Environ. Sci. Technol. 2000, 34, 4989-4994
Influence of pH and Other Modifying Factors on the Distribution Behavior of 4-Quinolones to Solid Phases and Humic Acids Studied by “Negligible-Depletion” SPME-HPLC HANS-CHRISTIAN HOLTEN LU ¨ T Z H Ø F T , * ,†,‡ W O U T E R H . J . V A E S , ‡,| A N D R E A S P . F R E I D I G , ‡,| BENT HALLING-SØRENSEN,† AND JOOP L. M. HERMENS‡ Section of Environmental Chemistry, Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark, and Research Institute of Toxicology (RITOX), University of Utrecht, P.O. Box 80176, 3508 TD Utrecht, The Netherlands
Flumequine, oxolinic acid, and sarafloxacin are 4-quinolone antimicrobials that are used in fish farming. The objective of this investigation was to study the influence of pH on the distribution behavior between buffer solutions and Aldrich humic acids (DOC) of these compounds. Negligibledepletion solid-phase microextraction coupled to highperformance liquid chromatography (nd-SPME-HPLC) was used to quantify the free concentration of the antimicrobials. It was shown that the distribution of the antimicrobials between buffer and the SPME fiber could be described as a function of pH and ionization constants. Distribution coefficients between buffer and DOC (DDOC) were established. It was not possible to describe this distribution using pH and ionization constants. The magnitude of the DDOC values (103.4-105.2) was found in the same range as for highly hydrophobic chemicals despite the low hydrophobicity of the 4-quinolones (KOW < 101.7).
Introduction Studies of partitioning of hydrophobic chemicals between organic and aqueous phases have shown that hydrophobicity usually has a strong influence on various parameters, e.g., bioconcentration, partitioning to dissolved organic carbon (KDOC), uptake rate, and lethality in guppies (1-4). Bioconcentration factors have been shown to decrease in the presence of DOC (5-7). KDOC was shown to decrease by increasing pH, which was explained by increased ionization of DOC (8). Little is known about the distribution of ionizable compounds, e.g., weak (carboxylic) acids and weak bases (9). Triorganotin compounds have been studied with respect to their distribution to 1-octanol (10), mineral surfaces (11), and humic acids (12); all revealing pH-dependent distribu* Corresponding author telephone: +45 35 30 64 59; fax: +45 35 30 60 13; e-mail:
[email protected]. † The Royal Danish School of Pharmacy. ‡ University of Utrecht. | Present address: TNO, Nutrition and Food Research, Zeist, The Netherlands. 10.1021/es000917y CCC: $19.00 Published on Web 10/21/2000
2000 American Chemical Society
tions. Also nitrophenols revealed a pH-dependent adsorption to mineral surfaces, which was approaching zero when pH was increased (13). Substituted phenols showed pH-dependent distribution to 1-octanol and liposomes (14). The free concentration is generally believed to be bioavailable and thus responsible for toxic effects (15). In this context, Cano et al. (16) showed that when the organic carbon content in the sediment increased, the LC50 values based on the concentration in the sediment increased as well, whereas the LC50 values based on the concentration in the overlying and interstitial water did not change. Several studies have shown the feasibility of solid-phase microextraction (SPME) using gas chromatography for measuring concentrations (1721). Negligible-depletion SPME (nd-SPME) is an application of SPME in which a negligible part of the freely dissolved concentration is extracted. Hence, it can be applied for measuring freely dissolved concentrations (22) and partition coefficients (23). Equilibrium dialysis with bovine serum albumine on one side of the dialysis membrane was used to validate the ndSPME method (22). Quantified by nd-SPME, the free concentration of 4-chloro-3-methylphenol at both sides of the dialysis membrane was equal. Using nd-SPME, Urrestarazu Ramos et al. (24) showed that with and without humic acids bioconcentration factors remained the same when free concentrations were used instead of total concentrations, whereas the use of total concentrations resulted in decreased bioconcentration factors in the presence of humic acids. The objective of this investigation was to study the influence of pH on the distribution behavior between Aldrich humic acids (DOC) and buffer solutions of 4-quinolones using nd-SPME coupled to high-performance liquid chromatography (nd-SPME-HPLC). Distribution to the SPME fiber may depend on the chemical speciation, e.g., protonated or deprotonated species or ion pair formation. Therefore, the influence of salinity, buffer capacity, and pH on the distribution to the SPME fiber was studied. The interactions between the 4-quinolones and DOC may depend on the degree of ionization; therefore, the binding to humic acids was studied at pH values covering different ionization states of the compounds. Realizing that Aldrich humic acids are not similar to natural humic acids, the study focused more on the changes and the magnitude of the results than on the exact values. The antimicrobials flumequine (FLU), oxolinic acid (OXA), and sarafloxacin (SAF) (for chemical structures, see Table 1) were chosen as model compounds for the 4-quinolone group of antimicrobials. 4-Quinolones are weak carboxylic acids and weak bases that are relevant ionizable chemicals for studying their distribution behavior between aqueous and organic phases. They are used to control bacterial infections and are present in the environment due to application in intensive fish farming (25-28). Furthermore, laboratory experiments with 4-quinolones in the microgram per liter range have been shown to inhibit the growth of algae (29).
