Article pubs.acs.org/JAFC
Comparative Study of the Selective Degradations of Two Enantiomers in the Racemate and an Enriched Concentration of Indoxacarb in Soils Yu-Ping Zhang, De-Yu Hu, Hu-Rong Ling, Lei Zhong, An-Xiang Huang, Kan-Kan Zhang, and Bao-An Song* State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, PR China S Supporting Information *
ABSTRACT: In this study, selective degradations of the two enantiomers of indoxacarb in the concentrate (2.33S/1R) and racemate (1S/1R) are examined. The absolute configurations of indoxacarb enantiomers were determined using X-ray diffraction. The results showed that in two alkaline soils, the S-(+)-indoxacarb was preferentially degraded in both the concentrate and racemate. In one acid soil, the two enantiomers degraded no-selectivity. In another acid soil and one neutral soil, the R(−)-indoxacarb was preferentially degraded in both the concentrate and racemate. Indoxacarb enantiomers were configurationally stable in the five soils, and no interconversion was observed during the incubation. Because no significant difference in degradation was observed after samples were sterilized, the observed enantioselectivity may be attributed primarily to microbial activity in soils. The results indicate that the selective degradation behavior was the same for both formulations that were tested. KEYWORDS: indoxacarb, enantiomers, absolute configuration, enantioselective degradation, soil
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INTRODUCTION Chiral pesticides account for more than a quarter of currently used pesticides, and the proportion of chiral pesticides is increasing.1,2 The pesticidal activity of a chiral pesticide is usually due to one or several of its stereoisomers. To lessen the application and thus reduce the amounts of chemicals released into the environment, significant efforts have been given to replace pesticide racemates (i.e., mixtures of enantio-/stereoisomers) by single biologically active stereoisomers or mixtures enriched in the latter. Indoxacarb is a commercially available chiral oxadiazine insecticide enriched in the (S)-enantiomer. Indoxacarb, (S)-methyl-7-chloro-2,5-dihydro-2[[(methoxycarbonyl)[4-(trifluoromethoxy)phenyl]amino]carbonyl]indeno[1,2-e][1,3,4]-oxadiazine-4a(3H)-carboxylate (Figure 1). It was introduced to the Chinese market 10 years ago by DuPont. Indoxacarb has a chiral carbon and consists of two enantiomers; the activity of this insecticide is mainly attributed to S-(+)-indoxacarb.3 Three major indoxacarb products of DuPont, namely, DPX-JW062, DPX-MP062, and DPX-KN128, are composed of mixtures of R-(−)-indoxacarb and S-(+)-indoxacarb at ratios of 1:1, 1:3, and 0:1, respectively.
Indoxacarbs are intensively used to control Lepidoptera insects in vegetable, tea, cotton, and rice in China. The main indoxacarb products in the Chinese market are mixtures of the two enantiomers enriched with the S-enantiomer (S/R = 2.2:1 to 3:1); therefore, both enantiomers are dissipated in the environment after application. The enantiomeric specific products of chiral contaminants have become important topics at the forefront of chemistry and toxicology research because the enantiomers of many pesticides show different toxicities, biological activities, and environmental fates.4−13 The degradation of indoxacarb in soils14−20 and in terrestrial plants21−23 has been reported. Today, enriched formulations of several companies and one enantiopure formulation of DuPont are sold in the Chinese markets; there is no racemate formulations in the Chinese markets. Sun et al.20 and Li et al.16 have only investigated the selective degradations of the enantiomers of indoxacarb racemate in soils. The indoxacarb is released into the environment in main forms enriched in the Senantiomer; therefore, it is important to study the selective degradations of enriched formulations and not just the racemate. Recently, Zhang et al. reported24 no significant enantioselectivity in the degradation of racemate-dufulin in the soil types studied; however, enantiopure S-(+)-dufulin degraded much faster than its antipode R-(−)-dufulin when applied to these same soils. No studies have reported whether the enantioselective degradation will be affected by the Received: Revised: Accepted: Published:
Figure 1. Chemical structures of indoxacarb enantiomers. © 2014 American Chemical Society
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Table 1. Sampling Sites and Characterization of the Soils Studied particle size no.
