Environ. Sci. Technol. 2000, 34, 3174-3179
Nonexhaustive Cyclodextrin-Based Extraction Technique for the Evaluation of PAH Bioavailability B R I A N J . R E I D , * ,† J O A N N A D . S T O K E S , ‡ KEVIN C. JONES,‡ AND KIRK T. SEMPLE‡ School of Environmental Sciences, University of East Anglia, Norwich, NR47TJ, U.K. and Department of Environmental Science, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, LA1 4YQ, U.K.
Traditionally, soil extraction techniques have been concerned with the determination of “total” organic contaminant concentrations, following an “exhaustive” extraction. However, in light of the increasing body of knowledge relating to organic contaminant availability and aging, such methods have little relevance to the amount of contaminant that may pose an ecological risk i.e., the “bioavailable” portion. Less exhaustive techniques have therefore been the subject of more recent approaches in the hope that they may access the “labile” or bioavailable pool. The use of an aqueous-based extraction technique utilizing hydroxypropyl-β-cyclodextrin (HPCD) is presented here for the extraction of PAHs from soil. The optimization of the method is described in terms of HPCD concentration, extraction time, and solution buffering. The procedure is then tested and validated for a range of 14C-labeled PAHs (phenanthrene, pyrene, and benzo[a]pyrene) added at a range of concentrations to a range of soil types. The amounts of soilassociated phenanthrene mineralized by catabolically active microorganisms were correlated with total residual phenanthrene concentrations (r 2 ) 0.889; slope of best fit line ) 0.763; intercept ) -5.662; n ) 24), dichloromethane (DCM)-extractable phenanthrene concentrations (r 2 ) 0.986; slope of best fit line ) 0.648; intercept ) 0.340; n ) 24), butan-1-ol (BuOH)-extractable phenanthrene concentrations (r 2 ) 0.957; slope of best fit line ) 0.614; intercept ) 0.544; n ) 24), and HPCD-extractable phenanthrene concentrations (r 2 ) 0.964; slope of best fit line ) 0.997; intercept ) 0.162; n ) 24). Thus, in this study, the microbially bioavailable concentrations of soil-associated phenanthrene were best predicted using the optimized HPCD extraction technique. In contrast, the DCM Soxhlet extraction and the BuOH shake extraction both overestimated phenanthrene bioavailability by, on average, >60%.
Introduction Traditionally, soil extraction techniques have been concerned with the determination of “total” organic contaminant concentrations in soils and sediments. Soils have therefore usually been “exhaustively” extracted, for example, by Soxhlet and saponification procedures (1, 2). However, in light of the increasing body of knowledge relating to contaminant * Corresponding author tel: +44 (0) 1603 592357; fax; +44 (0)1603 507719; e-mail:
[email protected]. † University of East Anglia. ‡ Lancaster University. 3174
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availability and aging, such methods may have little relevance in measuring the proportion of contaminant that may pose an ecological risk i.e., the “bioavailable” fraction (3-6). Less exhaustive techniques have therefore been the subject of more recent approaches in the hope that they may access the “labile” or bioavailable pool (3, 5, 6). For example, Kelsey et al. (5) correlated the extraction of phenanthrene using butan-1-ol (BuOH) against bacterial mineralization and earthworm uptake (extraction with and without agitation, respectively). This extraction technique did not however provide a good correlation for all of the pollutants tested. Other extractants were found to be more appropriate in other cases, e.g., a 1:1 methanol:water mixture for atrazine bioavailability evaluation to microbes and a 9:1 methanol:water mixture for atrazine bioavailability valuation to earthworms (5). While the use of less exhaustive techniques may be more appropriate than exhaustive extraction for contaminated soil risk assessments, they may still poorly mimic the processes inherent to bioavailability. Since bioavailability is not an allencompassing term but rather is organism- (3, 5, 6) and indeed species-specific (7, 8), a chemical method to mimic bioavailability must be shown to correlate with uptake or degradation by a specific organism or assay. The method presented here is tested for its ability to correlate with bioavailability to microorganisms capable of degrading the test