Environ. Sci. Technol. 1997, 31, 1960-1967
Conversion Reactions of Various Phenoxyalkanoic Acid Herbicides in Soil. 2. Elucidation of the Enantiomerization Process of Chiral Phenoxy Acids from Incubation in a D2O/Soil System
CHART 1. Structures of Phenoxyalkanoic Acid Herbicides Investigateda
HANS-RUDOLF BUSER* AND MARKUS D. MU ¨ LLER Swiss Federal Research Station, CH-8820 Wa¨denswil, Switzerland
The enantiomerization of chiral phenoxyalkanoic acid herbicides in soil under laboratory conditions in the presence of D2O was investigated. The compounds studied were 2-(4chloro-2-methylphenoxy)propionic acid (MCPP), 2-(2,4-dichlorophenoxy)propionic acid (DCPP) and, for comparison, the achiral 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Enantiomerization was studied by incubating racemic and enantiopure MCPP and DCPP in D2O/soil and following the formation of deuterated analogs using enantioselective highresolution gas chromatography (HRGC) and mass spectrometry/ mass spectrometry (MS/MS). MS/MS showed less interference from natural 13C isotopomers than conventional MS and allowed the exact localization of deuterium in the labeled compounds. The results indicated exclusive H-D exchange at C-2 (R-methin H) of MCPP and DCPP with formation of the 2-deuterio analogs. H-D exchange in MCPP proceeded with retention as well as inversion of configuration, forming both of the labeled enantiomers from each of the native ones at comparable rates. H-D exchange was also observed for MCPA and 2,4-D with formation of the 2-deuterio and 2,2-dideuterio analogs. The importance of racemization reactions of chiral compounds is discussed, and general conclusions are drawn on the environmental fate of chiral compounds and on consequences for the monitoring of such compounds in the environment using enantioselective techniques.
Introduction Phenoxyalkanoic acids are an important group of herbicides used in agriculture, industrial weed control, and forestry (1). Some of these compounds are among the most popular pesticides used worldwide. The compounds were originally developped at the end of World War II (2), and they have been extensively used since then. The cumulative global production is estimated at several million tons. Some of the compounds have become notorious because of their use (in the form of alkyl esters) as defoliants in Vietnam (agent orange) and because of their contamination with dioxin (3, 4). Of particular importance are the phenoxyacetic and the 2-phenoxypropionic acids. These compounds include 4-chloro-2-methylphenoxyacetic acid (MCPA), 2,4-dichlorophe* Corresponding author telephone: ++41 1 783 6286; fax: ++41 1 783 6439; e-mail:
[email protected].
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a The two enantiomers of MCPP and DCPP are shown in the top row with the methyl groups in front (R enantiomers) or in the back of the paper plane (S enantiomers). Note that the two R-hydrogens in MCPA and 2,4-D are not identical in that the one in front is pro-R; the one in the back is pro-S.
noxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2-(4-chloro-2-methylphenoxy)propionic acid (MCPP), 2-(2,4-dichlorophenoxy)propionic acid (DCPP), and others. The 2-phenoxypropionic acids (MCPP, DCPP) contain an asymmetrically substituted C-atom (C-2) and are chiral. They consist of two enantiomers with R and S configuration (see Chart 1). Only the R enantiomers are herbicidally active; the S enantiomers are devoid of herbicidal activity but are powerful anti-auxins (5). This different biological activity is not surprising since most biological reactions are enzymatically mediated and involve chiral centers. There are some chiral compounds that are configurationally instable under certain conditions and may undergo enantiomerization (or racemization) such as by inversion of the absolute configuration of an asymetrically substituted C-atom (6). This process may be problematic for pesticides if it occurred under environmental conditions, particularily if it occurred prior to deployment of the desired biological activity such as in pre-emergent applications. If enantiopure products were used, this process could not only decrease the amount of the active enantiomer (eutomer) and thus inactivate part of the product, it would also result in residues of the inactive enantiomer (distomer). On the other hand, an inactive enantiomer may partly be converted into the active one and thus simulate activity of an inherently non-active isomer. As the case may be, the biological activity of a racemic product may thus be higher or lower than that based on the content of the active enantiomer alone. If enantiomerization is fast, it would even be pointless to use enantiopure products since these may racemize prior to deployment of a desired biological activity. A detailed knowledge on the environmental behavior of biologically active chiral compounds must thus include some knowledge on enantiomerization and its magnitude relative to other processes such as degradation. In a previous study (7), we investigated the enantiomerization processes by incubation of racemic and enantiopure compounds followed by enantioselective analysis. In this study, we examine the enantiomerization process of the 2-phenoxypropionic acid herbicides MCPP and DCPP in soil under laboratory conditions in the presence of D2O. By studying the incorporation of deuterium into the residual compounds, a more detailed insight into the mechanisms of enantiomerization was obtained. For comparison, the achiral phenoxyacetic acid herbicides MCPA and 2,4-D were included in the study.