soil sitea
sand
silt
clay
soil texture
pHb
Corg(%)c
biomass C (mg/kg)d
alk-1 alk-2 neut-3 ac-4 ac-5
Haozhou Guiyang Jilin Hangzhou Haikou
39.7 16.8 25.5 10.8 58.1
50.3 72.5 68.4 80.1 32.7
10.0 10.7 6.1 9.1 9.2
silt loam silt loam silt loam silt sandy loam
8.29 7.73 7.22 5.65 5.01
1.68 3.49 2.12 5.56 0.26
213 218 178 236 26.4
a The cities in China. bSuspension of soil in water, 1:2.5 (w/w). cFollowing the potassium dichromate volumetric method. dFollowing the fumigation−extraction method.
characteristics (particle size, texture, pH, and organic carbon) are presented in Table1. Incubation of Indoxacarb Concentrate (2.33S/1R) and Racemate (1S/1R) in Soils. Separate incubation experiments with indoxacarb concentrate and with racemate compounds in five soils were conducted in 50 mL of polypropylene centrifuge tubes covered with aluminum foil. Before pesticide treatment, the soils were preincubated in the dark at 25 ± 2 °C for 7 days to activate the soil microorganisms. The soil sample (5.0 g, dry weight equivalent) was treated with 50 μg of indoxacarb (50 μL 1000 μg/mL stock solution in acetone) and then air-dried for 30 min before homogenization. The soil samples were then rehydrated by adding 1.2 mL of purified water [approximately 60% of the field holding capacity (w/w)] and incubated at 25 °C in the dark. The samples were weighed regularly, and purified water was added to the samples to compensate for water loss during the entire experiment. Triplicate samples were removed from each treatment at different time intervals (0, 1, 3, 5, 7, 10, 14, 21, 33, and 51 d) and immediately transferred into a freezer (−20 °C). Incubation of Enantiopure R-(−)- and S-(+)-Indoxacarb. Separation of the two pure indoxacarb enantiomers is necessary to understand the differences in their dissipation behaviors and detect potential enantiomerization of indoxacarb. Separate incubations were conducted with the enantiopure R-(−)- and S-(+)-indoxacarb in five soils. The tested soils were fortified with the pure enantiomers at the 10 μg/g level and incubated in the same way as the other soil experiments. Incubation of Indoxacarb Racemate (1S/1R) or Concentrate (2.33S/1R) in Soils under Sterilized Conditions. To determine whether the enantioselective degradation was a result of microbially mediated transformations, portions of the 5.0 g soil set in 50 mL polypropylene centrifuge tubes were sterilized with the use of an autoclave (twice) at 121 °C for 40 min with 24 h intervals to eliminate microbial activity. In a biologically clean workbench, the sterilized samples were treated with 50 μg of indoxacarb racemate or concentrate (50 μL of a 1000 μg/mL stock solution in acetone). After eliminating the acetone by air-drying for 20 min, 1.2 mL of sterile water was added to the samples. The samples were then sealed with a stopper and membrane to maintain sterile conditions. The samples were incubated at 25 °C in the dark. Three or two replicate samples were removed from each treatment at different time intervals (0, 5, 10, 20, 40, 60, and 93 d) and immediately transferred to a freezer (−20 °C). Extraction of Soil Samples. Three replicate samples were removed from each treatment at different time intervals after pesticide addition and immediately transferred into a freezer (−20 °C) to stop dissipation. For extraction, 25 mL of methanol/water (6:4, v/v) was added into a 50 mL polypropylene centrifuge tube containing 5 g (dry weight basis) of incubated soil sample. The sample was ultrasonically extracted for 20 min and centrifuged at 6000 rpm for 5 min. The extract was transferred to a 150 mL separatory funnel. The extraction was repeated by adding 15 mL of methanol/water (6:4, v/v) to the 50 mL polypropylene centrifuge tube. The tube was then stirred for 30 s on a vortex mixer and centrifuged at 6000 rpm for 5 min. The extracts were combined in the 150 mL separatory funnel and extracted two times using 40 mL of dichloromethane. Afterward, the organic phase (dichloromethane) was collected, concentrated to dryness, and reconstituted with 1.0 mL of methanol. Each sample was filtered
enantiomeric proportion of various enantiomeric compositions thus far. Moreover, microorganisms in soils are considered as the main cause of enantioselective processes,25−28 and enantiomeric degradation constantly changes due to soils with different microbial communities. Our study systematically investigated the selective degradations of two enantiomers in indoxacarb concentrate (2.33S/1R) and in its racemate (1S/ 1R) in five soils to determine the differences between the racemate and concentrate with respect to enantiomer degradation (In this article, mixtures of the two enantiomers enriched with the S-enantiomer (S/R = 2.33:1) are called concentrate). We also investigated the degradation of pure Rindoxacarb and pure S-indoxacarb applied to five soils. The steric configuration of S-(+)-indoxacarb and R-(−)-indoxacarb was first confirmed here by X-ray diffraction. The results provide new aspects of the enantioselective behavior of indoxacarb, which should be considered to achieve better understanding of the profiles and fate of chiral pesticides in the environment.
