Environ. Sci. Technol. 2000, 34, 4952-4957
Influence of Contact Time on Extractability and Degradation of Pyrene in Soils CHRISTOPHER J. A. MACLEOD AND KIRK T. SEMPLE* Department of Environmental Science, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, LA1 4YQ, U.K.
In this study, temporal changes in extractability of [14C]pyrene were followed in two soils with differing organic matter contents under sterile and nonsterile conditions over 24 weeks. The nonsterile pasture soil was the only incubation to show significant loss of [14C]pyrene-associated activity over the 24-week incubation. Sequential extraction using methanol:water (1:1), followed by 1-butanol and finally dichloromethane-Soxhlet showed changes in the relative proportions of extractability with increased soilPAH contact time. Significant decreases in methanol:water and 1-butanol extractability were recorded over the 24week incubation. The nonsterile pasture soil exhibited the greatest decrease in 1-butanol extraction. Significant nonextractable residues were formed with increased soilpyrene contact time in all soils, with the largest increase found in the nonsterile pasture soil. These residues were investigated by the alkaline extraction of the humic material and saponification of the resultant humin. After 24week soil-pyrene contact time, the bioavailability of the added [14C]pyrene was assessed by bacterial mineralization. A comparison was made between bioavailability and the amount of 14C activity extracted by the sequential scheme of solvents. Methanol:water significantly underestimated the bioavailable fraction, whereas 1-butanol overestimated the bioavailability of the [14C]pyrene-associated activity.
Introduction The chemical extractability and bioavailability of hydrophobic organic contaminants (HOCs), such as polycyclic aromatic hydrocarbons (PAHs), from soil have been shown to decrease with increasing contact time (1-7). A number of chemical extractants have been proposed to determine the fate and putative bioavailability of HOCs in aged soils (1, 3-11). The decrease in extractability may be controlled by physical sequestration of HOCs and limited mass transfer from soil to solvent (2-14) or by the action of a soil’s microbial community (8, 10, 14-17). This decrease in extractability and bioavailability has important implications for the risk assessment of HOCs in historically contaminated soil (3). The processes of HOC sequestration in soil are thought to be driven by partitioning into the soil organic matter (SOM) (13, 18-20) and sequestration into soil micropores (13, 21, 22). In soils with greater than 0.1% organic carbon (OC) content, partitioning into the SOM has been found to be the dominant process (13, 18-20). The sorption of HOCs by the * Corresponding author telephone: +44 (0) 1524 594534; fax; +44 (0) 1524 593985; e-mail:
[email protected]. 4952
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SOM is thought to occur in two distinct regions, the expanded (rubbery) region in which linear, rapidly reversible partitioning can occur and the condensed (glassy) region in which nonlinear, slowly reversible sorption may also occur (13, 2225). Soils with higher OC contents have a larger capacity to sequester added HOCs (15, 25), with regions of expanded SOM controlling rapid desorption (13, 25). Furthermore, increased soil-PAH contact time reduces the magnitude of the rapidly desorbing phase and the extent of biodegradation (2, 3, 13, 15), with the sorption of HOCs to sites in the condensed region controlling the slowly desorbing phase (13, 15, 22-25). Soil-HOC-microbe interactions are influenced by many parameters, either singly or in combination, including residence time in soil, HOC concentration, and microbial properties of the soil. The biotic removal of HOCs results from the activity of microorganisms and the ability to use HOCs as a source of carbon and energy for growth (17, 2629). A threshold concentration of substrate needs to be reached before growth and/or biomass maintenance can occur, and the rate of degradation is controlled by the mass transfer of the sorbed HOCs to the microorganisms (14). Desorption rates of freshly added PAHs have been found to be faster than observed mineralization rates (17). However, desorption rates of aged PAHs have been found to be equal to or slower than observed mineralization rates (15-17), indicating that the microbial community may have been adapted to use the HOCs as rapidly as they become available (15-17). The presence of degrading microorganisms has been shown to alter the desorption rates of contaminants from sorbed surfaces (28, 29). The aim of this study was to investigate the influence of contact time on the chemical extractability and degradation of pyrene in two similar soils under sterile and nonsterile regimes.