compound. It has been proposed that contaminant mass transfer, from soil solid phase to solution, governs microbial bioavailability (4, 9-12), in particular, the size of the rapidly desorbable fraction (13). The foundation of this proposal is that substrate uptake is far more extensive from fluid than from sorbed states (14, 15). Since nonpolar organic contaminants have low aqueous solubilities and strong affinities for soil organic matter, at any one time only a very small proportion of a given compound will be present in the soil solution. Thus, water itself is a poor choice of solvent for bioavailability assessment as a large labile (potentially bioavailable) pool of chemical will be present on the soil solid phase. The ideal extractant is therefore one that can access the entire labile fraction of the soil-borne contaminant. A method is described, optimized, and tested here for the extraction of three polycyclic aromatic hydrocarbons (PAHs)s phenanthrene, pyrene, and benzo[a]pyrenesat a range of concentrations and from a variety of soil types using aqueous hydroxypropyl-β-cyclodextrin solutions (HPCD). Performance of the HPCD extraction technique was then compared with other well-known/published techniques, and correlations between soil-associated compound extractability and mineralization were determined. Cyclodextrins are a group of macrocyclic contaminants comprising a torus of R-1,4-linked glucose units (16). These molecules have high aqueous solubilities because of the array of hydroxyl functional groups on the exterior of the torus. They also have a hydrophobic organic cavity in the interior, which is approximately 6.5 Å in diameter for β-cyclodextrin (16). It is therefore possible to form a 1:1 inclusion complex between the cyclodextrin macromolecule and an organic moiety (16-18). Aqueous solutions of cyclodextrin have been used to dissolve a range of contaminants of low aqueous solubility to greater than their aqueous solubility limits (e.g., phenanthrene, 2-methylphenanthrene, fluorene, 1-methylfluorene, 1,2-benzofluorene, and β-bromonaphthalene (16-18)). It has been shown that molecules that are too large to form 1:1 inclusion complexes with β-cyclodextrin can form 1:2 inclusion complexes, consisting of two macrocycles and one guest (19, 20). Alternatively, a larger cyclodextrin macrocycle can be used to accommodate larger guests (21). 10.1021/es990946c CCC: $19.00
2000 American Chemical Society Published on Web 06/30/2000
We hypothesized that an aqueous solution of cyclodextrin can be used to extract labile soil-borne nonpolar organic contaminants while strongly bound or sequestered molecules will not readily transfer to the aqueous phase and will therefore not be extracted. Thus, contaminant mass transfer mechanisms inherent to this extraction technique closely mimic the processes that dictate microbial bioavailability. It is proposed that this extraction may access the microbially bioavailable fraction of soil-borne nonpolar organic contaminants and, once fully tested and understood, may provide a chemical extraction technique to reliably determine the bioavailable portion of compound in soils and sediments.
Materials and Methods The paper is subdivided as follows: Experiment A. Optimization of the HPCD extraction procedure in terms of (i) HPCD concentration, (ii) extraction time, and (iii) buffering/pH. Experiment B. Testing the optimized procedure on (i) three different PAHs at a range of concentrations and (ii) three different soil types. Experiment C. Correlation of microbial contaminant bioavailability with total phenanthrene concentration and concentrations of phenanthrene extractable using dichloromethane, BuOH, and the optimized HPCD technique. Chemicals. Phenanthrene, pyrene, and benzo[a]pyrene and their 14C-radiolabeled analogues ([14C-9]-phenanthrene, [14C-4,5,9,10]-pyrene, and [14C-7]-benzo[a]pyrene) (radiochemical purity >98%, except for pyrene (= 95%)) were obtained from Sigma Aldrich Co. Ltd., U.K. Ethanol and toluene were obtained from Merck, U.K., and Rathburn Chemicals Ltd., U.K., respectively. HPCD was obtained from the Aldrich Chemical Co., U.K. KH2PO4 and K2HPO4 were obtained from Merck, U.K. Sample oxidizer scintillation cocktails (Carbosorb-E and Permafluor-E); the organic based combustion aiding solution (Combustaid); and the scintillation cocktails Ultima Gold and Ultima Gold XR were all obtained from Canberra Packard, U.K.