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MATERIALS AND METHODS
Chemicals and Reagents. The analytical standard of racemic indoxacarb was purchased from Dr. Ehrenstorfer GmbH. Indoxacarb powder enriched with the S-enantiomer (2.33S/1R, 97.3% purity) was provided by Yancheng Limin Agrochemical Co., Ltd. (Jiangsu, China). The two indoxacarb enantiomers were prepared by semipreparative high-performance liquid chromatography (HPLC) in our laboratory.29 HPLC-grade n-hexane and isopropanol (IPA) were purchased from Jiangsu Hanbang Science and Technology Co., Ltd. (Jiangsu, China). All other chemicals and solvents used were of analytical grade and purchased from commercial sources. Single-Crystal X-ray Investigation of Enantiopure S-Indoxacarb and Enantiopure R-Indoxacarb. The stereochemical configuration was determined using X-ray diffraction. Diffraction patterns were obtained at 293 K with a Bruker SMART II diffractometer using Mo-Kα radiation (λ = 0.71073 Å) under φ−ω scan. Empirical absorption correction (SADABS) was applied to raw intensities. The structures were determined by direct methods (SHELXS-97) and refined by full matrix least-squares procedures on F2 in the anisotropic approximation for all of the atoms except hydrogen. The absolute configurations were determined by anomalous dispersion effects and corresponding calculations of Flack’s absolute structure parameters.30 Soil Samples. Five soil samples representing different physicochemical properties and climatic environments were collected at 0 to 10 cm plow layer from geographically distinct regions of China (Haozhou city, Anhui Province, Northern China; Guiyang City, Guizhou Province, Southwest China; Jilin city, Jilin Province, Northern China; Hangzhou city, Zhejiang Province, Southeastern China; and Haikou City, Hainan Province, Southern China). No indoxacarb was found at detectable levels in soils. After collection, the soil samples were air-dried at room temperature, homogenized, passed through a 2 mm sieve, and then stored in the dark until use within a few days. Details about soil sites and specific physicochemical 9067
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Figure 2. Stereoview of the indoxacarb enantiomers (C22H17ClF3N3O7, M.W. = 527.83) (a) R-indoxacarb and (b) S-indoxacarb. through a 0.45 μm nylon membrane filter and analyzed by HPLC (20 μL final extract). Instrumental Conditions (Enantioselective HPLC Analysis of Indoxacarb). HPLC analysis of racemic indoxacarb was performed using an Agilent 1200 series apparatus composed of a quaternary pump, an autosampler, a diode array detector, a vacuum degasser, a column oven, and Agilent Chemstation software. The chiral analytical column amylose tris(3,5-dimethylphenyl carbamate) immobilized on silica-gel (Chiralpak IA) was purchased from Daicel (Tokyo, Japan). The dimensions of the column were 250 mm × 4.6 mm i.d., and it was packed with 5 μm particles. The samples were analyzed using a mixture of n-hexane/ isopropanol (70:30 v/v) at 1.0 mL/min flow rate on a Chiralpak IA column. The UV detection wavelength was 310 nm, and the temperature for separation was 30 °C. Under the selected conditions, the R-(−)-enantiomer eluted before the S-(+)-enantiomer. Method Validation. A series of rac-indoxacarb standard solutions (0.25, 2.5, 5, 50, and 100 μg/mL) was prepared for HPLC analysis to determine the linearity of the method and quantify the analyte. The obtained regression equations and respective correlation coefficients were A = 44.3880C − 1.8603 (R2 = 0.9999) for the R-(−)-enantiomer and A = 43.325x + 2.9917 (R2 = 0.9999) for the S-(+)-enantiomer. A series of blank samples fortified with rac-indoxacarb at 