S0013-936X(96)00783-3 CCC: $14.00
1997 American Chemical Society
TABLE 1. SRM Transitions Used for Detection of Phenoxyalkanoic Acids: EI MS Data EI MS data (m/z) compd MCPP-ME DCPP-ME MCPA 2,4-D-ME clofibric acid-ME
SRM transition native -d1 -d2 native -d1 -d2 native -d1 -d2 native -d1 -d2 native
169+ f 125+ 170+ f 125+ 171+ f 125+ 189+ f 145+ 190+ f 145+ 191+ f 145+ 155+ f 125+ 156+ f 125+ 157+ f 125+ 175+ f 145+ 176+ f 145+ 177+ f 145+ 169+ f 111+
loss monitored (mass loss) CH3CHO CH3CDO CH2DCDO CH3CHO CH3CDO CH2DCDO CH2O CDHO CD2O CH2O CDHO CD2O C3H6O
(44) (45) (46) (44) (45) (46) (30) (31) (32) (30) (31) (32) (58)
M+
(M - 59)+
phenol ion (phenoxonium ion)
228 229 230 248 249 250 214 215 216 234 235 236 228
169 170 171 189 190 191 155 156 157 175 176 177 169
142 142 142 162 162 162 (141) (141) (141) (161) (161) (161) 142
CHART 2. Hypothetical Pathways for Enantiomerization (Racemization) of 2-Phenoxypropionic Acidsa
a In pathway P1, one hydrogen (at C-2) is replaced via a carbanionic intermediate (or an enolic form); in pathway P2, two hydrogens (at C-2 and C-3) are replaced via an enoic acid intermediate.
Experimental Section Phenoxyalkanoic Acid Herbicides Analyzed. Reference Compounds. The compounds investigated were (rac)-MCPP, (rac)-DCPP, (R)- and (S)-MCPP, MCPA, and 2,4-D. The sources and the chemical and enantiomeric purities (where applicable) of the compounds are listed in ref 7. Deuterium oxide (D2O) with an isotopic purity of >99.8% was from Fluka (Buchs, Switzerland). 2-(4-Chlorophenoxy)-2-methylpropionic acid (clofibric acid) was used as an internal standard (see also ref 7). Incubation of Phenoxyalkanoic Acids in D2O/Soil. The soil used was from the same location and pretreated in the same manner as in the previous study (7). The water content of the sieved and carefully mixed soil was found to be 18%. A subsample of it was further dried to ≈2%. Experiments were initially carried out with (rac)-MCPP, (rac)-DCPP, MCPA, 2,4-D, and dicamba and later followed by separate incubations of (R)- and (S)-MCPP. Portions of 210 and 150 g of the 18% and 2% soils, respectively, were carefully mixed and fortified with 40 mL of a D2O solution containing 400 µg of each enantiomer or compound (1 ppm fortification level) yielding ≈400 g of soil mixture with a total water content (H2O plus D2O) of ≈20% and a protium/deuterium ratio of ≈1. After being carefully mixed, the fortified soil was incubated at 2023 °C as described in the preceding paper (7). Replicate samples were removed immediately after fortification, and single samples were removed periodically thereafter (up to 45 d). Prior to extraction, 10 µg of clofibric acid in 100 µL of methanol was added as an internal standard.
