Enantiomeric Selectivity in the Environmental Degradation of

The chiral herbicide dichlorprop (2,4-dichlorophenoxy-. 2-propionic acid), which is sold and applied as the racemic mixture, was observed to degrade ...
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Environ. Sci. Technol. 1996, 30, 2449-2455

Enantiomeric Selectivity in the Environmental Degradation of Dichlorprop As Determined by High-Performance Capillary Electrophoresis A . W . G A R R I S O N , * ,† P . S C H M I T T , ‡ D. MARTENS,‡ AND A. KETTRUP‡ National Exposure Research Laboratory, U.S. Environmental Protection Agency, 960 College Station Road, Athens, Georgia 30605-2700, and Institut fu ¨r O ¨ kologische Chemie, GSF-Forschungszentrum fu ¨ r Umwelt und Gesundheit, Neuherberg, D-85758 Oberschleissheim, Germany

The chiral herbicide dichlorprop (2,4-dichlorophenoxy2-propionic acid), which is sold and applied as the racemic mixture, was observed to degrade completely in soil within 31 days, with a half-life of 6.6 d. Degradation occurred with enantiomeric selectivity, indicating biologically mediated reactivity as opposed to strictly abiotic degradation. The S-(-)-isomer degraded significantly faster (t1/2 ) 4.4 d) than the R-(+)-isomer (t1/2 ) 8.7 d); this is contrary to other published results that show selective degradation of the R-(+)-enantiomer, although in other media. Soil samples taken from a field plot at increasing time intervals after application of Foxtril, a commercial herbicide formulation, were solvent-extracted and analyzed for total dichlorprop by capillary zone electrophoresis (CZE), using an acetate buffer at pH 4.7. Heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin, a chiral reagent, was then added to the buffer to effect separation of the (+)- and (-)-isomers of dichlorprop. Baseline resolution allowed calculation of relative concentrations (enantiomer ratios) of the two isomers. CZE is a fast and efficient technique for the analysis of ionic organic species (such as the anion of dichlorprop), including their enantiomers, in pesticide formulations as well as in environmental samples. It thus was possible to analyze Foxtril directly after dilution with water for ioxynil (2,6-diiodo-4-cyanophenol) as well as for dichlorprop. Ioxynil also was detected in the soil extract on the day of application. The hydrolysis product [methyl 2-nitro-5-(2,4dichlorophenoxy) benzoic acid] of bifenox methyl ester, another herbicide component of Foxtril, was detected in the soil samples taken at 17 and 31 d. * Corresponding author e-mail address: garrison.wayne@EPAMAIL. epa.gov. † National Exposure Research Laboratory. ‡ Institut fu ¨r O ¨ kologische Chemie.

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 1996 American Chemical Society

