Environ. Sci. Technol. 2005, 39, 6575-6583
Distribution of Aged 14C-PCB and 14C-PAH Residues in Particle-Size and Humic Fractions of an Agricultural Soil KIERON J. DOICK,† PETER BURAUEL,‡ KEVIN C. JONES,† AND K I R K T . S E M P L E * ,† Department of Environmental Science, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, LA1 4YQ, United Kingdom, and Agrosphere Institute, ICG IV, Forschungszentrum Ju ¨ lich, D-52425, Ju ¨ lich, Germany
Organic matter is considered to be the single most important factor limiting availability and mobility of persistent organic pollutants (POPs) in soil. This study aimed to characterize the distribution of 14C-PCB (congeners 28 and 52) and 14C-PAH (fluoranthene and benzo[a]pyrene) residues in an Orthic Luvisol soil obtained from two lysimeter studies initiated in 1990 at the Agrosphere Institute (Forschungszentrum Ju¨ lich GmbH, Germany). The lysimeter soils contained a low-density OM fraction, isolated during soil washing, which contained a significant fraction (3-12%) of the total 14C-activity. Soils were also fractionated according to three particle sizes: >20, 20-2, and (20 µm) for the PCBs. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 µm > ( (>20 µm) for the PAHs. The distribution of 14C-PCB or 14C-PAH residues in the organic and inorganic matrixes of the particle-size fractions was determined using methyl isobutyl ketone (MIBK). 14C-PCB and 14C-PAH-associated activities were primarily located in the humin fraction of the 20-2 and 50% of the total OC in a soil (6), and most nonextractable contaminant residues are associated with humin (7, 10). An important reason for this is the intractable nature of humin. Alkaline extractions traditionally used to remove HA and FA leaves the humin residues dispersed in the mineral fraction. Subsequently, detailed analysis of either the humin or the mineral component first requires removal of the other fraction, usually by additional chemical treatments. For example, HF/HCl treatment is used to dissolve the mineral component (allowing further study of the humin) (6), while hydrogen peroxide treatment is used to completely destroy OM (11) and thus allow further study of the mineral fraction. Undoubtedly, such harsh chemical treatments influence the chemical nature/structure of the nontarget fraction (i.e., the one to be studied). For example, humin has been shown to become soluble in alkaline solutions following treatment with HF/HCl (12). Alternative chemical techniques to extract POP residues (especially nonextractable residues) from soil intentionally split chemical bonds. For example, methanolic saponification (MSE) hydrolyzes labile ester bonds (4); therefore, these techniques may also induce a chemical bias to results. Increasingly, traditional chemical extractions are not considered appropriate because their chemically degrading nature may induce modifications to the OM or mineral structure and introduce unacceptable bias to the results (refs 4 and 13 and references therein). Instead, attention has been directed at the use of physical fractionation of soil according to size and density and characterization of the resultant phases by a variety of physicochemical techniques, including infrared, fluorescence, and photon correlation spectroscopy, diffuse reflectance Fourier transform infrared spectroscopy (DRIFT), and solid-state 13C-nuclear magnetic resonance (13C NMR) (13). In the absence of such analytical equipment and in the interest of comparisons to past studies, chemical fractionation techniques still have a role to play in advancing our understanding of the fate of POPs in soil. VOL. 39, NO. 17, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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second lysimeter. The aims of this study were to (i) characterize the distribution of aged PCB and PAH residues in soil (following ca. 13 years aging in an environmental setting) according to respective organic and inorganic matrixes within various particle-size fractions of the soils and (ii) to test the hypothesis that the fate of PCBs differs from that of PAHs in soil. To the best of our knowledge, this is the first study to apply humic material separation to particle-size fractionated soils to give a detailed description of aged PCB and PAH distributions in soils.
