SPECIATION AND TOTAL DETERMINATION OF ARSENIC, CHROMIUM AND SELENIUM IN FRESHWATER BODIES SEDIMENT NAZARATUL ASHIFA BINTI ABDULLAH SALIM A thesis submitted in fulfillment of the requirements for the award of the degree of Master of Science (Chemistry) Faculty of Science Universiti Teknologi Malaysia NOVEMBER 2009 iii To my beloved husband and parent whose endless love and support helped me get through all the obstacles. Little Sufi and Faris; always remind mama to work hard and achieve high. iv ACKNOWLEDGEMENTS All commendations to Almighty Allah, who imparted me resoluteness and fortitude for accomplishment of this work successfully. I would like to express my deep gratitude to my supervisor, Prof Alias Mohd Yusof for all his thought, guidance, encouragement and invaluable help given to me during my graduate research. I am also very thankful to my supervisory committee members in Nuclear Malaysia Agency, Dr. Abdul Khalik Hj. Wood, Dr. Mohd Suhaimi Hamzah, Puan Shamsiah Abdul Rahman and Md Suhaimi Elias for providing me with support and feedback throughout my studies. I also appreciate the help from Puan Mek Zum, En Hanan Basri, En. Ariffin Talib, Puan Pirmala Devi, Mr. Lim Ching Choah, Puan Jamaliah Mat Yatim, Puan Eeewiat, Cik Ezwiza Sanuri, Puan Zaleha Hashim and En. Burhanuddin Ahmad in chemicals purchasing, samples collection and preparation, chemical analysis, counting processes and discussion of my ideas in the research process. The financial support for this project from the Universiti Teknologi Malaysia in-cooperation with Malaysian Nuclear Agency under Reactor Interest Group Fund and Master scholarship from Malaysian Nuclear Agency are greatly acknowledged. I am indebted to my family members whose endless prayers and continuous moral support enabled me to get through the period of my academic journey. v ABSTRACT Arsenic (As) is generally known for its toxicity while chromium (Cr) and selenium (Se) at the appropriate amount are essential elements to man and becomes quite toxic in excessive amount. Anthropogenic activities such as industrialization, agricultural and urbanization have led to the contamination of toxic elements into aquatic that finally end up in the sediment system. Environmental process such as diagenetic process causes the toxic elements to migrate from the bedrock materials into the sediment surface and lastly into the water column. This process has been recognized to be the factor of As contamination in well water in several countries such as Bangladesh, Taiwan, USA and Canada. A number of samples of freshwater sediments from identified rivers and lakes in Johor Bharu area had been analyzed to determine the concentration level of As, Cr and Se using neutron activation analysis (NAA) technique. Certified reference material (CRM) namely IAEA Soil-7, Marine Sediment Reference Material BCSS-1 and PACS -2 were applied to provide good quality assurance control during analysis. The results obtained show that the concentrations of As in the rivers and lakes sediment fall between 10 to 34 g g-1 and 18 to 62 g g-1, respectively. The concentrations of Cr in the rivers ranged between 27 g g-1 to 125 g g-1, while in the lake sediments the concentrations ranged between 173 g g-1 to 301 g g-1. The river sediment showed the Se concentration ranged between 0.56 g g-1 to 1.91 g g-1 and in lake sediment the concentration ranged between 0.31 g g-1 to 1.08 µg g-1. The results of the As, Cr and Se concentrations were then compared to the ‘background value’ proposed by National Oceanic and Atmospheric Administration (NOAA), USA and several sediment quality guidelines. The As, Cr and Se concentrations were also correlated and normalized to iron content in order to evaluate and estimate the degree of contaminant enrichment in sediments. For speciation study, the inorganic species of As(III), As(V), Se(IV) and Se(VI) were studied by extracting the sediments using the microwave at the optimized time and power setting in the mild concentration of acid media. The As(III) and As(V) were preconcentrated and separated by ammonium pyrolidinedithiocarbamate (APDTC) into methyl isobuthyl ketone (MIBK) while the Se(IV) and Se(VI) were separated after co-precipitation of sodium dibenzyldithiocarbamate (Na-DBDTC) with phenolphthalein. The efficiencies of all extraction procedures were determined using standard solutions and several CRM. The results showed that As(V) and Se(VI) were the dominant species in the river and lake sediments. vi ABSTRAK Arsenik (As) telah diketahui umum akan ketoksikannya sementara kromium (Cr) dan selenium (Se) pada amaun yang berpatutan merupakan unsur keperluan kepada manusia dan menjadi toksik pada amaun yang berlebihan. Aktiviti pencemaran seperti perindustrian, pertanian, perbandaran telah mendorong kepada pencemaran unsur toksik ke dalam akuatik yang akhirnya terkumpul dalam sistem enapan. Proses sekitaran seperti proses diagenetik menyebabkan unsur toksik bergerak dari bahan dasar ke dalam permukaan enapan dan akhirnya ke dalam turus air. Proses ini dikenalpasti sebagai faktor pencemaran As di dalam air telaga di beberapa negara seperti Bangladesh, Taiwan, Amerika dan Kanada. Beberapa sampel enapan sekitaran air tawar dari beberapa sungai dan tasik yang dikenalpasti di kawasan Johor Bharu telah dianalisis untuk menentukan kepekatan As, Cr dan Se menggunakan teknik analisis pengaktifan neutron. Bahan rujukan piawai antaranya, IAEA Soil-7, Marine Sediment Reference Material BCSS-1 dan PACS -2 telah digunakan untuk memberi kawalan kualiti jaminan yang baik semasa analisis. Keputusan yang diperolehi menunjukkan kepekatan As di sungai dan tasik masingmasing ialah 10 hingga 34 g g-1 dan 18 hingga 62 g g-1. Kepekatan Cr di sungai berjulat antara 27 g g-1 hingga 125 g g-1 manakala kepekatannya di dalam enapan tasik berjulat antara 173 g g-1 hingga 301 g g-1. Kepekatan Se di dalam enapan sungai berjulat antara 0.56 g g-1 hingga 1.91 g g-1 dan kepekatannya di dalam enapan tasik antara 0.31 g g-1 hingga 1.08 g g-1. Kemudian keputusan kepekatan As, Cr dan Se dibandingkan dengan ‘nilai latar’ yang dicadangkan oleh National Oceanic and Atmospheric Administration (NOAA), Amerika dan beberapa panduan kualiti sedimen. Korelasi dan normalasi kepekatan As, Cr dan Se kepada kandungan besi dijalankan untuk menilai dan menganggar pengkayaan bahan cemar di dalam enapan. Bagi kajian penspesiesan, spesies bukan organik As(III), As(V), Se(IV) dan Se(VI) ditentukan dengan mengekstrak enapan menggunakan gelombang mikro pada aras kecekapan masa dan kuasa yang telah ditetapkan di dalam media asid berkepekatan rendah. Pra-pemekatan dan pemisahan As(III) dan As(V) dijalankan dengan mengunakan ammonium pirolidineditiokarbamat (APDTC) di dalam metil isobutil keton (MIBK). Sementara Se(IV) dan Se(VI) dipisahkan selepas kopemendakan natrium dibenzilditiokarbamat (Na-DBDTC) dengan fenolftalein. Kecekapan semua prosedur pengekstrakan telah ditentukan dengan menggunakan larutan piawai dan beberapa bahan rujukan piawai. Keputusan menunjukkan As(V) dan Se(VI) adalah spesies yang dominan di dalam enapan sungai dan tasik. vii TABLE OF CONTENTS CHAPTER TITLE PAGE THESIS STATUS DECLARATION SUPERVISOR’S DECLARATION TITLE PAGE i DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv ABSTRACT v ABSTRAK vi TABLE OF CONTENTS vii LIST OF TABLES xii LIST OF FIGURES xv LIST OF APPENDICES 1 xviii LIST OF SYMBOLS AND ABBREVIATIONS xx INTRODUCTION 1 1.0 Research Background 1 1.1 Problem Statement 5 1.2 Research Objectives 6 1.3 Scope of Research 7 1.4 Man, Environment and Toxic Elements 7 1.5 Arsenic 8 1.5.1 Geochemistry of Arsenic 10 1.5.2 The Toxicology of Arsenic 15 viii 1.6 Selenium 2 17 1.6.1 Geochemistry of Selenium 20 1.6.2 The Toxicology of Selenium 22 1.7 Chromium 23 1.7.1 Geochemistry of Chromium 25 1.7.2 The toxicology of Chromium 27 LITERATURE REVIEW 29 2.0 Sediment and Its Role in Freshwater Ecosystem 29 2.0.1 Sediment Contamination 2.0.2 Sediment Quality Issue and Assessment 2.1 32 Analytical Technique for Elemental Analysis 34 2.1.1 Neutron Activation Analysis 35 2.1.2 Principles of Instrumental Neutron 2.1.3 Activation Analysis 35 Nuclear Reactor 39 2.2 Elemental Speciation 40 2.3 Speciation Technique for Sediment Analysis 42 2.3.1 Extraction Technique 2.3.2 Usage of Microwave in Sample Digestion Prior to Extraction 2.3.3 2.3.4 3 30 43 46 Preconcentration and Separation Technique 48 Detection Technique 51 2.4 NAA in Speciation Study 56 MATERIALS AND METHODOLOGY 62 3.0 Introduction 62 3.1 Study Area 62 3.1.1 River 64 3.1.2 Lake 66 ix 3.2 Sediment Characteristic 68 3.3 Sampling Methodology 68 3.4 Chemicals and Reagents 69 3.5 Labware 70 3.6 Laboratory Apparatus 70 3.7 Sample Processing Equipment 71 3.8 Sample Preparation 72 3.9 Standard Solutions 72 3.9.1 Standard Solutions for As, Cr, Se and 72 Fe 3.9.2 Standard Solution for Arsenic (III) and Arsenic (V) 3.9.3 73 Standard Solution for Selenium (IV) and Selenium (VI) 3.10 Certified Reference Material (CRM) 74 74 3.11 Preparation of Sediment Sample For Total 75 As, Cr, Se and Fe 76 3.12 Arsenic Species 3.12.1 Microwave extraction 76 3.12.2 Preconcentration and Separation 77 3.12.2.1 Effect of pH 79 3.12.2.2 Repeatability 79 79 3.13 Selenium Species 3.13.1 Microwave extraction 80 3.13.2 Preconcentration and Separation 80 3.13.2.1 Effect of pH 81 3.13.2.2 Repeatability 82 3.14 Analysis Using Neutron Activation Analysis Technique 83 3.14.1 Limit of Detection 86 x 4 RESULTS AND DISCUSSION 87 4.0 Introduction 87 4.1 Water Quality of The Water Bodies 88 4.2 Data Quality 93 4.3 Total Concentration of Elements 95 4.4 Concentration of Arsenic, Chromium and Selenium in Rivers 4.5 Concentration of Arsenic, Chromium and Selenium in Lakes 4.6 96 101 Comparison in Elements Concentration to the Guideline Recommendation for Freshwater Sediments 4.7 107 Association of Arsenic, Chromium and Selenium to Iron Concentration 110 4.8 Evaluating Contaminant Impact 117 4.8.1 Enrichment Factor (EF) 118 4.8.2 Geoaccumulation Index (Igeo) 120 4.9 Speciation of Arsenic and Selenium in Sediment 4.9.1 4.9.2 122 Optimization of Arsenic Speciation Study 123 4.9.1.1 Microwave Extraction 124 4.9.1.2 Effect of pH and Repeatability 125 Optimization of Selenium Speciation Study 127 4.9.2.1 Microwave Extraction 127 4.9.2.2 Effect of pH and Repeatability 129 4.10 Concentration of Arsenic and Selenium Species in River and Lake 130 xi 5 CONCLUSIONS AND SUGGESTIONS 136 5.0 Conclusions 136 5.1 Suggestions 139 REFERENCES Appendices A - I 141 157 - 181 xii LIST OF TABLES TABLE NO. 1.1 TITLE PAGE Naturally occurring inorganic and organic arsenic species 11 1.2 Inorganic arsenic species in the environment 13 2.1 Major contaminants in sediments 31 2.2 Summary of analytical arsenic and selenium speciation in environmental samples 53 2.3 Speciation study using NAA method determination 60 3.1 Date of sampling carried out in the rivers and lakes 63 3.2 The coordinates and description of the river sampling site 66 3.3 Sampling coordinates at the lakes 67 3.4 Standard solutions of original concentration supplied 3.5 by Merck 73 The specification of the CANBERRA HPGe detector 84 xiii 4.1 Overall average of the water physico-chemical parameters in the rivers and lakes 89 4.2 Analytical results for As, Cr, Se and Fe in CRMs. 94 4.3 LOD for As, Cr, Se and Fe 94 4.4 Result of average concentration of arsenic, chromium and selenium in sediment of each sampling sites at river 4.5 Result of previous studies from several literatures for As and Cr in river sediment 4.6 96 98 Result of average concentration of arsenic, chromium and selenium in sediment of each sampling sites at lake 4.7 Result of previous studies from several literatures for As, Cr and Se in lake sediment 4.8 105 Several guidelines value for arsenic, chromium and selenium 4.9 102 108 Selenium guideline value derived from the Guidelines for Interpretation of the Biological Effects of Selected Constituents in Biota, Water and Sediment 4.10 109 Enrichment factors (EF) and Index of Geoaccumulation (Igeo) in sediment of rivers and lakes 117 xiv 4.11 The Igeo scale 4.12 Results of arsenic species under various microwave 121 condition and H3PO4 concentration (expressed as µg g-1 dry mass) 4.13 125 Results of selenium species under various microwave condition and HCl concentration (expressed as µg g-1 dry mass) 128 4.14 Arsenic speciation analysis in river and lake sediment 132 4.15 Selenium speciation analysis in river and lake sediment 134 xv LIST OF FIGURES FIGURE NO. 1.1 TITLE Structures of naturally occurring inorganic and organic arsenic species 1.2 31 Schematic diagram illustrating the sequence of events for a typical (n, ) reaction 2.3 26 Principal sources, fates and effect of sediment contamination in aquatic ecosystem 2.2 21 The Eh-pH diagram of chromium in water at 25C and one atmosphere 2.1 15 The Eh-pH diagram of selenium in water at 25C and one atmosphere 1.5 14 Simplified transformation pathway of inorganic arsenic in the environment 1.4 12 The Eh-pH diagram of arsenic in water at 25C and one atmosphere 1.3 PAGE 37 Schematic set-up of gamma ray spectrometer for use in INAA 38 xvi 2.4 Processes for speciation study in different samples 43 3.1 Sampling locations for river 65 3.2 Sampling locations for lake at University Teknologi Malaysia 3.3 Flow chart of the preconcentration and separation of As (III) and As (V) in APDTC – MIBK system 3.4 67 78 Flow chart separation-extraction procedure of Se (IV) and Se (IV) by co-precipitation of Na-DBDTC with phenolphthalein 4.1 82 Water Quality Status for River Basin of Peninsular Malaysia in 2006 with highlight of Sungai Skudai, Sungai Tebrau and Sungai Buluh (Kaw. Pasir Gudang) 4.2 Average concentration of arsenic in sediment of river sampling sites 4.3 100 Average concentration of arsenic in sediment of lake sampling sites 4.6 100 Average concentration of selenium in sediment of river sampling sites 4.5 99 Average concentration of chromium in sediment of river sampling sites 4.4 92 103 Average concentration of chromium in sediment of lake sampling sites 103 xvii 4.7 Average concentration of selenium in sediment of lake sampling sites 4.8 Concentrations of As, Cr and Se plotted against concentrations of Fe in river sediments 4.9 116 Extraction recovery of As (III, V) by APDTC-MIBK at different pH 4.13 115 Ratios of concentration of As, Cr and Se to concentrations of Fe in lake sediments 4.12 113 Ratios of concentration of As, Cr and Se to concentrations of Fe in river sediments 4.11 112 Concentrations of As, Cr and Se plotted against concentrations of Fe in lake sediments 4.10 104 126 Extraction recovery of Se (IV, VI) by co-precipitation technique at different pH 130 xviii LIST OF APPENDICES APPENDIX A TITLE The range of the water physico-chemical parameters B PAGE 157 National Water Quality Standard for Malaysia (NWQS) 158 C Certified Reference Material 160 D Results of As, Cr, Se and Fe concentration in sediments E Details on the international guideline for freshwater sediment F G 167 169 Statistic evaluation of As, Cr and Se correlation to Fe in sediments 174 a) The stability study of As species 175 b) The extraction efficiency of As (III) and As (V) to pH function c) 175 The repeatability of the extraction by APDTC-MIBK 176 xix H a) The stability study of Se species b) The extraction efficiency of 1 g mL-1 Se (IV) and Se (VI) to pH function c) a) 179 ANOVA : Analysis of arsenic species in river and lake c) 178 Results of As and Se inorganic species in river and lake sediments. b) 177 The repeatability of the co-precipitation technique by DBDTC-Pp I 177 181 ANOVA : Analysis of selenium species in river and lake 181 xx LIST OF SYMBOLS AND ABBREVIATIONS % - percent C - degree Celcius - microgram per gram - microgram per mililiter g L - microgram per liter mg kg-1 - milligram per kilogram mg L-1 - milligram per liter g kg-1 - gram per koligram ng m-3 - nanogram per meter cubic -3 - pikogram per meter cubic m - micrometer mm - millimeter min - minute km - kilometer kPa - kilopascal M - mol W - watt APDTC - ammonium pyrolidinedithiocarbamate AAS - atomic absorption spectrometry AFS - atomic fluorescence spectrometry BOD - biological oxygen demand COD - chemical oxygen demand CRM - certified reference material DIBK - diethyl isobutyl ketone DO - dissolved oxygen DOE - Department of Environmental g g-1 g mL -1 -1 pg m xxi EPA - Environmental Protection Agency ETAAS - electro thermal atomic absorption spectrometry GFAAS - graphite furnace atomic absorption spectrometry HPLC - high performance liquid chromatography HCl - hydrochloric acid HClO4 - perchloric acid HF - fluoric acid HNO3 - nitric acid H2O2 - hydrogen peroxide H3PO4 - ortho phosphoric acid H2S - hydrogen sulfate H2SO4 - sulfuric acid IAEA - International Atomic Energy Agency ICPMS - inductively coupled plasma mass spectrometer MIBK - methyl isobutyl ketone NAA - neutron activation analysis Na-DBDTC - sodium diethyldithiocarbamate NOAA - National Oceanic and Atmospheric Administration NWQS - National Water Quality Standard RSD - relative standard deviation UV - ultra violet WHO - World Health Organization CHAPTER 1 INTRODUCTION 1.0 Research Background Development in industrial and agricultural sectors as well as rapid increase of population and mining activities have adversely affected the quality of aquatic system. Industrial, domestic and municipal wastes that contain toxic contaminants including hazardous chemicals and pesticide residues may enter the aquatic environment and affect the natural system. Toxic contaminants such as heavy metals that are introduced into the aquatic system may also associate themselves with sediment components and can contribute to toxicity up to some extent. The Department of Environment (DOE) had, in 2006, monitored about 146 river basins in Malaysia. Out of those 146 river basins, 69 river basins had been reported and categorized as polluted based on water quality parameter assessment of biochemical oxygen demand and ammoniacal-nitrogen (NH3-N). The untreated and partially treated sewage and discharges from agro-based and manufacturing industries were the principal contributors to the major pollution in river system. Meanwhile, 42 rivers were recorded to be heavily polluted by earthwork and land clearing activities. In the aquatic system, the sediment acts as a sink to a major number of contaminants. It also serves as an important medium for the transformation and migration of toxicants. Beyond this environmental system, the chemistry of some chemicals may have changed due to environmental, biological and physical 2 processes. Some chemicals are not biodegradable but may be biotransformed through food chain that reaches human beings where they ultimately result in chronic and acute ailments and illnesses. Förstner and Wittmann (1983) grouped the existence of metals in water into two groups; inorganic and organic. Some of them might be soluble and others in particulate form. The metals in soluble state are present as ions, complex ions, chelate ions or molecule forms. The particulate metal form may occur as colloidal, precipitate or adsorbed to other constituents in the aquatic environment. In natural waters, both inorganic and organic metals may undergo oxidationreduction, precipitation-dissolution, adsorption-desorption or biochemical methylation reactions. All of these reactions control deposition, accumulation and mobilization of metals in the aquatic environment. These reactions do not only take place in the aqueous phase but also interact at the solid phase such as sediment, soil and geologic materials. Several metals can go through transformation and translocation by soil microbes and higher plants via their uptake mechanism. According to Förstner (1983), aquatic solids are composed of a mixture of material inputs from various sources, including eroded rocks and soils, sewage and solid waste particles, atmospheric fallout and other formation in the aquatic system such as inorganic precipitates, biogenic matter, adsorbates on particles from solution, complexed and colloidal matter. During periods of reduced flow rates, suspended materials settle to the bed of the river, lake and sea, becoming partly incorporated into the bottom sediment. 3 Sediment analysis is particularly useful in detecting pollution sources and in the selection of critical sites for routine water sampling for contaminants that, on discharge to surface water, do not remain solubilized but are rapidly adsorbed by particulate matter (Simpson et al., 2005). Local sources of pollution can be determined and evaluated with the lateral distribution analysis. Vertical sediment profiles are also useful, since they often uniquely preserve the historical sequence of pollution intensities and at the same time enable a reasonable estimation of background levels and the variation input of a pollutant over an extended period of time. Since three decades ago, research aspect into sediment has become apparent from the fact that the sediments with their contaminants are in a constant interrelation with the liquid phases and organisms which means that the sediments themselves represent another environmental contaminant. Several problem areas with regard to the presence of contaminated sediment in the environment including the potential availability of the contaminants in the sediments for aquatic life, the behaviour of contaminants in soil and sediment with respect to potential pollution of groundwater and uptake of contaminants by plants from polluted sediments on agricultural land have been highlighted, studied and discussed extensively by scholars (Kubota, 1983; Thornton, 1983). One of the well-known aquatic environmental studies ever conducted was conducted at The Great Lakes situated in United State and also in Canada region. The Great Lakes contain 20 % of the world's surface fresh water which cover more than 244,000 square kilometers of water (http://epa.gov/greatlakes/index.html). Contaminants are washed into the lakes via rivers and streams, or through pipes and discharge outlets. The lakes are also exposed to contaminants via atmospheric depositions. Contaminated sediments, primarily in harbors and industrialized segments of rivers and estuaries, contain pollutants that have been entering the lakes for decades. The sediment assessment study for metals, acid volatile sulfides, methylmercury, tributyltin, pesticides, polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs) and dioxins were conducted to evaluate the nature and extent of bottom sediment contamination of The Great Lakes 4 (Schumacher, 1994). Studies conducted on sediment from this area have demonstrated that contaminated sediments are of great concern to humans and wild life that live in the Great Lake Basin. EPA’s Great Lakes National Program Office has reported that polluted sediments are the largest major sources of contaminants in Great Lakes rivers and harbors which could eventually enter the food chain (EPA, 2004). The introduction of elemental contaminants into the aquatic system has various sources. The source of contaminants could be originated from two origins; direct and indirect sources. The direct sources could be coming from natural processes like weathering and volcanic activity that contributes to the redistribution of metals from bedrock into the surface environment. Through the water system runoff and leaching process, the metals in solution form are introduced into lakes, rivers, groundwater and sea. The elements enrichment can be attributed to human activities such as industrial processing of ores and metals, agricultural, industrial and domestic wastes. The serious effects of mine, industrial and domestic effluents on water quality in river and lakes, as well as on the organism, have been known and studied for many years. The indirect sources of contaminants could be originated from geochemical processes occuring in a particular system. The diagenesis process is a natural aquatic environmental process that contributes to the enrichment of toxic chemical in the sediment. This process may induce the vertical migration of multi oxidation state of element such as As from bottom of anoxic sediment to surface that can act as input of the toxic chemical to the water column. The diagenesis process may have developed in sediments of anoxic condition of the aquatic system occurring as a result of exhausted oxygen due to excessive utilization for degradation of biodegradable material such as municipal wastes. The diagenesis process usually occurs in marine, river, lake and ground water ecosystem which are highly contaminated by biodegradable materials such as sewage. Arsenic contamination in Bangladesh and North East India, are example of serious consequence of migration of multi oxidation state toxic element into water column in the anoxic sedimentary condition (McArthur et al., 2004). 5 1.1 Problem Statement In early civilizations, human culture and development were closely related to freshwater utilization in many aspects of life. Rivers and lakes were utilized for agriculture, transportation, daily usage and food resources. After the Industrial Revolution in early 19th century, rapid development in urbanization and industrialization took place including at some strategic areas close to the river or lake. Without proper planning, enforcement in environmental controlling, environmental education and awareness, man has allowed contaminants to enter the river and lake ecosystem. In Malaysia, most rivers and lakes are still providing raw water for daily consumption. But on the other hand, the contaminants entering the freshwater system are not deniable. Health issue concerning freshwater especially for drinking purpose has always been a top priority in the nation. Rapid development in industrial and agricultural sectors as well as rapid increase of population has affected the quality of aquatic system adversely. Toxic contaminants such as arsenic, chromium and selenium introduced into aquatic system may also associate with sediments components as well and contribute to some extent of toxicity. In the contaminated sediment, the chemistry of some contaminants may have changed due to environmental, biological and physical processes. Some contaminants are not biodegradable but may be biotransfromed through food chain, thereby reaching human beings where they produce chronic and acute ailments. While, some other toxic contaminants released from the sediment bound enter the water column when the changes of the aquatic system environment occur such as decline in dissolved oxygen or pH. 6 1.2 Research Objectives Sediment contamination issue is highly important in environmental concern. The sediment represents the largest reservoir for toxic contaminants within the aquatic system such as marine, river, lake and pond. The freshwater bodies such as river, lake and pond have great potential to be contaminated with toxic contaminant as they are situated inland closely to human settlement. Contamination of freshwater from anthropogenic activities or natural weathering may give hazardous impact toward drinking water consumption. The study and investigation of contaminants conducted at those stressful freshwater areas especially in sediments provides significant information regarding the possible sources of contaminant, mobility, and transportation behavioral, potential environmental and health risk particularly for some contaminants that have bioaccumulation capability. The research objectives can be outlined as follows: a) To determine and quantify the total concentration of arsenic, selenium,chromium and iron in river and lake sediments using neutron activation analysis (NAA) technique. b) To compare the quality of sediments particularly of arsenic, selenium and chromium concentration with several international Sediment Quality Guidelines. c) To develop a suitable analytical method for inorganic speciation of As (III), As (V), Se (IV), and Se (VI) in sediment samples using neutron activation analysis (NAA) as a principal detection technique. d) To depict and compare the availability of arsenic, selenium, chromium, inorganic arsenic and selenium of the river and lake system used in this study. 7 1.3 Scope of Research The main scope of research include sediment sampling at identified rivers and lake and collection of water parameter data such as pH, temperature and DO, analysis of sediment for the total concentration of arsenic, selenium, chromium and iron using nuclear analytical technique namely NAA and correlation study between the analysis results of arsenic, selenium and chromium to iron content. The data of total arsenic, selenium and chromium concentration obtained were compared to several International Sediment Quality Guidelines. An effort to apply Enrichment Factor and Geoaccumulation Index for the purpose of evaluating and estimating the contamination impact in sediment has been taken and completed. In addition, a speciation preparation method using microwave and preconcentration technique of inorganic arsenic and selenium species has been developed and arsenic, selenium, their respective species as well as chromium occurrence in two different environmental systems; river and lake, using statistical technique has been undertaken. 