Environ. Sci. Technol. 2002, 36, 221-226
Environmental Behavior of the Chiral Acetamide Pesticide Metalaxyl: Enantioselective Degradation and Chiral Stability in Soil HANS-RUDOLF BUSER,* MARKUS D. MU ¨ LLER, THOMAS POIGER, AND MARIANNE E. BALMER Swiss Federal Research Station, CH-8820 Wa¨denswil, Switzerland
Racemic metalaxyl is currently being replaced in many countries by metalaxyl-M, the fungicide enantiomerically enriched with the biologically active R-enantiomer. This “chiral switch” is expected to reduce the amount of pesticide released into the environment as well as potential sideeffects on nontarget organisms. Detailed knowledge of the environmental behavior of such chiral compounds should include information on the chiral stability (interconversion of enantiomers). In the present study, the degradation/ dissipation of metalaxyl and its primary carboxylic acid metabolite (MX-acid) in soil was investigated under laboratory conditions using enantioselective gas chromatographymass spectrometry (GC-MS). Racemic and the enantiopure R- and S-compounds were incubated in separate experiments. The degradation of metalaxyl was shown to be enantioselective with the fungicidally active R-enantiomer being faster degraded than the inactive S-enantiomer, resulting in residues enriched with S-metalaxyl when the racemic compound was incubated. The relatively high enantioselectivity suggests that degradation/dissipation was largely biological. The data indicated a conversion of 40-50% of metalaxyl to MX-acid, and the remaining metalaxyl being degraded via other pathways. The degradation of MX-acid was also enantioselective. Metalaxyl and MXacid were both configurationally stable in soil, showing no interconversion of R- to S-enantiomers, and vice-versa. Furthermore, the conversion of metalaxyl to MX-acid proceeded with retention of configuration. Degradation followed approximate first-order kinetics but showed significant lag phases.
Introduction Acetamide pesticides are important compounds used as selective herbicides, and as fungicides to control phytopathogenic fungi (peronosporales) in potatoes, sugar beets, and other crops (1). The compounds include metalaxyl {2[(2,6-dimethylphenyl)methoxyacetylamino]propionic acid methyl ester} as an important systemic fungicide. Metalaxyl is C-chiral due to the presence of an asymmetrically substituted C-atom in the carboxy alkyl moiety. The compound consists of a single pair of enantiomers with * Corresponding author phone: ++41 1 783 6286; fax: ++41 1 783 6439; e-mail:
[email protected]. 10.1021/es010134s CCC: $22.00 Published on Web 12/13/2001
2002 American Chemical Society
FIGURE 1. Structures (absolute configurations) of R- and S-metalaxyl; the absolute configurations of R- and S-MX-acid (replacement of the carboxy methyl group in metalaxyl by a carboxy group) are accordingly. 1′R- and 1′S-configuration showing levo- and dextrorotation, respectively (R- and S-metalaxyl; see Figure 1 for the absolute configurations) (2, 3). Metalaxyl was initially marketed as the racemic product, although its fungicidal activity is almost entirely from the R-enantiomer (3, 4). Currently, rac-metalaxyl is being replaced in many countries by metalaxyl-M, the product enriched with the R-enantiomer (M ) minus; levorotating) (4), although the use of generic rac-metalaxyl may continue in some countries. Metalaxyl-M typically consists of 97.5% of R-isomer and 2.5% of S-isomer and has the same biological activity at ≈50% of the use rate of rac-metalaxyl. This replacement is a further example of a “chiral switch” of a pesticide (5, 6). Metalaxyl is chemically stable but is readily biodegraded in soil, plants, and animals by cleavage of the ester and concurrent series of oxidative biotransformations (N-dealkylation, alkyl and aryl hydroxylation; see ref 7). A major metabolite of metalaxyl in soil is metalaxyl carboxylic acid {MX-acid; 2-[(2,6-dimethylphenyl)methoxyacetylamino]propionic acid}, and further metabolites are also formed via this primary hydrolysis product. MX-acid is also C-chiral. The use of enantio-/isomerpure products in place of racemic products (or isomer mixtures) not only allows lower application rates but also reduces the amounts of pesticides released into the environment, prevents deployment of an inactive isomer to the biosphere, and thus reduces potential side-effects on nontarget organisms. Furthermore, the use of enantioenriched products is also beneficial from the points of production, transport, and handling (8-10). Although many pesticides are chiral, the environmental behavior and fate of such compounds with respect to stereo-/ enantioselectivity so far has received little attention. In a previous study we showed that residues from the application of rac-metalaxyl in soil were enriched with the S-enantiomer, indicating that the biologically active R-enantiomer is more rapidly degraded (11). However, other aspects such as the chiral stability of the compound were not investigated in that study because single isomers, at the time, were not available. Under certain conditions some chiral compounds are configurationally unstable and may undergo enantiomerization/racemization, as was observed with the chiral phenoxy propionic acids mecoprop and dichlorprop [2-(4-chloro2-methylphenoxy)propionic acid and 2-(2,4-dichlorophenoxy)propionic acid, respectively] in soil (12). Enantiomerization, the stereochemical conversion of an enantiomer into its antipode, may influence efficacy and side effects. If enantiomerization is fast, it would even be pointless to use the enantiopure product, because it could racemize prior to deployment of the desired biological activity. A detailed knowledge of the environmental behavior of biologically active chiral compounds must, therefore, include some VOL. 36, NO. 2, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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knowledge on racemization, enantiomerization, and/or isomerization and their magnitudes relative to other processes such as degradation/dissipation. In this study, we investigated the degradation/dissipation of metalaxyl and its primary hydrolysis product, MX-acid, in soil with respect to their stereochemistries using the racemic and the enantiopure compounds in combination with enantioselective analyses.
