Article pubs.acs.org/est
The Chiral Herbicide Beflubutamid (II): Enantioselective Degradation and Enantiomerization in Soil, and Formation/Degradation of Chiral Metabolites Ignaz J. Buerge,* Markus D. Müller, and Thomas Poiger Plant Protection Chemistry, Swiss Federal Research Station (Agroscope), CH-8820 Wädenswil, Switzerland S Supporting Information *
ABSTRACT: Beflubutamid is a chiral soil herbicide currently marketed as racemate against dicotyledonous weeds in cereals. Biotests have shown that (−)-beflubutamid is at least 1000× more active than (+)-beflubutamid. Potential substitution of the racemate by (−)-beflubutamid should therefore be further considered. Here, we investigated the degradation behavior in soils and formation and degradation of two chiral metabolites. Laboratory incubation experiments were performed with an alkaline and an acidic soil. The compounds were analyzed by enantioselective GC-MS. Degradation rate constants were determined by kinetic modeling. In the alkaline soil, degradation of beflubutamid was slightly enantioselective, with slower degradation of the herbicidally active (−)-enantiomer. In the acidic soil, however, both enantiomers were degraded at similar rates. In contrast, degradation of a phenoxybutanamide metabolite was highly enantioselective. Chiral stability of beflubutamid and its metabolites was studied in separate incubations with the pure enantiomers in the same soils. In these experiments, (−)-beflubutamid was not converted to the nonactive (+)-enantiomer and vice versa. Significant enantiomerization was, however, observed for the major metabolite, a phenoxybutanoic acid. With regard to biological activity and behavior in soils, enantiopure (−)-beflubutamid definitively has the potential to substitute for the racemic herbicide.
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in some cases, a correlation was found with soil pH.11−13,19 Besides actual degradation, the enantiomers of chiral compounds may also be converted to their antipodes. For example, fast and preferential enantiomerization from the S- to the herbicidally active R-enantiomer has been observed for fluazifop,20 diclofop,21 and haloxyfop.22 Enantiomerization has also been reported for the herbicides dichlorprop and mecoprop.7,8 The enantiomer composition of residues of chiral compounds in soils is thus driven by enantioselective degradation and enantiomerization. Understanding these processes is relevant for pesticides that are absorbed through roots, in particular soil herbicides, and for compounds, where one enantiomer is clearly more active than the other. Previous studies on the degradation of beflubutamid in soils were performed with the racemic compound. Under laboratory conditions, half-lives of 5−100 days were determined.23 Two metabolites were identified, a primary amide (in the following denoted as bfl-amide) and the corresponding acid (bfl-acid, Figure 1). They were detected in molar amounts up to 7% and 26% of the applied active substance, respectively.24 Half-lives
INTRODUCTION Beflubutamid is a recently introduced soil herbicide for pre- and early postemergence control of dicotyledonous weeds in cereals.1 The compound, a fluorinated phenoxybutanamide (Figure 1), was developed in the nineties by Ube Industries. It inhibits the enzyme phytoene-desaturase, which is involved in the biosynthesis of carotenoids. Depletion of carotenoids results in photooxidation of chlorophyll and thus bleaching/ chlorosis of susceptible weeds.1 Beflubutamid is chiral due to an asymmetrically substituted C atom and consists of a pair of enantiomers (Figure 1). It is currently applied as the racemic mixture, but only one enantiomer, (−)-beflubutamid, is herbicidally active.2 Generally, enantiomers of chiral pesticides may differ with respect to biological activity, plant metabolism, environmental fate, and (eco)toxicity.3−5 Therefore, a potential substitution of racemic by enantiomer enriched compounds should always be considered as it may lower application rates at similar field performance, may result in lower residues in crops and environment, and may lead to less side effects on nontarget organisms.4 For many chiral pesticides, degradation in soil is enantio-/ stereoselective, for example, for dichlorprop and mecoprop,6−10 metalaxyl and benalaxyl,11,12 cyproconazole, epoxiconazole, triadimefon, triadimenol, and fenbuconazole,13−16 bifenthrin, cyfluthrin, cypermethrin, and permethrin.17,18 The degree of the observed enantioselectiviy often differs from soil to soil and, © XXXX American Chemical Society
Special Issue: Rene Schwarzenbach Tribute Received: May 11, 2012 Revised: September 5, 2012 Accepted: September 25, 2012
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dx.doi.org/10.1021/es301877n | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 1. Degradation pathway of the herbicide beflubutamid in aerobic soils (adapted from ref 24). The asterisks indicate the asymmetrically substituted C atom of the chiral compounds.
from Stähler, Stade, Germany, and 2,6-dichlorobenzamide (99.9%) was from Sigma-Aldrich, Steinheim, Germany. Pure enantiomers of beflubutamid, bfl-amide, and bfl-acid were prepared from the racemates by enantioselective, semipreparative HPLC as described in ref 2. Soils. Soil samples were collected in February and April 2011. Soil Dübendorf, an alkaline sandy clay loam, was from arable land and soil Steig, an acidic loam, from a forest. Further soil parameters are listed in Table 1. Standard equipment was used for sampling soil from the top 10 cm. The soils were kept in the dark at ≈4 °C. Incubation experiments were set up a few days after soil sampling.
