1 Evaluation of DAX-8 fractionation of soil DOM 2 DAX-8 fractionation of dissolved organic matter (DOM) from soils: 3 calibration with test components and application to contrasting soil 4 solutions 5 F. AMERY, C. VANMOORLEGHEM AND E. SMOLDERS 6 Section Soil and Water Management, Department Earth and Environmental Sciences, 7 K.U.Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium 8 Correspondence: F. Amery. E-mail: Fien.Amery@ees.kuleuven.be 1 9 Summary 10 Most methods to fractionate natural dissolved organic matter (DOM) rely on sorption of 11 acidified DOM samples onto XAD-8 or DAX-8 resin. Procedural differences among methods 12 are large and their interpretation is limited due to lack of calibration with DOM model 13 molecules. An automated column based DOM fractionation method was set up for 10 mL 14 DOM samples dividing DOM into hydrophilic (HI), non-neutral hydrophobic (nn-HO) and 15 neutral hydrophobic (n-HO) fractions. Fifteen DOM model components were tested in 16 isolation and in combination. Aliphatic low molecular weight acids (LMWA’s) and 17 carbohydrates were classified as HI DOM, but maleic, succinic and propionic acid showed 18 also a partial HO character. Aromatic LMWA’s and polyphenols partitioned in the nn-HO 19 fraction, menadion (quinon) and geraniol (terpenoid) in n-HO DOM. Molecules with log Kow 20 > 0.5 had negligible HI fractions. The HO molecules except geraniol had specific UV 21 absorbance (SUVA, measure for aromaticity) > 3 l g-1 cm-1 while HI molecules had SUVA 22 values < 3 l g-1 cm-1. The DOM of 8 different soil solutions was fractionated with this method. 23 Distributions ranged from 31-72% HI, 25-46% nn-HO and 2-28% n-HO of total dissolved 24 organic carbon with notable effects of DOM sampling method. The SUVA of the HI DOM 25 was lower compared to the HO DOM, but its variation coefficient was only slightly lower for 26 the nn-HO fraction and even larger for the HI fraction compared to the unfractionated DOM. 27 The calibration data allow interpreting results of the fractionation method. Our data show that 28 isolated soil DOM is not structurally more homogeneous than the unfractionated sample, 29 contrasting the general paradigm in fractionation approaches. 30 2 31 Introduction 32 Dissolved organic matter (DOM) has an important role in aquatic and soil systems for 33 contaminant transport, for biochemical and geochemical processes, and serves as a substrate 34 for microorganisms (Leenheer & Croué, 2003; Marschner & Kalbitz, 2003). The influence of 35 DOM on these processes does not only depend on the dissolved organic carbon (DOC) 36 quantity, but also on the DOM quality, i.e. the structural features (Raber et al., 1998; Kalbitz 37 et al., 2003). The composition of DOM in the soil solution varies with soil type, season, water 38 regime, soil depth and vegetation or crop. 39 Due to its complex and heterogeneous composition, soil DOM is commonly analyzed by 40 measuring bulk properties (e.g. spectroscopy) or by fractionating DOM based on chemical or 41 physical properties. The underlying idea of fractionation is that DOM fractions are 42 structurally more homogeneous than the bulk samples (Leenheer, 1981), however this is 43 rarely verified. A frequently used fractionation procedure is based on sorption of acidified 44 DOM onto the weakly hydrophobic resin XAD-8 dividing DOM into hydrophobic (HO) and 45 hydrophilic (HI) DOM fractions (Kaiser, 1998; Van Zomeren & Comans, 2007). The division 46 of DOM into the 2 fractions shows environmental relevancy. Metals transported in soil are 47 particularly associated with hydrophilic DOM (Guggenberger et al., 1994). This fraction has 48 also shown larger bioavailability (Marschner & Kalbitz, 2003), whereas polycyclic aromatic 49 hydrocarbons have larger affinity to hydrophobic DOM (Raber et al., 1998). Reported 50 distributions of soil DOM vary largely: 25 to 90% HI and 10 to 75% HO of total DOC (Table 51 1). The fractionation does not only change with soil depth, DOC concentration, sampling 52 method, land use and solution chemistry, but also depends on the fractionation method. 