1.4 Man, Environment and Toxic Elements The introduction of elemental contaminants into the aquatic system has various sources where the main point sources originate from anthropological activities and geochemical processes. Anthropogenic activities in industrial, agricultural, mining and transportation have led to many pathways of toxic elements into aquatic system and significantly contribute to the increase in contaminations of aquatic system. Some activities may include smelting process and fuel combustion via atmospheric fallout, pollution from leak, effluents, and land application of 8 sewage materials and leaching of garbage (Förstner and Whitman, 1983; MacDonald et al., 2003). Several elements may also originate from the natural weathering process of underlying mineralized rock or associated overburden. In water bodies, elemental contaminants are associated with the sediment underneath whereby sediments are often the ultimate repositories of contaminants leased to the environment. Toxic elements such as arsenic, cadmium, chromium, copper, mercury, selenium and other species tend to accumulate at the bottom of the sediment. The presence and addition of certain metals to sediments at such a level that would have harmful effects on organisms can be considered to constitute metal pollution. The compositions, quantities and the modes of these elemental inputs vary widely from one sediment to another and thus influence the contaminants behavior in affected sediments. Aquatic organisms such as fishes, clams and mussels are among the species closely associated with sediment. Under polluted water conditions, these organisms can take up and accumulate metals present in the polluted sediment. Toxic elements are then able to enter aquatic biota through the food chain and this provides a great concern in human health perspective. Therefore, uptake of the contaminants by aquatic organisms has become a subject of interest by several researchers in which several organisms have been used as bio-indicators for assessing water pollution (Yusof et al., 2004; Zanaton, 1994). 1.5 Arsenic Arsenic is a metalloid that is widely distributed in the earth’s crust at an average concentration of 2 µg g-1 (Merian, 1985). It ranks as the twentieth element in crustal abundance and has been listed to be contained in at least 23 most commonly naturally occurring minerals where the arsenargentite (Ag3As) was first described back in 1795, (Azcue and Nriagu, 1994). Arsenic occurs mostly in rocks, soil, water and air. 9 WHO, in the Environmental Health Criteria 224 for Arsenic and Arsenic Compunds (2001), reported that the mean As concentrations in air from remote and rural areas range from 0.02 to 4 ng m-3. The mean total As concentrations in urban areas range from 3 to about 200 ng m-3 and much higher concentrations (> 1000 ng m-3) have been measured in the vicinity of industrial sources. Arsenic is widely distributed in surface freshwaters, and concentrations in rivers and lakes are generally below 10 µg L-1, although the individual samples may range up to 5 mg L-1 near anthropogenic sources. Arsenic levels in groundwater average about 1–2 µg L-1 except in areas with volcanic rock and sulfide mineral deposits where As levels can be up to 3 mg L-1. Concentrations of As in open ocean seawater are typically around 1–2 µg L-1. In sediments, the As concentration may range from 3 µg g-1 to 4000 µg g-1 with the higher levels occurring in areas of contamination or pollution (Mandal and Susuki, 2002). Background concentrations in soil range from 1 to 40 µg g-1, with mean values often around 5 µg g-1 (Burguera and Burguera, 1997; Chu, 1994). Arsenic concentration is considerably higher in sediment or soil than in earth crust, because of its accumulation during weathering and tranlocation in colloid fractions. Elemental As is a member of Group 15 of the periodic table that includes nitrogen, phosphorus, antimony and bismuth. It has an atomic number of 33 and an atomic mass of 74.91. Arsenic can exist in four valency states: –3, 0, +3 and +5. Arsines and methylarsines are characteristic of As in the -3 oxidation state. Elemental As, As0 is formed by the reduction of arsenic oxide. Arsenic trioxide (As+3) is a product of smelting operations and is the material used in synthesizing most arsenicals. It is oxidized catalytically or by bacteria to arsenic pentoxide (As+5) or orthoarsenic acid (H3AsO4) (Eisler, 2000). Arsenic and its compounds occur in crystalline, powder, amorphous or vitreous forms. Arsenic accumulation in soils and sediments may be due to the use of arsenical pesticides, application of fertilizers, irrigation, oxidation of volatile arsines in air, dust from the burning of fossil fuels and disposal of industrial, municipal and animal wastes (Prohaska and Stingeder, 2005; Skonieczny and Hahn, 1978). 10 Historically, the use of arsenic-containing pesticides has left large tracts of agricultural land contaminated. Furthermore, soil treated with pesticides shows higher As concentration when compared to soil treated with herbicides, which are usually applied at lower rates (Prohaska and Stingeder, 2005). In the industry, elemental As is used as an additive in special alloys. Arsine is used in microelectronics and semiconductor industry. Some arsenic trioxide is used in the glass and ceramics industry as a decolorizing agent. Coal combustion and fuel burning contribute to As emission in the atmosphere. The degree of industrialization is also reflected in the amount of As in sewage sludge. The presence of As ions in the environment particularly in drinking water is regulated by environmental and public health agencies or authorities. In Malaysia, according to Ministry of Health, the maximum permitted level of As in drinking water is 0.05 mg L-1 (Food Act 1983 (Act 281) and Regulations 2, 2000). Whereas, the provisional guideline value established for As according to Guidelines for Drinking-water Quality by WHO is 0.01 mg L-1 (WHO, 2004). 1.5.1 Geochemistry of Arsenic The toxicity and mobility of As in the environment are dependent on the chemical form or species in which it exists. There are many As compounds of environmental importance. Representative marine As-containing compounds, of which some are found in terrestrial systems, are shown in Table 1.1; their molecular structures are shown Figure 1.1. Other inorganic As compounds of environmental significance are listed in Table 1.2. 11 Table 1.1: Naturally occurring inorganic and organic arsenic species. (WHO, 2001) (see Fig. 1.1 for structures 1–22) Name Structure Arsenate Arsenite Methylarsonic acid, MMA Dimethylarsinic acid, DMA Trimethylarsine oxide Tetramethylarsonium ion Arsenobetaine, AB Arsenocholine, AC Dimethylarsinoylribosides Trialkylarsonioribosides Dimethylarsinoylribitol sulfate 1 2 3` 4 5 6 7 8 9 – 19 20 - 21 22 Arsenic (III) and (V) are the most often determined species in environmental water, soil and sediment, while organic As species are common constituents of biological tissue and fluids. Organic As, MMA and DMA can also be found in dissolved forms in the water column. Under oxidizing and aerated conditions, the predominant form of As in water and soil is As (V). Under reducing (acid and mildly alkaline) and waterlogged conditions, As (III) can become the predominant As compounds. The rate of conversion is dependent on the Eh and pH of the soil as well as on other physical, chemical and biological factors. In brief, at moderate or high Eh, As can be stabilized as a series of pentavalent (arsenate) oxyanions, H3AsO4, H2AsO4–, HAsO42– and AsO43–. Figure1.2 shows the speciation of As under varying Eh and pH. 12 Figure 1.1: Structures of naturally occurring inorganic and organic arsenic species. (WHO, 2001) 13 Table 1.2: Inorganic arsenic species in the environment. (WHO, 2001) Name Synonyms Inorganic As, trivalent As(III) oxide Formula As trioxide, arsenous oxide, white As As2O3 (or As4O6) arsenenous acid arsenious acid HAsO2 As(III) chloride As trichloride, arsenous trichloride AsCl3 As(III) sulfide As trisulfide orpiment, auripigment As2S3 As pentoxide As2O5 arsenic acid ortho-arsenic acid H3AsO4 arsenenic acid meta-arsenic acid HAsO3 Inorganic As, pentavalent As(V) oxide arsenates, salts of ortho-arsenic acid H2AsO4–, HAsO42–, AsO43– Arsenic solubility is low under oxidizing conditions and the more typical form of As is arsenate. At high pH or under reducing conditions, arsenate is reduced to soluble arsenite and the amount of arsenite increases significantly under strong reducing conditions. Under moderate reducing conditions, As solubility is controlled by iron oxy-hydroxides or manganese oxides (Barrachina et al., 1999). With the presence of sulfide and under reducing conditions, it can lead to the precipitation of As2S3 in lake, river and marine sediments. The formation of sulfides in reducing conditions occurs simultaneously with the reduction of arsenate to arsenite. Figure 1.3 shows the transformation pathway of inorganic As in the environment. 14 Figure 1.2: The Eh-pH diagram of arsenic in water at 25C and one atmosphere (Kartinen and Martin, 1995 and WHO, 2001). Arsenic is released into the atmosphere primarily as As2O3 and mainly adsorbed by particulate matters. These particles are dispersed by the wind and are returned to the earth by wet or dry deposition. Arsines released from microbial sources in soils or sediments undergo oxidation in the air, reconverting the As to non-volatile forms, which then settle back to the soil. 15 Arsenate H3AsO4 reduction Arsenite H3AsO3 oxidation + S2sorption sorption AsS2- + Fe Adsorption and precipitation precipitation reduction (also microbial reduction) As2S3 FeAsO4 Figure 1.3: Simplified transformation pathway of inorganic arsenic in the environment (Prohaska and Stingeder, 2005). 1.5.2 The Toxicology of Arsenic Arsenic is a common environmental agent whose toxic properties have been known for centuries. Since ancient time, arsenicals have been used as agents of suicide and murder. Arsenic as a toxic, poisonous and killing agent was closely associated to the death of France’s famous ruler, Napolean Bonaparte. Humans are exposed to many different forms of inorganic and organic As species in food, water and other environmental media. Each As form has different physicochemical properties and bioavailability. Zielhuis (1985) had classified at least three groups of As-compounds: i) inorganic water soluble compounds; ii) inorganic non- or low-solubility compounds and iii) organic As used to distinguish the metabolic models and health risks of arsenic-compounds in order to establish the quality standard or recommended limits of As. 16 The chemical forms and oxidation states of As are important with regards to their toxicity which is directly related to their mobility in water and body fluids. The toxicity of As compounds decreases in the following order; arsines > inorganic arsenites (As (III)) > arsenoxides > inorganic arsenates (As (V)) > organic pentavalent compounds > arsonium compounds > elemental As (Mandal and Susuki, 2002; Prohaska and Stingeder, 2005). It appears that the stable, soluble inorganic arsenites and arsenates are readily absorbed by digestion tract, abdominal cavity and muscle tissue. Excretion of arsenate is faster than that for arsenite, mostly in urine (Förstner and Wittmann, 1983). Arsenate has a low order of toxicity than arsenite and does not inhibit any enzyme system due to its lack of affinity to hydroxo and thiol groups, but adenosine triphosphate (ATP) synthesis is inhabited by a AsO43- by uncoupling oxidative phosphorylation and replacing the stable phosphoryl group. In contrast, arsenite inhibits thiol-dependent enzymes, binds to tissue protein as keratin disulfides in nails, hair and skin and retained in the body for a prolonged period. Routes of As intake in vivo are considered respiratory for dust and fumes, and oral for As in water, beverages, soil, and food. People from many countries all over the world are suffering from the toxic effects of arsenicals as a result of natural groundwater contamination caused by industrial effluent and drainage problems. A number of As poisoning cases via contamination of groundwater in Argentina, Bangladesh, Chile, India, Taiwan and United State have also been reported (Mandal and Susuki, 2002). They also reviewed several As poisoning episodes from industrial sources and contamination in food and beverage all over the world. 17 McArthur et al. (2004) had carried out an extensive study on natural organic matter in sedimentary basin and its relation to As in anoxic groundwater. Water and sediment samples were collected from several wells in southern West Bengal. This investigation was aimed to explain the mechanism of As release to ground water by reductive dissolution of FeOOH. Human excreta in latrines penetrates the upper confining layer which has hydraulic continuity with the aquifer, may locally promote additional reduction of FeOOH and add to the As problem. Generally, the complete reduction of FeOOH contributes to high concentration of As (200-1180 g L-1). Several epidemiological studies have shown that acute and chronic exposure to As can cause a variety of adverse health effects to humans such as dermal changes, respiratory, pulmonary, cardiovascular, gastrointestinal, hematological, hepatic, renal, neurological, developmental, reproductive, immunologic, genotoxic, mutagenetic, and carcinogenic effects (Brown and Ross, 2002; Buchet, 2005; Leonard, 1985). Increased risks of lung and bladder cancer and arsenic-associated skin lesions have been reported to be associated with ingestion of drinking water at concentrations of As equal to and less than 50 µg L-1 (WHO, 2001). 1.6 Selenium Selenium is classified as a metalloid that lies between nonmetallic sulphur and metallic tellurium in Group 16 and between arsenic and bromine in Period IV of the Periodic Table. It has an atomic number of 34 and an atomic mass of 78.96. In physical state, Se occurs in grey metallic or red amorphous powder or vitreous form. Selenium can exist in four valency states: –2, 0, +4 and +6. 18 Selenium is an essential trace element to life. However at very low concentrations it can cause anomalies in organisms and at high concentrations it is toxic (Haygarth, 1994; Merian, 1985). Selenium exists at low abundance in the earth’s crust with concentration of 0.05 g g-1 (Haygarth, 1994). It is often associated with sulfur-containing minerals and usually accompanies sulfur in volcanic effluents which make the soils in the neighborhood of volcanoes tend to have enriched amounts of Se (Malisa, 2001; WHO, 2003b). Selenium has vast potential use in the industries. Selenium exhibits both photovoltaic action, where light is converted directly into electricity, and photoconductive action, where the electrical resistance decreases with increased illumination. These properties make Se useful in the production of photocells and exposure meters for photographic use, as well as solar cells. Selenium is also able to convert alternating current electricity to direct current, and is extensively used in rectifiers. Below its melting point, Se is a p-type semiconductor and is finding many uses in electronic and solid-state applications such as in xerography for reproducing and copying documents (Lide, 2007). Selenium is widely used in glass and ceramic industry for decolorizing and glazing. In heavy industry, it is used to improve the porosity of stainless steel casting and to improve the machinability of copper and copper alloy (Green and Turley, 1961). Selenium is also used as pigments in plastic and paint due to its resistant to heat, light and weathering. Selenium is an antioxidant, which makes it useful for inclusion in inks, mineral and vegetables oil, and lubricants (Haygarth, 1994). Early agricultural uses of Se compounds as pesticides were very limited and short-lived. The recent use of Se compounds as feed additives for the prevention of Se deficiency diseases in farm animals represents a source for environmental contamination (WHO, 1987). Selenium sulfide is used in shampoos as an anti-dandruff agent. Human activities, particularly mining, agricultural, petrochemical, and industrial manufacturing operations may result in environment contamination with Se. 19 The concentration of Se in igneous rocks is relatively low, usually much less than 1 µg g-1, and similar levels probably occur in metamorphic rocks. Sedimentary rocks, such as sandstone, limestone, phosphorite, and shales may contain from less than 1 and up to 100 µg g-1 of Se (WHO, 1987). The Se content in a soil may indicate, to some extent, the parent material from which the soil has been formed. Thus, in arid and semi-arid areas, soils with high Se content have been derived from sedimentary rocks, usually shales and chalks. The range of soil Se content is typically from 0.1 µg g-1 to 2 µg g-1. But at seleniferous area, an average of Se concentration may be found to be between 45 µg g-1 and 80 µg g-1 (Quinn, 1985). In Great Lakes, USA, Se concentrations from bottom sediments range from 0.35 µg g-1 to 0.75 µg g-1. While, oceanic study for sediment samples reported that the Se concentrations range from 0.34 µg g-1 to 4.8 µg g-1 (Eisler, 2000). The concentrations of Se which are equal to or greater than 4.0 µg g-1 in sediments are a great environmental concern because of the potential for bioaccumulation in fish and wildlife (Derveer and Canton, 1997). Under natural conditions, the concentration of Se in water usually ranges from a few tenths to 2 or 3 µg L-1 (WHO, 1987). Surface waters seem much less likely to contain excessive levels of Se than ground waters. Selenium concentrations with levels exceeding 50 µg L-1 have been documented in groundwater, especially in areas with seleniferous soils, in sewage wastes, in irrigation drain water and in water of flyash settling ponds (Eisler, 2000). The 2004 WHO International Standards for Drinking Water proposed a health-based guideline value of 0.01 mg L-1 for Se on the basis of human studies and following the recognition that biota accumulated Se from water. In Malaysia, under the Ministry of Health, the maximum permitted level of Se in drinking water is 0.01 mg L-1 (Food Act 1983 (Act 281) and Regulations 2, 2000). 20 1.6.1 Geochemistry of Selenium The concentration of Se in soil depends on the parent material, topography, climate, age of soil and man activities (Frankenberger and Karlson, 1994). Inorganic Se species enter natural waters, groundwaters, soil and sediment from many sources. The two most common sources of Se in the environment include the weathering of rock and soils and organic compounds from decayed plant tissue and agricultural products (Koll, 1993). Selenium also enters the geochemical cycle through human activities which include agricultural drain water, sewage sludge, fly ash from coalfired power plants, oil refineries and mining (Hamilton, 2004). The fate of Se in natural environments is affected by many physical, chemical and biological factors, which are closely associated with changes in its oxidation state. The concentration and speciation availability of Se are associated with the pH, redox potential, solubility, complexing ability of soluble and solid ligands, and microbial interaction (Belzile et al., 2000). Figure1.3 shows the availability of Se species under varying Eh and pH. Changes in the valence state of Se from -2 (hydrogen selenide) through Se0 (elemental selenium), +4 (selenite) and +6 (selanate) are associated with its geological distribution, redistribution and use. Most of the Se species found in soil and water are the inorganic selenite (SeO32-) and selenate (SeO42-) forms. Soluble selenates which occur in alkaline soil, are slowly reduced to selenites and are then readily taken up by plants and converted into organoselenium compounds, including selenomethionine, selenocycsteine, dimethyl selenide and dimethyl diselenide. At equilibrium, the typical form of Se in soil is elemental selenium. Referring to Figure 1.4 below, the biologically available Se is only present in relatively large amount in acidic areas or neutral soils and declines as the soils become more alkaline. The decline may be accelerated by active agricultural or industrial practices. In dry areas with alkaline soils and oxidizing conditions, elemental selenium and selenides in rocks and volcanic soils may oxidize sufficiently to maintain the availability of biologically active Se. 21 Figure 1.4: The Eh-pH diagram of selenium in water at 25C and one atmosphere (Séby et. al, 2001). Selenite (Se(IV)) would form mineral salts which are generally less soluble than selenate (Se (VI)) salts. Under acidic reducing conditions in soil, which may be waterlogged and rich in organic matter, elemental selenium and selinides are the dominant species. Elemental selenium, selinides and selenium sulfides are less biologically available for uptake. At high redox potential and in well-oxygenated alkaline soil, highly soluble selenate is the dominant species, and at neutral pH, selenite occurs in approximately equal concentrations to selenate. Selenate is stable in oxidized environments and is the form in which Se is most readily taken up by plants, which can cause great environmental concern. 22 1.6.2 The Toxicology of Selenium The toxicity of Se depends on the biologically active oxidized forms of 2- SeO3 or SeO42- (Uden, 2005). In oxidizing conditions or alkaline soil, Se may be sufficiently oxidized to maintain the availability of biological active forms that are readily absorbed by plants. In acidic or neutral soil, it tends to remain relatively insoluble and the amount of biologically active forms of Se available that can be absorbed by plants constantly decreases. Selenium dioxide is the primary problem involved with most industrial exposure to the element as the oxide is easily formed when Se is heated. The dioxide itself forms selenious acid when it mixes water or sweat and becomes an irritant. Selenium compound released during fossil fuel combustion may pose an inhalation hazard to people. Most probable forms of Se released during combustion are selenious acid and elemental selenium. Acute poisoning through inhalation may occur in industry which produces Se dust and fumes, selenium dioxide and hydrogen selenide as the by products. The considerable biological importance of Se was first recognized in the 1930s when it was discovered that certain well-defined and economically important farm animal diseases were actually the result of chronic Se poisoning. These animal diseases were restricted to agricultural areas in which large amounts of Se in the soil were available for uptake by the plants, which were then consumed by the animals. The farm animals showed severe signs of distress such as laboured breathing, abnormal movement and posture, prostration, and diarrhea. Death often followed within a few hours after the animals fell sick (WHO, 1987). The capability of Se to accumulate in living tissue is a great concern as it can cause adverse health effect. The Se capability to accumulate in living tissue is due to its propensity to bioaccumulate within the base of food webs: from water and sediment to aquatic plants and aquatic invertebrates (Hamilton, 2004). Tinggi (2003) had summarized some effects of Se exposure to human. For an example, industrial exposures in the manufacture of Se rectifiers have caused hypochromic anemia and 23 leucopenia, and damaged the nails of long-term workers employed in the industry. There have been a number of reported cases of acute and subacute Se poisoning in humans as a result of accidental ingestion of selenic acid and vitamin tablets that contained high levels of Se. An ingestion of Se at high level is reported to cause gastrointestinal disturbances (vomiting, diarrhea), hair and nail changes, and neurologic manifestations. Examination of exposed individuals showed increased levels of Se in the blood and urine (WHO, 1987). 1.7 Chromium Chromium has been identified both as an essential element and as a chemical carcinogen agent (Hoet, 2005). It is a transition element located in group 6 of the periodic table. It has an atomic number of 24 and an atomic mass of 51.996. Chromium demonstrates oxidation numbers of 2+, 3+, 4+, 5+, and 6+. Of these, the compounds of the trivalent chromium, Cr (III) are the most stable, the most abundant, less toxic and are mainly bound to organic matter in soil and aquatic environment. In acid media, hexavalent chromium, Cr (VI) compounds are strong oxidizing agent where Cr (VI) is readily reduced to Cr (III) at low pH. Chromium and its compounds are widely used in the industrial, manufacturing and commercial sectors (Katz and Salem, 1994). In metallurgical industries, Cr is used as an alloying element that imparts the resistance to corrosion property to stainless steels by the formation of a thin, transparent and protective film of Cr2O3. The film stability can be improved by increasing the Cr contents of the alloys and by the addition of nickel, molybdenum or other elements. Chromel is a nickel-chromium alloy used in the manufacture of resistance wire for electrical heaters and thermocouples. 24 Trivalent and hexavalent chromium compounds are widely used in manufacturing and industrial due to its chemical and physical properties. The colours of trivalent and hexavalent chromium compounds coupled with appropriate solubility characteristics make them the preferred pigments and colourants in the manufacture of paints, printing inks, floor coverings and vinyl sheeting. The inertness of the trivalent oxide makes Cr compounds useful as corrosion inhibitors and as agents for oxidizing and plating metals. The tendency of trivalent chromium to form complexes with basic oxygen and/or nitrogen atoms in protein makes it possible for the tanning of leathers to be completed in hours rather than in days. The leathers produced become more resistant to wear and heat. The oxidizing properties of hexavalent chromium compounds have found applications in the synthesis of organic dyestuffs. Chromium and its compounds have become ubiquitous in modern society. Chromium is found in all phases of environment, including air, water, rock and soil. Almost all of the sources of Cr in the earth’s crust are in the trivalent state. The concentration of Cr in rocks varies from an average of 5 µg g-1 in granitic rocks, to an average of 1800 µg g-1 in ultrabasic and serpentine rocks (WHO, 1988). Weathering of rocks produces Cr complexes that are almost exclusively in the trivalent state. In most natural soils, Cr occurs in low concentrations of an average of 5 µg g-1. The highest concentrations of Cr which are at 125 g kg-1 are always found in serpentine soils (Shanker et al., 2005). In freshwater, Cr concentrations generally range from 0.1 to 117 g L-1 whereas the values for seawater range from 0.2 to 50 g L-1 (Shanker et al., 2005). Chromium concentration varies widely in the atmosphere, from background concentration of 5 to 7 pg m-3 in air samples from remote areas such as Artic to at least 2 to 4 times higher concentration in urban areas than background concentrations (WHO, 2003a). 25 As a guideline, the WHO (2004) recommended a maximum level of 50 g L-1 Cr in drinking water. In Malaysia, the quality criteria for drinking water is set under the Food Act 1983 (Act 281) and Regulations 2 (2000) and the maximum permitted value for total Cr is 50 g L-1. Due to the fact that the health effects are determined largely by the oxidation state, WHO (2003a) proposed a different guideline values for chromium (III) and chromium (VI). However, the available toxicological data mainly in the study of carcinogenicity of Cr (VI) is yet not enough to support the derivation of a new value. 1.7.1 Geochemistry of Chromium Chromium compounds are highly varied and widely distributed in the occupational, domestic and natural environments. Environmental Cr concentrations reflect the element’s distribution in air, water, soil, plants and animals from both anthropogenic and natural sources. Chromium compounds are released from minerals containing Cr during the physical, chemical and biological weathering processes by which rocks are abraded or dissolved. Once released, the Cr compounds can be transported as wind or waterborne particulates. Volcanism is an alternative to weathering for the release of Cr compounds. The combustion of large quantities of coal and oil is also the major anthropogenic source of environmental Cr. Emissions from Cr chemical manufacturing facilities, from many cooling towers, and steel mills are other major sources of airborne Cr. Industrial and domestic wastes are also dispatched to landfills for disposal. Sewage sludge compost piles and steel mill slag heaps are additional wastes to be considered as potential sources of Cr for the contamination of soil and groundwater. 26 The distribution of compounds containing Cr (III) and Cr (VI) depends on the redox potential, pH, the presence of oxidizing or reducing compounds, the kinetics of the redox reactions and formation of Cr (III) complexes or insoluble Cr (III) salts (WHO, 2003a). In soils, Cr (III) predominates. In the environment, Cr (VI) occurs mostly as CrO42- or HCrO4- and Cr (III) as Cr(OH)2+. Figure 1.5 shows dissolved Cr species under varying pH and Eh. Figure 1.5: The Eh-pH diagram of chromium in water at 25C and one atmosphere (Beverskog and Puigdomenech, 1997). Most of the Cr in air will eventually settle and end up in waters or soils. Chromium in soils strongly attaches to soil particles and as a result it will not move towards groundwater. In water, Cr will be absorbed by sediments and become immobilized. Only a small part of the Cr that ends up in water will eventually dissolve.Chromium (VI) can easily be reduced to Cr (III) by organic matter. It is a moderately strong oxidizing agent and will react with organic matter or other reducing agents to form Cr (III). Therefore, in surface water rich in organic content, Cr (VI) will exhibit a much shorter lifetime (EPA, 1998). In water, Cr (III) is a 27 positive ion that forms hydroxides and complexes and is adsorbed at relatively high pH values. In surface waters, the ratio of Cr (III) to Cr (VI) varies widely, and relatively high concentrations of the latter can be found locally. In general Cr (VI) salts are more soluble than those of Cr (III), making Cr (VI) relatively mobile (WHO, 2003a). 