Introduction Phenoxy acid herbicides are important as selective preand post-emergence herbicides. Their toxicity, herbicidal effects, and environmental persistence have been studied in detail. For example, studies of the degradation kinetics of 2,4-D (2,4-dichlorophenoxy acetic acid) and dichlorprop (2,4-dichlorophenoxy-2-propionic acid) in various soils showed their half-lives to range from about 3 to 10 d (1, 2). Microbiological degradation of 2,4-D and dichlorprop in moist soils results in the formation of 2,4-dichlorophenol and 2,4-dichloroanisole, respectively, which are then rapidly transformed by both biological and nonbiological mechanisms (3, 4). Several of these herbicides are chiralsthose with the phenoxy substituent on the 2-position of propionic acid, for examplesand exist as a pair (racemic mixture) of optical antipodes called enantiomers. Chiral compounds show enantiomeric selectivity in reactions with biological systems. Biological activity in soil or water environments may result in the preferential reactivity of one enantiomer of a pesticide in terms of microbial degradation, biological uptake, metabolism, and/or toxicity (5-8). It was shown, for example, that only the (+)-isomers of dichlorprop and mecoprop (2-methyl-4-chlorophenoxy propionic acid) are herbicidally active (9). Ludwig et al. (10) reported that the (+)-isomer of dichlorprop and not the (-)-isomer was degraded by marine microorganisms, and Tett et al. (11) showed the same behavior for mecoprop degradation by mixed and pure soil bacteria cultures. Investigations of this preferred reactivity phenomenon could produce important results; manufacturers, for example, may be able to tailor pesticide formulations that are more selective for target organisms and vegetation, thereby reducing total chemical application significantly. In fact, it could be that the optical isomer with the most toxicity to the target species could be the least toxic to nontarget organisms; ideally, the isomer most toxic to nontarget species would be the least persistent in the environment. It thus is important to determine whether enantiomeric selectivity is involved in pesticide degradation. Incidentally, the occurrence of such selectivity implies the mediation of bacteria, enzymes, or other biological entities; abiotic degradation processes are not enantioselective. A variety of GC- and HPLC-based methods have been developed for analysis of the phenoxy acid herbicides in their commercial formulations and in environmental samples, e.g., for monitoring or for studying their fate in water, soil, and sediment matrices. More recently, chiral solid phases for GC and HPLC columns have permitted the chromatographic separation of optical isomers, including those of pesticides (7, 8, 12, 13). The enantiomers of dichlorprop and mecoprop have been separated by HPLC (10, 14). The recent advent of high-performance capillary electrophoresis (HPCE or CE) adds a separation tool of superior efficiency to the more conventional chromatographic instrumentation. CE is beginning to be applied to pesticide analysis and to other environmental problems (15-20). In addition, techniques for chiral separation of optical isomers by CE, usually involving the addition of chiral reagents such as cyclodextrins to the separation buffer (21, 22), are now

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also available and have been widely applied in the pharmaceutical and biomedical fields during the past few years (23-26). The phenoxy acids are excellent candidates for separation by HPCE. Their pKa values are such as to allow separation by the simplest form of CEscapillary zone electrophoresis (CZE)swhich separates only charged species. CZE already has been applied for the analysis of phenoxy acids (27, 28). The authors recently established conditions for the CZE separation of 2,4-D and three chiral phenoxy acid herbicidessdichlorprop, mecoprop, and fenoprop (2,4,5-trichlorophenoxy propionic acid) (29). Subsequent addition of a cyclodextrin chiral reagent to the CE separation buffer resulted in baseline separation of the two enantiomers of each of the chiral herbicides. There were two objectives to our research: (1) to determine whether the environmental degradation of dichlorprop, a representative phenoxy acid herbicide, is enantiomerically selective in the particular soil involved in this study and (2) to further demonstrate the applications of capillary electrophoresis to environmental problems. To these ends, soil samples from a field sprayed with Foxtril, a commercial herbicide formulation containing dichlorprop, ioxynil, and bifenox ester as the active ingredients, were taken at increasing time intervals, extracted, and analyzed by CZE for residual dichlorprop and its enantiomers. In addition, ioxynil and bifenox acid were detected by CZE in the Foxtril formulation and/or the soil extracts.