53 Most of the fractionation procedures are based on the (first step of the) fractionation 54 scheme of Leenheer (1981), that classifies dissolved organic molecules into hydrophobic and 55 hydrophilic fractions based on their pH dependent sorption onto the XAD-8 resin. As the 3 56 production of XAD-8 has been ceased, it is now replaced by SupeliteTM DAX-8, also a 57 polymethylmethacrylate resin. The sorption-desorption properties of the latter are slightly 58 more precise compared to the former (Peuravuori et al., 2002). Many modifications to this 59 method have been proposed, but generally the procedure is as follows. After acidifying the 60 DOM sample to pH 2 to neutralize all weak acid groups, the solution is pumped over a 61 column with DAX/XAD-8 resin. The column is rinsed with 0.01 M HCl. The DOM not 62 sorbed onto the resin is classified as hydrophilic. The hydrophobic DOM except for the 63 neutral molecules can be removed from the resin by rinsing with 0.1 M NaOH. The 64 interpretation of the fractionation in terms of the octanol water partition coefficient (Kow), 65 molecular weigth (MW) or acidity is, however, unknown, and the classification of the solutes 66 into hydrophilic and hydrophobic fractions depend on the procedure used (Peuravuori et al., 67 1997). Authors modify the procedure according to the volume, concentration and application 68 of the sample (Marhaba et al., 2003). In aquatic studies, the resin is frequently used to 69 concentrate and isolate DOM, thereby neglecting the hydrophilic part (Thurman & Malcolm, 70 1981). In contrast to aquatic samples, fractionation of soil solution DOM has often sample 71 volume limitations, requiring adapted fractionation techniques. 72 Despite the limited interpretation of fractionation, few studies have examined the 73 classification of specific organic molecules into the hydrophilic and hydrophobic fraction. 74 The hydrophobic fraction of natural DOM is assumed to contain lignin-derived, partly 75 aromatic molecules, nucleic acids, quinones, hydrocarbons and fatty acids. Hydrophilic DOM 76 consists of amino acids and sugars, small organic acids and carbohydrates and contains less 77 aromatic components than HO fractions (Cilenti et al., 2005; Simonsson et al., 2005). The list 78 of compounds to be found in each fraction is often based on spectroscopic measurements of 79 the fractions (Leenheer, 1981; Guggenberger & Zech, 1994; Kaiser et al., 2001a; Dilling & 80 Kaiser, 2002; Croué et al., 2003; Cilenti et al., 2005), instead of subjecting model DOM 4 81 components to the fractionation procedure. Among the exceptions are Qualls & Haines (1991) 82 who tested 5 compounds in their modified fractionation procedure. Over 90% of rutin and 83 quercitin (flavonoids) was found in the hydrophobic fraction, whereas over 85% of oxalic 84 acid, albumin and glucose phosphate was classified as hydrophilic DOM. David et al. (1989) 85 recovered over 89% of oxalic, succinic and citric acid as hydrophilic acids; over 95% of 86 benzoic and salicylic acid was found in the HO fraction. Thurman et al. (1978) determined 87 the capacity factors of 20 nonionic organic solutes on XAD-8 and concluded that the resin 88 favors aliphatic over aromatic over alicyclic components. The logarithm of the capacity factor 89 correlated inversely with the logarithm of the aqueous molar solubility, but it remains unclear 90 which components can be expected to appear in the hydrophilic fraction. 91 Numerous modifications to the fractionation procedures have been described varying in 92 resin/volume ratio, flow rate in the columns (often not reported) and in further fractionation of 93 HI or HO fractions. These modifications are known to affect the fractionation and it is striking 94 that no calibration steps are systematically adopted in modified procedures. We developed a 95 fractionation procedure with DAX-8 for only 10 ml of DOM solution. Fifteen soil DOM 96 model components were subjected to this fractionation scheme. Soil solutions from different 97 sources were subsequently fractionated. Specific UV absorbance (SUVA, 254 nm, Weishaar 98 et al., 2003) of the DOM and their fractions were measured to determine relationships with 99 the hydrophobicity and to study characteristics of the fractions. It was investigated if the 100 structural variability (linked to SUVA) of the bulk samples is lower in the corresponding 101 fractions. 102 Materials and methods 103 Fractionation procedure 5 104 The procedure to fractionate DOM solutions is based on preliminary experiments with water 105 extracts of soil. The first step of the fractionation scheme of Leenheer (1981) was adapted to 106 clearly separate hydrophilic and hydrophobic DOM of small volumes of soil solutions. 