Materials and Methods Materials. Nonlabeled and 14C-4,5,9,10-labeled pyrene (radiochemical purity >95%) were obtained from Sigma Aldrich Co. Ltd., U.K. The liquid scintillation cocktails (Ultima Gold and Ultima Gold XR) and the sample oxidizer cocktails (Carbosorb-E and Permafluor-E) were obtained from Canberra Packard, U.K. Teflon centrifuge tubes used in the sequential extraction were obtained from Merck, U.K. Soil Characteristics. Soil (Brown Earth) was collected from Meathop Wood, Cumbria, U.K. (SD 436795). A comparative soil was removed from the adjacent pasture with similar physiochemical properties (Table 1). Bulked soil samples were removed from the mineral Ah horizons (5-20 cm) over a 1-m2 area. The soils were homogenized by sieving (2 mm) to remove stones and plant roots and stored for 7 d at 4 °C in the dark. The organic matter content was determined by an acid hydrolysis followed by combustion at 450 °C for 4 h and by a Carlo Erba 1108 elemental analyzer. Soil Spiking and Microcosm Setup. Two sets of identical incubations were set up with soils (250 g) spiked with 14Clabeled (166.7 kBq kg-1) and nonlabeled pyrene (100 mg kg-1) at field moisture content (40-60% WHC) using acetone as the carrier solvent. The soils and pyrene spike were blended using a Waring commercial blender (model 35BL64). This method gave the most homogeneous distribution of the 14C label through the soil and the least disruption to the physical and biological components, after comparing several spiking procedures (30). After being spiked, the acetone was allowed to volatilize over 24 h, and the soils were stored in amber glass microcosms. Sterile microcosms were prepared in an 10.1021/es000061x CCC: $19.00
2000 American Chemical Society Published on Web 11/01/2000
TABLE 1. Physiochemical Properties of Soils mechanical analysis sand silt clay
soil
texture
pasture woodland
silt loam humose silt loam
a
Perkins elemental analyzer.
b
28 25
43 44
29 31
total
% OCa humic & fulvic
humin
pHb
CECc
WHC (%)d
CFUe
∑PAH f
pyrene f
4.5 10.0
1.3 3.4
3.2 6.6
4.6 6.1
25 19
35-40 35-40
3 × 106 1 × 107
200 400
40 80
0.01 CaCl2 (1:2). c mequiv g-1.
d
Percent of water holding capacity. e Nutrient agar. f ng g-1 (37).
identical manner and sterilized by γ-irradiation (2.5 Mrad) at Isototron plc (Bradford, U.K.) within 7 d of spiking. The sterility of the microcosms was tested using standard microbiological techniques. Determination of [14C]Pyrene Activity in Soils. A scheme of sequential extractions and oxidations was carried out in triplicate and is described below. The use of a sequential extraction scheme allowed the comparison of operationally defined chemical availability of aged [14C]pyrene with the bioavailability to a pyrene-degrading microbial inoculum. The choice of solvents used in this study was based on the chemical properties of pyrene and on the findings of previous studies (2-8, 10, 31). (i) Determination of Total Residual [14C]Pyrene Activity in Soils. At each time point, the total amount of 14C activity associated with the soil was determined by sample oxidation. The amount of 14C activity at each time point was divided by the total amount of [14C]pyrene initially added. Subsamples (1 g) of each of the soils from before and after the sequential extraction were combusted using a Packard 307 sample oxidizer. The combustion process was conducted over 3 min, aided by the addition of Combustaid (100 µL). Trapping efficiency of the sample oxidizer was determined prior to the combustion of soil samples and found to be >97%. The resultant 14CO2 was trapped, and the eluted solutions were counted by liquid scintillation counting using a Canberra Packard Tri-carb 2250CA liquid scintillation counter using standard parameters