Polyacrylate-Coated SPME Fibers as a Tool To Simulate Body

Environ. Sci. Technol. 2000, 34, 324-331

Polyacrylate-Coated SPME Fibers as a Tool To Simulate Body Residues and Target Concentrations of Complex Organic Mixtures for Estimation of Baseline Toxicity E R I C M . J . V E R B R U G G E N , †,‡ W O U T E R H . J . V A E S , †,§ THOMAS F. PARKERTON,| AND J O O P L . M . H E R M E N S * ,† Research Institute of Toxicology, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands, and Exxon Biomedical Sciences, Inc., CN2350, East Millstone, New Jersey 08875

The acute toxicity of narcotic organic chemical mixtures is related to the total molar concentration within organisms. In this study, the use of polyacrylate SPME fibers is investigated for the purpose of biomimetic extractions, a procedure used to simulate bioconcentration and to estimate total concentrations in aquatic organisms. Experimental SPME-water partition coefficients correlate well with octanol-water and membrane-water partition coefficients, indicating that these passive sampling devices provide a good surrogate for lipid partitioning. On the basis of these relationships, the total internal concentration resulting from exposure to an aqueous sample can be estimated from the total moles of chemicals that sorb to the fiber. The aquatic toxicity of the sample is then predicted by comparison to critical internal concentrations that elicit adverse effects. This procedure offers a number of practical advantages over previous biomimetic extraction techniques (e.g., Empore disk) due to faster sorption kinetics, smaller sample volumes, and applicability to volatile compounds.

Introduction Risk assessment of surface waters and effluents inevitably must deal with the problem of complex chemical mixtures. The toxic action of compounds in mixtures can be antagonistic, concentration- or response-additive, or synergistic. For chemicals that act by narcosis, it has been shown that effects are completely concentration-additive (1-5). Narcosis represents a nonspecific mechanism because each chemical has equivalent potency and effects occurs at the same internal concentration or body residue (6, 7). This concentration at which toxicity occurs is referred to as critical body residue (CBR) or lethal body burden (LBB). For lethality, the CBR is reported to range from 30 to 160 mmol/Llipid (6). Due to narcosis additivity, the total internal concentration of organic * Corresponding author tel: +31 30 253 53 37; fax: +31 30 253 54 77; e-mail: [email protected]. † Utrecht University. ‡ Present address: National Institute of Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands. § Present address: TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands. | Exxon Biomedical Sciences, Inc. 324

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chemicals, often referred to as the total body residue (TBR), is a relevant metric for ecotoxicity assessments. It is estimated that more than 50% of all organic micropollutants present in the environment are chemicals that act simply by narcosis. Moreover, also chemicals with a more specific mode of action will always contribute to the overall unspecific toxicity (7, 8). Especially in complex mixtures, the concentration of compounds may probably be too low to cause specific toxicity, but they still do contribute to narcotic effects. Biomimetic Extraction. Recently, the biomimetic extraction technique has been developed to estimate total body residues (9-11). In the first instance, Empore disks, coated with C18, have been applied by Verhaar et al. (9). Since the biomimetic extraction process is a simple physical partitioning, it cannot take into account effects such as bioaccumulation, which includes food chain uptake, and metabolism. The procedure thus mimics the bioconcentration process (passive diffusion) in organisms that do not metabolize. The biomimetic extraction procedure consists of two major steps. First, the bioconcentration process is simulated by a hydrophobicity-dependent extraction. The partitioning of organic compounds between water and the hydrophobic phase of Empore disks was shown to be equally dependent on hydrophobicity as the bioconcentration process (9). In the aquatic environment, no depletion due to bioconcentration occurs, and consequently, the biomimetic extraction technique has to be nondepletive. An appropriate low phaseratio between the hydrophobic (C18-coated Empore disk) phase and aqueous phase must be achieved (9). The second step is to measure the total molar concentration in the hydrophobic phase. After an equilibration period with the aqueous sample, the disk is extracted with cyclohexane. The total molar concentration of this extract is then determined by gas chromatography-mass spectrometry (GC-MS) or vapor pressure osmometry (VPO) (10). The measurement of total molar concentrations requires that the analytical response of chemicals is additive and that the molar response of different chemicals is approximately equal (9). For VPO (9, 10, 12) and GC-MS (10), these conditions are met for compounds that are considerably less volatile than the solvent. By using a normal solvent injection in the case of GC-MS, the solutes that are not considerably less volatile than the solvent are not separated from the solvent and pass the GC in the solvent delay. The Empore disk method has been applied to surface waters and effluents, resulting in large differences in the estimated total amount of accumulated chemicals (11) and demonstrating the feasibility of this method for analyzing real environmental samples. A disadvantage of the Empore disk method is that the equilibration times are relatively long. For example, the time needed to reach 90% of equilibrium is about 2 weeks for pentachlorobenzene (9). On the basis of this work, an extraction time of 2 weeks is chosen for this procedure. Furthermore, to minimize the depletion of compounds from the aqueous phase and thus fulfill the requirement for biomimetic extraction, a sample volume of 10 L is used. The sample is renewed after 1 week so that the total sample volume needed for a single analysis is 20 L (10). Consequently, these considerations make this biomimetic extraction procedure impractical for routine application. An alternative to the use of the Empore disk is the solidphase microextraction (SPME) technique (13), as has been suggested and used as a biomimetic tool by Parkerton (14). The advantage of SPME is the ability to use a small volume of hydrophobic phase. SPME is frequently applied in the analysis of environmental samples (13, 15-20) and may also 10.1021/es990616s CCC: $19.00