Experimental Section Soil. Soil (Dystrochrept) was collected from a rural hillside environment (Lancaster University, Hazelrigg, Field Station, U.K.; O.S. sheet 97 (493578)) below the root zone (5-15 cm) and used to conduct most of the experiments. Field moist soil was passed through a 10-mm sieve and air-dried (for 14 d). The soil was then passed through a 2-mm sieve to remove roots and other vascular material. The organic content of the soil was established to be 3.74 ( 0.15% (analysis by Carlo Erba EA1108 elemental analyzer, after inorganic carbon acid digestion). The water holding capacity (relative to soil dry weight) was 61.2%, and the pH was 5.1. Soil Spiking. Phenanthrene as both its nonradiolabeled and 14C-radiolabeled analogue ([14C-9]-phenanthrene) was introduced into the soil by a single-step spiking/rehydration procedure (22). A phenanthrene concentration of 10 mg kg-1wet soil, indicative of a marginally contaminated soil and a concentration used in other studies (23), was used. Moist soil (250 g) was generated by all spiking events (70% water holding capacity). PAH standards were prepared using a mixture of toluene and ethanol (to ensure PAH solubilization) such that 1 µL would deliver the required amount of contaminant per gwet soil and an activity of approximately 50 Bq g-1wet soil. By adding the phenanthrene to the water (in the blender) before the soil was added, greater compound homogeneity in the spiked soils was achieved (22). Soils were not sterilized in any part of this study. Assessment of Total Activity. The 14C-spiked soils were left for 1 d to allow the contaminant to become incorporated/ associated with the soil. After this conditioning period, samples (6 × 1 g) were removed for analysis and packed into
paper combustion cones. The samples were then combusted using a Packard 307 sample oxidizer. Carbosorb-E and Permafluor were used to trap CO2 and as a scintillant, respectively. The combustion process was conducted over 3 min, aided by the addition of Combust-aid (100 µL) injected onto the samples prior to oxidation. Trapping efficiency of the sample oxidizer was assessed prior to the combustion of soil samples and was >97%. The resultant solutions were counted using a Canberra Packard Tri-Carb 2250CA liquid scintillation analyzer for 10 min. Experimental blanks were prepared using rehydrated dry soil, to which no solvent or PAH had been added. Experimental Section A. Experiment A1: Optimization of HPCD Extraction Concentrations. HPCD solutions were prepared using Milli-Q water to provide a range of concentrations from 0 to 60 mM. Triplicate samples of [14C]phenanthrene-spiked soil (1.25 g) were weighed into Teflon centrifuge tubes (35 mL capacity), and HPCD solution (25 mL) was added to each. Samples containing unspiked soil were also prepared to provide analytical blanks. The tubes were sealed and placed on their sides on an orbital shaker (Janke and Kunkel, IKA-Labortechnik KS 250) and shaken at 150 revertants min-1 for 20 h. The tubes were centrifuged at 27000g (using a Beckman JA 21/2 centrifuge). The supernatants were then sampled (6 mL) and added to Ultima Gold XR scintillation fluid (14 mL). The 14C-labeled radioactivity in the resultant solutions was counted as described previously. A mass balance was determined on completion of the extraction by sample oxidation of the residual pellet after removal of the supernatant (correction was made for residual extraction solution in the pellet). Experiment A2: Optimization of Extraction Time. To ensure that the extraction procedure had been given sufficient time to come to completion, an extraction efficiency time series was determined. A solution of HPCD was prepared using Milli-Q water to provide a concentration of 50 mM (excess for optimal extraction at 20 h). Soil