Materials and Methods
FIGURE 1. Schematic of modified methyl isobutyl ketone (MIBK) fractionation procedure adopted for isolation of the humic acid, fulvic acid, humin-only, and mineral-only fractions, applied to the individual particle-size fractions (17). Particle-size fractionation is used to distinguish pools of different SOM composition and turnover rates (14, 15). For example, sand fraction SOM is mostly comprised of fresh or slightly decomposed plant material; silt-size fractions contain partially degraded residues, and the clay-fraction SOM is heavily characterized by strongly processed OM, predominated by aromatic and aliphatic structures that have a greater resistance to microbial degradation (14, 15). OC in silt- and clay-sized fractions is generally turned over more slowly than OC in the sand fraction. Coupled to the observation that organic contaminants are closely associated with SOM, the distribution of POPs among particle-size fractions has been proposed as a method of evaluating a contaminant’s potential to be recycled. Such studies have also been suggested as a method of assessing the relative importance of the different OM pools in a soil, with a view of determining potential contaminant bioavailability (14, 16). Rice and MacCarthy (17) have reported a novel variation of the alkaline extraction scheme whereby humin is obtained as a result of an active isolation step and is free from other insoluble materials. The methyl isobutyl ketone (MIBK) method sequentially partitions the humic substances back and forth between an aqueous phase and MIBK as a function of pH (Figure 1). The MIBK method has been used to achieve quantitative separation of HA, FA, humin, and the mineral-only fraction (11). The employment of alkaline and MIBK extractions and particle-size fractionation of soils has greatly enhanced our understanding of the fate of many POPs in soil and in some cases helped to elucidate mechanisms of degradation. However, studies utilizing 14C-labels are often short-term (less than 1 year duration), and studies employing environmental samples, although offering detail on deposition and (re-)distribution mechanisms, cannot elicit information on the bound residue (nonextractable) fraction. This work furthers investigations into two lysimeter studies initiated in 1990 at the Agrosphere Institute (Forschungszentrum Ju ¨ lich GmbH, Germany), with PCBs 28 and 52 in one lysimeter and PAHs fluoranthene (Flu) and benzo[a]pyrene (BaP) in a 6576
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Materials. GoldStar scintillation fluid was obtained from Meridian (Epsom, UK). Sample oxidizer cocktails (CarbosorbE, Permaflour-E, Combustaid) were obtained from Canberra Packard (UK). Extraction thimbles and cellulose nitrate membrane filters were obtained from Whatman (UK). Calcium chloride was obtained from Sigma Aldrich (Germany). Anhydrous sodium sulfate was obtained from BDH Chemicals, Ltd. (Poole, UK). Hydrogen peroxide was purchased from Fisher Scientific (Loughborough, UK). Soils. Soils containing aged PCB (congeners 28 and 52) or aged PAH (fluoranthene and benzo[a]pyrene) residues were obtained from two lysimeters at the Agrosphere Institute (Forschungszentrum Ju ¨ lich GmbH, Germany). The lysimeters was set up in July and September 1990 for the PCBs and PAHs, respectively, using Orthic Luvisol soil obtained from intensively cultivated agricultural land. The soil pH was 7.2, and the organic matter (OM) content was 2.2% (0-35 cm depth). The lysimeters (surface area 1 m2 and depth of 1.2 m) containing the soils were sunk to ground level, surrounded by a control cultivation area and were subject to normal environmental processes (9). The contaminants (12C- and 14C-analogues) were uniformly mixed into the Ap horizon (0-35 cm (18)). Bioavailability to plants was assessed annually by rotational cropping of carrots, winter wheat, spinach, potatoes, kale, or sugar beet (9, 18). Three cores (5 cm diameter and 50 cm deep) were sampled from each lysimeter on March 14, 2003, divided into 0-30 and 30-50 cm depths, and thoroughly mixed prior to analysis. Alkaline Extraction of Soils. Subsamples of soils were homogenized, ground with anhydrous sodium sulfate to facilitate solvent-soil interactions (19), and DCM-soxtec extracted (Foss Tecator Soxtec 2055 Avanti (Sweden) extraction unit) on a 30 min boil-150 min reflux cycle. Aliquots of DCM supernatant (3 mL) were analyzed for 