Experimental Section Standard Materials. Analytical standards (purities, >99%) of rac-metalaxyl, metalaxyl-M, and rac-MX-acid were obtained from Syngenta AG (Basle, Switzerland). Stock solutions at concentrations of 1-10 mg/mL were prepared in methanol or the mobile phases later used in high-performance liquid chromatography (HPLC). For analysis by gas chromatography-mass spectrometry (GC-MS), solutions were prepared in ethyl acetate at concentrations of 1-10 ng/µL. Alachlor and clofibric acid, the former used as an internal standard and the latter as a surrogate compound, were obtained from Monsanto (St. Louis, MO) and Aldrich (Buchs, Switzerland), respectively. Semipreparative HPLC of MX-Acid Enantiomers and Preparation of R- and S-Metalaxyl. Enantiopure R- and S-metalaxyl and the corresponding MX-acids were required to detect potential enantiomerization/racemization of the compounds. Enantioselective (chiral) HPLC showed exceptionally high resolution of the two MX-acid enantiomers which were isolated in high enantiomer purity (>99%). Enantiopure R- and S-metalaxyl were then prepared from these acids by methylation, as detailed below. Enantiomer assignment for metalaxyl, and for MX-acid after methylation, was done using enantioselective GC-MS and in reference to earlier work (see ref 2 and below). The enantiomers of rac-MX-acid were resolved on a Chiralcel OD-H HPLC (5-µm, 25 cm × 4.6 mm i.d.; Daicel Chemical Industries, Tokyo, Japan) column operated with 1.0 mL/min n-heptane/2-propanol (98:2) with 0.1% trifluoroacetic acid. The HPLC system consisted of a Jasco model PU-910 pump (Jasco Corp., Tokyo, Japan), a Jones model 7955 column chiller (Jones Chromatography, Hengoed, Wales, UK), and a Jasco model UV-975 UV/VIS detector set to 254 nm. Separations were performed at 10 °C because of an improved resolution at this lower temperature. The MXacid enantiomers eluted at retention times of 12.2 (R) and 25.0 min (S). Fractions were collected manually from multiple injections (1-100 µL) by observing the UV-signal and changing the collection vials accordingly. The two MX-acid enantiomers were isolated from the mobile phase and were then used for the incubation experiments, and in part for the subsequent preparation of R- and S-metalaxyl by methylation with diazomethane as outlined below. In this way, R- and S-MX-acid, and R- and S-metalaxyl were obtained in high (>99%) enantiomeric purity. Incubation in Soil. Garden soil (sandy loam; 1.6% organic carbon; pH value 7.0) from a location in Wa¨denswil was carefully air-dried at room temperature to a final water content of ≈15% and sieved (5 mm). The soil was then kept in a porous clay pot until used within a few days. The soil was from the same plot as used in previous investigations on acetamide and phenoxy acid pesticides (11, 12). Separate incubation experiments were carried out with the racemic and with the pure R- and S-compounds, respectively, using 750-mL wide-mouth clear glass jars covered with aluminum foil and lid. Portions of 300 g of the air-dried soil were placed in the jars and fortified with a solution containing ≈300-600 µg of rac-, R-, or S-metalaxyl dissolved in 10 mL of water, yielding a final water content of ≈18% in soil and a fortification level of 1-2 mg/kg (experiments S1, S2, and S3, respectively). The soil was 222
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carefully mixed and then incubated at 20-23 °C with daylight (but avoiding direct sunlight) for up to 3 months, while closed to exclude losses by evaporation. The jars were opened for a short time every 1-3 days for aeration to ensure aerobic conditions. Similar experiments were carried out with rac-, R-, and S-MX-acid (experiments S4, S5, and S6, respectively). Periodically, 10.0-g samples were removed and placed into 20-mL glass vials for further sample treatment; duplicate samples were taken immediately after fortification and mixing (t ) 0), single samples periodically thereafter. The good agreement for the initial concentrations (c0) measured for duplicate samples (see below) indicated good homogeneity of the fortified soils. Blank determinations of the soil prior to fortification revealed no metalaxyl or MX-acid present (detection limit, < 0.005 mg/kg). Extraction of Metalaxyl and MX-Acid from Soil. Derivatization of MX-Acid. The 10.0-g samples were mixed with 10 mL of methanol and then kept at 4 °C until extracted and analyzed. Immediately prior to extraction, 10 µg of clofibric acid and 10 µg alachlor in 100 µL methanol were added, and after vigorous shaking, the samples were centrifuged (4000 rpm for 30 min). The clear supernatants were removed and placed in 20-mL glass vials and carefully evaporated to dryness at room temperature using a stream of air. The residues were redissolved in 0.2 mL methanol and derivatized (see below) resulting in fraction #1 of a sample. The centrifugates were mixed with 5 mL of water and vigorously shaken and then 5 mL of acetone was added followed by vigorous shaking and centrifugation. The clear supernatants were removed and added to 5 mL of distilled water and acidified with dilute H2SO4 to pH ≈ 2. The analytes were re-extracted from the aqueous phase with three 5-mL portions of methylene chloride. The combined methylene chloride extracts were then carefully brought to dryness (see above). A small amount of methanol was then added, and the samples were derivatized (see below) resulting in fraction #2 of a sample. MX-acid cannot be directly analyzed by GC-MS but must be derivatized. All samples from experiments S1, S2, and S3 (incubation with metalaxyl) were treated with diazoethane (see ref 13 and cautionary notes in ref 14) to form the ethyl ester of any MX-acid present. In this way, the parent metalaxyl (methyl ester, not affected by diazoethane) and its metabolite, MX-acid (analyzed as the ethyl ester, MX-acid-Et) were distinguishable by GC-MS. All samples from experiments S4, S5, and S6 (incubation with MX-acid) were treated with diazomethane (see ref 14 and cautionary notes therein) to form the methyl ester (metalaxyl) because metalaxyl showed a better enantiomer resolution than MX-acid-Et. Fractions #1 were acidified with a few drops of 1% trifluoroacetic acid in methanol in order to ensure proper derivatization; fractions #2 (acidic from the partition procedure) were directly derivatized. After derivatization, the residues were dissolved in 5.0 mL of ethyl acetate. Aliquots of fraction #1 and #2 of a sample were then combined and analyzed. The two-step extraction procedure gave recoveries of ≈80% for metalaxyl and alachlor and ≈70% for MX-acid. No transesterification (formation of methyl and ethyl ester from the presence of methanol and ethyl acetate, respectively) was observed. However, when the samples were ethylated with diazoethane, small amounts (≈3%) of methyl ester were formed, presumably as a result of some diazomethane present in the reagent. Furthermore, there were small amounts of the respective MX-acids in R- and S-metalaxyl (≈3%), and the data were corrected accordingly. Enantioselective GC-MS Analysis. The samples were analyzed using a VG Tribrid magnetic sector mass spectrometer (VG Fisons, Manchester, England) under electronimpact ionization (EI, 70 eV, 180 °C) and full-scan (m/z 35435, 1.16 s/scan; mass resolution M/∆M ) 500) or selected-
ion-monitoring (SIM) conditions (m/z values, see below). A 25-m enantioselective PS086-BSCD (BSCD ) tert-butyldimethylsilyl-β-cyclodextrin; relative amount, 50%; BGBAnalytik, Adliswil, Switzerland) column was used. The column was temperature programmed as follows: 70 °C, 2-min isothermal, 20 °C/min to 120 °C, then at 3 °C/min to 230 °C, followed by an isothermal hold at that temperature. The PS086-BSCD column fully resolved metalaxyl into its enantiomers (enantiomer resolution, R ≈ 1.5) and showed acceptable, though lower enantiomer resolution (R ≈ 0.8) of the ethyl ester analogue, MX-acid-Et. The enantiomer elution sequence was S as first-eluted and R as second-eluted for both esters with the MX-acid-Et enantiomers eluting ≈1.3 min after the respective metalaxyl