Environ. Sci. Technol. 2002, 36, 5399-5404
Bioconcentration of Organic Chemicals: Is a Solid-Phase Microextraction Fiber a Good Surrogate for Biota? H E A T H E R A . L E S L I E , * ,†,‡ THOMAS L. TER LAAK,† FRANS J. M. BUSSER,† MICHIEL H. S. KRAAK,‡ AND JOOP L. M. HERMENS† Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands, and Department Aquatic Ecology & Ecotoxicology, University of Amsterdam, P.O. Box 94084, 1090 GB Amsterdam, The Netherlands
When organic chemicals are extracted from a water sample with solid-phase microextraction (SPME) fibers, the resulting concentrations in exposed fibers are proportional to the hydrophobicity of the compounds. This fiber accumulation is analogous to the bioconcentration of chemicals observed in aquatic organisms. The objective of this study was to investigate the prospect of measuring the total concentration in SPME fibers to estimate the total body residue in biota for the purpose of risk assessment. Using larvae of the midge, Chironomus riparius and disposable 15-µm poly(dimethylsiloxane) fibers, we studied the accumulation and accumulation kinetics of a number of narcotic compounds with a range of log Kow between 3 and 6. The fibers, which have a larger surface area-tovolume ratio, had consistently higher uptake and elimination rate constants (k1 and k2, respectively) than midge larvae and accumulated the chemicals 5 times faster. Comparison of the relationships of the partition coefficients KPDMS-water and Kmidge-water (lipid-normalized) to log Kow for all compounds yielded a factor of 28 for translating fiber concentrations to biota concentrations. This factor can be used to estimate internal concentrations in biota for compounds structurally similar to the compounds in this study. The exact chemical domain to which this factor can be applied needs to be defined in future research.
Introduction The hydrophobicity of many industrial chemicals is an important factor in determining their potential to bioaccumulate and reach target sites, which may result in ecotoxicological risk. It is mainly this property that drives organic chemicals in the aquatic environment to partition to and bioconcentrate in the hydrophobic phases of organisms, such as lipid bilayer membranes of cells. If certain critical concentrations of xenobiotics are reached in membranes, the membrane disturbance results in baseline * Corresponding author. Tel: +31-30-2535018. Fax: +31-302535077. E-mail:
[email protected]. † Utrecht University. ‡ University of Amsterdam. 10.1021/es0257016 CCC: $22.00 Published on Web 10/29/2002
2002 American Chemical Society
toxicity, a concept that has been widely applied in ecotoxicology (1-5). Because concentration addition applies to the narcotic mode of action (1, 6-8), the total body residue (TBR) represents a useful parameter to estimate the total contribution of baseline toxicity to the overall toxicity of a sample (9-13). Direct measurement of xenobiotic body residues in organisms themselves is complicated, first, by other bodily substances present that may interfere with analyses, making extra cleanup steps before analysis necessary. Moreover, in complex or unknown contaminant mixtures, it is impossible to detect and measure all compounds individually. Therefore, research has been focused on chemical extraction methods as an alternative to measuring contaminants directly in organisms. These methods can be employed even under adverse conditions (e.g., toxic, anaerobic, turbid, dark) in which no surviving organisms may be found for residue analysis. The first step in estimating TBR is to simulate the accumulation process in a surrogate phase. Methods that mimic accumulation in biota are referred to as “biomimetic” and rely on physicochemical partitioning of the compounds over aqueous and hydrophobic phases, such as the semipermeable membrane device (SPMD) (14, 15), Empore disks (9, 10, 16), or the recently introduced thin films (17). Solidphase microextraction with negligible depletion, nd-SPME, (11-13, 18, 19) is also based on the partitioning principle. The more hydrophobic the compound, the greater the volume ratio of aqueous to hydrophobic phases required