Experimental Section Instrumentation. The capillary electrophoresis instrument was a Beckman P\ACE 2100 Series system with Beckman System Gold Chromatography Software. The fused-silica CE column (75 µm i.d., 300 µm o.d., 50 cm length to the detector, total length 57 cm) was obtained from Beckman. Usual conditions for separation of the herbicides were temperature, 30 °C; voltage, 20 kV; hydrodynamic sample injection time, 5-15 s; detector wavelength, 230 nm. The column was rinsed between runs with 0.1 M NaOH for 2 min followed by water for 2 min. Reagents and Chemicals. The acetate separation buffer was 50 mM at pH 4.65 and was composed as follows: 0.05 M glacial acetic acid:0.05 M sodium acetate, 1:1, v:v. The cyclodextrin-containing buffer for enantiomeric separation was prepared by dissolving heptakis(2,3,6-tri-O-methyl)β-cyclodextrin (TM-B-CD) in the acetate separation buffer to a final concentration of 25 mM cyclodextrin. Concentrated herbicide stock solutions were prepared in pesticidegrade methanol, and then diluted to 4 µg/mL in distilled/ deionized water. This solution was used directly for CZE analysis. The Foxtril commercial formulation, which was a milky white aqueous dispersion produced by RhonePoulenc, contained 400 g/L dichlorprop, 187.5 g/L bifenox, and 57.5 g/L ioxynil. This formulation was diluted with distilled water to give a concentration of 40 µg/mL of dichlorprop; this solution was used directly for CZE analysis. Dichlorprop was obtained in greater than 99% purity from Dr. Ehrenstorfer GmbH, Augsburg, Germany. D-(+)dichlorprop [correctly named R-(+)-dichlorprop] was obtained from Riedel de Hae¨n, Munich, Germany. TM-B-CD was obtained from Sigma Chemie GmbH, Deisenhofen, Germany. All solvents were pesticide analysis grade. Sampling Procedure. A 100-ha field in an experimental farm near Scheyern, Germany, was sprayed with Foxtril at a nominal rate of 2 L/ha on May 18, 1992. The soil is a fine,

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loamy material of the Dystric Eutrochrept family with the following composition: sand, 65%; silt, 25%; clay, 10%; organic carbon, 1.15%; and pH, 6.2. Beginning on May 18, soil samples were collected at increasing time intervals of 0, 3, 9, 17, and 31 d from six sites of 10 m2 each located in representative areas of the field. Each sample was a composite of from 16 to 20 subsamples collected from depths of 0-5 cm. Samples were dried in the laboratory at room temperature, passed through a 2-mm seive, and stored at -80 °C until analyzed. The principal objective of this project was to investigate the environmental degradation of the herbicides in Foxtril by HPLC analysis (30). A sample from only one of the six sites was selected for analysis by CZE. Extraction Procedure. An extraction procedure (similar to that of ref 4) was developed to produce an extract suitable for HPLC analysis. Fifty grams of moist soil was weighed into a centrifuge tube and extracted for 2 h with 100 mL of an acetonitrile/water/glacial acetic acid mixture (80/ 20/2) in an ultrasonic bath. The mixture was centrifuged, and 50 mL of the clear extract was transferred to a 250-mL separatory funnel and shaken with 60 mL of dichloromethane and 100 mL of 1% HCl; the resulting aqueous layer was reextracted with 50 mL of dichloromethane. The combined dichloromethane phases were dried by passing the extract through a column of sodium sulfate, which was then washed with another 15 mL of dichloromethane. The combined extracts were then evaporated just to dryness. The residue was transferred to a 2-mL flask with a small amount of acetonitrile, 0.5 mL of water was added, and the extract was made to volume with acetonitrile for HPLC analysis. Recovery of the dichlorprop was 90%, as measured by HPLC analysis of soil freshly spiked with dichlorprop. By the time these extracts were submitted for CZE analysis, most of the solvent had evaporated, leaving a redbrown or dark green gummy or oily residue. This extract (or residue) had to be treated further to prepare it for CZE analysis. The residue was dissolved in 1 mL of methanol (a small amount of white solid sometimes remained insoluble), which was then diluted with 1 mL of water, after which a light brown precipitate formed. The solution was still light yellow to brown. This mixture was filtered through a 0.45-µm syringe filter (e.g., Acrodisc LC13 PVDF from Gelman Sciences, Ann Arbor, MI), and the filtrate was then ready for CZE analysis. For more details on the CZE analysis, refer to the authors’ recent work on CZE separation of phenoxy acid herbicides and their enantiomers (29).

Results and Discussion Analysis of the Commercial Formulation. Figure 1 shows the structures of the three herbicides in Foxtril and their degradation products that could be important to this study. Dichlorprop, ioxynil, 2,4-dichlorophenol, and bifenox acid are all ionizable organic acids and are thus amenable to analysis by CZE. Figure 2a shows the electropherogram of the Foxtril formulation, diluted 1-10 000 with distilled water. This electropherogram corresponds to a 5-s injection, or about 30 nL of sample injected, which represents 1.2 ng of dichlorprop and 0.17 ng of ioxynil. (The absorbance of dichlorprop in this electropherogram is 0.013 units.) Figure 2b provides confirmation of the identity of ioxynil by spiking with a standard; the identity of the dichlorprop peak was likewise confirmed by spiking.