1.7.2 The Toxicology of Chromium The importance of Cr is underscored by knowledge that it is an ubiquitous, naturally occurring element usually found in rocks, minerals and geological emissions. Chromium in excess amounts can be quite toxic, depending upon the chemical species of Cr and the route of exposure (Crounse et al., 1983). Exposure to Cr can occur from the large amounts of Cr released into the environment from the industries, mining and processing of chromites ores. Moreover, Cr may make its way into the human body from dietary intake and drinking water. Chromium is not known to accumulate in the bodies of fish, but high concentrations of Cr, due to the disposal of metal products in surface waters, can damage the gills of fish that swim near the point of disposal. In animals, Cr can cause respiratory problems, a lower ability to fight disease, birth defects, infertility and tumor formation (WHO, 1988). Chromium (III) is an essential nutrient for humans and Cr deficiency may cause heart conditions, disruptions of metabolisms and diabetes. But the uptake of too much of Cr (III) can cause adverse health effects as well, for instance skin rashes. Chromium (VI) compounds are, in general, more toxic than Cr (III) compounds with regards to acute and chronic oral toxicity, dermal irritancy and allergy, systemic effects, cytotoxicity, genotoxicity and carcinogenicity (Katz and Salem, 1994). Chromium (VI) is a danger to human health, mainly for people who work in the steel and textile industry. Some epidemiological data suggest that an excess of lung cancer has also occurred in the chromate-pigment industry (WHO, 1988). People who 28 smoke tobacco also have a higher chance of exposure to Cr. When present as a compound in leather products, it can cause allergic reactions, such as skin rash. Inhalation of Cr (VI) can cause nose irritations and nosebleeds. CHAPTER 2 LITERATURE REVIEW 2.0 Sediment and Its Role in Freshwater Ecosystem The particulate material that lie below the water in rivers, lakes, ponds, streams and other aquatic systems are called sediments (MacDonald et al., 2003). Sediments in aquatic system are primarily derived from rock weathering processes. Transportation of the sediments particles from its pint of origin will sort them into different size range and associated mineral fraction before they are deposited at the bottom of water body. Re-suspensions and further transfers can occur to the sediment before reaching at its ultimate resting point. Sediments represent a complex and dynamic environment. Sediments also represent essential elements of aquatic ecosystem because they support aquatic organisms. Nutrients collect in the sediments, and many aquatic ecosystems depend to a large extent on the cycling of nutrients back and forth between the sediment and water. Sediment are also the rooting medium for aquatic plants and home for invertebrates and benthic fish (Dixit and Witcomb, 1983). 30 Importantly, sediments cal also provide habitats for many wildlife species during portions of their life cycle (MacDonald et al., 2003). For example, a variety of fish species utilize sediments fro spawning and incubation of their eggs and larvae. Many amphibian species burrow into the sediments in fall and remain there throughout the winter period, such that sediment provides important overwintering habitats. Therefore, sediments play a variety of essential roles in terms of maintaining the structure and function of aquatic ecosystems. 2.0.1 Sediment Contamination Some of the multiple sources that contribute to sediment contamination are shown in Figure 2.1. Runoff and discharge from rivers, creeks, and drainage channels produce some major “nonpoint” contaminant sources, as they carry storm water and dry weather runoff from the upland watershed. Contaminants may also come from point source discharges, such as accidental spills or regular municipal wastewater and industrial discharges. Additional nonpoint contaminant sources include atmospheric deposition and groundwater. Though a large portion of these contaminants may be associated with suspended particles in the discharge or receiving water body, each source influences water and sediment quality on different spatial and temporal scales. This variety of sources, combined with various physical mixing processes such as currents, water flow rate, sedimentation, tidal exchange, and wave, can produce complex and widespread patterns of sediment contamination (Battelle Memorial Institute, 2003). Various contaminants from a variety of sources can be classified into several groups. United State EPA through an overview report on sediment quality (Lyman et al., 1987) had categorized five major types pf contaminants generally found in sediments. The contaminants were defined as in Table 2.1. 31 Table 2.1: Major contaminants in sediments. Contaminants Categories Type of Contaminants Nutrients Phosphorous and nitrogen compounds such as ammonia. Bulk Organics A class of hydrocarbons that includes oil and grease A group of chemicals that are very resistant to decay Halogenated Hydrocarbons or Persistent Organics like DDT and PCB A group of organic chemicals that includes several Polycyclic Aromatic Hydrocarbons (PAHs) petroleum products and by products Iron, manganese, lead zinc and mercury; Metals and Metalloids Arsenic and selenium (metalloids) Figure 2.1: Principal sources, fates and effect of sediment contamination in aquatic ecosystem (Bridges et al., 2005). 32 The properties of sediments can influence the ultimate fate and effects of the contaminants it contains. Sediment particles can vary from coarse sand with a diameter of about 1 mm to fine silts and clays with diameters less than 0.01 mm. Variations in the size and composition of these particles have an effect on the binding of contaminants to them. Finer particles generally contain higher contaminant concentrations due to a much greater surface area and greater number of chemical sorption sites (Förstner, 2004). Bottom sediments represent an important indicator of contamination because they tend to accumulate contaminants in aquatic system. They therefore help identify areas, which have become contaminated and reveal spread patterns from sources and contaminant input history. They show much less temporal and spatial variation than the water column data. Furthermore, metal levels in sediments are generally one or two order magnitude higher than the amount of dissolved form in water (Beckett et al., 1991). 2.0.2 Sediment Quality Issue and Assessment Sediments are being used increasingly to assess human impact on the aquatic environment. Sediments are frequently used to identify sources to contaminants, determine dispersion pathway and locate contaminants sink in aquatic systems due to their capability to transport and accumulate various contaminants (Simpson et al., 2005). Sediments are also economically attractive to use as a monitoring tool and are being increasingly employed in the early phase of environmental assessment of aquatic system (Birch et al., 2001). Contaminated sediments represent an important environmental concern for several reasons. First, contaminated sediments have frequently been demonstrated to be toxic to sediment-dwelling organism and fish. Exposure to contaminated sediments can result in decreased survival, reduced growth or impaired reproduction in aquatic organisms. Additionally, certain contaminants are taken up by benthic 33 organisms through a process called bioaccumulation. When large animals feed on these contaminated prey species, the contaminants are taken into their bodies and are passes along to other animals in the food chain in a process called biomagnifications. As a result of the effects of toxic and bioaccumulative substance, aquatic organism, birds and mammals can be adversely affected by contaminated sediments (MacDonald et al. 2002). Contaminated sediments can also adversely affect human health. Human health can be potential affected due to direct exposure to contaminated sediments during wading or swimming in affected water bodies. Consumption of contaminated fish and shellfish also poses a risk to human health (MacDonald et al., 2003). Sediment quality data are obtained using a variety of analytical technique depending on the contaminants of interest. For example of metal or metalloids contaminants, reliable and sensitive analytical methods have an important role in determining and evaluating the environmental impact of the pollutions which also cover other types of environmental samples including water, plants and animals (Förstner and Wittmann, 1983). Some reliable techniques such as inductively coupled plasma atomic emission spectrometry, inductively coupled plasma mass spectrometry and neutron activation analysis have virtually ideal characteristics for simultaneous multi-element determination. Reliable analytical data of sediments can provide information on the sediment quality conditions in order to evaluate the level of contamination and the effect of contaminated sediment on aquatic organism, wildlife and human health. According to MacDonald et al. (2003), assessing of sediment quality thorough evaluating the contaminants data is conducted for several reasons as following: - To support broad assessment of environmental conditions. - To support the identification and assessment of sites with contaminated sediments. - To evaluate the status and trends in environmental conditions. - To support ecological risk assessments. 34 - To assess the efficacy of point or non-point source pollution control efforts. - To assess the cumulative environmental effects of multiple facilities area. - To evaluate the feasibility of restoring aquatic habitats. - To assess the environmental impacts of various anthropogenic activities. Countries like Canada and several states in America had established guideline value for contaminants in the sediments. The guidelines documents are usually known as Sediment Quality Guidelines (SQG). For example, Canada had established Canadian Sediment Quality Guidelines for Protection of Aquatic Life which covers freshwater and marine sediment quality (Canadian Council of Ministers of Environment, 1999). SQG is one of usefully tool in assessing the sediments quality conditions (Wisconsin Department of Natural Resources, 2003). 2.1 Analytical Technique for Elemental Analysis Reliable and sensitive analytic methods have an important role in determining and evaluating the environmental impact of elemental pollution. It is realized that no single analytical technique can be used for all analysis. Several requirements for an analytical method to be acceptable are (1) sensitivity, specificity and accuracy; (2) rapidity and ease of operation; (3) the possibility of automation; (4) low cost of the equipment and (5) reliability of result (Förstner and Wittman, 1983). Instrumentation of analytical technique can be divided into nuclear and nonnuclear techniques. Activation analysis and X-ray fluorescence spectrometry are methods of analysis that are generally known for the nuclear analytical techniques. The non-nuclear techniques can be found in most analytical laboratory such as atomic absorption spectroscopy, inductively couple plasma mass spectrometry and chromatography techniques. 35 2.1.1 Neutron Activation Analysis Neutron activation analysis (NAA) is one of the popular methods in nuclear analytical techniques. Historically, following the development of nuclear reactor in the 1940s and sodium iodide scintillation detectors in the early 1950s, the possibilities for applying NAA for trace element analysis of samples from many disciplines have been recognized. Early development of NAA was taking place rapidly. Then, the invention of high resolution solid-state Ge(Li) detectors in the 1960s and more recent advancements of computers and automation during 1970s and 1980s have made this technique applicable to several research studies such as archeology, environmental science, forensics, geology, material science and life science which particularly involve very large numbers of samples (IAEA, 1990). 2.1.2 Principles of Instrumental Neutron Activation Analysis Neutron activation analysis has become the core tool for geochemical and life science trace element research because the technique possesses several important advantages. These include, substantial freedom from systematic errors, it is complementary to other methods, freedom from analytical blank and other problems related to dissolution, multi-element capability and sensitivity at sub-picogram amounts. These characteristics of nuclear methods have been widely exploited, particularly in research into trace element analytical methodology. NAA is being the premier analytical technique in this study as it gives convenient on solid sample preparation and analysis while providing high accuracy and sensitivity. The physical phenomena upon which NAA are based are the properties of the nucleus, radioactivity and the interaction of radiation matter. The NAA depends on the irradiation of a stable nuclide AX with neutron using the AX(n, ) A+1X nuclear reaction. The sequences of events during a typical (n, ) reaction are illustrated in Figure 2.2. When a neutron interacts with a target nucleus by a non-elastic collision, a compound nucleus is formed in a highly excited state. The high excitation energy 36 of the compound nucleus averages at 8 MeV due to the high binding energy of the neutron with the nucleus. The lifetime of the compound nucleus is typically 10-16 to 10-14s. This is long enough that no traces remain to identify the particular process of formation, but short enough that the nucleus can undergo a rapid de-excitation to a more stable configuration, in a number of different ways that usually involve emission of nucleus particles or prompt gamma rays. In most cases, the new nucleus is radioactive and will further de-excite by emitting decay gamma rays. Once the production A+1X is -ray emitter, a gamma-ray detection system can be employed to measure both intensity and energy of the -ray used to indicate the target nuclide AX and to determine the concentration of the element in the samples. About 70% of the elements have nuclides possessing properties suitable for NAA (IAEA, 1990). With the use of automated sample handling, gamma-ray spectrum measurement with solid-state detector and computerized data processing it is often possible to measure more than thirty elements without chemical separations. This application of purely instrumental procedures for trace element analysis is frequently called instrumental neutron activation analysis (INAA) (IAEA, 1990). The most common approach to NAA is the “comparator” method, which is commonly accepted as the most accurate way to quantify element concentrations. According to this method, samples are irradiated simultaneously with standards containing known amounts of the elements. After irradiation, both samples and standards are measured under identical geometrical conditions with the same detector. This procedure eliminates uncertainties in the nuclear parameters and detector efficiencies. 37 p PROMPT GAMMA-RAY TARGET NUCLEUS A Z A1 Z X RADIACTIVE NUCLEUS X STABLE NUCLEUS A 1 Z 1 Y INCIDENT NEUTRON COMPOUND NUCLEUS A1 Z X DECAY GAMMA-RAY d Figure 2.2: Schematic diagram illustrating the sequence of events for a typical (n, ) reaction. The measurement of induced radioactivity is being done by gamma-ray spectrometry detector. The basic set-up of a gamma-ray spectrometer for use in INAA is shown in Figure 2.3. It consists of a semiconductor detector with associated preamplifier, a high-voltage (HV) power supply, a spectroscopy amplifier, an analogue-to-digital converter (ADC), a multi-channel pulse height analyser (MCA) and a computer- system with input/output facilities. For application in INAA, only germanium detectors are of importance. Germanium semiconductor detector exists in two versions, Lithium drifted germanium detector or known as Ge(Li) detectors and Hyperpure (HP-Ge) or intrinsic germanium detectors. The basic element of a semiconductor detector is a single crystal of semiconductor material with P-I-N diode structure where N- and Prefers to the nature of the impurities in the crystal. N-impurities are pentavalent atoms that act as a electron donor, P-impurities are trivalent atom, acting as electron acceptor and I is the intrinsic layer. 38 For a Ge(Li) detector, lithium ions drifted into the crystal structure, compensate for impurity centers and forming an active region. One of the advantages of HP-Ge is that well-type detector can be more easily fabricated and repaired than ever was attainable with Ge(Li) material, resulting also at more practical large willdiameters (up to 25 mm) and at highly competitive prices. Semiconductor detectors are operated at liquid nitrogen (LN2) temperature (77K). The crystal is mounted in a vacuum cryostat that is thermally connected to a copper rod, the ‘cold finger’which transfers the dissipated heat from the crystal to the cooling medium. Ge(Li) detectors always need to be stored at LN2-temperature; warming up of the detectors can lead to almost irreparable damage. HP-Ge detectors can be stored at room temperature without damaging the crystal as long as the HV bias is removed. HIGH VOLTAGE SUPPLY PREAMPLIFIER SPECTROSCOPY AMPLIFIER ANALOGUE TO DIGITAL CONVERTER GERMANIUM DETECTOR COMPUTER MULTI CHANNEL PULSE-HEIGHT ANALYSER Figure 2.3: Schematic set-up of gamma ray spectrometer for use in INAA. One of the drawbacks of INAA, sometimes mentioned when considering its use in a monitoring program, is its relatively long turn-around time, when compared to other analytical techniques. Indeed, sometimes 3 or 4 weeks decay period is required to obtained highest sensitivity. However, many elements can often be determined with adequate sensitivity after shorter decay times. Moreover, INAA with short-half life nuclides may also provide a very rapid measurement. In general, INAA may be the first choice for materials research or study from the applied field especially for samples with are difficult to be converted into solution form for analysis, e.g., via AAS or ICPMS; and/or of which only milligram quantities are available. 39 2.1.3 Nuclear Reactor The research reactor is the most widely used source of neutrons for INAA, particularly with respect to the number of samples processed and number of elemental analysis performed. Five general types of research reactor are Slowpoke, Argonaut, TRIGA, Pool and Heavy Water. The types of reactors are not mutually exclusive and may overlap in some aspects, e.g., TRIGA reactors are the Pool type (IAEA, 1990). A research reactor is a major investment and it is also expensive to run. But, normally the reactor would have been built for some other purposes, such as nuclear research and training, material testing and isotope production. Besides that, neutron activation analysis is a useful facility of reactor operation and therefore the cost of irradiation can usually be kept to an affordable level. The TRIGA reactor is one of the typical reactors used for neutron activation analysis. It is designed as a general-purpose reactor and has incorporated in its design the facility for flux equalization system, which makes it very suitable for activation analysis. The TRIGA reactors range in power levels from 18 kW to 3 MW with 250 kW and 1 MW being the most common operating levels. These reactors operate with uranium-zirconium hydride homogenous solid fuel with an enrichment of either 10% or 70%. The reactors are light water cooled, graphite reflected and of the pool type. Most TRIGA reactors are capable of operating in the pulse mode and most have a rotary specimen rack called Lazy Susan containing 40 irradiation positions between the core and the reflector. The rotary specimen rack can be rotated during the irradiation in order to get homogenous flux for all samples. 40 2.2 Elemental Speciation Interest in chemical and elemental speciation procedures is expected to rapidly expand as more of the scientific community recognizes that assessments of environmental risk, toxicity, bioavailability, transport and metabolism must be done on the specific chemical form or species, rather than on the total elemental concentration. Speciation science strives to identify and characterize, as many as possible, the element species in order to understand the environmental and biological implications of individual element. It is a discipline which is a great relevance to groups that include environmentalist, soil and sediment scientists, remediation specialists, pharmaceutical scientists, and specialists in various fields of nutrition and medicine. While there are several well-documented examples of varying degrees of toxicity with regards to certain elements and tremendous advances in efforts to understand the biological transport and metabolic mechanism of some elements, there is still a vast wealth of knowledge yet to be gained in the area of elemental speciation. Determination of total metal concentration is not always the most suitable measurement of real toxicity level due to existence of many difference species. It is important to characterize and determine the forms of elements in a system since different forms can have totally different properties. Some elements can be highly toxic to various life forms while others are considered essential at a very small amount or concentration, but become toxic at higher doses. For an example, Cr (III) is an essential element while Cr (IV) is highly toxic (Kartz and Salem, 1994). Inorganic arsenic in the form of As (III) is considered about 10 times more soluble and mobile and also more toxic than As (V) (Herreweghe et al., 2003). On the other hand, organic arsenic compounds; methylarsonate and dimenthylarsinate are much less toxic than the inorganic arsenic compounds (Prohaska and Stingeder, 2005). Therefore, considering the adverse environmental and human health impact, a study of specific metal species with particular attention to the determination of the metals origin and the understanding of their geochemical behavior – diagenesis, mobility, transport and also biological availability has become increasingly important. 41 In nature, elemental species can be formed, transformed and transported between environmental compartments through various ways involving chemical, physical and biological processes. The elemental species in water, soil, sediment and air have various pathways to enter plants, animals and also human beings which often to be the last entity in the food chain. Exposure of toxic species to human through foods and water consumption, inhalation, or through permeation of the skin or mucous membranes has become great concern for toxicologists. The properties of the elemental species such as the size, oxidation state and solubility can influence the absorption of toxic species in human body. Further understanding of the route and dose exposure, accumulation mechanism, metabolic reaction of the species will help predict and model the fate, risk and effect towards human being. Speciation study has shown an increasing interest in the analytical niche during the last decade. It is well recognized that the total element concentration determination does not provide adequate information to understand the effects observed in the environment and in living systems. The International Union for Pure and Applied Chemistry (IUPAC) (Templeton, 2000) has defined elemental speciation in chemistry as follows: i. Speciation analysis. Analytical chemistry: analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample. ii. Chemical species. Chemical element: specific forms of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure. iii. Speciation of an element; speciation. Distribution of an element amongst defined chemical species in a system. The term speciation is widely used in dealing with numerous chemical forms. Szpunar et al. (1996) described speciation analysis as the ability to define which forms of a given element are present in a particular sample and at what levels precisely these species occur. Marin et al. (1997) defined the term chemical speciation as the identification and quantification of the different species, forms or phases present in a sediment material, or the description of them. In terms of metal 42 speciation, Ge (2002) referred to it as the distribution of physico-chemical forms that comprises the total concentration of a given metal in soil. In soil or sediment, metals exist in several different forms and are associated with a range of components. It is principally recognized that information about the physico-chemical forms of the metals is required for understanding their environmental behaviour such as mobility, pathways and bioavailability (Tack and Verloo, 1995). 2.3 Speciation Technique for Sediment Analysis Element speciation analysis of sediment can provide insightful information associated with risk assessment, fate, transport and chemical equilibria within the substrate. Many studies on elemental speciation are conducted to determine their inorganic or organic species. Speciation studies also cover other environmental samples including water, soil, biological and air particulate. The fundamental requirement in elemental speciation is the need to quantitatively determine each of the forms of a given element independently and without interference from the other forms. The study on metal speciation requires a specific separation technique followed by sensitive method for determining the inorganic compounds. The processes involved in speciation study are shown in Figure 2.4. Further discussions will focus on techniques applied during the process to determine inorganic species of arsenic and selenium in some solid samples. The brief description of various speciation techniques applied is stipulated in Table 2.2. 43 SAMPLES Gases Solids Liquids Soils Sediment Air particulate Tissue Air Waters Biological fluids TREATMENT PRECONCENTRATION - acidic media base hydrolysis organic solvent microwave radiation - solvent extraction precipitation coprecipitation chromatographic separation UV-vis DETECTION AAS HG-FAAS HG-GC-AAS HPLC-HG-AAS ETAAS ICPMS HPLC-ICPMS HPLC-HG-ICPMS ICP-AES OTHERS NAA HPLC-AFS CE HG-ICP HPLC-HG-ICP Figure 2.4: Processes for speciation study in different samples (Burguera and Burguera, 1997). 2.3.1 Extraction Technique Extraction is an important stage in the determination of elemental species in sediment. A challenge in speciation analysis is in establishing an appropriate extraction method that will provide good recoveries while maintaining the species integrity by avoiding transformations. Furthermore, at present, there are no soil or sediment certified reference materials for arsenic and selenium species, which makes any methodology development rather more challenging (Leermakers et al., 2006). 44 In metal speciation analysis, the extraction of inorganic and organic compounds from the samples is an important step since not only high recovery is required but the original chemical form of the compounds must also be preserved. So far this procedure has not yet been fully established and there is no generally acceptable method available that can be applied to determine various arsenic and selenium species in the matrices of interest. However, several successful and promising findings are available in the literatures (Gallardo et al., 2001; Hutton et al., 2005; Ochsenkühn-Petropoulou et al., 2003; Peters et al., 1999; Sathrugnan and Hirata, 2004; Thomas et al., 1997; Yusof et al., 1998). To get data on the availability of metal species from solid samples, dissolution or leaching in aqueous media is absolutely necessary. The concept of elemental speciation by leaching is based on the thought that a particular reagent is either specific to discrete phase or specific in its action (Hlavay et al., 2000). Various reagents such as the purified water, solvents, acid extraction with diluted hydrochloric and phosphoric acid have been proposed for the leaching of arsenic and selenium from solid environmental samples particularly soils and sediments. Extraction of arsenic and selenium inorganic species from solid samples in water media had been carried out by Koll (1993), Pongratz (1998) and Sun et al. (2004). Meanwhile, Cordos et al. (2006), Sathrugnan and Hirata (2004) and Ochsenkühn-Petropoulou et al. (2003) had proven that water alone provided the ‘weakest attack’ on the sample to extract arsenic and selenium species. Solvents like methanol and acetonitrile-water mix are usually employed for biological samples. Tukai et al. (2002) had successfully extracted inorganic and organic arsenic in marine macroalgae in methanol. Caruso et al. (2001) used an acetonitrile-water mix to extract arsenic species in freeze-dried apple. Phosphate buffer was applied to extract the mobilisable and bioavailable fraction of arsenic species from sediment (Huerga et al., 2005 and Georgiadis et al., 2006). Extraction of inorganic selenium species from sediments had been performed by using alkaline leaching (GómezAriza et al., 1998). 45 The use of diluted H3PO4 has been widely proposed to extract arsenic species for various types of soil and sediment samples (Ellwood and Maher, 2003; Gallardo et al., 2001; Hutton et al., 2005; Iserte et al., 2004; Ruiz-Chancho et al., 2005; Ruiz-Chancho et al., 2007; Sathrugnan and Hirata, 2004; Thomas et al., 1997). A range of 0.1 M to 2 M H3PO4 concentrations was studied that finally resulted in satisfactory extraction efficiency. The diluted HCl of 0.5 M has been used to extract selenium species in soil samples (Ochsenkühn-Petropoulou et al., 2003 and Bujdoš et al., 2005). Ochsenkühn-Petropoulou et al. (2003) recommended that HCl treatment should be the effective reagent for extracting selenium species in the soil sample due to the high concentration recoveries. Meanwhile, Chappell et al. (1995) extracted inorganic arsenic species from contaminated soil in 10 M HCl media and yielded 97% of the total arsenic present. Cordos et al. (2006) had also employed 1 M and 10 M HCl in single extraction of inorganic arsenic species in soil samples. The concentration of 1M obtained 39 - 80% while 10 M HCl produced better extraction efficiency of 77 - 94%. The concentrated media of HNO3 had been utilized for selenium species extraction in solid environmental samples (Moreno et al., 2000; Savard et al., 2006; Saygi et al., 2007; Wang et al., 2001; Yusof et al., 1997; Yusof et al., 1998, ). Approaches such as concentrated nitric acid or perchloric acid extraction in conjunction with hydrofluoric acid are too aggressive and lead to the destruction or alteration of studied species (Georgiadis et al., 2006; Thomas et al., 1997,). Mechanical devices have been applied in the extraction procedures as an aid to enhance and optimize the extraction efficiency. The shaking technique had been introduced using orbital or horizontal shaker at optimized extraction period (Bujdoš et al., 2005; Cordos et al., 2006; Ellwood and Maher, 2003; Georgiadis et al., 2006; Iserte et al., 2004; Pongratz, 1998; Sun et al., 2004). Caruso et al. (2001), GómezAriza et al. (1998), Huegra et al. (2005) and Moreno et al. (2000), had used sonication technique in the extracting process. 