24 and 72 h from the onset of treatments) and the root production of ethylene (after 24
and 72 h from the onset of treatments). These results were obtained in three independent
experiments.
On the other hand, HA root application caused a significant increase in the root activity of the
H+-ATPase that was also associated with changes in the root-shoot distribution of nitrate
concentration, which decreased in roots and increased in the shoot.
These changes in nitrate root-shoot distribution were associated with concomitants changes in
the root-shoot distribution of the main cytokinins (principally, isopentenyladenine and tZeatine Riboside) and polyamines (principally, putrescine), which increased in the shoot and
decrease in the roots. These effects were well correlated to significant increases in shoot
growth and shoot relative rates. A transient increase in ABA shoot concentration was also
observed.
Finally, it was noteworthy that all these effects, principally those concerning CK plant
distribution were also associated with changes in the root to shoot distribution of the
concentration of the main mineral nutrients, which experience a descrease in the root and an
increase in the shoot.
In consequence all these results, taken together, indicate that in the shoot promoting effect of
HA are directly involved significant effects on the plant distribution of CKs and PAs, which
in turn seem to be linked to a previous action on nitrate plant distribution. This effect of HA
on nitrate plant distribution is associated with significant increases in the root H+-ATPase
activity.
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15th IHSS Meeting- Vol. 3
Regarding the mechanism by which HA acts on H+-ATPase activity, it could be associated
with the above-mentioned effects on IAA, NO and / or ethylene root concentrations.
All these effects are presented in Figure 1.
Figura 1: HA action mechanism on cucumber development: working model
References
1. Y. Chen, M. De Nobili, and T. Aviad. Stimulatory effects of humic substances on plant growth.
Soil Organic Matter in Sustainable Agriculture. Boca Raton, Florida: CRC Press; (2004). p. 10329.
2. S. Nardi, D. Pizzeghello, A Muscolo, and A. Vianello. Soil Biol Biochem;34 (2002) 1527.
3. V. Rubio, R. Bustos, ML. Irigoyen, X. Cardona-Lopez, M. Rojas-Triana, and J. Paz-Ares. Plant
Mol Biol 69(2009) 361.
4. R. Pinton, S. Cesco, G. Iacolettig, S. Astolfi, and Z. Varanini. Plant Soil 215 (1999) 155.
5. E. Aguirre, D. Leménager, E. Bacaicoa, M. Fuentes, R. Baigorri, AM. Zamarreño , and JM
García-Mina. Plant Physiol Biochem 47 (2009) 215.
6. M. Garnica, F. Houdusse, JC. Yvin , and JM Garcia-Mina. J Plant Physiol 166 (2009) 363.
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Health and Medical Applications of Natural Organic Matter
and Humic Substances
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Inclusion Complexes of Aspirin in Fulvic Acid Offer Enhanced Dissolution,
Permeability, Stability and Better Pharmacodynamics
Khalid Anwera, b*, Aamir Mirzaa, Suraj P. Agarwala, Asgar Alia, Yasmin –Sultanaa
a
Dept. of Pharmaceutics, Jamia Hamdard (University), Hamdard Nagar, New Delhi-110062,
India; bAl-kharj College of Pharmacy, King Saud University, Saudi Arabia
E-mail: mkanwer2002@yahoo.co.in
1. Introduction
Shilajit, a wonder medicine of ayurveda, neither a plant nor animal origin, it is a mineral pitch
that comes out from the rocks of the Himalayas, as they become warm during summer months
[1–2]. Shilajit contains a variety of organic compounds that can be broadly classified into
humic and non-humic substances [2]. Humic substances are further classified into humic and
fulvic acid. Humic acid (HA) and fulvic acid (FAs) have relatively open, flexible structure
punctured by voids (micropores) of different diameters (200-1000Å) as reported in literature
[3-4]. The interior of these humic and FA hydrophobic and thus are capable of forming
inclusion complexes with non-polar solutes and unstable drug molecules [4].
Aspirin (acetylsalicylic acid) is very old drug but still havimg a very high market value. It
possesses antipyretic, anti-inflammatory, analgesic and anti-aggregatory activity due to
decreased production of prostaglandins and thromboxanes. The acetylsalicylic acid molecule
has a carboxyl group and an ester group. The ester group can be easily hydrolyzed, which
reduces the medicinal value and causes side effects on humans. A strategy designed how to
inhibit the hydrolytic decomposition and enhancement of dissolution of aspirin inside the void
of HA of shilajit. We propose to investigate the effects of FA as carrier on aspirin
(acetylsalicylic acid) in enhancing the dissolution rate and bioavailability, increasing the
stability and decreasing the toxicity of aspirin through complexation.
2. Experimental
Extraction of humic acid form Shilajit. Humic acid is extracted from shilajit by increasing the
polarity of solvent as reported in literature (6). The method consisted of successive extraction
of raw shilajit with hot organic solvents of increasing polarity to remove the bioactive
components. The residue (marc) was dissolved in 0.1 M NaOH with intermittent shaking in
the presence of nitrogen. The suspension was filtered and the filtrate was acidified to a pH of
less than 3 to precipitate the HAs. The resulting HA is dried, pulverized in glass mortar pestle
and stored in dessicator.
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Preparation of inclusion complexes. Complexes of Aspirin-FA in the molar ratio of 1:0.5, 1:1
and 1:2 were prepared by using solvent evaporation (rota evaporator), freeze drying
(lyophilizer) and spray drying (mini spray dryer) methods.
Characterization of Complexes. The complexes were characterized by using differential
scanning calorimetry (DSC), X-Ray diffraction (XRD), Fourier transform infra red
spectroscopy (FT-IR), scanning electron microscopy (SEM) and proton nuclear magnetc
resonance (1HNMR) methods.
Release study of aspirin from their complexes. The sample corresponding to 100 mg of aspirin
were placed in hard gelatin capsules. Dissolution medium was acetate buffer (pH 4.5). The
stirring speed was 50 rpm and temperature 37 ± 0.5 ºC. 5 mL samples were withdrawn at a
settled time interval using a syringe and analyzed by HPLC method.
Stability studies. All the complexes and ASA alone were packaged in well labeled sealed
polythene lined aluminium pouches and stored in stability chamber at 40 ± °C and 75 ± 5%
RH for 120 days. Samples were analyzed by HPLC for salicylic acid content at 0, 30, 60, 90
and 120 days.
Drug Permeation Study Across Rat Everted Gut Sac. In order to study the effect of
complexation on the intestinal permeability of aspirin and the permeability of aspirin-FA
spray dried complex (1:1) was compared with aspirin alone by the rat everted gut sac
technique.
Anti-Inflammatory and Gastric Ulceration studies. Anti-inflammatory activity was performed
using carrageenan induced rat hind paw edema model. However, pyloric ligation ulcer model
was used for gastric ulceration study.
3. Results and Discussion
Inclusion complex formation resulted in the production of an amorphous powder with
improved solubility, dissolution, permeability and stability of aspirin. Aspirin-FA system 1:1
spray dried complex was optimized according to spectral characterization, in vitro release and
stability studies. It is evident from the results that the complexation showed a significant
increase in the solubility of the drug, with the maximum increase in solubilization is observed
in the case of spray dried (1:1) ASA-FA complex (43 times) as compared to aspirin alone in
0.1 M HCl. The dissolution data indicated only 31.32% release was obtained with aspirin
alone at 30 minutes and a maximum of 99.7% release was obtained from 1:1 spray dried
fulvic acid complex in 25 minutes. The overall profile of ASA degradation in the complexes
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of fulvic acid were studied at 40 ± 2 °C and 75 ± 5% RH for 120 days as indicated by the rate
of appearance of salicylic acid. However, content of salicylic acid 4.31% was determined
after 120 days. The permeation of aspirin from aspirin -fulvic acid complex (1:1) prepared by
spray drying was found to be significantly higher (about 8 times) as compared to aspirin
alone. The anti-inflammatory effect of aspirin caused an inhibition of 22.92 % after four hours
while their optimized complex of fulvic acid inhibited edema 35.4% after four hours of
treatment. The spray dried complex prepared with fulvic acid (1:1) gave the lowest score of
ulcer index; 0.48 ± 0.08 as compared to aspirin alone 1.12 ± 0.08. Tablets prepared with
aspirin-FA (1:1 spray dried complex) have greater dissolution as compared to marketed
formulation containing aspirin in an uncomplexed form. The optimized tablets of aspirin-FA
complex wase subjected to accelerated stability studies to ascertain the chemical and physical
stability of formulations. The optimized tablets were kept at 400 ± 0.50 C and 75% ± 5% RH.
No significant changes in properties like hardness and disintegration time of formulation was
observed.
Figure 1: Comparative stomach lesions after treatment
4. Conclusions
Desirable enhancement in dissolution and stability of aspirin can be achieved through FA
complexation. However, such a novel approach appears to be beneficial to overcome the
problem of poor bioavailability. A highly significant anti-inflammatory and anti-ulcerogenic
action were evidenced by the treatment of optimized complex. This has potential for industrial
application in developing and improved dosage form of aspirin
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References
1. S. Ghosal, Shilalit: its origin and significance. Indian. J. Indg. Med 9 (1992) 1.
2. S.P. Agarwal, R. Khanna, R. Karmarkar, M.K. Anwer, and R. Khar, Shilajit: A Review.
Phytother. Res. 21 (2007) 401.
3. M.K. Anwer, S.P. Agarwal, A. Ali and Y. Sultana, Influence of fulvic acid and hydroxy propyl-βcyclodextrin on aspirin degradation”. Drug dev Indus Pharm. 2009, in press.
4. R. Khanna, M. Witt, M.K. Anwer, S.P. Agarwal, and B.P. Koch, Spectroscopic characterization of
fulvic acids extracted from the rock exudate Shilajit. Org. Geochem. 39 (2008) 1719.
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The Effect of Fulvic and Humic Acid Supplementation on the Intensity of
the Immune Response in Rats
Vucskits A. V.a*, Hullár I.a, Andrásofszky E.a, Hetényi N.a, Csicsor J.b, Móré A.c, Szabó J.a
a
Department of Animal Breeding, Nutrition and Laboratory Animal Science, Faculty of
Veterinary Science, Szent István University, H-1077 Budapest, István u. 2, Hungary; bOrganit
Ltd., H-8175 Balatonfűzfő, Ipari Park hrsz.: 1498/278; cAlpha-Vet Ltd., H-8000
Székesfehérvár, Homoksor 7
E-mail: vucskits.andras@aotk.szie.hu
1. Introduction
The immune system is one of the most important mechanisms for preserving the health of the
livestock animals. There are a lot of chemical substances that can enhance the immune
response, such as n-3 and n-6 polyunsaturated fatty acids (PUFA) [7], arginine [2], vitamins
[9], microelements [1, 4], etc. Humic substances [HSs] are new candidates in the field of
immune-based nutrition. It has been known that HSs have immune stimulating properties [6].
According to the latest findings HSs might help to inhibit the infectivity of certain diseases
(i.e. HIV) [3]. It is important to state that HSs are not chemically homogenous. Their two
most important ingredients are humic acid (HA) and fulvic acid (FA). Humic acid is soluble
in water under alkaline conditions and has a higher molecular weight than FA. It is
theoretically not absorbed from the intestines. Fulvic acid is soluble in water under all pH
conditions and it is absorbed well from the intestines [5]. The aim of this experiment was to
investigate the effect of purified FA and HA - originated from Dudarite - on the humoral
immune response of rats.
2. Materials and Methods
Thirthy Wistar CRL:[WI] BR, female, SPF rats were used in the experiment. After 4 days of
adaptation animals were randomly divided into 3 dietary treatment groups (10 animals in each
group) on the basis of their bodyweight. The diets were composed according to the AIN-93G
formula of the American Institute of Nutrition [8]. One group received the control diet, 1–1
treatment groups received FA 0.4% or HA 0.4% supplemented diets respectively. Animals
were housed in individual cages at 24 °C ambient temperature. Drinking water and diet was
provided ad libitum during the experiment. On the second day of the experiment animals were
immunized with ovalbumin incorporated into Freund's incomplete adjuvant (animals were
injected with 150 µg ovalbumin, 150 µL incomplete freund’s adjuvant 150 µL PBS
containing suspension sc.) On the 26th day of the experiment animals were euthanized (90
mg/BW CP Ketamin and 0.5 mg/BW Medetomidin) and insanguinated. Enzyme-linked
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immunosorbent assay (ELISA) was used to analyze the antibody titer of the serum samples.
Histometrical analysis was carried out on the mucus membrane of the small intestine and the
lymphoid cell zones if the spleen of 9 animals (3 animals from each group).
3. Results
Geometrical mean 2. week
Geometrical mean 4. week
Control
FA
709.68
1203.27
544.32
1969.83
Antibody titer against ovalbumin
HA
1269.92
1600.00
Histometrical analysis shows that the size of the lymphoid follicles (average size of the
centrum germinativum, consisting of lymphoblast cells) in the mucus membrane of the ileum
was much bigger (800–900 μm) in the FA and HA supplemented groups than in the control
group (300–400 μm). The average thickness of the marginal (“B”-dependent) cell-zone was
151–200 μm in the FA and HA supplemented group. This value was 100–150 µm in the
samples of the control group.
4. Discussion
The immunological and histometrical results show that the 0.4% supplementation of either
fulvic or HA has a strong immune-stimulatory effect. Our results also indicate that this effect
is mainly focused on the humoral immune response. The antibody titer analysis shows that
both FA and HA increase the longevity of the immune response, since the antibody titer
results of the 4th week are higher in the fulvic and HA supplemented groups than at 2 weeks.
Evaluating the results of the histological and the ELISA examinations together, it can be said
that both FA and HA are strong humoral immune stimulants and they also increase the
persistence of antibodies in the system.
Acknowledgements: The financial support of the Hungarian Scientific Research Fund
(OTKA, T 049116) is greatfully acknowledged.
References
1. A. Favier, Annales Pharmaceutiques Françaises (2006) 64, 390–6.
2. B. Lewis, B. Langkamp-Henken, J. Nutrition (2000) 130, 1827–1830.
3. C. E. van Rensburg, T. L. Smith, E. J. van Rensburg, J. Schneider, Chemotherapy (2002) 48, 138–
143.
4. K. E. Saker, Vet. Clin. N. A.: Small Animal Practice (2006) 36, 1199–224.
5. K. M. S. Islam, A. Schumacher, J. M. Gropp, Pak. J. Nutr. (2005) 4, 126–134.
6. N. Lange, S. Golbs, M. Kühnert, Archiv für experimentelle Veterinärmedizin (1987) 41, 140–146.
7. P. C. Calder, Lipids (2001) 36, 1007-24
8. P. G. Reeves, J Nutrit, (1997) 127, 838S-841S.
9. V. Badmaev, M Majeed, R. A. Passwater, R. A., Alternative Therapies in Health and Medicine
(1996) 2, 59-62, 65–7
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Evaluating Potential Nephrotoxicity of Compost Derived Humic Acid to
African Mud Catfish (Clarias Gariepinus) Grown in Static Water Culture
Iheoma Mary Adekunle* and Olawale Razaq Ajuwon
Department of Environmental Management and Toxicology,
University of Agriculture, P.M.B. 2240, Abeokuta, Nigeria
E-mail: imkunle@yahoo.com
1. Introduction
Composting of organic solid waste materials is seen as an economically feasible waste
management option in Nigeria and the resulting products (solid and liquid organic fertilizers)
are utilized in organic farming and horticultural purposes for urban greening in order to boost
food security, combat global warming and consequent climate change. These organic
fertilizers inherently contain humic acid (HA), a type of humic substance, which exists in
decomposed natural organic matter, compost, peat, lignite, soil organic matter, sediment and
all classes of water (groundwater and surface water). HA consists of closely related complex
aromatic polymers and oxygenated functional groups but the exact composition varies with
geographic location amongst other factors. On application of the organic fertilizers to the soil,
fate processes such as leaching
could transfer some
humic substance fractions to the
surrounding water bodies thereby adding to the existing dissolved humic matter in the
receiving waters and HA is
reported to influence a variety of processes in aquatic
ecosystem[1]. There are no readily available data on the toxicological effect(s) of HA
isolated from composted organic wastes of Nigeria origin to aquatic organisms. This study,
therefore, evaluated the potential nephrotoxicity of HA isolated from source segregated
composted municipal solid wastes generated in Abeokuta, southwest Nigeria, to African mud
catfish (Clarias gariepinus) grown in static water culture.
2. Materials and Methods
Source separated municipal solid wastes generated from Abeokuta city, Nigeria, consisting
largely of vegetable matter, crop and food residues were composted for 75 days via in-vessel
technique after the procedure of [2]. Humic acid was extracted from the cured product using
alkaline method, purified and characterized using the method of [3] with slight modification.
Three prognostic markers of kidney disease: plasma creatinine, urea and albumin levels of C.
garipinus exposed to different concentrations of HA (0, 100, 250, 500 and 1000 mg/L) in
controlled water culture for 45 days, were determined using standard clinical techniques.
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Creatinine and urea were determined using Cromatest kits while albumin was determined
using Randox test kit.
Twenty five juvenile fishes of average weight and length 43.2 ± 0.5g and 18.6 ± 0.8cm
respectively, fed with 2 mm copen fish feed, were utilized for the experiment at 5 per
aquarium of 45 L capacity. Temperature, electrical conductivity, nitrate, phosphate, dissolved
oxygen, total hardness and alkalinity of the process water were also analyzed using standard
procedures. Data obtained from study were subjected to one-way analysis of variance and
Pearson correlation using SPSS 15.0.
3. Results and Discussion
Humic acid characteristics: Infrared spectra and results from volumetric analyses showed
oxygenated functional groups and hydrocarbon skeleton, indicating content of phenolic
hydroxyl and carboxyl reactive sites, similar to the humic acid properties reported in our
previous published works [3,4,5]. The process water was suitable for aquaculture (Table 1)
and conforms to the reports of [6].
Serum album: Albumin level in the control group was 0.87 ± 0.19 g/dL and ranged from 0.43
to 0.87g/dL in the test groups. Correlations gave negative coefficients for albumin versus HA
concentrations (- 0.114; p > 0.10), indicating albumin depletion (Fig.1) with increasing HA
concentration in water. Serum albumin is the most abundant plasma protein in humans and
other mammals, being essential for maintaining the osmotic pressure needed for proper
distribution of body fluids between intravascular compartments and body tissues. It also acts
as a plasma carrier by non-specifically binding several hydrophobic steroid hormones and as a
transport protein for hemin and fatty acids. Albumin (when ionized in water at pH 7.4, as
found in the body) is negatively charged. The glomerular basement membrane is also
negatively charged in the body and some studies suggest that this prevents the filtration of
albumin in the urine. According to this theory, charge plays a major role in the selective
exclusion of albumin from the glomerular filtrate [7,8]. A defect in this property results in
nephrotic syndrome leading to albumin loss in the urine. Decreased serum albumin as found
in this study, may thus be a sign of kidney disease.
Creatinine: The creatinine level in the control group was 0.36± 0.09 mg/dL and ranged from
0.20 to 1.53 mg/dL in the test groups. Positive correlation was obtained for creatinine and
HA concentrations (+ 0.704; p > 0.10), indicating rising blood levels of creatinine (Fig.1) with
increasing HA concentration in water. Creatinine is produced naturally by the body, being a
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break-down product of creatine phosphate, which is found in muscle. It is freely filtered by
the glomerulus. There is little-to-no tubular reabsorption of creatinine. If the filtering of the
kidney is deficient, blood levels rise [7, 8]. Results from this study are thus predictive of renal
dysfunction.
Figure 1: Albumin, creatinine and urea concentrations in C. gariepinus in relation to humic acid
concentrations in water
Urea: The urea level in the control group was 5.61 ± 0.07 mg/dL and ranged from 1.95 to
5.21 mg/dL in the test groups. Correlations gave negative coefficients for urea versus HA
concentrations (- 0.586; p > 0.10), indicating a decreasing trend (Fig.1) with increasing HA
concentration in water. Urea is a break down product of amino acid catabolism. Similar to the
case of creatinine, if kidneys are not able to remove urea from the blood, the level rises,
predicting nephrotic dysfunction [7, 8] as was found in this study.
Inter-biomarker ratios: The ratios are presented in Table 1.
Table 1: Quality of process water used in the aquaria and ratios of the nephrotoxic biomarkers
Water quality
Temperature
27 oC
pH
6.4
Biomarker ratios
HA (mg/L)
3-
Ur-Cr
Cr-Ur
Alb-Cr
Cr-Alb
Alb-Ur
Ur-Alb
20 mg/L
0
15.58
0.064
2.42
0.414
0.155
6.45
NO3-
ND
100
26.05
0.038
4.20
0.238
0.161
6.20
Total
154 g/L
250
7.00
0.143
1.00
1.00
0.143
7.00
80 mg/L
500
1.27
0.785
0.38
2.040
0.385
2.60
80 mg/L
1000
2.39
0.419
0.55
1.813
0.231
4.33
PO4
hardness
Total
alkalinity
Dissolved
oxygen
Ur – urea, Cr- creatinine, Alb- albumin, ND – not detected
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A more complete estimation of renal function can be made when interpreting the blood
(plasma) concentration, using the ratios. This can indicate other problems besides those
intrinsic to the kidney. As an illustration, a urea level raised out of proportion to that of
creatinine may indicate a pre-renal problem such as volume depletion [7, 8].