 2000 American Chemical Society Published on Web 12/10/1999

TABLE 1. QSARs from Literature for BCF [L/kg] and LC50 [mM]a parameter

slope (a)

intercept (b)

r2

n

compounds

species

ref

log BCFw log BCFw log BCFw log BCFw log LC50 log LC50 log LC50

0.85 0.844 0.898 0.86 ( 0.30 -0.90 ( 0.02 -0.871 -0.91 ( 0.03

-0.70 -1.235 -1.315 -0.333 1.71 ( 0.06 1.87 1.72

0.897 0.692 0.925 0.968 0.92 0.976 0.984

55 34 20 12 150 50 19

miscellaneous miscellaneous miscellaneous chlorobenzenes miscellaneous miscellaneous miscellaneous

fish molluscs daphnids rainbow trout fathead minnow guppy Daphnia magna

38 39 39 40 6 41 42

a

Based on log Kow: log BCF/log LC50 ) a log Kow + b.

be used to measure freely dissolved concentrations of organic chemicals in the presence of biological matrixes, humic acids, or dissolved organic polymers (21-25). The SPME device consists of a fiber containing a hydrophobic coating. The fiber is placed in an aqueous solution. Thereafter, the extracted compounds are determined directly by thermal desorption of the fiber in the injector of a GC. Fibers are available with different coatings, e.g., polyacrylate (PA) and poly(dimethylsiloxane) (PDMS). The major advantages of SPME are the faster kinetics and the very small volume of the hydrophobic phase, with consequently much shorter equilibration times and smaller sample volumes in a negligible depletion extraction. Objectives. The objective of this study is to test the feasibility of SPME fibers for application in biomimetic extraction. Polyacrylate-coated fibers were selected, and partition coefficients were measured for a set of 28 chemicals with a wide variety of chemical structures. A second difference from the earlier work was the use of ion trap instead of quadrupole mass spectrometry for measuring total molar concentrations. Therefore, molar response factors were measured for the same set of chemicals. One of the crucial conditions of the biomimetic extraction is that the partition process should resemble the bioconcentration process. To be suitable for biomimetic extraction purposes, the solid phase should fulfill two requirements: 1. The partition coefficient to the SPME fiber (KSPME) must correlate well with the bioconcentration factor (Kb or BCF) or partition coefficient to the target site (Ktarget). 2. These partition coefficients must be proportional. In respect to the first requirement, these partition coefficients can only be compared indirectly by plotting them versus log Kow, since no direct relationship between log KSPME and log BCF is available. For species with a high lipid content, the bioconcentration factor is determined to a large extent by partitioning to storage lipids. Although for narcosis the lethal aqueous concentration (LC50) strongly depends on bioconcentration, BCF is not solely the partition coefficient to the target site. Because the toxic potency for narcosis is similar for all compounds, differences in LC50 directly express differences in partition coefficient to the target site, which is the cell membrane in the case of narcosis (26). Recently, it was shown that partitioning to artificial phospholipid membrane vesicles is a better descriptor for narcosis than log Kow (27). Therefore, the partition coefficients to the SPME fibers were alternatively compared with the partition coefficients to these L-R-dimyristoylphosphatidyl choline (DMPC) vesicles (KDMPC). The total concentration on the fiber can be correlated to the total concentration in membranes, which can be considered as a total target concentration (TTC). As an internal effect concentration, a critical target concentration (CTC) can be defined in the same way as it is usually done for the whole body of organisms, the critical body residue (28). To be suitable for biomimetic extraction, the partition coefficients to the SPME fibers must resemble membrane-water partition coefficients.

Regarding the second requirement, the slope of the loglinear relationship between KSPME and BCF or KDMPC has to be unity. The slope of BCF versus log Kow is not a fixed one but varies between 0.7 and 1. Based on theoretical considerations, the slope should approach unity (29). However, many QSARs have slopes of about 0.9, e.g., see Table 1. Therefore, as a reasonable criterion for biomimetic extraction, the slope of the log KSPME versus log Kow plot should be 0.9 ( 0.1.