46 Recently, microwave digester has also gained much attention in the extracting process in the speciation study. Low power heating, suitable operating time and appropriate extracting reagents have made microwave a reliable technique for extraction of arsenic and selenium species from solid samples. Zanaton (1994), Thomas et al. (1997), Tukai et al. (2002), Sathrugnan and Hirata (2004), RuizChancho et al. (2005) and Hutton et al. (2005) had used the microwave technique to extract arsenic species while Yusof et al. (1997), Yusof et al. (1998), Peters et al. (1999), Moreno et al. (2000), Wang et al. (2001) and Saygi et al. (2007) had applied similar technique for extracting the selenium species in solid samples. 2.3.2 Usage of Microwave in Sample Digestion Prior to Extraction Microwave technology is of considerable significance at home and in analytical laboratories. Microwave-assisted methods have been widely used in analytical chemistry and have attracted much attention in recent years. Luque-García and Castro (2003) reviewed latest advances in the use of microwaves in various analytical chemical fields including sample digestion for elemental analysis, solvent extraction, sample drying, moisture measurement, analyte adsorption and desorption, sample clean-up, chromagenic reactions, solid-phase retention, elution, distillation, microwave plasma atomic spectrometry and synthetic reactions. Apart from safety, the main advantage of using microwave is the ability to control the microwave energy release to the sample and programmable addition of solution (Nobrega et al., 2002). The merit of pressurized acid digestion in closed vessels with microwave heating, particularly the increased speed and reduced losses of volatile elements, are widely recognized (Yusof et al., 1994; Wen et al., 1997; Wang et al., 2001). This feature is important when it comes to volatile element such as arsenic and selenium. In the speciation analysis of solid samples, the time necessary for the quantitative isolation of the species from the sample matrix can be reduced from more than 24 hours to a few minutes by the application of microwave technology. At the same time solvent consumption is reduced and the species recoveries are improved (Szpunar et al., 1996). 47 Zanaton (1994) had obtained 99.3% recovery of inorganic arsenic species in marine biological samples by performing suitable heating program in 7 minutes at 30% power in concentrated HNO3 using microwave digester model CEM MDS-81D. The extraction of arsenic species in the marine macroalgae using microwave for 5 minutes at 60C had achieved efficiency greater than 88% with the appropriate methanol/water mixture (Tukai et al., 2002). They also found that extraction temperature and extraction time did not significantly influence the extraction of arsenic in macroalgae. Low power microwave extraction was successfully applied for arsenic extraction from various sediment and soils samples. Thomas et al. (1997) had extracted arsenic species using microwave in 2 M H3PO4 with 20% power for 20 minutes that obtained recoveries in sediment CRM 320 and IAEA Soil-7 of 82% and 87%, respectively. Sathrugnan and Hirata (2004) proposed low heating microwave power of 50 W for 10 minutes to extract arsenic and selenium species in sediment and coal fly ash. They obtained good extraction efficiency between 90 to 110% in diluted 0.1 M H3PO4 media for arsenic and 60% for selenium species. Ruiz-Chancho et al. (2005) and Ruiz-Chancho et al. (2007) established new approached of microwave-assisted (60W power, 10 minutes) for extracting arsenic species from soils by adding 0.5 M ascorbic acid together with 1M H3PO4 media and attained relatively higher extraction efficiency and minimizing As (III) oxidation during the extraction step. Hutton et al. (2005) extracted arsenic species using standard solution in microwave-assisted extraction at 40% power in 0.3 M H3PO4 media, obtaining recoveries between 90 to 110%. Gallardo et al. (2001) reported the total arsenic extraction yield of 96% for sediment CRM 320 and 99% for sewage sludge CRM 007-040 after microwave digestion at 40 W power for 20 minutes in 1 M H3PO4 . Inorganic selenium was extractable from the aquatic species using microwave digestion at low power heating of 30% in concentrated HNO3 media (Yusof et al. 1997). The similar approach was also used by Yusof et al. (1998) to extract the Se (IV) from marine sediment in media mixture of HNO3 (70%), HClO4 (60%) and HF (48%). The presence of selenium species in muscles tissues was 48 determined by hydrolyzing them in 6 M HCl using microwave heating (100C, 414 kPa, 10 minutes) (Peters et al., 1999). Moreno et al. (2000) was able to extract inorganic selenium species from sewage sludge after applying several steps of microwave programs at power range of between 475 W and 570 W in concentrated HNO3. Saygi et al. (2007) also applied several simultaneous heating programs with power range of between 250 W and 550 W for 23 minutes in order to extract inorganic selenium species from solid samples. Wang et al. (2001) studied the effect of several extracting reagents such as HNO3, H2SO4/H2O2, H3PO4 and HNO3/HClO4 in environmental and natural waters on selenium speciation. The digestion reagents such as HNO3 and H2SO4/H2O2, an appropriate selection of power program and a suitable heating time would help to minimize the changes of element oxidation state in digestion solutions. 2.3.3 Preconcentration and Separation Technique The concentration levels for inorganic or organic element species in the environmental samples are generally lower than detection limit of most detection techniques. The influence of the matrix components of real samples presents challenges in species determination. For many detection techniques, it is not possible to determine different oxidation states of the element species. Therefore, the separation and preconcentration techniques including solvent extraction, coprecipitation, solid-phase extraction and chromatographic have been proposed in order to address that problem. A number of reagents have been used by many researchers as the means of selectively removing the species from extracted samples of previous procedure and also as the means of preconcentration prior to the quantification by ETAAS or NAA methodology (Burguera and Burguera, 1997; Yusof et al., 1994). The preconcentration techniques such as chelation, solvent extraction and coprecipitation are frequently employed in conjunction with NAA technique (Alfassi and Wai, 1991). A selective extractions and preconcentration of inorganic arsenic species 49 using ammonium pyrrolidinedithiocarbamate (APDTC) into chloroform and APDTC into methyl isobuthyl ketone (MIBK) has been reported (Mok et al., 1986; Mok and Wai, 1988; Shi, 2000; Zanaton, 1994). Yusof et al. (1997) used a mixture of APDTC and chloroform to preconcentrate the inorganic selenium species from aquatic species. Chappell et al. (1995) extracted As (III) as arsenic trichloride into chloroform from a solution with highly concentrated HCl followed by backextraction of the arsenic into water. An extensive study for solvent stability has been evaluated on six types of solvents, namely chloroform, cyclohexane, DIBK, MIBK, toluene and o-xylene for extracting As (III), Sb (III) and Se (IV) using APDTC (Shi, 2000). MIBK is considered to be the most effective solvent for the extraction purpose due to its higher boiling point, lower solubility in water and acceptable density difference with water. Apart from that, it does not have very strong odor and interfering elements in it. Abdullah et al. (1995) had successfully obtained concentrated Se (IV) from seawater by extracting it in APDTC complex with chloroform and back-extracting the fraction into nitric acid. Meanwhile, the total selenium had been extracted by coprecipitation with tellurium in an acidified sample. Simultaneous determination of arsenic, selenium and antimony in natural water had been studied by Sun and Yang (1999). Arsenic (III), selenium (IV) and antimony (III) can be simultaneously coprecipitated with Pb(PDC)2 (PDC=pyrrolidinedithiocarbamate) from pH 2 to 4, while quantitative co-precipitated of the higher oxidation state forms can be subsequently accomplished by reduction with titanium (III) chloride followed by Pb(PDC)2 co-precipitation. They also found that pyrrolidinedithiocarbamate to be the more suitable co-precipitant than oxine and thionalide for the studied species. The co-precipitation technique for Se (IV) dibenzyldithiocarbamate complex with phenolphthalein had been carried out by Saleh et al. (1990) and Yusof et al. (1998) from water and marine sediment samples, respectively. 50 A selective complex formation of Se (IV) with Bismuthiol-II, which was subsequently adsorbed on activated carbon had been successfully applied by Sakai et al. (1994) to determine selenium species in natural water. Pyrzynska et al. (1998) had proposed a speciation procedure for inorganic selenium in natural water samples based on solid phase extraction on activated alumina and determined by graphite furnace AAS. The application of solid phase extraction for inorganic selenium in environmental samples was performed by Saygi et al. (2007). The selenium species were determined by graphite furnace AAS after extraction on Diaion HP-2MG adsorption resin. The automated separation techniques such as hydride generation and chromatographic column incorporated with detection system like AAS and ICPMS have been employed in speciation study. Cordos et al. (2006) determined the content and fraction of As (III), (V) and total arsenic leachable in water, 1M HCl and 10M HCl in soil samples by hydride generation-quartz furnace atomic absorption spectrometry. Ion chromatographic separation technique integrated with ICPMS detection was successfully applied in speciation of the inorganic arsenic in drinking water, soils and plants (Mattusch et al., 2000; Gallagher et al., 2001; Gault et al., 2003). A liquid chromatographic separation of inorganic arsenic and selenium by HPLC using mixed ion-pairing reagents followed by ICPMS had been performed by Sathrugnan and Hirata (2004). The chromatographic separation allows direct separation of metal ions using ion exchange columns or by adsorption (reversed- or normal-phase) liquid chromatography if the metal species are complexed with organic ligands. 51 2.3.4 Detection Technique A number of detection techniques for speciation of arsenic and selenium compounds are available. Spectrometric methods such as UV, visible, atomic absorption, atomic emission, graphite furnace and inductively coupled plasma had been employed for the determination of arsenic (Jain and Ali, 2000). Some other reliable detection techniques including X-ray spectrometry, neutron activation analysis (NAA), capillary electrophoresis, atomic fluorescence spectrometry and online combinations of some separation technique with the detection method or socalled hyphenated technique had also been applied for speciation study (Burguera and Burguera, 1997). Hyphenated techniques such as gas chromatography-mass spectrometry, liquid chromatography or hydride generation integrated with detection system like inductively coupled plasma mass spectrometry (ICPMS) and AFS for speciation study has also been widely reported. A number of researchers had reported on the use of liquid chromatography particularly HPLC integrated with ICPMS instrument as the detection method (Caruso et al., 2001; Ellwood and Maher, 2003; Ochsenkühn-Petropoulou et al., 2003; Rodas et al., 2005; Sathrugnan and Hirata, 2004). This method showed good accuracy, repeatability, robustness and high selectivity. Recently, some studies on simultaneous determination of arsenic, selenium and chormium species conducted using HLPC-ICPMS method achieved good recovery and low detection limit (Martínez-Bravo, et al., 2001). The combination of both liquid chromatographic and hydride generation separation followed by AFS detection had been applied in the speciation of arsenic in soil and sediment samples by Gallardo et al. (2001), Georgiadis et al. (2006), Huegra et al. (2005), Hutton et al. (2005) and Thomas et al. (1997). The main drawback of ICPMS instrumentation is that since arsenic has only one isotope (m/z 75), the detection process can suffer interference from the ArCl polyatomic ion produced during extraction of the plasma through the interface. For the detection of selenium with ICPMS, the most abundant isotopes 80Se and 78Se are not accessible for evaluation because of interference from polyatomic argon. The m/z 52 82 was not used because of the random presence of 82Kr as a contaminant in the Ar cylinders. The other isotope 77Se can be subjected to interference from the formation of the ArCl polyatomic species in chloride-rich samples (Bravo, et al., 2001). Therefore much effort should be given to chromatographically separating the Clfrom arsenic and selenium species to eliminate the interference. Moreover, due to the high cost of the combination techniques and of their maintenance, they are not available in many laboratories (Burguera and Burguera, 1997). Table 2.2: Summary of analytical arsenic and selenium speciation in environmental samples. Analyte Matrix Extraction procedure Separation procedures Detection Ref. As (inorganic) Soil Hydride generation (HG) AAS Chappell et al. (1995) As (inorganic and organic) As (inorganic and organic) As (inorganic and organic) As (inorganic) As (inorganic) As (inorganic and organic) As (inorganic) Soil and sediment Solvent Extraction In 10M HCl followed into chloroform, back-extraction in water Microwave 20% Power, 20 min in 2M H3PO4 Shaking 2hrs in water Chromatographic - HPLC ICPMS Thomas et al. (1997) Chromatographic - HPLC ICPMS Pongratz (1998) Microwave 40W, 20 min in 1M H3PO4 Shaking 1hr in 0.5M H3PO4+ 0.1M hydroxylamine hydroloride Sequential extraction Chromatographic - HPLC HG-AFS Gallardo et al. (2001) Chromatographic - HPLC ICPMS Chromatographic - IC ICPMS Ellwood and Maher (2003) Gault et al. (2003) Sediment Sonication 2hrs phosphate buffer and Chromatographic - HPLC HG-AFS Huerga et al. (2005) Soil Chromatographic - HPLC HG-AFS Hutton et al. (2005) As (inorganic and organic) As (inorganic) As (inorganic) As (inorganic) Soil Microwave 40% Power, 25 min in 0.3M H3PO4 Microwave 60W, 10 min in 1M H3PO4+ 0.5M Ascorbic acid Shaking at 20C in water, 1M HCl and 10M HCl Shaking at 300rpm in phosphate buffer Microwave 60W, 10min in 1M H3PO4+ 0.5M Ascorbic acid Chromatographic - LC HG-AFS Ruiz-Chancho et al. (2005) Hydride generation (HG) QFAAS Cordos et al. (2006) Chromatographic - HPLC HG-AFS Georgiadis et al. (2006) Chromatographic - LC HG-AFS Ruiz-Chancho et al. (2007) Soil Sediment and sewage sludge Sediment Sediment Soil Soil and sediment Soil in water 53 Table 2.2: (continued) Analyte Matrix Extraction procedure Separation procedures Detection Ref. As (inorganic and organic) Se (inorganic) As and Se (inorganic) As and Se (inorganic and organic) As (organic) As (inorganic and organic) As (inorganic and organic) As (inorganic) As (inorganic and organic) As, Se, Cr (inorganic) Se (inorganic and organic) Se (inorganic and organic) Sediment Shaking 1hr in 0.3 H3PO4 Chromatographic - HPLC ICPMS Iserte et al. (2004) Sediment and coal fly ash Sediment Microwave 50W, 10 min in 0.1M H3PO4 Shaking 2 hrs in water Chromatographic - HPLC ICPMS Reverse electroosmotic flow Capillary electrophoresis Sathrugnan and Hirata (2004) Sun et al. (2004) Plant tissue Shaking 2hrs in bidistilled water Chromatographic - IC ICPMS Mattusch et al. (2000) Apple Sonication with acetonitrile –water mix Chromatographic - HPLC ICPMS Caruso et al. (2001) Marine macroalgae Microwave At 60C, 5 min in methanol-water mix Chromatographic - HPLC ICPMS Tukai et al. (2002) Water Chromatographic - IC ICPMS Gallagher et al. (2001) Water Chromatographic - HPLC ICPMS Rodas et al. (2005) Water Chromatographic - HPLC ICPMS Anion-exchange resin and derivatise using 4-chloro-ophenylenediamine Chromatographic - LC GCMS Martínez-Bravo, et al. (2001) Gómez-Ariza et al. (1998) Sediment Sonication 2hrs in alkaline solution 2M NaOH Soil Water 20C, 24hrs; Hotwater 55C; Methanol 20C, 24hrs and 0.5M HCl 4C ICPMS Ochsenkühn-Petropoulou et al. (2003) 54 Table 2.2: (continued) Analyte Matrix Extraction procedure Separation procedures Detection Ref. Se (inorganic) Se (inorganic and organic) Se (inorganic) Soil Shaking 1hr in 0.5M HCl Microwave 100C, 10min in 6M HCl Hydride generation (HG) QFAAS Bujdoš et al. (2005) Chromatographic - HPLC ETAAS Peters et al. (1999) Waste water Microwave 98C, 5min in 4M HCl Reducing agent 12M HCl on heating plate 98C, 55min Hydride generation (HG) AFS Moreno et al. (2000) Sewage sludge i) Environmental and natural water Microwave with 5 steps programme between 10min to 15 min in HNO3, H2SO4/H2O2, H3PO4 Microwave with 5 steps programme, 23min in concentrated mixture HNO3 + H2O2 Reducing agent 4M HCl in microwave for 13 min Chromatographic - HPLC Fluorimetric detection Wang et al. (2001) Solid phase extraction on the Diaion HP-2MG adsorption resin GFAAS Saygi et al. (2007) Se (inorganic and organic) Se (inorganic) Muscle tissue Water, garlic, onion, rice, wheat, haxelnut Microwave with 2 steps programme in HNO3 media ii) Sonication 45 min in HNO3 media 55 56 2.4 NAA in Speciation Study The nuclear analytical technique namely neutron activation analysis (NAA) is well-known for its most reliable technique for trace-element determination in environmental samples (Mok et al. 1986). In the decade of 1980s, some significant studies on speciation of several elements were highlighted with particular interest in the usage of the solvent extraction for the extraction and preconcentration steps followed by neutron activation analysis (Paiva, 2004). The extraction and preconcentration steps are complementary to NAA because direct irradiation of samples without separation will not distinguish between the oxidation states of the various species involved. It was reported that the use of large volumes of samples for irradiation without preconcentration was unfeasible due to small concentration factor involved (Mok et al. 1986). Several separation techniques such as solvent extraction, chelation and ion exchange and coprecipitation have been used in order to preconcentrate the relevant species prior to NAA determination. Several speciation studies in conjunction with NAA determination are showed in Table 2.3. Solvent extraction or liquid-liquid extraction involves the partition of one or more components between two liquids of limited miscibility that is caused by the different solubilities of a given substance in the two phases. Solvent extraction was a versatile, useful and popular separation technique because of its simplicity, speed and applicability to both major and trace components (Holzbecher et al. 1976). If there are constituents of the samples interfering with the analytical instrument, the analyte or, alternatively the interfering species can be separated by solvent extraction. In addition, large distribution ratios also allow for the determination of analytes below the detection limit that increases the sensitivity of the analysis. If separation and preconcentration were simultaneously required, solvent extraction could be offered as a potential tool (Paiva, 2004). 57 One example of the solvent extraction application in speciation study is the work that had been extensively carried out by Mok et al. (1986) to determine As (III) and As (V) from natural waters. Quantitative extraction of the arsenic species was achieved by extracting them in APDTC at pH 1-1.5 into chloroform followed by a nitric acid back extraction and then subjected to NAA. The study showed that the advantage of using dithiocarbamic acid derivatives for extraction was that the interfering matrix species including the alkali metals, the alkaline-earth metals and the halogens t could be effectively eliminated. High selectivity of solvent extraction can easily be obtained if the conditions of the solutions are properly arranged. This can be done by adjusting the pH of the solution where the extraction efficiencies of many metal-dithiocarbamate complexes depend strongly on acidity. APDTC was much more stable in acidic solution than Na- diethylthiocarbamate and therefore, it was recommended as a solvent extraction reagent of many metal ions from acidic media (Cheng et al. 1982). Co-precipitation has found applications mainly for the separation of minor components or trace amounts of substances. The isolated precipitate was possible either to determine the trace amount in the presence of the collector or to remove the collector (Holzbecher et al. 1976). One of the important features of a co-precipitation method lied in its potential for achieving extremely high concentration factors (103-104 fold) (Mizuike, 1983; Sun and Yang, 1999). The co-precipitation process provides a solid sample favorable for NAA. This procedure also eliminates most of the alkali metals, alkaline earth metals and halogens. It indirectly decreased the background level of irradiation and the radiation dose received by personnel (Sun and Yang, 1999). Some metals like As, Bi, Sn, Sb and Te have the tendency to interfere with selenium signal determined by hydride generation technique. However, the use of NAA would overcome this problem due to the selective nature of the gamma-rays analysis (Yusof et al., 1998). Saleh et al. (1990), Sun and Yang (1999) and Yusof et al. (1998) had applied coprecipitation technique in the determination of inorganic selenium species prior to analyse by NAA method. 58 Several solvent extraction and co-precipitation procedures apply additional agent such as ethylenediaminetetraacetic acid (EDTA) as a masking agent for the extraction. Mok et al. (1986) reported the use of EDTA as a masking agent that could reduce the interferences caused by other metal ions present in complex natural water systems such as seawater and acid mine waters. Zanaton (1994), Yusof et al. (1997) and Yusof et al. (1998) applied EDTA as a masking agent in speciation study for marine biota, marine sediment, seawater and other aquatic biota samples. EDTA had also been used in the AAS for the purpose of masking and increasing sensitivity (Cheng et al. 1982). The speciation study using NAA method usually allows for preconcentration and separation of lower oxidation state species. The determination of higher oxidation state species is accomplished with a previous reduction to lower oxidation state by a reducing agent before the application of the extraction procedures. The reducing agents make it possible to determine the total concentrations of the elements and the concentration of the higher oxidation state species can be indirectly obtained by subtractions. Mok et al. (1986), Zanaton (1994) and Yusof et al. (1997) used sodium thiosulfate as the reducing agent for As (V) at low pH condition. Meanwhile, Sun and Yang (1999) applied titanium (III) chloride to simultaneously reduce As (V), Sb (V) and Se (VI) in natural water systems. Shi (2000) studied the ability of three different reducing agents, namely Lcysteine, potassium thiosulfate and potassium iodide to reduce As (V), Sb (V) and Se (VI) to As (III), Sb (III) and Se (IV), respectively. L-cysteine and potassium thiosulfate can quantitatively reduce As (V) and Sb (V) at pH 1. The reduction yield of Se (VI) to Se (IV) by the proposed reducing agents was found to be very low. The concentrated HCl ranges between 4 M to 12 M and followed by heating at boiling bath or hot plate to temperature nearly of 100C was was found to be associated with reducing Se (VI) to Se (IV) in several studies (Gómez-Ariza et al., 1998; Moreno et al., 2000; Sakai et al., 1994; Savard, 2006). 59 The method for the determination of metal species particularly arsenic and selenium which consists of selective separation and preconcentration followed by NAA determination has been well established. NAA provided fairly low detection limits with good accuracy and high sensitivity for small sample size of at least 100 mg (Saleh et al., 1990; Yusof et al., 1997). NAA also offers some advantages over GFAAS and ICP-MS such as eliminating the desorption step and recalibration. It was also non-destructive, less time consuming than GFAAS and had no interference unlike ICP-MS (Savard, 2006). NAA has been chosen as the primary method in speciation study due to the availability and reliability of its technique. Contaminated sediments are usually associated with high level concentration of several elements. In this case, NAA technique serves an advantage as no dilution step is required to be carried out and it offers high specificity on the interest element without any interference during analysis. However, the optimization procedure for extraction and preconcentration steps must be conducted in order to obtain the optimum condition and high recovery of particular species. The extraction and preconcentration steps are also ideal to create an interference-free environment in the matrix during the activation period. Table 2.3: Summary of speciation study using NAA as a method of determination. Analyte Matrix As (III) and As (V) Extraction Separation and preconcentration Reducing agent Ref. Natural Water Solvent extraction APDTC in chloroform + EDTA at pH1 – 1.5, back extract with nitric acid Sodium thiosulfate Mok et al. (1986) Se (IV) Water Solvent extraction Co-precipitation of metal dibenzyldithiocarbamate with phenolphthalein Se (IV) and Se (VI) Natural Water Solvent and solid phase extraction Se (IV)/ Bismuthiol-II complex and adsorption on activated carbon 4M HCl + NH2OH.HCL at water bath 99C Sakai et al. (1994) As (III) and As (V) Marine biota and seawater Microwave with 2 steps programme, 7min, 30% power in concentrated HNO3 Solvent extraction APDTC in chloroform + EDTA at pH1 – 1.5, back extract with nitric acid Sodium thiosulfate Zanaton (1994) As (III) and As (V) Marine biota and seawater Microwave 5min, 48C, 30% power in concentrated HNO3 Solvent extraction APDTC in chloroform + EDTA at pH1 – 1.5, back extract with nitric acid Sodium thiosulfate Yusof et al. (1994) Se (IV) and Se (VI) Aquatic biota Microwave 7min, 48C, 30% power in concentrated HNO3+H2O2 Solvent and solid phase extraction APDTC in chloroform + EDTA back extract with nitric acid Sodium thiosulfate Yusof et al. (1997) Se (IV) Marine sediment Microwave 10min, 48C, 30% power in concentrated HNO3+HClO4+HF Solvent extraction Co-precipitation of metal dibenzyldithiocarbamate with phenolphthalein Saleh et al. (1990) Yusof et al. (1998) 60 Table 2.3: continued Analyte Matrix As (III,V), Se (IV,VI) and Sb (III,V) Extraction Separation and preconcentration Reducing agent Ref. Natural Water Solvent extraction Co-precipitation of Pb-APDTC at pH 2-4 Titanium (III) chloride Sun and Yang (1999) As (III,V), Se (IV,VI) and Sb (III,V) Natural Water Solvent extraction APDTC and back extract with nitric acid L-cysteine and potassium thiosulfate Shi (2000) Total Se Geological material Preconcentration using thiolcotton fiber 6M HCl at boiling bath 95-100C, 30min Savard et al. (2006) Digestion block in HNO3+H2O2+HF at 55-60C 61 CHAPTER 3 MATERIALS AND METHODOLOGY 3.0 Introduction This chapter describes the materials and the methodologies used for the determination of total arsenic, chromium, selenium and iron concentration and species determination of arsenic and selenium in the contaminated freshwater sediments. The study area of this project is also described. The rational for the selection of sites sampling, parameters measured, sampling frequency, sampling methodology, method of analysis and statistical analysis are also explained. 3.1 Study Area This study was focused to two types of freshwater systems, river and lake which are situated at the southern region of Peninsular Malaysia. The potential sites for this study were surveyed prior to commencement of sampling. The information such as industrial area, land use patterns and other human activities that could impact the aquatic system at the proposed sampling sites are considered in the site selection criteria. Briefly, the sampling sites were based on the following criteria: 1) Accessibility and ability to sample under all weather conditions; 2) Homogeneity of the sediment, and 3) Location of the industrial and settlement areas. 