for bfl-amide were not determined, those for bfl-acid were 1− 18 days.23 Both metabolites contain the asymmetrically substituted C atom and are thus chiral (Figure 1). Chiral aspects, however, were hardly investigated. In a few samples from soil incubation studies, the enantiomer composition of beflubutamid and bfl-acid was determined,24 but a kinetic analysis was not undertaken. In this study, the enantioselectivity of degradation of beflubutamid and the formation and degradation of the two metabolites was investigated with laboratory incubation experiments. Two soils were selected, an alkaline sandy clay loam and an acidic loam. A total of 18 experiments were carried out with racemic beflubutamid, bfl-amide, and bfl-acid, and also with their pure enantiomers to study possible enantiomerization of the chiral compounds. Pure enantiomers, prepared from racemic standards by enantioselective, semipreparative HPLC, were available from an accompanying study.2 After extraction from soil, the compounds were analyzed with GC-MS using two different cyclodextrin-based chiral columns (bfl-acid was methylated with diazomethane2). Kinetic parameters for degradation and isomerization of individual enantiomers were derived by kinetic modeling. As parent beflubutamid and metabolites, both as racemates and single enantiomers, were incubated in the same soils, it was possible to obtain a comprehensive insight into the behavior of individual enantiomers in soil, that would not have been accessible by incubation experiments with the (racemic) parent compound alone. The results of this study will be relevant for the assessment of a possible substitution of the racemate by (−)-beflubutamid.
Table 1. Sampling Sites and Characterization of the Soils Used for Incubation Experiments coordinates land use texture, USDA classification Corg [%] pH (CaCl2)a moisture [%]b
soil Dübendorf
soil Steig
47°23′34″N/ 8°35′59″E arable land sandy clay loam
47°32′10″N/ 8°37′01″E forest loam
1.7 7.4 14−18/24−31
4.9 3.7 48−49/61−62
a
Suspension of soil in 0.01 M CaCl2, 1:2.5 (w/w). bIn g water per 100 g dry soil/in % of the maximum water holding capacity.
Incubation. Aerobic incubation experiments were carried out in Erlenmeyer flasks closed with air-permeable cellulose plugs. Portions of 2 mm sieved, field-moist soil (200 g) were filled into the flasks and fortified by addition of 10 mL of a solution containing 200 μg of the respective enantiomer in water/methanol (98:2) using a spray bottle. The spike levels were 1 μg/g moist soil, and were thus slightly higher than expected concentrations resulting from typical application rates of beflubutamid of up to 250 g/ha (0.33 μg/g, assuming a uniform incorporation in the top 5 cm soil layer and a bulk density of 1.5 g/cm3). Nine separate incubations were carried out per soil, with racemic beflubutamid, bfl-amide, and bfl-acid as well as the pure (+)- and (−)-enantiomers. Incubation
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EXPERIMENTAL SECTION Chemicals. Beflubutamid (2-[4-fluoro-3-(trifluoromethyl)phenoxy]-N-(phenylmethyl)butanamide, purity, 99.5%), flutolanil (N-[3-(1-methylethoxy)phenyl]-2-(trifluoromethyl)benzamide, 99.5%), and cloprop (2-(3-chlorophenoxy)propanoic acid, 99.0%) were from Ehrenstorfer, Augsburg, Germany, bfl-amide (2-[4-fluoro-3-(trifluoromethyl)phenoxy]butanamide, purity unknown) and bfl-acid (2-[4-fluoro-3(trifluoromethyl)phenoxy]butanoic acid, 99.0%) were obtained B
dx.doi.org/10.1021/es301877n | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
experiments with sterilized soil were not performed, since in earlier studies it was shown that degradation of beflubutamid is biologically mediated24,25 and because degradation was enantioselective (see results). After spiking, the soils were thoroughly mixed and incubated at 20 °C in the dark for up to 22 days. The soil moisture (14− 49 g water per 100 g dry soil, corresponding to 24−62% of the maximum water holding capacity, Table 1) was maintained by regular addition of distilled water. At appropriate time intervals, aliquots of 10 g soil were removed for extraction and analysis. Extraction. Soil samples were mixed with 10 mL of methanol, and internal standards (a mixture of 10 μg flutolanil, 2,6-dichlorobenzamide, and cloprop in 100 μL methanol) were added. Internal standards were selected based on their structural similarities to the analytes (beflubutamid, bfl-amide, and bfl-acid, respectively, Figure 1 in ref 2). After vigorous shaking (≈1 min), the samples were centrifuged (≈1500g for 5−10 min) and the supernatants were transferred to 40 mL glass vials. This procedure was repeated with 10 mL of acetone/distilled water (1:1) and 10 mL of distilled water to improve extraction of bfl-acid. The combined extracts (methanol/acetone/water) were acidified with H2SO4 to pH ≈2 and partitioned three times with 5 mL portions of dichloromethane. The combined dichloromethane phases were evaporated at room temperature with a gentle draft of air, and the residues were redissolved in 5 mL of dichloromethane. GC-MS Analysis. Aliquots of 0.5 mL of these extracts were transferred into 2 mL glass vials and carefully evaporated to dryness. The residues were then dissolved in ≈50 μL of methanol, and bfl-acid and cloprop were derivatized using diazomethane in diethyl ether.26,27 After derivatization and evaporation of the solvents, the residues were dissolved in 0.2− 0.5 mL of ethyl acetate. The compounds were analyzed by gas chromatography− mass spectrometry (GC-MS) using two chiral columns according to methods described in ref 2. Representative chromatograms of soil extracts are shown in Figure 3 of ref 2. Recoveries of beflubutamid, bfl-amide, and bfl-acid from soils Dübendorf and Steig, determined immediately after fortification, were 91−99% and 73−87%, respectively. Concentrations in soil Steig were corrected for recoveries. Limits of detection (signal-to-noise ratio, 3) were ≈ 0.01, 0.005, and 0.005 μg/g for beflubutamid, bfl-amide, and bfl-acid, respectively. In blank soils (and soils immediately after spiking), none of the (other) compounds could be detected, except for soils spiked with bflamide, where traces of bfl-acid (
