Nonexhaustive β-Cyclodextrin Extraction as a Chemical Tool To

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Environ. Sci. Technol. 2008, 42, 8419–8425

Nonexhaustive β-Cyclodextrin Extraction as a Chemical Tool To Estimate Bioavailability of Hydrophobic Pesticides for Earthworms T H O M A S H A R T N I K , * ,†,‡ J O H N J E N S E N , § AND JOOP L. M. HERMENS| BioforsksNorwegian Institute for Agricultural and Environmental Research, Frederik A. Dahls Vei 20, 1432 Ås, Norway, Department for Plant and Environmental Sciences, Norwegian University of Life Sciences, P. O. Box 5003, 1432 Ås, Norway, Department of Terrestrial Ecology, National Environmental Research Institute, University of Aarhus, Vejlsoevej 25, 8600 Silkeborg, Denmark, IRASsInstitute for Risk Assessment Sciences, Utrecht University, Yalelaan 2, P. O. Box 80176, 3508 TD Utrecht, The Netherlands

Received March 31, 2008. Revised manuscript received August 15, 2008. Accepted September 2, 2008.

Chemical methods to assess bioavailability in soil and sediment often use synthetic polymers that mimic uptake of organic compounds in organisms or microbial degradation. In this paper we have assessed a biomimetic extraction method using hydroxyl-β-cyclodextrin (HP-β-CD) to estimate uptake of the two insecticides R-cypermethrin (R-CYP) and chlorfenvinphos (CFVP) in the earthworm Eisenia fetida. Additionally, a novel approach was developed to estimate the efficiency of biomimetic extractions. The study revealed that HP-β-CD is a suitablesurrogateforestimatingthebioaccessibilityofhydrophobic chemicals in soil. If one uses a 3.5 times higher amount of HPβ-CD than soil, effective and reproducible extractions can be achieved within 48 h. At these conditions, inclusion of dissolved chemicals by HP-β-CD mimics uptake of a given compound into earthworms and takes into account sorption-related aspects that control biological uptake. The data indicate that, with increasing hydrophobicity, the affinity of organic chemicals to HPβ-CD does not increase to the same degree as to soil organic matter. Therefore, a high surplus of HP-β-CD is necessary to provide a sufficient extraction capacity in biomimetic extractions.

Introduction During recent years, biomimetic extractions have increasingly become popular to assess bioavailability in soils and sediments. The term biomimetic indicates that these chemical approaches try to mimic uptake mechanisms that occur in organisms. Biomimetic extractions often use a synthetic polymer that samples the bioavailable fraction of a compound in water, soil, or sediment. The polymer acts either as a * Corresponding author phone: Phone: +47 92694021; fax: +47 63009410; e-mail: [email protected]. † Bioforsk. ‡ Norwegian University of Life Sciences. § University of Aarhus. | Utrecht University. 10.1021/es8008908 CCC: $40.75

