Cu mobility in soils is affected by dissolved organic matter

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Evaluation of DAX-8 fractionation of soil DOM
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DAX-8 fractionation of dissolved organic matter (DOM) from soils:
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calibration with test components and application to contrasting soil
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solutions
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F. AMERY, C. VANMOORLEGHEM AND E. SMOLDERS
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Section Soil and Water Management, Department Earth and Environmental Sciences,
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K.U.Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium
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Correspondence: F. Amery. E-mail: Fien.Amery@ees.kuleuven.be
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Summary
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Most methods to fractionate natural dissolved organic matter (DOM) rely on sorption of
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acidified DOM samples onto XAD-8 or DAX-8 resin. Procedural differences among methods
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are large and their interpretation is limited due to lack of calibration with DOM model
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molecules. An automated column based DOM fractionation method was set up for 10 mL
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DOM samples dividing DOM into hydrophilic (HI), non-neutral hydrophobic (nn-HO) and
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neutral hydrophobic (n-HO) fractions. Fifteen DOM model components were tested in
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isolation and in combination. Aliphatic low molecular weight acids (LMWA’s) and
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carbohydrates were classified as HI DOM, but maleic, succinic and propionic acid showed
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also a partial HO character. Aromatic LMWA’s and polyphenols partitioned in the nn-HO
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fraction, menadion (quinon) and geraniol (terpenoid) in n-HO DOM. Molecules with log Kow
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> 0.5 had negligible HI fractions. The HO molecules except geraniol had specific UV
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absorbance (SUVA, measure for aromaticity) > 3 l g-1 cm-1 while HI molecules had SUVA
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values < 3 l g-1 cm-1. The DOM of 8 different soil solutions was fractionated with this method.
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Distributions ranged from 31-72% HI, 25-46% nn-HO and 2-28% n-HO of total dissolved
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organic carbon with notable effects of DOM sampling method. The SUVA of the HI DOM
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was lower compared to the HO DOM, but its variation coefficient was only slightly lower for
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the nn-HO fraction and even larger for the HI fraction compared to the unfractionated DOM.
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The calibration data allow interpreting results of the fractionation method. Our data show that
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isolated soil DOM is not structurally more homogeneous than the unfractionated sample,
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contrasting the general paradigm in fractionation approaches.
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Introduction
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Dissolved organic matter (DOM) has an important role in aquatic and soil systems for
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contaminant transport, for biochemical and geochemical processes, and serves as a substrate
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for microorganisms (Leenheer & Croué, 2003; Marschner & Kalbitz, 2003). The influence of
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DOM on these processes does not only depend on the dissolved organic carbon (DOC)
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quantity, but also on the DOM quality, i.e. the structural features (Raber et al., 1998; Kalbitz
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et al., 2003). The composition of DOM in the soil solution varies with soil type, season, water
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regime, soil depth and vegetation or crop.
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Due to its complex and heterogeneous composition, soil DOM is commonly analyzed by
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measuring bulk properties (e.g. spectroscopy) or by fractionating DOM based on chemical or
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physical properties. The underlying idea of fractionation is that DOM fractions are
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structurally more homogeneous than the bulk samples (Leenheer, 1981), however this is
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rarely verified. A frequently used fractionation procedure is based on sorption of acidified
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DOM onto the weakly hydrophobic resin XAD-8 dividing DOM into hydrophobic (HO) and
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hydrophilic (HI) DOM fractions (Kaiser, 1998; Van Zomeren & Comans, 2007). The division
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of DOM into the 2 fractions shows environmental relevancy. Metals transported in soil are
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particularly associated with hydrophilic DOM (Guggenberger et al., 1994). This fraction has
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also shown larger bioavailability (Marschner & Kalbitz, 2003), whereas polycyclic aromatic
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hydrocarbons have larger affinity to hydrophobic DOM (Raber et al., 1998). Reported
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distributions of soil DOM vary largely: 25 to 90% HI and 10 to 75% HO of total DOC (Table
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1). The fractionation does not only change with soil depth, DOC concentration, sampling
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method, land use and solution chemistry, but also depends on the fractionation method.
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Most of the fractionation procedures are based on the (first step of the) fractionation
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scheme of Leenheer (1981), that classifies dissolved organic molecules into hydrophobic and
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hydrophilic fractions based on their pH dependent sorption onto the XAD-8 resin. As the
3
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production of XAD-8 has been ceased, it is now replaced by SupeliteTM DAX-8, also a
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polymethylmethacrylate resin. The sorption-desorption properties of the latter are slightly
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more precise compared to the former (Peuravuori et al., 2002). Many modifications to this
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method have been proposed, but generally the procedure is as follows. After acidifying the
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DOM sample to pH 2 to neutralize all weak acid groups, the solution is pumped over a
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column with DAX/XAD-8 resin. The column is rinsed with 0.01 M HCl. The DOM not
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sorbed onto the resin is classified as hydrophilic. The hydrophobic DOM except for the
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neutral molecules can be removed from the resin by rinsing with 0.1 M NaOH. The
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interpretation of the fractionation in terms of the octanol water partition coefficient (Kow),
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molecular weigth (MW) or acidity is, however, unknown, and the classification of the solutes
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into hydrophilic and hydrophobic fractions depend on the procedure used (Peuravuori et al.,
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1997). Authors modify the procedure according to the volume, concentration and application
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of the sample (Marhaba et al., 2003). In aquatic studies, the resin is frequently used to
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concentrate and isolate DOM, thereby neglecting the hydrophilic part (Thurman & Malcolm,
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1981). In contrast to aquatic samples, fractionation of soil solution DOM has often sample
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volume limitations, requiring adapted fractionation techniques.