was then extracted as described earlier, but the extraction was terminated after 3, 6, 12, 18, and 24 h. Experiment A3: The Influence of pH Buffering. A phosphate buffer of pH 8 was prepared by combining KH2PO4 (0.2 M) and K2HPO4 (0.2 M) solutions in a ratio of 1:17.9. HPCD solutions were prepared in phosphate buffer to give concentrations in the range of 0-20 mM. Extraction of soilassociated phenanthrene and quantification of the extracted activity was conducted in an identical manner to that employed in the unbuffered extractions (A1). The soil was also extracted (in triplicate), using water only and phosphate buffer only, to provide control values. Experimental Section B. Experiment B1: Testing the Optimized Procedure on Different PAHs at a Range of Concentrations. Soil was spiked as before by a single step spiking/rehydration procedure (22) with phenanthrene, pyrene, and benzo[a]pyrene and left for 1 d prior to analysis. Phenanthrene was spiked into the soil at concentrations of 25, 50, 100, and 200 mg kg-1. Pyrene was spiked into the soil at concentrations of 12.5, 25, 50, and 100 mg kg-1. Benzo[a]pyrene was spiked into the soil at concentrations of 6.25, 12.5, 25, and 50 mg kg-1. In all cases, both nonradiolabeled and 14C-radiolabeled analogues were introduced to provide an activity of approximately 50 Bq g-1wet soil. The spiked soil (1.25 g) was then extracted (in triplicate) using the optimized technique (determined in section A, i.e., using 50 mM HPCD, in Milli-Q water for an extraction time of 20 h). The resultant extracts were handled and analyzed as described previously. Experiment B2: Testing the Optimized Procedure on a Range of Soil Types. Three contrasting soil types (Table 1) were spiked with phenanthrene to a concentration of 25 mg kg-1. After 1 d, the soils (1.25 g) were then extracted (in triplicate), as described previously, using HPCD solutions VOL. 34, NO. 15, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Physical and Chemical Characteristics of the Contrasting Soil Types Used in This Investigation soil location
USDA soil taxonomy
sand (%)
silt (%)
clay (%)
organic carbon (%)
pHa
WHCb (% WHC used)
generic description
Countesswells Burren Insch
Haplorthod Rendoll Dystrochrept
69.9 52.1 57.7
9.8 43.2 18.9
16.2 4.7 20.6
3.8 24.5 2.9
5.9 7.8 6.4
25.5 (50) 52.6 (50) 68.2 (50)
sandy, low OCc sandy-silt, high OCc clay, low OCc
a
pH determined in water.
b
WHC, water holding capacity (% water of dry wt). c Organic carbon.
produced in Milli-Q water for 20 h. HPCD concentrations of 50, 70, and 90 mM were used to ensure that an excess of HPCD was present. The resulting extracts were handled and analyzed as described previously. Experimental Section C: Correlation of Microbial Contaminant Bioavailability with Total Phenanthrene Concentrations and Concentrations of Phenanthrene Extractable Using Different Solvents/Extraction Techniques. Soil Spiking, Storage, and Sampling. Soil was spiked as described previously (22) to obtain phenanthrene concentrations of 25 and 50 mg kg-1wet soil. The soils were aged in sealed amber glass jars (in the dark) for 1, 42, 84, and 322 d. After these aging times, the soils were analyzed as outlined below. Determination of Total Residual Phenanthrene Concentrations. Samples (6 × 1 g) were packed into paper combustion cones. The samples were then combusted and analyzed as described previously. Determination of Phenanthrene Residues Extractable Using Different Solvents/Extraction Techniques. Soil was extracted into DCM by Soxhlet extraction (1) and into BuOH (3-6) and aqueous solutions of HPCD by shake extraction. Soxhlet extraction represents a harsh, exhaustive method of extraction (1), while the other two extractions methods are nonexhaustive. Alexander and co-workers have proposed the BuOH