FIGURE 1. Structures of the herbicides in Foxtril (ioxynil, dichlorprop, and bifenox ester) and some of their degradation products.

The pH of the separation buffer determines both the degree of ionization of the analyte as well as the extent of protonation of the silanol groups on the fused silica column surface. At lower pHs, the column surface is less negatively charged because of increased protonation, resulting in decreased electroosmotic flow (EOF) and lower flow rates. For complete ionization and maximum electrophoretic mobility of anionic analytes, the pH should be 2 or more units above the pKa of the analyte. The 50 mM acetate buffer of pH 4.65 used in these analyses resulted in most (about 70%) of the ioxynil and almost all (about 97%) of the dichlorprop being ionized, while providing sufficient EOF for reasonably fast migration. Although higher pH would result in greater EOF and faster migration times, because both herbicides would be completely ionized at pHs above about 7 (pKa values are 4.3 and 3.2), separation between them would be decreased because their charge-to-mass ratios would be closer. The order of migration of these two herbicides is in accordance with their pKa values (Figure 2). Because ioxynil, with a higher pKa, has a lower average charge at the buffer pH of 4.65 and is also a heavier molecule than dichlorprop, it has a lower charge-to-mass ratio and, therefore, a shorter migration time (10.3 min) than dichlorprop (11.7 min). The pKa of 2,4-dichlorophenol is 7.69, so it was not ionized under these conditions; it therefore migrated at the velocity of the

EOF, as did all other neutral components of the sample and buffer to form the “neutral peak”. Figure 2c shows the electropherogram of Foxtril after the addition of 25 mM TM-B-CD to the pH 4.65 acetate separation buffer. The chiral dichlorprop separated with baseline resolution into its (-) and (+) enantiomers. The (-)-isomer has a shorter migration time (29). The presence of the TM-B-CD resulted in a shift of the migration time of ioxynil from 10.3 to 9.5 min, while its electrophoretic mobility (µep) decreased from -0.01255 to -0.00611 cm2 V-1 min-1; the dichlorprop isomers migrate at 15.7 and 16.2 min (µep ) -0.01203 and -0.01231 cm2 V-1 min-1) as opposed to the racemate, which migrated at 11.7 min (µep ) -0.01421 cm2 V-1 min-1). Analysis of Soil Samples. Figure 3a is the electropherogram of the extract of the soil sample taken on day 0, i.e., just after the application of Foxtril. Dichlorprop is the most prominent peak, but a small peak corresponding to the migration time of ioxynil was also detected; the concentrations of the two compounds are in a similar ratio to that of the commercial formulation (Figure 2). The large peak at just over 4 min is the “neutral peak”, which includes all neutral species in the injected extractsmostly methanol. Of additional interest is the broad peak centered at about 10.5 min; this probably consists of extracted soil organic macromolecules, including humic materials (31). The CZE conditions developed for these analyses provide for good separation from these humic substances, which would probably cause interferences in GC or HPLC analysis. Note that the migration times of dichlorprop and ioxynil are much less than in the Foxtril formulation (Figure 2), even though the buffer and other CZE conditions are the same. One possible cause of this decrease in migration times is an increase in EOF within the CZE column because of a different sample matrix. The Foxtril formulation most likely contains a dispersing agent that might react with the column walls to lower the EOF and increase migration times. Such an agent could also form micelles that would complex with the analytes and change their electrophoretic mobility. Figure 3b shows the same day 0 extract analyzed under identical conditions as in Figure 3a except that 25 mM of the chiral reagent, TM-B-CD, has been added to the separation buffer. The dichlorprop peak has split into two componentssthe two enantiomers. The average migration time and µep of this pair (7.4 min and -0.0132 cm2 V-1 min-1) is again different from that of the racemate peak

FIGURE 2. Electropherograms of the Foxtril formulation: (a) Foxtril, diluted 1:10 000 with distilled water, 5 s injecton time; (b) Foxtril spiked with ioxynil; (c) Foxtril after addition of 25 mM TM-B-CD to the separation buffer. See Experimental Section for CZE conditions.