to avoid depletion (cf., refs 9 and 18). Due to the small size of SPME fibers, the method is characterized by faster accumulation kinetics than SPMDs or Empore disks. When simulating accumulation, not only the end result of the partitioning process but also the kinetics of the accumulation in fibers should be characterized to give information about the time needed to reach equilibrium (often the sampling point of preference). SPME fibers were used in the present study to perform biomimetic extractions of hydrophobic organic chemicals in static test systems for comparison with the accumulation by test organisms. These SPME fibers were coated with a 15-µm layer of poly(dimethylsiloxane) (PDMS). This polymer is a viscous liquid at room temperature through which compounds can quickly diffuse (20). According to partitioning theory, when fibers are exposed to test water, the test compound diffuses from the aqueous phase into the polymer coating. This is a passive process driven by the differences in fugacities of the chemical on both sides of the phase interface. As shown by Mayer et al. (19), the organic compounds are not adsorbed, but absorbed into the PDMS layer so that neither saturation nor competition between compounds will occur in the PDMS coating in aqueous solutions (20). In earlier work, measured body residues of individual compounds were compared with body residues estimated with the biomimetic SPME technique (21). In the present study, we set out to collect experimental data for chemicals with various Kow values in order to investigate the accumulation kinetics and the relationship between the KPDMS-water and BCF. The overall aim was to establish whether the sum concentration of different chemicals in PDMS fiber can be used to predict TBRs in biota. To determine calibration factors to translate CPDMS to Cbiota, we exposed disposable PDMS fibers and Chironomus riparius fourth instar midge larvae to a set of nonpolar and polar narcotic compounds with log Kow values ranging from 3 to VOL. 36, NO. 24, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Overview of KPDMS-water, Wet Weight Kmidge-water, and k2 (h-1) Values for Fibers and Midges with Standard Errors and Correlation Coefficients (r2) Calculated from Model Fita log Kow
K
SE
24DCNB
3.09
4C3MP 12DCB 245TCA
3.10 3.43 3.69
234TCNB 123TCB 2356TeCA 1234TeCB
3.74 4.14 4.46 4.64
PeCB HCB
5.18 5.73
83.1 124.6 70.2 163 120.1 122 450 1382 1613 7959 7107 26380 68320
2.5 4.4 5.0 16 3.3 10 13 74 48 296 272 768 2312
compound
log Kow
K (ww)
SE
24DCNB 4C3MP 245TCA 1234TeCB PeCB HCB
3.09 3.10 3.69 4.64 5.18 5.73
23.3 20.0 109.6 1257 3048 29170
1.7 1.8 4.2 51 342 5329
compound
PDMS Fibers log K k2 1.92 2.10 1.85 2.21 2.08 2.09 2.68 3.14 3.21 3.90 3.85 4.42 4.84
r2
k1
Caq
0.67 2.5 0.92 0.102 1.9 2.0 0.24 0.046 0.064 0.038 0.016 0.012 0.0012
0.821 0.671 0.640 0.526 0.867 0.753 0.916 0.927 0.948 0.943 0.956 0.975 0.985
246 1296 207 32.7 1281 1245 804 398 860 1862 784 2659 730
0.290 0.352 2.509 0.109 0.081 0.109 0.0984 0.0376 0.0489 0.00615 0.0173 0.00338 0.00292
k2
SE
r2
k1
Caq
1.93 0.818 0.421 0.0483 0.0168 0.00326
0.88 0.372 0.099 0.0073 0.0045 0.00090
0.569 0.668 0.906 0.959 0.922 0.988
45.0 16.4 46.2 60.8 51.1 95.2
0.290 0.231 0.081 0.00615 0.00338 0.00292
2.96 10.4 2.95 0.201 10.7 10.2 1.79 0.288 0.533 0.234 0.110 0.101 0.0107
Midges log K (lipid) 3.37 3.30 4.04 5.10 5.48 6.47
SE
a k values calculated as K/k , Log K 1 2 PDMS, log Kmidge(lipid weight), and average aqueous exposure concentrations, Caq (µM) also shown. Log Kow values from De Bruijn et al. (31).
6 for different time periods to make uptake curves. An advantage of these PDMS fibers, which have previously been used for analyzing pore water concentrations (22), is that they are disposable and can easily be exposed simultaneously with organisms in a toxicity test. The measured KPDMS-water values for the chemicals were compared to the Kmidge-water measured in the same water. Comparisons of kinetic rate constants were made between fibers and midges. On the basis of the data, we discuss the possibilities for application of chemical concentrations in PDMS fibers to predict the concentrations in exposed organisms.