4. Conclusions
The present results show that exposure of C. gariepinus to up to 100 mgHA/L did not evoke
significant adverse effects on the three biomarker levels in the blood but beyond this
concentration, from 250 to 1000 mg/L, significant nephrotic dysfunction was predicted from
albumin and creatinine levels but based on urea levels, nephrotoxicity was not induced. Study
showed that nephrotic syndrome was HA concentration dependent, indicating safety at levels
≤ 100 mg/L, which lie in an environmentally realistic range.
References
1. F.J. Stevenson, Humus Chemistry, Wiley, New York: 1994.
2. I.M Adekunle, Temperature effect on water extractability of cadmium, copper, lead and zinc from
composted organic solid wastes of South-West Nigeria, Int. J. Environ. Res. Public Health 2009,
6, 2397-2407; doi:10.3390/ijerph6092397
3. I.M. Adekunle ., T.A. Arowolo., N.P. Ndahi, B. Bello and D.A. Owolabi, Chemical characteristics
of humic acids in relation to lead, copper and cadmium levels in contaminated soils of southwest
Nigeria, Annals of Environmental Science, North Eastern University, Boston, Massachusetts USA,
2007, 1, 23-34.
4. I.M. Adekunle., S.O. Olagundudu and O.O. Ogunleye, Influence of humic acid on plant metal
uptake and translocation. Frimmel, F.H., Abbt-Braun, G. (Eds). Proceedings of the 13th
International Conference of the International Humic Substance Society (IHSS), held at University
of Karlsruhe, Germany, July 30 to August 4, 2006, 45 (I), 433-436.
5. I.M. Adekunle., O.A. Akinola and M.A. Kuyoro, Role of humic acid on metal accumulation in
plant tissue. Frimmel, F.H., Abbt-Braun, G. (Eds). Proceedings of the 13th International
Conference of the International Humic Substance Society (IHSS), held at University of Karlsruhe,
Germany , July 30 to August 4, 2006, 45 (II), 817- 820.
6. I.M. Adekunle., T.A. Arowolo., I.T. Omoniyi and Olubambi. O.T, Risk assessment of nile tilapia
(Oreochromis niloticus) and African mud catfish (Clarias gariepinus) exposed to cassava effluent
(2007). Chemistry and Ecology 23 (5), 383 -392.
7. National Kidney Foundation. K/DOQ1 clinical practice guidelines for chronic kidney disease:
evaluation, classification and stratification. Am J Kidney Dis 2002, 39 (Suppl 1):S1-266.
8. Spencer K. Analytical reviews in clinical biochemistry: the estimation of creatinine. Ann Clin
Biochem 1986, 23,1-25.
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Treatment of Pilonidal Sinus by Salts of Humic Acid
Mümin Dizman
Oluşum Kimya, Kandıra, 41633, Turkey
E-mail: mumindizman@hotmail.com
1. Introduction
This study relates to application of curing material containing humic acid and its salts for pilonidal
sinus disease. In this study humic acid and sodium humate or potassium humate provide to cure
pilonidal sinus disease by applying topically to skin without any need of surgical application. Humic
acid containing polyphenol is produced from Afşin-Elbistan lignite gyttja [1]. This treatment method
guarantees and provides to cure patient with the logic of “healing of intermittent wound” without
affecting the daily life standard and recurrence probability. There is no study related with pilonidal
sinus disease which can be rubbed with agent or avoided by a material from the skin. There was no
other alternative solution suggested except from phenol and silver nitrate (AgNO3) because the only
medical treatment is known as surgical intervention up until now.
2. Materials and Methods
Humic acids, sodium humate or potassium humate are used as a cosmetic product to solve out
pilonidal sinus problem. Humic acids and salts that include polyphenolic components are
prepared from the extraction of Afşin-Elbistan gyttja with sodium hydroxide or potassium
hydroxide at 70-150 degrees centigrade, pH between 10 to 12 interval and in 12-24 hours with
continuously stirring. Humic acid originated polyphenols are effective as the products that is
found as anti-viral, anti-microbial active agent for preventing and reducing microbes and
viruses inside the human skin [2].
3. Results and Discussion
Polyphenols that are originated from humic acid treat pilonidal sinus as healing of intermittent
wound principle by supporting phagocytosis [8] and building up collagen synthesis [3]. If the
deficient factors in condition deforming wound healing are submitted to wound, healing will
increase [4]. Because of very high collagen synthesis effect of humic acid polyphenols,
treatment of damaged skin is quickened [7]. With the impact of the humates containing
polyphenol hydroxyls, building up three units of α-bond is strengthened before the formation
of collagen [5]. At this point, three collagen α-bond encounters alteration period. This period
includes hydroxylization of lysine amino acids by polyphenols and formation of fibrin
(glicosylation). Polyphenols quickens the linking of α-bonds as knits [6]. This knit is trio
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spiral curve called procollajen monomer. Propeptids of N- and C-terminal are separated by
the effects of polyphenols to special peptidase enzymes. As a result, processed collagen starts
to make multiple molecular clusters. These clusters are aligned end to end so as to form
striped fibers. Cross-links between the special amino acids determines and provides stretching
resistance of collagen fibers. Skin enriched by the collagen provides to eliminate damaged
cell by its flexible tough character [7].
4. Conclusions
One hundred-and ninty two patients with chronic and acute pilonidal sinus were treated by a
new chemical technique [9]. It was found to be simple, time-saving and to minimize the
postoperative morbidity and without need to stay in hospital. Results of this new technique
were compared with those of other methods in the literature and were found to be superior to
them with a minumum recurrence.
Acknowledgements
I thank to Prof. Dr. Ahmet Tutar, Department of Chemistry, University of Sakarya, for his
assistance in the preparation of this article.
References.
1. Mümin Dizman, Turkish Patent 2007 00973.
2. Ricard J. Laub, Laub Biochem. Corp., US Patent 6,534,049.
3. Gert J. van Klinken and Robert E. M. Hedges, Experiments on Collagen-Humic Interactions:
Speed of Humic Uptake, and Effects of Diverse Chemical Treatments, Radiocarbon Accelerator
Unit, University of Oxford, September 19 ,1994, Abstract.
4. Dr. Sevda CİĞER, Alsancak Devlet Hastanesi Dermatoloji Kliniği Uzmanı, Yara İyileşmesi ve
Büyüme Faktörleri, www.dermaneturk.com/yara_online/buyume_faktor.doc, p.6.
5. Scutt, S. Meghji, J. P. Canniff and W. Harvey, Cellular and Molecular Life Sciences, Volume 43,
Number 4/April, 1987, p.391.
6. Tamara Stipcevic, Jasenka Piljac and Dirk Vanden Berghe, Plant Foods for Human Nutrition,
Volume 61, Number 1/March, 2006, p.27.
7. U. N. Riede, I. Jonas, B. Kirn, U. H. Usener, W. Kreutz and W. Schlickewey, Archives of
Orthopaedic and Trauma Surgery, Volume 111, Number 5 / September, 1992, p.259.
8. Inglot AD, Zielińska-Jenczylik J, Sypuła A., Arch Immunol Ther Exp (Warsz). 1993; 41(1):87.
9. Hasan Mete Aksoy, Berna Aksoy, Didem Egemen, Effectiveness of topical use of natural
polyphenols for the treatment of sacrococcygeal pilonidal sinus disease: a retrospective study
including 192 patients, European Journal of Dermatology; Volume 1, Number 1, October 2008.
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Stabilization of Iron Oxide Magnetic Nanoparticles with Different
Morphology in Aqueous Suspensions Using Humic Substances
A.Yu. Polyakova, A.E. Goldta, T.A. Sorkinab, E.A. Goodilina, b, I.V. Perminovab*
a
Department of Material Science, Lomonosov Moscow State University, 119991, Moscow,
Leninskie Gory 1, building 73, Russia; bDepartment of Chemistry, Lomonosov Moscow State
University, Leninskie Gory 1-3, 119991, Moscow, Russia
E-mail: iperminova@gmail.com; iperm@org.chem.msu.ru
1. Introduction
A use of magnetic liquids for therapy of cancer diseases is an important branch of modern
biomedical technologies. The magnetic nanoparticles suitable for those applications should be
biocompatible and stable in aqueous suspension. Ferric oxides represent one of the most
biocompatible magnetic phases which forms a variety of different morphologies.
Humic acids (HA) are a complex mixture of natural macromolecular compounds with vast
functional periphery dominated by carboxyl and hydroxyl groups. They possess
polyelectrolite properties and distinct surface activity. Thereupon, they can bind nanoparticles
both by electrostatic surface interactions and by complex formation [1]. Humic acids were
reported to be suitable stabilization agents for magnetite (Fe3O4) suspensions [2]. However
Fe3O4 could be oxidized to FeIII-compounds in physiological medium and this can lead to
adverse consequences. At the same time δ-FeOOH and γ-Fe2O3 do not have such
shortcomings and possess necessary magnetic properties.
Thereupon, the objective of this work was to synthesize feroxyhyte (δ-FeOOH) and
maghemite (γ-Fe2O3) nanoparticles and to study the stability of their suspension in the
presence of HAs.
2. Materials and Methods
Synthesis and characterization of iron oxide nanoparticles. Nanoparticles of δ-FeOOH and
γ-FeOOH were synthesized by oxidation of “green rust” under different conditions.
Nanoparticles of γ-Fe2O3 were obtained by annealing of γ-FeOOH at 200-250oC. Phase
composition of obtained nanoparticles was proven by X-ray phase analysis (Rigaku
D/Max-2500 diffractometer). Morphology of the nanoparticles was characterized by using
Hitachi H-8100 transmission electron microscope (TEM). Magnetic properties of synthesized
compounds were studied by using Faraday balance magnetometer.
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Preparation of humic materials and suspensions. Humic materials used in this work were
HAs isolated from leonardite. The HA were dissolved in 1M NaOH under ultrasonic
treatment. Then the solution was diluted with distilled water and pH was set to 7.00–7.05.
Magnetic nanoparticles were added directly into HA solutions with subsequent ultrasonic
treatment. Suspensions were kept at 4–5oC and their stability was monitored. Suspensions of
the nanoparticles in distilled water were used as blank experiments.
Iron (III) concentration in supernatant was monitored as main parameter of suspensions
stability. Sample preparation was carried out according to conventional thiocyanate technique
[3]. Optical density were measured by Varian Cary 50 Probe spectrophotometer at λ = 480
nm.
Size of colloidal particles and salt tolerance of suspensions were tested by using Zetasizer
(Malvern, UK) apparatus based on dynamic light scattering (DLS).
3. Results and Discussion
Synthesis of magnetic nanoparticles. Two different morphologies were investigated in this
work. We have synthesized δ-FeOOH nanospheres (average diameter ~ 30–40 nm, associated
into 200 nm aggregates) and γ-Fe2O3 nanorods (~ 200–250 nm length, ~ 10–15 nm width)
(Fig. 1).
a)
b)
Figure 1: TEM images of δ-FeOOH – a) and γ-Fe2O3 – b)
Nanoparticles of both iron oxides synthesized in this work were monophase as determined by
X-ray phase analysis. Monophase δ-FeOOH has lattice parameters a=2.956(2); c=4.519(3)
and monophase γ-Fe2O3 has lattice parameter a=8.3395(3).
Magnetic measurements show that both δ-FeOOH and γ-Fe2O3 display ferromagnetic
properties. Feroxyhyte δ-FeOOH has saturation magnetization (Ms) ~ 18 emu/g and coercive
force Hc = 110 Oe. Maghemite γ-Fe2O3 has Ms = 45 emu/g and Hc = 182 Oe. At the same
time magnetite (Fe3O4), which often is synthesized for hyperthermia experiments, has
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saturation magnetization up to 190 emu/g and coercive force from 32 to 65.5 Oe [4]. Thus, the
synthesized nanoparticles possess magnetic properties comparable to those of magnetite and
are suitable for biomedical application.
Study of magnetic suspensions stability in water. During storage magnetic suspensions
gradually coagulate and large colloid particles precipitate. Thereupon, stability of the prepared
suspensions was characterized by measurements of iron (III) concentration and colloidal
particles size in supernatant.
The stabilisation of the obtained nanoparticles with HAs was studied in the broad range of
concentrations. It was shown that the concentration of 100 mg/L of HA in solution provides
the best conditions for stabilization of synthesized magnetic phases. At this concentration, up
to 14.7 mg/L of iron (III) in the form of feroxyhyte nanoparticles were still stabilized after 4
days of observation (Fig. 2a). At the same time stabilization of maghemite nanoparticles was
much worse (Fig. 2b). The difference between two oxides could be explained by the presence
of large amount of hydroxyl groups on surface of feroxyhyte nanoparticles, whereas hydroxyl
groups on maghemite surface are lost during annealing. In addition, γ-Fe2O3 nanoparticles
have rather big size and shape anisotropy. Thus, morphology of magnetic nanoparticles and
presence of surface hydroxyl groups have a significant impact on stability of suspensions.
a)
b)
Figure 2: Content of stabilized iron in presence of HA: a) – δ-FeOOH nanospheres, b) – γ-Fe2O3
nanorods.
The DLS data show that nanoparticles quickly aggregate in suspensions without HA, whereas
the presence of HAs sustains initial size of dispersed nanoparticles. Of particular importance
is that the size of humic colloids without and with iron oxide nanoparticles does not differ
significantly. This allows for suggestion that magnetic nanoparticles are captured into
branched structure of HAs which play a role of “nanocontainer” (Fig. 3).
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Figure 3:Supposed mechanism of stabilization of magnetic nanoparticles suspensions by HA
Magnetic suspensions for biomedical applications must be stable in physiological salt
solution, whereas ionic strength has a significant impact on stability of suspensions [2].
Thereupon, we have characterized salt tolerance of the prepared magnetic suspensions by
coagulation kinetic measurements in the presence of different concentrations of NaCl.
Suspensions stabilized by HAs are stable in presence of 150 mmol/L NaCl (concentration of
physiological salt solution); whereas suspensions of magnetic nanoparticles in distilled water
possess very low salt tolerance (~ 30 mmol/L NaCl).
4. Conclusions
Characterization of magnetic suspensions stability displays that HAs are suitable stabilization
agent for iron oxide nanoparticles particularly for feroxyhyte (δ-FeOOH) and maghemite
(γ-Fe2O3). Measurements of iron (III) concentration in supernatant of suspensions show that
feroxyhyte isotropic (spheroidal) nanoparticles with large amount of hydroxyl groups at their
surface are stabilized much better than anisotropic annealed maghemite nanorods. According
to salt tolerance measurements magnetic suspensions stabilized by HAs are stable in
physiological salt solution. These results show a good promise for development of new
biologically active magnetic preparations based on ferric-humic interactions.
References
1. R.S. Swift, In: Humic Substances II. Hayes M.H.B., MacCarthy P., Swift R.S. (Eds.) John Wiley
& Sons Ltd., 1989. p. 468-495.
2. E. Illes, E. Tombacz, The effect of humic acid adsorption on pH-dependent surface charging and
aggregation of magnetite nanoparticles, Journal of Colloid and Interface Science, 295 (2006), 1,
115-123.
3. Pa Ho Hsu, Determination of Iron with Thiocyanate, Soil Sci. Soc. Am. J., 31 (1967), 353–355.
4. Dong-Lin Zhao, Xian-Wei Zeng, Qi-Sheng Xia, Jin-Tian Tang, Preparation and coercivity and
saturation magnetization dependence of inductive heating property of Fe3O4 nanoparticles in an
alternating current magnetic field for localized hyperthermia, Journal of Alloys and Compounds,
469 (2009), 215–218.
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Neutralisation of the Anticoagulant Effect of Naturally Occurring Humic
Acids and Synthetic Humic Acid-Like Polymers by Protamine Sulfate
Hans-Peter Klöckinga*, Nicolle Mahra, Susanne Kunzea, Renate Klöckingb
a
University of Jena, Institute of Pharmacology and Toxicology/Working Group Erfurt,
Nordhäuser Str. 78, D-99089 Erfurt, Germany; bZittau/Görlitz University of Applied
Sciences, Research Institute for Peat and Natural Products, Theodor-Körner-Allee 16,
D-02763 Zittau, Germany
E-mail: hpkloecking@gmx.net
1. Introduction
Previous studies have shown that negatively charged polymers such as naturally occurring
humic acids (HA) and synthetic HA-like polymers prolong the clotting time of blood in vitro
and in vivo [1, 2]. This anticoagulant effect is based on their ability to inhibit the coagulation
factors IIa, VIIa and Xa [3]. Bleeding induced by unfractionated heparin (UFH) can be
antagonized by protamine sulfate as shown by normalisation of thrombin time and aPTT [4].
Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced
anticoagulation by neutralisation negatively charged groups [5].
In order to learn about the neutralisation capacity of protamine sulfate against the anticoagulant effects of HA and synthetic HA-like polymers, an in vitro study was performed.
2. Materials and Methods.
Test substances: Nine negatively charged polymers of different origin were employed: Peat
humic acid A1 (PAH-A1) and peat humic acid A2 (PAH-A2) isolated according to Kirsch [6]
from the Altteich Moor in Saxony (Germany); sodium humate (NaHS) isolated according to
Klöcking et al. [7] from a rainmoor peat of the coastal region of Mecklenburg-Vorpommern
(Germany); commercially available brown coal humic acid (Aldrich HA; Sigma-Aldrich
Chemie GmbH, Steinheim, Germany); the synthetic HA-like polymer KOP 409/85 (caffeic
acid oxidation product) [8]; Melanoidin M1 synthesized from glycine, phenylalanine and
xylose and Melanoidin M42 synthesized from glutamic acid and xylose, both prepared by
Pompe et al. [9]; HS 5, an oxidation product of hydroquinone and glycine, and HS 136, an
oxidation product from hydroquinone and L-lysine, both prepared by Herdering [10].
Biochemicals: Thrombin was obtained from Kallies Feinchemie AG Sebnitz, Germany;
Fibrinogen (Haemokomplettan®) from Behringwerke Marburg, Germany, and protamine
sulfate from Merck, Darmstadt, Germany.
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Test procedure: 50 µl protamine sulfate solution over a concentration range of 0,01 to 100
µg/ml, 100 ml fibrinogen solution (1%) and 50 µl of a fixed test substance concentration
which has to exceed the concentration necessary for doubling thrombin time of the control,
were incubated for 2 minutes at 37°C. After this, 50 µl thrombin solution (3 NIH/ml) were
added to start the coagulation process. The control value of thrombin time was determined in
the same way; instead of protamine sulfate solution, 50 µl Tris buffer pH 7.4 were added. The
coagulometer CL4 (Behnk Elektronik, Norderstedt, Germany) was used for all the
measurements.
3. Results and Discussion
We started our experiments with investigating the influence of protamine sulfate (0.01-100
µg/ml) on the thrombin-fibrinogen reaction (Fig. 1). Concentrations below 7 µg/ml protamine
sulfate did not influence the thrombin-fibrinogen reaction. Higher protamine sulfate
concentrations caused a thrombin time-shortening activity (procoagulant effect), this mean
that they could not be used in the neutralisation experiment.
50
45
Thrombin time (s)
40
35
30
25
20
15
10
5
0
0
0,01
0,1
0,5
1
3
5
7
10
20
40
60
80
100
Protamine sulfate (µg/ml)
Figure 1: Influence of protamine sulfate on the thrombin-fibrinogen reaction, n = 4
In the next experiment, we investigated the effect of protamine sulfate on the thrombin timeprolonging activity of HA and HA-like substances. As shown in Table 1, protamine sulfate
produced varying degrees of neutralisation of the tested HA and HA-like polymers. The
thrombin time-prolonging activity of NaHS, Aldrich HS, KOP, Melanoidin type M1 and
M42, HS 5 and HS 136 was completely inhibited by protamine sulfate. The ratios differ from
1 : 0.2 up to 1 : 1.3 in accordance with the anticoagulant activity of the test substances. HS 5
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140
Thrombin time (s)
120
100
80
60
40
20
0
Blank
0
0,02
0,2
2
Protamine sulfate (µg/ml)
Figure 2: Partial neutralisation of the anticoagulant effect of humic acid PHA-A1 (200 µg/ml) by
protamine sulfate, n = 3
Table 1: Influence of protamine sulfate on the prolongation of thrombin time by different naturally
occurring humic acids and synthetic HA-like polymers
Test substance
PHA-A1
PAH-A2
Substance concentration (µg/ml)
necessary for
doubling clotting time
121.9 a)
Test substance
used for the
neutralisation
experiment (µg/ml)
200
Neutralisation ratio
Test substance :
protamine sulfate
(w/w)
1 : 0.1
600
1 : 0.03
120
1 : 0.3
410.3 a)
b)
Na HS
85.7
Aldrich HS
28.9 b)
75
1 : 0.4
KOP 409/85
22.5 b)
30
1 : 0.8
106.4 b)
130
1 : 0.3
7.0 b)
25
1 : 0.7
27.2 b)
40
1 : 1.3
135.6 b)
140
1 : 0.2
Melanoidin M1
Melanoidin M42
HS 5
HS 136
a) thrombin-plasma reaction; b) thrombin-fibrinogen reaction
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(1 : 1.3) proved to be the most active substance, in contrast, HS 136 showed only minimal
activity (1 : 0.2). The thrombin time-prolonging effect of PHA-A1 and PHA-A2 was only
partially neutralised (70-80 %, see Fig. 2 and Table 1), although the effect of these peat humic
acids is very weak.