Published on Web 10/22/2008

 2008 American Chemical Society

surrogate for an organism or specifically accumulates the fraction of a compound that can be taken up by organisms. Among biomimetic extractions of organic compounds two main approaches can be distinguished: those that are equilibrium-based and negligibly change the partitioning of a compound between a solid and aqueous phase and those that instantaneously capture all of a compound that is released from the solid phase into the aqueous phase within a certain time period. The first approach measures chemical activity of a compound in soil or sediment; the second, the bioaccessible fraction (1). Examples for the first group are negligibly depleting solid-phase microextraction (ndSPME) (2, 3), extraction with polyoxymethylene strips (4, 5), triolein-embedded cellulose acetate membrane (6), and hollow fiber supported liquid membrane (7). Examples for the second group are extractions with Tenax beads (8, 9) and with β-cyclodextrin (10). Both approaches can be implemented in ecological risk assessment of contaminated sites as outlined by Jensen and Mesman (11). Depleting biomimetic extractions are based on the principle that water is the exchange medium in bioaccumulation (independent of the exposure route) and that organisms take up compounds from the aqueous phase. Thereby they might temporarily deplete the aqueous phase, which is more or less rapidly replenished by compound desorbing from the solid phase (12). Depletion of the aqueous phase occurs if desorption rates from soil to water are lower than uptake rates in organisms or biodegradation rates. It is assumed that the “labile” fraction that predominantly comprises of the rapidly desorbing fraction is a mechanistically sound and methodologically robust estimateofthebioaccessiblefractioninsoilandsediment(9,13). In this paper we concentrate on an extraction method that determines the labile, bioaccessible fraction in soil using cyclodextrin extraction. According to Reid et al. (10) and subsequent studies (14-16), there exists a 1:1 relationship between biodegradability and β-cyclodextrin extractability, which enables the accurate determination of the biodegradable fraction of a compound in soil. Cyclodextrins are a group of cyclic oligosaccharides that contain six to eight glucose units and consist of a polar exterior and a nonpolar cavity. These molecules have received considerable attention as versatile complexing agents for hydrophobic compounds that are encapsulated in the cavity of the cyclodextrin and thereby enhance the water solubility of hydrophobic compounds such as phthalic esters (17), organophosphorus pesticides (18), 1,1,1-trichlor-2,2-bis(pchlorophenyl)ethane (DDT), chlorobenzenes (19), and polycyclic aromatic hydrocarbons (PAHs) (14, 20). While extraction with cyclodextrin has been predominantly used to study microbial bioavailability for mono- and polycyclic aromatic hydrocarbons, information is rare for other compound groups and organisms of higher trophic level. This study specifically aims at examining if extraction with cyclodextrins estimates bioaccessible fractions of hydrophobic insecticides for earthworms. The extraction conditions were optimized according to Reid et al. (10). The primary objective, however, was to get more detailed insight into the efficiency of the cyclodextrin extraction. The extraction efficiency will depend on the extraction capacity of cyclodextrin and on the sorption capacity of soil. Extraction of the fast desorbing fraction is only possible if the extraction capacity of the cyclodextrin is much larger than the sorption capacity of the soil or sediment. Otherwise, the system will equilibrate, and the extractant is not able to extract more than a certain amount of a compound from soil or sediment. VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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The investigations were performed with two widely used insecticides chlorfenvinphos (CFVP), an organophosphate, and R-cypermethrin (CYP), a synthetic pyrethroid, in two soils. Both compounds and soils differ considerably in their physical-chemical properties (Tables S1 and S2 in the Supporting Information).

Extraction Capacity of Cyclodextrin versus Sorption Capacity of Soil. To understand the underlying processes for the extraction with cyclodextrin and to test the feasibility of cyclodextrin as a good extractant for biomimetic extractions, two parameters are introduced: the extraction capacity of cyclodextrin and the sorption capacity in soils that are derived from the equation of a three-phase equilibrium model (soil, water, and cyclodextrin): Ai,H2O VH2O

)

Ai,CD Ai,soil ) MCDKi,CD MsoilKi,OC fOC

(1)

where Ai, CD, Ai,soil, and Ai, H2O are the amount of a chemical i in respectively cyclodextrin, soil, and water and MCD, Msoil, and VH2O are the masses of cyclodextrin and soil and the water volume in the extraction. Ki, CD and Ki, OC are the partition coefficients between cyclodextrin and water, and organic carbon and water, respectively, while fOC is the portion of organic carbon in soil. The extraction capacity (EC) and sorption capacity (SC) are defined as follows: SC ) Msoil fOCKi,OC

(2)

EC ) MCDKi,CD

(3)

The total amount of a compound (that is initially bound to soil) can be expressed as Ai,tot. ) Ai,soil + Ai,CD + Ai,H2O

(4)

where Ai, tot. is the total amount of a chemical. For hydrophobic compounds with low water solubility, Ai, H2O is negligible, and eq 4 can be written as Ai,tot. ) SC(Ci,H2O) + EC(Ci,H2O)

(5)

where Ci, H2O is the freely dissolved concentration of a compound in water. Equation 5 can be further transformed to derive the maximum extractable fraction (MEF) of a compound in soil. Given a certain EC and SC, cyclodextrin can only extract a certain portion of a compound from soil until equilibrium is reached in the system. MEF )