for 14C counting and automatic quench correction. (ii) Determination of [14C]Pyrene Activity Extractable into Sequential Solvents. Methanol:water (1:1, v:v) (24 mL) was added to 8 g (dry weight) of soil in 35-mL Teflon-lined centrifuge tubes. The tubes were shaken for 24 h on a tumble shaker at 100 rpm at 21 ( 2 °C and then centrifuged at 2600g for 20 min on a Beckman Centaur 2 centrifuge. The supernatant was carefully removed, the volume was determined, and a 3-mL aliquot was added to 17 mL of Ultima Gold XR scintillation fluid and counted, as described previously. The centrifuged soil pellet was loosened, and 24 mL of 1-butanol was added. The tubes were shaken for 24 h on a tumble shaker and then centrifuged, as described previously. The supernatant was carefully removed, sampled, and counted, as described previously. The soils remaining in the centrifuge tubes were carefully transferred to preweighed cellulose extraction thimbles, and the mass was determined. The thimbles were extracted with dichloromethane (DCM; 35 mL) for 3 h using a Soxtet extractor (Tecator Soxtec system HT 1043) set for a 30-min boil and a 2.5-h rinse. The extracting solution was found to consist of one phase, and its volume was determined before being sampled and counted, as described previously. DCM quantified the total extractable [14C]pyrene-associated activity remaining in association with the soil. (iii) Determination of Nonextractable [14C]Pyrene Residues in Soils. Fractionation of Soil Organic Matter. The soil (3 g dry wt) remaining after the sequential extractions was shaken with 40 mL of 0.1 M Na4P2O7:0.1 M NaOH (1:1) at a ratio of 1:20 for 24 h under nitrogen gas and then centrifuged at 2600g for 20 min. The supernatant was removed, and the
soil pellet was resuspended in 40 mL of 0.1 M Na4P2O7:0.1 M NaOH (1:1) and centrifuged a further three times until a clear supernatant was obtained. To establish the [14C]pyrene associated activity in each fraction, an aliquot of the humic and fulvic acids and a subsample of the residual humin was sample oxidized and counted, as described previously. Each fraction was dried (50 °C) until constant weight, and the organic carbon content was determined by a Carlo Erba 1108 elemental analyzer. This procedure allowed the extraction of humic and fulvic acids from the SOM, leaving the higher molecular weight humin fraction (32). Saponification of the Humin Fraction. The remaining humin fraction was subjected to a methanolic saponification (2). To the extracted soil, 2.8 mL of methanol and 0.2 mL of 2 M NaOH were added and mixed thoroughly. The samples were incubated in sealed tubes for 1 h in an oven at 95-99 °C, then allowed to cool, and centrifuged at 2600g for 20 min. The supernatant was carefully removed, and its volume was accurately determined. An aliquot (1 mL) was analyzed by liquid scintillation counting, as described previously. The release of humin-associated HOCs by saponification was used to study the binding of [14C]pyrene activity to SOM (2, 10). Mineralization of Aged [14C]Pyrene-Associated Activity in Soils. Subsamples of the soils were incubated with a pyrene-degrading inoculum, PAS1, to assess the availability of [14C]pyrene-associated activity in the soils, after 24-week contact time with the soils. PAS1 was a pyrene-degrading consortium extracted from the nonsterile pasture soil used in the experiment. The inoculum was grown in mineral basal salts medium (MBS) with pyrene as the sole carbon source (33), and the cell suspension was centrifuged at 10000g for 10 min. The cell pellet was then resuspended in quarterstrength Ringers solution (2.25 g of NaCl, 0.105 g of KCl, 0.12 g of CaCl2, and 0.05 g of NaHCO3 L-1) and centrifuged as before. The evolution