14C-activity by liquid scintillation counting (LSC; Tri-Carb 2300TR liquid scintillation counter, Canberra Packard). Pre-extracted soils (6-8 g of dry wt) were subsequently treated with 0.5 M NaOH (30 mL) in Teflon centrifuge tubes (35 mL capacity) and shaken to extract HA and FA (100 rpm, 24 h; Janke and Kundel orbital shaker (IKA-Labortechnik KS 201)). HA was recovered by precipitation at pH 1 and centrifugation (3100g, 1 h, Beckman Centaur 2 centrifuge). HA was redissolved in a minimum volume of 0.5 M NaOH (typically 1 mL), and 14C-activity was quantified by LSC. The supernatant containing the FA was decanted, and an aliquot was taken for 14C-quantification (by LSC, as stated previously). Residual 14C-activity not extracted (i.e., humin-mineral component) was quantified by combustion and LSC (1 g samples, Sample Oxidizer, model 307, Packard, Berkshire, England). Particle-Size Fractionation of the Soils. Prior to fractionation, subsamples of soil were analyzed for residual 14C-PCB- and 14C-PAH-associated activity in the soil pore water and labile fractions (sum of dissolved, soluble, rapidly desorbing, and dissolvable OM-associated compound) (in triplicate for PCBs, singularly for PAHs). Pore water analysis involved placing soil (2 ( 0.1 g) in a small plastic syringe (5 mL volume), which had been pre-plugged with a little glass wool, and adding 1 mL of dH2O to aid displacement of the
FIGURE 2. Schematic of the soil fractionation procedure. Soils were separated into >20, 20-2, and 12 h to normalize the effects of chemiluminescence. All procedures were performed in triplicate unless otherwise stated. Following blank-correction, statistical analysis of the results was performed in SigmaStat for Windows (Version 2.03, SPSS Inc.) using ANOVAs (Tukey test, p < 0.05) and student t-tests (p < 0.05).
Results and Discussion Data Description and Background Information. In this study, lysimeter soils were spiked with either PCBs 28 and 52 or PAHs fluoranthene (Flu) and benzo[a]pyrene (BaP), and in each case using both 12C- and 14C-analogues. Data presented herein are based entirely upon 14C-activities. The tetra-chlorinated biphenyl 52 was applied to the lysimeter at a higher concentration than the tri-chlorinated biphenyl 28 when the lysimeter was set up in 1990. At the time of this study, extractable PCB 28 and 52 (parent compound) concentrations were 0.73 and 1.13 mg kg-1, respectively (23); previous studies of these lysimeter soils did not detect degradation products for the PCBs (9, 18). Therefore, 14C-activity detected in this study probably represented slightly more PCB 52 than PCB 28 and is collectively referred to as 14C-PCB-associated activity. In the second lysimeter, loss rates differed considerably between the two PAHs, a reflection of their different physicochemical properties and microbial lability, and degradation products were likely to form a significant proportion of nonextractable 14C-PAH-associated activity (9, 18). At the time of this study, extractable Flu and BaP (parent compound) concentrations were 0.36 and 0.42 mg kg-1, respectively; degradation products were not determined (23). Therefore, 14C-PAH data presented herein represent the sum of 14C-Flu- and 14C-BaP-associated activities plus unspecified degradation products and are collectively referred to as 14C-PAH-associated activity. Sodium Hydroxide Extraction of Bound Residues in Particle-Size Soil Fractions. Soil was subjected to a harsh chemical extraction (DCM-soxtec) to allow an investigation of the nonextractable (bound residue) fraction. Mean extractable fractions of 14C-activity were 62 ( 4 and 63 ( 13% (errors are 1 standard error of the mean (SEM)) for the PCBs (0-30 and 30-50 cm deep, respectively) and 8 ( 1 and 8 ( 2% for the PAHs (0-30 and 30-50 cm deep, respectively) (values are percentages of the total 14C-activity measured at each soil depth during this study). DCM extractabilities did not significantly vary between the 0-30 and the 30-50 depth soils, for either the 14C-PCB- or the 14C-PAH-associated activities (t-tests, p > 0.05, data not shown). The observation VOL. 39, NO. 17, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Percentage Recovery of 14C-PCB- and 14C-PAH-Associated Activities in Porewater, 0.01 M CaCl 2 Solution, and Low-Density Organic Matter Associated Fractions, According to Soil Depth fraction recovered (%)a contaminant PAHs PCBs
soil depth (cm)
pore water
CaCl2 ext.