Ai,CD EC ) Ai,tot. SC + EC

(6)

Affinity Coefficient of a Compound for Cyclodextrin. can be calculated according to

CD

Ci,free 1 ) Ci,tot. 1 + Ki,CDCCD

(7)

where Ci, tot. is the concentration of a dissolved and encapsulated compound in water and CCD is the concentration of cyclodextrin in water. The stability of cyclodextrin complexes is conventionally given by the stability constant. However, the partition coefficient is better suited for the calculation of the extraction capacity than the stability constant of cyclodextrin complexes, since it is applicable for all kinds of polymers. For 1:1 complexes (one cyclodextrin molecule complexes one compound molecule) the stability constant 8420

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Scyp-CD )

Ci-CD2 CfreeCCD2

)

KCD CCD

(8)

where Ci-CD2 is the concentration of cyclodextrin encapsulated compound.

Theory

Ki,

(Scyp-CD) and affinity coefficient (KCD) are equivalent; for 2:1 complexes the relationship is expressed according to

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Materials and Methods Chemicals. Radiolabeled [carboxyl-14C] R-CYP and [ethyl1-14C2] CFVP were purchased from the Institute of Isotopes (Budapest, Hungary) and had a specific activity of 35.5 and 8.3 µCu/µmol, respectively. Unlabeled R-CYP (97.6% purity) and CFVP (94.2% purity) was kindly provided by Inter-Trade Denmark (Bindslev, Denmark), and BASF (Wa¨denswil/Au, Switzerland), respectively. 2-Hydoxypropyl-β-cyclodextrin (>98% purity) was purchased as Cavasol W7 HP Pharma from Wacker-Chemie GmbH (Burghausen, Germany). Acetone, 2-propanol, and cyclohexane (all Merck LiChroSolv, >99.7% purity) were purchased from VWR Norway (Oslo, Norway). Sample oxidizer cocktails (Carbosorb-E, Permaflour-E, Combustaid), scintillation cocktail (Optima Gold), and oxidizer sample trays were obtained from Perkin-Elmer (Hvidovre, Denmark). Soils. The experiments were performed with a forest soil with a total organic carbon (TOC) content of 5.5% that was sampled at Steinskogen in Norway about 15 km northwest from Oslo. The other soil was an agricultural soil from Askov in Denmark with a TOC content of 1.4% (see Table S2 in the Supporting Information for additional soil properties). Both soils were air-dried for 48 h and sieved through a 2 mm mesh prior to use. The soils were stored at 4 °C until use. The soils were spiked with a mixture of 14C-labeled and unlabeled R-CYP or CFVP. The compounds were dissolved in acetone to achieve final concentrations of 5, 20, and 100 mg/kg and a 14C acitivity of 15 µCi/kg for R-CYP and 0.4, 1.6, 8, and 20 mg/kg and a 14C activity of 9 µCi/kg for CFVP. Solutions were added to soil at a soil/acetone ratio of 5:1. For all test soils, the chemical was mixed thoroughly into the soil and placed under a fume hood for evaporation of acetone. After 24 h, deionized water was added to the soil to obtain 60% of the water holding capacity, and the soil was mixed again. Uptake Experiments with Earthworms. Accumulation of 14C-labeled R-CYP and CFVP in earthworm was studied in both test soils at the above-mentioned concentrations by exposing 10 earthworms (Eisenia fetida) to 400 g spiked soil (dw). Ten replicates per concentration were prepared, and uptake kinetics of the compounds in earthworms were studied by sampling at 10 exposure times ranging from 1 to 28 days. However, only bioaccumulation data at 28 days (where internal concentrations were highest) were used for the comparison between earthworm uptake and cyclodextrin extractability. The kinetic aspects, including biotransformation of these two compounds, are the subject of another study (21). The worms were fed with up to 5 g of ground cow manure weekly, and water was replenished to maintain the initial water content. Optimization of the CD-Extraction Procedure with r-Cypermethrin. All extractions were performed in triplicate in 24 mL glass vials with poly(tetrafluroethylene) (PTFE) lined caps.The effect of HP-β-CD addition on extraction efficiency was determined for Askov soil that was spiked with 5 mg/kg 14C-labeled R-CYP (aged for 14 days). Different amounts of HP-β-CD were added to 0.6 g of soil to achieve HP-β-CDto-soil ratios between 0 and 7 by weight. A 15 mL aliquot of a 10 mM sodium azide (NaN3) solution was added, and the suspensions were shaken on a one-dimensional shaker at a speed of 150 rpm for 48 h. Afterward, the suspensions were