The soil samples were extracted, methylated, and cleaned up as before (7) except that the sample extracts after methylation were passed through a small silica column (0.7 g of silica gel 60, Merck, Darmstadt, FRG; deactivated with 5% water; 5-mm i.d. Pasteur pipet) topped with 10 mm of sodium sulfate. The methyl esters (ME) of the phenoxyalkanoic acids were eluted with 10 mL of n-hexane/methylene chloride (4:1). After careful concentration and dilution to 200 µL with n-hexane, 2-µL aliquots were used for analysis by high-resolution gas chromatography/mass spectrometry/ mass spectrometry (HRGC/MS/MS). Blank determinations of the soil prior to fortification revealed no phenoxy acids present (detection limit, [S]) are consistent with the faster net degradation of the S enantiomers. The SRM chromatograms for the labeled compounds show the presence of small (≈2.5% relative intensity) peaks prior to incubation from the 13C isotopomers (see panels c and g; ER ≈ 1.0) but much larger signals (≈25 and 35% relative intensity) after incubation from the presence of labeled MCPP and DCPP (see panels d and h). The ER values of the labeled compounds were similar (1.42 and 2.26 for MCPP-d1 and DCPP-d1, respectively) to those of the remaining native ones. No formation of dideuterio analogs (104 (initial ER values >100:1 and kRS and thus K ) kRS/kSR < 1, as was observed in the previous non-labeled experiments (7). The ratio of labeled to native enantiomers was 0.748 and 0.801 for (R)- and (S)-MCPP,
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FIGURE 5. SRM chromatograms (169+ f 125+, panels in top row; 170+ f 125+ panels in bottom row) showing elution of native MCPP-ME (top row) and of deuterated analogs (bottom row) prior to (panels a, c, e, and g) and after (panels b, d, f, and h) incubation in D2O/soil. Panels a-d, chromatograms from the 2-d incubation of (R)-MCPP; panels e-h, chromatograms from the 2-d incubation of (S)-MCPP. Note enantiomerization of the native stereoisomers as shown by the chromatograms in panels b and f and the formation of deuterated analogs as indicated by the chromatograms in panels d and h. Intensities normalized to that of the monoisotopic transition in top row except for vertical expansion as indicated. Small peaks in chromatograms c and g are due to 13C isotopomers.
FIGURE 6. Degradation of (a) (rac)-MCPP and (b) (rac)-DCPP and formation of the deuterated analogs from incubation in D2O/soil. Note the degradation of the native isomers (S faster degraded than R) and the concurrent formation of labeled R and S enantiomers. Normalized concentrations (100 × c/c0, logarithmic scale) are plotted versus incubation time (d). respectively, and indicated 75-80% incorporation of deuterium relative to protium in the inverted compounds. Kinetic of Phenoxyalkanoic Acid Degradation in D2O/ Soil. The results from the D2O/soil experiment with the racemic compounds are shown in Figure 6a,b. The data for the native MCPP and DCPP stereoisomers show degradation to follow first-order kinetics with a slower initial reaction (0-30 d) followed by a more rapid one (30-45 d), although with a less pronounced change of the rates than in the previous study (7). In Table 2, we list rates of degradation (k, d-1) for MCPP, DCPP, and MCPA in both phases, as determined from regression plots ln(c/c0) ) -kt. The rates for the inital, slower phase ( (R)-MCPP > (R)-DCPP with rates corresponding to half-lives [τ ) ln (2/k)] of 8-22 d. The order is thus the same as in the previous study (experiment S1 in ref 7). Again, the S enantiomers were faster degraded than the R enantiomers, indicating enantioselective degradation in the same sense (S > R) for both MCPP and DCPP, as observed in the previous study (7). The rate of degradation for (rac)-MCPP, as calculated by summing concentrations of the native enantiomers, was higher than that for DCPP (corresponding to half-lives of 10.6 and 12.7 d, respectively). In the second phase (>30 d), the rates of degradation for (R)- and (S)-MCPP
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TABLE 2. Net Rate Constants (10-3 d-1) and Half-Lives (in Parentheses, τ, d) for Degradation of MCPP, DCPP, and MCPA in D2O/Soil compound
slow phasea
fast phaseb
(R)-MCPP (S)-MCPP (R)-DCPP (S)-DCPP MCPA
54 (12.8) 77 (9.0) 32 (21.7) 83 (8.1) 75 (9.2)
105 (6.6) 121 (5.7) 105 (6.6) 107 (6.5) 262 (2.6)
slow phase native/labeledc 36 (19.3) 62 (11.2) 17 (40.8) 74 (9.4)
a 0-30 d data. b 30-45 d data. c Rate constants as determined from the summation of the respective native and labeled stereoisomers, simulating the rates in a “protium-only” environment (see text).