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Despite the limited interpretation of fractionation, few studies have examined the
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classification of specific organic molecules into the hydrophilic and hydrophobic fraction.
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The hydrophobic fraction of natural DOM is assumed to contain lignin-derived, partly
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aromatic molecules, nucleic acids, quinones, hydrocarbons and fatty acids. Hydrophilic DOM
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consists of amino acids and sugars, small organic acids and carbohydrates and contains less
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aromatic components than HO fractions (Cilenti et al., 2005; Simonsson et al., 2005). The list
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of compounds to be found in each fraction is often based on spectroscopic measurements of
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the fractions (Leenheer, 1981; Guggenberger & Zech, 1994; Kaiser et al., 2001a; Dilling &
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Kaiser, 2002; Croué et al., 2003; Cilenti et al., 2005), instead of subjecting model DOM
4
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components to the fractionation procedure. Among the exceptions are Qualls & Haines (1991)
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who tested 5 compounds in their modified fractionation procedure. Over 90% of rutin and
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quercitin (flavonoids) was found in the hydrophobic fraction, whereas over 85% of oxalic
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acid, albumin and glucose phosphate was classified as hydrophilic DOM. David et al. (1989)
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recovered over 89% of oxalic, succinic and citric acid as hydrophilic acids; over 95% of
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benzoic and salicylic acid was found in the HO fraction. Thurman et al. (1978) determined
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the capacity factors of 20 nonionic organic solutes on XAD-8 and concluded that the resin
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favors aliphatic over aromatic over alicyclic components. The logarithm of the capacity factor
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correlated inversely with the logarithm of the aqueous molar solubility, but it remains unclear
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which components can be expected to appear in the hydrophilic fraction.
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Numerous modifications to the fractionation procedures have been described varying in
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resin/volume ratio, flow rate in the columns (often not reported) and in further fractionation of
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HI or HO fractions. These modifications are known to affect the fractionation and it is striking
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that no calibration steps are systematically adopted in modified procedures. We developed a
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fractionation procedure with DAX-8 for only 10 ml of DOM solution. Fifteen soil DOM
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model components were subjected to this fractionation scheme. Soil solutions from different
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sources were subsequently fractionated. Specific UV absorbance (SUVA, 254 nm, Weishaar
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et al., 2003) of the DOM and their fractions were measured to determine relationships with
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the hydrophobicity and to study characteristics of the fractions. It was investigated if the
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structural variability (linked to SUVA) of the bulk samples is lower in the corresponding
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fractions.
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Materials and methods
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Fractionation procedure
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The procedure to fractionate DOM solutions is based on preliminary experiments with water
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extracts of soil. The first step of the fractionation scheme of Leenheer (1981) was adapted to
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clearly separate hydrophilic and hydrophobic DOM of small volumes of soil solutions.
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SupeliteTM DAX-8 resin was cleaned according to the method of Thurman & Malcolm
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(1981). The resin was extracted with 0.1 M NaOH, replacing the solution and decanting off
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fines daily. After 5 days, the resin was soxhlet-extracted for 24 hours with methanol,
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acetonitrile, diethyl ether and again methanol. The resin was stored in methanol/water until it
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was packed in an 8.0 cm x 1.6 cm column, giving a bed volume (BV) of 16 ml. The column
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was connected to a compact liquid chromatographic system (Äkta Prime Plus, GE Healthcare)
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with fraction collector and online pH, conductivity and UV (254 nm, path length 0.2 cm)
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detectors. The void volume (VV) between the injection valve and the detectors was 8 ml. The
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resin was rinsed with ultrapure water until the DOC concentration of the column effluent was
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<0.5 mg l-1. Before and after each fractionation procedure, the resin was rinsed with
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successive cycles of 3 VV’s of 0.01 M HCl and 3 VV’s of 0.1 M NaOH (both 2 VV at 30 VV
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h-1 and one VV at 7.5 VV h-1). This rinsing was repeated until the DOC concentration was
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less than 0.5 mg l-1 and the UV absorbance of the effluent stabilized.