Theoretical Considerations Uptake Profiles to SPME Fiber. The kinetics of the partitioning to an SPME fiber was described in detail by Vaes et al. (22) using a one-compartment model to determine the uptake profile:
[X]SPME,t )
k1 [X] (1 - e-k2t) k2 W,0
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TABLE 1. Chemical Structures and Some Physical Chemical Properties of the 4-Quinolonesa chemical
a
structure
molecular formula
MW (g/mol)
FLU
C14H12FNO3
261.25
OXA
C13H11NO5
261.23
SAF
C20H17F2N3O3
385.37
S (mg/L)
MW, molecular weight; S, aqueous solubility at pH 7. b From ref 39. c From ref 40. d From ref 33,
where [X]SPME,t is the concentration of X on the SPME fiber at time t; k1 and k2 are the uptake and elimination rate constants, respectively (from which KSPME can be calculated as k1/k2); and [X]W,0 is the concentration of X in the aqueous phase at time zero. A minimum sampling time (ts) can be calculated using the following equation, which is a balance between accuracy and time efficiency (22):
ts g -
(
0.05 1 ln k2 k2 + 0.05
)
(2)
Ka
1
Ka
2
(3)
where A refers to either the deprotonated state of the carboxylic acid in FLU and OXA or the deprotonated states of the carboxylic acid and the amine function in the piperazine moiety of SAF; AH refers to either the protonated state of the carboxylic acid in FLU and OXA or the protonated state of the carboxylic acid and the deprotonated state of the amine function in the piperazine moiety of SAF; and AH2 refers to the protonated state of both functions in SAF. On the basis of eq 3, the following equations can be derived:
Ka1 )
[AH]W[H+]W
Ka2 ) D′ )
[AH2]W [A]W[H+]W [AH]W
[AH2]HP + [AH]HP + [A]HP [AH2]W + [AH]W + [A]W
(6)
D′ )
D′ )
4990
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1.72c
4.1b
6.9d
0.68e
-f
-
-
From ref 41.
f
-, no data available.
DAH2 1 + 10pH-pKa1
DAH2 + DAH × 10pH-pKa1 1 + 10pH-pKa1
DAH2 + 10pH-pKa1(DAH + DA × 10pH-pKa2) 1 + 10pH-pKa1(1 + 10pH-pKa2)
(7)
(8)
(9)
Having only one ionizable function, eqs 7 and 8 are valid by substituting AH2 with AH, AH with A, and pKa1 with pKa. The above equations are not taking the distribution of ion pairs into account but only the ionization properties of the species. Distribution Coefficients to Aldrich Humic Acids. When introducing a third phase to the system, like DOC, distribution of the 4-quinolones is not only limited to the SPME fiber but also interactions with humic material will occur. The distribution coefficient (DDOC) is defined as
DDOC )
CDOC(X) Cfree(X)
(10)
where CDOC(X) is the concentration of X bound to the humic material and Cfree(X) is the concentration of unbound X, the free concentration, in the aqueous phase. The free fraction (f) is defined as follows:
f) Ka1 and Ka2 are the respective acid dissociation constants,
e
D′ )
(4)
(5)
log KOW
and D′ is the apparent distribution coefficient. When having two ionizable functions, three subcases can be considered: (i) Only AH2 partitions to the hydrophobic phase. (ii) Both AH2 and AH partition to the hydrophobic phase. (iii) AH2, AH, and A partition to the hydrophobic phase. On the basis of eqs 4-6, the following pH-dependent distribution coefficients can be derived, analogous to the Henderson-Hasselbalch equations, referring to subcases i, ii, and iii, respectively:
Distribution Coefficients to SPME Fiber. The distribution of the antimicrobials between the aqueous phase (W) and a hydrophobic phase (HP) can be considered with regard to their ionizable properties. The general scheme is given in eq 3:
AH2 {\} AH {\} A
p Ka 6.4c
71b
Cfree(X) Ctotal(X)
)
1 1 + DDOCC(DOC)
(11)
where Ctotal(X) ) CDOC(X) + Cfree(X) and C(DOC) is the concentration of DOC (in kg/L). Fitting experimental data to eq 11 reveals DDOC. Nonlinear regressions, as well as linear regressions, were performed using GraphPad Prism version 2.01 (1996) for Windows, GraphPad Software, San Diego CA (www.graphpad.com).