Experimental Section Chemicals. All chemicals used for partitioning experiments (see Table 2) were of high purity and were obtained from either Fluka (Buchs, Switzerland), Aldrich-Chemie (Steinheim, Germany), Riedel-de Hae¨n (Seelze, Germany), Merck (Darmstadt, Germany), Shell Nederland Chemie (Rotterdam, The Netherlands), J. T. Baker (Deventer, The Netherlands), or Accu Standards (New Haven, CT). Solvents used were cyclohexane and methanol (both J. T. Baker, resi analyzed) and pure Milli-Q water (Millipore, Bedford, MA). As internal and external standard 2,4,5trichlorotoluene was used (Janssen Chimica, Geel, Belgium, 98%). Chemical Analysis. For quantification, a Varian (Walnut Creek, CA) GC-MS combination was used consisting of a 8200 CX autosampler, a Star 3400 CX GC, and a Saturn 2000 ion trap MS. A short, thin-film column was used (J&W, Folsom, CA; DB-1, length, 10 m; i.d., 0.25 mm; film thickness, 0.1 µm) with a nonpolar retention gap (deactivated fused silica retention gap, J&W, length, 1 m; i.d., 0.25 mm). Peak integration was performed using a Varian Saturn GC-MS workstation, version 5.1. The GC-MS analyses were performed under the following conditions. Solvent injections were carried out splitless with an injection volume of 1.0 µL. An insert liner of 0.8 mm φ was used in combination with a programmed temperature vaporizing injection (PTV) from 85 to 320 °C in 2 min. A rapid column temperature program was used, starting at 40 °C for 2 min to 290 °C at a speed of 30 °C/min. A solvent delay of 2.5 min was used. Fibers were desorbed splitless for 5 min at 275 °C. The same column temperature program was used as in the case of the solvent injections. The solvent delay for the injection of the fibers was 18 s. The ionization mode was EI (EI energy 70 eV), and a scan range of 34-650 m/z was used. All concentrations were determined by an average of the quantities analyzed by fullscan and selective ions, except for 2,3-dichlorophenol, hexachlorobenzene, aldrin, and dibutyl phthalate, for which only the quantities based on selective ions were used. The selective ions for each compound are listed in Table 2. The scan time was 0.36 s, and the autogain control target value was set to 20 000. The temperatures of the transfer line, the manifold, and the ion trap were set to 220, 110, and 210 °C, respectively. Helium was used as carrier gas with a linear velocity of about 1 m/s (determined by injection 5 µL of an argon/methane mixture). VOL. 34, NO. 2, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Selective Ions for Quantification of Each Chemical, the Concentration of the Chemicals in the Methanol Solutions (CMeOH) Used for Addition to Water, the Ratio between the Actual Concentration in Water (Ca) and the Nominal Concentration (Ca,nom), and the Relative Molar Response (RMR) compound

a

selective ions

2(3H)-benzothiazolone 2-chloroaniline benzene 4-chloronitrobenzene 3-chlorophenol atrazine toluene

Mixture 1 151 + 96 + 123 127 + 129 78 157 + 127 + 111 128 + 130 + 100 200 + 173 + 216 + 215 91 + 92 + 65

2,3-dichlorophenol chlorobenzene 1,2-dichlorobenzene 1,4-dichlorobenzene 1,3-dichlorobenzene 2,4,5-trichlorophenol 1,2,4-trichlorobenzene

162 + 164 + 63 112 + 114 + 77 146 + 148 + 111 146 + 148 + 111 146 + 148 + 111 198 + 196 + 97 182 + 180 + 184

1,2,3-trichlorobenzene fluoranthene 1,3,5-trichlorobenzene dibenzo-p-dioxin phenanthrene 1,2,4,5-tetrachlorobenzene 1,2,3,4-tetrachlorobenzene

182 + 180 + 184 202 182 + 180 + 184 184 + 128 + 92 178 216 + 214 + 218 216 + 214 + 218

1,2,3,5-tetrachlorobenzene dibutyl phthalate pentachlorobenzene endrin hexachlorobenzene aldrin di(2-ethylhexyl) phthalate

Mixture 4 216 + 214 + 218 149 250 + 248 + 252 245 + 243 + 263 + 265 + 261 + 281 284 + 286 + 282 + 288 263 + 261 + 265 + 293 + 291 149 + 167

Molar response relative to 2,4,5-trichlorotoluene.

n

9

Ca/Ca,nom

RMRa

27.4 14.5 11.5 9.2 6.4 8.8 9.7

1.08 0.93 ndb 0.96 0.92 1.24 nd

0.47 0.31 nd 0.48 0.35 1.21 nd

10.3 9.8 15.5 11.7 10.9 11.4 14.9

0.39 nd 0.46 0.47 0.35 0.73 0.32

0.65 nd 0.52 0.59 0.50 0.58 0.75

10.6 9.9 13.1 4.0 10.8 13.0 11.6

0.85 0.94 0.85 0.96 1.03 0.88 0.86

0.88 0.94 0.96 1.19 0.88 1.05 0.72

11.0 11.2 10.7 6.5 1.5 10.5 6.3

0.53 1.07 1.10 0.70 0.50 0.86 nd

1.46 1.99 2.08 1.36 2.80 2.94 nd

Mixture 2

Mixture 3

nd, could not be determined, see text.