centrifuged at 2000 rpm for 20 min. A 1.75-2 mL aliquot of the supernatant was mixed with scintillation cocktail Ultima Gold and measured by liquid scintillation counting (LSC) on a Tri-Carb 2300TR liquid scintillation counter (Canberra Packard Ltd., Manchester, U.K.). Additionally, the effect of extraction time was studied at a fixed HP-β-CD-to-soil ratio of 3.5 (w/w) by sampling the solution after 1, 3, 6, 9, 12, 24, 48, and 120 h. The optimized procedure used an extraction time of 48 h and a HP-β-CD amount that was 3.5 higher than the amount of soil. This optimized procedure was applied on R-CYPand CFVP-contaminated Askov and Steinskogen soils that were used for the earthworm uptake experiments. Determining the Extraction and Sorption Capacities at Different HP-β-CD Concentrations. The partitioning coefficient of the cyclodextrin-insecticide complexes was determined by measuring the concentration of complex bound and unbound compound in the aqueous phase. The Ki, CD was determined using the radiolabeled compound in the case of CFVP and the unlabeled compound in the case of R-CYP. Spiked soil was extracted with different amounts of HP-β-CD in the presence of poly(dimethylsiloxane) (PDMS) coated SPME fibers. These were added to estimate the freely dissolved concentration of pesticides in the aqueous phase according to ter Laak et al. (22). HP-β-CD was added in amounts to achieve HP-β-CD-to-soil ratios between 0.35 and 7 (w/w). For14C-labeled CFVP, 2 g of spiked soil (0.4 and 20 mg/kg CFVP in Askov soil, 1.6 mg/kg in Steinskogen soil) were shaken with 35 mL of HP-β-CD solution at 150 rpm for 24 h. Afterward, two 7.5 cm long SPME fibers coated with 30 µm of PDMS were added and the suspensions carefully shaken on a rock’n-roll-shaker (Snijders, Tilburg, The Netherlands) for 7 days. This time was found to be sufficient to reach equilibrium between water and fibers. Suspensions were centrifuged (2000 rpm, 20 min), and SPME fibers were removed, cleaned with a wet paper tissue, and extracted in 5 mL of cyclohexane for 48 h. The SPME extract and an aliquot of the particle-free CD supernatant were measured for their 14C acitivity by LSC. For unlabeled R-CYP, the extractions were performed in the same manner as for CFVP; however, 0.6 g of Steinskogen soil (25 and 500 mg/kg) was extracted with 15 mL of cyclodextrin solution. One 5 cm long SPME fiber (30 µm PDMS coating) was added to the suspensions and exposed for 14 days, which is sufficient to reach equilibrium in the system. R-CYP in the SPME extracts and in the CD supernatant were analyzed on a gas chromatograph equipped with an electron capture detector (GC-ECD; Varian 3600CX, Varian Chromatography Systems, Walnut Creek, CA). The chromatographic conditions are described in the Supporting Information. Determination of the PDMS-Water Partition Coefficient (KPDMS). The KPDMS for R-CYP was determined according to the dilution method described by ter Laak et al. (23). The experiment was performed with 14C-labeled compound with five replicates. One preloaded SPME fiber (5 cm long, 30 µm PDMS coating) with a 14C activity of 2920 ( 14 dpm/SPME (corresponding 0.71 µg/µL R-CYP) was gently shaken in 100 mL of deionized water (containing 10 mM NaN3) for 14 days. Afterward, the 14C activity in the fibers, the aqueous phase and the amount that could be extracted from the vial wall with cyclohexane, was measured by LCS and the KPDMS and recovery were determined according to eq S1 in the Supporting Information For CFVP, the “classical” approach was chosen: 10 mL of deionized water was spiked with a mixture of labeled and unlabeled CFVP to achieve a 14C activity of 100 dpm/mL (corresponding to 7.5 µg/L CFVP). One clean SPME fiber (5 cm long, 30 µm PDMS coating) was added and the solution