and for (R)- and (S)-DCPP are surprisingly similar (see Table 2), corresponding to half-lives of 5.7-6.6 d. Since there is a continuous removal of the deuterated analogs from the pool of the native compounds (see next paragraph), the rates cannot be directly compared to those of the previous study (7). The data in Figure 6a,b also document the formation of deuterated analogs of MCPP and DCPP in this experiment. The concentrations of the labeled enantiomers increased to
maxima of 16% and 8% for MCPP and to 27% and 9.5% for DCPP (concentrations of R and S analogs relative to initial concentrations of native ones) and then decreased again approaching those of the respective native ones with further incubation. The rates of degradation for the labeled enantiomers were approaching those for the native ones, and there seems to result a “dynamic” equilibrium with constant ER values for native and labeled compounds, a situation similar to the one previously observed for the native compounds (7). In the final phase, the relative amounts of deuterated to native compounds (summed concentrations) were 0.75 and 0.88 for MCPP and for DCPP, respectively, indicating an incorporation of 75-88% deuterium relative to protium. The final ER values ([R]/[S]) for the labeled compounds were ≈3 and ≈6.5 for MCPP and DCPP, respectively. The curves for a native stereoisomer and its labeled enantiomer show very similar trends as those previously observed when single isomers were incubated (experiments S2 and S3, see ref 7). The graphs in Figure 6a,b thus resemble a combination of the graphs from experiments S2 and S3 of that study with “racemization points” at ≈24 and ≈18 d for (S)-MCPP and (S)-DCPP in soil, respectively. When the concentrations of the R stereoisomers (native plus labeled) and those of the S stereoisomers were summed, the rates thus determined simulate the degradation of MCPP and DCPP in a “protium-only” environment (see Table 2). The net rates thus calculated corresponded to half-lives of 9-41 d and amounted to 52-75% of those of the racemic compounds in the previous study (7), indicating slower degradation in D2O/ soil. The data in Figure 6a,b indicate higher rates for the formation of the labeled R enantiomers than for the labeled S enantiomers. The rates were estimated from regression plots ln[(c0 - c)/c] ) kt where c0 is the initial concentration of a native stereoisomer (precursor) and c is the concentration of the labeled enantiomer (0-5 d data). The rates of formation were 1.4 × 10-2 and 8.7 × 10-3 d-1 for labeled (R)- and (S)MCPP, respectively, and 1.8 × 10-2 and 1.2 × 10-2 d-1 for labeled (R)- and (S)-DCPP, respectively. The higher rates of formation of the R enantiomers reflect higher rates of enantiomerization of the S enantiomers (kSR > kRS); the ratios of these rates of 1.61 and 1.5 for MCPP and DCPP, respectively, reflect equilibrium constants (reciprocal values, K ) kRS/kSR) of 0.62 and 0.67 for MCPP and DCPP, respectively. Whereas the value for MCPP is similar to the one from the previous study (K ) 0.78, see ref 7), the value for DCPP deviates from the previous one (K ) 0.37) for unknown reasons. Nevertheless, the values indicate a preference for the conversion of the S into R enantiomers for both compounds.