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The DOM solution was acidified to pH 2 with 2 M HCl and filtrated (0.45 µm). The
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column was rinsed with 0.01 M HCl until pH was stable. The UV absorbance signal was set
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to zero. Ten ml of the filtered DOM solution was injected onto the column using a superloop
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(22.5 VV h-1). After the sample, the column was rinsed with 4.5 VV of 0.01 M HCl (22.5 VV
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h-1). All DOM collected before the end of this rinsing was classified as hydrophilic. The flow
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direction was subsequently reversed, and the non-neutral hydrophobic (nn-HO) DOM was
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eluted with 2.25 VV of 0.1 M NaOH, followed by 4 VV of ultrapure water (7.5 VV h-1). After
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1 VV, the eluate pH increased from 2 to >10 within 0.1 VV. All eluting solution was collected
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in 30 subfractions with a volume varying from 2 to 5 ml. All DOM subfractions were
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acidified to pH 2 before DOC analysis and inorganic C was removed by sparging samples
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with O2 for 30 seconds. Concentrations of DOC in the subfractions were measured by
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catalytic combustion (800°C) and infrared measurement of formed CO2 (Multi N/C 2100S,
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AnalytikJena). The DOC present in the neutral hydrophobic fraction (n-HO, not eluted by 0.1
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M NaOH) was calculated by difference between DOC in the original sample and the DOC
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quantities in fractions recovered.
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Test components
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Fifteen components were subjected to the fractionation procedure (Table 2). The components
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were selected because of their presence in soil solutions or because they are a model for
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compound classes present in soil solutions. Molecules were also selected to obtain a range in
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Kow, acidity constant (pKa) and MW. The components were dissolved in or diluted with
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ultrapure water to obtain DOC concentrations of approximately 20 mg l-1. Beside fractionation
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of the isolated components, 3 of them were also fractionated in combination: 11.0 mg DOC l-1
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oxalic acid, 5.0 mg DOC l-1 rutin and 3.3 mg DOC l-1 geraniol, giving a combined
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concentration of 19.3 DOC mg l-1. Also a solution of Suwannee River Fulvic Acid (SRFA), a
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fulvic acid isolated by the International Humic Substances Society (IHSS), was fractionated.
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After acidifying the DOM samples to pH 2 with 2 M HCl and filtration (0.45 µm), DOC
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concentrations (AnalytikJena) and UV absorbance (254 nm, path length 1 cm, Perkin-Elmer
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Lambda 20) were measured. Fractionations were performed in duplicate.
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Fractionation of soil solutions
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The topsoil of 4 uncontaminated soils (1-4) was sampled, air-dried (25°C), sieved (< 2 mm)
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and stored dry for various periods (4-12 years). The air-dried soils were wetted with deionized
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water to field capacity (pF 2), covered in plastic reservoirs and incubated moist during various
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days at 21°C (Table 3). Topsoil 5 was not dried after sieving (4 mm) but immediately
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incubated moist during 4 days. After incubation soil solutions were obtained by centrifugation
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(pore water) or by extraction. Pore water was sampled by the ‘double chamber method’. A 30
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mL syringe without plunger was filled with quartz wool and approximately 60 g of moist soil.
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The syringe was placed in a 50 ml centrifuge tube and centrifuged during 30 minutes (2500
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g). The obtained pore water was filtered (0.45 µm). Extractions were performed in a 50 ml
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centrifuge tube filled with 5 g moist soil and 20 ml 1 mM CaCl2. After shaking end-over-end
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for 24 hours, the suspensions were centrifuged during 30 minutes (2500 g). The supernatant
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was filtered (0.45 µm). Soil solutions were sometimes diluted before fractionation to obtain
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DOC concentrations below 150 mg l-1. Fractionations were performed in duplicate, except for
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the pore water of soil 5.
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Specific UV absorbance
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The specific UV absorbance (SUVA, l g-1 cm-1) of DOM was calculated by
SUVA 
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A 254
b  DOC
(1)
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where A254 is the UV absorbance at 254 nm measured by a spectrophotometer (Perkin-Elmer
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Lambda 20 or online with GE Healthcare Äkta Prime Plus) (dimensionless), b the path length
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of the quartz cell of the spectrophotometer in cm and [DOC] is in g l-1. The SUVA of DOM
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with DOC concentration < 1.5 mg l-1 was not calculated because of accuracy problems. The
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specific UV-absorbance is used as an estimate of the aromaticity of DOM (Weishaar et al.,
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2003).
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The SUVA of a collected subfraction was calculated by equation (1) using the average
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UV absorbance of the eluting subfraction. The SUVA of the HI or nn-HO fraction (SUVAfr, l
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g-1 cm-1) was calculated by
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 A
n
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SUVA fr 
i 1
n
254
i
 Vi

b   DOCi  Vi 
(2)
i 1
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where n is the number of subfractions in the fraction fr and A i254 , Vi and [DOC]i the average
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UV absorbance (-), volume (l) and DOC concentration (g l-1) of subfraction i, respectively.
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Results and discussion
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Fractionation of the test components
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The fractionation of the 15 test components varied largely (Table 2). Carbohydrates, proteins
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and aliphatic LMWA’s showed a (partial) HI character, whereas aromatic LMWA’s and
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polyphenols ended up in the HO fraction. Quinons and terpenoids did not desorb from the
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resin during base rinsing and were therefore classified as n-HO molecules. The peak of the
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DOC concentration of the HI fraction of oxalic acid and other pronounced HI components
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was situated only after 2 VV with DOC concentrations dropping to zero only after 5 VV
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(Figure 1). This was also the case for the HI fraction of soil DOM (see below). The DAX-8
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resin showed affinity for the HI molecules as well, resulting in retardation of the components.