Materials and Methods Test Chemicals. Compounds tested, with abbreviations, included the following: 1,2,3,4-tetrachlorobenzene (1234TeCB), pentachlorobenzene (PeCB), 2,4,5-trichloroaniline (245TCA), 1,2,3-trichlorobenzene (123TCB) (all Pestanal, Riedel-de Hae¨n, 99%); 2,3,5,6-tetrachloroaniline (2356TeCA), hexachlorobenzene (HCB) (both Pestanal, Riedel-de Hae¨n, 98%); 4-chloro-3-methylphenol (4C3MP) (Aldrich, 99%); 2,4dichloronitrobenzene (24DCNB) (Fluka Chemicals, 98%); 1,2dichlorobenzene (12DCB) (Fluka Chemicals, 99%); and 2,3,4trichloronitrobenzene (234TCNB) (Aldrich, 97%). Aqueous solutions of the test compounds were prepared using 2-propanol (Baker Resi-analyzed) as carrier solvent in copper-free Utrecht tap water (hardness: DH 7, pH 7.6) giving a 2-propanol concentration of 0.01% (v/v). The test concentration for each compound was chosen to be under the LC10 for baseline toxicity in order to preclude toxic effects leading to test organism mortality. (See Table 1 for log Kow values and exposure concentrations.) Still, because of concentration addition, testing several compounds together near the LC10 would lead to significant mortality of the larvae. Therefore, to avoid lethality and still have sample concentrations that are well above detection limits, it was necessary to test the compounds either alone or in smaller groups. (The following compounds were tested with fibers twice: 24DCNB, 245TCA, and 1234TeCB). The volume of the test solution was chosen to be sufficiently large relative to the volume of the fiber and midges 5400
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to satisfy the conditions for biomimetic nd-SPME (9). Tests were carried out in half-filled, airtight 250-mL glass Erlenmeyer flasks. The most hydrophobic compounds, PeCB and HCB, required a larger volume to avoid depletion of aqueous concentration and were tested separately in 2-L glass bottles. Leaving air in the containers supplied oxygen for midge larvae. The temperature during the exposures was 24 °C, and no food or substrate was added. Four fourth instar C. riparius midge larvae (three for HCB test) and three 2-cm-long 15-µm poly(dimethylsiloxane), PDMS fibers (Fiberguide Industries) were exposed to the test solutions for different exposure periods. The volume of PDMS per fiber was 202 nL. Midge larvae were ∼5 mg, wet weight. Fibers and midges were exposed to test compound solutions together in the same test vessels except for 12DCB, 234TCNB, 123TCB, and 2356TeCA, which were tested with fibers only. At the end of each time interval, a different test vessel was opened and sampled. The PDMS fibers were collected, and each was extracted separately in 0.4 mL of hexane (Baker Resi-analyzed) with an internal standard from the test set, but which was not present in the exposure in question. Live larvae from the Erlenmeyer were blotted dry, weighed together, and stored frozen (-20 °C) until extraction. For the determination of internal concentrations in larvae, the frozen larvae were extracted with hexane, with an internal standard from the test set, as for the fibers. Also, different volumes of hexane (3-4 mL) were used, depending on the chemical. At the time of hexane addition, 1 mL of 2 M KOHaq was added and the samples were placed in an ultrasonic bath for 1 h (at room temperature) to speed breakdown of the larval body. The KOH and hexane phases were separated by centrifuging the midge extracts (3000g, 10 min). Lipid analysis could not be performed in the same larvae whose body residues were measured; therefore, lipid content was measured in fourth instars that had been kept for 4 days in similarly closed, half-headspace test systems with four different concentrations of TeCB. Sampled larvae were pooled into groups (n ) 14) with g30 mg of biomass for analysis. Lipid content was measured using a procedure developed by Verweij (23). In short, the lipids were extracted with a
hexane-2-propanol mixture (v/v 3:2), hydrolyzed to fatty acids with sulfuric acid, colored with vanillin reagent (vanillin, Merck, in H3PO4, 85% Riedel-de Hae¨n), and measured spectrophotometrically with cholesterol (BDH Biochemicals) as standard. No correlation was found between exposure concentration and lipid content, and the average lipid content for the midge samples was 0.96% of wet weight (SD 0.37). Therefore, to express body residue results in terms of lipid weight, 1% of wet weight was assumed for all larvae. Chemical concentrations in the midge extracts were measured by GC-ECD using a Carlo-Erba 5360 gas chromatograph, equipped with an electron capture detector and a 15-m capillary column, J&W type DB5.625, i.d. 0.32 mm, film thickness 0.25 µm. Injections of the hexane solutions (1 µL) were carried out in split mode with an isothermal (170 °C) temperature program. The data were processed with a Fisons Chromcard data system. The lowest measured concentrations in hexane were 10 pg/µL for all compounds except 4C3MP, which was detected at 40 pg/µL. The hexane extracts of PDMS fibers and water samples, taken at t ) 0 and at the end of each exposure in duplicate, were similarly analyzed by GC-ECD. Standards of all test compounds in hexane were injected for calibration. Fiber, water, and midge extraction recoveries were between 97 and 102%, so no corrections were made, with the exception of extractions of aqueous solution (water and midge extracts only) of 4C3MP (54%). Uptake of the chemicals by fibers and larvae after different exposure periods was plotted. These data were fitted using a one-compartment model assuming first-order kinetics, Chp ) CaqK(1 - e-tk2), where Chp is the concentration of chemical in the hydrophobic phase, Caq is the concentration in the aqueous phase, K is the equilibrium partition coefficient between the hydrophobic phase and water (and which is equal to k1/k2), k2 is the elimination rate constant, k1 is the uptake rate constant, and t is time. The difference in K for fibers and midges determines the calibration factor for body residue estimates. A comparison of k2 values () k1/K) indicates whether the fibers or the larvae reach equilibrium concentrations faster (k2 indicates the time to equilibrium since from the model, t1/2 ) ln 2/k2). Fiber and midge data were summarized in the plots of log K versus log Kow, log k1 versus log Kow, and log k2 versus log Kow.
Results The decrease in water concentrations from the start to the end of the exposures was small enough (