The ratio between humic acid and protamine sulfate was estimated to be 1 : 0.1 and 1 : 0.03,
respectively.
4. Conclusions
The results suggest that the anticoagulant activities of HA and HA-like polymers can be
antagonised by protamine sulfate in vitro. These studies warrant further in vivo assessment to
validate the relative neutralisation profile of HA and HA-like substances.
References.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
H.P. Klöcking, Arch. Toxicol. Suppl. 14 (1991) 166.
H.P. Klöcking, B. Helbig and R. Klöcking, TELMA, 24 (1994) 153.
H.P. Klöcking, N. Mahr, R. Klöcking, K.H. Heise and W. Herdering, in L. Martin-Neto,
D.M.B.P. Milori and W.T.L. da Silva (Eds.), Humic Substances and Soil and Water Environment:
12th International Meeting of IHSS, São Pedro, São Paulo, Embrapa Instru-mentação
Agropecuária, 2004, p. 504.
K. Andrassy, V. Eschenfelder and E. Weber, Thrombosis Research 73 (1994) 85.
A. Racanelli, J. Fareed, J.M. Walenga, E. Coyne, Seminars in Thrombosis and Hemostasis 11
(1985) 176.
F. Kirsch, Diploma Theses, University of Applied Sciences, Zittau/Görlitz, Germany.
R. Klöcking, B. Helbg, P. Drabke, Pharmazie, 32 (1977) 97.
K.I. Hänninen, R. Klöcking and B. Helbig, Science of the Total Environment, 62 (1987) 201.
S. Pompe, M. Bubner, M.A. Denecke, T. Reich, A. Brachmann, G. Geipel, R. Nicolai, K.H. Heise
and H. Nitsche, Radiochimica Acta, 74 (1996) 135.
W.
Herdering,
http://analytik.chemie.uni-hamburg.de/rosig/Modelle.html,1998.
Modellsubstanzen für ROS.
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Protolytic Properties of Alkoxysilylated versus Natural Humic Materials
Aimed at Use as Stabilizers for Magnetic Fluids
Sorkina T.a*, Goldt A.b, Polyakov A.b, Dubov A.b, Toth I.c, Hajdu A.c, Goodilin E.b,
Tombacz E.c, Perminova I.a
a
Department of Chemistry, Lomonosov MSU, Leninskie Gory 1-3, 119991, Moscow, Russia;
Department of Material Science, Lomonosov MSU , Leninskie Gory 1-3, 119991, Moscow,
Russia; cDepartment of Colloidal Chemistry, Univ. of Szeged, Aradi Vt. 1, Szeged H-6720
E-mail: sorkina@org.chem.msu.ru
b
1. Introduction
Biocompatible magnetic fluids (MF) receive currently a lot of attention due to broad
applications in biomedical technologies such as hyperthermia, drug delivery, tomography, and
others. Magnetic fluids of this note are water based colloidal suspensions composed of
ferromagnetic or superparamagnetic nanoparticles. The main problem to solve is aggregation
of nanoparticles in aqueous solutions under physiological conditions. The main requirements
to modifiers are non-toxicity and ability to form stable coating on magnetic nanoparticles. In
this regard, of particular advantage can be a use of humic substances (HS). Application of HS
as stabilizing agent for magnetic fluid has been previously reported [1]. However, the humic
coating obtained was very sensitive to changes in pH and salinity. In this work, the
proptolytic properties were investigated of native HS against the specifically modified HS
with incorporated alkoxysilyl-groups providing high affinity of these humic materials for
mineral surfaces. It was hypothesized that these humic derivatives will form stable coating on
the surface of iron oxide particles due to formation of Si–O–Fe linkages. The selected
magnetic nanoparticles were superparamagnetic γ-Fe2O3 and ferromagnetic δ-FeOOH. The
magnetic nanoparticles possessed different morphology, which will be stable at physiological
conditions after appropriate coating and possess reliable magnetic properties.
Thus the goal of the research was to evaluate stabilizing properties of alkoxysilylated humic
derivative versus natural non-modified HS with respect to magnetic nanoparticles of different
micromorphology presented by γ-Fe2O3 and δ-FeOOH, and to conclude on their applicability
for producing biocompatible MF suited for biomedical applications.
2. Materials and methods
Four samples of HS from different sources were studied as stabilizing agents for MF. Two
IHSS samples of aquatic SR FA and SR NOM, one sample of natural leonardite humic acids
(CHA-Pow), extracted from Powhumus (Humintech, Germany) and one sample of modified
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leonardite HA (CHA-APTS-20) were tested as stabilizing agent for biocompatible MF. The
CHA-APTS-20 was obtained as described in [2] using modification of parent humic materials
with organosilane 3-aminopropyltriethoxysilane (APTS) in DMF solution. Maghemite
γ-Fe2O3 nanoparticles were obtained in microspheres using aerosol spray pyrolysis and in
nanotubes: 200-300 nm long and ~ 10-15 nm thick. Feroxyhyte δ-FeOOH was synthesized in
the form of spheres with average diameter of ~ 30-40 nm associated into aggregates (~ 200–
300 nm).
Humic based MF were obtained by dispersion of iron oxide dry powder in water solution of
humic samples with pH 7.0–7.15 as described in [3]. The pH-dependent surface charge state
of HS was determined from acid-base titration under CO2-free condition using background
electrolytes (NaCl) to maintain the constant ionic strength of 0.01 M as described in [4].
The two IHSS samples were dissolved in MQ water; the leonardite samples were converted to
suspensions using technique described in [5]. Equilibrium titration was performed by means
of a self-developed titration system (GIMET1) with 665 Dosimat (Metrohm) burets, nitrogen
bubbling, magnetic stirrer, and high-performance potentiometer. Powder sample was added to
0.01 M NaCl solution equilibrated with electrolyte to reach a starting pH 3.5. After nitrogen
purging for 15 min suspensions were titrated by standard NaOH solution up to pH 10.5 and
then by standard acid solution down to pH 3.5.
3. Results and discussions
The set of humic materials used in this study included the samples of aquatic FA and NOM
with low aromaticity and significant content of aliphatic oxidized structures and the sample of
leonardite humic acid (CHA-Pow-05) with the highest content of aromatic fragments and
lowest – of carbohydrate groups and directly modified leonardite humic acid (CHA-APTS-20)
with 20% modified carboxylic groups. Direct modification of humic backbone using APTS
increases the affinity of humic materials for mineral surfaces by incorporation of methoxysilyl
groups into their structure. The latter produce covalent bonds with hydroxyl carrying surfaces
of silica and metal oxides. Conversion of carboxyl groups into amides can lead to significant
changes in the acidic properties of HS.
Furthermore, complexing properties of HS depend on carboxyl groups content. Interaction
between humic acids and iron oxides occurs via hydroxyl group on the surface of iron oxide
and acidic groups on HS, so one of the most important parameters is the acidic groups
content. To characterize acid-base properties of the humic samples used in this study, the
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method of direct potentiometric titration was used. The pH-dependent net proton surface
excess curves calculated from acid-base titration data are presented in the Fig. 1.
Net H+ excess surface,
mmol/gC
3
4
5
6
7
8
9
10
11
2
0
-2
-4
-6
CHA-APTS-20
-8
CHA-Pow-05
-10
SR FA
-12
SR NOM
-14
-16
pH
Figure 1. pH-dependent net surface H+ excess curves for IHSS SR NOM (black rhombuses) and SR
FA (small white triangles), for the natural leonardite sample CHA-Pow-05 (white rhombuses) and
modified sample CHA-APTS-20 (grey triangles)
According to the obtained net proton surface excess vs. pH curves, modified humic material
has acid-base properties substantially different from those of the parent humic material. The
negative and positive values of net proton surface excess indicate the presence of negatively
and positively charged groups such as e.g., deprotoneted –COO- and protonated –NH3+ at the
given pHs, respectively. In particular, this referred to the appearance of the inflection points
on the titration curves as well as to the positive values of net proton surface excess.
Comparing the measured points at pH~8, the given difference might be connected to the
conversion of carboxyl groups into amide ones, which was caused by the undertaken
modification. The modified sample had also lower water solubility as compared to the parent
humic materials that indicates an increase in their hydrophobocity.
The acidic group content calculated according to Ritchie&Perdue (2003) is given in the Table
1. The results are presented as quantity of functional group in mmol per mass of carbon in
samples.
Table 1: Titration results: humic substances acidic group content
Sample
-COOH, mmol/g C
-OH, mmol/gC
IHSS SR FA
11.57
3.54
IHSS SR NOM
10.25
4.48
CHA-Pow-05
6.61
3.52
CHA-APTS-20
3.32
2.88
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The results of acid-base titration of the parent leonardite humic acids (CHP-Pow-05) and its
derivative show substantial change in acid-base properties of the modified HS and an increase
in its hydrophobicity may be caused not only by a significant decrease in –COOH content, but
also by the presence of residual organic solvent (DMF) in the composition of the obtained
compound.
4. Conclusions.
Natural and modified humic acids are perspective stabilizing agent for water based MF for
biomedical application. The obtained iron oxides nanoparticles γ-Fe2O3 and δ-FeOOH
possessed necessary magnetic properties and were suitable for stabilization with HS due to the
presence of hydroxyl groups on their surface. As APTS modification of parent humic material
leads to increasing sample’s hydrophobicity and decreasing ability for complexing surface FeOH sites of iron oxides, efficiency of natural HS as stabilizing agents of water based MF is
higher in the case of common chemisorption procedure. Directly modified humic samples can
be applied for preparation of stable humic coating on mineral surfaces in solid organo-mineral
sorbents. Modification of parent humic material using silicon organic compounds should to be
provided in other non-toxic and less hydrophobic solvent. At present sample CHA-Pow-05 of
natural humic acids from leonardite at concentration 100 mg/L have shown to be the most
effective stabilizing agent against iron oxide nanoparticles in water solutions from tested
samples.
Acknowledgements
Tatiana Sorkina would like to acknowledge the International Humic Substances Society for
financial support of her stay in the research group of Prof. Etelka Tombacz at the University
of Szeged (Humgary) within the IHSS Training Award - 2009.
References
1. Illés E., Tombácz E. 2006. The effect of humic acid adsorption on pH-dependent surface charging
and aggregation of magnetite nanoparticles. J Colloid Interface Sci. 295:115–123.
2. Perminova, I.V., Karpiouk, L.A., Shcherbina, N.S., Ponomarenko, S.A., Kalmykov, St.N.
Hatfield, K. 2007. Preparation and use of humic coatings covalently bound to silica gel for Np(V)
and Pu(V) sequestration. J. Alloys Comp., 444–445, 512–517.
3. Chekanova A.E., Sorkina T.A., Nikiforov V.N., Davidova G.A., Selezneva I.I., Goodilin E.A.,
Dubov A.L., Trusov L.A., Korolev V.V., Aref'ev I.M., Perminova I.V., Tretyakov Y.D. 2009.
New environmental nontoxic agents for the preparation of core-shell magnetic nanoparticles.
Mendeleev Commun., 19, 1–4.
4. Tombacz E., Szekeres M. 2001. Interfacial Acid-Base Reactions of Aluminum Oxide Dispersed in
Aqueous Electrolyte Solutions. 1. Potentiometric Study on the Effect of Impurity and Dissolution
of Solid Phase, Langmuir, 17, 1411–1419.
5. Ritchie J., Perdue M. 2003. Proton-binding study of standard and reference fulvic acids, humic
acids, and natural organic matter, Geochim.Cosmochim. Acta, Vol. 67, No. 1, pp. 85–96.
Vol. 3 Page - 374 -
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Halogen-free Preparation and Preliminary Characterization of Humic
Substances from Different Substrates
Carola Kleiner, Claudia Barthel, Ralf Junek, Roland Schubert, Juergen I. Schoenherr,
Renate Klöcking*
Research Institute for Peat and Natural Products, Hochschule Zittau/Görlitz – University of
Applied Sciences, Theodor-Körner-Allee 16, D-02763 Zittau, Germany
E-mail: rkloecking@hs-zigr.de
1. Introduction
The potential use of peat-derived humic substances (HS) in medicine, veterinary medicine and
body care requires adequate peat sources as well as sophisticated methods for isolation of
active ingredients. Despite various modifications and additional purification steps, F. K.
Achard’s classical method for isolation of humic acids (HA) from peat, i.e. the alkaline
extraction of HS followed by the acid precipitation of HA [1], represents the core of most HA
isolation procedures so far. However, some HS solubilising, precipitating and purifying
agents currently used are capable to interact with HS and thus may cause problems of
toxicological concern. For example, different chlorine species including chlorine itself and
hypochlorite, but also chlorite, chlorate and even chlorides may produce potentially genotoxic
organic halogen compounds [2, 3].
To avoid any contact of halogens with HS during the HA preparation process, the aim of this
study is twofold: a) to identify halogen-free organic compounds suitable to separate HA and
fulvic acids (FA) and b) to use these agents for the isolation of HA from different substrates.
In addition, the isolated HA will be characterized by elemental analysis, AOX determination,
high performance size exclusion chromatography (HPSEC) and isoelectric focusing (IEF)..
2. Materials and Methods
Humic sources and designation of isolated humic substances (in brackets): Peat from the
Altteich Peatland situated in the Upper Lusatia, Saxony, Germany (HA-AP), digestate of a
biogas plant north of Zittau, Saxony, Germany (HA-DBP), autumn foliage from Alnus
glutinosa (HA-AGL, and commercially obtained roasted coffee (HA-RC) were used.
IHSS Reference humic substances: Waskish Peat humic acids 1R107H (HA-WP) and Waskish
Peat fulvic acids 1R107F (FA-WP) were purchased from IHSS.
Halogen-free isolation procedure for peat humic acids: After homogenization of the peat
sample, ultrapure water was added at a ratio of 1:10. The pH value of the peat suspension was
adjusted with one of the usual extracting agents (e.g. NaOH, KOH) to 9 and kept constant for
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15th IHSS Meeting- Vol. 3
2 hours. The extracting temperature was 30° C. After finishing the extraction, the mixture was
centrifuged at 3500 g for 15 minutes. Peat HA were precipitated in the supernatant by
addition of the selected halogen-free organic acid (0.5 mol/l) in a ratio of 3:1. After
precipitation has been completed, HA were separated from FA by centrifugation at 11000 g
for 10 min. The resultant pellet was washed and centrifuged several times (at least 4 to 5
times) with water and then freeze-dried.
Except for the peat HA AP-A which was precipitated with hydrochloric acid according to
standard procedures (Table 1), the halogen-free isolation method has also been employed for
HA from the other above-mentioned sources.
High performance size exclusion chromatography (HPSEC): Freeze-dried HA samples were
dissolved in a buffered saline solution adjusted with 0.2 mol/l NaOH to pH 10. Water-soluble
alkali humates were dissolved in ultrapure water. HPSEC analysis was carried out using a
high-performance liquid chromatograph from Varian (Varian ProStar series) equipped with
the Diode Array Detector Agilent 1100 (Series G1315B) and fitted with a PSS Hema Bio
column (Polymer Standard Service Mainz, Germany). Data capture occurred at a wavelength
of 240 nm. Moreover, UV/vis spectra were recorded from each HA sample and its major SEC
fractions (Data not shown in the abstract).
Isoelectric focusing (IEF): Precast SERVALYT® PRECOTE® polyacrylamide gels,
pH 3-10, from SERVA (Heidelberg, Germany) were used. The run was performed in a
horizontal electrophoresis chamber at 5° C for 3500 Vh and at a final voltage of 1860 V. To
increase the detection sensitivity for low HA concentrations, HA bands were stained with
Alcian blue-tetrakis (methylpyridinium) chloride (Sigma-Aldrich Chemie, Steinheim,
Germany, Cat. No. A4045) dissolved in 10 % acetic acid.
3. Results and Discussion
In search of alternative, halogen-free precipitating agents for HA, we found polyprotic
organic acids with pKa values between 1.2 and 3.2 the most promising candidates. Two of a
total of 14 acids were considered appropriate for further investigations. Table 1 contains data
of the elemental analysis and of the determined adsorbable organic halogen compounds
(AOX) in peat HA precipitated with 0.5 mol/l hydrochloric acid (A), oxalic acid (B) and citric
acid, repectively. The results show that the precipitation agents do not considerably influence
the elemental analysis of the isolated HA. In contrast, insufficient washing of HA after HCl
precipitation causes a reversible increase of the AOX value.
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15th IHSS Meeting- Vol. 3
Table 1: Elemental analysis and AOX content of three Altteich Peat humic acids (n=3) precipitated
with different agents (A hydrochloric acid, B oxalic acid, C citric acid); 2 x W, 10 x W = Number
of washing steps
Humic acids from Altteich Peat (HA-AP)
C%
N%
C/N Ratio
H%
S%
AOX (mg/kg)
AP-A
2xW 10xW
AP-B
2xW 10xW
AP-C
2xW 10xW
58.8
1.4
41.1
5.3
0.3
724
59.4
1,3
44.6
5.2
0.2
424
54.9
1.3
43.2
5.3
0.2
428
60.7
1.5
40.1
5.4
0.3
438
57.1
1.5
38.3
5.6
0.4
436
59.7
1.3
46,5
5.1
0.3
434
IHSS Reference
Humic Acids
from Waskish
Peat
54.7
1.5
36.5
4.0
0.2
Not detected
Figure 1: HPSEC of humic substances isolated from Altteich Peat (HA-AP), from the digestate of a
biogas plant (HA-DBP) and from autumn foliage of Alnus glutinosa (HA-AGL). Precipitating agent:
oxalic acid
Figure 1 exemplifies the molecular size distribution of three HA from different substrates.
The relation of molecular size classes of HA of the same origin was found to be relatively
constant and widely independent of the precipitation agents used.
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15th IHSS Meeting- Vol. 3
Figure 2 provides IEF images of
HA from the digestate and the
pI
roasted
coffee,
both
prepared
according to the halogen-free
6.0
isolation method as described. IEF
images
from
IHSS
reference
substances are shown for compa5.3
5.2
rison. As a fingerprint technique,
4.2
fied, alcianblue-positive bands, the
3.5
IEF shows a lot of still unidentiposition of which allows assessing
the more or less acid character of
HS.
1
2
3
4
5
6
7
Figure 2: Isoelectric focusing of HS from different
substrates. 1, 2 = HA-WP; 3 = HA-DBP; 4, 5 =
HA-RC; 6, 7 = FA-WP.
4. Conclusions
With regard to prospective applications of humic substances in medicine and cosmetics, a
concept for the halogen-free preparation of HA was developed. Preliminary preparative and
analytical results make polyprotic organic acids the most promising candidates for replacing
hydrochloric acid as HA–precipitating agent.
Acknowledgements
The authors want to thank Stefanie Mey for their assistance in conducting IEF experiments.
The financial support of the study by the German Federal Ministry for Education and
Research (FH³ program, FKZ 1746 X06) is greatly acknowledged.
References
1. F.K. Achard, Crells Chem. Annalen 2 (1786) 391-403.
2. D. Feretti, I. Zerbini, E. Ceretti, M. Villarini, C. Zani, M. Moretti, C. Fatigoni, G. Orizio, F.
Donato, S. Monarca, Water Res., 42 (2008) 4075-4082.
3. I.J. Fahimi, F. Keppler, H.F. Schöler, Chemosphere. 52 (2003) 513-520.
Vol. 3 Page - 378 -
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Young Researchers in Humic Substances and Natural Organic Matter
(IHSS Travel Award)
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Influence of Surface Chemistry and Structure of Activated
Carbon on Adsorption of Fulvic Acids from Water Solution
T. V. Poliakova, L. A. Savchynaand N. A. Klymenko
Institute of Colloid Chemistry and Chemistry of Water, Ukrainian National Academy of
Sciences, 42 Vernadsky Avenue, Kiev 03680, Ukraine
E-mail: PoliakovaT@ukr.net
1.Introduction
The adsorption of fulvic acids (FAs) on activated carbon (AC), which contain fractions of
different molecular weight with various functional groups, is greatly affected by AC surface
chemistry. The presence of oxygen-containing groups on the AC surface affects on the
mechanism of interaction between sorbate and sorbent, changing the value of the free energy
of adsorption, and creates prerequisites for the manifestation of catalytic properties of AC.
The adsorption capacity of AC can be changed by modifying its surface by oxygen-containing
groups.
Natural organic matters which are containing in surface source of water supply are a mixture
of compounds with very different adsorption characteristics.