Measurement of Molar Response Factors and SPME Partition Coefficients. Four mixtures of seven chemicals in water were prepared by adding stock solutions of these chemicals dissolved in methanol to water (dilution factors of stock in methanol with water were 2000, 20 000, 200 000, and 1 000 000 for the four mixtures, respectively). The resulting nominal aqueous concentrations were well below aqueous solubility for all compounds. Furthermore, a solution in cyclohexane of each mixture was prepared by adding 100 µL of each compound dissolved in methanol to 100 mL of cyclohexane. The actual concentrations of the aqueous solutions were determined by extraction of aqueous samples with 10 mL of cyclohexane. These samples were compared with 10 mL of cyclohexane, containing the same amount of chemicals, equilibrated with an equal water volume to correct for the extraction recovery of the compounds. These 10-mL extracts and 10 mL of the cyclohexane solutions were concentrated to 0.5 or 1 mL by slow evaporation under nitrogen. It has been shown that losses due to evaporation are almost negligible (10). Before analysis, 50, 100, or 500 µL of a 0.040 mM solution of 2,4,5-trichlorotoluene in cyclohexane was added as the internal standard. The relative (to 2,4,5trichlorotoluene) molar response (RMR) based on the fullscan peak areas could be derived from analysis of the concentrated cyclohexane solutions. SPME extractions were performed in a 250-mL flatbottomed round flask with a screw cap, closed with a PTFE seal (Phase Separations BV, Waddinxveen, The Netherlands) without headspace. This flask was placed on a magnetic stirrer (Ikamag REQ, Janke & Kunkel, Staufen, Germany), using a spin bar of 2 cm. The whole uptake experiment was carried 326

CMeOH [mM]


out with the same polyacrylate fiber (Supelco, Bellefonte, PA). Polyacrylate fibers, with a length of 1 cm, have an internal diameter of 110 µm on which a coating of 85 µm of partially cross-linked polyacrylate is applied. The calculated volume of the polyacrylate phase on such a fiber is 0.521 µL. During extraction, the stirrer was operated at maximum speed (∼1200 rpm). The extraction times were 5, 10, 15, 30, 45, 60, and 75 min for mixtures 1-3 and were 30, 60, 90, 120, and 150 min for mixture 4. Before and after analysis of the fibers, 1 µL of a 0.040 mM solution of 2,4,5-trichlorotoluene in cyclohexane was injected as the external standard, in duplicate and triplicate, respectively. The concentrations on the fiber were calculated from the RMR values, and the ratio of the peak areas of the compounds, and the external standard. To determine the partition coefficient, an uptake curve was fitted to the data. For the uptake curves, a onecompartment model with first-order kinetics was used:

k1 Cfiber,t ) Ca (1 - e-k2t) k2

(1)

In this equation, t is the time of the partitioning process [min], Cfiber,t is the concentration on the fiber at time t [mM], Ca is the concentration in the aqueous phase [mM], k1 is the first-order uptake rate constant [(Lwater/LPA) min-1], and k2 is the first-order elimination rate constant [min-1]. KSPME is defined as the ratio of k1 and k2. To obtain a standard error for log KSPME, data were also fitted by the following equation:

Cfiber,t ) Ca × 10log KSPME(1 - e-k2t)

(2)

TABLE 3. Calculated Values for the Energy of the Highest Occupied Molecular Orbital (EHOMO), the Energy of the Lowest Unoccupied Molecular Orbital (ELUMO), the Molecular Volume (MV), the Most Negative Charge on Any Non-Hydrogen Atom (Q-), the Most Positive Charge on Any Hydrogen Atom (Q+), log Kow, and the Estimated Partition Coefficients to Artificial Membranes (KDMPC) EHOMO [eV]

ELUMO [eV]

MV [Å3]

Q- [au]

Q+ [au]

log Kowa

log KDMPCb [La/LDMPC]

2(3H)-benzothiazolone 2-chloroaniline benzene 4-chloronitrobenzene 3-chlorophenol atrazine toluene

-8.84 -8.62 -9.65 -10.47 -9.30 -9.41 -9.33

-0.183 0.285 0.555 -1.344 0.039 0.038 0.520

Mixture 1 121 107 83 119 104 193 99

-0.508 -0.953 -0.092 -0.492 -0.493 -0.984 -0.239

0.326 0.387 0.092 0.123 0.343 0.375 0.104

1.76 1.90 2.13 2.39 2.50 2.61 2.73

2.31 2.31 2.10 2.57 3.06 2.72 2.57


-9.39 -9.56 -9.60 -9.52 -9.68 -9.32 -9.62

-0.249 0.155 -0.142 -0.216 -0.158 -0.511 -0.469

Mixture 2 118 96 110 110 111 131 124

-0.512 -0.163 -0.096 -0.111 -0.202 -0.516 -0.153

0.350 0.116 0.102 0.087 0.121 0.358 0.101

2.84 2.89 3.43 3.44 3.53 3.72 4.02

3.41 2.89c 3.42 3.39 3.52 4.27 3.97

1,2,3-trichlorobenzene 1,3,5-trichlorobenzene dibenzo-p-dioxin phenanthrene 1,2,4,5-tetrachlorobenzene 1,2,3,4-tetrachlorobenzene fluoranthene