of 14CO2 was followed in modified 250mL Erlenmeyer flasks (34). These incorporated a Teflon-lined screw cap and a CO2 trap containing 1 M sodium hydroxide (1 mL) loaded onto a GF/A filter paper contained within a glass scintillation vial. To each respirometer, 5 g of soil (dry wt), 15 mL of MBS, and an inoculum of PAS1 [6 × 106 cells (g of soil)-1] were added. The respirometers were shaken at 100 revertants min-1 on an orbital shaker (Janke and Kunkel, IKA-Labortechnik KS 250). The trapped 14CO2 was determined daily by adding Ultima Gold (6 mL) and subsequent liquid scintillation counting (as described previously) for 21 d after which the evolution of 14CO2 had ceased. This procedure provided data for the comparison between the chemically extractable [14C]pyrene-associated activity with that available to a pyrenedegrading consortium. Data Analysis. To compare the amount of [14C]pyrene associated activity extracted from soil after various contact times and to compare lag times and extents of mineralization, an analysis of variance was conducted. If the F-statistic from the analysis of variance showed a significant difference, Tukey least significant difference was used to determine which samples differed. VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. [14C]Pyrene-Associated Activity (%) Extracted by a Sequential Extraction Scheme from Soils over Time total 14C activity (%) of initial activity soil sterile pasture
nonsterile pasture
sterile woodland
nonsterile woodland
a
aging time (weeks)
methanol: water
1-butanol
DCM
nonextractableresidues
0 4 8 16 24 0 4 8 16 24 0 4 8 16 24 0 4 8 16 24
7.1Aa 5.1B 5.1B 4.9B 4.9B 7.1A 5.5B 4.5B 3.2B 2.2B 4.0A 3.1A 2.3A 2.1A 2.1A 4.0A 2.9B 3.2B 1.9C 2.1C
85.9A 74.6B 62.8C 59.7C 57.5C 85.9A 74.3B 54.6C 44.3D 25.5E 89.9A 74.7B 63.6C 54.9D 53.2D 89.9A 74.2B 56.9C 53.9C 50.6C
6.5A 19.0B 21.2B 22.2B 21.7B 6.5A 18.8B 21.8C 15.8C 11.1C 6.7A 20.5B 23.8B 24.2B 23.3B 6.7A 21.1B 26.9B 21.2B 20.6B
0.6A 1.4A 10.9B 13.1B 16.0C 0.6A 1.3A 9.0B 13.7C 20.0D 0A 1.7A 7.3B 11.5C 13.1C 0A 1.8A 8.0B 12.4C 17.6D
Values in columns for the same soil followed by the same letter are not statistically different (P > 0.05).
FIGURE 1. Mass balance of [14C]pyrene-associated activity in sterile pasture (b) and woodland (1) soils as well as nonsterile pasture (O) and woodland (∇) soils over 24-week soil-pyrene contact time. Values are the mean ( SD (n ) 3).
Results Temporal Changes in [14C]Pyrene Residues in Soils. The amount of [14C]pyrene-associated activity remaining in the sterile and nonsterile pasture and woodland soils was assessed after 0-, 4-, 8-, 16-, and 24-week soil-pyrene contact time, with the only significant loss (P < 0.01) of 14C activity being found in the nonsterile pasture soil. There was no corresponding loss in the nonsterile woodland soil (Figure 1). As no 14C activity was lost in the sterile pasture soil, loss from the nonsterile soils was due to the soil microflora. Determination of Temporal Changes in the Extractability of [14C]Pyrene-Associated Activity in Soils. (i) Methanol:Water Extraction. The amount of [14C]pyrene-associated activity extracted from each of the soils over 24 weeks decreased with increasing soil-pyrene contact time (Table 2). Furthermore, there was a significant decrease (P < 0.01) in the 14C activity extracted from all of the soils during the incubation. The nonsterile pasture soil displayed the greatest decrease in [14C]pyrene-associated activity extracted. Consistently less 14C activity was extracted from the sterile woodland soils as compared to the sterile pasture soils at all time points. Significantly more (P < 0.05) 14C activity was extracted from the sterile pasture soil than the nonsterile 4954