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FIGURE 3. Electropherograms of the extract of the soil sample taken on day 0: (a) the extract; (b) the extract after addition of 25 mM TM-B-CD to the separation buffer; (c) same as panel b but spiked with (+)-dichlorprop.

FIGURE 4. Electropherograms of (a) the day 17 soil sample extract; (b) the day 31 extract. No chiral reagent was added to the buffer.

(8.0 min and -0.01701 cm2 V-1 min-1, Figure 3a). The identity of the (+)-isomer of dichlorprop as the second peak of the pair was confirmed by spiking with the pure R-(+)-isomer (Figure 3c). Finally, the broad peak in Figure 3a, speculated to be humic substances, has disappeared from Figure 3b and c. The humic substances may have formed a complex with the cyclodextrin; multiple complexes between various cyclodextrins and organic macromolecules have been demonstrated to occur (32). The electropherogram of the extract of the soil sample taken 3 d after application of Foxtril again showed a very definitive peak for dichlorprop, the presence of which was confirmed by spiking with a standard. Subsequent addition of the cyclodextrin produced the expected two peaks for the dichlorprop enantiomers, the first of which was smaller relative to the second than in the day 0 sample. Selective degradation of the (-)-isomer had begun to be apparent. The day 17 extract (without cyclodextrin, Figure 4a) shows that dichlorprop is still present in the soil. However, there also occurs a new component, bifenox acid (confirmed by spiking). This is the herbicidally active hydrolysis product of the bifenox ester (Figure 1), an ingredient in

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Foxtril. The ester does not show up on these CZE electropherograms because it is a neutral species. Finally, in the day 31 extract (Figure 4b)sthe last of the seriess dichlorprop is not detectable (i.e., less than 0.05 µg/g), whereas there is more bifenox acid present than on day 17. No attempt was made to quantitate the bifenox acid. These CZE results are in line with those based on HPLC data (30). HPLC shows that bifenox acid just begins to appear on day 3, and there is a very small peak at the corresponding migration time on the day 3 CZE electropherogram. HPLC shows the concentration of bifenox acid to increase about 4-fold from day 17 to day 31; CZE results also indicate this increase. HPLC shows a decrease in concentration of racemic dichlorprop from day 0 to day 9 of about 2-fold, with almost complete disappearance by day 31. CZE shows a corresponding decrease from day 0 to day 9 with no dichlorprop detectable by day 31 (Figure 4b). Enantiomeric Ratios and Degradation Rates. Figure 5a shows changes in the relative concentrations of the (-)and (+)-isomers of dichlorprop in the soil samples with increasing time. The (-)-isomer peak is slightly smaller

FIGURE 5. (a) Electropherogram peaks for (-)- and (+)-dichlorprop, as separated by TM-B-CD, in Foxtril and in extracts of soil samples taken with increasing time. (b) Decrease in total dichlorprop concentration as measured by HPLC.