-9.78 -9.92 -8.57 -8.62 -9.65 -9.73 -8.63

-0.365 -0.402 -0.134 -0.409 -0.731 -0.652 -0.929

Mixture 3 124 125 161 170 138 137 189

-0.171 -0.249 -0.324 -0.145 -0.058 0.056 -0.130

0.122 0.048 0.149 0.120 0.073 0.097 0.118

4.14 4.19 4.38 4.47 4.60 4.64 5.16

4.11 3.89c 4.16 4.29 4.50 4.65 5.00


-9.76 -10.41 -9.79 -9.95 -9.91 -9.51 -10.29

-0.684 -0.719 -0.891 -0.482 -1.041 -0.266 -0.669

Mixture 4 137 277 151 244 164 236 404

-0.199 -0.539 -0.137 -0.345 -0.004 -0.155 -0.549

0.058 0.113 0.064 0.125 0.000 0.119 0.131

4.66 4.72 5.18 5.20 5.73 6.50 7.45

4.42 4.05c 4.96 4.68 (5.34) (5.94) (6.24)

Phenols from Ref 30 -1.185 113 -0.567 0.426 108 -0.497 0.402 123 -0.498 -0.245 119 -0.499 0.314 157 -0.509

0.408 0.337 0.336 0.341 0.321

1.79 1.94 2.23 3.06 3.31

2.83 2.41 2.64 3.59 3.51

compound

2-nitrophenol p-cresol 3,4-dimethylphenol 2,4-dichlorophenol 4-tert-butylphenol a

-9.91 -8.88 -8.82 -9.27 -9.29

Selected values (ClogP star list) from ref 32.

b

Calculated values (see text for further details). c Experimental KDMPC available.

Uptake curves were fitted using GraphPad Prism, version 2.01 (GraphPad Software, San Diego, CA). Calculation of Membrane-Water Partition Coefficients. For the chemicals from this study and those reported by Dean et al. (30), L-R-dimyristoylphosphatidyl choline (DMPC, artificial phospholipid membrane vesicles)-water partition coefficients (KDMPC) were calculated as described by Vaes et al. (31) from log Kow (32) and five calculated descriptors: energy of the highest occupied molecular orbital (HOMO), energy of the lowest unoccupied molecular orbital (LUMO), molecular volume (MV), most negative charge on any nonhydrogen atom (Q-), and most positive charge on any hydrogen atom (Q+). These descriptors were calculated using the quantum chemical package Spartan (33) running on an IBM RS/6000 workstation. Further details are described by Vaes et al. (31). The values of these parameters are presented in Table 3. If available, experimental values for log KDMPC were used (22, 31).

Results and Discussion Molar Response Factors. The molar response of each chemical relative to the internal standard could be determined for all chemicals except for benzene, toluene, and chlorobenzene because these three compounds could not be separated from the solvent peak. For these compounds,

an RMR value of 0.5 was assumed for calculating the concentration on the fiber. The relative molar responses (RMR) for the full-scan signal are listed in Table 2. Initially, we observed a very high RMR value for di(2-ethylhexyl) phthalate (DEHP), which is probably caused by the fact that this compound is detected in many samples as a contaminant. However, the sample in cyclohexane with added water contained a much lower amount of DEHP than the other two samples, which would correspond to an RMR value of 5.18. In further calculations this value was used. The rest of the RMR values show a log-normal distribution. This distribution is shown in Figure 1. As can be seen from Figure 1, the deviations are somewhat larger than previously reported data using quadrupole MS (10), but also from this dataset it is estimated that for 90% of the compounds the RMR value is between 0.32 and 2.43 (mean log RMR ) -0.053, SD ) 0.267, n ) 24). Both datasets show that the average of the molar response relative to 2,4,5-trichlorotoluene is very close to unity. Therefore, for both GC-MS systems, the fullscan signal relative to that of a known amount of 2,4,5trichlorotoluene can be used to estimate the total molar amount of unknown chemicals. Kinetics of Uptake to SPME Fibers. The actual aqueous concentrations are given in Table 2 as a percentage of the nominal concentrations. If the actual concentration of a VOL. 34, NO. 2, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Distribution of molar responses relative to 2,4,5trichlorotoluene (RMR). (9) and (s), this research; (b) and (- -), data from van Loon et al. (10). Note that the value of RMR for 2,4,5trichlorotoluene itself should be 1. compound was higher than the concentration in the control sample, the nominal concentration was taken as a value for the aqueous concentration. For most chemicals, a onecompartment first-order kinetics model could be fitted through the data (Table 4). In Figure 2, the rate constants k1 and k2 are shown as a function of log KSPME. As can be seen, k1 is constant and k2 decreases for compounds with log KSPME values of 3.5 and higher, which means that diffusion through the aqueous phase becomes the rate-limiting step. Previously reported data on kinetics of the polyacrylate fibers show that the resistance in the hydrophobic phase is the rate-limiting step for uptake to the fiber for compounds with log KSPME values up to 3.5, because constant log k2 values were observed (34). Furthermore, rate constants in this system (250 mL) seem to be in accordance with the previously reported rate constants (34) that were determined in 1.5-mL GC vials. Values for six compounds that were not reported in the original study (31) were also included (Table 4). These values were determined by using an automated agitating device instead of stirring with a spin bar (31). However, the kinetic constants still are in very good agreement with the values determined with a spin bar. For the least hydrophobic compounds (mixture 1), the partitioning process is fast. This leads to a larger uncertainty in the values of k1 and k2 and accordingly a lower correlation coefficient (Table 4). Nevertheless, the estimation of log KSPME is still reliable because equilibrium is reached almost completely within the period of the uptake experiment. For highly hydrophobic compounds, the log k2 value becomes very small and the error in log k2 and log KSPME increases. For endrin, the curvature of the data was not sufficient to obtain a value for k2 or KSPME (Table 4). The only reliable value for endrin is that of log k1 because this value is determined solely by the initial uptake from water. Correlation of Log KSPME with Log Kow and Log KDMPC. In Figure 3, log KSPME data from Table 4 are shown as a function of log Kow (all selected values from the CLogP star list) (32). Also included in this plot are values from literature (30, 34). Although the scatter of the results is rather high, it can be seen that log KSPME increases with increasing hydrophobicity. However, the log KSPME values for compounds with log Kow higher than 5.2 (endrin) seem to decrease. When this would be caused by an experimental artifact, such as reduced bioavailability, this would lead to erroneous low k1 values. However, k1 values are almost constant over the range of log Kow values between 3.5 and 7.5. The value of k2 only depends on the relative amount on the fiber and not on the aqueous concentration or the relative molar response. For all compounds, including DEHP, background contamination on the fiber was not significant, and consequently, experimental 328