The characterization of multicomponent adsorption equilibrium for natural organic matters
using a “conventional component” was successfully effected by applying the ideal adsorption
solution theory (IAST) [1]. In accordance with the approaches developed in [2], in the case of
adsorption of natural organic matters from a multicomponent solution, adsorption isotherm in
the logarithmic coordinates of the Freundlich equation has two or three regions of principle as
straight lines: (1) nonadsorbable part of NOM, which is expressed by a vertical line even at
large adsorbent doses; (2) slightly adsorbable part of NOM, where at medium adsorbent doses
the highly adsorbable component and the proportional part of the fraction of the more slightly
absorbable part of NOM. Еach region is described by individual values of ni and Kf,i.
Thus, the aim of this work was to estimate the characteristics of adsorption of FAs from
aqueous solutions in accordance with the approaches considered and to determine the
variation of the adsorption characteristics of sorbents as a function of the conditions of
carrying out the process.
2. Materials and Methods
The FA have been obtained according to the Forsith’s method [3] from a high-moor peat were
used as a sorbate.
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15th IHSS Meeting- Vol. 3
The object of investigation was KAU carbon, which is obtained by treatment of crushed fruit
stones with concentrated alkali and hot hydrochloric acid (after washing with water), washing,
carbonization and activation with steam.
Carbons were oxidized according to the procedures described in [4]. KAU carbon was
oxidized with nitric acid during three and nine hours (KAU-N3 and KAU-N9) and hydrogen
peroxide (KAU-O).
3.Results and Discussion
Figure 1 shows adsorption isotherms of FAs on KAU, KAU-O, KAU-N3 and KAU-N9
active carbons in logarithmic coordinates of the Freundlich equation.
100
a (mgC/g)
10
КАU
КАU-N9
1
1
КАU-N3
10
100
КАU-O
0,1
Ceq (mgC/l)
Figure1: Isotherms of adsorption of fulvic acids on KAU, KAU-O, KAU-N3 and KAU-N9 in the
coordinates of the Freundlich equation
In accordance with the proposed approaches [1, 2] we have been divide the adsorption
isotherm into two regions: (1) for low adsorbable part and (2) for high adsorbable part of FA.
Table 2 lists values of Freundlich equation constants calculated from a single averaged
straight line without division into regions (Kav, Hav), and n1 and n2 values corresponding to
two FA adsorbability regions.
As it is evident from Table 2, change in surface chemistry, i.e. increase in surface
heterogeneity, and the appearance of additional lactonic, phenolic groups and basic properties
on the surface changes greatly the adsorption characteristics of AC concerning adsorption of
FAs. Although the Freundlich equation is empirical, a physical meaning is attached to its
constants in some works. KF is regarded as an adsorption capacity factor, and the exponent n
characterizes the heterogeneity of energy centers on the surface and is related to the driving
force of adsorption (i.e. adsorption energy).
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Table 2: Change of coordinates of the Freundlich equation in dependence of properties surface of AC
Constants of the
Freundlich equation
Acidic
(mg/gAC)
Basic
(mg/gAC)
Carboxy-lic
(mg/gAC)
Phenol-lic
(mg/gAC)
Lacto-nic
(mg/gAC)
Sample
КF
n
n1
n2
KAU
1.41
1.03
0.21
1.13
0.15
0.05
0.46
0.10
0.05
4.8
KAU-О
0.21
0.74
0.26
1.01
0.85
0.20
0.44
0.55
0.20
2.6
KAU-N3
0.50
0.81
0.18
1.06
0.85
0.30
0.49
0.50
0.30
3.0
KAU-N9
0.81
0.75
0.21
0.85
1.0
0.30
0.40
0.70
0.30
3.5
pH
In accordance with this approach, it can be concluded from Table 2 that the oxidation of AC
reduces adsorption capacity for FAs as a whole due to an increase of energy surface inhomogeneity. In this case, the adsorbability of FA is most likely affected adversely by
increase in the percentage of lactonic and phenolic surface groups (i.e. negatively charged
groups). However the adverse effect on the absorbability of FA on KAU-N3 and KAU-N9 is
weakened as compared with KAU-O. This adverse effect on the adsorbability of FA is
understandable if the above-mentioned chemical nature of the functional groups of FA is
taken into consideration.
The exponent n for the highly adsorbable components (n2) are larger than exponent for the
low adsorbable components (n1). This points to the fact that increase in energy surface inhomogeneity especially of basic properties increases the driving force of adsorption for well
adsorbable compounds.
Table 3 lists data concerning the determination of adsorption equilibrium constants Ka and
changes in the free energy of adsorption -ΔGa0 for two "conventional component" of FA.
Table 3: Changes of adsorption equilibrium constants (Ka) and free energy of adsorption (-ΔGao)
for two “conventional component” of FA solutions in dependence of type of AC
(-ΔGa0), kJ/mol
Ka
First “conventional
component”
(-ΔGa10)
11.42
Second
“conventional
component”
(-ΔGa20)
18.97
KAU
153
Second
“conventional
component”
Ka2
4258
KAU-О
35
3881
8.08
18.76
KAU-N3
150
3767
11.38
18.69
KAU-N9
251
3761
12.54
18.69
Sample
First “conventional
component”
Ka1
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As it is evident from Table 3, the lowest adsorption energy of the low adsorbable component
is characteristic for KAU-O, and for the high adsorbable component this parameter is
practically invariable for all types of AC.
Thus, increase in the energy surface in-homogeneity of AC affects mainly on the adsorption
of the low adsorbable part of FA. This effect may be due to the fact that the adsorption of this
part of FA is brought about mainly by Van der Waals forces, and that the screening of a part
of the surface by functional groups reduces the total adsorption energy. The change in surface
chemistry does not practically affect on the more high adsorbable fraction of FA, indicating
that there is a specific interaction between the functional groups of FA and the surface.
Besides, it should be noted that change in the structure and surface chemistry of AC leads to a
change in concentration limits whereby two FA components. This is evident from the data in
Table 4.
Table 4: Change in concentration limits of conditional division FA on slightly adsorbable fraction and
highly adsorbable fraction
Samples
Range of equilibrium concentration on the
isotherm that apropriate slightly adsorbable
fraction (mg C/l)
KAU
Up to 3.40
KAU-О
KAU-N3
KAU-N9
Up to 6.25
Up to 4.40
Up to 4.17
4.Conclusions
1. The oxidation of AC reduces the adsorption capacity for FAs in whole due to an increase of
energy surface in-homogeneity.
2. The data obtained allow the conclusion to be that the change in AC energy surface inhomogeneity due to oxidation leads mainly to a decrease in FA adsorption energy and to
increase of concentration range of the conventional portion of the low adsorbable fraction,
especially in the case of adsorption on AC oxidized by hydrogen peroxide.
References
1. E.H. Smith and W.I. Weber, Water, Air, Soil Pollut., 53 (1990) 279.
2. E.H. Smith Water Res., 28 (1994) 1693.
3. L.N. Aleksandrova, Organic Matter of Soil and Processes of its Transformation. Science,
Leningrad, 1980, p 288.
4. M.F. Pereira and S.F. Soares, J.J.M. Orfao, J.L. Figueiredo, Carbon. 41 (2003) 811.
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Studies by Chemometric Methods of the Interaction Between
Pb(II) and Humic Acids
Silvia Orsetti, Estela Andrade, Fernando Molina*
INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires Ciudad
Universitaria, Pabellón II, Buenos Aires C1428EHA, Argentina
E-mail: fmolina@qi.fcen.uba.ar
1. Introduction
Humic substances are important components of natural organic matter in groundwaters and
soils, where they have a fundamental role in the fate of pollutants such as heavy metals. In
particular, retention and transport of hazardous substances by humic and fulvic acids have
been frequently studied in the last few years.
Fluorescence spectroscopy, combined with multi - way data analysis techniques, is a powerful
tool to obtain information regarding humic substances and their interaction with heavy metals,
such as Pb(II). In this kind of analysis, excitation emission matrices (EEM) are used: they are
obtained by combination of emission spectra measured at different excitation wavelengths. In
general, humic acids (HA) show a broad emission peak between 300 and 700 nm and a broad
excitation peak between 300 and 450 nm. However, the location and shape of these peaks
strongly depend of the origin of the humic material.
Excitation emission matrices provide wide information, and until recently EEMs
characterization techniques were focused on visual identification and shape of peaks, leading
to qualitative analysis. Recently, multi - way data analysis techniques have been introduced in
the study of fluorescence signals of natural organic matter. One of these techniques is parallel
factor analysis (PARAFAC), which is able to decompose the fluorescence signal into the
underlying fluorescent individual phenomena [1, 2].
PARAFAC models three – way data by using eq. 1, fitting the equation by minimizing the
sum of squares of the residuals (εijk):
F
xijk = ∑ a if b jf c kf + ε ijk
i = 1,..., I
j = 1,..., J
f =1
k = 1,..., K
(1)
xijk is an element of the three - way array with dimensions I, J and K. In the case of EEMs, xijk
is the fluorescence intensity of sample i, measured at the emission wavelength j and excitation
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wavelength k. The term εijk accounts for the unexplained signal (experimental error). The
output of the model are the parameters a, b and c. These represent, ideally, the concentration,
emission spectra and excitation spectra of the underlying fluorophores, respectively, and they
are usually referred to as scores (a) and loadings (b, c). In this way, important information
about the number of fluorophores, their fluorescence profiles and their behavior in presence of
the metal is obtained; the last subject, from the relative concentration of each fluorophore in
each individual sample, with a characteristic Pb(II) concentration [3].
The aim of this work is to study EEMs of HA at several Pb(II) concentrations, analyzing them
with PARAFAC as a whole to estimate the number of individual components of the sample
(fluorophores), their emission and excitation fluorescence spectra, as well as the relative
concentration of those in each sample.
The effect of pH value and ionic strength is also analyzed.
2. Materials and Methods
The EEMs of two different HA where measured in absence and presence of several
concentrations of Pb(II). These measurements were conducted for Elliot Soil HA (EHA,
reference material of the IHSS) and other commercially available from Fluka, previously
purified (FHA). Experiments were performed at pH 4.0 and 5.5, and at two values of ionic
strength: NaClO4 0.1 and 0.02 M. The samples were prepared by dissolving the HA in the
NaClO4 (aprox. concentration 30 mg L-1) using a minimum quantity of NaOH, and then the
pH was adjusted with HClO4. This sample was separated in 25.0 mL aliquots, and different
volumes of a stock solution of Pb(II) were added to these. The EEMs of those aliquotes were
measured, as well as the EEM of the HA sample without Pb(II). In all cases, the samples were
under N2 atmosphere.
Data processing: after elimination of the scattering zones in the EEMs, PARAFAC was used
indicating number of components from 2 to 5, adding a non negativity constrain for the b and
c loadings (which account for emission and excitation spectra respectively). From the values
of core consistency and SD residuals, the number of components was estimated following Bro
and Kiers [4], considering that a high core consistency value (over 50%) means that the model
presents high spectral resolution of components. From there a, b and c were obtained.
3. Results and Discussion
A total of 3 independent components were estimated for both HA. In Fig. 1 the relative
concentrations of fluorophores (a score) in each set of samples is plotted for EHA.
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From Fig. 1 it can be noticed that all 3 components have a different behavior towards the
increment of Pb(II) concentration, being the component A the most affected in its
fluorescence intensity. The difference between components B and C is more marked at higher
ionic strength (a and b of Fig. 1), component C showing less sensitivity towards Pb(II) at pH
= 4.0 and I = 0.10 M (a in Fig. 1). The fluorescence decrease does not correlate with the
fraction of carboxylic and phenolic groups bound to Pb(II) ions as predicted by the NICA –
Donnan model, thus Pb(II) induced aggregation is proposed as the cause of fluorescence
6
2.5x10
Fluorescence Intensity Scores (a. u.)
Fluorescence Intensity Scores (a. u.)
quenching.
a
6
2.0x10
6
1.5x10
6
1.0x10
5
5.0x10
1E-5
1E-4
b
6
3.0x10
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5.0x10
1E-6
1E-5
2+
8.0x10
Fluorescence Intensity Scores (a. u.)
[Pb ] added (M)
6
Fluorescence Intensity Scores (a. u.)
2+
c
6
6.0x10
6
4.0x10
6
2.0x10
1E-5
[Pb ] added (M)
6
2x10
6
1x10
0
1E-4
2+
d
6
3x10
1E-5
[Pb ] added (M)
1E-4
2+
[Pb ] added (M)
Figure 1: a scores (Fluorescence Intensity Scores, in arbitrary units) are shown as a function of Pb(II)
concentration. squares: component A; circles: component B; triangles: component C
pH 4.0 and I = 0.10 M (a); pH 5.5 and I = 0.10 M (b); pH 4.0 and I = 0.02 M (c) and pH 5.5 and I =
0.02 M (d)
0.4
0.3
0.2
0.1
0.0
200
300
400
500
600
700
b
0.3
spectral loading
spectral loading
a
0.2
0.1
0.0
200
300
400
500
λ (nm)
λ (nm)
600
700
Figure 2: emission (continuous line) and excitation (dotted line) spectra of components for EHA (a)
and FHA (b), both at pH = 5.5, I = 0.1 M. Black: component A; red: component B; blue: component C
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The loadings (b and c of PARAFAC) for EHA at pH = 5.5, I = 0.1 M are shown in Fig. 2. The
shape and location of these emission and excitation spectra are typical of humic substances
[5,6]. The spectra are shifted to the red (not shown) as the ionic strength decreases, especially
at pH = 5.5. This was observed for most components.
4. Conclusions
In both EHA and FHA 3 fluorophores can be estimated; one of them is strongly deactivated in
presence of Pb(II) wile the others show less sensitivity. All present emission and excitation
spectra typical of humic substances. The fluorophores which are most affected have their
emission and excitation spectra shifted to the red, thus might be attributed to groups
containing condensed rings. It is considered that humic molecules would aggregate with the
metal as a bridge, presenting no–radiative deactivation in the aggregated form and thus
quenching of their fluorescence intensity. The deactivation depends on the ionic strength and
pH, especially for the components B and C.
Regarding the effects of pH and ionic strength values, it was observed that emission and
excitation spectra shifted towards the red with lower ionic strength, being this shift higher at
superior pH.
References
1.
2.
3.
4.
5.
6.
C. M. Andersen and R. Bro, J. Chemometrics, 17, (2003) 200.
C. A.Stedman and R. Bro, Limnol. Oceanogr.: Methods, 6 (2008) 572.
T. Ohno, A. Amirbahman and R. Bro, Environ. Sci. Technol., 42 (2008) 186.
R. Bro and H. A. L. Kiers, Journal of Chemometrics, 17 (2003) 274.
T. Ohno and R. Bro, Soil Sci. Soc. Am. J., 70 (2006) 2028.
M. M. D.Sierra, M. Giovanela, E. Parlanti and E. J. Soriano–Sierra, Chemosphere, 58 (2005) 715.
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Seasonal Dynamics of Biomass and Copper Concentrations in Ectohumus
of Forest Soils Impacted by Copper Industry in South-West Poland
Agnieszka Medyńska*, Cezary Kabala
Institute of Soil Science and Environmental Protection
University of Environmental and Life Sciences, Wroclaw, Poland
E-mail: jagamedynska@yahoo.com
1. Introduction
Humus plays an important role in the functioning of forest ecosystems. It acts as a link
between above- and below-ground ecosystem compartments. The fall and decomposition of
organic matter return minerals and energy to the soil biota. The forest floor is the most
dynamic part of soil organic matter (Yanai et al. 2003). Previous studies about nutrient release
from forest litters, formed under various tree species, show significant differences in
dynamics of nutrients. The release of nutrient elements during decomposition of forest floor is
an important internal pathway in nutrient flux of forested ecosystems. Nutrients may be
released from ectohumus horizons by leaching or mineralization (Regina, 2000). Nutrient
release from decomposing litter affects the primary productivity of ecosystem, since these
nutrients become available for plant uptake and are not lost in the ecosystem. The rate at
which nutrients are released depends on several factors: the chemical composition of the
forest litter, the structural nature of the nutrient in the litter matrix, the microbial demand for
the nutrient, and the availability of exogenous substance sources for example from
anthropogenic activity (Brown et al., 1999). Litter quality affects the rates of mass loss, but
also the patterns and rates of nutrient immobilization or release. Forest litter highly
contaminated with heavy metals may effect nutrient cycling throughout the whole ecosystem
(Tyler, 1974). Many reports have shown that short-term or long-term exposure to toxic metals
results in the reduction of microbial diversity and activity which is mainly observed in
inhibiting litter decomposition and increase of undecomposed organic matter in humus layer
(Berg et al., 1991). From the other hand, forest litter acts like a sink for contaminants,
regulating the amount of heavy metals leaching to deeper soil layers (Medyńska and Kabała,
2007 Laskowski et al. 1995). Dynamics of heavy metals in contaminated forest floor is
exceptionally investigated, and in particular - on recently afforested soils, where the
ectohumus layer has been already formed.
The aim of present study was to determine the seasonal dynamics of biomass of forest litters
in ecosystems impacted by copper industry as an important factor determining actual
concentration of copper in forest floor.
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2. Materials and Methods
Study sites. The investigation was carried on two study areas. The first one was located in a
surrounding of the large copper smelter near town Legnica, in Lower Silesia region. The
copper smelter Legnica is a part of the mining and metallurgy complex founded in 1951,
which currently includes 4 mines, 3 ore enrichment plants and 3 smelters. The complex has
been producing approximately 500,000 tons of copper annually, one fourth of that is produced
in the Legnica smelter. Copper smelting was connected in the past with a large emission of
metal-containing dust, significantly reduced during the 1980s and 1990s (Byrdziak et al.
2005). Long-term copper smelting in the Legnica area has however resulted in an extensive
soil contamination with number of trace elements. To exclude crop production in
neighborhood of the smelter, the so called “protective zone” was delimited in late 80s around
the smelter. All the area has been successively planted with poplar trees. The other study area
was located in the surrounding of copper ore tailings impoundment Żelazny Most, located
near the town Lubin, 30 km north of Legnica smelter. The over-ground impoundment has
been in operation since 1977. Over 368 mln m3 of tailings from copper ore flotation are
assembled on the area of 1,390 ha. Wind expose, dry metal-bearing tailings are blown about
to surrounding areas leading to soil and plant contamination.
Methodology. Two study areas were projected as separate experiments. On each study site, 3
plots were located. The plots at the Cu smelter Legnica were arranged along decreasing
concentration of Cu in soil, at distances of 0.3, 1.5 and 2.1 km, north of the smelter. All plots
are on similar soils (Haplic Luvisols) and in the poplar stands of the same age. The plots near
tailings impoundment are located at the same distance from impoundment, on similar soils
(Brunic Arenosols), but under pine stands varying in age, from 8 to 50 years old. On each site
samples of litter were collected in 4 replicates using a stainless-steel cylinder (d=23 cm).
Samples were collected every month between November 2007 and October 2008. Dry matter
of litter was determined in all samples, followed by high-pressure sample digestion with aqua
regia (HCl:HNOs ratio 3:1), and measurements of total Cu concentration by the atomic
absorption spectroscopy (AAS) technique.
3. Results
Forest floor biomass estimation. Biomass of forest floor (as dry matter) and Cu
concentrations were calculated for standard area of 1m2. On both sites biomass of litter varied
between the plots (Fig. 1 and 2). The highest mass of ectohumus in poplar stands near Cu
smelter, and the largest thickness (ca 7 cm) was observed on the plot 1 located 0.3 km from
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the smelter. This phenomenon should be considered with the highest concentration of Cu in
litter – circulating around 11,300 mg/kg (Fig. 3). High concentrations of heavy metals can
suppress the decomposition rate and lead to accumulation of undecomposed organic matter,
because of the toxicity effect on soil microorganism (Tyler 1972, Rühling and Tyler 1973;
Strojan 1978; Berg et al. 1991, Niklińska et al. 1998). The highest mass of ectohumus (litter)
in forest stands surrounding the tailings impoundment was observed in the oldest (50-yearsold) oak-pine stand. Changes of a litter mass during studied period of time mostly depended
on all plots on biomass input pattern. Input of leaf biomass changed seasonally, and was the
highest in late fall (October-November) and during the drought periods (February, March,
August, and September), when trees lost leafs partly as a water stress effect. Seasonal changes
of ectohumus biomass are also considered with the intensity of decomposition process. In
studied litters, the highest intensity of decomposition process was observed during winter
time. Unreceptively decomposition played a secondary role in biomass changes, this can be
considered with an inhibiting effect of heavy metals on decomposers in studied ectohumus
horizons.
Fig. 1 Biomass (mg/m2 of d.m.) of litter layer in poplar
stands in the surrounding of copper smelter Legnica
Fig. 2 Biomass (mg/m2 of d.m.) of litter layer in pine
stands in the surrounding of tailings impoundment
Żelazny Most
Figure 4: Copper concentration in litter of forest
stands in the surrounding of tailings impoundment
Żelazny Most
Figure: 3: Copper concentration in litter of forest
stands in the surrounding of copper smelter Legnica
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Changes of copper concentration in ectohumus. Significant impacts of copper smelting plant and
tailings impoundment were observed in studied forest soils and ecosystems. Both emission sources
cause air pollution, partially filtrated by forest stands planted in surroundings of the industrial objects.