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pasture after 16- and 24-week contact time. Methanol:water was also found to extract similar amounts of 14C activity from the sterile and nonsterile woodland soils. (ii) 1-Butanol Extraction. The amount of [14C]pyreneassociated activity extracted from each of the soils decreased with increasing soil-pyrene contact time (Table 2). There was a significant decrease (P < 0.01) in the 14C activity extracted from the sterile and nonsterile pasture and woodland soils by 1-butanol over the incubation. As with the methanol:water extractions, the nonsterile pasture soil displayed the greatest decrease in [14C]pyrene-associated extraction. Interestingly, the amount of [14C]pyrene-associated activity extracted from each of the soils was very similar with the exception of the nonsterile pasture soil at 24 weeks. (iii) Dichloromethane (DCM) Extraction. The amount of [14C]pyrene extracted from each of the soils over 24 weeks significantly increased (P < 0.01) with increasing soil-pyrene contact time (Table 2). A higher proportion of 14C activity was extracted by DCM from the sterile woodland soil as compared to the sterile pasture soil. During the first 8 weeks of incubation, similar amounts of 14C activity were removed from the sterile and nonsterile pasture soils. However, after 16- and 24-week contact time, there was significantly more (P < 0.05) 14C activity extracted by DCM from the sterile as compared to that of the nonsterile pasture soil. In contrast, there were similar amounts of 14C activity removed from the sterile and nonsterile woodland soils at all time points. Determination of Nonextractable [14C]Pyrene-Associated Residues in Soils. As soil-pyrene contact time increased, there was a significant increase (P < 0.01) in nonextractable [14C]pyrene-associated residues (Table 2). After 16- and 24week soil-pyrene contact time, there was significantly smaller (P < 0.05) nonextractable residues associated with the sterile woodland soil as compared to the sterile pasture soil. In the nonsterile pasture soil, there was significantly more (P < 0.05) 14C activity as solvent nonextractable residues as compared to the nonsterile woodland soil after 16- and 24week contact time. There was a significant decrease (P < 0.05) in the total activity extracted by the sequential solvent scheme from the nonsterile soils at 24 weeks as compared to the sterile soils. However, there were higher levels of 14Clabeled residues in both of the nonsterile soils. (i) Humic and Fulvic Acid Fraction. A significant increase (P < 0.01) in the 14C activity associated with the humic and
TABLE 3. [14C]Pyrene-Associated Activity (%) Associated with Nonextractable Residues in Soils over Time
soil sterile pasture
nonsterile pasture
sterile woodland
nonsterile woodland
ageing time (weeks)
residual fraction
humic and fulvic acids fraction
humin fraction
fraction released by saponification
0 4 8 16 24 0 4 8 16 24 0 4 8 16 24 0 4 8 16 24
0.6 ( 0.5Aa 1.4 ( 0.2A 10.9 ( 1.2B 13.1 ( 0.8C 16.0 ( 0.3D 0.6 ( 0.5A 1.3 ( 0.1A 9.0 ( 1.7B 13.7 ( 0.2C 20.0 ( 1.6D 0.0 ( 0.0A 1.7 ( 0.A2 7.3 ( 1.4B 11.5 ( 0.2CD 13.1 ( 0.5D 0.0 ( 0.0A 1.8 ( 0.A3 8.0 ( 1.5B 12.4 ( 0.1C 17.6 ( 0.7D
NDb ND 3.5 ( 0.2A (32.1)c 3.8 ( 0.5A (29.0) 4.6 ( 0.1B (28.8) ND ND 3.9 ( 0.5A (43.3) 4.9 ( 0.3B (35.8) 6.8 ( 0.3C (34.0) ND ND 2.8 ( 0.2A (38.4) 1.6 ( 0.1B (13.9) 1.3 ( 0.5B (9.9) ND ND 3.5 ( 0.3A (43.8) 2.2 ( 0.4B (17.7) 3.7 ( 0.2A (21.0)
ND ND 7.4 ( 1.2A 9.3 ( 0.4B 11.4 ( 0.2C ND ND 5.2 ( 1.4A 8.8 ( 0.4B 13.2 ( 1.9C ND ND 4.5 ( 1.2A 9.9 ( 0.1B 11.7 ( 0.2C ND ND 4.6 ( 1.2A 10.2 ( 0.4B 13.9 ( 0.6C
ND ND 1.5 ( 0.4A (20.3)d 1.9 ( 0.2AB (20.4) 2.2 ( 0.3B (19.3) ND ND 1.3 ( 0.2A (25.0) 1.6 ( 0.1A (18.2) 2.4 ( 0.2B (18.2) ND ND 1.1 ( 0.3A (24.4) 1.5 ( 0.1AB (15.2) 1.9 ( 0.2B (16.2) ND ND 0.9 ( 0.1A (19.6) 1.2 ( 0.2AB (11.8) 1.8 ( 0.6B (12.9)