9


FIGURE 2. First-order rate constants for uptake (k1) [(La/(Lfiber) min-1] and elimination (k2) [min-1] for partitioning to polyacrylate SPME fibers: (9) log k1 from this study, (0) data from Vaes et al. (34) including data from Table 4, (b) log k2 from this study, and (O) data from Vaes et al. (34) including data from Table 4. artifacts will probably not affect k2 values. For the compounds in the log Kow range between 3.5 and 5.5, k2 decreases with increasing hydrophobicity. However, for the three most hydrophobic compounds, k2 increases slowly with hydrophobicity. This may indicate that the relatively low KSPME values of the highly hydrophobic chemicals are caused by a real physical effect, which may be explained by a strongly increasing activity coefficient for these compounds in the partially cross-linked polyacrylate phase. A similar effect is also suggested to occur in the highly structured bilayers of membranes (35, 36). Membrane-water partition coefficients indeed tend to be constant or even decrease at very high hydrophobicity (37). By omitting these highly hydrophobic compounds, the following relationship between log KSPME and log Kow can be derived:

log KSPME ) 0.92 ((0.06) log Kow + 0.01((0.20) r 2 ) 0.813

SE ) 0.53 (n ) 51) (3)

For the chemicals from this study and those reported by Dean et al. (30), log KDMPC values were calculated (Table 3). For the chemicals from Vaes et al., experimental values are available (22, 31). The corresponding set of (calculated and experimental if available) values of log KDMPC show a similar relationship with log Kow as was found for log KSPME:

log KDMPC ) 0.92 ((0.05) log Kow + 0.33((0.14) r 2 ) 0.893

SE ) 0.38 n ) 51 (4)

In Figure 4, log KSPME is plotted versus log KDMPC. The scatter of the data is somewhat reduced in comparison with Figure 3, which suggests that log KDMPC correlates better to log KSPME than log Kow does. Another important feature is that the slope of this regression is almost unity:

log KSPME ) 0.98 ((0.05) log KDMPC - 0.25((0.18) r 2 ) 0.868

SE ) 0.45 n ) 51 (5)

This equation is based on different datasets. If only the data from this study are used, for which actual aqueous concentrations were determined, the correlation coefficient is considerably increased (r 2 ) 0.916, SE ) 0.32, n ) 24). For this set of chemicals, log KSPME is also significantly better correlated to log KDMPC than to log Kow (P < 0.05, one-tailed). If KDMPC is considered as membrane-water partitioning, SPME polyacrylate fibers are an appropriate surrogate phase

TABLE 4. First-Order Rate Constants for Uptake (k1) and Elimination (k2), the Partition Coefficient (KSPME), and the Correlation Coefficient of the Curve Fit (r) for Partitioning to Polyacrylate SPME Fibers and Partition Coefficients to Artificial Membranes (KDMPC) compound

log k1 [(La/Lfiber) min-1]

log k2 [min-1]

log KSPME [La/Lfiber]