FIGURE 1. Effect of extraction time (using a HP-β-CD-to-soil ratio of 3.5) (A) and HP-β-CD concentration (using 48 h extraction time) (B) on extractability of r-CYP (5 mg/kg) from Askov soil. Error bars represent standard deviations of triplicates. gently shaken for 7 days. This was conducted in triplicate. The determination of the KPDMS and the recovery is the same as that for R-CYP. For determining the KOC of R-CYP and CFVP, the experimental setup used was the same as that for the determination of the Ki, CD but with water instead of CD solution. Extraction of Soil and Earthworm and Measurement of 14C Activity. Concentrations of the compounds in homogenized soil and depurated earthworms were measured as radioactivity in the samples. Worms were depurated for 18 h on wet filter paper. The total amount of pesticides in soil and earthworm was measured in triplicate by combusting single earthworms (300-500 mg in weight) or 0.5 g of soil in a sample oxidizer, model 307, Packard (Berkshire, England). Produced 14CO2 was captured in Carbosorb-E and Permaflour-E and 14C activity measured by LSC. The efficiency of the sample oxidizer was >93%. To differentiate between parent compound, metabolites, and nonextractable residues, soils were exhaustively extracted with cyclohexane/acetone (50/50, v/v) and extracts fractionated on a HPLC. This fractionation revealed that more than 90% of the 14C activity could be attributed to parent R-CYP in all soil samples that were taken during 28 days exposure time (21). For CFVP 98% of the 14C activity in soil could be related to parent CFVP. Earthworm and soil concentrations are based on total 14C activities in worms and soils.

Results and Discussion Optimization of the Extraction Method. Experiments that were conducted to optimize the extraction method revealed that, with increasing extraction time, R-CYP extractability increases and converges to 70% after 40 h (Figure 1A). The extraction yield after 48 h extraction did not significantly differ from that after 120 h. Increasing concentrations of HPβ-CD increased the amount of R-CYP that was extracted from soil (Figure 1B); however, at a HP-β-CD-to-soil ratio of 3.5, the curve levels off and the extraction yield converges to 70%. No significant difference in extraction yield could be found at HP-β-CD-to-soil ratios between 3.5 and 7. When interpreting the converging of the extraction efficiencies at increased extraction times, it has to be considered whether the steady state is due to equilibrium in the extraction solution or due to slow desorption kinetics VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Partition coefficient of r-CYP and CFVP between HP-β-CD and water. Circles display the HP-β-CD-water partition coefficients measured in Askov soil: diamonds, in Steinskogen soil. (see also the discussion about extraction and sorption capacity). In the case of equilibrium, probably not the entire labile fraction is extracted, and higher extraction yields can be obtained at higher cyclodextrin concentrations. However, the results show that a HP-β-CD-to-soil ratio of 3.5 was sufficient to continuously deplete the aqueous phase up to 48 h since the extraction efficiency did not increase at higher HP-β-CD-to-soil ratios (Figure 1B). The converging of the extraction yield with increasing extraction time might be explained by the fact that further release of compound from soil is kinetically hampered after the easily accessible fraction was extracted from soil. From these experiments it can be concluded that extraction of soil for 48 h with a HP-β-CD-to-soil-ratio of 3.5 is appropriate for an effective and reproducible extraction of the labile fraction of R-CYP and CFVP in soil. The extraction time is twice as long and the HP-β-CD-to-soil ratio is twice as high as that suggested by Reid et al. (10). The chosen extraction conditions are expected to result in extraction yields similar to methods using other polymers (20). Extraction Capacity of β-Cyclodextrin versus the Sorption Capacity of Soil. The KPDMS for R-CYP was determined as (3.53 ( 0.40) × 105 (average ( SD), and the recovery was 92% in the test. For CFVP a KPDMS of 800 ( 23 was found. Here, the recovery was 101%. These values are about 1 order of magnitude lower than the Kow and agree with observations made by Mayer et al. (24). The KPDMS was used to calculate the concentrations of freely dissolved compound and thus the Ki, OC and Ki,CD. log Ki,OC values were 6.14 ( 0.09 and 6.10 ( 0.04 for R-CYP and 3.42 ( 0.09 and 3.44 for CFVP in Askov and Steinskogen soils, respectively. The values were used to calculate the SC and the MEF. When soil was simultaneously extracted with different amounts of HP-R-CD and SPME fibers, the freely dissolved concentrations of R-CYP and CFVP considerably decreased with increasing cyclodextrin concentration. For CFVP, the partition coefficient between encapsulated and freely dissolved compound (KCD) was fairly constant at different cyclodextrin concentrations (262 ( 54 L/(kg of HP-β-CD)) (Figure 2) For R-CYP, the value increased from 10000 to approximately 230000 L/(kg of HP-β-CD) with HP-β-CDto-soil-ratios increasing from 0.35 to 7. This observation might be explained by the fact that CFVP forms 1:1 inclusion complexes with HP-β-CD, while R-CYP forms 2:1 complexes (two molecules of CD encapsulate one molecule of CYP). For 1:1 complexes, the partition coefficient is constant with changing HP-β-CD concentrations, while it changes for 2:1 8422