63 A total of 15 sediment sampling locations constituted of three major rivers in Johor Bahru and three main lakes in the Universiti Teknologi Malaysia campus. The sampling locations consist of eight and seven sites at the river and lake respectively. The samplings were carried out for a total number of three series at both rivers and lakes within a period of five months from August to December 2006 (Table 3.1). Like other parts of Malaysia, the climate of the sampling areas is equatorial monsoon where uniform and high temperatures are observed throughout the year. The mean ambient temperature over a period of six months from July to December 2006 was about 26.7C based on the meteorological measurement recorded from the nearest meteorological stations, Hospital Johor Bahru (01 ° 28 ' N, 103 ° 45 ' E) and Senai Airport (01 ° 38 ' N, 103 ° 40 ' E). The total rainfall over the particular period was averagely 1860 mm and the highest rainfall was observed during the month of December. The relative humidity was averagely 82%. (Malaysian Meteorological Services, 2006). Table 3.1: Date of sampling carried out in the rivers and lakes. Location Frequency Date River 1 22nd August 2006 2 20th November 2006 3 14th December 2006 1 22nd August 2006 2 21st November 2006 3 13th December 2006 Lake 64 3.1.1 River Three main rivers namely Sungai Skudai, Sungai Tebrau and Sungai Buluh have their estuaries towards Johore Straits. The rivers were located in polluted and slightly polluted river area which was categorized by the Department of Environmental (2007) in Malaysian Environmental Quality Reports 2006. The sediments were collected from three sampling points at Sungai Skudai and Sungai Buluh respectively and two sampling points at Sungai Tebrau (Figure 3.1). The sampling points at each river were situated approximately within 10 km to 20 km along the respective river. The order of samplings was done starting from upstream and moving towards downstream of the river. The coordinates and description of the sampling sites were summarized in Table 3.2. The first sampling point at Sungai Skudai was located at Kg. Jaya Sepakat which was situated near the Singapore Public Utilities Board Complex (PUB) at Skudai. This area is surrounded by oil palm plantation and squatter settlement and it was formerly a water intake point by PUB. Industries almost do not exist in this area of the river. The second sampling point was located at Kg. Laut beside a settlement and shop houses where some lorry and car workshops were actively operating. Additionally, the river was also polluted with domestic wastes from the presence of squatter areas around the sampling point. The third sampling point was located at Kg. Teluk Serdang, near the Taman Perling commercial area. A small Malay village is located along the river. Fishing activities were also noticed in the area because the location was close to the river mouth. Similar to Sungai Skudai, the sampling points at Sungai Tebrau were close to industries, palm oil plantation and housing estates. The first sampling point was located near Ladang Tebrau where large scale land works at the palm oil plantation area was being carried out during the sampling period. From the notice board erected at the work area, it was believed that the land has been developed as a housing estate. The second sampling point was located at Pandan, near a low cost housing estate. A squatter area was also found along this location. 65 The third sampling area at Sungai Buluh was located at Pasir Gudang area. Pasir Gudang is very well known as an industrial town where a lot of heavy industries such as petrochemical, oleo chemical, biodiesel, machinery and chemical were located. The first and second sampling points were located approximately 2 km from each other and situated close to fat and oil factory and an oxidation pond. The third sampling point was located under the highway bridge near Shell plant and Feoso oil storage. Figure 3.1: Sampling locations for river. 66 Table 3.2: The coordinates and description of the river sampling site Name of Sampling location point Sungai Skudai (SGS) 1 N 01 33’ 21.6” E 103 39’ 36.3” Kg. Jaya Sepakat, near PUB Oil palm plantation and squatter 2 N 01 31’ 51.5” E 103 40’ 14.6” Kg. Laut Settlement, shop houses and squatter 3 N 01 29’ 56.5” E 103 41’ 00.4” Kg. Teluk Serdang, near Taman Perling Village and fishing activities 1 N 01 32’ 53.4” E 103 44’ 24.1” Ladang Tebrau Housing estate development, formerly oil palm plantation 2 N 01 31’ 22.7” E 103 46’ 05.5” PandanHousing estate, squatter 1 N 01 26’ 39.8” E 103 54’ 03.3” 2 N 01 27’ 01.2” E 103 53’ 20.0” 3 N 01 27’ 23.2” E 103 54’ 05.0” Sungai Tebrau (SGT) Coordinates Description of site Near to fat and oil factory Sungai Buluh (SGSB) Near to oxidation pond, Near to highway bridge, Shell plant and Feoso storage 3.1.2 Lake Sediment samples were collected from several sampling points in each of 3 lakes located around Universiti Teknologi Malaysia campus (Figure 3.2). The lakes had an average area of 0.5 km2 and its depth ranged from 0.5 m to 5 m. The first lake sampled (known as TKR) was located behind a deer cage. This lake had a fountain that provided continuous water aeration. The second lake sampled (known as TUP) surrounded by student residencies whose rain drainage system ended into the lake. Previously, small scale horticulture activity was conducted by students at 67 one side of the lake bank. The final lake sampled (known as TB) was used for water recreation activities. It also acted as a water catchments pond as it was situated at the low land of the hilly campus area. Table 3.3 shows the coordinates of sampling points lakes. Figure 3.2: Sampling locations for lake at Universiti Teknologi Malaysia. Table 3.3: Sampling coordinates at the lakes Location Name of location Coordinates Lake TKR N 01 33’ 13.2”, E 103 38’ 09.1” TUP N01 33’ 39.7”, E 103 37’ 34.5” TB N 01 33’ 117.9”, E 103 38’ 16.2” 68 3.2 Sediment Characteristic The sediment collected from the rivers and lakes consist of fine- to mediumgrained size. It is generally accepted that fine-grained suspended and bottomsediment particles accumulate greater concentration of contaminants than coarse particles. The fine-grain also supports a large part of the benthic community by supplying the food in sediment organic matter associated with fine-grained particles (Forstner, 2004). Several samples collected from the lake were observed for living biota in the sediment. Worms had been noticed in the sediment samples of lake TKR and TUP. Similar biota was also sighted in the sediment collected from Sungai Buluh at sampling point number two which was located near the oxidation ponds. The flaky upper layer of the sediment approximately 0 to 3 cm depth usually has lighter colour than the underneath layer. Most of the sediments had light brown colour associated with high iron content. The sediment from Lake TUP had dark brown colour and Sungai Buluh sediments were dark brown to black in colour. This might be associated with organic matter input into the water body. This river sediment also had a slightly odor that smell of H2S which signified the anoxic condition in the sediment. 3.3 Sampling Methodology The coordinates of sampling sites were obtained by using global positioning remote sensing (GPRS) device. The quality of water bodies at the respective sampling points was checked by measuring on-site physical parameters such as pH, temperature and dissolved oxygen. Then, the sediment samples were collected at a distance of about 1 m from the bank and at a depth of 0-15 cm using PVC corer sampler of diameter 71 mm. The samples were scooped up with a polypropylene spade into polyethylene bags. Air was purged out manually from the bags to 69 minimize air content and later kept airtight. The sample identification and date of sampling were noted on the bag using marker pen. Next, the samples were transported in ice loaded container to the laboratory on the same day and stored in frozen state. 3.4 Chemicals and Reagents Only acids of ultra high-purity grade were used during the course of this study. The types of acids used were Concentrated HCl acid (Merck, Darmstadt, Germany), H2SO4 acid (Merck, Darmstadt, Germany), H3PO4 (orthophosporic) acid (Merck, Darmstadt, Germany) and HNO3 acid (Merck, Darmstadt, Germany). All chemicals used were of analytical grade. The types of chemicals used are Arsenic trioxide (As2O3) (BDH, Poole, England), sodium arsenate (AsHNa2O4.7H2O) (Fluka, Steimheim, Switzerland), selenous acid (H2SeO3) (Aldrich, Steinheim, Germany), sodium slenate (Na2O4Se) (Fluka, Steimheim, Switzerland), ammonium pyrolidinedithiocarbamate (APDTC) (Fluka, Steimheim, Switzerland), MIBK (Merck, Darmstadt, Germany), methanol (Merck, Darmstadt, Germany), sodium dibenzyldithiocarbamate (Na-DBDTC) (Fluka, Steimheim, Switzerland), phenolphthalein (Pp) (Riedel-de Haën, Germany), ethylenediamminetetraacetic acid (EDTA) (Merck, Darmstadt, Germany) and sodium thiosulfate (Na2S2O3. 5H2O) (GCE Lab. Chem.). Deionized distilled water (DDW) with a quality of at least 18M resistivity was used for preparation of solutions, dilution and for final rinsing of the acid cleaned labware. The water was prepared using a Barnstead (USA) model Easypure RF –ultra pure water system by passing distilled water through a mixed bed of anion and cation exchange resins. 70 3.5 Labware For the determination of trace levels of elements, contamination and loss are of prime consideration. The typical potential contamination sources include improperly cleaned laboratory apparatus and general contamination within the laboratory environment from dust. Sample containers can introduce positive and negative errors in the determination of trace elements by contributing contaminations through surface desorption processes. All reusable labware (glass, polyethylene, Teflon, etc.) including the sample container were cleaned prior to use. Labware were soaked for overnight and thoroughly washed with laboratory-grade detergent and water, rinsed with water and additionally soaked for 24 hours in 10% dilute nitric acid, followed by rinsing with DDW and oven drying. 3.6 Laboratory Apparatus A number of 250 mL conical flasks, 50 mL conical flasks, 100 mL and 200mL beakers, measurement cylinders, 50 mL, 100 mL and 1000 mL volumetric flasks, 200 mL separating funnels, filter funnels, assorted Gred A calibrated pipettes and 50 mm watch glass were used in this experiment. High quality plastic filter (Nalgene, USA) assembly with suction flask of 1000 mL capacity connected to a vacuum pump aspirator model A-3S (Eyela, Japan), 0.45 m Whatman (cellulose nitrate) membrane filter disc, 150 mm Whatman filter paper, 200 mL capacity of PTFE sample storage bottles (wide mouth bottles) with polypropylene screw cap, 200 mL CEM Teflon PFA vessels and its assemblies for microwave extraction and 3 mL polyethylene irradiation vials (Kartell, Italy) were used. Apart from that, high quality disposable pipette tips and Pasteur pipettes were used in order to prevent samples from being contaminated and to increase precision. 71 3.7 Sample Processing Equipment A number of laboratory equipments were used for various measurements of related physical parameter analysis and sediment sample preparation prior to analysis. A HORIBA Water Checker model U-10 (UK) was used to take in-situ measurements of pH, temperature and DO of the bottom water body at each sampling locations. The instrument was calibrated with buffer solution prior to use. The coordinates of sampling sites were obtained through GPRS device, eTrex Summit GPS (Garmin Ltd., USA). The frozen sediment samples were thawed in a MBRAUN Unilab, (Germany) glove box and transferred to freeze-dryer container. A freeze-dryer (ilShin Lab Co., Ltd, Seoul, Korea) was employed for drying of sediments at approximately -40C before grinding it into powder with a planetary ball mill grinder model Fritsch Pulverisette 5 (Laval Lab, Quebec, Canada). A stainless steel siever tray was used to sieve the ground sediment samples and to obtain particle size fractions of < 200 mesh ( 75 m). A laboratory analytical balance, capable of weighing accurately up to 0.1 mg was used throughout the experiment. The balance was calibrated daily with 200 mg standard weight (KERN & Sohn GmbH, Germany) prior to use. A conventional microwave oven Panasonic model NN-C20035 (Japan) which was operated at low power setting was used to extract species of interest from sediment samples in diluted acid media. A HANNA PH211 pH meter, capable of proper calibration and reading accuracy of 0.2 provided the pH measurement of the extracted samples before separation and preconcentation procedure. The pH meter was also calibrated daily prior to use with pH 7 and pH 4 buffer solutions (Hanna Instrument, Hungary). The solution of extracted samples was stirred using a magnetic stirrer model CAT M6/1 with maximum speed of 1400 rpm during the separation and preconcentration procedure. 72 3.8 Sample Preparation The sediment samples used for analysis were prepared in dried form. Drying, grinding and sieving processes were included in the handling operation of sample drying. Frozen sediment samples were thawed and transferred into freeze-dryer container where these procedures were conducted in a glove box unit. Then, the samples were placed on the freeze-dryer chamber with drying settings at approximately -40C and pressure of 10 mTorr for 48 to 72 hours. Next, the dried sediments were ground with grinder mill for at least 15 minute in order to obtain homogenous powdered form of approximately < 200 mesh ( 75 m). Sieving of ground sediment to obtain particle size fractions of < 200 mesh was carried out and the sediments were stored in polyethylene bottles in preparation for future analysis. 3.9 Standard Solutions Standard solutions for the experiment consist of standard solutions for total determination of arsenic, chromium, selenium and iron and stock solutions for arsenic and selenium species analysis. All stock solutions were stored in Teflon bottles and maintained at 4C. Appropriate dilutions of the stock solutions to the require concentration were conducted daily. 3.9.1 Standard Solutions for As, Cr, Se and Fe Single element stock solutions of As, Cr, Se and Fe were supplied by Merck (Darmstadt, Germany) and used for standard chemical solutions for neutron activation analysis method. The standard solutions provided by Merck were treaceable mainly to NIST certified standard solution. The standards of As, Cr and Se were prepared by pipetting aliquots of 10 or 100 g mL-1 of the standard solution 73 onto high quality Whatman filter paper which was cut in small round shape and placed inside the polyethylene vials and heat sealed. The standard of Fe was prepared by pipetting aliquots of 1000 g mL-1 and then followed by the same procedure as for the standards of As, Cr and Se described previously. Table 3.4 shows the original concentration of the standard solutions used. Table 3.4: Standard solutions of original concentration supplied by Merck. Element Concentration Traceability (mg L-1) (Certificate no.) As 1002 5 NIST SRM 3103a Cr 1002 5 NIST SRM 3112a Se 1002 5 NIST SRM 3149 Fe 10000 10 NIST SRM 3126a 3.9.2 Standard Solution for Arsenic (III) and Arsenic (V) A standard solution of As(III) with concentration of 1000 mg L-1 was prepared by dissolving 0.1321 g arsenic trioxide (As2O3) in 1 mL HCl in 100 mL volumetric flask. Then, the DDW was added to the mark. A standard solution of As (V) with the same concentration was prepared by dissolving 0.4164 g of sodium arsenate (AsHNa2O4.7H2O) in concentrated H2SO4 and diluted with 100 mL of DDW. All stock solutions were maintained at 4C. Appropriate dilutions of the stock solutions to the required concentration were conducted daily. 74 3.9.3 Standard Solution for Selenium (IV) and Selenium (VI) A standard solution of Se (IV) in concentration of 1000 mg L-1 was prepared by dissolving 0.1632 g of selenous acid (H2SeO3) with 100 mL of DDW in volumetric flask. Next, a standard solution of Se (VI) with the same concentration was prepared by dissolving 0.2392 g of sodium slenate (Na2O4Se) with 100 mL of DDW. 3.10 Certified Reference Material (CRM) Certified reference materials (CRM) have a wide variety of uses in analytical chemistry (Venelinov and Sahuquillo, 2006). The main role of RMs is to evaluate the accuracy as well as the precision of measurements. They are treated in exactly the same way as the test materials and can be used to characterize the performance of the analytical system. Thus they are regarded as important tool in implementing internal quality control in analytical laboratories. They can also be used for methodology and instrumental calibration purposes. The CRM is employed in most measurement system as they have already been technically certified with regards to their property values as well as homogeneity and stability. According to International Organization for Standardization (ISO), reference materials is a substance for which one or more properties are established sufficiently well for the purpose of calibrating a chemical instrument, validating a measurement process or for assigning values to materials (Venelinov and Sahuquillo, 2006). Taylor (1987) had defined 4 types of reference material. An internal reference material is a material developed by a laboratory for its own internal use. An external reference material is one provided by someone other than the end user. A CRM is a reference material certified by an organization which establishes its traceability to an 75 accurate realisation of the unit in which the property values are expressed by and uncertainty at a stated level of confidence. A standard reference material (SRM) is a certified reference material issued by the National Institute of Standards and Technology (NIST). The most important criterion when choosing a CRM is that it should be a reasonably close match to the samples to be analysed (IAEA, 1990). To provide quality assurance controls, the reference materials having almost similar matrix were used in the analysis. CRM of Marine Sediment Reference Material BCSS-1 and Marine Sediment Reference Material PACS-2 provided by the National Research Council of Canada (NRCC) and IAEA Soil-7 supplied by International Atomic Energy Agency were applied throughout the analysis. 3.11 Preparation of Sediment Samples for Total As, Cr, Se and Fe Powdered samples with weights between 0.1 g to 0.15 g were loaded into polyethylene irradiation vials (1 cm inside diameter and 2 cm height) for the determination of total concentration of As, Cr, Se and Fe. The samples were prepared in triplicates and then heat-sealed. Two vials containing CRM with the same matrix as the sediment samples were included in the batch. The CRM used were Marine Sediment Reference Material BCSS-1 and IAEA Soil-7. Aliquots of the appropriate single element standard solution were also irradiated along with the samples. For blank correction, an empty vial and a vial pelletized with filter paper were also irradiated. 76 3.12 Arsenic Species Arsenic species from solid samples were determined by extracting the species using microwave oven in a mild acid media. The preconcentration and separation procedures were performed using APDTC into MIBK. The procedures applied in this study followed that of Shi (2000). 3.12.1 Microwave Extraction About 1.00 g of powdered sample was weighed in triplicate into Teflon microwave digestion vessels. Then 15 mL H3PO4 were added. A total of 6 vessels were placed on the turntable for simultaneous extraction under close system using a conventional microwave oven which was operated at low power microwave setting. The samples were allowed to cool to ambient temperature before being filtered with a filter paper and finally made up to 100 mL with DDW for subsequent analysis. These extracts were preserved by acidification to pH 2, refrigeration at 3-4C and then preconcentration procedure was conducted within 24 h. The optimization of microwave extraction process was carried out in order to obtain the optimum condition for good recovery while maintaining As species integrity. The effect of the microwave power, time and H3PO4 concentration on the recovery was investigated by extracting the CRM of Marine Sediment Reference Material BCSS-1 at different settings. Experiments on simulated extracts using standard solution of 0.25 µg mL-1 were also carried out to study the stability of the As species using the power, time and media concentration setting which obtained high recovery from previous experiment. 77 3.12.2 Preconcentration and Separation The arsenic species were determined by preconcentration and separation procedures using APDTC into MIBK. The extraction solution was prepared by dissolving 5 g of APDTC in 100 mL of DDW and was then filtered to remove the insoluble materials. Next, MIBK was added into the solution to remove bromine and other impurities. 12% (w/v) of EDTA solution was used as a masking agent. All extraction solutions were prepared fresh daily. Prior to the As (III) extraction, 100 mL of sample aliquot from microwave extraction was adjusted to optimum pH value using concentration of 2 M HNO3. Two mL of APDTC, 4 mL of EDTA solution and 10 mL of MIBK were added into the sample aliquot. The samples were stirred vigorously using magnetic stirrer for 10 min and allowed to stand for 10 min for phase separation. The organic phase was washed a few times with 5 mL DDW. Then 8 mL of organic phase was transferred to a flask and 3 mL of 4 M HNO3 was added into organic phase to back extract arsenic. The samples were stirred for 10 min and allowed to stand for 10 min for phase separation. Finally after phase separation, 1 mL of the solution of HNO3 was transferred into polyethylene vials which were heat-sealed and packed into larger vials for neutron irradiation. For the determination of As (V), a second aliquot of 100 mL sample was adjusted to pH 1 with nitric acid. The reduction of As (V) to As (III) was achieved using 1 mL of 25% sodium thiosulfate. The solution was shaken for 1 min and allowed to stand for 5 min. Next, the same procedure for As (III) extraction process was also carried out for As (V). This was followed by similar irradiation procedures which included CRMs, standard solution and blank were applied for determination of arsenic species. The difference of arsenic concentrations from the two sample aliquots represents the total As (V) in the sample. The flow chart of the preconcentration and separation of As (III) and As (V) can be viewed in Figure 3.3. 78 Sample aliquot 1, 100 mL (As (III)) Sample aliquot 2, 100 mL (As (V)) Adjust pH value Adjust pH value 1 mL sodium thiosulfate (25%) As (V) As (III) Stir, 1 min and stand, 5 min 2 mL APDTC (5%) + 4 mL EDTA (12%) + 10 mL MIBK Stir, 10 min and stand, 10 min Organic phase As (III) Wash a few times with DDW Aqueous phase (remove) 8mL Organic phase + 3mL 4 M HNO3 Stir, 10 min and stand, 10 min Aqueous phase (back extract) of As (III) Organic phase (remove) Pipette (~1.0 mL) and irradiate Figure 3.3: Flow chart of the preconcentration and separation of As (III) and As (V) in APDTC – MIBK system 79 3.12.2.1 Effect of pH The extraction efficiency of As (III) and As (V) from APDTC-MIBK system as a function of the pH was studied. 100 mL solution that contained 2 g mL-1 of As species was adjusted to pH range of 1 to 10. Each pH solution was prepared in duplicate. Similar procedure in extracting As (III) was applied throughout the experiments. 3.12.2.2 Repeatability Repeatability is important when assessing the performance of the extraction procedure. Based on ISO definition, repeatability refers to the closeness of agreement among a number of consecutive measurements made under the same measurement procedure or operating conditions by the same operator (EURACHEM, 2002). Under the optimized preconcentration conditions, the repeatability of the extraction by APDTC-MIBK was studied using a standard solution of As (III) and As (V) at 2 µg mL-1. Data of seven extraction runs that were carried out on the same day was collected. Low value of the relative standard deviations (RSD < 15%) suggests that sufficient repeatability for As extraction has been achieved. 3.13 Selenium Species Microwave extraction was applied to extract Se species from solid samples in a mild acidic media. The preconcentration and separation of Se (IV) and Se (VI) by co-precipitation technique were performed using Na-DBDTC with phenolphthalein. Saleh et al. (1990), Yusof et al. (1998) had also suggested this particular procedure for the extraction of Se species. 80 3.13.1 Microwave Extraction About 1.00 g of powdered sample was weighed in triplicate into Teflon microwave digestion vessels followed by the addition of 15 mL of HCl. Six vessels were placed on the turntable for simultaneous extraction under a close system using a conventional microwave oven which was operated at low power microwave setting. The samples were allowed to cool to ambient temperature before being filtered with a filter paper and finally made up to 100 mL with DDW for subsequent analysis. These extracts were preserved by acidification to pH 2, refrigeration at 34C and the co-precipitation procedure was conducted within 24 hr. Optimization of microwave extraction process was carried out in order to obtain the optimum condition for good recovery while maintaining Se species integrity. The effect on the recovery with regards to the microwave power, the period of microwave usage and HCl concentration was investigated by extracting the CRM of Marine Sediment Reference Material PACS-2 at different setting. Experiments on simulated extracts using standard solution were carried out to study the stability of the Se species using the power, time elapsed and media concentration settings which obtained high recovery from previous experiment. 3.13.2 Preconcentration and Coprecipitation Speciation of Se (IV) and Se (VI) were determined after co-precipitation with sodium dibenzyldithiocarbamate (Na-DBDTC) and phenolphthalein (Pp). The extraction solution was freshly prepared by dissolving 2 g of Na-DBDTC in 100 mL methanol. The 5% Pp was prepared by dissolving 5 g of substance in methanol followed by dilution to the 100 mL of DDW and 12% EDTA solution was prepared by dissolving 12 g of the sodium form in 100 mL of DDW. 81 About 100 mL of sample aliquot from microwave extraction was first adjusted to optimum pH value with 2M of HCl. Then, 2 mL of 2% DBDTC, 4 mL of 12% EDTA and 5 mL of 5% Pp were added to the sample aliquot. The sample was stirred with a magnetic stirrer for 10 min and allowed to stand for 1 hr. The precipitate formed was separated by filtration through a 0.45m membrane filter and freeze-dried for 15 hr prior to irradiation. Approximately 0.15 g to 0.20 g of the precipitate was transferred into polyethylene vials which were heat-sealed for neutron irradiation. Sodium thiosulfate of 25% acted as a reducing agent for Se (VI) into Se (IV) and 1 mL was used into the second aliquot of 100 mL sample. Then, the same procedures applied for Se (IV) extraction process was then also carried out for Se (VI). Next, similar irradiation procedures which included CRMs, standard solution and blank were applied for the determination of selenium species. The difference of the two selenium concentrations represents the total Se (VI) in the sample. Figure 3.4 shows the flow chart for co-precipitation of Se (IV) and Se (VI). 3.13.2.1 Effect of pH The extraction efficiency of Se (IV) and Se (VI) in the co-precipitation procedure as a function of the pH was studied. About 100 mL solution containing 1 g mL-1 of Se species was adjusted to pH range of 1 to 10. Each pH solution was prepared in duplicate. Similar procedure was used to extract Se (IV) throughout the experiments. 82 3.13.2.2 Repeatability Under the optimized co-precipitation conditions, the repeatability of the extraction step by DBDTC-Pp was studied using standard solutions of Se (IV) and Se (VI) at 1 µg mL-1. Data of seven extraction runs carried out on the same day was collected. Low values of the relative standard deviations (RSD < 15%) suggests sufficient repeatability for Se extraction. Sample aliquot 1, 100 mL (Se (IV)) Sample aliquot 2, 100 mL (Se (VI)) Adjust pH value 1 mL sodium thiosulfate (25%) Se (VI) Se (IV) Adjust pH value Stir, 1 min and stand, 5 min 2 mL Na-DBDTC (5%) + 4 mL EDTA (12%) + 6 mL phenolphthalein (5%) Stir for 10 min and Stand for 1 hr Filter by 0.45 m membrane Freeze-dry for 15 hours Weight (~0.15- 0.2 g) and irradiate Figure 3.4: Flow chart separation-extraction procedure of Se (IV) and Se (IV) by co-precipitation of Na-DBDTC with phenolphthalein. 83 3.14 Analysis Using Neutron Activation Analysis Technique The NAA is based on gamma counting of the neutron activated elements in the samples to be analysed. The irradiations were performed using a TRIGA Mark II nuclear research reactor at Malaysian Nuclear Agency. The samples were irradiated for 6 hours at 750kW power with a neutron flux of 2 x 1012 neutron cm-2 s1. The standards, samples, CRM and blank were all irradiated together at the same time in proper positioning in the nuclear reactor by the same irradiation interval. For arsenic, the cooling period took at least 3 days while chromium, selenium and iron underwent 3 to 4 weeks of cooling before counting commenced. After proper decay time, the batch of irradiation samples was counted in identical conditions by the same detector and at the same geometry. A high resolution coaxial CANBERRA HP-Ge with a resolution of 1.9 keV at 1332 keV gamma-rays line of Co-60 was used for counting over a period of 3600s. The detailed specification of the gamma-detector is shown in Table 3.5. Each element of As, Cr, Se and Fe was observed for their specific gammarays energy. The signals from the 559.0 keV gamma-rays of 76As, 264.7 keV and 136.0 keV of 75Se were used to determine the elemental concentration in the standards and samples. Chromium was analysed via the 320.1 keV of 51Cr, while, iron was analysed through the 1099 keV and 1292 keV photopeak of 59Fe. The applicability of the analytical technique for As, Cr, Se and Fe were evaluated by analyzing the CRM. The spectral data from the unknown standards, samples, and CRM were processed using the spectrum analysis software for gamma peak search and peak area analysis. This software allows the determination of the centroid channel (energy), FWHM (full width-half max), net integrated peak area, and background. 