low-density OM
0-30 30-50 0-30 30-50
n.db n.d n.d n.d
20 µm) (Table 2). However, particle-size fractions varied in their percentage contribution to the bulk-soil composition and contained different quantities of OM. A relative affinity (RA) value of a contaminant for a given particle-size fraction was therefore determined using percentage 14C-activity associated with a fraction divided by the percentage OM of the whole soil in that fraction (10). Relative affinity (RA) values
were also determined as percentage 14C-activity associated with a fraction divided by the percentage contribution of that fraction to the bulk soil. According to this scale, a RA value of one reflects no special affinity of the contaminant(s) for that fraction, and the binding can simply be attributed to homogeneous distribution throughout the SOM (10). A RA value greater or less than one shows a higher or lower affinity, respectively, for that fraction as compared to the other particle-size/humic fractions in the soil (10). Organic matter normalized RA values varied in the order (20-2 µm) > (20 µm) in both top- (0-30 cm) and subsoils (30-50 cm) for the PCBs. Organic matter normalized RA values varied in the order (20-2 µm) > ( (>20 µm) in both top- (0-30 cm) and subsoils (30-50 cm) for PAHs. Furthermore, the 20-2 µm fraction displayed a statistically greater RA value in subsoil than in topsoil for both the PCBs and the PAHs (ANOVA, p < 0.001; Table 2). For both contaminant classes, OM normalized RA values changed the order of significance of the different particlesize fractions relative to the unnormalized data, although this was not the case for the grain-size normalized RA values (Table 2). Assessing the dynamics of OC associated with different particle-size separates, researchers have demonstrated that OC in silt and clay is generally turned over more slowly than sand OC (i.e., it is more stable) and hence can show an accumulation of recalcitrant compounds (ref 15 and references therein). However, it is unclear whether silt-OC or clay-OC is the more stable (15). The unnormalized distributions of contaminants observed in this study were comparable to those reported by Krauss and Wilcke for urban and peri-urban soils, which decreased in the general order of silt > clay > coarse silt/sand (14). Our study and that of Krauss and Wilcke (14) indicated the importance of silt (as well as clay) in the partitioning of PAHs in soil. Interestingly, Amellal et al. (16, 33) employing particlesize fractionation to investigate the role of aggregate sizes on PAH bioavailability in soil concluded that because bacteria and PAH concentrations were most numerous in the 20-2 and 20 µm fraction, whereas, the pattern was reversed in all other particle size fractions (Figure 4A). Second, the fraction of 14C-PAH-associated activity oxidized to 14CO2 was far greater than for the 14C-PCB-associated activity. Furthermore, losses to 14CO2 were approximately equal for each of the particle-size fractions with only the 20 µm PAH topsoil displaying significantly greater release than respective fractions (ANOVA, p < 0.05, data not shown). Third, 14C-PAH-associated activity was recovered in significantly greater proportions in the gas and supernatant fractions than the HPCD-extractable fraction. However, the total proportion lost to the gas phase as opposed to the supernatant phase varied across particlesize fraction and top-/subsoil. OM is suggested to vary in quality between various particle-size fractions of soil (14-16). Nevertheless, the H2O2 treatment was expected to be insensitive to differences in OM composition/quality or quantity as the H2O2 treatment was prolonged and extensive, and H2O2 was typically used in the complete destruction of OM (11, 14-16). Therefore, to determine the quantity of 14C-activity-associated with OM, the assumption was made that total release to gas, supernatant, and HPCD-extractable fractions was a product of OM decomposition. The sum of these fractions in comparison to the total residual (nonextractable) fraction is given in Table 3. In summary, H2O2 treatment released ca. 50% of the 14C-PCB-associated activity from all soil fractions investigated; 6580