( SE

r2

-1.29 -1.18 -0.96 -1.01 -1.37 -1.34 -0.94

1.87 2.22 1.15 2.54 2.51 1.95 1.83

0.02 0.03 0.09 0.04 0.03 0.02 0.06

0.96 0.89 0.33 0.77 0.96 0.97 0.48

-1.75 -0.84 -1.41 -1.33 -1.41 -2.02 -1.60

3.71 2.12 3.47 3.51 3.55 4.23 3.83

0.07 0.05 0.03 0.03 0.02 0.13 0.04

0.98 0.50 0.96 0.94 0.98 0.99 0.98

-1.73 -1.70 -2.07 -2.30 -2.20 -2.13 -2.25

3.79 3.73 4.23 4.45 4.40 4.45 4.53

0.07 0.06 0.17 0.23 0.21 0.22 0.20

0.98 0.98 0.98 0.99 0.99 0.98 0.99

-2.38 -1.84 -2.61 nd -2.65 -2.38 -2.14

4.56 3.81 4.64 nd 4.82 4.53 3.96

0.20 0.03 0.44 nd 0.39 0.33 0.23

0.98 0.98 0.97 0.96 0.98 0.93 0.87

-0.83 -0.89 -1.17 -1.08 -1.20 -1.05

1.00 1.77 3.04 2.84 2.86 3.27

0.03 0.02 0.04 0.04 0.05 0.04

0.91 0.95 0.98 0.96 0.97 0.95

Mixture 1 2(3H)-benzothiazolone 2-chloroaniline benzene 4-chloronitrobenzene 3-chlorophenol atrazine toluene

0.58 1.04 0.19 1.53 1.14 0.61 0.89


1.96 1.28 2.06 2.18 2.14 2.22 2.23

1,2,3-trichlorobenzene 1,3,5-trichlorobenzene dibenzo-p-dioxin phenanthrene 1,2,4,5-tetrachlorobenzene 1,2,3,4-tetrachlorobenzene fluoranthene

2.06 2.03 2.16 2.15 2.21 2.32 2.29


2.18 1.97 2.03 2.37 2.17 2.14 1.82

diethyl malonate diethyl adipate dimethyl-2-amino-p-phthalate methyl-4-chloro-2-nitrobenzoate dibutyl succinate dibutyl phthalate

0.18 0.88 1.87 1.76 1.66 2.21

Mixture 2

Mixture 3

Mixture 4

Data from Ref 31

for membranes and thus a rational choice as a tool to perform biomimetic extractions, because the site of toxic action of narcosis is probably located in the cell membranes (26). Calculation of CBR or CTC for Narcosis. Two different approaches can be chosen to derive a value for the concentration in an organism at which a certain effect occurs. In the traditional approach, the internal concentration in organisms at which lethality occurs is directly measured or calculated as the product of BCF and LC50 (6, 7). The resulting value of CBR for chemicals acting by narcosis is ∼50 mM (30-160 mM), based on lipid weight of the organism (6). Alternatively, the critical target concentration (CTC) is calculated as the concentration in the DMPC membranes related to a certain effect concentration in the aqueous phase. This approach is based on the assumptions that the cell membrane is the target for narcosis and that partitioning to artificial DMPC membranes and cell membranes is equal. From the data by Vaes et al. (27) for 18 narcotic chemicals (quinoline excluded), the concentration in DMPC membranes at lethal aqueous concentrations can be calculated from values of log KDMPC and log LC50 for guppy. The resulting log CTC for lethality is equal to 2.02 (( 0.28, n ) 18). A value for CTC can also be derived from QSARs for log 1/LC50 on basis of log Kow. Both the slope (a) and the intercept (b) of these QSARs are remarkably constant (Table 1). If LC50 is determined at steady state, 1/LC50 is equal to Ktarget/CTC.

Assuming that Ktarget is equal to KDMPC, the QSARs for log 1/LC50 with the constants a and b can be rewritten as:

log KDMPC ) a log Kow + b + log CTC

(6)

Forcing eq 4 to have exactly similar slopes as the three QSARs for 1/LC50 from Table 1, log CTC can be determined by combining eqs 4 and 6 with a and b from Table 1. The resulting mean value for log CTC derived from three QSARs for fathead minnow, guppy, and Daphnia magna is equal to 2.16 (( 0.15). The CTC values for narcosis by both approaches are mutually consistent. Moreover, the range of 50-200 mM is also very similar to the CBR for narcosis, which is between 30 and 160 mmol/Llipid (6). Summarizing, for critical internal concentrations, two alternative values may be used. The first value of 50 mM is for CBR, representing concentrations in lipids deduced from experimental data for aquatic organisms. The second value of 125 mM is for CTC, based on membrane partitioning to artificial DMPC membranes. Calculation of TBR or TTC from Total Molar Concentrations on SPME Fibers. The same two approaches for deriving the critical internal concentrations can be followed to correlate the total internal concentration to the total concentration on the fiber. There are no direct relationships between KSPME and BCF. As was described for the Empore VOL. 34, NO. 2, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 5. Comparison of Biomimetic Extraction Performed by Empore Disks and Polyacrylate SPME Fibers with Pentachlorobenzene as an Example Empore disk

polyacrylate fiber

ratio of vol hydrophobic 1.1 × 10-6 2.0 × 10-6 phase (Vh) vs aqueous phase (Va) depletion of PeCB after 21% after 2 weeks, 9% after extraction time calculated including 1 day renewal of sample after 1 week (43) time to reach 90% of 15 days, 12 days due 16 h equilibrium for PeCB to depletion (43)

FIGURE 3. Partition coefficients to polyacrylate SPME fibers (KSPME) [La/Lfiber] versus log Kow: (9) log KSPME from this study, (2) data from Vaes et al. (34) including data from Table 4, and (b) data from Dean et al. (30).