enantiomers. The elution temperatures were as follows: clofibric acid methyl ester, 161.9 °C; clofibric acid ethyl ester, 167.6 °C; alachlor, 201.8 °C; S-metalaxyl, 204.8 °C; R-metalaxyl, 205.3 °C; S-MX-acidEt, 208.7 °C; R-MX-acid-Et, 209.0 °C. Split-splitless injection (SSL; splitless time, 60 s; injection volume, 1 µL) was used with a heated (250 °C) injector. No enantiomerization/ racemization was observed for metalaxyl and its ethyl ester analogue using SSL. The amounts of analyte were determined from SIM peak area ratios relative to the internal standard (alachlor) and in reference to suitable standards. Methylation (or ethylation) of clofibric acid served as a control. Enantiomer ratios (ER) were defined as ER ) pS/pR where pS and pR are the peak areas of the S- and R-enantiomers, respectively. rac-Metalaxyl, expectedly, showed the presence of both enantiomers in a 1:1 ratio (ER ) 1.03 ( 0.02). Metalaxyl-M from a commercial formulation showed a product highly enriched in R-metalaxyl and an enantiomer composition (R: S) 97.5:2.5. Expectedly, the mass spectra of R- and S-metalaxyl were identical and showed molecular ions ions (M•+) at m/z 279; likewise, the mass spectra of R- and S-MX-acid-Et were identical (M•+ at m/z 293). Both compounds yield fragment ions of the same exact mass, m/z 220.134, which were used for quantification in the SIM analyses. These ions correspond to the M•+ - 59 ion (M+ - COOCH3) of metalaxyl and to the M•+ - 73 ion (M•+ - CH2OCH3 - CO and/or M•+ - COOC2H5) of MX-acid-Et, respectively. The relative response ratio metalaxyl:MX-acid-Et at m/z 220.134 was 0.41. Further ions (m/z) used for confirmatory purposes and to monitor other target analytes were as follows: 248.129 (MX-acid-Et); 249.136 (metalaxyl); 188.108 (alachlor); 228.055 (clofibric acid methyl ester); 242.071 (clofibric acid ethyl ester). In experiments S1, S2, and S3, the degradation/dissipation of metalaxyl and the formation and subsequent degradation of MX-acid were followed. In experiments S4, S5, and S6, the degradation/dissipation of MX-acid was followed. Duplicate determinations were made for c0 (agreement, (3% for metalaxyl and MX-acid) in all experiments, single determinations thereafter. For the quantitative evaluation of the kinetics, the program AQUASIM (EAWAG, Du ¨ bendorf, Switzerland; see ref 15) was used assuming firstorder kinetics and applying lag phases with lower rates when necessary.
Results and Discussion Degradation/Dissipation of Metalaxyl in Soil. Metalaxyl eventually degraded to levels R). A plot of ln(ER) versus t was initially roughly linear but eventually (>60 d) leveled off, indicating some loss of enantioselectivity during the later phase of the experiment (see Figure 3d). The initial rate difference ∆k calculated from this plot is 0.038 d-1, which is in reasonable agreement with above data (kR - kS ) 0.045 d-1). The overall half-life of rac-metalaxyl estimated from the data of exp. S1 by summing the concentrations of both enantiomers is ≈30 d and is similar to the one observed in 1995 (≈32 d, see ref 11) with soil from the same location. As a measure of enantioselectivity (or stereoselectivity; ES) we previously defined the excess of the rate of the faster over the slower degraded enantiomer (stereoisomer) in a particular medium (11). In exp. S1, the degradation of racmetalaxyl is highly enantioselective with an ES value of 0.60 as expressed by ES ) (kR - kS)/(kR + kS). This relatively high ES value suggests a predominantly biotic pathway. In the previous study, an ES value of 0.37 was determined (11). In a completely chemically mediated (nonenantioselective) reaction (∆k ) 0), ES is 0, and hence the enantiomer composition is not changed. Chiral Stability of Metalaxyl in Soil. In Figure 4a-d, EI SIM chromatograms (m/z 220) show the elution of metalaxyl and MX-acid from samples of experiments S2 and S3 (incubation with R- and S-metalaxyl), prior to and following incubation (upper and lower panels, respectively). The chromatograms in panels a and c document the high enantiomeric