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This emphasizes the need for sufficiently rinsing of the resin and the importance of reporting
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cumulative resin/solution ratio in the procedure for the distribution of DOM in HI and HO
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fractions. When no or few rinsing is applied, part of the retarded HI molecules will be
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classified as HO. The choice for rinsing with 4.5 VV in this method was based on preliminary
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fractionation experiments with soil solutions. Concentrations of DOC dropped only below 0.5
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mg l-1 after rinsing for more than 4 VV with 0.01 M HCl. However 3 of the aliphatic
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LMWA’s (maleic, succinic and propionic acid) showed an even larger retardation on the
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column at pH 2 (Figure 1), resulting in a partial HI and partial HO classification based on this
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fractionation procedure. This can be related to the Kow and pKa of the components (see
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below). Aromatic LMWA’s, e.g. salicylic acid, were almost completely retained by the resin
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at pH 2 but were eluted with NaOH (Figure 1). This was also the case for polyphenols (e.g.
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rutin). Geraniol and menadion were classified as n-HO, but also some HI and especially some
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nn-HO components showed a partial n-HO character (Table 2). This was most pronounced for
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components with high molecular weight (e.g. ovalbumin and tannic acid). Peuravuori et al.
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(1997) suggested already that the sorption mechanism of the DAX/XAD-8 resin is partly
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based on molecular size of the solute. Qualls & Haines (1991) also found rutin in the
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hydrophobic fraction and oxalic acid and albumin in the hydrophilic fraction. David et al.
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(1989) also classified oxalic acid as hydrophilic and salicylic acid as hydrophobic, but they
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found over 89% of succinic acid in the hydrophilic fraction. They used the fractionation
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method of Leenheer (1981) who only rinsed the XAD-8 resin with 1 BV compared to 4.5 VV
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(=2.25 BV) in our method. This should lead to a smaller HI DOC amount, however they
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found higher DOC recovery in their HI fraction compared to this study (59%).
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Fractionation of the combined components (oxalic acid, rutin and geraniol) gave similar
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results as their isolated fractionations. The HI and nn-HO fractions were slightly lower (49.5
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± 1.3% and 16.4% ± 0.1% of total DOC respectively, average values ± standard deviation)
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compared to the calculated values based on the isolated fractionation results (53.8 ± 1.1% and
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21.1 ± 1.6%). These lower values can be an indication of minor interactions of the
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components. The neutral hydrophobic geraniol can interact with oxalic acid and rutin,
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increasing slightly their n-HO fraction. Almost all DOM of the SRFA appeared in the HO
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fraction: 76.2 ± 0.4% in the nn-HO and 23.5 ± 0.0% in the n-HO fraction. The negligible part
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of DOM in the HI fraction (0.3 ± 0.4%) can be explained by the SRFA isolation method of
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the IHSS. All SRFA DOM originates from desorption from the XAD-8 resin with 0.1 M
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NaOH after percolating Suwannee River water at pH 2 over the resin. Surprisingly, 23.5% of
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the sorbed SRFA on the DAX-8 resin could not be desorbed in our method. A possible
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explanation is the use of different resins: XAD-8 by the IHSS method and DAX-8 in our
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method. The method to desorb DOM from the resin also varies: 3 VV with 0.1 M NaOH at
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350 ml min-1 in the IHSS method (Malcolm et al., 1995 and E.M. Perdue, personal
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communication) compared to 2.25 VV with 0.1 M NaOH and 4 VV with MQ water at 1 ml
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min-1 in our method. Structural conformations of the isolated fulvic acid during further IHSS
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sample preparation (passing through cation exchange resin, hydrogen saturation and freeze
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drying) are also possible.
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The affinity of the DAX-8 and XAD-8 resin for components is assumed to be related to
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the hydrophobicity of the components. A negative linear relationship was found between the
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logarithm of aqueous solubility and the logarithm of the capacity factor of molecules on
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XAD-8 (Thurman et al., 1978). It was investigated if the Kow of the test components, a
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measure for the hydrophobicity, showed a relationship with the classification of the molecule
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into the HI, nn-HO and the n-HO fraction (Table 2 and Figure 2). Components with log Kow <
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-1.5 appeared almost completely in the HI fraction. An exception is rutin which has a low log
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Kow value (-2.02) because of the carbohydrate part of the molecule. The sorption by the DAX-
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8 resin at pH 2 is probably due to the aromatic part (polyphenol). This was also the case for
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tannic acid which has a quite low log Kow (-0.19) as well because of its carbohydrate part.
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Maleic acid, succinic acid and propionic acid have intermediate Kow values (-1 < log Kow <
240
0.5) and have some affinity for the DAX resin, resulting in a partly HI and partly HO
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character. Of these 3 molecules maleic acid is less retained, probably because it is charged at
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pH 2 (pKa1 = 1.83), as is oxalic acid. Succinic acid and propionic acid are not ionized at pH 2
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(pKa1 = 4.16 and pKa = 4.87 respectively). The Kow of a charged molecule is more than 100
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times smaller compared to that of the non-dissociated molecule (Schwarzenbach et al., 1993).