84 Table 3.5: The specification of the CANBERRA HP-Ge detector. Specifications Geometry : Closed end coaxial Crystal Diameter : 62.0 mm Crystal Length : 41.0 mm Active Volume : 123.8 cm3 Active Area : 30.2 cm2 Operating Voltage : 4000 V Relative Efficiency : 30% Resolution: Energy – 122 keV; FWHM – 0.70 keV Energy – 1332 keV; FWHM – 1.9 keV Peak to Compton Ratio – 58 : 1 The associated electronics with HPGe detector: - A pre-amplifier from CANBERRA built in with the detector. - An amplifier, to amplifying and shaping the signal. - A high voltage power supply to supply detector with the needed bias. - Minibin and power supply - Multi-channel analyzer card (MCA Card) - Personal computer with Genie 2000 Software for gamma analysis. - Printer. 85 The two peak areas of the two photo-peaks of interest for standard and sample respectively are proportional to the activation rate and the amount of the element in the standard and the sample after decay correction. The concentration of elements in the samples was determined by performing calculation on equation 3-1 and 3-2 as listed below: Cs = Where: AS k S LC WC * * * * CC AC k C LS WS As ( 3-1) = Corrected net count of selected peak for interested element in sample Ac = Corrected net count of selected peak for interested element in standard ks = Correction in counting due to dead time for sample kc = Correction in counting due to dead time for standard Ls = Counting live time for sample Lc = Counting live time for standard Ws = Weight of sample used Wc = Weight of standard used Cc = Concentration of interested element in standard (e.g. ppm, ug g-1, mg kg-1) Cs = Concentration of interested element in sample (e.g. ppm, ug g-1, mg kg-1) Corrected net count A is calculated from the following equation: A = Ao exp ( 0.693 * Dt / T1/2 ) Where: Ao ( 3-2) = net count of peak during counting T1/2 = the half life of the radionuclide (in day or hour) Dt = the decay period (in day or hour) 86 3.14.1 Limit of Detection The limit of detection is a measure of the lowest level at which a detector can distinguish sample activity from background with no sample present. The lower limit of detection is often calculated using Currie’s formula based on equation 3-3 (IAEA, 2001): LOD = 2.71 + 4.65 Where: B (3-3) LOD = limit of detection B = background under the gamma-ray peak However, the limit of detection is depending on many parameters such as: The amount of material to be irradiated and to be counted. The neutron fluxes. The duration of the irradiation time. The total induced radioactivity that can be measured The duration of counting time. The detector size, counting geometry and background shielding. CHAPTER 4 RESULTS AND DISCUSSION 4.0 Introduction Sampling and analyses during the duration of the study proceeded successfully and no major problems were encountered. This chapter explains and discusses results of the experiments consisting of three sampling sessions that spanned almost two years for the analysis of total As, Cr, Se and Fe, including the analysis of inorganic As and Se species in two freshwater systems of the rivers and lakes situated at the southern region of Peninsular Malaysia. The samples were analyzed using NAA techniques conducted at the Malaysian Nuclear Agency, Bangi, Selangor for elemental concentrations. The discussion includes an overview of the river and lake water quality based on three parameters measured in-situ and the determination of As, Cr and Se in both freshwater system and then they were applied for the correlation study to the concentration of Fe present in the sediment. The total concentrations of those elements were compared to several international guidelines for freshwater sediments. This chapter also includes the discussion on the preliminary study of As and Se species in optimizing the extraction procedures for sediment samples. Besides that, the availability of the species in both freshwater systems were determined and discussed. The trends and patterns of the elements of interest and species in rivers and lakes were made and compared using statistical analysis procedures. 88 4.1 Water Quality of The Water Bodies The physico-chemical parameters of the water column such as pH, temperature and DO are important because they have a significant effect on the water quality. The three parameters are also the major factors influencing physiology of water organisms and possibly contribute to metal forming in water (Forstner and Wittmann, 1983). The measurements of those parameters were taken in-situ to gather information on the current water quality and the environmental conditions of the sampling locations. The results of the parameter measurements at the rivers and lakes are presented in Appendix A. The overall average of the parameter values at the six sampling locations are presented in Table 4.1. pH plays a critical role in the chemistry of freshwater systems. A fall in pH may allow the release of toxic metals that would otherwise be adsorbed to sediment and essentially removed from the water system. A decrease in pH would increase metal availability, lending itself to greater uptake by organisms and can cause physiological damage to aquatic life (Connell and Miller, 1984). pH values of surface waters usually range between 6.5 – 8.5, and only rarely are outside the range of 4 – 9. Surface water has lower buffering capacity and consequently the pH of surface water is more changeable. In water that is too acidic or too alkaline, there could be very limited number of aquatic life. The acidification of surface water may accelerate the leaching out of heavy metals and radionuclide from the bottom of the sediments. The pH levels recorded at the rivers ranged from 6.34 to 6.81. The average level of pH measured from all the river sampling sites were within the National Water Quality Standard for Malaysia (NWQS) threshold range for all classes (DOE, 2007; refer also Appendix B). In terms of pH level, the river system is still suitable for the support of aquatic life. The high pH level at Sungai Buluh which is situated at Pasir Gudang industrial area was a result of industrial discharge from several chemical and oleo factory effluents nearby the sampling point. Previous study on Sungai Skudai reported pH ranging from 5.26 to 7.15 and the mean pH value was found to be 6.2. (Thanapalasingam, 2005). Elsewhere, pH values from continuous 89 water quality monitoring station managed by DOE, pH value of 6.3 was recorded at Sungai Selangor, pH 6.4 at Sungai Terengganu and pH 6.6 at Sungai Melaka (DOE, 2007). Table 4.1: Overall average of the water physico-chemical parameters in the rivers and lakes Water Parameters Sites pH Temperature (C) Dissolve Oxygen (mg L-1) SGS 6.34 0.45 28.2 1.7 5.10 0.56 SGT 6.45 0.24 28.6 1.0 5.10 0.36 SGB 6.81 0.19 29.6 1.2 5.07 0.29 TKR 5.98 0.50 29.1 2.5 6.87 0.71 TUP 5.92 0.36 30.8 2.5 6.57 0.68 TB 6.76 0.54 30.3 1.3 6.64 0.42 Slightly lower pH level was observed from two sampling sites at the lake. TKR and TUP show average pH value of 5.98 and 5.92, respectively compared to TB site which showed an average value of 6.76. TKR site is located near a deer cage where the low pH may result from the high organic contents coming from the manure and food wastes entering the lake through surface run off. TUP site may also exhibit similar factor where previously small scale horticulture activity was conducted by students near the site. The surplus from the fertilizer used promotes microbial activity in the lake. pH of the lake water generally affect by ground water seepage, aquatic flora coverage and microbial activity (Kutty et al., 2005). The temperature of water is one of the important characteristics that determines, to a considerable extent, the trends and tendencies of changes in the river water quality. Increased water temperature decreases the solubility of dissolved oxygen and water temperatures above 27C are “unsuitable” for public use. At above 32C it would be considered “unfit” for public use (Chapman, 1992). Higher 90 temperature and combination of low DO levels may increase the toxicity of many substances such as heavy metals, whilst the sensitivity of the organisms to toxic substances also increases. Generally, there was no significant difference in temperature measured at all the rivers ranging from 28.2C to 29.6C. A slightly higher temperature of 29.6C was observed at Sungai Buluh as a result of industrial wastes and effluents that enter the river system. In a previous study of Sungai Skudai, the temperature that was recorded ranged from 26.5C to 29.6C (Thanapalasingam, 2005). The lakes temperature showed higher value than the temperature in rivers. This can be explained where tree cover is less at the particular lakes which allow higher level of incident sunlight and finally increase the water temperature. However, a fountain at the lake TKR site gives a cooling effect and this helps to reduce the temperature in the lake water. The temperature recorded at the lakes was found to be similar to other study conducted at Tasik Chini, Pahang which ranging from 27.9C to 31.9C (Kutty et al., 2005). The oxygen content or DO is an important indicator of the pollution of a water body. Oxygen is an absolute requirement for the metabolism of aerobic organism and also influences inorganic chemical reaction. The amount of DO is highly dependent on temperature. Apart from amount of degradable pollutants, the amount of oxygen that can dissolve in pure water is inversely proportional to the temperature of water. In a typical water system, the warmer the water, less DO can be measured in it. However, DO also depend on several other factors such as the amount of rainfall, saltiness of the water, the amount of decomposition in the water, the amount of aquatic plant and presence of pollutants. The aquatic life would be put under stress if the DO levels in the water drop below 5.0 mg L-1. Oxygen levels that remain below 1 to 2 mg L-1 for a few hours can result in large fish kills. The NWQS criteria for human consumption and aquatic life require that the DO ranging from 3 to 7 mg L-1 (DOE, 2007; refer also Appendix B). 91 The average levels of DO obtained from the rivers and lakes are within the threshold values set by the NWQS. The DO levels at the rivers were relatively lower than the lakes. The lowest DO was measured at Sungai Buluh. A low level of DO is contributed from the discharge of organic matter and waste from the sewerage plants, industries and agriculture activity into the water that can be observed along the rivers. A previous study indicated that DO recorded from Sungai Skudai ranged from 3.82 mg L-1 to 6.49 mg L-1 (Thanapalasingam, 2005). Elsewhere, pH recorded from the downstream of Sungai Langat ranged from 3.02 mg L-1 to 3.89 mg L-1 where low DO concentrations had been related to sewage outfalls (Azrina et al., 2006). On the contrary, the DO levels in the lakes showed higher DO level compared to the DO levels from Tasik Chini obtained by Kutty et al. (2005) with an average value of 3.76 mg L-1. Previous studies conducted by DOE in 2006 showed that several rivers in southern region of Peninsular Malaysia were categorized as heavily polluted river (Figure 4.1). This includes Sungai Buluh which is located at Pasir Gudang area and also Sungai Tebrau. Meanwhile, Sungai Skudai is categorized under slightly polluted river. The Water Quality Index (WQI) is used to evaluate the status of the river water quality consists of parameters such as DO, pH, BOD, COD, Ammoniacal Nitrogen (AN) and Suspended Solid (SS). Sungai Buluh had indicated the lowest WQI value of 32, lower than Sungai Tebrau (WQI 57) and Sungai Skudai (WQI 64). The water of Sungai Buluh was classified as Class IV, while Sungai Tebrau and Sungai Skudai were in Class III (Refer to Appendix B for water class definition). The state of health in water system would be an indicator on the quality of the sediment in the water bodies system. 92 Figure 4.1: Water Quality Status for River Basin of Peninsular Malaysia in 2006 with highlight of Sungai Skudai, Sungai Tebrau and Sungai Buluh (Kaw. Pasir Gudang) (DOE, 2007) 93 4.2 Data Quality The applicability of the analytical procedures for analysis of As, Cr, Se and Fe was examined by analysing CRM. Certified reference material namely IAEA Soil-7 supplied by International Atomic Energy Agency and Marine Sediment Reference Material BCSS-1 and PACS-2 provided by the National Research Council of Canada (NRCC) were applied as quality control material of each analytical regiment implemented. Data of the certified values of these CRM are shown in Appendix C. The accuracy of the analytical result of the elements in the CRM was evaluated using Z-score calculation (Crosby et al., 1995). The Z-score of an element concentration is computed based on the following equation (4-1): Z score xc u x2 u c2 (4.-1) where x is the analytical result; c is the certified value, ux is the uncertainty of analytical results, and uc is the uncertainty of certified value. The uncertainty of the analytical results is based on counting statistics whilst the uncertainty of the certified value is based on the certificate. For acceptance of results: -2 < z < 2 is anticipated. However, if z < -3 or z > 3, it is considered that the result is “out-of-control” and corrective action will be introduced. The accuracy of the analytical results of the elements in the CRM was evaluated using the Z-score calculation and showed As, Cr, Se and Fe are within the acceptance criteria (Table 4.2). The results also show a fair level of consistency between the certified and the measured value where the recovery ratios of As, Cr, Se and Fe were 93 - 117%. Analytical results disclosed that the precision of the analysis was satisfactory; generally better than 16.7% RSD. Limit of detection for elements of interest is presented in Table 4.3. Table 4.2: Analytical results for As, Cr, Se and Fe in CRMs. Values are given in dry weight basis and correspond to the average and standard deviation. IAEA Soil - 7 BCSS - 1 PACS - 2 As Cr Sea As Cr Se Fea Se (g g-1) (g g-1) (g g-1) (g g-1) (g g-1) (g g-1) (%) (g g-1) Certified value 13.4 0.4 60 7 0.40 0.10 11.1 0.7 123 7 0.43 0.06 3.80 0.10 0.92 0.22 Measured value 13.7 0.5 70 4 0.37 0.03 11.5 0.8 126 7 0.48 0.08 3.78 0.11 0.98 0.10 (n = 20) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) Z-score 0.50 1.24 0.29 0.37 0.30 0.50 0.13 0.25 Recovery (%) 102 117 93 104 103 112 99 107 RSD (%) 3.3 5.7 8.4 7.1 5.6 16.7 3.0 10.2 a x – Information value Table 4.3: LOD for As, Cr, Se and Fe Elements LOD As (g g-1) Cr (g g-1) Se (g g-1) Fe (g g-1) 0.2 1 0.02 20 94 95 4.3 Total Concentration of Elements Arsenic, chromium and selenium are among the elements widely studied in environmental samples. The excessive presence of these elements is often associated with anthropogenic activity and they are classified as toxic to human being if exposure levels are sufficiently high. In the ionic form, As, Cr and Se are capable of entering and adsorbing in life tissues including plants and animals. Aquatic sediment can be the preeminent media for those elements to accumulate and finally enter the food chain through benthic life which depend on the sediment as their habitats. The concentrations of As, Cr, Se and Fe in sediment from 15 sampling points from the rivers and lakes system were obtained. The results of As, Cr, Se and Fe concentrations in rivers and lakes for each sampling site were presented in Appendix D. A great deal of research has been directed towards the determination of toxic elements in sediments so as to identify unusual occurrences of these elements within the environment in association with urbanization and industrial development. Some summaries of the main areas with which this study is associated were presented in Table 4.5 and Table 4.7. The table should not be considered as exhaustive or complete, it simply identifies those contamination sources and those toxic elements for which extended occurrences has been studied. Results from previous findings listed in the table were then used as a comparison in this study. Iron was included in this study in order to relate any association of As, Cr and Se in the presence of Fe and hence interpret natural or anthropogenic contribution of these elements in river and lake system. The results of As, Cr and Se were then compared with several sediment quality guidelines established internationally. The contamination impact of the elements in sediments was evaluated and estimated using enrichment factor (EF) and geoaccumulation index (Igeo) tools. Genuinely, this study is hoped to generate the baseline data that can either be future reference or as reference for authority to take necessary action for preventing any anthropogenic input. The data may also useful for initiating a guideline criterion for freshwater sediment quality. 96 4.4 Concentrations of Arsenic, Chromium and Selenium in Rivers All the average concentrations of As, Cr and Se in surface river sediments are presented in Table 4.4. Meanwhile, the graphical forms of the results are illustrated in Figures 4.2 - 4.4 for the concentration of As, Cr and Se, respectively. The concentrations of As in the sediment taken from all 8 sampling sites from the rivers ranged from 10.17 g g-1 to 33.80 g g-1. The highest average concentration was obtained from sampling site SGS3 (Sungai Skudai) and the lowest concentration was obtained from sampling site SGB2 (Sungai Buluh). The sediment of Sungai Skudai has the highest concentration of As followed by Sungai Buluh. The As level showed an incremental trend towards the downstream in all rivers (Figure 4.2). The high As contents of the sediment can be attributed to the mobilization and transportation process of As along the river depositing it near the river mouth. Table 4.4: Results of average concentration of arsenic, chromium and selenium in sediment of each sampling sites at river. Element Concentration (g g-1) Sites As Cr Se SGS1 19.40 0.80 122 14 1.31 0.14 SGS2 30.59 1.30 125 16 1.08 0.05 SGS3 33.80 1.25 103 9 0.88 0.02 Average 27.92 7.60 117 11 1.09 0.20 SGT1 12.26 0.63 50.36 5.50 1.72 0.19 SGT2 13.46 0.85 77.04 8.83 1.91 0.24 Average 12.86 1.00 63.90 19.00 1.82 0.10 SGB1 10.90 0.91 26.83 7.32 0.72 0.04 SGB2 10.17 0.75 50.82 7.81 0.93 0.12 SGB3 24.29 1.46 59.27 8.85 0.56 0.06 Average 15.12 7.00 46.04 15.00 0.74 0.20 97 Previous studies as presented in Table 4.5 indicated that As concentrations in contaminated river sediments ranged from 1 g g-1 to 100 g g-1 (Barringer et al., 2007; Doong et al., 2008; Karageorgis et al., 2003). Soil usually contains As between 0.001 g g-1 and 0.04 g g-1 in the absence of industrial or agricultural contamination (Reilly, 1991). This clearly shows that the As concentrations obtained from the three river system is highly contaminated. The industrial effluents, domestic waste inputs and agricultural activities along the river have led to high enrichment of As in the river sediments. Sediment samples taken from all the rivers in this study showed the concentration of Cr of between 26.83 g g-1 at sampling site SGB1 (Sungai Buluh) to 125 g g-1 at SGS2 (Sungai Skudai). The highest level of Cr was found in sediment of Sungai Skudai of average 117 g g-1 followed by Sungai Tebrau and Sungai Buluh with average value of 63.90 g g-1 and 46.04 g g-1, respectively (Figure 4.3). Previous study by Thanapalasingam (2005) reported that average Cr level in Sungai Skudai from 16 sampling points is 135.57 g g-1. This was explained by high concentration of Cr in the effluent from the active zones along the Sungai Skudai, especially electroplating and printing factories. It was reported elsewhere, that the Cr concentrations in river sediments associated to some anthropogenic activities ranged from 2 g g-1 to 2714 g g-1 as indicated in Table 4.4 (Boszke et al., 2004; Doong et al., 2008; Karageorgis et al., 2003; Zhou et al., 2004). Table 4.5 : Result of previous studies from several literatures for As and Cr in river sediment. Location As Cr General source of contamination Reference Gao-ping River, Taiwan 5.12 – 25.8 g g-1 35.7 – 2714 g g-1 Domestic and industrial wastewater, flooding event Doong et al., 2008 Wallkill River, New Jersy 5.2 – 100 g g-1 Ex-mining site, agriculture, municipal sewage plant Barringer et al., 2007 Middle Odra River, Poland 1.57 – 47.5 g g-1 Mining, heavy industry, agriculture Boszke et al., 2004 Pearl River Estuary, China 58.1 – 117.8 g g-1 Industrial wasterwater, domestic sewage, mining Zhou et al., 2004 39 -180 g g-1 Tailings, industrial effluents, agriculture activity Karageorgis et al., 2003 Axios River, Greece 1 – 40 g g-1 98 99 The Se level at each sampling sites is indicates in Figure 4.4. The concentration of Se in the river sediments were ranged from 0.56 g g-1 at SGB3 (Sungai Buluh) to 1.91 g g-1 SGT2 (Sungai Tebrau). The highest level of Se was found in the sediment of Sungai Tebrau of average 1.82 g g-1. Meanwhile the lowest Se in sediment was attained from Sungai Buluh of average 0.74 g g-1. Sungai Skudai contained on the average 1.09 g g-1 of Se in the sediment and the level decreased towards the downstream. Higher levels of Se in sediments of Sungai Tebrau and Sungai Skudai as compared to Sungai Buluh could be due to sewerage effluent from housing estates and squatter settlement sprawled along the rivers. Effluents from industrial plants located along Sungai Buluh discharged into the oxidation ponds which help improve on the effluent quality, thus reducing the amount of Se later discharged into the river. 40.0 -1 As concentration (ug.g ) 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 SGS1 SGS2 SGS3 SGT1 SGT2 SGB1 SGB2 SGB3 River Figure 4.2: Average concentration of arsenic in sediment of river sampling sites. 100 150.0 -1 Cr concentration (ug.g ) 125.0 100.0 75.0 50.0 25.0 0.0 SGS1 SGS2 SGS3 SGT1 SGT2 SGB1 SGB2 SGB3 River Figure 4.3: Average concentration of chromium in sediment of river sampling sites. 2.00 -1 Se concentration (ug.g ) 2.50 1.50 1.00 0.50 0.00 SGS1 SGS2 SGS3 SGT1 SGT2 SGB1 SGB2 SGB3 River Figure 4.4: Average concentrations of selenium in sediment of river sampling sites. 101 4.5 Concentrations of Arsenic, Chromium and Selenium in Lakes The average concentrations of As, Cr and Se in surface lake sediment at each sampling sites is presented in Table 4.6. The graphical forms of the results are illustrated in Figures 4.5 - 4.7 for the concentration of As, Cr and Se, respectively. The concentrations of As in the lake sediment taken from the 7 sampling sites ranged from 17.62 g g-1 to 61.60 g g-1. The highest average concentration was obtained from sampling site TKRC and the lowest concentration was obtained from sampling site TBB (Figure 4.5). The arsenic level at lake TKR showed the highest average concentration of 56.81 g g-1 compared to the other two lakes. Previous studies in several countries reported levels of As in lake sediment ranging from 1 g g-1 to 201.5 g g-1 as presented in Table 4.7 (Linge and Oldham, 2002; Nikolaidis et al., 2004; Sompongchaiyakul and Sirinawin, 2006). This shows that high levels of As contamination occurred in the lake sediment studied. It is believed that usage of fertilizers, pesticide and herbicides for landscaping activities around the campus are responsible for the general enrichment of As concentrations in the lake sediment. The sediment taken from the lake system contained Cr ranging from 173 g g-1 to 301 g g-1. The sampling site of TUPA contained highest average concentration of As while the lowest concentration found to be at TKRA to be (Figure 4.6). The lake TUP showed the highest Cr level in the sediment with average value 273 g g-1 followed by lake TB and TKR. The Cr concentrations in contaminated lake sediment ranged from 8 g g-1 to 130 g g-1 as reported by other previous studies in Table 4.7 (Gatti et al., 1999; Loska and Wiechula, 2003; Nikolaidis et al., 2004; Rosales–Hoz et al., 2000; Sompongchaiyakul and Sirinawin, 2006; Wang et al., 2003). This clearly indicates that the Cr concentrations obtained from all the sediments of lake system is highly contaminated due to various sources of human activities. 102 Table 4.6 : Results of average concentrations of arsenic, chromium and selenium in sediment of each sampling sites at lake. Element Concentration (g g-1) Sites As Cr Se TKRA 48.49 3.60 173 19 0.55 0.11 TKRB 60.34 5.11 198 21 0.31 0.04 TKRC 61.60 8.43 195 23 0.42 0.07 Average 56.81 7.23 189 14 0.43 0.12 TUPA 38.86 2.67 301 22 0.87 0.10 TUPB 44.07 3.59 244 17 0.94 0.14 Average 41.46 3.70 273 40 0.91 0.05 TBA 45.68 2.51 242 15 0.35 0.07 TBB 17.62 1.60 188 17 1.08 0.10 Average 31.65 15.00 215 39 0.72 0.25 High amount of Cr in the sediment is always associated with industrial pollution such as metallurgical, electroplating industry, dyeing or paint operations (Kaushik et al., 2008; Kotaś and Stasicka, 2000). However, there was no industrial activity existing near the lakes. According to Rosales-Hoz et al. (2000), Cr as well as As contamination were closely associated with domestic wastewater effluents. Therefore, high contamination of Cr may come from wastewater that flows from the student residential drainage system or undetected leakage of underground sewage system which enters the lake through water run off during the wet season. 103 80.0 -1 As concentration (ug.g ) 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 TKRA TKRB TKRC TUPA TUPB TBA TBB Lake Figure 4.5: Average concentration of arsenic in sediment of lake sampling sites. 360 -1 Cr concentration (ug.g ) 320 280 240 200 160 120 80 40 0 TKRA TKRB TKRC TUPA TUPB TBA TBB Lake Figure 4.6: Average concentration of chromium in sediment of lake sampling sites. 104 -1 Se concentration (ug.g ) 1.50 1.20 0.90 0.60 0.30 0.00 TKRA TKRB TKRC TUPA TUPB TBA TBB Lake Figure 4.7: Average concentration of selenium in sediment of lake sampling sites. The concentration of Se in the lake system ranged from 0.31 g g-1 to 1.08 g g-1. Sampling site TBB recorded the highest level of Se in sediment while sampling site TKRB obtained the lowest concentration (Figure 4.7). The Se level at lake TUP showed the highest average concentration of 0.91 g g-1. The high Se content in the sediment of lake TUP was suspected originated from previous horticulture activities especially on the usage of fertilizer and herbicide. Several previous studies reported that Se concentrations in contaminated sediment ranged from 0.48 g g-1 to 12 g g-1 as presented in Table 4.7 (Lemly, 1997; Peters et al., 1999; Wang et al., 2003; Zhang and Moore, 1997). This clearly shows that the lake sediment from the sampling sites are considerably contaminated with Se. Investigation carried out by Hillwalker et al. (2006) at two types freshwater physical characteristic namely stagnant and flowing water, showed a significant difference of Se distribution in the sediment. The sediment in the stagnant water such as pond, lake and well has high potential to accumulate Se compared to the flowing water such as rivers. Table 4.7 : Result of previous studies from several literatures for As, Cr and Se in lake sediment. Location As Cr Songkhla Lake, Thailand 4 – 26 g g-1 Maine Lake, Greece 201.5 g g-1 Yangebup Lake, Australia General source of contamination Reference 8 -73 g g-1 Runoff water, wastewater Sompongchaiyakul and Sirinawin, 2006 51.33 g g-1 Landfill of municipal and commercial waste Nikolaidis et al., 2004 Village development, townshop industry Wang et al., 2003 Effluent from industry Linge and Oldham, 2002 68.6 g g-1 Taihu Lake, China Se 0.48 g g-1 21.8 g g-1 Chapala Lake, Mexico 66.12 g g-1 Urbanization, agricultural, industrial development Rosales –Hoz et al., 2000 Infernao Lake, Brazil 80.7 g g-1 Industrialization Gatti et al., 1999 Peters et al., 1999 Macquarie Lake, Australia 0.8 – 1.4 g g-1 Urban and industrial – lead-zink smelter, fertilizer plant Belew Lake, North Carolina 4 -12 g g-1 Wastewater from coal plant Lemly, 1997 105 Table 4.7 : Continued Location As Cr Benton Lake, Montana Rybnik Reservoir, Poland Oolitic Pond (standing water) Lee Creek (flowing water) Se General source of contamination Reference 2.38 – 3.29 g g-1 Agricultural, seleniferous lake basin Zhang and Moore, 1997 Long-range transport, atmospheric precipitation Loska and Wiechula, 2003 129.8 g g-1 0.63 – 8.2 g g-1 Hillwalker et al., 2006 0.37 – 3.6 g g-1 106 107 4.6 Comparison in Elements Concentration to the Guideline Recommendations for Freshwater Sediments Several sediment quality guidelines were established to estimate the possible toxicological significance of contaminants concentrations in the sediments. The Canadian Council of Ministers of Environment (1999) had used the sediment quality guidelines in ranking areas that warranted further detailed study of the actual occurrence of adverse effects such as toxicity. They were also intended for use in ranking chemicals that might be of potential concern. The Wisconsin Department of Natural Resources (2003) had employed the sediment quality guidelines for the following aspects:i. To provide a reliable basis for assessing sediment quality conditions in freshwater ecosystems. ii. To identify hot spots with respect to sediment contamination. iii. To determine the potential for and spatial extent of injury to sedimentdwelling organisms. iv. To evaluate the need for sediment remediation. v. To support the development of monitoring programs to further assess the extent of contamination and the effects of contaminated sediment on sediment-dwelling organisms. Results of current study were made comparison to several guideline values (Table 4.7 and Table 4.8). The background value for freshwater sediment is derived from NOAA (Buchman, 1999; Appendix E). The probable effect level in freshwater sediment is established under the Canadian Environmental Quality Guidelines (Appendix E) and the severe effect level is derived from New York State Department of Environmental Conservation (Appendix E). The probable effect level represents the lower limit of the range of chemical concentrations that are usually or always associated with adverse biological effects. Sediments with measured chemical concentrations equal to or greater than the probable effect level are considered to represent significant and immediate hazards to exposed organisms. Biological assessment is recommended at these sites to determine the nature and 108 extent of effects that are being manifested as a result of the sediment associated contaminants (Canadian Council of Ministers of Environment, 1999). The severe effect level values is also known by the 50th percentiles or median which represents the concentrations above which biological effects frequently occur and the analysed sediments are considered to be severely impacted if exceeded the criterion value (New York State Department of Environmental Conservation, 1999). The range in concentration of As, Cr, and Se from both rivers and lakes found to exceed the background values for freshwater sediment. Several sampling sites at the river were below the probable effect level of 17 g g-1 for As and 90 g g-1 in Cr concentrations. However, concentration of As and Cr reached 102% and 114% of the severe effect level, respectively, in very few samples. Table 4.8 : Several guidelines value for arsenic, chromium and selenium Arsenic (g g-1) Chromium (g g-1) Selenium (g g-1) Observed ranged in river system 10.17 – 33.80 26.83 - 125 0.56 – 1.91 Observed ranged in lake system 17.62 - 61.60 173 – 301 0.31 – 1.08 1.10 7 - 13 0.29 Canadian probable effect levels for freshwater sediment b 17 90 na Severe Effect Level c 33 110 na Background a na – not available a - Buchman (1999) b - Canadian Council of Ministers of Environment (1999) c- New York State Department of Environmental Conservation (1999) 109 Arsenic concentrations in the range of 17.62 g g-1 to 61.60 g g-1 for most lake sediments were substantially elevated over the probable and severe effect level of 17 g g-1 and 33 g g-1, respectively. Similar indication was also observed for Cr concentrations in lake sediment. The results indicated that the level of As and Cr contamination in the lake sediment is potentially significant with regard to aquatic life. Table 4.9 : Selenium guideline values derived from the Guidelines for Interpretation of the Biological Effects of Selected Constituents in Biota, Water and Sediment Medium No effect Level of concern Toxicity threshold Sediment (g g-1) <1 1–4 >4 Guidelines for Interpretation of the Biological Effects of Selected Constituents in Biota, Water and Sediment was established by USDOI (1998) which was based on three levels of selenium threshold; the no effect, the level of concern and the toxicity threshold. The no effect level indicates that concentration less than the given value do no produce discernible adverse effects on fish or wildlife and are typical of the background concentrations in uncontaminated environment. The range of elemental concentrations in the level of concern rarely produces discernible adverse effects on some fish or wildlife species while any concentration values greater than the toxicity threshold levels seem to produce adverse effects on biological life. The Se concentrations found showed low level of contamination in rivers and lakes sediment by referring to the guidelines (Table 4.9). Only one sampling sites at the lake, TBB exceeded the no effect level. Meanwhile, all the sampling sites of Sungai Buluh exhibited Se concentrations below the no effect level. Se concentrations in sediments at the other river and lake sampling sites lies between levels of concern. 