even in the Foxtril formulation, with an enantiomeric ratio (ER) of 0.95, and there is an additional slight decrease relative to the (+)-isomer just after field application (day 0, ER ) 0.88). (ER ) peak area of the first eluting enantiomer/peak area of the later eluting enantiomer.) From that point, there is a regular decrease in the (-)isomer relative to the (+)-isomer through day 17 (ER ) 0.18), which is the last day’s sample for which the (-)isomer was detectable. There were no samples taken between day 17 and day 31. By day 31, both isomers had completely degraded. Figure 5b shows the total dichlorprop in each day’s soil sample as previously measured by HPLC. The degradation of dichlorprop was observed as a firstorder reaction; the half-life calculated for degradation of total dichlorprop is 6.6 d; that for the (-)-enantiomer is 4.4 d and that for the (+)-enantiomer is 8.7 d. Preferential degradation of the S-(-)-enantiomer is somewhat contrary to the results of two previous studies. Ludwig et al. (10) showed that the R-(+)-enantiomer, exclusively, is degraded by marine microorganisms to produce 2,4-dichlorophenol; the S-(-)-enantiomer is unaffected after 21 days. Tett et al. (11) reported that the R-(+)enantiomer of mecoprop is degraded and the S-(-)enantiomer unaffected by mixed and pure soil bacterial cultures. Reversal of enantiomeric selectivity in different media should not be so surprising, however. Jantunen et

al. (33) reported reversal of the ER ratios for R-hexachlorocyclohexane in different water bodies; water samples from the Bering and southern Chukchi seas showed preferential degradation of (-)-R-HCH, while the (+)-R-HCH enantiomer degraded faster in more northern waters. Variation in Migration Times. The changes in migration times upon complexation with the cyclodextrin are confusing at first. The electrophoretic mobility, µep, of a species is a weighted function of its free and bound forms (34). The decrease in absolute mobility (the actual mobilities of anions are always negative) of dichlorprop upon addition of the cyclodextrin reflects the formation of complexes between the herbicide enantiomers and the cyclodextrin. If its size permits, and the change in migration time indicates that it does, ioxynil will also form an inclusion complex with the cyclodextrin and its mobility will decrease, even though ioxynil is achiral and there are no enantiomers to separate. The observed (net) mobility of a species (µn) is the vector sum of the species’ mobility (µep) and the mobility due to electroosmotic flow (µeo) in the column: µn ) µep + µeo (or µep ) µn - µeo). The µep values for dichlorprop and ioxynil given earlier in this paper were calculated by subtraction of µeo, which can be calculated from the migration time of the neutral peak, from µn, which can be calculated from the observed migration time of the analyte. As seen in the electropherograms in the various figures, there was always a neutral peak from which to calculate µeo; this peak was caused by methanol from the extraction procedure and other neutral compounds that may have been in the various sample matrices. The critical point is that the calculated absolute mobilities of dichlorprop and ioxynil always decreased upon complexation with the cyclodextrin as expected, because of the decrease in charge to size ratio of the complex relative to the uncomplexed dichlorprop or ioxynil, but the net mobility could change in either direction because of variations in µeo. We have no experimental evidence to definitively explain the large changes in migration times with different sample matrices. The volume of sample injected into the CZE column is very small compared to the volume of separation buffer, which fills the column, yet the sample composition can play an important role in separation efficiency and migration characteristics of analytes. “Stacking”, for example, is a well-known process that improves peak shape and lowers detection levels by simple dilution of the sample with water or with buffer of lower ionic strength than the running buffer (35). More directly related to our study is the research by Garcia and Shihabi (36) in which they examined sample matrix effects in CZE. They found that the presence of proteins (e.g., albumin), ion (NaCl or borate) concentration, and sample volume injected had significant effects on peak heights and migration times of small molecules in serum samples. The observed changes in migration times in changing from the matrix of diluted Foxtril to the soil extracts may be explained by the presence of a dispersing agent or detergent in the formulation, as mentioned above, and/or by the presumed different ionic strengths in the soil extracts. The increase in migration times of dichlorprop and its enantiomers from one soil extract to another (i.e., day 0 to day 3 to day 17) may be caused by a gradual coating of the column; for example, with co-extracted organic macromolecules. Although the column was treated with 0.1 M NaOH after each sample, which is a standard practice in CZE, that may not be sufficient reconditioning for all types