107 SupeliteTM DAX-8 resin was cleaned according to the method of Thurman & Malcolm 108 (1981). The resin was extracted with 0.1 M NaOH, replacing the solution and decanting off 109 fines daily. After 5 days, the resin was soxhlet-extracted for 24 hours with methanol, 110 acetonitrile, diethyl ether and again methanol. The resin was stored in methanol/water until it 111 was packed in an 8.0 cm x 1.6 cm column, giving a bed volume (BV) of 16 ml. The column 112 was connected to a compact liquid chromatographic system (Äkta Prime Plus, GE Healthcare) 113 with fraction collector and online pH, conductivity and UV (254 nm, path length 0.2 cm) 114 detectors. The void volume (VV) between the injection valve and the detectors was 8 ml. The 115 resin was rinsed with ultrapure water until the DOC concentration of the column effluent was 116 <0.5 mg l-1. Before and after each fractionation procedure, the resin was rinsed with 117 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 118 h-1 and one VV at 7.5 VV h-1). This rinsing was repeated until the DOC concentration was 119 less than 0.5 mg l-1 and the UV absorbance of the effluent stabilized. 120 The DOM solution was acidified to pH 2 with 2 M HCl and filtrated (0.45 µm). The 121 column was rinsed with 0.01 M HCl until pH was stable. The UV absorbance signal was set 122 to zero. Ten ml of the filtered DOM solution was injected onto the column using a superloop 123 (22.5 VV h-1). After the sample, the column was rinsed with 4.5 VV of 0.01 M HCl (22.5 VV 124 h-1). All DOM collected before the end of this rinsing was classified as hydrophilic. The flow 125 direction was subsequently reversed, and the non-neutral hydrophobic (nn-HO) DOM was 126 eluted with 2.25 VV of 0.1 M NaOH, followed by 4 VV of ultrapure water (7.5 VV h-1). After 127 1 VV, the eluate pH increased from 2 to >10 within 0.1 VV. All eluting solution was collected 128 in 30 subfractions with a volume varying from 2 to 5 ml. All DOM subfractions were 6 129 acidified to pH 2 before DOC analysis and inorganic C was removed by sparging samples 130 with O2 for 30 seconds. Concentrations of DOC in the subfractions were measured by 131 catalytic combustion (800°C) and infrared measurement of formed CO2 (Multi N/C 2100S, 132 AnalytikJena). The DOC present in the neutral hydrophobic fraction (n-HO, not eluted by 0.1 133 M NaOH) was calculated by difference between DOC in the original sample and the DOC 134 quantities in fractions recovered. 135 Test components 136 Fifteen components were subjected to the fractionation procedure (Table 2). The components 137 were selected because of their presence in soil solutions or because they are a model for 138 compound classes present in soil solutions. Molecules were also selected to obtain a range in 139 Kow, acidity constant (pKa) and MW. The components were dissolved in or diluted with 140 ultrapure water to obtain DOC concentrations of approximately 20 mg l-1. Beside fractionation 141 of the isolated components, 3 of them were also fractionated in combination: 11.0 mg DOC l-1 142 oxalic acid, 5.0 mg DOC l-1 rutin and 3.3 mg DOC l-1 geraniol, giving a combined 143 concentration of 19.3 DOC mg l-1. Also a solution of Suwannee River Fulvic Acid (SRFA), a 144 fulvic acid isolated by the International Humic Substances Society (IHSS), was fractionated. 145 After acidifying the DOM samples to pH 2 with 2 M HCl and filtration (0.45 µm), DOC 146 concentrations (AnalytikJena) and UV absorbance (254 nm, path length 1 cm, Perkin-Elmer 147 Lambda 20) were measured. Fractionations were performed in duplicate. 148 Fractionation of soil solutions 149 The topsoil of 4 uncontaminated soils (1-4) was sampled, air-dried (25°C), sieved (< 2 mm) 150 and stored dry for various periods (4-12 years). The air-dried soils were wetted with deionized 151 water to field capacity (pF 2), covered in plastic reservoirs and incubated moist during various 152 days at 21°C (Table 3). Topsoil 5 was not dried after sieving (4 mm) but immediately 7 153 incubated moist during 4 days. After incubation soil solutions were obtained by centrifugation 154 (pore water) or by extraction. Pore water was sampled by the ‘double chamber method’. A 30 155 mL syringe without plunger was filled with quartz wool and approximately 60 g of moist soil. 156 The syringe was placed in a 50 ml centrifuge tube and centrifuged during 30 minutes (2500 157 g). The obtained pore water was filtered (0.45 µm). Extractions were performed in a 50 ml 158 centrifuge tube filled with 5 g moist soil and 20 ml 1 mM CaCl2. After shaking end-over-end 159 for 24 hours, the suspensions were centrifuged during 30 minutes (2500 g). The supernatant 160 was filtered (0.45 µm). Soil solutions were sometimes diluted before fractionation to obtain 161 DOC concentrations below 150 mg l-1. Fractionations were performed in duplicate, except for 162 the pore water of soil 5. 