110 Generally, level of Se in the sediment was in the acceptable and tolerant limits to aquatic organisms, while the sites where Se was observed to be at the level of concern should have continuous assessment conducted as some aquatic organisms may show adverse effects at this level. Toxic dietary exposure study showed that thresholds for dietary toxicity in animal for Se level are generally reported as 2 to 5 g g-1 (USDOI, 1998). Continuous exposure to high Se content by the aquatic organisms may also affect human dietary as Se has the characteristic of bioaccumulating in the tissues of organisms. 4.7 Association of Arsenic, Chromium and Selenium to Iron Concentration Particulate metals from natural and anthropogenic sources accumulate together in sediment. In order to determine the level of metal pollution in a particular area the two metal proportions need to be isolated and this can pose problems because sedimentary metal loads can vary by several orders of magnitude, depending on the mineralogy and grain size distribution. Usually, a normalization procedure is suggested to overcome this problem (Cheevaporn and Diego-McGlone, 1997; Whalley et al., 1999). With this procedure, the natural component of the total concentration can be calculated by defining the ratio of natural concentrations to that of some normalizing factor whose concentration is unaffected by human activities which would then enable to detect and quantify any anthropogenic metal contribution to the system. Aluminum, iron, scandium, total organic carbon, and grain size can be used in particular cases as geochemical normalisers (Breslin and Sanudo-Wilhelmy, 1999; Cheevaporn and Diego-McGlone, 1997; Yusof and Wood, 1993). They will often show strong relationships with site contaminants. Normalization based on Fe, Al or Sc, as reference conservative elements having supposed natural distributions, is a useful tool in estimating the anthropogenic over natural contribution in the chemical content of soils and sediments. Presuming an existing linear dependence between the 111 conservative element and the heavy metal, it is possible with the use of simple linear regression to define the heavy metal geochemical background and to isolate natural and/or anthropogenic outliers. Iron was chosen as an element of the normalization in this study because Fe is abundant in crustal-derived sediments and has a reactive fraction associated with particle surfaces similar to contaminant metals. Anthropogenic Fe is usually small compared to the amount of Fe naturally present (Breslin and Sanudo-Wilhelmy, 1999). This is in agreement with the study conducted by Kaushik et al. (2008) whose reported that the enrichment value of Fe in sediment of River Yamuna, India is less than 1 indicating insignificant effect of anthropogenic flux of this element into sediment even though Fe concentrations ranged from 4.15 g kg-1 16.2 g kg-1 was found. Normalisation of the specific heavy metal concentrations in the bottom sediments under aerobic conditions to Fe can also be used as a correction factor for comparative assessment of sampling sites and river basin contamination by heavy metals (Boszke et al., 2004). The concentrations of As, Cr and Se in sediments are plotted against Fe concentrations presented in Figure 4.8 and Figure 4.9 for river and lake, respectively. The results of statistical evaluations were presented in Appendix F. Investigations show that Fe has significant effects on the particular metals content in bottom sediments of both the river and lake system. A study conducted by Sompongchaiyakul and Sirinawin (2006) indicated that the major geochemical factor that controls the dispersal and accumulation in the lake system is Fe in its oxides form. Iron usually studied in the geochemistry of a aquatic sediment due to its high adsorption capacities when compared to other oxides such as Al and Si oxides. Iron highly affects trace metal cycling in lake sediment because they participate under oxic conditions and dissolve under anoxic condition (El Bilali et al., 2002). 112 40 35 -1 Total As (ug g ) 30 25 20 15 10 5 R² = 0.6721 0 10000 15000 20000 25000 30000 35000 40000 45000 -1 Total Fe (ug g ) 160 140 -1 Total Cr (ug g ) 120 100 80 60 40 R² = 0.8754 20 0 10000 15000 20000 25000 30000 35000 40000 45000 -1 Total Fe (ug g ) 2.5 -1 Total Se (ug g ) 2 1.5 1 0.5 R² = 0.0007 0 10000 15000 20000 25000 30000 35000 40000 45000 -1 Total Fe (ug g ) Figure 4.8: Concentrations of As, Cr and Se plotted against concentrations of Fe in river sediments. 113 80 70 -1 Total As (ug g ) 60 50 40 30 20 10 0 20000 R² = 0.6973 25000 30000 35000 40000 45000 50000 55000 -1 Total Fe (ug g ) 320 300 260 -1 Total Cr (ug g ) 280 240 220 200 180 160 R² = 0.1681 140 120 20000 25000 30000 35000 40000 45000 50000 55000 -1 Total Fe (ug g ) 80 70 -1 Total As (ug g ) 60 50 40 30 20 10 0 20000 R² = 0.6973 25000 30000 35000 40000 45000 50000 55000 -1 Total Fe (ug g ) Figure 4.9: Concentrations of As, Cr and Se plotted against concentrations of Fe in lake sediments. 114 Positive correlation levels of Fe were significant with As and Cr in river sediment for correlation coefficient value of 0.82 and 0.94, respectively. A study by Whalley et al. (1999) at Humber Estuary, UK showed that As has statistically significant relationship with Fe concentrations in the sediment (p < 0.001). Strong correlation was found between Cr and Fe in river sediment. No statistically correlation was observed between Se and Fe concentrations (p < 0.95). The result indicates that Se concentrations in river sediment are not controlled by the amount of Fe present. Iron levels were found to be significantly correlated with As (r = 0.76, p< 0.02) in sediments of lake system. Nikolaidis et al. (2004) found that the correlation between As and Fe in contaminated lake sediments was 0.92 and large fraction of As was bound to Fe oxide. Therefore, strong correlation between As and Fe in both lake and river sediment indicate that the sediment is a significant sink for As. Correlation analysis shows significant relationship between Se and Fe concentrations (p< 0.005). Se showed an inverse association to Fe concentrations in lake sediment with correlation coefficient value of -0.81. However, there is no clear explanation to this phenomenon. Even though Fe particularly in its oxyhydroxide form has strong affinity for Se, but adsorption of Se to Fe is also controlled by other factors such as pH, redox state and particle concentration (Balistrieri and Chao, 1990). Changes in salinity and pH may also cause Se exchange to pore water and organisms may accumulate Se at a greater rate that result in the reduction of Se concentrations in sediment (Miao et al., 2006; Peters et al., 1999). Chromium was weakly correlated and demonstrated an inverse association to Fe concentrations (r = -0.24, p< 0.36). The result signifies that non-lithogenic factor like human activity may also influence the amount of Cr in the lake sediment. The distribution of element-to-Fe ratios in the sediment of river and lake sampling sites is shown on Figure 4.10 and 4.11, respectively. In order to show detail of the distribution ratios, the element-to-Fe ratios were then normalized with specified factors (Miller and Mcpherson, 2001). Arsenic to Fe ratio is multiplied by 50 000, Cr and Se to Fe are multiplied by 10 000 and 1 000 000, respectively. The highest ratio for Se was observed at Sungai Tebrau, possibly due to anthropogenic 115 input into the river system which may come from domestic and industrial activities along the river. The ratio for Cr is relatively consistent at all sampling site which is in agreement with its correlation to Fe that signifies natural background occurrence. At several sites, As showed high ratio to Fe concentrations. This finding suggested that As is prone to accumulate near the river mouth as a result of As transportation from upstream and deposition at the river mouth. Ratio of As, Cr and Se concentrations to Fe concentration 100 As/Fe 90 Cr/Fe 80 Se/Fe 70 60 50 40 30 20 10 0 SGS1 SGS2 SGS3 SGT1 SGT2 SGB1 SGB2 SGB3 River Figure 4.10: Ratios of concentration of As, Cr and Se to concentrations of Fe in river sediments. 116 Arsenic, chromium and selenium ratio to Fe concentrations showed a uniform level at lake TKR sampling sites. At sampling site TUPA, the three elements were observed for the highest ratio compared to other sampling site. Previously, the lake bank nearby the sampling site was used as a horticulture learning center by the university. Usage from fertilizer and pesticide was suggested to contribute to the higher levels of Cr, As and Se in the sediment at the respective site. Ratio of As, Cr and Se concentrations to Fe concentration 140 (As/Fe) 120 (Cr/Fe) (Se/Fe) 100 80 60 40 20 0 TKRA TKRB TKRC TUPA TUPB TBA TBB Lake Figure 4.11: Ratios of concentration of As, Cr and Se to concentrations of Fe in lake sediments. 117 4.8 Evaluating Contaminant Impact Several methods have been put forward to quantifying the degree of contaminant enrichment in sediments (Battelle Memorial Institute, 2003; Abrahim and Parker, 2008). Enrichment factor (EF) and geoaccumulation index (Igeo) are the most convenient methods to evaluate and estimate the contamination impact in sediments by giving a broad descriptive bands of contamination ranging from low to high intensity. The value of EF and Igeo in the sediment analyzed from the river and lake sampling sites is presented in Table 4.10. Table 4.10: Enrichment factors (EF) and Index of Geoaccumulation (Igeo) in sediment of rivers and lakes Arsenic Sites Chromium Selenium EF Igeo EF Igeo EF Igeo SGS1 SGS2 SGS3 Average 12.3 21.2 20.7 18.1 2.5 2.9 3.0 2.8 1.0 1.2 0.9 1.0 1.8 1.9 1.7 1.8 11.8 10.6 7.7 10.0 1.1 0.9 0.7 0.9 SGT1 SGT2 Average 15.1 12.0 13.6 2.0 2.1 2.1 0.8 0.9 0.9 0.9 1.4 1.2 29.9 24.1 27.0 1.4 1.5 1.5 SGB1 SGB2 SGB3 Average 20.2 12.1 20.9 17.7 1.9 1.8 2.7 2.1 0.7 0.8 0.7 0.7 0.3 1.0 1.1 0.8 18.9 15.7 6.8 13.8 0.5 0.8 0.2 0.5 TKRA TKRB TKRC Average 33.3 30.4 35.6 33.1 3.4 3.6 3.6 3.5 1.6 1.3 1.5 1.5 2.2 2.3 2.3 2.3 5.9 2.6 3.8 4.1 0.3 -0.2 0.1 0.1 TUPA TUPB Average 37.3 32.7 35.0 3.2 3.3 3.3 3.9 2.4 3.2 2.7 2.5 2.6 12.5 10.4 11.5 0.8 0.8 0.8 TBA TBB Average 27.5 16.2 21.9 3.3 2.4 2.9 2.0 2.3 2.2 2.5 2.3 2.4 3.5 14.7 10.9 -0.1 1.0 0.5 River Lake 118 4.8.1 Enrichment Factor (EF) Enrichment factor is defined by the ratio of chemical concentration of an element in a weathered material to that in its fresh parent material. It corresponding to each sediment sample as the ratio of the interest metal concentration in the sample to the normalizing metal concentration in the sample, divided by the same ratio in the parent rock (Battelle Memorial Institute, 2003). The enrichment factor is usually applied in evaluating and estimating to which extend that sediment are impacted by anthropogenic sources. Metal ratios for the parent rock usually are determined by referring to published metal concentration data. Wedepohl (1995) continental crust derived values were applied in this study for calculating the ratio of crustal abundance metals. Sediment metal enrichment factors have been previously calculated using either Al (Pekey, 2006), Fe (Abrahim and Parker, 2008; Breslin and SanudoWilhelmy, 1999) or Sc (Yusof and Wood, 1993) as the normalizing element. In this study, the enrichment factor is expressed as (4-1): EF = (M / Fe) sediment / (M / Fe) crust (4-1) where: (M / Fe) sediment = the ratio of the metal concentration detected in a sample to the Fe (normalizing metal) concentrations detected in the same sample. (M / Fe) crustal = the ratio of the average metal concentration in the crustal to the average Fe concentration in the crustal. A value of EF ≤2 can be considered to be of lithogenic origin for a metal whereas EF > 2 indicates the addition of an anthropogenic component and/or a biogenical enrichment process (Grousset et al., 1995). Meanwhile, Pekey (2006) divided the enrichment of element into three major groups with respect to their corresponding median EF: elements without enrichment (EF < 10), elements with 119 medium-level enrichment (10 < EF < 100) and finally highly enrich elements (EF > 100). However, the evaluation of EF proposed by Grousset et al. (1995) was take into consideration to be used in this study as the elements were analyzed based on the average concentrations and not divided into their corresponding median EF group. The results in Table 4.10 show EF for As and Se at the rivers were elevated more than the factor of 2. This indicates the anthropogenic inputs are present in the study area. The effluent discharged from the industries and the used of agricultural chemical containing arsenic in the palm oil estate near the rivers could contribute to high enrichment of As and Se in the sediment (Thanapalasingam, 2005). Sungai Skudai is highly enriched with As while Sungai Tebrau has the highest enrichment of Se. A study on the elemental EF in the sediment in the costal areas of South Johore found that the maximum concentration of 41.4 g g-1 for As in the bay area and reflected high EF value of between two to five magnitudes higher than that of the reference sediment (Yusof and Wood, 1993). High enrichment of As in the sediment of the bay area could be introduced from the effluents directly flow from in land or transported by rivers that meet the near-coast marine system. The current study on the three rivers meet the same costal area which studied by Yusof and Wood (1993) were highly enriched with As contribute to high concentration of As in the coastal sediment. Chromium shows an EF less than 2 in all the rivers. The EF values suggest that the high level of Cr concentrations in sediments could be originated from the weathering of litogenous material of the river bed and along the river bank. Therefore, anthropogenic inputs play insignificant factor in the present of Cr in river sediment. 120 Similarly in the river, As and Se also show high EF values in the sediment of the lakes. The EF value for As in the lake ranged from 21.9 to 35. The EF for Se in the lake is found to range from 4.1 to 10.9. Lake TUP was observed to have a high enrichment of As, Cr and Se. A high EF value has proven that the suggested reason of high element-to-iron ratio at the site arose from previous the horticulture activities. The As enrichment at lake TKR could be explained by an excessive input of deer manure or their food waste through run-off process. Meanwhile, there is no clear explanation on the As and Se enrichment at lake TB. However, the location of the lake at the low land of the hilly area could promote the input of the elements through the run-off process from the surrounding area through application of the fertilizer and pesticide in conjunction with landscaping activities. There may be also an additional source that accounts for the higher levels of As in the lake sediments. According to Durant et al. (2004), it is possible that vertical transport of sedimentary As from deeper sediment layers to surface layers could be occurring by molecular diffusion or due to advection by groundwater entering the lake. 4.8.2 Geoaccumulation Index (Igeo) An approach of geoaccumulation index, Igeo was used to quantify the degree of anthropogenic contamination and compare different metals that appear in different ranges of concentration in contaminated sediments (Abrahim and Parker, 2008; Lokeshwari and Chandrappa, 2006). This index is calculated as follows (4-2): Igeo where: Cm = log2 Cm / 1.5 Bm (4-2) = measured concentration at sampling point; Bm = background concentration value for element; 1.5 = the background matrix correction factor due to lithogenic effects. 121 The geoaccumulation index scale consists of seven grades ranging from unpolluted to very highly polluted. The description of seven Igeo scale is presented in Table 4.11. In this study, the background value was taken from the value establish by NOAA as presented in Table 4.8. (Buchman,1999). Table 4.11: The Igeo scale Igeo value Igeo class Designation of sediment quality >5 6 Extremely contaminated 4–5 5 Strongly to extremely contaminated 3–4 4 Strongly contaminated 2-3 3 Moderately to strongly contaminated 1–2 2 Moderately contaminated 0–1 1 Uncontaminated to moderately contaminated 0 0 Uncontaminated The pollution levels of As in the river system expressed in terms of the geoaccumulation indices indicate that the river sediments is moderately to strongly contaminated. Anthropogenic inputs from agricultural, industrial and urbanization along the rivers contribute to excessive amount of As in sediments. The level of As contamination in the river sediment is potentially significant with regard to the health of the river ecosystem. Meanwhile, Cr and Se at most rivers were found to be in Igeo Class 1 and 2, respectively with uncontaminated and moderate contaminated indication. Present of Cr and Se in the river sediments may be attributable to combination of natural and anthropogenic factors. Sediments from Sungai Skudai recorded the highest contamination level of As and Cr. 122 The lakes sediment can be described as strongly contaminated with As based on the high Igeo value. Arsenic contamination in the lake sediments was associated with fertilizer and herbicide application for landscaping activity and domestic wastewater discharge from student’s residential blocks into the drainage system. Chromium was laid in Igeo Class 3 with moderate to strong contaminated indication. Chromium contamination in the lake was likely caused by domestic wastewater or from the weathering of litogenous material of the lake bed or around the bank particularly at site TKR where the EF value showed less than the factor of 2 indicative of the enrichment originating from weathering of parent materials. Even though the lake sediments showed an occurrence of Se enrichment, the negative Igeo value found at a few sites are the result of relatively low levels of contamination (Abrahim and Parker, 2008). The lake TUP shows the highest contamination level of As, Cr and Se compared to the other two lakes. Besides human factor, another possible source of As and Cr were due to the vertical migration of the elemental species resulted from diagenetic process that normally takes place in anoxic sedimentary conditions as notified from the characteristic of the sediment for example the odor due to H2S and the greasy appearance of the column of the sediment (Wood et al., 1997). Investigation conducted by El Bilali et al. (2002) over 20 lakes in Ontario, Canada showed that the trace elements enrichment in the surface sediment may not be solely attributed to anthropogenic mobilization but also to the role played by Fe, and Mn oxides and organic matter during burial and diagenesis process. 4.9 Speciation of Arsenic and Selenium in Sediments Speciation of arsenic and selenium in environmental samples is gaining increasing importance, as the toxic effect of the As and Se are related to its oxidation state. Inorganic As and Se; As (III), As (V), Se (IV) and Se (VI) are the predominantly species found in contaminated sediment or soil (Goh and Lim, 2004). Inorganic species of As and Se from solid samples have been separated and 123 quantified using various methods as presented in Chapter 2 : Table 2.2. Even though at present there are no CRM available specifically for As and Se species in sediment, the determination of the species were made possible by validating the proposed analytical method (Hutton et al., 2005; Leermakers et al., 2006; Ruiz-Chancho et al., 2005). In this study, the CRM which certifies the total arsenic and selenium concentration was applied to investigate on the recovery of the species during the microwave extraction under the proposed range of media concentration, microwave power and time of extraction. The results of total inorganic species found by microwave extraction were compared with the certified values of the reference material. The standard species was spiked into the extraction media to trace on the repeatability of the analysis and any loss or inter-conversion of the species. The effect of pH on the extracted media was studied in order to assess the extraction efficiency during the preconcentration step. The optimum conditions of the validated studies were then applied for subsequent experiments of As and Se species in the river and lake sediments. 4.9.1 Optimization of Arsenic Speciation Study Several studies have shown that H3PO4 in the presence of low-power microwave creates promising conditions for the extraction of As species in soil and sediment. However, the extraction yields from several CRM of sediment varied between 80 and 110% (Gallardo et al., 2001; Hutton et al., 2005; Sathrugnan and Hirata, 2004; Thomas et al., 1997). Therefore, a microwave-assisted H3PO4 extraction was investigated, with microwave specific power, time and H3PO4 concentration in order to optimize an extraction procedure that extracts inorganic As species from sediment samples. A sediment CRM BCSS-1 and spiked solution of the As species of interest were studied to check for extraction efficiency and species stability. The extraction efficiency of As (III) and As (V) for separation and preconcentration procedure using APDTC-MIBK system was also investigated as a function of the pH. 124 4.9.1.1 Microwave Extraction In order to determine the optimum condition for sediment extraction using microwave, the CRM BCSS-1 was treated using H3PO4 concentration of 0.25 M, 0.5 M and 1 M for 10 and 20 min at 20 and 25% microwave power. The certified value for total As in BCSS-1 is 11.1 g g-1. The mean values for As species extractable from various acid concentration and microwave conditions was summarized in Table 4.12. These test revealed that at low concentration of 0.25 M H3PO4 up to 43% of total As was extracted at all time and microwave power applied. Good recovery of 92% for total As was achieved at an operating time of 20 min treated at 25% power in 1 M H3PO4. The respective microwave conditions and optimum acid media were employed for all subsequent studies. The stability study of As species was carried out at the optimum condition of 20 min at 25% power in 1M H3PO4 using standard solution of 0.25 µg mL-1 As (III) and As (V). No significant loss or inter-conversion of As species was observed with a high recovery percentage of 97 to 99% and RSD of 2.5 to 3.4% (Appendix G). This test was carried out on six samples. 125 Table 4.12: Results of arsenic species under various microwave conditions and H3PO4 concentration (expressed as µg g-1 dry mass). H3PO4 (M) 0.25 0.5 1.0 As extracted Power (%) Time (min) 20 25 10 20 10 20 As (III) As (V) Total As Recovery from total (%) 0.240.01 3.330.13 3.57 0.300.01 3.140.20 3.44 0.430.01 3.960.20 4.39 0.700.06 4.100.21 4.8 32 31 40 43 As (III) As (V) Total As Recovery from total (%) 0.370.02 3.020.20 3.39 0.450.05 3.910.17 4.36 1.460.10 5.120.30 6.58 1.500.10 7.430.36 8.93 31 39 59 81 As (III) As (V) Total As Recovery from total (%) 0.610.06 3.600.30 4.21 1.120.10 4.250.21 5.37 1.740.14 5.780.31 7.52 2.220.17 7.980.42 10.2 38 48 68 92 n=3 4.9.1.2 Effect of pH and Repeatability The investigation of extraction efficiency of As (III) and As (V) as a function of pH showed that pH range affects the extraction efficiency. Results shown in Figure 4.12 demonstrate that As (III) can be quantitatively extracted by APDTCMIBK in the pH range of 4 to 9 whereas As (V) is not extracted at all at pH 4 to 10. As (V) can be extracted at pH 1 to 3 with a low efficiency of up to 10%. Therefore, these two species can be separated from each other at pH 4 to 9 and pH 6 was employed for all subsequent investigations. The detail results of extraction efficiency of As (III) and As (V) are presented in Appendix G. 126 This study is consistent with those of Mok et al. (1986) and Shi (2000) who reported that As (III) can be quantitatively extracted by APDTC-MIBK in the pH range of 0.6 to 9. A small fraction between 2% to 6% of As (V) was found in the extraction at pH 1 (Mok et al., 1986). However, Shi (2000) found that up to 25% of As (V) was extracted at pH 2 - 5. 120 Recovery, % 100 80 60 40 As (III) 20 As(V) 0 1 2 3 4 5 6 7 8 9 10 pH Figure 4.12: Extraction recovery of As (III, V) by APDTC-MIBK at different pH Under the optimized preconcentration conditions, the repeatability of the extraction by APDTC-MIBK had been studied using standard solution of As (III) and As (V) at 2 µg mL-1. The standard deviations of extractable As concentration were calculated from NAA data of seven extraction runs carried out on the same day. The RSD of approximately 7% and 5% were obtained for As (III) and As (V), respectively and low RSD values suggest that sufficient repeatability for As extraction is achieved. 127 4.9.2 Optimization of Selenium Speciation Study Similar procedures like As speciation study were applied in validating the extraction method for Se species in sediment. A sediment CRM oPACS - 2 and spiked solution of the Se species of interest were investigated to check for extraction efficiency and species stability in low concentration of HCl media. The extraction efficiency of Se (IV) and Se (VI) for separation and co-precipitation procedure was also studied as a function of pH. Several studies have shown that mild HCl extracts significant amount of Se species in solid environmental samples such as soil and muscles tissue using different mechanism of extraction (Bujdoš et al., 2005; Ochsenkühn-Petropoulou et al., 2003; Peters et al., 1999). About 94 % extraction efficiency was achieved by Ochsenkühn -Petropoulou et al. (2003) using 0.5 M HCl in temperature of 4C where the results also indicate the combination of other organic Se species.such as selenourea, selenoethionine and selenomethionine. Sathrugnan and Hirata (2004) obtained about 60% of extraction efficiency for inorganic Se in coal fly ash but no quantitative amount of Se species was extract from sediment using microwave extraction in 0.1 M H3PO4. Meanwhile, some other studies applied microwave extraction method for inorganic Se determination in a mixture of concentrated acid but no further information on the extraction efficiency was available (Yusof et al., 1997; Yusof et al., 1998). 4.9.2.1 Microwave Extraction The optimum condition for sediment extraction using microwave was studied using the CRM PACS-2 in HCl concentration of 0.1 M, 0.25 M, 0.5 M and 1 M for 10 and 20 min at 20 and 25% microwave power. The certified value for total Se in CRM PACS-2 is 0.92 g g-1. 