In studied forest ecosystems through fall of leaf biomass was the main source of copper input into the
ectohumus horizons. With an increase of through fall, also concentration of studied metal in forest
litters, increased rapidly. Despite decomposition processes and copper leaching with rain waters
observed during the year of study, copper concentrations on all studied plots increased successively
with time (Fig. 3 and 4). This can be concluded as evidence that in young forest ecosystems impacted
by heavy metal pollution, already developing ectohumus horizon may accumulate heavy metals,
therefore play a role of a natural sink for anthropogenic contamination.
4. Conclusions
1.
The dynamics of forest litter biomass and Cu concentrations depend strongly on leaf input during
seasons.
2.
Decomposition processes play a secondary role in biomass changes and Cu release. Inhibiting
effect of heavy metals on decomposition rate was observed in poplar stands in the vicinity of
copper smelter.
3.
In studied young forest ecosystems, developing ectohumus horizons play a role of a natural sink
for Cu contamination.
References
1. Berg B., Ekbohm G., Söderström B., Staff H. 1991: Reduction of decomposition rates of Scots pine needle
litter due to heavy metal pollution. Water, Air, Soil Pollut. 59: 165–177.
2. Brown S.L., Schroeder P., Kern J.S. 1999: Spatial distribution of biomass in forests of the eastern USA:
Forest Ecology and Management 123: 81–90.
3. Laskowski, R., Niklińska, M., Maryanski, M., 1995. The dynamics of chemical elements in forest litter.
Ecology 76, 1393–1406.
4. Byrdziak H., Jędrzejewski J., Kierdel Z., Mizera A. 2005: Environmental Protection – Bulletin 2002–2004.
KGHM CUPRUM, Lubin, Poland: 1–170.
5. Medyńska A., Kabała C. 2007: Heavy metal concentration in ectohumus horizons of forest soils under
6.
7.
8.
9.
10.
11.
12.
impact of copper ore tailings impoundment Żelazny Most: Environmental Protection and natural resources
31: 137–144.
Niklińska M., Laskowski R., Maryańska M. 1998: Effect of heavy metals and Storage time on two types of
Forest Litter: Basal respiration rate and exchangeable metals: Ecotoxicol. Environ.Safety 41: 8–18.
Regina I. 2000: Biomass estimation and nutrient pools in four Quercus pyrenaica in Sierra de Gata
Mountains, Forest Ecology and Management 132: 127–141.
Rühling A, Tyler G. 1973: Heavy metal pollution and decomposition of spruce needle litter. Oikos 24: 402–
416.
Strojan C.L. 1978: Forest litter decomposition in the vicinity of a zinc smelter. Oecologia 32: 203–212.
Tyler G. 1972: Heavy metal pollution and mineralization of nitrogen in forest soils, Ambio 1(2): 52–57.
Vandecasteele B., De Vos B. , Muys B., Tack F. 2005: Rates of forest floor decomposition and soil forming
processes as indicators of forest ecosystem functioning on a polluted dredged sediment landfill: Soil Biol.
Biochem. 37 761–769.
Yanai R.D., Currie W.S., Goodale C.L. 2003: Soil carbon dynamics after forest harvest: an ecosystem
paradigm reconsidered: Ecosystems 6: 197–212.
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Limitation for Study of Humic Substances or NOM Using High Resolution
and Accuracy Mass-Spectrometry
Gleb Vladimirova,b, Eugene Nikolaev a,b*
a
The Institute for Biochemical Physics RAS, Moscow, Russia; bThe Institute for Energy
Problems of Chemical Physics RAS, Moscow, Russia
E-mail: ennikolaev@rambler.ru
1. Introduction
Stoichiometric formulas determination using high resolution and accuracy mass-spectrometry
(ICR-FT mass-spectrometry) for molecules of humic substances (HS) or natural organic mater
(NOM) is a powerful tool which show full scale of molecular complexity of HS and NOM
[1,4]. However, using this method you should take into account its limitations: selectivity of
ionization process and limitations caused by the physics of mass determination processes.
That leads to correct work of method only within a certain m/z range and for differences in
components concentrations limited by method dynamic range.
2. Materials and Methods
Suwannee River Reference Fulvic acid (SRFA) spectra (sample id 1S101F) was obtained on
commercial mass-spectrometer 7 Tesla Finnigan LTQ FT (Thermo Electron, Bremen,
Germany) equipped with electro spray ion source (Finnigan Ion Max Source) located at the
facilities of the Emmanuel Biochemphysics Institute of RAS (Russia). The spectra was used
for determination of ion cloud distribution for estimation of method limitation. The following
conditions were used for electrospray: flow rate 1 ul/min, negative ion mode, needle voltage
3.4 kV; tube lens voltage 130 V; heated capillary temperature 250 oC. Filtering linear ion trap
was set up to collect ions in m/z range from 200 to 2000 Da, the number of accumulated ions
1·106. The average spectrum was calculated from 100 coadded scans. Time domain was set
up for resolution R=400000 at m/z 400. LTQ FT calibration mix was used for external mass
calibration. All acquired spectra were internally recalibrated by solvent impurities to mass
measurement error <0.5 ppm. Analytical approaches [2] and simulation of realistic ions cloud
behavior [3] was used to determine operating range of m/z and restrictions of dynamic range.
3. Results and Discussion
We saw 9597 peaks for SRFA spectrum. Capacity of ICR trap in this mode is 106
charges (it is confirmed by estimates of traps capacity for using trapping voltage). If assumes,
that intensities of peaks proportional to number of ions created this peak, and that all ions are
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singly charged ions (it is true, because of a little fraction of multiply charged ions) then we
have: a minimum number of ions observed in ion cloud is 27, the largest number of ions in a
cloud is 1870 ions. Therefore we observe dynamic range for different SRFA compounds
equal to 70. (Although dynamic range can be considered as ratio of total number of ions to
minimum number of ions in observable peak 106/27= 37 000, but that ratio doesn’t reflect
observable dynamic range of SRFA compounds.)
Figure 1: Statistics of ion cloud numbers and numbers of elementary charges in ion cloud for spectrum
of SRFA
The statistics of cloud charge distribution (see Fig. 1) shows that: in case of SRFA spectrum
roughly the same number of elementary charge are in ion clouds of different charge value,
while number of ion clouds with a small number of charges increases greatly during reducing
of number of charges in ion clouds. High charge ion clouds are not observed - the maximum
number of elementary charges in a cloud is 1870 charges, and it is ~ 500 times smaller than
trap capacity. Therefore we can conclude that for spectra like SRFA the main problem is
detection of clouds with a minimum number of ions (detection limit) and destruction of
clouds by a huge number of small clouds during detection (dynamic range limit).
Therefore increasing of dynamic range for work with spectra like SRFA demands decreasing
of minimum detectable number of ions value:
There are several different estimations of minimum number of ions [4], which is necessary to
provide detectable signal. It should be about 50 (Group Dick Smith) or 100 (Marshall)
elementary charges in a cloud of one m/z to make signal from such m/z detectable for single
spectrum.
Minimum number of ions (that is minimum number of elementary charges in the cloud of a
certain m/z) which is necessary to ensure detectable signal for given m/z can vary for different
mass-spectrometers, because of influence of amplifier and preamplifier construction, for
example using of QSUID detector provides an opportunity to receive signal from one single
charged ion rather than from group of 50 single charges ions for traditional scheme.
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Definition ions minimal number which can provide detectable m/z in spectrum is complicated
by fact that it depends on level of noise in amplifier system, therefore using sum of several
different spectra of the same sample obtained at identical conditions allows to reduce noise in
the system, thus it increases minimum number of ions in which this m/z are detectable.
Sensitivity of mass-spectrometer depends on the distance from the ion cloud to the walls of
mass-spectrometer cell, reducing this distance we increase the sensitivity of the massspectrometer, but the stability of the ion cloud is decreased due to greater anharmonicity of
trapping potential for higher radius orbit. Anharmonicity Value for trapping potential for cell
determined by trap design: it could be improved by use of newer harmonized cell design.
Resolving power requirements of mass-spectrometer for work with complex samples like
SRFA are determined by necessity to resolve doublet of C13/C12H (dm = 0.00447 Da) for
work with Cx Hy Oz stoichiometric formulas. Work with Cx Hy Oz Nm Sn stoichiometric
formulas requires resolving a greater number of doublets for combination of C12=12.00000
Da, C13=13.00335 Da, H1=1.007825 Da, O16=15.99491, N14=14.00335 Da, S32=31.9720 Da,
but resolution required for this doublets is comparable with C13/C12H doublet. Resolution of
mass-spectrometer for work in the same mode decreases with increasing of measured m/z
therefore for doublet mass difference we have R (C13/C12H) = M (Da) * 224. On other hand
resolving power on frequency of any m/z is determined by signal duration and m/z frequency.
Signal duration determined by destruction of ion clouds: due to dependence of cyclotron
frequency from amplitude for inharmonic trapping potential, collisions of ions with residual
gas and ion cloud-ion cloud collision. Therefore for B = 7T; t = 1 and 3 s; m/z = 100 Da we
obtain resolving power about R ~ V [Hz] t [sec], finally we can determine m/z above which
resolving power will be insufficient for work with complex spectra like SRFA (above this m/z
impossible to resolve doublets):
M/z (C13/C12H, B = 7T t = 1 sec) = 750 Da
M/z (C13/C12H, B = 7T t = 3 sec) = 1250 Da
Therefore 200–800 Daltons range could be considered suitable for work with such complex
samples like SRFA on 7 Tesla ICR mass-spectrometers. Bulk part of components distribution
will fit to this range for majority of complex samples such HS or NOM for such massspectrometers.
Resolving power is determined by the duration of detection signal, but there is phenomenon
of coalescence [3] (merging different m/z peaks due to space charge clouds interaction). This
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phenomenon leads to limit of resolution, and it shows itself for ion cloud with large number
of elementary charges or very close m/z. Coalescence is observed in numerical simulations
[3], there are several analytical estimations [2] that show the boundary of coalescence
condition.
For coalescence limit in case of C13/C12H doublet in 7T, 2 inch sell, 30% of sell cyclotron
orbit radius according to formula N = 5.6 a2R B2 dm/(m2k) [2] we can find m/z above which
C13/C12H doublet will be unresolved due to coalescence:
m/z(C13/C12H, cloud size 10000 charges, B=7T ) = 1200 Da
m/z(C13/C12H, cloud size 1000 charges, B=7T ) = 3800 Da
m/z(C13/C12H, cloud size 100 charges, B=7T ) = 12000 Da
Therefore in our case resolution limit due to coalescence is less significant compared to
resolving power limit due to length of detection time.
4. Conclusions
Studding of samples like SRFA using ICR-FT mass-spectrometers with 7 Tesla magnetic
field strength possible in mass range up to 800 Da. Working in higher mass range demands
using higher magnetic fields.
Dynamic range for work with HS or NOM is restricted by limiting of minimum number of
ions needed to make signal detectable. Increasing the minimum number of ions which are
necessary for detection firstly requires improvement of trap design for harmonization of
trapping potential.
References
1. E.V. Kunenkov, A.S. Kononikhin, I.V. Perminova, N. Hertkorn, A. Gaspar, P. Schmitt-Kopplin,
I.A. Popov, A.V. Garmash and E.N. Nikolaev, Anal. Chem., (2009) in print.
2. I.A. Boldin, Nikolaev E.N. Rapid Commun. Mass Spectrom. 2009; 23: 3213–3219.
3. E.N. Nikolaev, G.N. Vladimirov, I.A. Boldin, R.M. Heeren, C. Hendrickson, G. Blakney, A.G.
Marshall 57th ASMS Conference on Mass Spectrometry 2009 Instrumentation: FTMS – poster
278.
4. A. Kononikhin, G.N. Vladimirov, E.V. Kunenkov, I.A. Popov, I.V. Perminova, A. Garmash, G.
Karpov, S. Varfolomeev, E.N. Nikolaev 56th ASMS Conference on Mass Spectrometry
2008 Hydrocarbon and Petrochemical - poster 141.
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A Study of Interaction Between Pharmaceuticals and Humic Substances
Ansone Lb., Klavins M.a*
a
Faculty of Chemistry, Univ. of Latvia, Kr. Valdemara iela 48, Riga, LV 1013, Latvia; Dept.
of Environmental Sciences, Univ of Latvia, 19 Raina Blvd., Riga, LV 1586, Latvia
E-mail: linda_ansone@inbox.lv
1. Introduction
The role of pharmaceuticals in the urban water cycle is of rising concern. The problem is
increasing with population growth and increased use of pharmaceuticals, with strong impact
on aquatic ecosystems. It has been found that loading of pharmaceuticals in urban regions can
be of the same level as loading of pesticides. First degradation stage of excreted
pharmaceuticals and human metabolism products, takes place in wastewater treatment
facilities. During treatment of wastewaters as well as in waterways the binding of
pharmaceuticals and their degradation products to natural organic matter —humic substances
(HS) plays the major role.
An important and increasingly used group of pharmaceuticals contains bulky hydrophobic
structures, however the behavior of these substances in the environment has not been much
studied. Considering the wide application of some drugs, it may be particularly important to
study drugs containing tricyclic structures (adamantane derivatives), such as Rimantadine
(widely used in the prophylaxis and treatment of influenza A virus infections) and many
others. Substitutions at various locations on the ring will determine the pharmacokinetics and
biotransformation which could contribute to the overall activity including side effects [1].
Adamantane ring-containing substances are extraordinarily stable compounds, thus the study
of their fate in the environment could be of high importance during intensive use, for example,
during flu epidemics which may result in high concentrations in the environment [2].
2. Materials and Methods
The studied pharmaceuticals are summarized in Table. Binding of studied pharmaceuticals to
humic substances has been studied by fluorescence spectroscopy, FT IR, 1H NMR. To prove
the impact of the character of interaction between pharmaceuticals and humic substances also
biotests has been used.
To quantitatively characterize the interaction, fluorescence quenching approach has been used [3].
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15th IHSS Meeting- Vol. 3
Table: The studied pharmaceuticals
Substance
Formula
Rimantadine hydrochloride
Structure
H3C
C12H21N · HCl
1-(1-adamantyl)ethylamine
NH2
HCl
hydrochloride
NH2
2-Aminoadamantane hydrochloride
C10H17N · HCl
Phenibut
HCl
H
H 2N
C10H13NO2 · HCl
(4-Amino-3-phenyl-butanoic acid
HCl
hydrochloride)
3-Aminoquinuclidine dihydrochloride
C7H14N2 · 2HCl
NH2
N
2HCl
OH
1-Hydroxyadamantane
C10H16O
The intensity of the fluorescence quenching is supposed to be proportional to the
concentration of the formed humic-pharmaceutical complex according to the Stern-Volmer
equation. The fluorescence of the humic acids quenching mechanism by pharmaceuticals is
considered in the 1:1 interaction model. The binding constants are obtained by steady-state
fluorescence quenching measurements and are given as a slope in the Stern-Volmer plot and
can be calculated estimating I0/I - fluorescence intensity ratio of the initial substance and
fluorescence in the presence of quencher.
3. Results and Discussion
Fluorescence intensity quenching shows significant interaction between pharmaceuticals and
HS, which increase with HS concentration and solution pH. The character of the relationships
from modified Stern-Volmer plots (Fig. 1) offer strong support for the 1:1 complex (r2 >
0.99). Fluorescence quenching of 4-amino-3-phenyl butanoic acid, 1-hydroxyadamantane and
2-aminoadamantane by dissolved humic acid was described by nonlinear Stern-Volmer plots.
Vol. 3 Page - 398 -
15th IHSS Meeting- Vol. 3
45
2
R = 0,995
40
35
2
R = 0,992
30
2
I0/I
25
R = 0,990
20
15
10
5
0
0
10
20
30
40
50
60
CHA, mg/l
2-aminoadamantane HCl
4-amino-3-phenyl-butanoic acid HCl
1-Hydroxyadamantane
Figure 1: Stern-Volmer plots: the ratio of I0/I of Aldrich HA–pharmaceuticals as a function of CHA.
Cpharmaceuticals = 0.5 mmol/L
If the linear Stern-Volmer plot is indicative of a single class of fluorophores with equal
accessibility to the quencher [4], then a combination of dynamic and static quenching
typically produces a nonlinear Stern-Volmer plots as we found in our case.
Influence of ionic strength shows salting-out effect. The interaction with pharmaceuticals
much depends on the origin and structure of humic substances as it has been demonstrated
comparing humic acids from aquatic sources, soil, peat and reference materials. The
synchronous-scan fluorescence excitation emission spectra of humic substances are shown in
Figure 2 and they differ significantly depending on the origin of the humic acids: spectra of
highly humified HAs (IHSS reference humic acids: Leonardite HA, Pahokee peat HA, as well
as the industrially produced Aldrich HA) were characterized by two major fluorescence
peaks.
Aldrich HA
160
140
Intensity
100
80
Pahokee HA
Gagu HA
120
Leonarditte HA
Daugava HA
60
40
20
0
250
300
350
400
450
500
550
600
Wavelength, nm
Figure 2: Synchronous-scan fluorescence spectra of HA used in the study (CHA = 25 mg/L, pH 7)
Vol. 3 Page - 399 -
15th IHSS Meeting- Vol. 3
Aquatic humic acid isolated from water of the River Daugava was characterized by one peak
(~ 375 nm), but peat humic acid (isolated from Gagu Sphagnum bog peat) was characterized
by two peaks (~ 345 and 375 nm). Synchronous scan spectra of humic substances isolated
from highly humified organic material (leonardite, coal) had an intensive fluorescence peak ~
475 nm that was determined by the presence of conjugated unsaturated bond systems bearing
carbonyl and carboxyl groups (substituting aromatic core structures), but its intensity differed
with respect to aromaticity of the humic acid selected [5].
We have determined the binding constants between HA and selected pharmaceuticals by
fluorescence quenching technique. It is found that amongst the studied adamantine group
pharmaceuticals and their precursors the Rimantadine has markedly larger binding constants
than their structural analogues. This difference is explained by electrostatic interaction
between HA and studied pharmaceuticals. Our findings suggest that an electrostatic
interaction plays a dominant role in the complex formation between humic acids and
pharmaceuticals. The importance of the electrostatic attraction between humic acids and
pharmaceuticals was also confirmed by a salt effect and pH dependence of the fluorescence
quenching. The electrostatic interaction between cationic pharmaceuticals (phenibut) and HA
is weakened at low pH, resulting in decrease in the binding constants.
4. Conclusions
It can be supposed the hydrophobic pharmaceuticals can cause aggregation of humic
molecules, changing their micellar behavior. The obtained results support development of
understanding of fate of pharmaceuticals in the environment as well as development of
analytical methods for analysis of pharmaceuticals in waters and medical wastewater
treatment approaches.
5. References
1. Hoffman C.E. Structure, activity and mode of action of amantadine HCl and related compounds.
Antibiot. Chemother. 27, (1980), 233–250.
2. Scholtisek C. & Webster R.G. Long-term stability of the anti-influenza A compounds-amantadine
and rimantadine. Antiviral Res. 38, (3), (1998), 213–215.
3. Nakashima K., Maki M., Ishikawa T., Yoshikawa T., Gong Y.K. & Miyajima T. Fluorescence
studies on binding of pyrene and its derivatives to humic acid. Spectrochim. Acta Pt. A. 67,
(2007), 930–935.
4. Gadad P., Lei H. & Nanny M.A. Characterization of noncovalent interactions between 6propionyl-dimethylaminonapthalene (PRODAN) and dissolved fulvic and humic acids. Water.
Res. 41, (2007), 4488–4496.
5. Peuravuori J., Koivikko R. & Pihlaja K. Characterization, differentiation and classification of
aquatic humic matter separated with different sorbents: synchronous scanning fluorescence
spectroscopy. Water Res. 36 (18), (2002), 4552–4562.
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15th IHSS Meeting- Vol. 3
Characterization of Soil Organic Matter of Treated Sewage Effluent
Irrigated Areas
Bruno Henrique Martinsa,b*, Larissa Macedo dos Santosa,c, Débora Marcondes Bastos Pereira
Miloria, Ladislau Martin-Netoa, Célia Regina Montesd
a
b
Embrapa Instrumentação Agropecuária, C.P.741, CEP: 13560-970, São Carlos, SP, Brasil;
Universidade de São Paulo, Instituto de Química de São Carlos (IQSC/USP), C.P.780, CEP:
13560-250, São Carlos, SP, Brasil; cUniversidade Federal de São Carlos (UFSCar),
Departamento de Química, CEP: 13565-905, São Carlos, SP, Brasil; dUniversidade de São
Paulo, Centro de Energia Nuclear na Agricultura (CENA/USP), C.P. 96, CEP: 13416-000,
Piracicaba, SP, Brasil;
E-mail: brunohm@cnpdia.embrapa.br; larissa@cnpdia.embrapa.br;
debora@cnpdia.embrapa.br; martin@cnpdia.embrapa.br, crmlauar@usp.br
1. Introduction
The increasing on water sources demand in the cities has done man seek different sources for
irrigation of crops, since agricultural activity consumes a large amount of this resource.
According to data from [6], it is estimated that approximately 65% of the water amount
available in the national territory is targeted to the practice of irrigation of crops, while only
about 17% is aimed for human consumption. This situation is worrying, once Brazil is a
country with intense agricultural activity.