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Propionic acid has the largest log Kow (0.33) and showed most retardation by the resin.
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Components with log Kow > 0.5 were all classified as HO. Although 2,4,6-trihydroxybenzoic
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acid is ionized at pH 2 (pKa1 = 1.68) it was also found in the HO fraction. The 2 molecules
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classified as n-HO, geraniol and menadion, showed a very large log Kow (3.47 and 2.20
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respectively). Salicylic acid is classified as nn-HO despite its larger log Kow (2.26) compared
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to menadion. This is probably due to the presence of the carboxyl group with pKa = 2.98 in
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the salicylic acid molecule. All other molecules classified as nn-HO have also pKa < 11,
12
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resulting in a charged molecule during rinsing with 0.1 M NaOH causing desorption from the
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DAX-8 resin.
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The SUVA of the eluted DOM showed similar values as the SUVA of the DOM
255
measured in the unfractionated DOM solution. Exceptions were tannic acid and especially
256
2,4,6-trihydroxybenzoic acid which showed 25 times larger SUVA values in the 0.1 M NaOH
257
eluate. This was probably because of the deprotonation of the carboxylic groups in the
258
alkaline conditions resulting in resonance and therefore larger UV absorption. The SUVA of a
259
molecule can also be an indicator of the hydrophobic character. Test components with SUVA
260
< 3 l g-1 cm-1 were classified as (partly) hydrophilic while hydrophobic components had
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SUVA > 3 l g-1 cm-1. Exceptions were maleic acid and geraniol. Maleic acid has a SUVA of
262
12.0 l g-1 cm-1 because of the resonance possibilities of the double bond, but the short chain
263
and the presence of carboxylic groups give the molecule a partial hydrophilic character.
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Geraniol has a very low SUVA (0.6 l g-1 cm-1) because of the lack of resonance, but the long
265
aliphatic chain renders the molecule hydrophobic. The relationship of SUVA and the
266
retention by the resin can be explained by the presence of aromatic structures in the
267
molecules. Aromaticity increases the hydrophobic character, and SUVA is a good measure for
268
aromaticity (Weishaar et al., 2003).
269
From these relationships it is clear that the affinity of the DAX-8 resin for molecules is
270
primarily based on hydrophobic structures in the components. Aromatic and long aliphatic
271
parts sorb onto the resin at pH 2, even if the molecules have hydrophilic parts (e.g. rutin).
272
Hydrophobic components can be desorbed from the resin if they are ionized during base
273
rinsing. Potentiometric titration of the resin between pH 2 and 13 revealed that no charges are
274
formed on the resin, even after extensive use (details not shown).
275
Fractionation of the soil solutions
13
276
The DOC concentration and SUVA of DOM in the eluate of the DAX-8 column changed
277
during the fractionation of the pore water DOM of the 83 days incubated soil 1 (Figure 3).
278
The DOC concentration and SUVA patterns were similar for the other fractionations. As
279
mentioned before, DOC concentrations of the hydrophilic fraction peaked after 2 VV’s and
280
dropped to zero only after 5 VV’s. This demonstrated the retardation of hydrophilic
281
components by the resin. The SUVA of this DOM increased gradually during elution. This
282
confirms that the hydrophilic fraction has a heterogeneous character and that retardation of
283
hydrophilic DOM increases with aromaticity of the hydrophilic components. The
284
hydrophobic DOC peak also tailed for several VV’s. The hydrophobic DOM is also
285
heterogeneous, demonstrated by the changing SUVA during elution. The first eluted
286
hydrophobic DOM had the highest SUVA values. This was probably because DOM with the
287
highest hydrophobicity and aromaticity was sorbed onto the first DAX-8 particles of the
288
column. This DOM comes first out of the column during base rinsing because the flow
289
direction was inversed after the acid rinsing.
290
Characteristics and fractionation results of all soil solutions are presented in Table 3.
291
Distribution of HI and HO DOC are within the ranges found in other studies (Table 1): 31-
292
72% HI, 25-46% nn-HO and 2-28% n-HO of total DOC. The DOC concentration was lowest
293
in the pore water of soil 5 which was not air-dried but sampled freshly from the field. This
294
DOM had also the smallest fraction of HI DOC. Soils that were stored air-dry for several
295
years and rewetted before incubation showed extremely high DOC concentrations in the pore
296
water (> 1000 mg l-1). This was less expressed in soil 1, the soil that was kept air-dry for the
297
shortest time (4 years). The high amount of DOM probably originated from cell lysis of soil
298
biomass due to soil drying (Merckx et al., 2001). Microbial biomass is assumed to consist of
299
simple molecules like sugars, amino acids and nucleotides (Merckx & Martin, 1987). This
300
was reflected in the relatively low SUVA values (< 11 l g-1 cm-1) and high HI fraction (>
14
301
49%) of the DOM in pore water of shortly incubated soils (3-13 days). The fractionation of
302
the test components showed already the hydrophilic character of carbohydrates and proteins.