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complexes with changing HP-β-CD concentrations according to eq 8. The calculated stability constant for R-CYP is fairly constant ((1.15 ( 0.29) × 106 M-2). The fact that R-CYP forms 2:1 complexes with HP-β-CD means that the extraction capacity for R-CYP changes with the square of the HP-β-CD concentrations, while, for CFVP, it changes only linearly. Differences in the extraction capacity result in differences in the MEF for a given soil according to eq 6. The values for the extraction and sorption capacity are listed in Tables S3 and S4 in the Supporting Information. With increasing HPβ-CD concentration the MEF for R-CYP changes more than for CFVP (Figure 3A,B versus Figure 3C,D). For R-CYP, the difference in MEF between a HP-β-CD-to-soil ratio of 1.75 and 3.5 is 30% in Steinskogen soil, while it is 12% for CFVP. The MEF in the low organic Askov soil was generally higher than in the high organic Steinskogen soil due to a lower sorption capacity (Figure 3B,D versus Figure 3A,C). This is particularly pronounced for low HP-β-CD concentrations. The MEF is the maximum fraction of a compound in soil that can be extracted with a certain HP-β-CD concentration. For example, a HP-β-CD-to-soil ratio of 1.75 does not result in higher extraction efficiencies than 60% for R-CYP in Steinskogen soil. As long as the accessible fraction of the compound in soil is lower than the 60%, the HP-β-CD-tosoil ratio of 1.75 estimates the “correct” accessibility. However, if the accessible fraction is higher, not the entire accessible fraction will be extracted because the soil-waterHP-β-CD system is in equilibrium. The actually extracted percentages (using the optimized extraction procedure) of R-CYP and CFVP from the soils used in the earthworm uptake experiments are shown in the column graphs in Figure 3A-D. Both for HP-β-CD-to-soil ratios of 3.5 and 7, the MEF exceeds the actual extracted fraction of both compounds in both soils. For a ratio of 1.75, the actual extracted amount of both compounds is on the same level as the MEF, while for lower HP-β-CD concentrations, the actual extracted amount exceeds the MEF. This result indicates that HP-βCD-to-soil ratios up to 1.75 might not have the capacity to extract the entire accessible fraction because the soil-waterHP-β-CD system reaches equilibrium before this. For ratios of 3.5 or higher, the extraction capacity is high enough to extract the accessible fraction. The actually extracted fraction is lower than the MEF because further release of the compound is impeded due to strong sorption to organic matter and retarded diffusion from micropores of organic matter. The difference between actually extracted fraction and MEF is larger for the more hydrophobic R-CYP than for

FIGURE 3. Maximum extractable and actual extracted fractions for r-CYP (A,B) and CFVP (C,D) in Steinskogen and Askov soil. The line graphs show the MEF calculated according to eq 6; the column graphs, the actual extracted fractions from soils used in the earthworm uptake study. Actual extracted fractions were determined according to the optimized extraction procedure with a HP-β-CD-to-soil ratio of 3.5. Error bars in the figure represent standard deviations.