To [4] water represents a development limiting natural resource, both in agricultural and
industrial activities, and has its quality breakdown by misuse and pollution, largely generated
by direct discard of raw and treated effluents in water courses.
According to research accomplished by SABESP (Companhia de Saneamento Básico do
Estado de São Paulo), only the city of São Paulo, by its STS (sewage treatment station),
generates nearly 3,000 L·s-1 of TSE (treated sewage effluent). However, in the National Policy
of Hidric Resources, there is no regulamentation about the use of waste water (such as TSE)
in any activities.
In this way, the purpose of the following study is to evaluate the soil organic matter (SOM) of
irrigated areas, comparing to non-irrigated area, analyzing about the sustainability of the TSE
use in agricultural soils instead of water, as a contribution in a bigger thematic project.
2. Materials and Methods
This study is part of a multidisciplinary research group of thematic project about use of
sewage effluent treated by biological process (stabilization pounds, UASB reactor / activated
sludge) in agricultural soils, sponsored by Fundação de Amparo à Pesquisa do Estado de São
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15th IHSS Meeting- Vol. 3
Paulo – and coordinated by Prof. Dr. Adolpho José Melfi (CENA-ESALQ/USP).
The project was initiated in January, 2003, and the experimental field was installed near to the
STS in the city of Lins – SP, operated by SABESP, under cultivation of grazing and
sugarcane. In this study were analyzed soil samples from the sugarcane area. The city STS is
Australian kind (primary treatment in anaerobic pounds and secondary treatment in optional
photosynthetic pounds) with flow rate of 140 L·s-1 and with mostly domestic sewage. The
experimental arrangement of the sugarcane area was comprised by five treatment blocks with
four repetitions. The irrigation with TSE was performed according to soil humidity, as it
follows: SI: soil non-irrigated with TSE; 100: soil irrigated with TSE and soil humidity in the
same level of field capacity; 125: soil irrigated with TSE and soil humidity 25% above field
capacity; 150: soil irrigated with TSE and soil humidity 50% above field capacity and 200:
soil irrigated with TSE and soil humidity 100% above field capacity. In this study were
analyzed samples of the SI, 100 and 200 conditions. The soil samples were randomly
collected, in three repetitions by analyzed condition, in May, 2006, in the depth till 100 cm,
dried at room temperature and subsequently sieved at 0.5 mm mesh.
The carbon content analyses were carried out by dry combustion [5] in a LECO CN-2000
instrument, belonging to the Laboratório de Biogeoquímica of CENA/ESALQ. The LIF
(laser-induced fluorescence) analyses were carried out according [3], in an instrument
belonging to Embrapa Instrumentação Agropecuária.
3. Results and Discussion
The carbon content analyses for samples for the three different conditions examined showed
decrease, in all depth consider, being more pronounced in areas subject to TSE irrigation,
mainly in the 200 condition. The data obtained are illustrated in Table 1. This decrease is
probably attributed to labile carbon fraction degradation, caused by the increase in microbial
activity related to the action of TSE in the soil.
According to [1], the use of TSE as irrigation source may alterate the organic matter
degradation rate, causing a decrease in the soil carbon content. The authors also remind that it
may cause an alteration in the soil carbon cycling process.
Table 1: Carbon content obtained for soil samples subjected to three different types of treatment
SI
100
200
0–10
0.97±0.01
0.88±0.01
0.86±0.01
10–20
0.96±0.01
0.86±0.01
0.82±0.01
20–40
0.74±0.01
0.70±0.02
0.70±0.01
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40–60
0.64±0.01
0.54±0.01
0.58±0.02
60–80
0.54±0.01
0.53±0.01
0.52±0.01
80–100
0.46±0.01
0.42±0.03
0.44±0.01
15th IHSS Meeting- Vol. 3
This situation is worrying since represents, among other factors, loss of SOM, that may cause
limitations in soil fertility and structure (taking into consideration its importance to the soil and culture
and when it comes to a soil with less than 1% carbon content), and possible carbon loss as CO2,
causing increase in atmospheric greenhouse gases concentration, negatively contributing to the global
warming scenario.
The obtained data by LIF spectroscopy, showed by Table 2 and Figure 1, are complementary to the
carbon content data obtained, achieving excelent data correlation.
Table 2: Humification degree (HFIL) obtained by Laser-Induced Fluorescence for soil samples
subjected to three different types of treatment
SI
100
200
0–10
527±5
548±6
574±4
10–20
487±4
566±9
635±5
20–40
718±43
831±16
795±42
40–60
990±7
1269±6
1141±15
60–80
1254±29
1289±29
1328±44
80–100
1568±77
1868±112
1711±154
In this way, its suggested that, in this case, the TSE use as irrigation source leads to an alteration in the
soil organic matter degradation process, probably due to an increase in the soil microbial activity and,
consequently, more labile carbon fraction degradation (as seen in the carbon content results),
remaining the most recalcitrant organic matter fraction, harder to degrade. This most recalcitrant
fraction leads to an increase in the organic matter humification degree, as detected by LIF.
Figure 1: Humification degree (HFIL) graphics obtained by Laser–Induced Fluorescence for soil
samples subjected to three different types of treatment
However, the decrease in the organic matter carbon content was verified even with conventional
irrigation with water, comparing to irrigation with TSE, in the same experimental field. In this way, it
may be suggested that the soil itself has this intrinsic characteristic of organic content loss, probably
due to increase in the soil microbial activity (even when irrigated with water), which is more
accentuated when irrigated with TSE.
Otherwise, in his study about stability of organic carbon in deep soil layers controlled by fresh carbon
Vol. 3 Page - 403 -
15th IHSS Meeting- Vol. 3
supply, [2], observed that changes in agricultural practices, increasing the distribution of fresh carbon
at depth, could lead to a loss of ancient soil carbon, which affects the present soil carbon content,
promoting the priming effect in soil.
4. Conclusions
Proper weights and discussions about the results for the samples analyzed, it is concluded that, to soil
conditions analyzed, the employment of TSE replacing the water used in agricultural activities for
irrigation of crops is worrying and may bring limitations on soil structure and fertility, as evidenced by
more pronounced decrease on the contents of SOM in samples for the areas under adding TSE, taking
into account that is a soil with less than 1% carbon content and has negative response even to
conventional irrigation with water.
It is suggested that, in this case, irrigation with TSE causes the priming effect in soil, observed by
more accentuated decrease of carbon content in samples of the irrigated areas, comparing to the nonirrigated area, what may become an environmental problem.
Between the three irrigation conditions analyzed, it is verified a more pronunciated effect in the
organic matter of samples belonging to the 200 condition (soil irrigated with TSE and soil humidity
100% above field capacity), as it showed by the lower carbon content and higher humification degree,
comparing to the SI and 100 conditions.
However, the experiments must continue to confirm and validate the initials tendencies detected, and
to search new alternatives for soil and culture tillage to make possible the TSE use and application in
sustainable conditions.
References
1. Falkiner, R.A.; Smith, C.J. 1997. Change in soil chemistry in effluent-irrigated Pinus radiata and
Eucalyptus grandis. Australian Journal of Soil Research, 35: 131–147.
2. Fontaine, S.; Barot, S.; Barré, P.; Bdioui, N.; Mary, B.; Rumpel, C. 2007. Stability of organic
carbon in deep soil layers controlled by fresh carbon supply. Nature, 450: 277–281.
3. Milori, D.M.B.P.; Galeti, H.V.A.; Martin-Neto, L.; Dieckow, J.; González-Pérez, M.; Bayer, C.;
Salton, J. 2006. Organic matter study of whole soil samples using laser-induced fluorescence
spectroscopy. Soil Science Society American Journal, 70: 57–63.
4. Montes, C. R.; Fonseca, A. F.; Melfi, A. J.; Gloaguen, T.; Mendonça, F. C.; Pivelli, R. P.; Herpin,
U.; Santos, A. P. R.; Forti, M. C.; Lucas, Y.; Mounier, S.; Carvalho, A.; Almeida, V. V.;
Cardinalli, C. G.; Steffen, T.; Monteiro R. C. 2004. Agricultural use of stabilization pond effluent:
a case study in the city of Lins (SP, Brazil). In: Martin-Neto, L.; Milori, D. M. B. P.; Silva, W. T.
L. (Eds.). Humic substances and soil and water environment, São Carlos: EMBRAPA. p. 732–
734.
5. Nelson, D.W.; Sommers, L. E. 1996. Total carbon, organic carbon, and organic matter. In: Sparks,
D. L. (Ed.). Methods of soil analysis: chemical methods, Madison: Soil Science Society of
America/American Society of Agronomy. p.961–1010.
6. Tucci, C. E. M. 2001. Gestão de água no Brasil. Brasília, UNESCO. 156 p.
Vol. 3 Page - 404 -
15th IHSS Meeting- Vol. 3
Modelling Differential Absorbance Spectra of SRFA During Complexation
with Copper and Lead
Deborah J. Dryera*, Gregory V. Korshina, Marc F. Benedettib
a
Department of Civil and Environmental Engineering, University of Washington, Box
352700, Seattle, Washington, 98195-2700, USA; bUniv. Paris Diderot-Paris 7-IPGP,
Laboratoire de Géochimie des Eaux, Paris, 75205, France
E-mail: ddryer@u.washington.edu
1. Introduction
Natural organic matter (NOM) is a component of all natural and engineered environmental
systems. NOM controls the speciation and distribution of many heavy metals in aquatic
systems and exerts a strong influence on the release of metal corrosion by-products in
drinking water systems [1, 2]. In order to understand the interactions of NOM with metals,
precise measurements of NOM properties like stability constants and binding capacities of
NOM for protons and metal cations are required. The complex nature of NOM makes this a
very difficult task. NOM has properties which are strongly dependent on local
biogeochemical and treatment conditions and contains a diverse array of functional groups
and molecular sizes [3, 4]. NOM complexation behavior has been studied by a wide variety of
techniques. However, because these techniques frequently require large amounts of preconcentrated NOM samples, a need remains for an efficient means to quantify important
NOM properties and behaviour in situ.
Because NOM is the major light-absorbing component of natural waters in the 200-800 nm
range [5], optical spectroscopy has long been used to assess the NOM content of many water
sources. However, the wide variety of light-absorbing functional groups (chromophores) in
NOM results in absorbance spectra which are broad and featureless [6]. Consequently,
interpretation of these spectra has traditionally been limited to using particular spectral
parameters (such as absorbance at 254 nm, absorbance ratios, or SUVA254) as surrogates for
NOM properties like concentration, aromaticity, and molecular size [7-9].
Differential absorbance spectroscopy (DAS) is an alternative method for the interpretation of
absorbance spectral data that focuses on changes in spectra associated with evolving reaction
conditions which affect NOM chemistry. This technique reveals changes which are not
apparent, or do not exist, in the raw absorbance spectra. DAS is capable of detecting very
subtle changes in NOM chemistry and this high sensitivity eliminates the need for sample pretreatment and makes it a promising technique for in situ elucidation of NOM behaviour. In
prior research, this technique has been applied to understanding NOM protonation behaviour
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15th IHSS Meeting- Vol. 3
[10]. In this study, DAS is applied to the exploration of the complexation behaviour of
standard Suwannee River fulvic acid.
It is also desirable to have a means to model and predict the metal complexation behaviour of
fulvic acids. While many models have been developed and utilized, the NICA-Donnan model
has emerged as a very useful means for fitting and predicting complexation behavior of fulvic
acids [11, 12]. This model uses a continuous distribution of binding site affinities to model the
direct complexation of metal cations to NOM binding sites, and models the indirect
electrostatic interactions between NOM and metal cations through distribution of potential
throughout a uniform Donnan gel phase. This study uses the NICA-Donnan model for metal
complexation to fulvic acids to fit the differential absorbance spectra generated during
complexation of SRFA with metals.
2. Methods
A Suwannee River fulvic acid (SRFA) sample was obtained from the International Humic
Substances Society (Sample # 1S101F). SRFA solutions were prepared at DOC
concentrations of 5 mg/L, and background ionic strength was established by including 0.01 M
NaClO4. The pH of NOM solutions during titrations was controlled through addition of
appropriate amounts of HClO4 or NaOH, and the volume changes associated with these
additions of acid and base were corrected for in the final data analysis. Metal concentrations
were adjusted between 0 – 1000 μg/L Cu2+ or Pb2+ through addition of small volumes of
dilute CuSO4 and PbClO4 solutions. Absorbance spectra were recorded in a 5 cm quartz cell
on a Perkin-Elmer Lambda 18 UV/Vis Spectrophotometer. Differential absorbance data
fitting to model calculations was done using visual MINTEQA.
3. Results and Discussion
Absorbance spectra for SRFA at varying metal concentrations retain the same broad and
featureless shape throughout the entire range of the titrations. However, the differential
spectra for these titrations show the emergence of clear features, as illustrated by Figure 1.
They indicate that subtle changes of the absorbance of SRFA can be detected even at copper
concentrations as low as 5 µg/L. At higher CuTOT values, the presence of several bands
becomes notable, the most prominent of which have maxima at 245 and 390 nm.
Correlations between the amount of copper bound by SRFA and changes in absorbance were
calculated using by examining the relative change of the ratio of absorbances at 350 and 390
nm (denoted as (ΔRA/RA), The selection of the wavelength of 390 nm is due to the presence of
Vol. 3 Page - 406 -
15th IHSS Meeting- Vol. 3
a distinct Cu-differential spectra feature of SRFA at all pH with maximum close 390 nm.
ΔR A
RA
The ΔRA / RA parameter is defined as:
In this expression, terms
( A390 / A350 )Cu
⎛ ⎛ A390
⎜⎜
⎜ ⎜⎝ A350
= ⎝
and
⎞
⎛ A
⎟⎟ − ⎜⎜ 390
⎠ Cu ⎝ A350
⎛ A390 ⎞
⎜⎜
⎟⎟
⎝ A350 ⎠ 0
⎞ ⎞
⎟⎟ ⎟
⎠ 0 ⎟⎠
( A390 / A350 )0 correspond to the ratio of absorbances
at 390 and 350 nm at any selected total copper concentration and its absence, respectively.
This ratio is introduced in order to more clearly illustrate the emergence of the particular band
with a maxima near 390 nm which emerges only for Cu concentrations greater than ~40 μg/L.
0.005
-1
0.004
-1
differential absorbance (cm mg L)
0.0045
0.0035
0.003
0.0025
1000 μg/L
0.002
500 μg/L
0.0015
200 μg/L
0.001
5 μg/L
0.0005
0
200
250
300
350
400
450
wavelength (nm)
Figure 1: Differential absorbance spectra of SRFA with selected copper concentrations at pH 6
To understand the nature of these phenomena in more detail, the complexation of copper by
SRFA was modeled using visual MINTEQA program that incorporates generic SRFA
protonation and complexation constants. The SRFA-bound copper determined in these
calculations was predominated the copper bound by the operationally defined phenolic and
carboxylic groups, while the copper bound via Donnan gel interactions was determined to be
negligible in comparison. The strong correlation between the modeled bound copper
concentration and the (A390/A350)Cu ratio is illustrated in Figure 2.
Change of A390/A350 ratio
0.21
0.18
0.15
R2 = 0.97
pH 5
pH 6
pH 7
pH 8
0.12
0.09
0.06
0.03
0.00
0.0E+00
2.0E-06
4.0E-06
6.0E-06
8.0E-06
1.0E-05
1.2E-05
Total NOM-bound copper (M)
Figure 2: Correlation between total predicted NOM-bound copper and changes in ΔRA/RA index
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15th IHSS Meeting- Vol. 3
4. Conclusions
This study examined the differential absorbance spectra of metal complexation with
Suwannee River fulvic acid. While absorbance spectra show a very subtle change in intensity
with increasing metal concentrations, they retain their characteristic featureless shape
throughout the titrations. Differential absorbance spectra, on the other hand, show a number
of features that are not present in the traditionally interpreted absorbance spectra. These
features can be analyzed to reveal information about the nature of complexation-active
chromophores in NOM. The differential absorbance spectra of SRFA can be successfully
correlated to bound metal concentrations calculated based on the NICA-Donnan model
theory. This study demonstrates that differential absorbance spectroscopy is a useful method
to examine in situ the complexation behaviour of NOM. DAS can potentially provide detailed
information about the emergence and contribution of different complexation-active functional
groups and has the potential to elucidate the presence of functionalities which may not be
detectable in traditional potentiometric experiments. In principle, this technique could allow
the obtainment of detailed in situ information about NOM chemistry and reactivity caused by
a wide variety of environmental and water treatment processes.
References
1. Korshin, G. V.et. al. Influence of NOM on copper corrosion,Journal of the American Water
Works Association 1996, 88, 36–47.
2. Edwards, M.; Sprague, N. Organic matter and copper corrosion by-product release: A mechanistic
study,Corrosion Science 2001, 43, 1–18.
3. Croue, J.-P.et. al. Isolation, fractionation, and characterization of natural organic matter in
drinking water, AWWA Research Foundation, 2000.
4. Leenheer, J. A.; Croue, J.-P. Characterizing aquatic dissolved organic matter,Environ. Sci.
Technol. 2003, 37, 18A–26A.
5. Stewart, A. J.; Wetzel, R. G. Fluorescence: Absorbance ratios –a molecular-weight tracer of
dissolved organic matter,Limnol. Oceanogr. 1980, 25, 559–564.
6. Del Vecchio, R.; Blough, N. V. On the origin of the optical properties of humic
substances,Environ. Sci. Technol. 2004, 38, 3885–3891.
7. Chin, Y.-P.et. al. Molecular weight, polydispersity, and spectroscopic properties of aquatic humic
substances,Environ. Sci. Technol. 1994, 28, 1853–1858.
8. Novak, J. M.et. al. Estimating the percent aromatic carbon in soil and aquatic humic substances
using ultraviolet absorbance spectroscopy,J. Environ. Qual. 1992, 21, 144–147.
9. Peuravouri, J.; Pihlaja, K. Molecular size distribution and spectroscopic properties of aquatic
humic substances,Analytica Chimica Acta 1997, 337, 133–149.
10. Dryer, D. J.et. al. In situ examination of the protonation behavior of fulvic acids using differential
absorbance spectroscopy,Environ. Sci. Technol. 2008, 42, 6644–6649.
11. Kinniburgh, D. G.et. al. Metal ion binding by humic acid: Application of the NICA-Donnan
model, Environ. Sci. Technol. 1996, 30, 1687–1698.
12. Benedetti, M. F.et. al. Metal ion binding by natural organic matter: From the model to the
field,Geochim. Cosmochim, Acta 1996, 60, 2503–2513.
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15th IHSS Meeting- Vol. 3
Behavior of Soil Carbon in Amended Areas with Sewage Sludge
Bruno Henrique Martinsa,b, Tânia Leme de Almeidaa*, Sérgio Gaiadc, Débora Marcondes
Bastos Pereira Miloria, Ladislau Martin-Netoa
a
b
Embrapa Instrumentação Agropecuária, C.P.741, CEP: 13560-970, São Carlos, SP, Brasil;
Instituto de Química de São Carlos, Univ. de São Paulo (IQSC/USP), C.P. 780, CEP: 13560250, São Carlos, SP, Brasil;cEmbrapa Florestas, C.P. 319, CEP: 83411-000, Colombo, PR,
Brasil
E-mail: brunohm@cnpdia.embrapa.br; tlalmeida@yahoo.com.br; gaiad@cnpf.embrapa.br;
debora@cnpdia.embrapa.br; martin@cnpdia.embrapa.br
1. Introduction
The Greenhouse Effect, an Earth’s natural and essential phenomenon, lately has been
increased by the anthropogenic intervention, by fossil fuel burning, deforestation, and mostly
wrong agricultural tillage, leading to an increase in the atmospheric gases that causes this
effect.
Correct soil tillage and forestry practices are considered important tools to promote decrease
of emissions of GHG and mitigating of Greenhouse Effect through soil carbon sequestration.
Soil represents is the third greater pool of carbon in the planet. It is estimated that there is
approximately 2300 Petagrams of carbon in soils, which represents nearly three times the
atmospheric carbon concentration.
Sewage sludge (SS) plays an important role as soil fertility improver because it contains high
levels of organic matter and nutrients [1]. However, its application requires careful
monitoring to avoid soil contamination and changes in organic matter that could cause serious
implications for the crop where it is applied.
It is related that SS plays an important role as soil fertility improver, hence it is a wastewater
treatment product and has the potential to enhance soil productivity, as it contains high levels
of organic matter and nutrients [1].
In this way, the purpose of the following study is to evaluate the SOM of SS amended areas,
comparing to no amended area, analyzing about the sustainability of its use in forestry
systems as a tool for mitigating GHG emissions and sequestrating atmospheric carbon.
2. Materials and Methods
The experimental field under eucalyptus plantation is installed in two different farms (Entre
Rios and Areona) in the city of Itatinga, São Paulo state. Each farm was divided in two
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different areas, according to the SS amendment. The soil profile in the Entre Rios farm was
characterized as an Oxisoil, with a clay content varying between 16 and 20%, while the
Areona farm soil is characterized as Quartzarenic Neosoil, with a clay content varying
between 5 and 12%. The sample identification according to the SS amendment was performed
as it follows: ER 60 (Entre Rios farm and SS amended), ER 228 (Entre Rios farm and no SS
amended), AN 254 (Areona farm and SS amended) and AN 36 (Areona farm and no SS
amended).