303
Dissolved organic matter released by drying-wetting cycles is easily degradable (Merckx et
304
al., 2001; Amery et al., 2007). This was also observed in this study as prolonged incubation
305
of soil 1 resulted in lower DOC concentrations and higher SUVA of DOM (Table 3). The
306
fraction of HO DOC increased during incubation as a result of smaller degradation of the HO
307
fraction (-21%) compared to the HI fraction (-54%). Incubation experiments with extracts of
308
soil and plant material showed that resistance of DOM to microbial degradation is mostly
309
found in the HO DOC fraction (Kalbitz et al., 2003).
310
Extracts had lower DOC concentrations compared to corresponding pore water, but
311
extractions sampled more DOC when expressed in mass DOC extracted per kg soil. As this
312
DOM had higher SUVA compared to pore water, it is assumed that the extra DOM sampled
313
by extraction originates from the solid organic matter which is more humified and has higher
314
aromaticity (Amery et al., 2007). The extracts of soil 2 and 3 contained relatively more HO
315
DOC compared to the pore waters, demonstrating the hydrophobic character of the extra
316
DOM that is released by soil extraction. Again, this shows that soil extraction should be
317
discouraged as it dissolves DOM not present in solution as stated before (Amery et al., 2007).
318
The average SUVA of the HI DOM was lower compared to the SUVA of nn-HO DOM
319
(Table 3). This was expected on the basis of the fractionation of the test components as
320
aromatic compounds ended up in the HO fraction. Studies dealing with forest floor and
321
aquatic DOM showed also higher specific UV absorbance for the HO compared to the HI
322
fraction (Martin-Mousset et al., 1997; Imai et al., 2001; Dilling and Kaiser, 2002; Croué et
323
al., 2003; Simonsson et al., 2005). The SUVA of the HI DOM varied largely from 2 to 17 l g-
324
1
325
(Table 3). Not only the distribution of DOM into the HI, nn-HO and n-HO fractions differed
cm-1 and even overlapped the range of SUVA of the nn-HO DOM, from 14 to 35 l g-1 cm-1
15
326
among the soil solutions, but also the characteristics of one fraction depended on the DOM
327
source. This shows that soil solution DOM is not a varying composition of different fractions
328
with constant properties since there is considerable variation in composition and quality
329
within fractions. The statement that the DAX/XAD-8 fractionation procedure isolates similar
330
groups of compounds (Leenheer, 1981) is not proven. The variation coefficient of the SUVA
331
values of the nn-HO DOM (31%) is only slightly smaller than that of the unfractionated DOM
332
(41%). The variation coefficient of the SUVA of HI DOM is even much larger (72%). The
333
standard deviation of the SUVA of the nn-HO DOM is larger (7.5 l g-1 cm-1) and of the HI
334
DOM is smaller (5.0 l g-1 cm-1) compared to the standard deviation of the SUVA of the bulk
335
DOM (6.0 l g-1 cm-1). This is in contrast with Dilling & Kaiser (2002) who found large
336
similarities in the chemical composition of HI and HO fractions of 3 forest floor extracts
337
despite the large range in distribution within the bulk samples (37-71% HI DOC). As a result,
338
the HO DOC concentration could be estimated well using the SUVA of the bulk sample. The
339
attempt of Simonsson et al. (2005) to estimate HO DOC fractions and concentrations of floor
340
leachates based on UV absorbance (210-300 nm) was, however, not very successful due to
341
non-consistent UV absorptivity of the HI and HO DOM. No significant correlation was found
342
between the SUVA of the bulk soil DOM and the fraction HO DOC in this study. These
343
findings question the practical role of DOM fractionation in predicting DOM properties. We
344
did not found smaller SUVA variation in the HI and nn-HO fraction compared to the
345
unfractionated DOM. Fractionation is probably useful to interpret properties of the DOM such
346
as metal and hydrophobic contaminant affinity, biodegradability etc. Predictions of these
347
characteristics based on fractionation can only be successful if these structural properties are
348
more homogenous within the fractions, however our SUVA data show that the variation
349
coefficient and standard deviation can be even larger within a fraction.
350
Conclusions
16
351
A DAX-8 fractionation procedure for small volumes of DOM solution was developed.
352
Subjecting 15 soil DOM model components to this fractionation method showed that Kow,
353
pKa and SUVA of the components can be used to predict the hydrophobic character of the
354
molecule. Components with aromatic or long aliphatic parts sorb onto the DAX-8 resin even
355
if the molecule has negative log Kow values due to hydrophilic parts. Hydrophilic organic
356
components also showed DAX-8 resin affinity, resulting in retardation on the column.
357
Researchers using a different fractionation method are encouraged to subject DOM model
358
components to their procedure as well. In this way, calibration, understanding of the
359
fractionation mechanism and comparison of methods is possible.