FIGURE 4. Relationship between earthworm accumulation and total concentration in soil (A) or β-cyclodextrin extractable concentrations (B). Concentrations express equivalents of the parent compound. The data points represent average values of three replicates. For the single regression lines, 95% CI bands are shown (and can be hardly seen for CFVP Askov and r-CYP Steinskogen). CFVP. This might be explained by a higher percentage of slowly or very slowly desorbing compound for R-CYP than

for the less hydrophobic CFVP and is in line with observations made by Cornelissen et al. (25). As for the MEF, also the actual extracted fractions of both compounds are lower in Steinskogen soil than in Askov soil. For CFVP, 77-86% was extracted from Askov soil versus 63-79% from Steinskogen soil, and for R-CYP, 64-81% was extracted from Askov soil versus 36-52% from Steinskogen soil. This is obviously due to the approximately four times higher sorption capacity of Steinskogen soil that sorbs both compounds more strongly than Askov soil. Uptake of Chlorfenvinphos and r-Cypermethrin in Earthworm. Earthworm accumulated approximately five times more CFVP than R-CYP after 28 days exposure in soil (Figure 4A). Distinct differences in earthworm uptake were also found between soil types. Earthworms accumulated 30-40% less of both compounds from Steinskogen soil (with a high organic content) than from Askov soil. The results of this study show that uptake of CFVP and R-CYP in earthworm is not well predicted by total concentrations in soil. Earthworm uptake differs considerably between soils and between different concentrations of chemical in the soil (Figure 4A). When earthworm uptake is related to HP-R-CD extractability instead of total soil concentrations, the difference in compound uptake between the soil types was reduced for CFVP and almost disappeared for R-CYP (Figure 4B). In addition, the 95% confidence intervals for the single compounds and soils were reduced considerably. For R-CYP, the regression lines for the two soils almost coincide. This implies that the relationship between earthworm uptake and HP-β-CD extractability is independent of soil type. The clearly reduced 95% confidence intervals indicate that differences in accumulation at different soil concentrations are reduced and that the relationship between earthworm uptake and HP-β-CD extractability is independent of the soil concentrations. For CFVP, the correlation between HP-β-CD extractability and biological uptake is also fairly good; however, not all differences in uptake can be eliminated between the two test soils. VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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The results show that uptake of chemicals in earthworm is strongly affected by sorption of the chemical in soil, and cyclodextrin extractability seems to take into account sorption related aspects that govern uptake of the selected pesticides in earthworm. Desorption of chemicals into the aqueous phase and removal by cyclodextrin seems to cover the most important uptake mechanisms of earthworms. Differences in bioaccumulation between R-CYP and CFVP cannot be significantly reduced by relating earthworm uptake to CD extractability instead of total soil concentrations. Because chemical tools cannot account for biological factors that affect uptake of chemicals, these tools have to be calibrated in bioassays for the specific compound and organism in question. A more detailed analysis of biotransformation of these two compounds is the subject of another study (21). Determination of Cyclodextrin as a Suitable Extractant for Bioaccessibility Measurements. Cyclodextrin has the ability to dissolve high amounts of hydrophobic compounds that in principle are only slightly water soluble. Contrary to other polymers, HP-β-CD has the ability to selectively encapsulate freely dissolved organic molecules with a diameter similar to the membrane permeation limit of 9.5 Å (26). That makes HP-β-CD also suitable for sampling in environments containing high concentrations of dissolved organic carbon (e.g., in sewage sludge). Besides, cyclodextrins do not only encapsulate hydrophobic compounds but also polar organic compounds such as carboxylic acids and dissociated organic molecules (27). Thissignificantlyincreasestheapplicationrangeofcyclodextrins. Saturation of HP-β-CD by contaminant is not likely to occur at HP-β-CD-to-soil ratios of 3.5. In soil with 100 mg/kg R-CYP, only 0.02% of the HP-β-CD is occupied by contaminant (on a molar basis assuming 2:1 inclusion complexes); the rest is unbound. Besides, the affinity of HP-β-CD for R-CYP did not change with increasing amounts of R-CYP extracted from soil (see Figure S1 in the Supporting Information). To be a suitable polymer for biomimetic extractions, cyclodextrins have to be capable of extracting freely dissolved compound that is released into the aqueous phase. The extractant has to keep the concentration of dissolved compound in the aqueous phase low enough to enable continuous release of the particle-sorbed compound. Furthermore the driving force of the extraction, the extraction capacity of the cyclodextrin, should not become saturated within the extraction time. The MEF that relates the extraction capacity of the extractant to the sorption capacity of the soil is a useful parameter to interpret the results of a biomimetic extraction. Information about the partition coefficient between soil and water and extractant and water is therefore necessary. In some cases the extraction capacity of cyclodextrin might not be sufficient to determine the labile fraction in soil. This might be illustrated by an example from Reid et al. (10), where benzo(a)pyrene and phenanthrene (among other PAHs) were extracted with HP-β-CD from spiked soil with 3.8% organic carbon. The HP-β-CD-to-soil ratio was 1.75. For benzo(a)pyrene, 14% could be extracted; for phenanthrene, 72%. The authors explain the lower extraction efficiency for benzo(a)pyrene with a lower availability of benzo(a)pyrene for transfer to the aqueous phase or with steric hindrance at the cyclodextrin cavity that hampers complexation of benzo(a)pyrene by HP-β-CD. However, if the maximum extractable fraction is calculated on the basis of literature data for soil-water and cyclodextrin-water partition coefficients (19, 28, 29), a maximum extractable portion of 27% for benzo(a)pyrene and 67% for phenanthrene can be calculated for the specific experimental conditions. These estimated numbers are close to the experimental ones. 8424