163 Specific UV absorbance 164 The specific UV absorbance (SUVA, l g-1 cm-1) of DOM was calculated by SUVA 165 A 254 b DOC (1) 166 where A254 is the UV absorbance at 254 nm measured by a spectrophotometer (Perkin-Elmer 167 Lambda 20 or online with GE Healthcare Äkta Prime Plus) (dimensionless), b the path length 168 of the quartz cell of the spectrophotometer in cm and [DOC] is in g l-1. The SUVA of DOM 169 with DOC concentration < 1.5 mg l-1 was not calculated because of accuracy problems. The 170 specific UV-absorbance is used as an estimate of the aromaticity of DOM (Weishaar et al., 171 2003). 172 The SUVA of a collected subfraction was calculated by equation (1) using the average 173 UV absorbance of the eluting subfraction. The SUVA of the HI or nn-HO fraction (SUVAfr, l 174 g-1 cm-1) was calculated by 8 A n 175 SUVA fr i 1 n 254 i Vi b DOCi Vi (2) i 1 176 where n is the number of subfractions in the fraction fr and A i254 , Vi and [DOC]i the average 177 UV absorbance (-), volume (l) and DOC concentration (g l-1) of subfraction i, respectively. 9 178 Results and discussion 179 Fractionation of the test components 180 The fractionation of the 15 test components varied largely (Table 2). Carbohydrates, proteins 181 and aliphatic LMWA’s showed a (partial) HI character, whereas aromatic LMWA’s and 182 polyphenols ended up in the HO fraction. Quinons and terpenoids did not desorb from the 183 resin during base rinsing and were therefore classified as n-HO molecules. The peak of the 184 DOC concentration of the HI fraction of oxalic acid and other pronounced HI components 185 was situated only after 2 VV with DOC concentrations dropping to zero only after 5 VV 186 (Figure 1). This was also the case for the HI fraction of soil DOM (see below). The DAX-8 187 resin showed affinity for the HI molecules as well, resulting in retardation of the components. 188 This emphasizes the need for sufficiently rinsing of the resin and the importance of reporting 189 cumulative resin/solution ratio in the procedure for the distribution of DOM in HI and HO 190 fractions. When no or few rinsing is applied, part of the retarded HI molecules will be 191 classified as HO. The choice for rinsing with 4.5 VV in this method was based on preliminary 192 fractionation experiments with soil solutions. Concentrations of DOC dropped only below 0.5 193 mg l-1 after rinsing for more than 4 VV with 0.01 M HCl. However 3 of the aliphatic 194 LMWA’s (maleic, succinic and propionic acid) showed an even larger retardation on the 195 column at pH 2 (Figure 1), resulting in a partial HI and partial HO classification based on this 196 fractionation procedure. This can be related to the Kow and pKa of the components (see 197 below). Aromatic LMWA’s, e.g. salicylic acid, were almost completely retained by the resin 198 at pH 2 but were eluted with NaOH (Figure 1). This was also the case for polyphenols (e.g. 199 rutin). Geraniol and menadion were classified as n-HO, but also some HI and especially some 200 nn-HO components showed a partial n-HO character (Table 2). This was most pronounced for 201 components with high molecular weight (e.g. ovalbumin and tannic acid). Peuravuori et al. 202 (1997) suggested already that the sorption mechanism of the DAX/XAD-8 resin is partly 10 203 based on molecular size of the solute. Qualls & Haines (1991) also found rutin in the 204 hydrophobic fraction and oxalic acid and albumin in the hydrophilic fraction. David et al. 205 (1989) also classified oxalic acid as hydrophilic and salicylic acid as hydrophobic, but they 206 found over 89% of succinic acid in the hydrophilic fraction. They used the fractionation 207 method of Leenheer (1981) who only rinsed the XAD-8 resin with 1 BV compared to 4.5 VV 208 (=2.25 BV) in our method. This should lead to a smaller HI DOC amount, however they 209 found higher DOC recovery in their HI fraction compared to this study (59%). 