extraction method as a means of determining the bioavailabile fraction of soil-associated phenanthrene, while the aqueous HPCD extraction method is proposed here to be more appropriate for this purpose. Determination of Phenanthrene Residues Extractable into Dichloromethane. Soil (2.5 g) (in triplicate) was ground with sodium sulfate (10 g). The samples were then transferred to cellulose extraction thimbles and extracted for 3 h (30 min boil, 150 min rinse) into DCM (initial volume 40 mL) using a Tecator Soxhlet extraction system. The resultant extracts were made up to a total volume of 40 mL using DCM, and a 10-mL aliquot was added to Ultima Gold XR liquid scintillation fluid (10 mL). The activity in the resultant samples was determined by liquid scintillation counting (as described previously). To obtain a mass balance, the residual pellets were weighed (after overnight drying), and a portion of the residue (1.6 g) sample was oxidized and analyzed as previously described. Determination of Phenanthrene Residues Extractable Using BuOH. This method was adapted from the work of Alexander and co-workers (3, 5, 6). Triplicate samples from each soil system (3.5 g) were placed in polycarbonate centrifuges tubes. BuOH was then added to the tubes (35 mL). The tubes were sealed, placed on their sides on a flat bed shaker (Janke and Kunkel, IKA-Labortechnik KS 250), and shaken at 150 revertants min-1 for 12 h. The tubes were then centrifuged at 10000g (using a Centaur 2, MSE centrifuge). The supernatants were sampled (10 mL) and added to Ultima Gold XR scintillation fluid (10 mL). The radioactivity in the resultant solutions was determined by liquid scintillation counting (described previously). A mass balance could not be determined by sample oxidation due to the high flammability of BuOH in the pellets. Determination of Phenanthrene Residues Extractable Using Aqueous Solutions of HPCD. Soil samples (1.25 g) (in triplicate) were extracted using the optimized HPCD extraction tech3176
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nique, i.e., 50 mM HPCD solution (25 mL) over a 20-h extraction time. Determination of Phenanthrene Bioavailability Using Respirometry. Mineralization assays were conducted to assess contaminant bioavailability in modified Erlenmeyer flasks (250 mL) (adapted from ref 24). These incorporated a Teflonlined 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 (7 mL). Soil from each of the soil systems (10 g) was added to the flasks, and an inorganic minimal basal salts solution (MBS) (25 mL) (25) was added. An inoculum (5 mL) of phenanthrene-degrading bacteria (105-107 bacteria g-1wet soil), identified as a Pseudomonas sp. was then added to the slurries. 14C-Labeled activity in the traps was assessed daily by the addition of Ultima Gold liquid scintillation fluid (6 mL) and subsequent liquid scintillation counting (as described previously). Cumulative mineralization was determined, and sampling continued until 14CO2 evolution had plateaued (10 d). The phenanthrene-degrading pseudomonad was obtained by an enrichment culture technique from a sample of PAH-contaminated soil using MBS (25). The MBS contained phenanthrene (100 mg L-1) as the sole carbon source. The strain was isolated by growth on MBS-phenanthrene agar plates (phenanthrene was added to the molten agar prior to the plates being poured). The plates were incubated at 23 °C for 7 d. A microbial inoculum was prepared for the following experiment by transferring a single colony from the MBS-phenanthrene plates into MBS-phenanthrene liquid media (phenanthrene 100 mg L-1). After 4 d of incubation on an orbital shaker (100 rpm), the culture was centrifuged at 10000g for 10 min. The supernatant was poured off, and the cells were resuspended in fresh MBS (repeated twice).