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of samples. On the other hand, different ionic strengths or concentrations of dissolved organic macromolecules in the various soil extracts could produce changes in migration times and peak shapes and heights (36). The addition to the sample of buffer with lower ionic strength than the run buffer or adjustment of pH of the sample might improve migration time reproducibility, as might a buffer with higher ionic strength than the sample. At any rate, migration times change with the sample matrix and/or with column conditions, even when other conditions are constant, so it is essential to identify peaks of interest by matching retention times with a standard in the same matrix or, better yet, by spiking the sample with the analyte standard. This was done with the soil extracts for both dichlorprop and ioxynil. In addition, an EOF (neutral) marker (methanol in our case) is essential for tracking changes in migration times for what may be the same analyte. Reproducibility and Detection Limits. Analytical statistics for CZE analysis of the enantiomers of dichlorprop in the Foxtril formulation show good run-to-run precisions migration times, peak areas, and peak heights for both enantiomers are all reproducible to less than 3% relative standard deviation (n ) 3). For total dichlorprop (i.e., without enantiomeric separation) in the day 9 field sample, reproducibility of migration time for successive runs is less than 1%, but reproducibilities of peak area and peak height are not as good: 7.3 and 8.5% respectively (n ) 4). The latter values are still acceptable, considering the complexity of the matrix, but are not as good as routinely achieved by GC or HPLC, or even by CZE; the hydrodynamic injection technique used here usually gives 1-3% relative standard deviation. An internal standard similar in properties to the analyte should be spiked into the sample before injection to improve quantitation. The detection limit for dichlorprop in the diluted Foxtril (the injected solution) was estimated to be 0.1 µg/mL, based on data in the above section on Analysis of the Commercial Formulation. This compares favorably with the detection limit in aqueous standards of 0.05 µg/mL measured earlier (29). The detection limit for dichlorprop in the soil samples should be higher because of matrix effects in the extract. We estimate the limit to be about 0.5 µg/mL or lower in this extract (i.e., in the injected solution). Since the concentration factor between a soil sample and its extract was about 10 (the final extract, equivalent to 25 g of soil, was made to a volume of 2 mL), the CZE detection limit in the soil was about 0.05 µg/g.

Conclusions This demonstration of enantiomeric selectivity in the disappearance of dichlorprop confirms the occurrence of biologically mediated degradation, the expected process. An unexpected result, contrary to some published data for phenoxy acid herbicides (7, 10, 11), is that the (-)-isomer degrades faster than the (+)-isomer; this is another example of a recent (33) demonstration of the reversal of enantiomeric selectivity with different environmental media. Because this is the first published application of CZE to follow the degradation of a pesticide in field samples, operational factors, advantages, and problems peculiar to CZE need to be recognized. CZE was shown to be practical for this purpose; it provides speed, efficiency, and simplicity for the analysis of ionic organic species. Particular advantages were realized in the analysis of a commercial

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herbicide formulation without the need for sample preparation and in the separation of herbicide enantiomers, which only required the addition of a chiral reagent to the operating buffer system. Sample preparation and cleanup are less involved and time-consuming than with GC or HPLCsa simpler procedure than used here, which was devised for HPLC analysis, would probably suffice for CZE. Humic substances and other naturally occuring organic macromolecules appear to interfere less with CZE. The major problem encountered in this work was variation in migration times of the analytes. Shifts in migration times can occur under two conditions: (1) upon complexation with cyclodextrins, they can shift in either direction, depending upon the EOF of the column relative to the electrophoretic mobilities of the complexed and uncomplexed analytes and (2) with changes in sample matrices, shifts can occur because of stacking or unstacking effects or, conceivably, because of interactions of sample components with the capillary wall. Such excursion of migration times can be tolerated if an EOF marker is used to track the changes. It is also essential to verify the suspect analyte peak by spiking aliquots of the sample matrix with a standard.

Acknowledgments The authors thank W. Sinowski for data on soil properties and J. M. Long and S. W. Karickhoff of NERL-Athens for SPARC calculations of pKa values for dichlorprop and ioxynil. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the U.S. Environmental Protection Agency.

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Received for review July 24, 1995. Revised manuscript received March 29, 1996. Accepted April 4, 1996.X ES950552V X

Abstract published in Advance ACS Abstracts, June 15, 1996.

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