Experimental Section Chemicals. Chemicals were purchased from the following companies: flumequine (FLU), 9-fluoro-6,7-dihydro-5methyl-1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid (>99.9%), Sigma Chemical Co. (St. Louis, MO); oxolinic acid (OXA), 5-ethyl-5,8-dihydro-8-oxo-1,3-dioxolol[4,5-g]quinoline-7-carboxylic acid (>98%), Unikem A/S (Copenhagen, Denmark); sarafloxacin hydrochloride (SAF), 6-fluoro-1-(4fluorophenyl)-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid (88.5%), Abbott Laboratories (North Chicago, IL); HPLC grade methanol (GC assay 99.9%), Labscan (Dublin, Ireland). Buffer solutions were made of citric acid, KH2PO4, TRIZMA (tris[hydroxymethyl]aminomethane hydrochloride), and NaHCO3 and adjusted with NaOH to the desired pH values. Buffer substances and NaOH (analysis grade) were purchased either from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO). Stock solutions of the test compounds were made in NaOH. All aqueous solutions were made in Milli-Q water. Aldrich humic acid sodium salt, Lot No. 01816-054, was purchased from Aldrich Chemie (Zwijndrecht, The Netherlands). The organic carbon content of the Aldrich humic acids used was determined as 34% (30). Chemical structures and some physical chemical properties of the test compounds are shown in Table 1. HPLC Procedure. The HPLC system consisted of a Varian 9012 solvent delivery system (Varian, Walnut Creek, CA); a Supelco Discovery C18 column (Supelco, Bellefonte, PA), 150 × 4.6 mm, 5 µm particle size stationary phase; and a Merck Hitachi L-4000 UV detector (Merck, Darmstadt, Germany) operated at a wavelength of 260 nm. To minimize the physical effect on the fiber from the solvent flow, the flow rate was linearly increased from 0.2 mL/min at 0.3 min to 1.0 mL/min at 1.0 min and kept there throughout the analysis. The sample was injected at 0.3 min. The solvents used were Milli-Q water (A), methanol (B), and 2 mM H3PO4, pH 2.8 (C). The composition of the mobile phase A:B:C was programmed from 0:20:80 within 1 min to 0:40:60, within 3 min to 0:85:15 where it was ramped to 0:100:0 and kept there 1 min, where it was programmed to 100:0:0 and kept there for 1 min to condition the fiber and eliminate any residual solvent in an aqueous environment. The analysis was followed by an equilibration time of 6 min while the mobile phase returned to its original composition. Data were acquired during analysis and equilibration. The fiber was left in the desorption chamber of the SPME-HPLC interface (Supelco, Bellefonte, PA) overnight at calibrating conditions to obtain better performance and longer lasting fibers. All analyses were conducted at ambient/laboratory temperature, viz. approximately 25 °C. Calibration curves were obtained by
injecting 20-µL aliquots of 4-quinolones in buffer solutions of pH 3 by a Spark Holland Marathon autosampler (Spark Holland, Emmen, The Netherlands). These calibrations were used for the quantification of 4-quinolones after desorption from the fiber. nd-SPME Procedure. The carbowax/divinylbenzene (CW/ DVB), carbowax/templated polymer resin (CW/TPR), and polyacrylate (PA) SPME fibers (Supelco, Bellefonte, PA) were used in a preliminary test to find the most suitable fiber. Fibers were conditioned in the mobile phase for at least 30 min at operating flow rate followed by the gradient as described for the HPLC procedure. The CW/DVB fiber turned out to be noncompatible with organic solvents under pressure, and the PA fiber exhibited longer equilibration times as compared to the CW/TPR fiber. Therefore, the CW/TPR fiber was chosen for the rest of the experiments with the following procedure. Septum closed vials containing 1.6 mL of test solution were brought into vigorous stirring, approximately 1300 rpm, by means of a Teflon-coated magnetic stirring bar. The septum was pierced by the SPME needle, and depending on