Results and Discussion Experiment A1: Optimization of HPCD Extraction Concentrations. With increasing concentrations of HPCD, there were corresponding increases in the amounts of phenanthrene extracted from the soil up to 40 mM HPCD (Figure 1A). Above a HPCD concentration of 40 mM, there was no additional increase in extraction efficiency (Figure 1A). The maximum amount of soil-associated phenanthrene extracted (average of values using 40, 50, and 60 mM HPCD) was 68.4 ( 7.2 (n ) 9) after 1 d aging in the soil. Sample oxidation of the residual pellets, after extraction at excess concentrations of HPCD, indicated that all of the introduced contaminant could be accounted for (mass balance ) 100 ( 10.6%; n ) 21). Experiment A2: Optimization of Extraction Time. There was an initial increase in extraction efficiency with time up to 6 h (Figure 1B). After 6 h, the extraction efficiency plateaued and no more compound could be removed after 24 h extraction than after 6 h extraction. To ensure extractions with other soils and compounds were complete, an extraction time of 20 h was employed in the other parts of this investigation. Experiment A3: Optimization of Buffering and pH Conditions. As observed in the unbuffered HPCD extraction, there was a corresponding increase in the extent of extraction with increasing HPCD concentration when the phosphate buffer (containing HPCD) was used (data not plotted). However, the buffered extraction required a far lower HPCD
FIGURE 1. Optimization of soil-associated phenanthrene (spiked concentration, 10 mg kg-1wet weight): (A) using increasing concentrations of aqueous HPCD solutions, over 20-h extraction period; (B) over increasing extraction time, using aqueous HPCD solutions (50 mM). concentration (20 mM) than the unbuffered extraction (50 mM) before extraction efficiency plateaued. The extent of extraction where the HPCD phosphate buffer extraction was used was 103 ( 11%, significantly greater that the 68.4 ( 7.2% extraction where the unbuffered HPCD extraction was used. The phosphate buffer alone (i.e., without HPCD) removed 18.8 ( 0.7% of soil-associated phenanthrene and basically accounted for most of the difference in extraction efficiencies. The buffered extracts were highly colored; their absorbance at 440 nm (using a Pye-Unicam PU8610 UV/vis kinetics spectrophotometer) was 35 times higher for the soil extracts obtained using the phosphate buffer (with HPCD) relative to soil extracts obtained using water (with HPCD). Color was indicative of the organic matter content in the extracts (26). Nonpolar organic molecules interact strongly with soil organic matter (27). Given the extraction of organic matter from the soil under buffered conditions, the use of unbuffered (water only) HPCD solutions are advocated. Experiment B1: Testing the Optimized Procedure on Different PAHs at a Range of Concentrations. PAH concentration did not appear to alter the proportion (%) of soil associated contaminant extracted (after 1 d of aging) (Figure 2A). In the case of phenanthrene, 71.8 ( 6.3% (average of all applied concentrations) was extracted over almost an order of magnitude range in concentration (25-200 mg kg-1). In the case of pyrene, a lower extraction efficiency of 35.4 ( 4.8% (average of all applied concentrations) was observed, again over almost an order of magnitude range in concentration (12.5-100 mg kg-1). The lower extraction efficiency in the case of pyrene may be attributable to a lower availability of the contaminant for transfer to the aqueous phase. Lower availability/bioavailability has been suggested by others to be governed by molecular properties such as aqueous solubility and KOW (28, 29). In the case of benzo[a]pyrene, an even lower extraction efficiency of 14.1 ( 2.8% (average of all applied concentrations) was observed, again over almost an order of magnitude range in concentration (6.25-50 mg kg-1). This even lower extraction efficiency for benzo[a]pyrene may in part be attributable to a lower availability of the contaminant for transfer to the aqueous phase as governed by its aqueous solubility and KOW value (28, 29). Another influencing factor may have been that the HPCD cavity is