In December, 2009, soil samples were collected in three repetitions in the depths 0–10 and
10–20cm. They were dried at room temperature and subsequently sieved at 0.5 mm mesh. The
SS amendment occurred ten days before the plantation, at a volume between 1500 and 2000
kilograms per hectare.
Carbon content measurements were carried out by an elemental analyzer Carlo Erba EA1110 instrument. The measurements were made in triplicate, taking into account depth and
condition analyzed in both farms.
3. Results and Discussion
The carbon content results for Entre Rios and Areona farm, as well as some soil physical
characteristics and eucalyptus plantation beginning, are shown in Table 1.
In the case of the Entre Rios Farm, probably SS amendment increased the microbial activity
in soil by high availability of fresh organic matter [4] what, in a second stage, must have
triggered a soil carbon decrease by degradation of stable fractions [3].
According to [3], in their study about stability of organic carbon in soils, increasing of fresh
organic matter at depth, could lead to a loss of ancient soil carbon, which leads a decreasing
of total soil carbon content as a function of time, promoting the priming effect in soil.
This situation in the Entre Rios farm is worrying since represents, among other factors, loss of
soil organic matter by microbial activity, which may cause limitations in soil fertility and
structure, and possible carbon loss as CO2, causing increase in atmospheric GHG
concentration, negatively contributing to the global warming scenario.
The results obtained for the samples of the Areona farm showed an inverse behavior
comparing to the samples of the Entre Rios farm. It was observed that in the SS amended area
the values of carbon content were higher than in the no amended area, in all analyzed depths.
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Table 1: Carbon content results obtained for soil samples from Entre Rios and Areona Farm. ER 228:
no SS amended, ER 60: SS amended, AN 36: no SS amended and AN 254: SS amended
Eucalyptus
SAMPLE
C Content
Soil Profile
Clay Content
ER 228 0–10 I
1.85±0.02
Oxisoil
16 – 20 %
Nov/2004
ER 228 0–10 II
0.90±0.01
Oxisoil
16 – 20 %
Nov/2004
ER 228 0–10 III
0.98±0.03
Oxisoil
16 – 20 %
Nov/2004
ER 228 10–20 I
1.21±0.01
Oxisoil
16 – 20 %
Nov/2004
ER 228 10–20 II
0.80±0.04
Oxisoil
16 – 20 %
Nov/2004
ER 228 10–20 III
0.67±0.02
Oxisoil
16 – 20 %
Nov/2004
ER 60 0–10 I
0.84±0.03
Oxisoil
16 – 20 %
Apr/2004
ER 60 0–10 II
0.84±0.02
Oxisoil
16 – 20 %
Apr/2004
ER 60 0–10 III
1.18±0.04
Oxisoil
16 – 20 %
Apr/2004
ER 60 10–20 I
0.37±0.02
Oxisoil
16 – 20 %
Apr/2004
ER 60 10–20 II
0.45±0.01
Oxisoil
16 – 20 %
Apr/2004
ER 60 10–20 III
0.60±0.03
Oxisoil
16 – 20 %
Apr/2004
AN 36 0–10 I
0.57±0.03
Quartzarenic Neosoil
5 – 12 %
Mar/2008
AN 36 0–10 II
0.67±0.02
Quartzarenic Neosoil
5 – 12 %
Mar/2008
AN 36 0–10 III
0.70±0.01
Quartzarenic Neosoil
5 – 12 %
Mar/2008
AN 36 10–20 I
0.41±0.05
Quartzarenic Neosoil
5 – 12 %
Mar/2008
AN 36 10–20 II
0.46±0.01
Quartzarenic Neosoil
5 – 12 %
Mar/2008
AN 36 10–20 III
0.45±0.02
Quartzarenic Neosoil
5 – 12 %
Mar/2008
AN 254 0–10 I
0.71±0.01
Quartzarenic Neosoil
5 – 12 %
May/2008
AN 254 0–10 II
0.74±0.03
Quartzarenic Neosoil
5 – 12 %
May/2008
AN 254 0–10 III
0.75±0.02
Quartzarenic Neosoil
5 – 12 %
May/2008
AN 254 10–20 I
0.53±0.03
Quartzarenic Neosoil
5 – 12 %
May/2008
AN 254 10–20 II
0.61±0.04
Quartzarenic Neosoil
5 – 12 %
May/2008
AN 254 10-20 III
0,54±0,01
Quartzarenic Neosoil
5 – 12 %
May/2008
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Plantation
15th IHSS Meeting- Vol. 3
Anyway, these results showed an interesting behavior. The soil in Areona is characterized as
Quartzarenic Neosoil (a recently generated soil with amounts of quartz in its composition).
This kind of soil has a major fraction of sand and low carbon concentration in your profile.
This way, it was not expected strong interaction between SS and the organic matter of this
soil. However, it was noted that soil tillage with SS amendment in the Areona farm leads to a
better carbon accumulation when compared with the same tillage in the Entre Rios farm.
It is important to note that the decrease in the carbon content in the Entre Rios farm is
potentialized and accentuated by the SS amendment, probably, due to an increase in the
microbial activity in soil.
However, it is important to evaluate if this difference was due to different soil properties for
each farm or if may be attributed to period of the experiment. Eucalyptus plantation began in
2004 in Entre Rios Farm and in 2008 in the Areona farm.
4. Conclusion
Studies of soil amendment using SS are very important to avoid negative environmental
consequences, as soil contamination or decreasing of carbon content in soil. This kind of
residues management can be positive depending on the situation, and for this reason each case
needs to be carefully studied.
Thus, field experiments must continue in both farms to confirm and validate the initials
tendencies detected. New alternatives for soil tillage in forestry systems can be suggested to
make possible SS soil amendment in sustainable conditions with environmental benefits.
References
1. Arraigada, C.; Sampedro, I.; Garcia-Romero, I.; Ocampo, J. Sci. Total Environ., 407 (2009).
2. EMBRAPA, Brazilian System of Soil Classification, National Center of Soil Research, Rio de
Janeiro, 1999. p. 412.
3. Fontaine, S.; Barot, s.; Barré, P.; Bdioui, N.; Mary, B.; Rumpel, C., Nature, 450 (2007) 8.
4. Martins, E. L.; Coringa, J. E. S.; Weber, O. L. S., Acta Amazonica, 39 (2009) 3.
Vol. 3 Page - 412 -
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Gold(III) and Nanogold Interaction with Humic Acids: Spectrophotometry,
Capillary Electrophoresis and Mass Spectrometric Study
Nagender Reddy Panyalaa, Eladia Mª Peña-Méndezb, Josef Havel a,c*
a
Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 61137
Brno, Czech Republic; bDepartment of Analytical Chemistry, Nutrition and Food Chemistry,
Faculty of Chemistry, University of La Laguna, Campus de Anchieta, 38071 – La Laguna,
Tenerife, Spain; cDepartment of Physical Electronics, Faculty of Science, Masaryk
University, Kotlarska 2, 61137 Brno, Czech Republic
E-mail: havel@chemi.muni.cz
1. Introduction
Humic substances (HS) naturally occurring in soils and waters are usually divided into humic
acids (HA) soluble at high pH and insoluble in acids, fulvic acids (FA) soluble at all pH
values and insoluble humin. HA are extensively studied [1-3] but their real structure is still
not completely known. HA easily aggregate, show high complexing ability towards all metal
ions but also interact with various organics and xenobiotics [3], are also interacting with
minerals and gold as it is known for a long time [4, 5]. High attention is paid to the role of HA
in the transport of the elements including platinum group metals and gold in the environment
[6]. Gold in various forms interacts with HA [7] and the very first remark about this is known
since 1900 [8]. However, the interaction is by far not sufficiently explained and not
completely understood. Therefore, the aim of this work is to study and elucidate the
interaction of HA with Au(III) and metallic gold in the form of gold nano-particles, as this
knowledge might be quite important to explain gold migration in the environment and for
gold mining industry.
2. Materials and Methods
The HA used in this work were Soil HA standard (IS102H) and Leonardite HA standard
(IS101H) from International Humic Substances Society (IHSS) and coal-derived Czech HA
standard [9]. Gold(III) chloride as HAuCl4·3H2O was from Sigma-Aldrich (Steinheim,
Germany). Gallic acid was from Lachema (Brno, Czech Republic). Deionized water was
double-distilled from a quartz apparatus purchased from Heraeus Quartzschmelze (Hanau,
Germany). All other reagents were of analytical grade purity. Mass spectra were measured
using MALDI instrumentation of Kratos Shimadzu (Manchester, UK) and/or MALDI-TOF
Auto-flex mass spectrometer of Bruker Daltonics (Bremen, Germany). CE was carried out on
the SpectraPhoresis 2000 Thermo Separation Products (Fremont, CA, USA).
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3. Results and Discussion
Interaction of Au(III) with HA. HA are reducing Au(III) to metallic gold quite rapidly. An
example of kinetic spectra concerning reduction process is given in Figure 1 A, B. Figure 1 B
is showing that in the first stage the reaction is faster and later on slower. Figure 2 illustrates
details of kinetic spectra (for another HA preparation) [9]. Two isosbestic points and two new
absorption bands developed (550 and 580 nm) indicate that at least two different reactions
are going on.
B
2.5
‫ג‬1 max ‫ג‬2 max
2
A
2
Time
1.5
1
Absorbance
Absorbance (AU)
2.5
391nm
500nm
568nm
580nm
640nm
1.5
1
0.5
0.5
0
HA only
0
400
450
500
550
600
650
700
0
50
100
150
200
250
300
time (min)
750
Wavelength (nm)
Figure 1: A, B The kinetic spectra concerning of the reduction of HAuCl4 by HA. Conditions:
pH = 4.6, Total concentrations: HA = 0.12 and Au(III) = 1.6 mM
1.6
A
B
1.1
1st isosbestic point
Absorbance
Absorbance
1.5
1.4
1.2
1.3
1.2
1
2nd isosbestic point
0.9
1.1
1
253
258
263
268
0.8
265
Wavelength (nm)
275
285
295
305
315
325
Wavelength (nm)
Figure 2: A, B. The kinetic spectra concerning the reduction of HAuCl4 by HA (Chemapex standard).
pH = 2.9; Total concentrations: HA = 0.03 and Au(III) = 0.3 mM
Electrophoretic study of nanogold interaction with HA. Gold (III) and aqueous nanogold
solutions were mixed with HA in various ratios. Nanogold was prepared using gallic acid
(model compound of HA) as a reducing agent. The electrophoretic separation was done in
borate buffer (pH 9.6). Selected examples of electropherograms are given in Fig. 3. HA
show mostly just one high “hump” of the HA aggregate, in agreement with the literature
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15th IHSS Meeting- Vol. 3
[9]. Even if HA are negatively charged they migrate towards the cathode due to high
electroosmotic flow (EOF). Figure 3 B concerning a mixture of nanogold and HA solution
shows that there are several peaks observed. This is probably due to the interaction of
different size nanogold with different HA fractions.
0.009
0.007
A
0.012
0.007
Absorbance (mAU)
Absorbance (mAU)
0.017
EOF
0.002
-0.003
0.5
1.5
2.5
3.5
4.5
5.5
Migration time (min)
6.5
7.5
0.005
0.003
B
EOF
0.001
-0.001 0.5
1.5
2.5
3.5
4.5
5.5
6.5
7.5
-0.003
Migration time (min)
Figure 3: A Capillary electrophoretic separation of HA. B Electropherogram concerning a mixture
(nanogold+HA). Conditions: 20 mM borate buffer (pH 9.6); Total concentrations: HA = ~ 0.16 and
Au(III) = ~ 1.6 mM)
Mass spectrometry. Interaction of gold (III) and nano-gold with HAs was followed by mass
spectrometry using a commercial MALDI instrumentation where mass spectra were obtained
in Laser Desorption Ionization (LDI) mode, i.e. using no matrix. However, under these
conditions in LDI mass spectra only the formation of Aun clusters and adducts of gold with
low molecular weight HA fragments (215 and 347 Da) were observed. It seems that either the
stability of {Aun, HA} is low or such adducts are decomposed in the process of ionization.
4. Conclusions
It was found by spectrophotometric kinetic study that the reduction of Au(III) with HA is
relatively fast in wide pH range. The redox reaction proceeds at least in two main steps and
formed nanogold is stabilized in HA solution similar like with citrate but at elevated gold
concentration a kind of {Aun, HA} aggregate is anyway precipitated. The interaction of
nanogold with HA as studied by electrophoresis was found to be relatively weak. But the
formation of several peaks indicates the complexicity of the reaction mixture. Mass
spectrometry indicates that nanogold is forming adducts of gold with some low molecular
weight compounds. Possible explanation is that supramolecular {Aun, HA} aggregates might
be decomposed in the process of laser desorption ionization.
Taking into account available literature data and the results achieved we can conclude that the
both reaction of Au(III) with HA and interaction of nanogold with HA are quite complex. We
suggest that reduction of Au(III) with HA goes in several steps via Au(III) and Au(II) and
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Au(I) to at least 2 different size of Aun clusters. Complexation reaction with Au(II) and Au(I)
with HA fractions is not excluded. However, to prove that and to prove also possible
formation of {Aun, HA}supramolecules needs further intensive research.
Acknowledgements
Grant of the Academy of Sciences of the Czech Republic (project KAN 101630651) and of
the Ministry of Education, Youth and Sports of the Czech Republic (projects
MSM0021622411 and LC 06035) are acknowledged.
References
1. F. J. Stevenson (Ed.), Humus Chemistry, Willey-Interscience, New York, 2nd edn., 1994, Chap.
12, p. 288.
2. J. Havel, D. Fetsch, in: Wilson I. (Ed.), Encyclopedia of Separation Science, Academic
3. Press Ltd., London, UK, 2000, p. 3018.
4. M. L. Pacheco, E. M. Peña-Méndez, J. Havel, Chemosphere 51 (2003) 95.
5. F. W. Freise, Econ. Geol., 26 (1931) 421.
6. W. G. Fetzer, Econ. Geol., 41 (1946) 47.
7. S. A. Wood, Ore Geol. Rev., 11 (1996) 1.
8. M. L. Machesky, W. O. Andrade, A. W. Rose, Chem. Geol., 102 (1992) 53.
9. E. E. Lungwitz, t. prakt. Geol., (1900) 71.
10. L. Pokorná, D. Gajdošová, S. Mikeska and J. Havel, in: E. A. Ghabbour, G. Davies, (Ed.)
Versatile Components of Plants, Soils and Water, The Royal Society of Chemistry (RSC),
Cambridge, 2000, p. 299.
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Abiotic Treatment of Rice Bran Using an Accelerator Including OrganoIron Compound
Hikari Kanno*, Naoya Tachibana, Masami Fukushima
Laboratory of Chemical Resources, Division of Sustainable Resources Engineering, Graduate
School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan
E-mail: d-kethik@beach.ocn.ne.jp
1. Introduction
Because raw organic waste (ROW) produced during food processing contains organic matter
and plant nutrients, it represents a potentially useful material for use as a soil amendment.
Composting typically involves allowing ROW to be converted to compost via the
fermentation by microbial growth. However, this technique is time consuming and may lead
to several adverse effects on soils and plants due to the presence of insufficiently digested and
unstable organic matter in the ROW [1]. These effects include an increase in the rate of
mineralization of native soil organic carbon through extended microbial oxidation, the
development of anaerobic conditions as the result of the mineralization of large amounts of
non-stabilized organic carbon with associated extended oxygen-consumption, and alteration
in soil pH [2]. Therefore, for optimum use, ROW needs to be converted into chemically stable
compounds, i.e., humic substances.
It is generally accepted that humic substances are formed via polycondensation reactions of
plant, animal and microbial decay products, such as amino acids, phenols and sugars
(humification processes). Iron in clay minerals can serve as a Lewis acid, which facilitates the
polycondensation of phenolics and amino acids [3]. In the present study, we describe a system
that allows the efficient treatment of ROW, in which an organo-iron compound is used to
accelerate the process and an instrument that reduces the volume of wet ROW by heating. In
this study, rice bran was used for a model ROW, because of its homogeneous chemical
composition (e.g., lipids, phenols, proteins and vitamins). The quality of humic acid (HA) in
the compost-like material (CLM) samples can serve as an indicator of the maturity of a CLM
[4]. In this study, HAs were extracted from CLM samples and then purified. To optimize the
conditions for treatment by the proposed technique, the degree of humification for the HAs
were determined, in terms of elemental composition, acidic functional group content,
molecular weight, Uv-vis absorption and FTIR spectroscopy.
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2. Materials and Methods
The preparation of the CLM sample. The CLM sample was prepared by mixing rice bran, an
extending agent (sawdust or Akadama soil i.e., tuff loam) and an accelerator (an organ-iron
compound, Daiso KET Institute Co., Ltd). The mixture was first incubated at 60 ºC for either
1 day or 5 days. The mixture was subsequently transferred to the instrument for garbage
processing (MS-N10, Panasonic Co., Ltd.), and the CLM samples were prepared by ustulating
by incubating for 1, 3 or 15 days. Detailed conditions used for the treatment are summarized
in Tables 1 and 2.
Table 1: Ratio (%) of rice bran, extending agents and accelerator
CLM samples
Rice bran
Akadama soil
Sawdust
Accelerator
A
50
50
1
B
50
50
1
C
100
1
D
50
50
E
50
50
Table 2:Processing periods (day) for preparing CLM samples
Condition
S
M
L
LL
Processing time (day)
Incubator
Garbage processor
1
1
1
5
3
5
15
LL : Half quantity of raw materials were divided to garbage processor
The refinement and analysis of the HA. The HAs were isolated from the prepared CLMs by
extraction with aqueous 0.1 M NaOH and purified according to the procedure a recommended
by the International Humic Substances Society [5]. The HA samples were analyzed by the
methods described in a previous report [4], for elemental composition (C, H, N, S, and ash),
acidic functional groups (carboxylic acids and phenolic hydroxyl groups), UV-vis absorption
spectra, FTIR spectra and molecular weight by HPSEC.
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3. Results and Discussion
Yields and elemental compositions of Has. As shown in Fig. 1, the yields of HA that was
prepared in the presence of Akadama soil as an extending agent (B and E) were significantly
larger than samples produced in the presence of saw dust (A and D). Thus, the Akadama soil
is preferable for use as an extending agent. On the other hand, elemental analyses of the HAs
Figure 1 The yield of HAs in each treatment condition
(Table 3) showed that the H/C atomic ratio decreased and the O/C and N/C atomic ratios
increased with increasing incubation time. These results show that a longer incubation results
in the formation of HAs with a higher degree of humification.
Table 3:Elemental (C, H, N, O, S and ash) compositions of humic substances
Sample
B
E
Condition
S
M
L
LL
S
M
L
LL
%C
62.85
67.27
45.52
39.30
64.04
66.94
57.28
39.54
%H
9.81
10.38
6.00
5.31
10.00
10.39
8.15
5.97
%N
1.15
0.95
2.27
2.25
1.31
1.13
1.96
1.18
%O
21.28
19.50
42.33
49.45
20.97
18.75
28.85
49.92
%S
0.69
0.67
0.28
0.80
0.19
0.75
0.30
0.49
% ash
4.22
1.24
3.60
2.89
3.49
2.04
3.46
2.90
Alteration of structural features. Figure 2 shows FTIR spectra of the HAs, isolated from CLM
samples that contained added Akadama soil (B with the accelerator; E without the
accelerator). The peaks at 2900–2800 cm-1 , corresponding to alkyl C–H stretching (b in Fig.
2) decreased with increasing incubation time. In addition, the following peaks also increased
with incubation time: 3400 cm-1 for phenolic O–H stretching and/or amine N–H stretching (a
in Fig. 2); 1600 cm-1 for aromatic C=C ring stretching (c in Fig. 2); 1200–1000cm-1 for C–O
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stretching of alcohols and/or ethers (d in Fig. 2). Significant alterations in these peaks were
observed in the case of the incubation conditions for B-L and E-LL. These results are
consistent with the elemental analysis data, which suggest that a longer incubation time leads
to the formation of HAs with a higher degree of humification.
Figure 2: The FTIR spectra of the HAs from CLM samples
4. Conclusions
(i) The extent of humification is dependent on the incubation time used, and adding an
accelerator is effective in shortening the required incubation time.
(ii) For incubation pattern B, the intensity of the FT-IR peak for the incubation pattern of L
was similar to that for LL. Thus, the incubation period for pattern L is sufficient to produce a
matured CLM.
(iii) Because of the higher yield of HA, Akadama soil is useful as an extending agent.
References
1. F. Sellami, S. Hachicha, M. Chtourou, K. Medhioub and E. Ammar, Bioresour. Technol., 99
(2008) 6900.
2. W. Shi, J. M. Norton, B.E. Miller and M.G. Pace, Appl. Soil Ecol., 11 (1999) 17.
3. A. Miura, R. Okabe, K. Izumo and M. Fukushima, Appl. Clay Sci. 46 (2009) 277.
4. M. Fukushima, K. Yamamoto, K. Ootusuka, T. Komai, T. Aramaki, S. Ueda, S. Horiya, Biores.
Technol., 100 (2009) 791.