purity of R- and S-metalaxyl prior to incubation; the chromatograms in panels b and d indicate degradation of R- and S-metalaxyl and the concurrent formation of the respective MX-acids after incubation. Experiments S2 and S3 indicated negligible (74 d) with S-MX-acid (see chromatogram in Figure 2c). Degradation of MX-Acid in Soil. In Figure 5, EI SIM chromatograms (m/z 220) show the elution of MX-acid in the soil samples from experiments S4, S5, and S6, prior to and following incubation (upper and lower panels, respectively). The chromatogram from the incubation of the racMX-acid (exp. S4; see panel b) illustrates the more rapid degradation of the first-eluted S-enantiomer, leading to residues enriched in R-MX-acid. The chromatograms from the incubation of R- and S-MX-acid (exp. S5 and S6, respectively) revealed degradation of the compounds but no formation to the respective antipodes (see panels d and f). Experiments S5 and S6 indicated negligible ( S. A plot of ln(ER) versus time was roughly linear (0-42 d). The slope indicated a rate difference ∆k (kRacid - kSacid) of -0.013 d-1 (see Figure 6d), which is in reasonable agreement with the above data (kRacid - kSacid ) -0.016 d-1). The fact that the two enantiomers of MX-acid degrade at different rates (kSacid > kRacid) and the ES value of ≈ 0.36 calculated from these rates suggest again that degradation is clearly biotic. Agricultural and Environmental Consequences. The degradation of rac-metalaxyl in the soil studied is clearly enantioselective with R-metalaxyl being faster degraded than S-metalaxyl (residues enriched in S-metalaxyl). This enantioselectivity is the same as was observed previously (1995, see ref 11) and shows that the data are consistent. That this enantioselectivity may not be unique just to our situation is indicated by the fact that R-metalaxyl is faster degraded than rac-metalaxyl in other soils (17). This, however, may not imply that the same enantioselectivity is to be expected under all conditions. For example, a reversed enantioselectivity was observed in the anaerobic degradation in sewage sludge which resulted in residues enriched in R-metalaxyl (11), and in the degradation of other compounds enantioselectivity is reported to be influenced by environmental and climatic changes (18). Our data also showed that the degradation of metalaxyl to its primary metabolite, MX-acid, proceeded with retention of configuration and that both compounds, metalaxyl and MX-acid, are configurationally stable in soil. VOL. 36, NO. 2, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 7. Plot of predicted EEs for various compositions (EE0) as a function of incubation time, assuming an enantioselectivity as observed for rac-metalaxyl in exp. S1 (kR - kS ) 0.038 d-1). Note that EEs for rac-metalaxyl, metalaxyl-M, and combinations thereof decrease with increasing incubation time to eventual compositions S > R (EE < 0). FIGURE 6. Degradation of MX-acid in soil experiments S4, S5, and S6 with rac-, R-, and S-MX-acid, respectively. (a) Degradation of rac-MX-acid showing faster degradation of the S-enantiomer (exp. S4), (b) degradation of R-MX-acid (exp. S5), and (c) degradation of S-MX-acid (exp. S6). Normalized concentrations (100 c/c0) plotted versus incubation time; modeled concentrations using the rates from Table 1. (d) Plot of ln(ER) from exp. S4 (incubation of racMX-acid) versus incubation time showing rough linearity in the initial phase. Product activity is thus not affected by enantiomerization/ racemization. The use of metalaxyl-M (R:S ) 97.5:2.5) in place of the racemic product is expected to result in significantly lower environmental concentrations because of the near absence of the environmentally more persistent S-metalaxyl. Degradation of rac-metalaxyl in the soil studied resulted in residues with a composition S > R; degradation of metalaxyl-M expectedly results in residues with a composition R > S though with a relatively higher contribution of S-metalaxyl than in the product itself. The “chiral switch” from racmetalaxyl to metalaxyl-M will thus expectedly result in residues with a changed enantiomer composition, as outlined next. If initial product composition and the degradation kinetics are known, the enantiomer composition (ER) of environmental residues can be determined as a function of time according to eq 4. Because ERs show a high variability for compositions that are low in one isomer (e.g. metalaxyl-M), we preferred expressing enantiomer composition as enantiomer excess (6), defined here as the excess of R-metalaxyl (active) over S-metalaxyl (inactive)