a Values (mean ( SD) in columns for the same soil followed by the same letter are not statistically different (P > 0.05). b Not determined. c The numbers in parentheses represent the percentage of the humic and fulvic acid fraction in the residual fraction. d The numbers in parentheses represent the percentage of the saponification fraction in the humin fraction.
fulvic fraction with increasing contact time was found in the sterile and nonsterile pasture soils (Table 3). After 16- and 24-week contact time, there was significantly more (P < 0.05) 14C activity in the humic and fulvic fraction of the sterile pasture soil as compared to the sterile woodland soil. However, the percentage of humic and fulvic fraction relative to the total residues actually decreased in all of the soils from 8- to 24-week soil-pyrene contact time. The sterile pasture and woodland soils were also found to have significantly less (P < 0.05) 14C activity associated with the humic and fulvic fraction of the SOM as compared to the nonsterile pasture and woodland soils, respectively, after 16- and 24-week contact time. (ii) Humin Fraction. The [14C]pyrene-associated activity found in the humin fraction was found to significantly increase (P < 0.01) in all the soil incubations over 24 weeks (Table 3). In the sterile soils, a similar amount of 14C activity was associated with the humin fraction of the pasture and woodland soils after 16- and 24-week contact time. Also, there was a significant difference (P < 0.05) in the 14C activity associated with the humin fractions of the sterile and nonsterile pasture soils. The soil humin was subjected to a methanolic saponification resulting in [14C]pyrene associated activity being released from all the soils after 8-week soil-pyrene contact time (Table 3). There was consistently less 14C activity released from the sterile woodland soil as compared to the pasture soil, although no significant (P > 0.05) difference was detected. However, there was a significant increase (P < 0.01) in the 14C activity released from the nonsterile pasture soil between 8- and 24-week contact time. Furthermore, in the nonsterile pasture soil there was significantly more (P < 0.05) 14C activity released as compared to the nonsterile woodland soil after 8-, 16-, and 24-week contact time. In both the sterile and nonsterile pasture soils, there was a decrease in the saponified content of the humin fraction between 8 and 24 weeks. However, in the sterile and nonsterile woodland soils, there was only a decrease in the saponified fraction between 8 and 16 weeks. After 24-week contact time, there was no significant difference (P > 0.05) in the amounts of 14C activity released from the nonsterile soils as compared to the sterile soils.
Determination of [14C]Pyrene Bioavailability. The lag time before significant mineralization (>5% of 14CO2 evolution) occurred was significantly shorter (P < 0.05) in the sterile pasture soil, which was 6 days, as compared to 13, 11, and 12 d in the nonsterile pasture and sterile and nonsterile woodland soils, respectively. After 21 d, 18.9, 7.6, 15.6, and 15.7% of the added [14C]pyrene has been mineralized in the sterile and nonsterile pasture and woodland soils, respectively. There was no significant (P > 0.05) difference in the extent of mineralization between the sterile pasture and woodland soils. However, in the nonsterile pasture soil, significantly less (P < 0.05) 14C activity was mineralized as compared to the nonsterile woodland soil. Further, there was significantly more (P < 0.05) 14C activity mineralized in the sterile pasture soil as compared to the nonsterile pasture soil.