360
Fractionation of 8 different soil solutions resulted in a large variation of the distribution:
361
31-72% HI, 25-46% nn-HO and 2-28% n-HO of total DOC. Soil solution DOM of soils
362
stored air-dry for several years before moist incubation had large HI fractions. Soil solutions
363
sampled by extraction had larger HO fractions compared to isolating soil solution by
364
centrifugation of the same soil. Within one soil solution the SUVA of the hydrophilic DOM
365
was always lower than the SUVA of the hydrophobic DOM. However the SUVA values of
366
the fractions of the different soil solutions were not constant. The standard deviation of the
367
SUVA of the nn-HO DOM was larger than that of the unfractionated DOM and the relative
368
variation in SUVA of the hydrophilic DOM was also larger compared to the unfractionated
369
DOM. Fractionation reveals environmentally relevant information, however it is unlikely to
370
predict structural or functional properties of DOM in isolation.
371
Acknowledgements
372
This research was funded by the Onderzoeksfonds K.U.Leuven under the project number
373
GOA/2006/07-TBA, and was supported by the fund for Scientific Research-Flanders
374
(F.W.O.) through a doctoral fellowship awarded to F. Amery.
17
375
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23
491
FIGURE CAPTIONS
492
Figure 1 Average DOC concentration (mg l-1) in DAX-8 column eluates versus elution
493
volume (VV) of fractionation of oxalic acid (■), maleic acid (◊) and salicylic acid (∆).
494
Standard deviations are represented by error bars (2 replicates). Left from the dashed line
495
eluates the hydrophilic DOM, right the hydrophobic DOM.
496
497
Figure 2 Average percentage hydrophilic (HI) DOC of total DOC versus log Kow of the
498
test components (no Kow data for starch and albumin, value of log Kow = -0.48 used for
499
maleic acid). Standard deviations are represented by error bars (2 replicates).
500
501
Figure 3 Average DOC concentration (□) and SUVA (■) of the subfractions of the eluent
502
of the DAX-8 column versus elution volume (VV) of the fractionation of the ½ diluted
503
pore water of soil 1 incubated for 83 days. Standard deviations are represented by error
504
bars (2 replicates). Left from the dashed line eluates the hydrophilic DOM, right the
505
hydrophobic DOM.
24
FIGURE 1
25
Hydrophilic DOM
-1
DOC concentration /mg l
506
Hydrophobic DOM
20
15
oxalic acid
10
maleic acid
salicylic acid
5
0
0
2
4
6
8
Elution volume /VV
10
12
25
FIGURE 2
100
80
%HI
507
60
40
rutin
20
tannic acid
0
-4
-2
0
2
4
log Kow
26
FIGURE 3
25
Hydrophilic DOM
-1
70
Hydrophobic DOM
60
-1
20
15
40
10
30
20
5
-1
50
SUVA /l g cm
DOC-concentration /mg l
508
10
0
0
0
2
4
6
8
Elution volume /VV
10
12
27
509
TABLES
510
Table 1 Reported distributions of soil solution DOM into the HI and HO fraction (% of total
511
DOC) using XAD-8 resin.
Soil solution
Column leachate O+A horizon of
Spodosol
Column leachate O+A+B horizon
of Spodosol
Water extract Ap horizon of Haplic
Luvisol
Water extract O horizon of acidic
forest soil
Water extract non-saline soil
Water extracts 2 saline soils
Pore water A horizon of 7
agricultural soils
Water extract A horizon of
agricultural soil
Leachate A horizon of forest soil
Pore water 90 cm depth of Arenosol
with Scots pine
Pore water 90 cm depth of Leptosol
with European beech
Lysimeter leachate of brown earth
Lysimeter leachate of micropodzol
Lysimeter leachate of peaty gley
Water extract A horizon of 3
agricultural soils
Water extract 3 agricultural topsoils
in a fen area
Water extract forest topsoil in a fen
area
Extract surface oxidized peat soil
Extract subsurface reduced peat soil
[DOC]
/mg l-1
58.6
%HI
/%
41
%HO
/%
68
Reference
4.7
66
36
Guggenberger et al. (1994)
25.1
48
53
Raber & Kögel-Knabner (1997)
100.8
37
63
Kaiser (1998)
38-66
73
75-89
41-67
27
25-11
33-59
Cilenti et al. (2005)
Cilenti et al. (2005)
Raber et al. (1998)
22
69
33
Raber et al. (1998)
12
9.3-37.2
40
47-53
50
47-53
Qualls & Haines (1991)
Kaiser et al. (2001b)
6.4-35.6
63-90
10-37
Kaiser et al. (2001b)
variable
variable
variable
7.2-13.9
56
87
31
45-55
44
13
69
45-55
Tipping et al. (1999)
Tipping et al. (1999)
Tipping et al. (1999)
Kalbitz et al. (2003)
18.4-40.5
33-52
48-67
Kalbitz et al. (2003)
75.7
25
75
Kalbitz et al. (2003)
296
21
43
64
57
36
Chow et al. (2006)
Chow et al. (2006)
Guggenberger et al. (1994)
28
Table 2 Test components of the fractionation procedure with chemical characteristics (octanol water partition coefficient Kow, acid
constants pKa, molecular weight MW, SUVA at pH 2), average percentage of total DOC in the hydrophilic (HI), non-neutral
hydrophobic (nn-HO) and neutral hydrophobic (n-HO) fraction (2 replicates, standard deviation in parentheses) and classification of
the component according to the fractionation in isolation. Each component or class of the component has been identified in natural
DOM according to the corresponding reference.