EE ) ([R] - [S])/([R] + [S])
(7)
Using this definition, EEs will vary from +1 (enantiopure R) to -1 (enantiopure S); the EE for rac-metalaxyl is 0. Metalaxyl-M, with ≈97.5% of R-metalaxyl and ≈2.5% Smetalaxyl, thus has an EE of ≈0.95. The relationship between EE and ER is given as EE ) (1 - ER)/(1 + ER). In Figure 7, we plotted predicted EEs for the residues as a function of time for varying initial product compositions (EE0 ) 0-0.98), assuming an enantioselectivity as observed for rac-metalaxyl in exp. S1 (∆k ) 0.038 d-1). The plots show a continuous decrease of EEs with time, suggesting eventual compositions S > R (EEs < 0) from applications of rac-metalaxyl, metalaxyl-M, and all combinations thereof (EE0 g 0). The enantiomer composition of the initial product (EE0), or the mixture used, can be determined from the EEs of environmental residues only if exposure time t is known. If this is unknown, or the residues originate from different 226
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applications, EE0 cannot be determined unambiguously. However, if only rac-metalaxyl was used, and assuming an enantioselectivity as observed in this study, the residues expectedly show compositions S > R (EEs< 0), and residues with compositions R > S (EEs > 0) must originate from applications that included metalaxyl-M.
Acknowledgments This study was sponsored by Syngenta AG (Basle, Switzerland). Fruitful discussions were held with Hans Egli (Syngenta AG) and are kindly acknowledged.
Literature Cited (1) The Pesticide Manual, 9th ed.; Worthing, C. R., Hance, R. J., Eds.; British Crop Protection Council: Farnham, U.K., 1991. (2) Buser, H. R.; Mu¨ller, M. D. Environ. Sci. Technol. 1995, 29, 20232030. (3) Hubele, A.; Kunz, W.; Eckhardt, W.; Storm, E. In Proceedings of the 5th IUPAC Congress on Pesticide Chemistry, Mijamoto, Japan; Kearney, P., Ed.; Pergamon Press: New York, 1988; Vol. 2, pp 233-242. (4) Nuninger, C.; Watson, G.; Leadbitter, N.; Ellgehausen, H. Proc. Brit. Crop Prot. Conf. 1996, 1, 41-46. (5) Blaser, H. U.; Buser, H. P.; Coers, K.; Hanreich, R.; Jalett, H. P.; Jelsch, E.; Pugin, B.; Schneider, H. D.; Spindler, F.; Wegmann, A. Chimia 1999, 53, 275-280. (6) Buser, H. R.; Poiger, T.; Mu ¨ ller, M. D. Environ. Sci. Technol. 2000, 34, 2690-2696. (7) Roberts, T. R., Hutson, D. H., Eds. Metabolic Pathways of Agrochemicals, Part 2: Insecticides and Fungicides; The Royal Society of Chemistry: Cambridge, U.K., 1999. (8) Williams, A. Pestic. Sci. 1996, 46, 3-9. (9) Crosby, J. Pestic. Sci. 1996, 46, 11-31. (10) Stereoselectivity of Pesticides, Biological and Chemical Properties, Chemicals in Agriculture 1; Ariens, E. J., van Rensen, J. J. S., Welling, W., Eds.; Elsevier: Amsterdam, 1988. (11) Mu ¨ller, M. D.; Buser, H. R. Environ. Sci. Technol. 1995, 29, 20312037. (12) Mu ¨ller, M. D.; Buser, H. R. Environ. Sci. Technol. 1997, 31, 19531959. (13) Organic Syntheses; Baumgarten, H. E., Ed.; Wiley: New York, 1973; Collect. Vol. V, p 351. (14) Vogel, A. Vogels’s Textbook of Practical Organic Chemistry, 4th ed.; Longman: London, 1978. (15) Reichert, P. Water Sci. Technol. 1994, 30, 21-30. (16) Droby, S.; Coffey, M. D. Ann. Appl. Biol. 1991, 118, 543-553. (17) Egli, H. Syngenta AG: Basle, Switzerland, 2001; personal communication. (18) Lewis, D. L.; Garrison, A. W.; Wommack, K. E.; Whittemore, A.; Steudler, P.; Melillo, J. Nature 1999, 401, 898-901.
Received for review May 10, 2001. Revised manuscript received September 26, 2001. Accepted October 22, 2001. ES010134S