Discussion Impact of Contact Time on the Behavior of Pyrene in Soils. The pasture and woodland soils used in this study were found to contain low background levels of pyrene as well as other PAHs (Table 1). These findings combined with a detailed history of the site, indicating no large-scale nearby sources of PAHs (35) and the rural position 200 m downwind from the sea, support the assumption that the site has not previously experienced high levels of PAHs. The addition of 100 mg of pyrene (kg of soil)-1 resulted in significant removal (P < 0.01) of [14C]pyrene only in the nonsterile pasture soil during the 24-week incubation (Figure 1). Similar development of HOC catabolic ability has been found in other pristine soils when exposed to similar levels of HOC addition (10). The influence of an active microbial community on the extractability and bioavailability of the added [14C]pyrene is discussed further in this paper. In this study, the influence of soil-pyrene contact time was examined in two similar soils under sterile and nonsterile regimes. The findings that the extractability of pyrene by methanol:water decreased with increasing contact time agrees with previous studies (5, 7). Methanol:water and similar polar extractants have been shown to remove nonpolar parent compounds and more polar metabolites associated with the aqueous phase of the soil (5, 31). In this VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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study, however, only small amounts of [14C]pyrene-associated activity were removed by methanol:water during the incubation, which was attributed to the low solubility of pyrene. 1-Butanol removed the largest amount of [14C]pyreneassociated activity from all of the soils. It has been suggested that 1-butanol removes the rapidly desorbing fraction of HOCs in the soil (5, 7). In this study, the significant aging trend in decreasing amounts of [14C]pyrene-associated activities extracted by 1-butanol, with increased soil-pyrene contact time, mirrors the findings of other studies (5, 7). Harsh extractants, such as DCM, are generally used to quantify the total amount of HOC in the soil in a single extraction step. In this study, DCM was used as a final sequential extractant on methanol:water and 1-butanol preextracted samples. Interestingly the amount of [14C]pyreneassociated activity extracted by DCM increased significantly (P < 0.01) during the initial aging period in all the soils, indicating that a fraction of the added pyrene becomes more strongly sorbed during this period (13). This suggests the time-dependent formation of more strongly soil-associated residues, further supporting the aging phenomenon (1-7). The two soils used in this study had different SOM contents. Methanol:water removed significantly more (P < 0.01) [14C]pyrene-associated activity from the pasture soil as compared to the woodland soil, suggesting that the 100 mg kg-1 dose of pyrene resulted in more freely available pyrene in the pasture soil due to its lower SOM content. It has been suggested that there are a finite number of sorption sites to sequester added HOCs per unit of organic carbon in soils and sediments (7, 25). The woodland soil had a larger amount of organic carbon as compared to the pasture soil (Table 1), indicating that it may have a larger capacity to sequester added HOCs (7, 25). This hypothesis is supported by the findings that lower levels of pyrene (35.5 µg kg-1) addition to the pasture and woodland soils resulted in less [14C]pyreneassociated activity being extracted by methanol:water and 1-butanol as compared to higher levels (100 mg kg-1) used in this study (36). The influence of active microbial communities on the aging of persistent organic pollutants in the soil is an area of growing interest (8, 17). The use of methanol:water to extract freely available and more polar metabolites of PAHs has been proposed (5). The extractability of pyrene and its metabolites from nonsterile pasture soil was found to decrease faster than the sterile pasture soil, indicating that the presence of microbes resulted in the formation of a larger nonextractable residual fraction. Currently, the influence of microorganisms on the formation of solvent nonextractable residues is not fully understood (8, 10). This study found extensive formation of solvent nonextractable residues with increased soil-pyrene contact in all the soils. There is much interest in the formation of solvent nonextractable residues, since these are not thought to be bioavailable (3-7). The extent of residue formation, reported here, agrees with previous studies, which found the formation of extensive