the analyte, the fiber was exposed to the test solution for an appropriate period of time, ts, followed by a 3-min static desorption step in the desorption chamber of the SPME-HPLC interface. The interface was described in detail by Chen and Pawliszyn (31). The desorption solution had the same composition as the initial composition of the mobile phase in the HPLC analysis. The fiber was left in the desorption chamber for a dynamic desorption step throughout the analysis. After equilibration of the fiber, it was replaced with a broken one, and the chamber was sealed in order to fill it with initial mobile phase for later desorption. Experiments were conducted at ambient/laboratory temperature, viz. approximately 25 °C. Uptake Kinetics to Fiber. The fiber was exposed to individual vials containing 7.66 µM test compound for different time periods (n ) 10). pH 3 was used for FLU and OXA, whereas pH 5.5 was used for SAF, as the maximum uptake to the fiber appeared at these pH values. Uptake profiles and kinetic constants were obtained by fitting the data to eq 1, and sampling times were derived in accordance with eq 2, see Table 2. pH-Dependent Partitioning to the SPME Fiber. According to sampling times derived from eq 2 for the respective antimicrobials (see Table 2), the fiber was exposed to individual vials containing 7.66 µM test compound at pH values in the range of 3-11 (FLU, n ) 18; OXA and SAF, n ) 19). Influence of pH on DDOC. To individual flasks containing five different nominal concentrations of DOC of the same lot of Aldrich humic acids (for FLU and OXA: 1, 5, 12.5, 25, and 50 mg/L; SAF: 1, 3, 5, 9, and 12.5 mg/L) at six different pH values (3-8) FLU, OXA, and SAF were added to obtain a total 4-quinolone concentration of 7.66 µM. Test flasks were shaken thoroughly for at least 20 min before analysis. No significant difference was observed in the free fraction when equilibration times in the range of 20-140 min were used (5% significance level: P ) 0.058). The free fraction was calculated
TABLE 2. Effects on Fiber Kinetics Due to Exposure to DOCa chemical
n
exposure to DOC
k1( SD (min-1)
k2( SD (min-1)
KSPME ( SD
R2
ts (min)
depletion (%)
FLU
10
OXA
10
SAF
10
before after before after before after
27.6 ( 2.2 28.1 ( 2.5 22.4 ( 3.4 31.5 ( 2.5 42.5 ( 2.8 45.1 ( 1.9
0.49 ( 0.06 0.46 ( 0.06 0.93 ( 0.18 1.11 ( 0.11 0.27 ( 0.03 0.28 ( 0.02
56.0 ( 2.7 61.4 ( 3.4 24.0 ( 1.7 28.7 ( 0.9 155.3 ( 8.6 159.1 ( 5.5
0.97 0.97 0.88 0.97 0.98 0.99
4.84 5.06 3.19 2.84 6.83 6.70
0.8 0.9 0.4 0.4 2.1 2.1
a Results of data fitting for the uptake profiles to the CW/TPR SPME fiber according to eq 1, calculation of minimum sampling time (t ) according s to eq 2, and the magnitude of depletion. SD, standard deviation. n, number of experiments. R 2, correlation of the fitting.
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FIGURE 1. Uptake profiles to the CW/TPR SPME fiber. Open symbols represent experiments performed before exposure to humic acids, and closed symbols represent experiments performed after exposure to humic acids. Symbols represent single measurements and lines represent fitting to eq 1. Experiments were performed in 7.66 µM solutions at pH 3 for FLU (0/9) and OXA (O/b), whereas SAF ()/() was performed at pH 5.5. as the free concentration in the solutions containing DOC divided by the concentration in the solution without DOC. Finally, DDOC was calculated with eq 11. Procedure To Determine Uptake Kinetics to DOC-Exposed Fiber. First, uptake kinetics for the antimicrobials to the unexposed fiber were established as described above. Second, the pH-dependent DDOC experiments described above were performed for FLU. Third, according to the above-mentioned procedure uptake kinetics to the DOC exposed fiber were established for the very same antimicrobial. Subsequently steps 2 and 3 were conducted with OXA and SAF as well.