5. R.S. Swift, In, Methods of Soil Analysis Part 3, Soil Science Society of America, Madison, 1996,
p. 1018.
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Effect of Humic Substances on Uranium Removing by Bacterium
Bacillus polymyxa IMV 8910 from Aqueous Solution
I. Shevchuk*, N. Klymenko
A.V. Dumansky Institute of Colloid Chemistry and Chemistry of Water, National Academy
of Sciences of Ukraine. 42 Vernadsky Avenue, Kyiv 03680, Ukraine
E-mail: fjord_n@ukr.net
1. Introduction
Uranium is a long-lived radionuclide that is an ecological and human health hazard. The
mining and processing of uranium for nuclear power plants and nuclear weapons production
have resulted in the generation of significant amounts of radioactive wastes. The treatment of
low charge effluents, conciliating economic and technical constraints is impossible with
traditional physical-chemical processes. It has been suggested that biomass could be used to
decontaminate these wastes and to concentrate metals. Biological methods are the most
ecologically appropriate techniques. The major advantages of microbial treatment are selfreproducibility, adaptability, recyclisation of bioproducts, specificity, and good cost/benefit
ratio.
In most aquatic systems, species of natural organic matter (NOM), such as humic and fulvic
acids, constitute an important pool of ligands for complexing metals. NOM is a
polyfunctional, polyelectrolytic, heterogeneous amalgam of organic molecules of varying
molecular weight and size. Its physical and chemical properties can be a function of the
nominal molecular weight (e.g., [1]); properties will also vary from one source to the next [2].
Although the chemical and physical properties of NOM have been extensively studied and its
metal binding capability is undisputed [2], there still remain many questions regarding its role
in metal binding in heterogeneous systems.
In the process of water purification humic substances (HS) are partially destroyed and can
form toxic soluble complex compounds with radionuclides, that increase their migration to
drinking water [3–6]. It is known that, due to the high ability to complexation of actinides, the
effect of dissolved organic matter on the sorption of these radionuclides is manifested to a
greater extent than in the case of radionuclides of alkali and alkaline earth elements [5].
Existing in the literature are several experimental and modeling studies that have examined
U(VI) binding by NOM [5–8]. In general, each of these studies [5–8] concluded that NOM
has a strong affinity for uranium (VI).
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In previous studies [9, 10] was shown that Bacillus polymyxa IMV 8910 cells may be using
for removing of uranium (VI) from aqueous solutions. In this paper, we present results of
investigation the influence of humic substances on the efficiency of extraction of uranium
(VI) from aqueous solutions by biosorbent based on Bacillus polymyxa IMV 8910 cells.
2. Materials and Methods
Bacterium and growth conditions. The strain of Bacillus polymyxa IMV 8910 used in this
study was obtained from Institute of Microbiology and Virology, National Academy of
Sciences of Ukraine. The bacteria were subcultured in the laboratory using a meat-peptone
broth with addition of glucose (30 g/l) at 30°C with agitation. Cultures were harvested at 24 h
by centrifugation at 10 000×g for 15 min. Cells were triple washed by distilled water (dH2O)
and resuspended in a minimal volume of dH2O at a concentration of approximately 0.6 g/l.
Metal uptake experiments. Biosorption experiments were carried out in triplicate series.
Uranium is provided as uranyl sulfate (UO2SО4·3H2O). The pH was adjusted with 1 M HCl
and 1 M NaOH before the addition of cells suspension aliquot. The experiments were
performed at room temperature. As sorption equilibrium was reached, biomass was removed
by centrifugation at 10 000×g for 15 min. The residual concentration of uranium in solution
was determined by arsenazo III method. Sorption capacity is calculated by:
a = (Co-Ce)V/m;
where a is the sorption capacity (μmol/g of biomass), Co the initial metal concentration, Ce is
the residual metal concentration in solution (μmol/l), V the volume of solution (l) and m the
sorbent mass (g).
Effect of humic substances on sorption of uranium (VI). For investigation of the impact of
humic substances on sorption of uranium by the microbial sorbent we used humic substances
isolated from the Dnipro River, which brought in a solution of uranium (CU = 100 μmol/l, I =
0.01) in concentrations of 10, 25, 50, 100, 200 mg/l; initial pH of humic substances solution
and pH changes that occurred were recorded. Age of microbial culture was 24 h, duration of
the experiment – 1 h.
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15th IHSS Meeting- Vol. 3
3. Results and Discussion
It is known that complexation of uranium (VI) with HS is almost independent of the type
(peat, lake, etc.) [11] and ionic strength solution [11, 12], but it is intensified with pH
increasing [11, 13, 14]. It was shown that within the concentration of humic acids from 10 to
50 mg/l values of sorption of uranium by B. polymyxa IMV 8910 cells is slightly lower. In
case of 200-fold increase of their content the intensity of sorption was significantly decreased,
due to the formation of complex compounds, which are not practically adsorbed by biomass
(Fig. 1).
160
8
140
6
100
80
4
60
1
2
40
pH
a, μmol/g
120
2
20
0
0
10
25
50
100
200
CHS, mg/l
Figure 1: Effect of humic substances on sorption of uranium (VI) by B. polymyxa IMV 8910 cells: 1 curve of U(VI) adsorption; 2 - curve of pH ¡ (CU(VI) = 100 μmol/l, m = 0.03 g, INaCl = 0.01)
Also, it was observed a slight shift in pH from 6.0 to 7.0 in case of humic substances content
increasing in the model solution. It also leads to decrease in the values of uranium (VI)
adsorption by microbial sorbent based on B. polymyxa IMV 8910 cells.
4. Conclusions
It is shown that the presence of NOM in natural aqueous systems will significantly influence
on uranium speciation and must be accounted for in a proper assessment of U(VI) behavior in
Vol. 3 Page - 423 -
15th IHSS Meeting- Vol. 3
environmental settings. In the presence of high concentration of humic substances decreasing
the effectivity of uranium (VI) removal by biosorbent from aqueous solutions.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
J. A. Davis and R. Gloor, Environ. Sci. Technol., 15 (1981) 1223.
J. Buffle, Complexation Reactions in Aquatic Systems, Ellis Horwood, Chichester, 1990.
V.Moulin and C. Moulin, Radiochim. Acta, 89 (2001) 773–778.
P. Crancon and J. van der Lee, Radiochim. Acta, 91 (2003) 673–679.
P.K. Appelband, D.C. Baxter and J.O. Thunberg, J. Environ. Monit., 1(1999) 211–217.
W. C. Li, D.M. Victor and C.L. Chakrabarti, Anal. Chem, 52 (1980) 520–523.
V.Moulin, J. Tits and G. Ouzounian, Radiochim. Acta, 58/59 (1992) 179.
J. J. W. Higgo et al., Radiochim. Acta, 61 (1993) 91.
L.M. Spasonova et al., J. Water Chem. Technol., 28 (2006) 61–68.
Shevchuk, N. Klymenko, J. Water Chem. Technol., 31 (2009) 324–328.
M.A. Glaus, M. Hummel and L.R. Van Loon, Appl. Geochem., 15 (2000) 953–973.
G. Montavon et al, Radiochim. Acta., 88 (2000) 17–24.
J.J. Lenhart et al, Radiochim. Acta., 88 (2000) 345–353.
M. Kalin, W.N. Wheeler and G. Meinrath, J. Environ. Radioact., 78 (2005) 151–177.
Vol. 3 Page - 424 -
15th IHSS Meeting- Vol. 3
Humic Acids Inspired Hybrid Materials as Heavy-Metal Adsorbents
Panagiota Stathi*, Yiannis Deligiannakis
Laboratory of Physical Chemistry, Department of Environmental and Natural Resources
Management, University of Ioannina, Seferi 2, 30100, Agrinio, Greece
E-mail: me01791@cc.uoi.gr
1. Introduction
Humic substances are naturally occurring biogenic heterogeneous organic materials that
complex strongly with heavy metals and organic compounds1,2.
In order to study the sorption characteristics, natural humic acid are isolated, purified, and use
in laboratory studies1,2. However, these studies are often thwarted by the fact that it is difficult
to separate the humic acid from other moieties present in the solution. A way to avoid this
complication is the immobilization of the humic substances on inorganic or water insoluble
organic particles that can be separate from their suspension. Covalent grafting of HA on SiO2
provides a promising technique for the production of a stable zero-leaching hybrid material3.
The aim of the present work was (a) to develop SiO2 materials bearing covalent immobilized
i.e –COOH and Phenolic group as models for the metal binding groups of humic acids (b) to
study heavy metal binding by SiO2-COOH, SiO2-phenolic materials (c) to prepare SiO2immobilized humic acid SIO2-HA and study the metal ion binding of this material. (d) Based
o the SiO2–COOH and SiO2-Phenolic materials to parameterize theoretically the role of each
functional group for metal binding by humic acids.
2. Materials and Methods
Covalent Immobilization of Humic Acid onto SiO2. Humic acid obtained from Aldrich and
used after purification this humic acid was characterized in detailed previously4. Covalent
immobilization of humic acid on aminopropyl silica has been achieved by the method of
Koopal et al 3, by formation of amide bonds between the amino groups of the aminopropyl
silica and the carboxylic groups of HA activated by EDC.
Preparation of the SiO2-COOH Material. Activated silica was modified by –(CH2)3CN
groups following the method of Clark et.al.5. Hydrolysis by H2SO4 results in modified silica
which bears carboxylic acids as functional groups.
Preparation of SiO2-Gallic Acid. Immobilization of Gallic Acid on silica has been obtained
by formation of amide bonds between the amino group of the aminopropyl silica and the
carboxylic group of Gallic Acid activated by EDC6.
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15th IHSS Meeting- Vol. 3
3. Results and Discussion
Metal uptake. Figures 1-3 present the metal-uptake by the three SiO2-based materials, as a
function of pH. Control metal-uptake data by unmodified SiO2 are also included for
comparison. From Figures 1-3 we notice that the metal uptake was significantly increased
relative to the unmodified silica. Importantly, we observe that the modification of silica
results in an enhancement of adsorption at all pH values for all metals. Noticeably the
maximum adsorption by SiO2-HA was 10 times higher than the maximum metal adsorption
by SiO2-COOH or SiO2-GA. In the following we proceed in detailed analysis of the results
based theoretical fit of the experimental data. In all Figures the open symbols and lines
represent the theoretical calculations. Herein the theoretical calculations were performed
using the program FITEQL7. For the fit of the metal adsorption on SiO2-HA we used five
discrete pK for the protonation reaction of each functional groups i.e. five for carboxyl, five
SiO2
5
6
7
100
90
80
70
60
50
40
30
20
10
0
(B)
8
SiO2-COOH
SiO2
4
5
8
-6 Free Cd
-7
SiO2-COO-Cd
-8
Cd(OH)
-9
-10 SiO-Cd
-11
-12
-13
4
5
6
(C)
SiO2-COOH
SiO2
4
5
6
7
8
pH
-5
log(conc.)(M)
Log(Con.)(M)
pH
7
pH
pH
-4
SiO2-COO-Pb
-5
-6
-7
Free Pb
-8
-9
Pb(OH)
-10
-11 SiO-Cu
-12
-13
-14
-15
-16
4 5 6 7 8
6
100
90
80
70
60
50
40
30
20
10
0
7
8
pH
log(conc)(M)
SiO2-COOH
% cu Adsorbed
100 (A)
90
80
70
60
50
40
30
20
10
4
% Cd Adsorbed
% Pb Ads orbed
for Phenolic.
-4
Free Cu SiO -COO-Cu
2
-5
-6
-7
-8
Cu(OH)
-9
-10
-11
SiO-Cu
-12
-13
4 5 6 7 8
pH
Figure 1: Adsorption of Metals on SiO2-COOH . (A) Adsorption edge for Pb(II) onto SiO2-COOH,
solid squares and on to SiO2 solid circles. (B) Adsorption edge for Cd(II) onto SiO2-COOH, solid
squares and on to SiO2 solid circles. (C) Adsorption edge for Cu(II) onto SiO2-COOH, solid squares
and on to SiO2 solid circles. (Initial metal concentration 4.5μM )
SiO2-COOH. The Figure 1 (left panel) shows the pH edge adsorption for Pb (Fig 4A), Cd (Fig
2B) and Cu (Fig 2C) on the SiO2-COOH material. From Figure 2 we notice that the metal
uptake by SiO2-COOH was significantly increased relative to the unmodified silica i.e.
compare solid squares with solid circles in Figure 2, left panels.
The adsorption of metal to silica gel was previously studied for many authors and was
reported quite low. Our data confirm these observations.
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15th IHSS Meeting- Vol. 3
SiO2-GA. The SiO2-GA material has been studied for the adsorption Pb2+, Cd2+, and Cu2+. The
results of adsorption experiments onto to SiO2-GA as a function of pH are presented at Figure
3. The pH edge on the unmodified silica is included for comparison. From Figure 3 we notice
that Pb2+ adsorption was 1.7 times higher than Cd2+ and Cu2+ adsorption was 1.46 times
higher than Cd. For example at pH 8 almost 95% of the 4.5μΜ of Pb2+ were bound by SiO2GA.
The experimental data in Figure 2 show that the metal uptake increased at alkaline pH vales,
for both the SiO2-GA as well as for unmodified SiO2. More particularly, at pH 4 Pb2+
adsorption was 0.52 mmol/Kgr of SiO2-GA and at pH 7 was 2.29 mmol/kgr. For Cd2+ the
adsorption at pH 4 was 0.45mmol/Kgr and 1.35 at pH 7, while the adsorption for Cu2+ was
SiO2-GA
SiO2
4
5
6
7
100
90
80
70
60
50
40
30
20
10
0
log(Conc.)(M)
log(Conc.)(M)
SiO2-GA
SiO2
4
5
6
7
pH
8
pH
-5
-6
Free Pb
-7
SiO2-GA-Pb
-8
SiO2-Pb
-9
-10
-11
Pb(OH)
-12
-13
-14
-15
4 5 6 7 8
(B)
% Ads
(B)
-5
-6
Free Cd
-7
SiO 2-GA-Cd
-8
SiO 2-Cd
-9
-10
-11
-12
Cd(OH)
-13
-14
4 5 6 7 8
pH
pH
8
100
90
80
70
60
50
40
30
20
10
0
(B)
SiO2-GA
SiO2
4
5
6
7
8
pH
log(conc.)(M)
100
90
80
70
60
50
40
30
20
10
0
% Cd Adsorbed
% Pb Adsorbed
1.01mmol/Kgr and 1.98 mmol/Kgr at pH 4 and pH 7 respectively.
-5
-6
-7 SiO2-GA-Cu
-8 SiO -Cu Free Cu
2
-9
Cu(OH)
-10
-11
-12
-13
-14
-15
4 5 6 7 8
pH
Figure 2: Adsorption of Metals on SiO2-GA . (A) Adsorption edge for Pb(II) onto SiO2-GA, solid
squares and on to SiO2 solid circles. (B) Adsorption edge for Cd(II) onto SiO2-GA, solid squares and
on to SiO2 solid circles. (C) Adsorption edge for Cu(II) onto SiO2-GA, solid squares and on to SiO2
solid circles. (Initial metal concentration 4.5μM
SiO2 –HA. Figure 3 shows the adsorption of Pb2+, Cd2+, Cu2+ on SiO2-HA as a function pH.
According to the Figure 4-6 the adsorption of metals to immobilized humic acid was higher
compared with the adsorption to other materials. For example the Pb2+ adsorption at pH 7 was
3.88 mmol/Kgr for the SiO2-COOH material, 2.23mmol/Kgr for SiO2-GA and 34.65
mmol/Kgr for SiO2-HA material. For Cd2+ the obtained adsorption on SiO2-COOH was 2.07
mmol/Kgr at pH 7, 1.35 mmol/Kgr SiO2-GA and 14.22 mmol/Kgr on SiO2-HA. In analogous
manner the Cu adsorption on three materials was 2.57 mmol/kgr, 1.98 mmol/Kgr,
35.95mmo/kgr respectively. The experimental data in Figure 3 show that the Pb2+ by uptake
SiO2-HA increased at alkaline pH values.
Vol. 3 Page - 427 -
SiO2-HA
4
5
6
7
8
100
90
80
70
60
50
40
30
20
10
0
pH
log(conc.)(M)
-4.5
Free Pb RO--Pb
-5.0
-5.5 COO-Pb
-6.0
-6.5
-7.0
-7.5
4
5
6
4
-4
Pb(OH)
-8.0
SiO2-HA
5
6
7
8
100
90
80
70
60
50
40
30
20
10
0
(C)
4
SiO2-HA
5
pH
log(conc.)(M)
-4.0
(B)
% cu Adsorbed
(B)
7
-5
-6 COO-Cd
-7
-
RO -Cd
-9
-10
pH
4
5
6
7
8
7
-
RO -Cu
-5
COO-Cu
-6
-7
Free Cu
-8
-9 Cu(OH)
-10
Cd(OH)
-11
8
-4
Free-Cd
-8
6
pH
log(conc.)(M)
100
90
80
70
60
50
40
30
20
10
0
% Cd Adsorbed
% Ads
15th IHSS Meeting- Vol. 3
8
pH
-11
1
2
3 4
5
pH
Figure 3: Adsorption of Metals on SiO2-HA . (A) Adsorption edge for Pb(II) onto SiO2-HA. (B)
Adsorption edge for Cd(II) onto SiO2-HA, solid squares. (C) Adsorption edge for Cu(II) onto SiO2HA. (D) Adsorption edge for Zn(II) onto SiO2-HA.(E) Adsorption edge for Mg(II) onto SiO2-HA,
solid squares. (Initial metal concentration 45μM )
In analogous manner the adsorption of Cd2+ at pH 4-6 is due to the deprotonated COO- groups
while adsorption at pH>6 is due to binding of Cd2+ to Phenolic groups with pKa 8-10. The
Cd adsorption is 2.5 times lower than Pb2+. For example at pH 7 the adsorption of Cd2+ was
14.22mmol/Kgr and the adsorption was 34.64mmol/kgr for Pb2+
Figure 3c shows the adsorption of Cu2+ adsorption on SiO2-HA the amount of Cu2+ adsorb
was 35.95 mmol/Kgr see table 2. The main adsorb species from pH 4-6 are COO-Cu and from
higher pH the main species is RO--Cu.
4. Conclusions
The data presented herein show that all SiO2-based materials show significant improvement
for metal uptake, compared to unmodified silica. This enhancement was observed at all pH
values and can be attributed to adsorption of metals to deprotonated form of functional groups
(COOH, GA, HA).
Of particular importance is the observation that for SiO2-HA material the adsorption is 10
times higher compared with other two materials. This is a result of high concentration of
functional groups per Kgr of humic acid. The maximum amount of metals absorbed per Kgr
of materials shows in Figure 4 The carboxyl groups appear to be responsible for the
adsorption at pH 4-7 and phenolic groups for the adsorption at higher pH. In this respect the
SiO2-HA shows the typical behaviour of HA in solution.In addition, from the adsorption data
we conclude that SiO2-COOH and SiO2-GA are a good model for the adsorption properties of
Vol. 3 Page - 428 -
15th IHSS Meeting- Vol. 3
by
phenolic
the
groups
–COOH
of
and
HA
respectively. More particularly, we
observe that metal adsorption on
the three materials increased by the
same order [Cu]>[Pb]>[Cd]>[Mg].
The present data provide the first
direct experimental proof that HA
can be viewed and modeled as a
combination of -COO and R-OH
functional groups.
Ads. Metal (mmol/Kgr)
metals
35
SiO2
30
SiO2-GA
SiO2-COOH
SiO2-HA
25
20
15
10
5
0
Pb
Cd
Cu
Zn
Mg
a
Figure 4: Amounts (mmol/Kgr) of Heavy Metals
Adsorbed by Materials at pH=7
Moreover in natural environment sorption of inorganic and organic compounds to mineral
bound humic acids is influenced by the chemical properties and the conformation of the
humic acids. The present work shows that SiO2-COOH, SiO2-GA, and SiO2-HA can be useful
in physicochemical study of geochemical cycles of metals in natural environment.
References
1. Stevenson J. F., Humus chemistry: Genesis, Composition, Reactions, John Willey & Sons Inc:
New York, 1994.
2. Tipping E., Cation Binding by Humic Substances, Cambridge University Press, Cambridge, 2002.
3. Koopal L. K.; Yang Y.; Minnard A .J.; Theunissen P. L. M.; Van Riemsdijk W. H.; Colloids and
Surfaces A: Physicochem . Eng. Aspects 1998,141, 385.
4. Drosos M.; Jerlykiewich M.; Deligiannakis Y.; J. Colloid and Interface Sci., 2009, 332, 78.
5. Butterworth A.J.; Clark J.H.; Walton P. H.; Barlow S.J.; Chem.Comm. 1996, 1859.
6. Stathi P.; Louloudi M.; Deligiannakis Y.; Chemical Physicis Letters, 2009, 472, 85.
7. Dzombak D.A.; Morel F.M.M.; Surface Complexation Modeling, Jonh Willey & Son, New York,
1990.
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loursponsor
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