210 Fractionation of the combined components (oxalic acid, rutin and geraniol) gave similar 211 results as their isolated fractionations. The HI and nn-HO fractions were slightly lower (49.5 212 ± 1.3% and 16.4% ± 0.1% of total DOC respectively, average values ± standard deviation) 213 compared to the calculated values based on the isolated fractionation results (53.8 ± 1.1% and 214 21.1 ± 1.6%). These lower values can be an indication of minor interactions of the 215 components. The neutral hydrophobic geraniol can interact with oxalic acid and rutin, 216 increasing slightly their n-HO fraction. Almost all DOM of the SRFA appeared in the HO 217 fraction: 76.2 ± 0.4% in the nn-HO and 23.5 ± 0.0% in the n-HO fraction. The negligible part 218 of DOM in the HI fraction (0.3 ± 0.4%) can be explained by the SRFA isolation method of 219 the IHSS. All SRFA DOM originates from desorption from the XAD-8 resin with 0.1 M 220 NaOH after percolating Suwannee River water at pH 2 over the resin. Surprisingly, 23.5% of 221 the sorbed SRFA on the DAX-8 resin could not be desorbed in our method. A possible 222 explanation is the use of different resins: XAD-8 by the IHSS method and DAX-8 in our 223 method. The method to desorb DOM from the resin also varies: 3 VV with 0.1 M NaOH at 224 350 ml min-1 in the IHSS method (Malcolm et al., 1995 and E.M. Perdue, personal 225 communication) compared to 2.25 VV with 0.1 M NaOH and 4 VV with MQ water at 1 ml 226 min-1 in our method. Structural conformations of the isolated fulvic acid during further IHSS 11 227 sample preparation (passing through cation exchange resin, hydrogen saturation and freeze 228 drying) are also possible. 229 The affinity of the DAX-8 and XAD-8 resin for components is assumed to be related to 230 the hydrophobicity of the components. A negative linear relationship was found between the 231 logarithm of aqueous solubility and the logarithm of the capacity factor of molecules on 232 XAD-8 (Thurman et al., 1978). It was investigated if the Kow of the test components, a 233 measure for the hydrophobicity, showed a relationship with the classification of the molecule 234 into the HI, nn-HO and the n-HO fraction (Table 2 and Figure 2). Components with log Kow < 235 -1.5 appeared almost completely in the HI fraction. An exception is rutin which has a low log 236 Kow value (-2.02) because of the carbohydrate part of the molecule. The sorption by the DAX- 237 8 resin at pH 2 is probably due to the aromatic part (polyphenol). This was also the case for 238 tannic acid which has a quite low log Kow (-0.19) as well because of its carbohydrate part. 239 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 241 character. Of these 3 molecules maleic acid is less retained, probably because it is charged at 242 pH 2 (pKa1 = 1.83), as is oxalic acid. Succinic acid and propionic acid are not ionized at pH 2 243 (pKa1 = 4.16 and pKa = 4.87 respectively). The Kow of a charged molecule is more than 100 244 times smaller compared to that of the non-dissociated molecule (Schwarzenbach et al., 1993). 245 Propionic acid has the largest log Kow (0.33) and showed most retardation by the resin. 246 Components with log Kow > 0.5 were all classified as HO. Although 2,4,6-trihydroxybenzoic 247 acid is ionized at pH 2 (pKa1 = 1.68) it was also found in the HO fraction. The 2 molecules 248 classified as n-HO, geraniol and menadion, showed a very large log Kow (3.47 and 2.20 249 respectively). Salicylic acid is classified as nn-HO despite its larger log Kow (2.26) compared 250 to menadion. This is probably due to the presence of the carboxyl group with pKa = 2.98 in 251 the salicylic acid molecule. All other molecules classified as nn-HO have also pKa < 11, 12 252 resulting in a charged molecule during rinsing with 0.1 M NaOH causing desorption from the 253 DAX-8 resin. 254 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 261 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. 264 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 References 376 Amato, M. 1983. Determination of carbon 12C and 14C in plant and soil. Soil Biol. Biochem., 377 378 15, 611-612. Amery, F., Degryse, F., Degeling, W., Smolders, E. & Merckx, R. 2007. The copper- 379 mobilizing-potential of dissolved organic matter in soils varies 10-fold depending on soil 380 incubation and extraction procedures. 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Environmental Science & Technology, 37, 490 4702-4708. 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