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Thus, it cannot be ruled out that the low extraction efficiencies for benzo(a)pyrene and phenanthrene are due to an insufficient extraction capacity at the used HP-β-CD-to-soil ratio and not necessarily due to low availability. It should be kept in mind that the affinity of a chemical to cyclodextrin is supposed to change differently with hydrophobicity than affinity to soil particles. While the log KOC increases with increasing log KOW with a slope of approximately 1 for many hydrophobic chemicals (30), it is not sure that the Ki, CD develops in the same manner. The fact that the cavity of β-cyclodextrin has a polarity similar to that of ethanol (19) indicates that the Ki, CD increases with a slope considerably less than 1 with increasing log KOW. In that case, the capability of HP-β-CD to extract hydrophobic chemicals from soil decreases with increasing hydrophobicity of the compound. A HP-β-CD-to-soil ratio of 3.5 provides a working methodology. Where compounds are very hydrophobic or where the matrix contains a high organic matter content, this working procedure should be reviewed accordingly to ensure the sufficient extraction capacity of the HP-β-CD. To be sure that the extraction capacity of HP-β-CD is high enough to estimate the labile fraction of a compound in soil, the extraction capacity should be 10 times higher than the sorption capacity. The experimental conditions chosen in this study (48 h extraction time, HP-β-CD-to-soil ratio of 3.5 by weight) seem to be capable of estimating the accessible fraction of CFVP and R-CYP in soil. However, at lower HP-β-CD-to-soil ratios, the distribution of chemicals between cyclodextrin and soil seem to approach equilibrium, as is shown by the comparison between actual extracted and the MEF.

Acknowledgments This work was funded by the Norwegian Research Council (Project 153444/S30). The authors thank Frans Busser at IRAS for his assistance in GC analysis and Lis W. de Jonge at the Research Center in Foulum (Aarhus University) for the opportunity to perform analysis at the sample analyzer. Furthermore, we thank Thomas ter Laak for the fruitful discussions and his valuable comments on the paper.

Supporting Information Available Additional information on physical-chemical properties of the soils and chemicals and data on extraction and sorption capacities. This material is available free of charge via the Internet at http://pubs.acs.org.

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