FIGURE 2. Testing the optimized HPCD extraction procedure: (A) extraction of a range of PAHs present in soil at a range of concentrations, using 50 mM HPCD in water: phenanthrene (white bars), pyrene (black bars), and benzo[a]pyrene (hatched bars). (B) Extraction of phenanthrene present in a range of soil types at a concentration of 25 mg kg-1wet weight using a range of HPCD concentrations in water, over a 20-h period. Soil type (see Table 1): Countesswells (white bars), Burren (black bars), and Insch (hatched bars). too small to fully accommodate the benzo[a]pyrene molecule (17). Thus, the extraction efficiency would be much lower due to steric restraints (17). However, as noted earlier, molecules that are too large to form 1:1 inclusion complexes with β-cyclodextrin have been shown to form 1:2 inclusion complexes, consisting of two macrocycles and one guest (19, 20). It is possible that 1:2 inclusion complexes formed under these experimental constraints also. It is likely that larger cyclodextrin cavities, e.g., γ-cyclodextrin, would be more suitable for the extraction of larger molecules. The 1:1 inclusion complexes between, for example, γ-cyclodextrin and benzo[a]pyrene have been reported (21). In summary, the data in this section indicates that reproducible and consistent extraction efficiencies are obtained using the extraction procedure. However, steric restraints may limit the application of the technique for the extraction of larger molecules. Experiment B2: Testing the Optimized Procedure on a Range of Soil Types. After 1 d soil-compound contact time, the type of soil with which phenanthrene was associated did influence the amount of compound extracted (Figure 2B). However, phenanthrene was extracted to the same extent where 50, 70, and 90 mM HPCD solutions were used, from all of the soils. The Countesswells (sandy soil with low organic matter content (Table 1)) yielded the highest extraction efficiency (89.1 ( 1.2%) (average of all samples) of the soils tested. The Burren (high silt fraction low organic matter content) and Incsh (high clay fraction with high organic matter) had lower extents of extraction than the Countesswells soil (70.6 ( 13.6% and 74.8 ( 7.9%, respectively) (average of all samples). The lower extraction efficiencies are consistent with what might be expected in relation to sorbent pore size and soil organic matter (13, 30, 31). For example, Hatzinger and Alexander (30) noted that contaminant entrapment within nanopores greatly influenced microbial bioavailability. VOL. 34, NO. 15, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Correlation of phenanthrene mineralization (as determined using catabolically active microorganisms) with (A) total residual phenanthrene concentration; (B) Soxhlet DCM-extractable phenanthrene; (C) butan-1-ol-extractable phenanthrene; and (D) HPCD-extractable phenanthrene. Correlation of Soxhlet DCM-extractable phenanthrene with (E) butan-1-ol-extractable phenanthrene and (F) HPCD-extractable phenanthrene. Assessments were made after 1 (b), 42 (9), 84 (2), and 322 d (() for soil systems originally spiked to concentrations of 25 mg kg-1wet weight (black) and 50 mg kg-1wet weight (white). The dashed lines on each plot are of a 1:1 slope. The solid lines on each plot are the lines of best linear fit for the data points. Cornelissen et al. (13) indicated that soil organic matter significantly increased the size of the slowly desorbing pool of PCBs and chlorobenzenes. Similarly Brusseau et al. (31) provided strong evidence that intraorganic matter diffusion was responsible for the nonequilibrium sorption exhibited by a wide range of hydrophobic organic chemicals. Experiment C: Correlation of Microbial Contaminant Bioavailability with Total Phenanthrene Concentrations and Concentrations of Phenanthrene Extractable Using Different Solvents/Extraction Techniques. Linear correlations were calculated between the amounts of mineralized phenanthrene and total residual phenanthrene concentrations (r 2 ) 0.889) (Figure 3A), Soxhlet DCM-extractable 3178
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phenanthrene concentrations (r 2 ) 0.986) (Figure 3B), BuOHextractable phenanthrene concentrations (r 2 ) 0.957) (Figure 3C), and HPCD-extractable phenanthrene concentrations (r 2 ) 0.964) (Figure 3D). Clearly these plots indicate strong correlation in all cases. However, these correlations alone do not provide sufficient information on which to establish the suitability of an extractant to determine the mineralization of soil-associated organic contaminants. To determine the mineralizable fraction of a soil-associated contaminant reliably by a chemical extraction technique, the slope and the intercept of the best-fit line must be considered. If direct prediction of soil-associated organic contaminant bioavailability is to