Results and Discussion HPLC Procedure. The autosampler injected calibration curves for FLU, OXA, and SAF showed linearity over the measured concentration range (0.12-7.66 µM). Limits of quantification were 0.15, 0.10, and 0.36 µM, respectively, calculated as 10 times the standard deviation of the regression line divided by the slope. The carryover on the fiber was determined to be less than 7% for the highest concentrations. Uptake Kinetics to Fiber. To test whether preexposure to DOC affects the uptake kinetics of the antimicrobials, uptake profiles were established both before and after the fiber was exposed to DOC, see Figure 1. Experiments were performed as described in the Experimental Section. A summary of the data fittings, calculated minimum sampling times, and magnitude of depletion is given in Table 2. For each antimicrobial uptake and elimination rate constants derived for the two uptake profiles were compared by means of F tests at a P level of 0.05 (not shown) revealing no significant difference, except a minor effect on k1 for OXA (P ) 0.0487). Since k1 considers the resistance in the aqueous phase, this difference is not assumed to affect the overall
FIGURE 2. pH-dependent distribution coefficient (D′) for the test compounds between the CW/TPR SPME fiber and buffer. Symbols indicate single measurements. The curves for FLU (0) and OXA (O) represent data fitted to eq 8, and the curve for SAF ()) represents data fitted to eq 9. picture. If preexposure to DOC would have any influence on the partitioning to the fiber, this would influence the diffusion characteristics in the fiber phase and thus k2 (32). Preexposure as described in the Experimental Section of the SPME fiber to DOC does therefore not seem to affect the uptake kinetics. Similar conclusions were drawn by Urrestarazu Ramos et al. (24). Minimum sampling times were calculated according to eq 2. The depletion in the vials after the exposure of the sample to the fiber was determined to be less than 2.1%. Influence of Modifying Factors on Partitioning to SPME. The pH-dependent distribution of the 4-quinolones between the CW/TPR SPME fiber and buffer solution is shown in Figure 2. The experimental data for FLU and OXA were fitted to eqs 7 and 8, whereas data for SAF were fitted to eq 9 as well. Best fits were chosen based on F tests. Table 3 represents the statistics and the values of the nonlinear regression. The pKa values of 6.5 and 6.9 derived for FLU and OXA agree with data from literature (33). For SAF, the regression resulted in pKa values of 4.1 and 6.8 that are assigned to the tertiary amine of the piperazine moiety and the carboxylic group, respectively. The lower value for the amine, compared to literature values for related structures (34), may be due to electron-withdrawing effects from the fluorine groups in the structure. In experiments (not shown) under environmentally relevant pH values of 5-8, the variation of salinity (0-500 mM) and buffer capacity (0-200 mM) showed minor influence on the partitioning to the fiber. Only the pH has a strong influence and must be well controlled for chemical analysis of 4-quinolones by nd-SPME-HPLC. All together, this technique provides a method to measure macroscopic pKa values. Measuring Distribution Coefficients to Humic Acids and the Influence of pH. The distribution of FLU, OXA, and SAF between DOC and buffer at various concentrations (nominal) of DOC (1-50 mg/L) was studied in the pH interval 3-8. These experimental DOC concentrations are comparable to
TABLE 3. Results of Nonlinear Regression of Distribution Data to the CW/TPR SPME Fiber at Different pH Valuesa D ( SD chemical
pKa( SD
FLU OXA SAF
6.5 ( 0.1 6.9 ( 0.1 4.1 ( 0.1 6.8 ( 0.1
a
AH2
AH
A
F value
p value
best-fit eq
8.4 ( 7.1
48.8 ( 0.8 21.8 ( 0.4 101.3 ( 4.2
3.5 ( 1.1 1.8 ( 0.7 9.9 ( 3.0
8.27 6.85 103.9
0.012 0.019 OXA > SAF, whereas the binding to DOC on the contrary is SAF > OXA > FLU (see Figures 3 and 4).