Conclusions This and the previous study (7) indicated considerable chiral instability of MCPP and DCPP in soil. In the previous study, enantiomerization was detected when enantiopure compounds (single isomers) were studied, but it could not be observed when using the racemic compounds because of concurrent enantioselective degradation. Enantiomerization and degradation were shown to be predominantly biologically mediated because enantiomerization was not observed in sterilized soil and degradation was much slower (7). In this study now, the D2O/soil experiments gave a more detailed insight in the underlying mechanism of enantiomerization. In this approach, the labeled, enantiomerized products were distinguished from the native precursors by enantioselective HRGC and MS/MS detection techniques. This approach allowed enantiomerization to be observed even when using the racemic products. The results now confirm the enantiomerization of MCPP and DCPP to proceed in both
directions, from R to S and from S to R, with equilibrium constants (K ) kRS/kSR < 1) favoring the herbicidally active R enantiomers. The results indicated enantiomerization of 2-phenoxypropionic acids to proceed via the exchange of the R-methin H at C-2, in a process such as the hypothetical pathway P1 described in Chart 2. Furthermore, the results indicate H-D exchange to proceed with retention and inversion of configuration. In this way, both labeled enantiomers were formed from each of the native stereoisomers in very similar ER ratios despite the fact that the initial enantiomeric composition of the native ones varied >104 times. H-D Exchange was also observed with MCPA and 2,4-D whereby both R-H’s eventually can be exchanged. Incorporation of deuterium proceeded easiest with the 2-phenoxypropionic acids and less with the phenoxyacetic acids. The data further document the different fate of pesticide enantiomers in soil, the compartment which through its dense microbiological population is able to absorb and degrade pesticides released into the environment. The biologically mediated conversions observed in this study result in residues of MCPP and DCPP that do not mirror the enantiomeric composition of the products used. The actual occurrence of these reactions under environmental conditions is expected but remains to be determined.
Acknowledgments We greatfully acknowledge the experienced help of Verena Buser for all the sample preparations and we thank H. P. Kohler and C. Zipper of the Swiss Federal Institute of Environmental Science and Technology (EAWAG, Du ¨bendorf) for fruitful discussions and the gift of (S)-MCPP.
Literature Cited (1) Humburg, N. E.; Colby, S. R.; Hill, E. R.; Kitchen, L. M.; Lym, R. G.; McAvoy, W. J.; Prasad, R. Herbicide Handbook of the Weed Society of America, 6th ed.; Weed Society of America: Champain, IL, 1989. (2) Allen, H. P.; Brian R. C.; Downes, J. E.; Mees, G. C.; Springett, R. H. Selective Herbicides. In Jealott’s Hill, Fifty Years of Agricultural Research; Peacock, F. C., Ed.; ICI: Jealott’s Hill, UK, 1978; pp 35-41. (3) Young, A. L.; Kang, H. K.; Shepard, B. M. Environ. Sci. Technol. 1983, 17, 530A-540A. (4) Ramel, C. Chlorinated Phenoxy Acids and Their Dioxins; Ecological Bulletins, Swedish Natural Science Research Council: Stockholm, Sweden, 1978. (5) Loos, M. A. Phenoxyalkanoic Acids. In Herbicides: Chemistry, Degradation and Mode of Action; Kearney, P. C., Kaufman, D. D., Eds.; Marcel Dekker Inc.: New York, 1975; Vol. 1, pp 2-101. (6) Testa, B.; Trager W. F. Chirality 1990, 2, 129-133. (7) Mu ¨ ller, M. D.; Buser, H. R. Environ. Sci. Technol. 1997, 31, 19531959. (8) Buser, H. R.; Mu ¨ ller, M. D. Environ. Sci. Technol. 1994, 28, 119128. (9) Eliel, E. L.; Wilen, S. Stereochemistry of Organic Compounds; John Wiley: New York, 1994; Chapter 7. (10) Kenyon, G. L.; Hegeman, G. D. Biochemistry 1970, 9, 4036. (11) Hutt, A. J.; Caldwell, J. J. Pharm. Pharmacol. 1983, 35, 693-704 (12) Kohler, H. P. Personal communication, 1996. (13) Budzikiewicz, H.; Djerassi, C.; Williams, D. H. Mass Spectrometry of Organic Compounds; Holden-Day, Inc.: San Francisco, CA, 1967; pp 237-247. (14) Smith, H. L.; Hill, R. L.; Lehmann, I. R.; Lefkowitz, R. J.; Handler, P.; White, A. Principles of Biochemistry: General Aspects, 7th ed.; McGraw Hill: New York, 1983; pp 378-379. (15) Lynen, F. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1953, 12, 683-693.
Received for review September 11, 1996. Revised manuscript received February 20, 1997. Accepted February 26, 1997.X ES960783H X
Abstract published in Advance ACS Abstracts, May 1, 1997.
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