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FIGURE 4. Hydrogen peroxide treatment of PCB (A) and PAH (B) lysimeter soils, fractionated according to particle size and topsoil (TS) or subsoil (SS) depth (0-30 and 30-50 cm, respectively). Particle-size fractions represent total sand plus coarse silt (>20 µm), medium and fine silt (20-2 µm), and clays ( 0.05; Table 3). H2O2 treatment released ca. 40-85% of the 14C-PAH-associated activity and variedswith no clear trendsbetween particle size and top-/subsoils fractions (Table 3). Discussion of these results is given next, where a comparison to the MIBK measurement of OM has been made. MIBK Separation of OM Fractions in Particle-Size Soil Fractions. Isolation of humic fractions was performed using the MIBK technique of Rice and MacCarthy (17) on the particle-size fractionated soils. Figure 5A,B, respectively, displays the fraction of 14C-PCB- and 14C-PAH-associated activities recovered from each humic fraction (as a percentage of the total 14C-activity per particle-size fraction). 14C-PCB recovery in FA and HA ranged 0.01-0.14% and varied little between particle-size fractions, soil depths, or humic fraction, only displaying a statistically higher recovery in HA in the 20 (µm)
20-2 (µm)
20 (µm)
20-2 (µm)
50%) was not expected and cannot be compared to previous experiments. The recovery of 14C-activity from this mineral fraction could have been related to incomplete extraction of the humic material from the soil during the alkaline extraction. However, pre-studies showed that this was unlikely as proportionately VOL. 39, NO. 17, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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more 14C-activity was recovered for each additional gram of soil extracted up to the 5 g employed, indicating quantitative recovery (data not shown). Furthermore, recovery from the mineral-only fraction decreased with decreasing particle size (i.e., increasing OM content), suggesting that the methodology was probably as efficient at extracting OM when present in large as well as small quantities. However, previous studies have demonstrated that the mineral component of humin may contain up to 4% OM by weight (6, 11). Thus, it is hypothesized that the mineral component of the soil also may have contained a residual, unremoved quantity of OM, and it is recommended that future studies test this hypothesis. Cumulatively, these results suggest that some 14C-activity was directly associated with the mineral component (and not with the OM), and this possibility is discussed next. Comparison of OM Treatments. In this study, a comparison was sought between the behavior of PCBs and PAHs in soil, with respect to their association with SOM, using two techniques. First, quantification of the aged HOC residues associated with the OM fraction was attempted based on the H2O2 decomposition of the OM. Traditionally, analysis of the mineral component of soil has followed removal of the humic and fulvic acids by alkaline extraction (10, 17) and the subsequent complete destruction of the humin (which is dispersed within the mineral component) through H2O2 treatment (11). Second, individual humic fractions were quantified using the MIBK technique (17). Table 3 shows a comparison of the total 14C-activities recovered from OM fractions using the two techniques. Results obtained for the H2O2 treatment suggested that the aged 14C-PCB residues were approximately equally distributed between organic and inorganic components of the soil. This result is in contrast to the MIBK extraction technique, which displayed an increase in 14C-PCB residues associated with OM with decreasing particle-size fractions. Given that the OM content increased with decreasing particle size (data not shown), the trend exhibited by the MIBK was expected. Percentage recovery in the 20-2 and