Environmental Effects This investigation shows that the presence of DOC may result in declining free concentrations of 4-quinolones. Primarily DOC concentrations of 2-10 mg/L are observed in aquatic ecosystems (5, 6). At pH 7, a decrease in the free concentration of 10-35% can be expected for SAF, whereas the decrease for FLU, the most hydrophobic chemical tested, only ranges
TABLE 4. Experimental Log DDOC ( SD for the Test Compounds at Different pH Values Obtained by Fitting Data to Eq 11a pH chemical
3
4
5
6
7
8
FLU OXA SAF
3.44 ( 0.27 3.87 ( 0.05 4.77 ( 0.01
3.65 ( 0.03 4.00 ( 0.03 4.86 ( 0.08
4.23 ( 0.05 4.37 ( 0.02 5.19 ( 0.07
4.39 ( 0.02 4.50 ( 0.02 4.98 ( 0.02
4.23 ( 0.04 4.48 ( 0.02 4.74 ( 0.03
4.36 ( 0.08 4.37 ( 0.03 4.52 ( 0.06
a
SD, standard deviation.
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from 3 to 15%. In a study of the photodegradation pathway of fluoroquinolones, it was found that the piperazine ring was degraded within hours, whereas the quinolone structure was degraded within days (37). However, when chemicals are bound to DOC, they are not available for various processes, e.g., biodegradation or photodegradation. The photodegradation half-life of an SAF analogue thus doubled in the presence of DOC (38). Furthermore, the bioavailability of DOC-bound chemicals is influenced. In a toxicity study with a surfactant toward Hyalella azteca, it was shown that an increasing organic carbon content decreases sediment toxicity (16). In a bioaccumulation study with the highly hydrophobic chemicals pentachlorobenzene and PCB 177, it was shown that the bioconcentration factor was significantly lower in the presence of DOC (24). On the other hand, the binding to DOC will result in accumulation of the compound in environmental compartments such as water and sediments and reduce degradation. In case of discharge of uncontaminated water to a stream, free concentrations will be maintained according to the DDOC values due to desorption from DOC. Thus, the experiments revealed that the interaction with humic acids can be of significant importance in the environmental risk assessment of ionizable organic chemicals, since the studied 4-quinolones were shown to have log DDOC values of 3.4-5.2, which are in the same order of magnitude as for known highly hydrophobic chemicals.
Acknowledgments The grammatical and technical help and assistance provided by Klavs Mulvad, Eric Verbruggen, Frans Busser, and Theo Sinnige are gratefully acknowledged. The contribution concerning the equations in the Theoretical Consideration, performed by En ˜ aut Urrestarazu Ramos, is appreciated. This investigation was partly funded by a grant from the Danish Centre for Sustainable Land Use and Management of Contaminants, Carbon and Nitrogen under the Danish Strategic Environmental Research Programme, Part 2, 19972000. Sarafloxacin hydrochloride was kindly supplied by Abbott Laboratories, North Chicago, IL.
Literature Cited (1) Urrestarazu Ramos, E.; Vermeer, C.; Vaes, W. H. J.; Hermens, J. L. M. Chemosphere 1998, 37, 633-650. (2) McCarthy, J. F.; Jimenez, B. D. Environ. Sci. Technol. 1985, 19, 1072-1076. (3) Saarikoski, J.; Lindstro¨m, R.; Tyynela¨, M.; Viluksela, M. Ecotoxicol. Environ. Saf. 1986, 11, 158-173. (4) Ko¨nemann, H.; Musch, A. Toxicology 1981, 19, 223-228. (5) Day, K. E. Environ. Toxicol. Chem. 1991, 10, 91-101. (6) Kukkonen, J.; Oikari, A. Water Res. 1991, 25, 455-463. (7) Kukkonen, J.; Oikari, A. Sci. Total Environ. 1987, 62, 399-402. (8) Jota, M. A. T.; Hassett, J. P. Environ. Toxicol. Chem. 1991, 10, 483-491. (9) Ottiger, C.; Wunderli-Allenspach, H. Eur. J. Pharm. Sci. 1997, 5, 223-231. (10) Arnold, C. G.; Weiderhaupt, A.; David, M. M.; Mu ¨ ller, S. R.; Haderlein, S. B.; Schwarzenbach, R. P. Environ. Sci. Technol. 1997, 31, 2596-2602. (11) Weiderhaupt, A.; Arnold, C. G.; Mu ¨ ller, S. R.; Haderlein, S. B.; Schwarzenbach, R. P. Environ. Sci. Technol. 1997, 31, 26032609.
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Received for review January 21, 2000. Revised manuscript received August 11, 2000. Accepted August 24, 2000. ES000917Y