solvent nonextractable residues after similar aging periods (1, 2, 10-12). Recently, the nature of SOM has been well characterized, containing an expanded (rubbery) phase and a condensed (glassy) phase (13, 22-25). Since the solvents used to determine the extent of the residues only extracted a small amount of the SOM, the [14C]pyrene-associated activity within these two phases was studied. Current thinking suggests that the humic and fulvic fraction equates to the expanded SOM, which controls the rapidly desorbing phase (13, 22-25). Furthermore, alkaline extraction of SOM results in the removal of humic and fulvic acids, leaving the higher molecular weight humin fraction (32). In this study, the baseextracted fraction contained less [14C]pyrene-associated activity than that found in the humin fraction. However, there 4956
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was about twice the amount of OC associated with the humin as compared to the humic and fulvic fraction in both soils (Table 1). Interestingly, there were significant increases (P < 0.05) in the 14C activity released from the nonsterile pasture soil as compared to the sterile pasture soil, indicating that microbial degradation of pyrene resulted in the formation of more polar metabolites associated with this fraction (9). The relevant distribution of OC and 14C activity in the humic and fulvic and humin fractions was similar to those reported in previous studies (8, 9). The humin fraction is thought to be perforated with subnanometer size holes; these may operate as sorption sites for HOCs, such as PAHs (7, 13, 25). White et al. (7) found that the removal of humic acid fraction caused a decrease in the initial rate of phenanthrene desorption from two soils with contrasting SOM contents, although not a corresponding decrease in bioavailability as determined by biodegradation. Release of humin-associated HOCs by saponification has been used to study the binding of HOCs to the SOM (2, 10). In this study, methanolic saponification of the humin resulted in the significant release of [14C]pyrene-associated activity. Eschenbach et al. (2) found that a significant portion of PAHs could be recovered by saponification following a harsh solvent extraction. Even though the woodland soil was found to have a larger amount of OC released by saponification, there was no associated increase in 14C activity released as compared to the pasture soil. Richnow et al. (10) proposed that nonsolvent extractable residues are due to the covalent bonding of PAH metabolites to the SOM. However, when the ester/covalent bonds were cleaved, only a small proportion of the 14C activity was released (10). The interesting finding that there was no significant difference (P > 0.05) between the sterile and the nonsterile pasture soils indicates that putative microbial metabolites of pyrene did not form ester/ covalent bonds with the soil humin. Pyrene Bioavailability. The bioavailability of HOCs in soils has been shown to decrease with increasing soil-HOC contact time (4-7). This study found significantly less mineralization of the added [14C]pyrene after 24 weeks in the nonsterile pasture soil as compared to the other soil treatments. This difference was attributable to the removal of available [14C]pyrene in the nonsterile pasture soil by its incumbent microflora (Figure 1). In comparing the amount of [14C]pyrene-associated activity mineralized and extracted by the sequential extraction, we can say that the bioavailable fraction was greater than the amount extracted by methanol: water but less than that extracted by 1-butanol. Previous studies have indicated that mild chemical extractants, such 1-butanol, can account for the bioavailable fraction of aged HOCs in soil (5, 6). However, a recent study reported that 1-butanol overestimated the microbially available fraction of PAHs in aged soils (11). In agreement with this paper, our results indicated that the methanol:water extraction underestimated the microbially available fraction of aged [14C]pyrene-associated activity, while the 1-butanol extraction overestimated this fraction in the aged soils.
Acknowledgments This work was funded by NERC, U.K. (GR4/96/113 and GR9/ 03281). We thank P. Ineson, B. J. Reid, and G. L. Northcott for discussions relating to the design of the experiment and K. C. Jones for assistance with reviewing the manuscript. We are also grateful to M. Howsam for the PAH analysis of the soils.
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Received for review March 20, 2000. Revised manuscript received August 23, 2000. Accepted August 30, 2000. ES000061X
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