By forcing the slope of eq 5 to be exactly unity, the resulting intercept is equal to -0.32 (- log 2.07), and TTC can be related to the total concentration on the SPME-fiber:

TTC = CDMPC ) 2.07Cfiber

(8)

By comparing eqs 7 and 8 and the corresponding critical internal concentrations, it can be concluded that, for equal concentrations on the fiber, the two approaches yield equal ratios of the total internal concentration and the critical internal concentration (TBR/CBR ≈ TTC/CTC). Therefore, both equations are in good agreement and indicate the validity of the derived relationships. comparison of body residue approach and target concentration approach

FIGURE 4. Partition coefficients to polyacrylate SPME fibers (KSPME) [La/Lfiber] versus partition coefficients to artificial membranes (KDMPC) [La/LDMPC]: (9) log KSPME from this study, (2) data from Vaes et al. (34) including data from Table 4, and (b) data from Dean et al. (30). disk (9), QSARs for log BCF can be used to derive a relationship between the total concentration on the fiber (Cfiber) and the total concentration in biota (TBR). For application in biomimetic extraction, the slope of the log KSPME vs log Kow plot should be close to that of QSARs for log BCF on basis of log Kow. As can be seen from Table 1, QSARs reported in the literature for log BCF and log 1/LC50 generally exhibit a slope of about 0.9, similar to eq 3. For this reason, it is expected that polyacrylate fibers are suitable for biomimetic extraction. However, the intercept of the QSARs for log BCF, based on wet weight of the organisms, vary widely (-0.3 to -1.3). This difference reflects the difference in lipid content between different species, e.g., daphnids and rainbow trout. It is generally assumed that, if BCF is expressed on lipid weight basis, the slope of such relationships varies between 0.9 and 1.0 while intercept values are about 0, which is equal to the correlation between log KSPME and log Kow (eq 3). Therefore, the total concentration on the fiber can be used as an estimate of the total concentration in the lipids of biota (TBR):

TBR ) Cfiber

(7)

Alternatively, the total concentration in membranes, the total target concentration (TTC), can be deduced from the total concentration in artificial phospholipid membranes (CDMPC). 330

9


body residues for narcosis are constant: Cbiota ) BCF × LC50 bioconcentration factors are equal to SPME-water partition coefficients critical body residue CBR 50 mM total body residue TBR ) ∑Cbiota log BCFlipid ) (0.8-1.0) log Kow + 0 log KSPME ) 0.92 log Kow + 0.01 Cbiota ) CSPME

target for narcosis is the cell membrane: Cm ) Kmw × LC50 membrane-water partitioning is equal to DMPC-water partitioning critical target concentration CTC 125 mM total target concentration TTC ) ∑Cm log Kmw ) log KSPME + 0.32

Cm ) 2.07CSPME

Implications for Application in Biomimetic Extraction. The SPME fibers have some major advantages in comparison with the Empore disk. The biomimetic extraction can be performed faster because kinetics are much faster. An extraction time of 1 day would be sufficient for the fibers as compared to 2 weeks for the Empore disk. The water volume can be reduced from 10 L to 250 mL due to the much lower depletion by the SPME fiber. For pentachlorobenzene, these two advantages are illustrated in Table 5. Additionally, the hands-on time can be substantially decreased since the SPME fiber is injected directly in the gas chromatograph, while the Empore disk must be extracted with an organic solvent first. Due to the absence of a solvent, also the volatile compounds that are not separated from the solvent and pass the GC in the solvent delay in a normal solvent injection are taken into account. Some examples of these compounds are benzene, toluene, and chlorobenzene from the set of compounds from this study. Biomimetic extraction with SPME fibers provides a powerful analytical tool for quantifying the bioavailability of complex narcotic chemical mixtures that can be practically applied in risk assessment of surface waters and effluents. In the future, it should be investigated if the application of this technique can also be extended to risk assessment of soils and sediments.

Acknowledgments The authors thank Gert-Jan de Maagd from the Institute for Inland Water Management and Waste Water Treatment

(RIZA), Lelystad, The Netherlands, and Philipp Mayer from the Research Institute of Toxicology, Utrecht University, The Netherlands, for helpful discussions.

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Received for review May 27, 1999. Revised manuscript received October 11, 1999. Accepted October 25, 1999. ES990616S

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Polyacrylate-Coated SPME Fibers as a Tool To Simulate Body

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