be made by a chemical means, an intercept of 0 and a slope of 1 for the line of best fit are ideal. The correlation between total residual phenanthrene concentration and mineralization gave an intercept of -5.662 and a slope of 0.763 (n ) 24) (Figure 3A). As can be seen in Figure 3A, total residual concentrations of soil-associated phenanthrene overestimated mineralization, both in the early and later stages of aging. Where correlation between DCM-extractable phenanthrene concentrations and mineralization was made, an intercept of 0.340 and a slope of 0.648 were determined (n ) 24) (Figure 3B). In contrast to the correlation between total residual phenanthrene concentrations with mineralization, the Soxhlet DCM extraction indicated good agreement between amounts mineralized and amounts extracted, where phenanthrene extractability was low (in later stages of aging) (thus the intercept was close to 0). However, where phenanthrene extractability was high (in early stages of aging), the DCM extraction overestimated mineralization (thus the slope of the best-fit line was not close to 1). On average, the DCM extraction overestimated mineralization by 66 ( 44%. These results agree with the findings of others (3, 5, 6, 32) by indicating that the use of “harsh extractions” does not provide a good estimate of organic contaminant bioavailability. However, these results also confound their observations that the use of a harsh DCM extraction of soil-associated organic contaminants did not correlate strongly with bioavailable fractions of organic contaminants (an r 2 value of 0.986 was determined in this study). The correlation between BuOH-extractable phenanthrene concentrations and mineralization gave an intercept of 0.54 and a slope of 0.614 (n ) 24) (Figure 3C), almost identical to those for the Soxhlet-DCM extraction against mineralization. Indeed, correlation between the Soxhlet-DCM and BuOH extraction data yielded a line of best fit with slope 1.030, intercept 0.15 and associated r 2 ) 0.987 (Figure 3E). Thus, these two extraction procedures determined the same amount of extractable soil-associated phenanthrene throughout the study. On average, the BuOH extraction overestimated mineralization by 68 ( 36%. It was concluded therefore, that the BuOH shake extraction was not ideal for evaluating the microbial bioavailability of soil-associated phenanthrene in this study. The optimized HPCD-extraction when plotted against mineralization (Figure 3D) gave an intercept of of 0.162 and a slope of 0.977. In contrast to the correlation between total residual phenanthrene concentrations and mineralization, the HPCD extraction provided good agreement with the amount of mineralization where low extractability was observed (i.e., after extensive aging). Additionally, and in contrast to all of the other procedures, the optimized HPCD extraction gave good agreement with mineralization even where extractability was high (i.e., after minimal aging). Correlation between DCM-extractable phenanthrene concentrations and HPCD-extractable phenanthrene concentrations yielded a line of best fit with a slope of 0.639, an intercept of 0.664, and associated r 2 ) 0.952 (Figure 3F), thus indicating that the extractable fractions of soil-associated phenanthrene were different, although related. In summary then, the optimized HPCD extraction procedure performed better than the other techniques tested against the mineralization assay. The HPCD extraction described in this study marks a significant deviation away from the use of organic solvents to extract soil-associated nonpolar organic contaminants from soil, a practice recently criticized (33). The HPCD extraction technique more closely mimics the mass transfer mechanisms that govern the bioavailability of nonpolar organic contaminants (i.e., transfer to the aqueous phase). It thereby provides a more relevant, process-based extraction method for the determination of
soil-associated nonpolar organic contaminant microbial bioavailability. Once fully tested and understood, the aqueous HPCD extraction technique may provide a reliable means to determine the bioavailable fraction of a range of soil-associated organic contaminants and be applicable in the assessment of risk and contaminated land bioremediation potential.
Acknowledgments We are grateful to the Natural Environment Research Council, U.K. (GR4/96/113 and GR9/03281), and the Nuffield Foundation (AT/100/98/0284) for funding this work and to Dr. Graeme I. Paton at the Univeristy of Aberdeen, U.K., for supplying the characterized soils used in this investigation.
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Received for review August 12, 1999. Revised manuscript received May 9, 2000. Accepted May 9, 2000. ES990946C VOL. 34, NO. 15, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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