reference
component
class
log Kow
pKa1a
pKa2a
Strobel (2001)
Strobel (2001)
Strobel (2001)
Strobel (2001)
Strobel (2001)
Strobel (2001)
Strobel (2001)
Oxalic acid
Maleic acid
Succinic acid
Propionic acid
Phthalic acid
Salicylic acid
2,4,6-trihydroxybenzoic acid
Glucose
Aliphatic LMWA
Aliphatic LMWA
Aliphatic LMWA
Aliphatic LMWA
Aromatic LMWA
Aromatic LMWA
Aromatic LMWA
-1.74b
-0.48c;0.46c
-0.59c
0.33c
0.73c
2.26c
1.10b
1.23
1.83
4.16
4.87
2.89
2.98
1.68
Carbohydrates
-3.24c
Starch
Carbohydrates
Ovalbumin
Geraniol
Rutin
Kaiser &
Guggenberger (2005)
Kaiser &
Guggenberger (2005)
Schulze (2005)
Leenheer et al. (2003)
Qualls & Haines
(1991)
Page et al. (2002)
Kaiser &
Guggenberger (2005)
Stevenson (1994)
a
Tannic acid
Coniferyl alcohol
(subunit lignin)
Menadion
SUVApH=2
/L g-1 cm-1
2.1
12.0
0.3
0.4
13.7
6.1
4.1
%HI
/%
93 (1)
63 (0)
58 (2)
25 (2)
1 (1)
0 (0)
0 (0)
%nn-HO
/%
2 (0)
29 (2)
42 (1)
65 (1)
105 (16)
90 (2)
85 (4)
%n-HO
/%
5 (1)
8 (2)
0 (3)
10 (2)
10 (2)
15 (4)
classif.
4.19
6.07
5.61
5.51
13.6
-
MW
/g mol-1
90
116
118
74
166
138
170
-
-
180
0.2
97 (0)
2 (1)
1 (1)
HI
-
-
-
105-108
1.2
86 (0)
5 (1)
9 (1)
HI
Proteins
Terpenoids
Polyphenols
(flavonoids)
Polyphenols
Polyphenols
3.47c
-2.02b
8-10.5d
-
~45,000
154
610
1.1
0.6
55.9
73 (3)
3 (4)
1 (0)
2 (-)
6 (7)
73 (4)
25 (3)
91 (7)
26 (5)
HI
n-HO
nn-HO
-0.19b
1.19b
8-10.5d
9.4e
-
1701
180
51.7
30.2
1 (1)
1 (1)
68 (4)
78 (1)
31 (4)
22 (1)
nn-HO
nn-HO
Quinons
2.20c
HI
HI/nn-HO
HI/nn-HO
HI/nn-HO
nn-HO
nn-HO
nn-HO
172
101.1
0 (-)
7 (-)
93 (-)
n-HO
Weast & Astle (1980); EPISuite: estimate based on chemical structure with KOWWIN v1.67; c Experimental Kow database in EPISuite v3.20; d Not measured,
b
pKa estimations based on pKa of phenols; e Rasmussen et al. (1997).
29
Table 3 Characteristics of the soils used for soil solution sampling, the incubation time (IT), and the average percentage of total DOC in the soil
solution and SUVA (L g-1 cm-1) of DOM in the hydrophilic (HI), non-neutral hydrophobic (nn-HO) and neutral hydrophobic (n-HO) fraction (2
replicates, standard deviation in parentheses).
Soil
Origin
pHa
Org. Cb
1
Boris (Denmark)
5.6
1.3
2
Ochamps (Belgium)
5.8
5.7
3
Rhydtalog (U.K.)
4.2
12.9
4
5
Zegveld (The Netherlands)
Ter Munck (Belgium)
4.7
6.9
23.3
0.9
a
IT
/days
3
83
13
13
13
13
13
4 (fresh)
Soil solution
Pore water
Pore water
Pore water
Extract
Pore water
Extract
Pore water
Pore water
DOC
/mg l-1
77.8
42.8
1192
102.9
1335
111.0
1152
26.4
SUVA
/L g-1 cm-1
10.9
22.9
9.9
19.4
7.0
17.9
7.0
20.1
/%
72 (5)
60 (0)
49 (1)
33 (1)
52 (0)
36 (1)
61 (2)
31 (-)
%HI
SUVA
5 (0)
17 (0)
3 (0)
8 (0)
2 (0)
6 (0)
2 (0)
12 (-)
%nn-HO
/%
/SUVA
25 (5)
23 (1)
38 (1)
31 (1)
38 (1)
18 (0)
39 (1)
35 (0)
36 (1)
14 (1)
39 (2)
33 (0)
27 (0)
16 (0)
46 (-)
26 (-)
%n-HO
/%
3 (7)
2 (1)
12 (1)
28 (2)
13 (0)
25 (3)
12 (2)
23 (-)
Measured in 0.01 M CaCl2; b Organic C content was measured by a wet combustion technique using K 2Cr2O7 and H2SO4 followed by CO2 analysis (Amato, 1983).
30
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