128 The mean value for Se species extractable from various acid concentration and microwave conditions was presented in Table 4.13. The study shows that at low concentration of 0.1 M HCl the total Se species extracted ranged from 32% to 36% at all operating times and microwave power applied. High recovery of 86% for total Se species was achieved at an operating time of 20 min treated at 25% power in 1 M HCl. The respective microwave conditions and optimum acid media were employed for all subsequent studies. Table 4.13: Results of selenium species under various microwave condition and HCl concentration (expressed as µg g-1 dry mass). HCl (M) 0.1 Se extracted Se (IV) Se (VI) Total Se Recovery from total (%) Power 20 (%) Time 10 20 (min) 0.0730.006 0.0990.004 0.2280.020 0.2220.010 0.301 0.321 33 35 25 10 20 0.1010.010 0.1930.020 0.294 32 0.1210.010 0.2110.010 0.333 36 0.25 Se (IV) 0.1070.002 0.1240.005 0.1290.005 0.1430.010 Se (VI) 0.2580.020 0.2780.003 0.2480.030 0.3750.020 Total Se 0.365 0.402 0.377 0.518 Recovery from total (%) 40 44 41 56 Se (IV) 0.1380.002 0.1420.030 0.1520.010 0.1700.003 Se (VI) 0.3000.010 0.3300.004 0.3740.020 0.4770.040 Total Se 0.438 0.472 0.526 0.647 Recovery from total (%) 48 51 57 70 Se (IV) 0.1520.009 0.1570.010 0.1660.002 0.1810.010 Se (VI) 0.3360.010 0.3760.020 0.4710.020 0.6080.030 Total Se 0.488 0.533 0.637 0.789 Recovery from total (%) 53 58 69 86 0.5 1.0 n=3 129 This study achieved better extraction efficiency as compare to Ochsenkühn Petropoulou et al. (2003) who obtained 43% for total Se (IV) and Se (VI) extract in soil sample from 0.5 M HCl media. In mine tailing sample, Bujdoš et al. (2005) managed to extract 5.8% to 23.5% of Se mobile fraction using 0.5 M HCl. Meanwhile, Sathrugnan and Hirata (2004) and Iserte et al. (2004) were unable to extract quantitative amount of inorganic Se from sediment sample using mild H3PO4 media. The stability study of Se species using a 0.25 µg mL-1 standard solution of Se (IV) and Se (VI) was carried out at the optimum condition of 20 min at 25% power in 1M HCl. No significant loss or inter-conversion of Se species was observed with a satisfactory Se (IV) recovery percentage of 97% and RSD of 10% while Se (IV) achieved a high recovery of 99%. (Appendix H). This test was carried out on six samples. 4.9.2.2 Effect of pH and Repeatability The investigation of extraction efficiency of Se (IV) and Se (VI) by coprecipitation technique as a function of the pH showed that the pH range affects the extraction efficiency. The effects of pH on the extraction of Se (IV) and Se (VI) after co-precipitation of Na-DBDTC with phenolphthalein are shown in Figure 4.13. Good quantitative extraction of Se (IV) was observed at pH 2 and 3 with recovery of 101% and 91%, respectively. Above pH 3, the efficiency of extraction of Se (IV) begins to decrease at consistent level. Saleh et al. (1990) also observed similar pattern of extraction efficiency of Se (IV) in the same co-precipitation system where high recovery was obtained at pH 2 - 3 and it decreased rapidly after pH 4. According to literature study, Se (VI) cannot be co- precipitated by NaDBDTC even at low pH (Cheng et al., 1982). No significant amount of Se (VI) was found in the standard at all pH range. Therefore, these two species can be easily separated from each other and a pH value of 2 was employed for all subsequent studies. The detail results of extraction efficiency of Se (IV) and Se (VI) are shown in Appendix H. 130 120 Revovery, % 100 80 60 Se (IV) 40 Se (VI) 20 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 pH Figure 4.13: Extraction recovery of Se (IV, VI) by co-precipitation technique at different pH. The repeatability of the co-precipitation technique using Na-DBDTC with phenolphthalein had been studied using standard solutions of 1 µg.mL-1 Se (IV) and Se (VI) under the optimum conditions for co- precipitation. The standard deviations of extractable Se concentrations were calculated from NAA data of five extraction runs carried out on the same day. The RSD of approximately 4% and 3% were obtained for Se (IV) and Se (VI), respectively and low RSD values suggest that sufficient repeatability for Se extraction is achieved. 4.10 Concentration of Arsenic and Selenium Species in River and Lake The results obtained for As species in river and lake sediments are shown in Table 4.14. The results of each sampling session are presented in Appendix I. These results clearly indicated that the majority of As was present as As (V), with As (III) accounting for < 18% of the total As. The extraction efficiency, relative to the total As concentrations, varied from 68 to 99%. Some of the values were lower than the 131 values obtained during the optimization of the extraction, suggesting that the sediment contained a slightly different matrix than those used for optimization. However, extraction efficiencies were still at least as good as those described in studies carried out by Gallardo et al. (2001), Hutton et al. (2005), Thomas et al. (1997). Hutton et al. (2005) also discovered that low recoveries of the As species in the sediment samples were not due to interferences from that matrix or during the analysis stage. But, it was more likely that some of the As is strongly bound or complexed. According to Ellwood and Maher (2003), the presence of humic material and mineral As like arsenopyrite have little effect on As extraction efficiency. A longer extraction process would possibly yield better recoveries, but this could lead to inter-conversions or loss of the species. The majority of As extracted was As (V) which shows the predominance of oxidized As phases at the sampling sites. Low ratios of As (III) to As (V) had been observed at all sampling sites ranging from 0.03 to 0.24. By using ANOVA (Appendix I), the difference in the ratio of As (III) to As (V) in lake and river sediment were found to be statistically insignificant at = 0.05 (P =0.3). This study is consistent with those of Pettine et al. (1992) and Abdul-Moati (1990) who found that arsenate was the predominant arsenic species in the river and lake respectively. Pettine et al. (1992) reported that the ratio between oxidized and reduced species appears to be significantly influenced by the presence of iron and manganese oxides. As (V) are the more stable species and can be strongly adsorbed onto clay, iron and manganese oxides (Mandal and Suzuki, 2002). Abdel-Moati (1990) monitored arsenic in the Nile delta lakes and found As (V) to be the dominant arsenic species (85–95%). Increased As (III) (14–33%) was found near local sewage discharge points. 132 Table 4.14: Arsenic speciation analysis in river and lake sediment As (III) g g-1 As (V) g g-1 Recovery from totals (%) Ratio As (III)/ As (V) SGS1 SGS2 SGS3 Average 1.10 0.31 0.73 0.26 1.79 0.47 1.21 0.54 13.3 1.3 27.0 3.4 30.1 5.0 23.5 4.0 74.1 90.6 94.3 86.3 0.08 0.03 0.06 0.06 SGT1 SGT2 Average 0.88 0.33 1.99 0.58 1.44 0.48 11.0 3.0 11.4 1.0 11.2 0.3 96.8 99.6 98.2 0.08 0.17 0.13 SGB1 SGB2 SGB3 Average 0.69 0.23 0.55 0.17 1.07 0.27 0.77 0.19 7.4 0.9 7.2 1.1 19.6 1.1 11.4 2.2 74.3 76.4 85.2 78.6 0.09 0.08 0.05 0.07 TKRA TKRB TKRC Average 2.45 0.11 3.54 0.64 2.23 0.20 2.74 0.70 40.1 1.2 45.3 2.4 40.6 4.3 42.0 2.9 87.7 81.0 69.6 79.4 0.06 0.08 0.06 0.06 TUPA TUPB Average 6.68 0.44 3.04 0.32 4.86 0.60 27.6 1.7 35.7 0.7 35.7 2.5 88.2 87.9 88.0 0.24 0.09 0.16 TBA TBB Average 2.83 0.39 0.94 0.15 1.89 0.50 28.2 1.9 11.1 0.9 19.7 1.7 68.0 68.3 68.2 0.10 0.08 0.09 River Lake Sampling locations like SGT2 and TUPA had shown slightly higher percentage of 15% and 18% As (III), respectively while other locations constituted < 7% As (III) of the total As. Sediments at both sites may suggest undergoing an early sign of anaerobic condition. Sewage discharge from housing estate and drinking water factory in the vicinity of SGT2 site could contribute to the biological activity in the sediment and create anoxic environment which most likely encourage the reduction process of As (V) to As (III). The site at TUPA, however was formerly used for horticulture activities where an excessive application of pesticide and fertilizer may suggest for the presence of higher levels of As (III) in the sediment. 133 The environmental conditions of the sediment have a greater influence on As speciation and distribution than does the total concentration of arsenic in the sediment. Arsenic from-enriched sediments can be released to water column under anaerobic conditions making the existing conditions conducive to the reduction of As (V) to As (III) (Bhumbla and Keefer, 1994). The presence of sulfides and the hydrous oxides of iron and manganese act as important sinks through adsorption process and modes of transport for arsenic that is directly controlling the mobilization of arsenic in an aquatic environment (Mok and Wai, 1994). Selenium species concentrations in river and lake sediments are shown in Table 4.15 and the results of each sampling session are presented in Appendix I. The dominant species of Se was present as Se (VI) which was found to be about 61% to 86% of the total Se. Meanwhile, Se (IV) accounts for < 24% of the total Se concentrations. The extraction efficiency, relative to the total Se concentrations, varied from 71% to 94%. Some of the values were lower than the values obtained during the optimization of the extraction procedure, suggesting that the sediment contained a slightly different matrix than those used for the optimization step. Low recoveries of inorganic Se in the samples may be contributed from the presence of other Se species like organic Se compound that can also be found in the sediment. Gomez-Ariza et al. (1998) obtained significant amount of organic Se compounds such as dimethylselenide, dimethyldiselenide, dietyselenide and diethyldiselenide in sediments collected from areas in the Southwest of Spain. Selenium species of selenourea and selenoethionine was also extractable in 0.5 M HCl media from sediment at warm springs area of Thermopyles, Freece (Ochsenkühn -Petropoulou et al., 2003). Since the sediment obtained varies in nature, from high in organic matters to mostly clay, it was anticipated that the determination of Se (IV) and Se (VI) was likely suppressed by the interference of dissolved organic carbon as reported by Workman and Soltanpour (1980) when they found out that recoveries of inorganic Se in some extracts were poor. 134 Table 4.15: Selenium speciation analysis in river and lake sediment Se (IV) g g-1 Se (VI) g g-1 Recovery from totals (%) Ratio Se (IV)/ Se (VI) SGS1 SGS2 SGS3 Average 0.17 0.02 0.15 0.04 0.14 0.02 0.15 0.02 1.11 0.09 0.80 0.10 0.61 0.18 0.84 0.26 93.8 84.3 80.4 86.1 0.16 0.18 0.23 0.19 SGT1 SGT2 Average 0.22 0.05 0.16 0.01 0.19 0.05 1.33 0.20 1.54 0.13 1.43 0.15 86.9 86.2 86.5 0.18 0.11 0.15 SGB1 SGB2 SGB3 Average 0.17 0.06 0.13 0.02 0.13 0.08 0.14 0.02 0.60 0.10 0.72 0.10 0.43 0.04 0.58 0.15 91.4 86.2 89.3 89.0 0.27 0.20 0.31 0.26 TKRA TKRB TKRC Average 0.04 0.01 0.07 0.01 0.10 0.02 0.07 0.01 0.40 0.01 0.25 0.02 0.29 0.02 0.31 0.05 70.9 87.9 83.3 80.7 0.09 0.29 0.36 0.25 TUPA TUPB Average 0.15 0.01 0.12 0.03 0.13 0.02 0.66 0.03 0.81 0.02 0.7 0.10 87.7 94.0 90.9 0.23 0.15 0.19 TBA TBB Average 0.05 0.03 0.14 0.02 0.10 0.01 0.21 0.03 0.80 0.04 0.51 0.05 77.1 83.0 80.1 0.27 0.18 0.22 River Lake Low ratios of Se (IV) to Se (VI) had been observed at all sampling sites with values ranging from 0.09 to 0.36. The low Se (IV) to Se (VI) ratios suggest that oxidizing environment prevails in both freshwater body systems thus promoting the formation of the Se (VI) than the Se (IV) species. By using ANOVA (Appendix I), the difference in the ratio of Se (IV) to Se (VI) in river and lake sediment were found to be statistically insignificant at = 0.05 (P =0.7). The ratio of both Se species was fairly uniform in the river and lake sediment samples. 135 The distribution of Se in contaminated aquatic sediment is very much influenced by its chemical form and speciation. According to Goh and Lim (2004), Se (IV) is strongly adsorbed by soil or sediment while Se (VI) in only weakly sorbed and leaches easily. Selenite is present in mildly oxidizing and neutral pH environments while Se (VI) is predominant in oxidized and under ordinary alkaline conditions. CHAPTER 5 CONCLUSIONS AND SUGGESTIONS 5.0 Conclusions Since sediments are the ultimate repository of most of the contaminants that enter freshwater, it is appropriate that regulatory attention addresses the ecological risks that these sediment contaminants might pose. There is an increasing public awareness and concern for the health of our waterways and ecosystem, and an expectation that water quality will be improved, but any improvement in water quality must address sediments as an important component of aquatic ecosystem and a source of contaminants to the benthic food chain. Freshwater resources such as rivers, lakes and groundwater are the vital elements in human lives. Sediments act as a useful component in monitoring and assessing any physical and chemical changes which are related to contamination in the aquatic environment. Assessment and management of sediment quality conditions contributes to preserving and preparing future remediation plan for freshwater resources. Data on the respective study can serve as a baseline for long term studies in predicting the trends of water and sediment quality in water resources. It can also provide a better understanding of the behavior of the sediment quality and the important factors influencing them. The study of three water parameters; pH, temperature and DO at rivers and lakes demonstrated that human activities slightly affected the quality of the water bodies at several sampling locations. Sungai Skudai, Sungai Tebrau, lake sampling 137 sites of TKR and TUP showed an average pH parameter which was lower than the NWQS optimal Class I value of 6.5 to 8.5 except for Sungai Buluh and lake TB which were within the threshold value. Sungai Skudai and Sungai Tebrau met the threshold level for Class IIB while lake TKR and TUP were in Class III. Meanwhile, DO levels did not comply with the NWQS threshold level for Class 1 at all sampling sites but met the threshold value for Class IIA and IIB. However, the temperature of the water bodies at all sampling locations showed insignificant variation towards local studies conducted previously. The applicability of the analytical procedures using NAA technique for analysis of As, Cr, Se, Fe and species of As and Se has been successfully examined by analyzing CRM. The analytical results achieved recovery above 93% and with precision of generally better than 16.7% RSD. All results of elements studied were within the Z-score accepted criteria. Arsenic, chromium and selenium were present in the sediment of all sampling locations and the concentrations exceeded the background values for freshwater sediment criteria established by NOAA. The results obtained showed that the range of concentrations of As in the rivers and lakes were 10 - 34 g g-1 and 18 62 g g-1, respectively. The concentrations of Cr in the rivers ranged between 27 g g-1 to 125 g g-1, while in the lake sediments the concentrations ranged between 173 g g-1 to 301 g g-1. The Se concentrations of the river sediment and lake sediment ranged between 0.56 g g-1 to 1.91 g g-1 and 0.31 g g-1 to 1.08 g g-1 respectively. Human related activities such as agricultural, industrial, urbanization, landscaping may have been contributed significant amount of As, Cr and Se into the freshwater sediments. Arsenic was found to be significantly correlated with Fe in sediments of river and lake system (r = 0.82 and r = 0.76; respectively). Strong correlation between Cr and Fe (r = 0.94) suggested that Fe fractions played an important role in the sorption of Cr in river sediments. This indicated that the sediment was a substantial sink for As and Cr. 138 Enrichment factor (EF) and geoacculumation index (Igeo) were employed for quantifying the degree of As, Cr and Se enrichment in the sediments. Arsenic and selenium showed values of EF > 2 in river and lake sediment which indicates an excessive anthropogenic input entering the aquatic system. A value of EF≤ 2 for Cr in river sediment could be signified by lithogenous material input originated from natural weathering processes and sedimentation. The contamination levels of As in the river system expressed in terms of Igeo indicated that the river sediments were moderately to strongly contaminated and are categorized in Igeo Class 3. Chromium and selenium in rivers were categorized in Igeo Class 1 and 2 indicating uncontaminated and moderate contamination levels. The sediments of Sungai Skudai showed the highest contamination levels of As and Cr. The Igeo value of Class 4 indicated that the lakes sediment was strongly contaminated with As. Chromium and selenium in lakes were found to be in in Igeo Class 1 and 3, respectively. The sediments of lake TUP showed high values of Igeo for As, Cr and Se. Arsenic and selenium were determined for their inorganic species in both river and lake sediments. Significant amounts of the inorganic species of As (III), As (V), Se (IV) and Se (VI) in the sediments were extracted using the microwave digestion at the optimized time and power setting in mild concentration acid media. Combination of organic separation and NAA technique have been applied to determine the As and Se species of interest. A satisfactory recovery of extractable inorganic As species was achieved using microwave digestion at an operating time of 20 min and 25% power treated in 1 M H3PO4. The As (III) and As (V) were successfully separated by APDTC into MIBK media with high extraction efficiency achieved at pH 6. Meanwhile, a sufficient amount of extractable Se species was also obtained under microwave digestion procedure in 1 M HCl media at an operating time of 20 min and 25% power. Both inorganic Se species, the Se (IV) and Se (VI) were separated after coprecipitation of Na-DBDTC with phenolphthalein at an optimum media condition of pH 2. 139 Investigation of the As and Se inorganic species in river and lake sediments showed that As (V) and Se (VI) were the dominant species. There was statistically insignificant difference in the ratio of As (III) to As (V) and Se (IV) to Se (VI) between lake and river sediments. Low ratios of As and Se species in surface sediments suggested that oxidizing environment prevailed in both freshwater body systems thus promoting the formation of the oxidized species. 5.1 Suggestions An excessive amount of As, Cr and Se found in freshwater sediments in the populated areas should be made a strong factor for the recommendation of the development of a suitable mechanism to monitor, evaluate and manage any contaminants input into the aquatic ecosystem. An identification of geological background, point and non-point sources of contamination, and assessment of the physical as well as chemical interactions of contaminants in sediment body provide a better understanding of distribution, mobilization, transportation and bio-availability of the contaminants particularly in aquatic environment. These can be conducted in future research work. Currently there is no sediment quality guideline established locally. An establishment of a national sediment quality guidelines would be useful in gaining better estimation of contaminants impact and assessment in the level of pollution by taking into consideration factors such as local climate, geological and environmental conditions. The formulation of an integrated framework for planning, designing, implementing and interpreting the results of sediment quality investigations should be governed by several bodies such as DOE, water management districts, universities, and county governments through cooperative instigation. 140 Speciation studies have become an important tool in characterizing and determining the forms of elements in a sediment system. Speciation analysis requires suitable reference materials particularly for sediment samples to be available to verify accuracy and to meet quality assurance needs. There is a clear need to produce sediment CRMs by local or international body for elemental species to validate methods in general and to evaluate their accuracy, in particular. Various approaches can be developed for speciation studies and to intensify future research work in the related field. 141 REFERENCES Abdel-Moati, A. R. (1990). Speciation and behaviour of arsenic in the Nile Delta Lakes. Wat. 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Occupational and Environmental Standard Setting for Metals: More Questions than Answers. In Merian, E., Frei, R. W., Hardi, W., and Schlatter, C. (Eds.). Carcinogenic and Mutagenic Metal Compounds. (pp. 477-490). New York: Gordon and Breach Science Publisher. 157 APPENDIX A The range of the water physico-chemical parameters. RIVERS Water Parameters Sites pH Temperature (C) Dissolve Oxygen (mg L-1) 22nd Aug. 20th Nov. 14th Dec. 22nd Aug. 20th Nov. 14th Dec. 22nd Aug. 20th Nov. 14th Dec. SGS 6.59 7.00 6.12 6.60 5.785.97 28.830.6 29.429.9 26.428.1 4.775.33 4.254.64 5.465.77 SGT 6.626.74 6.326.59 6.176.23 28.429.6 27.828.9 26.928.7 4.885.27 4.754.98 4.905.83 SGB 6.626.82 6.776.92 6.817.14 28.229.5 29.430.8 28.431.4 4.985.39 4.775.25 4.625.18 LAKES Water Parameters Sites pH Temperature (C) Dissolve Oxygen (mg L-1) 22nd Aug. 21st Nov. 13th Dec. 22nd Aug. 21st Nov. 13th Dec. 22nd Aug. 21st Nov. 13th Dec. TKR 6.53 6.71 5.86 6.14 5.435.74 31.532.0 29.229.6 26.026.2 6.296.31 6.496.85 7.467.81 TUP 5.596.28 6.016.34 5.605.74 32.032.1 31.933.4 28.128.3 6.306.36 5.715.96 7.027.47 TB 6.997.54 6.446.69 6.076.28 30.431.2 31.131.5 28.428.7 6.526.74 6.176.19 6.957.34 158 APPENDIX B NATIONAL WATER QUALITY STANDARDS FOR MALAYSIA (NWQS) Parameters (Units) Classes l llA llB lll lV V Ammonical Nitrogen mg/l 0.1 0.3 0.3 0.9 2.7 >2 BOD mg/l 1 3 3 6 12 > 12 COD mg/l 10 25 25 50 100 > 100 DO mg/l 7 5-7 5-7 3-5 <3 <1 pH - 6.5-8.5 6.5 - 9.5 6-9 5-9 5-9 TCU 15 150 150 mmhos/cm 1000 1000 - 6000 - Floatables - N N N - - - Odour - N N N - - - /oo 0.5 1 - - - - - N N N - - - Total Dissolved Solids mg/l 500 1000 - - - - Total Suspended Solids mg/l 25 50 50 150 300 > 300 C - Normal +2 - Normal +2 - - NTU 5 50 50 - - - Faecal Caliform* counts/100ml 10 100 400 Total Coliform counts/100ml 100 5000 50000 Colour Electrical Conductivity o Salinity Taste o Temperature Turbidity Note:- N No visible floatable materials/debris or No objectionable odour or No objectionable taste * Geometric Mean @ Maximum not to be exceeded 5000 5000 (2000)@ (2000) 50000 50000 >50000 159 Class l Uses Conservation of natural environment Water supply l practically no treatment necessary (except by disinfection of boiling only) Fishery l - very sensitive aquatic species llA Water supply ll - conventional treatment required Fishery ll sensitive aquatic species llB Recreational use with body contact lll Water supply lll - extensive treatment required Fishery lll common, of economic value and tolerant species lV Irrigation V None of the above 160 APPENDIX C Certified Reference Material 161 162 163 164 165 166 167 APPENDIX D Results of As, Cr, Se and Fe concentration in river and lake sediments. RIVERS Sites SGS1 SGS2 SGS3 SGT1 SGT2 SGB1 SGB2 SGB3 Sampling session 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 NA – Not Available Elements Concentration As (g g-1) Cr (g g-1) Se (g g-1) Fe (%) 19.9 0.7 15.1 0.9 23.0 2.8 35.4 0.3 30.1 1.0 24.2 0.6 35.2 1.0 29.2 0.5 36.4 0.8 10.8 0.1 17.4 0.7 8.6 0.2 13.4 1.5 12.0 0.8 15.8 2.9 8.8 0.6 NA 13.0 0.6 8.6 0.4 9.5 0.2 12.5 1.4 29.6 0.8 26.8 2.9 16.5 0.8 140 6 117 4 110 6 130 8 79 4 166 2 111 4 93 4 107 4 27 1 31 3 19 1 71 10 59 5 102 5 28 1 NA 27 2 93 6 32 2 28 2 71 5 56 2 52 2 1.20 0.05 1.47 0.15 1.40 0.05 0.90 0.03 1.15 0.04 1.35 0.08 1.10 0.14 0.71 0.03 1.00 0.09 1.70 0.14 2.46 0.24 1.20 0.05 1.30 0.05 2.37 0.20 2.23 0.07 0.80 0.12 NA 0.70 0.04 1.40 0.04 0.94 0.08 0.60 0.11 0.80 0.07 0.37 0.04 0.70 0.04 4.31 0.20 3.73 0.19 3.98 0.12 3.39 0.19 3.00 0.10 4.63 0.15 4.08 0.20 3.68 0.20 4.67 0.21 2.20 0.08 2.31 0.17 1.99 0.05 2.76 0.39 2.60 0.05 3.21 0.18 1.18 0.06 NA 1.56 0.06 3.15 0.16 1.78 0.82 1.48 0.08 2.90 0.41 3.14 0.18 2.82 0.06 168 LAKES Sites TKRA TKRB TKRC TUPA TUPB TBA TBB Sampling session 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Elements Concentration As (g g-1) Cr (g g-1) Se (g g-1) Fe (%) 29.2 1.3 83.5 1.3 45.6 2.1 67.0 6.4 61.2 4.3 56.1 0.5 69.2 5.5 58.0 1.0 52.5 2.2 16.2 1.1 14.6 0.2 85.8 3.7 31.8 1.2 43.4 6.1 57.0 0.8 46.4 2.3 47.2 1.4 43.4 2.5 18.6 0.3 22.3 1.5 12.0 0.8 124 7 211 5 183 9 173 9 206 9 215 10 162 5 245 7 177 16 338 5 315 12 249 24 284 14 134 6 314 9 233 9 255 9 239 5 147 2 254 17 162 7 0.79 0.01 0.37 0.05 0.67 0.06 0.41 0.06 0.35 0.03 0.34 0.02 0.47 0.07 0.42 0.03 0.51 0.08 1.02 0.05 0.85 0.07 0.90 0.09 1.01 0.05 1.08 0.08 0.88 0.19 0.21 0.02 0.39 0.06 0.45 0.05 1.06 0.04 1.09 0.09 1.24 0.02 3.15 0.11 4.20 0.18 3.77 0.18 4.57 0.20 5.17 0.18 5.39 0.23 3.98 0.10 4.83 0.17 4.37 0.22 1.89 0.09 1.74 0.09 3.74 0.17 2.66 0.15 3.54 0.13 4.34 0.20 4.08 0.20 4.48 0.18 4.36 0.16 3.03 0.17 2.35 0.15 2.41 0.11 169 APPENDIX E Details on the international guideline for freshwater sediment. 170 171 172 173 Buchman, M. F., 1999. NOAA Screening Quick Reference Table, NOAA HAZMAT Report 99-1, Seattle WA, Coastal Protection and Restoration Division, National Oceanic and Atmospheric Administration, 12 pages. 174 APPENDIX F Statistical evaluation of As, Cr and Se correlation to Fe in sediments. RIVERS Correlation and Regression Analysis As Cr Se r 0.8198 0.9354 0.0258 R2 0.6721 0.8750 0.0007 Adjusted R2 0.6175 0.8541 -0.1659 Standard Error 5.7271 13.8407 0.5152 P-value 0.0127 0.0006 0.9517 8 8 8 Observations LAKES Correlation and Regression Analysis As Cr Se r 0.7537 - 0.2408 -0.8049 R2 0.6973 0.1681 0.8216 Adjusted R2 0.6368 0.0017 0.7859 5280.5590 44.6910 0.1438 0.0194 0.3610 0.0049 7 7 7 Standard Error P-value Observations 175 APPENDIX G a) The stability study of As species Test sample As (III) (g g-1) As (V) (g g-1) 1 0.247 0.251 2 0.255 0.239 3 0.235 0.242 4 0.246 0.253 5 0.234 0.254 6 Average Recovery (%) RSD (%) 0.237 0.243 0.010 97 % 3.4 % 0.245 0.247 0.010 99 % 2.5 % b) The extraction efficiency of As (III) and As (V) to pH function As species pH Amount found (g g-1) Recovery (%) As (III) 1 1.65 0.1 83 2 1.68 0.08 84 3 1.75 0.06 88 4 1.85 0.01 93 5 1.97 0.02 99 6 2.00 0.02 100 7 2.00 0.20 100 8 9 1.97 0.05 1.83 0.09 99 92 10 1.40 0.30 70 176 As species pH Amount found (g g-1) Recovery (%) As (V) 1 0.083 0.003 4.13 2 0.084 0.010 4.18 3 0.011 0.010 0.56 4 0.006 0.030 0.28 5 < 0.005 0 6 < 0.005 0 7 < 0.005 0 8 9 < 0.005 < 0.005 0 0 10 < 0.005 0 c) The repeatability of the extraction by APDTC-MIBK Test sample As (III) (g g-1) As (V) (g g-1) 1 1.77 1.88 2 1.87 1.96 3 1.88 2.12 4 1.85 1.92 5 1.95 2.08 6 2.13 1.89 7 Average Recovery (%) RSD (%) 2.05 1.93 0.12 97 7 2.03 1.98 0.1 99 5 177 APPENDIX H a) The stability study of Se species Test sample Se (IV) (g g-1) Se (VI) (g g-1) 1 0.275 0.240 2 0.245 0.261 3 0.221 0.255 4 0.214 0.252 5 0.266 0.241 6 Average Recovery (%) RSD (%) 0.233 0.243 0.03 97 % 10 % 0.236 0.248 0.01 99 % 4% b) The extraction efficiency of 1 g mL-1 Se (IV) and Se (VI) to pH function Se species pH Amount found (g g-1) Recovery (%) Se (IV) 1 0.430 0.003 43 1.5 0.620 0.010 62 2 1.01 0.01 101 2.5 0.98 0.03 98 3 0.91 0.02 91 3.5 0.89 0.01 89 4 0.88 0.02 88 5 6 0.88 0.03 0.88 0.03 88 88 < 0.0002 0 Se (VI) 1-6 178 c) The repeatability of the co-precipitation technique by DBDTC-Pp Test sample Se (IV) (g g-1) Se (IV) (g g-1) 1 0.89 0.93 2 0.90 0.92 3 0.96 0.96 4 0.97 0.98 5 Average Recovery (%) RSD (%) 0.92 0.93 0.04 93 4 0.95 0.95 0.02 95 3 179 APPENDIX I a) Results of As and Se inorganic species in river and lake sediments. RIVER Elements Concentration Sites Sampling session As (III) -1 SGS1 SGS2 SGS3 SGT1 SGT2 SGB1 SGB2 SGB3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 As (V) -1 Se (IV) -1 Se (VI) (g g ) (g g ) (g g ) (g g-1) 1.220.24 0.750.15 1.330.11 0.600.05 1.080.26 0.580.06 2.110.57 1.450.43 1.820.19 0.670.05 1.280.28 0.700.03 1.460.02 2.250.73 2.380.37 0.670.04 NA 1.000.13 0.490.05 0.540.07 0.590.04 1.190.15 0.690.13 1.20.2 13.30.8 11.60.3 14.40.3 29.52.0 27.80.6 22.42.7 33.61.2 24.41.7 33.42.0 7.490.97 13.43.3 9.370.35 12.11.2 11.00.6 11.51.1 6.620.76 NA 8.130.83 6.990.65 6.240.26 8.440.74 20.90.4 26.02.0 14.10.8 0.190.10 0.140.02 0.170.01 0.110.04 0.180.02 0.160.03 0.190.01 0.090.02 0.150.01 0.200.06 0.250.01 0.210.02 0.150.01 0.150.01 0.180.05 0.130.01 NA 0.100.01 0.140.02 0.120.01 0.120.02 0.130.02 0.080.03 0.170.09 0.880.10 1.240.13 1.200.11 0.700.08 0.810.10 0.890.04 0.690.19 0.540.06 0.590.06 1.130.19 1.950.37 0.900.13 0.970.06 1.810.01 1.820.18 0.580.01 NA 0.560.01 1.090.11 0.690.09 0.390.09 0.560.01 0.250.03 0.460.01 180 LAKE Elements Concentration Sites TKRA TKRB TKRC TUPA TUPB TBA TBB Sampling session 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 As (III) As (V) Se (IV) Se (VI) (g g-1) (g g-1) (g g-1) (g g-1) 1.540.12 3.260.10 2.550.10 2.960.73 4.840.90 2.830.30 1.430.15 2.590.10 2.680.34 2.530.18 2.340.15 15.171.00 1.820.27 3.650.57 3.660.12 3.520.78 2.520.29 2.460.10 0.850.19 1.850.25 0.120.02 25.21.0 56.31.4 38.61.2 50.02.1 52.72.6 33.32.5 46.55.6 42.23.6 33.13.7 11.71.1 9.980.51 61.13.5 12.70.7 11.50.8 16.90.7 23.11.6 26.51.4 35.12.5 12.10.9 12.71.1 8.520.70 0.036 0.006 0.0120.001 0.0680.002 0.0730.003 0.0620.014 0.0890.012 0.100.02 0.110.01 0.100.01 0.170.01 0.160.02 0.120.01 0.140.04 0.130.01 0.100.01 0.0450.002 0.0610.034 0.0540.007 0.130.01 0.120.02 0.180.02 0.510.01 0.230.01 0.450.01 0.310.02 0.210.03 0.240.02 0.250.01 0.310.01 0.300.02 0.640.01 0.610.03 0.730.02 0.850.02 0.860.01 0.730.20 0.120.01 0.260.01 0.260.03 0.660.26 0.810.07 0.940.03 181 b) ANOVA : Analysis of arsenic species in river and lake SUMMARY Groups River Lake Count 8 7 ANOVA Source of Variation Between Groups Within Groups SS 0.868738 8.913634 Total 9.782372 Sum Average Variance 0.647635 0.080954 0.001849 3.943392 0.563342 1.483448 df 1 13 MS 0.868738 0.685664 F P-value F crit 1.267002 0.280682 4.667193 14 c) ANOVA : Analysis of selenium species in river and lake SUMMARY Groups River Lake Count 8 7 ANOVA Source of Variation SS Between Groups 0.001099 Within Groups 0.078722 Total 0.079821 Sum 1.639476 1.554642 df 1 13 14 Average Variance 0.204935 0.004071 0.222092 0.008371 MS 0.001099 0.006056 F P-value F crit 0.181485 0.677069 4.667193