0.1, 1.0, and 10 mg/kg was prepared for method validation and analyzed by the described procedure. The recovery and precision data for the samples in five soils are summarized in Table S2 (Supporting Information). For indoxacarb enantiomers, the recovery ranges in low, intermediate, and high spiked levels were 86.1−98.3, 86.3−99.2, and 95.5−103%, respectively, and each recovery value was acceptable for enantiomer determination. The limit of quantitation (LOQ) for each indoxacarb enantiomer was 0.05 μg/g in soil samples based on the acceptable RSDs of 5.14% to 10.2% for the two enantiomers. The limit of detection (LOD), defined as the concentration that produces a signalto-noise (S/N) ratio of 3, was estimated to be 0.01 μg/g for each enantiomer by analyses of spiked samples at low concentration levels. Kinetic Analysis and Calculation. The kinetic study of indoxacarb in soil was performed by plotting the residue concentration against time, and the corresponding rate constants k for the S(+)-enantiomer and R-(−)enantiomer were calculated using the firstorder kinetic eq 1 by regression analysis; the half-life (t1/2) was determined using eq 2.
Ct = C0e−kt
(1)
t1/2 = ln 2/k
(2)
where C0 is the initial concentration (μg/g) of the enantiomer, Ct is its concentration (μg/g) at time t (d), and k is the degradation rate constant. The enantiomer fraction (EF) was used to express enantioselectivity as defined by the equation EF = peak area of the (+)-enantiomer/ [(+)-enantiomer + (−)-enantiomer]. EF values ranged from 0 to 1, with an EF value of 0.50 indicating a racemic mixture and EF value of 0.70 indicating indoxacarb enriched with the S-enantiomer (2.33S:1R). Enantioselectivity (ES), which reflects the overall trend in enantioselective dissipation, was defined as ES = (kS − kR)/(kS + kR) in a previous study,31 where kS is the degradation rate constant of S-indoxacarb, and kR is the degradation rate constant of R-indoxacarb. Positive values (0 < ES ≤1) indicate a more rapid degradation of the S-enantiomer, whereas negative values (−1 ≤ ES 99.0% were obtained by enantioselective separation of the indoxacarb racemate using semipreparative HPLC.29 Single crystals of enantiopure Sindoxacarb and enantiopure R-indoxacarb were obtained by recrystallization from dimethyl sulfoxide and ethanol mixture at room temperature. The molecular structures of R-indoxacarb and S-indoxacarb are illustrated in Figure 2, and the crystallographic data are summarized in Table S1, Supporting Information. Both compounds crystallize in the monoclinic crystal system in space group C2, and absolute structure parameters are −0.09 (13) and 0.10 (15), respectively. On the basis of these results, the first eluted enantiomer on the Chiralpak IA column was confirmed as the R-enantiomer followed by S-indoxacarb. The complete set of experimental and computational data was deposited in the Cambridge Crystallographic Data Center (CCDC# 911685 for S-indoxacarb and CCDC# 921088 for Rindoxacarb). Melting points were measured on a Tech X-4 melting point apparatus, and optical rotations were measured on a WZZ-ZS automatic polarimeter, and they were not corrected. The isolated enantiomers were characterized as follows: Rindoxacarb, [α] 20 D = −68.4 (menthol) and mp 86 to 89 °C; 9068
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Table 2. Kinetic Data for Dissipation of the Two Enantiomers of Indoxacarb Concentrate (2.33S/1R) in Soils soil no.
a
soil origins
alk-1
Haozhou
alk-2
Guiyang
neut-3
Jilin
ac-4
Hangzhou
ac-5
Haikou
enantiomer R-(−) S-(+) R-(−) S-(+) R-(−) S-(+) R-(−) S-(+) R-(−) S-(+)
regressive function C C C C C C C C C C
= = = = = = = = = =
−0.0687t
2.8216 e 6.5825 e−0.0829 t 2.8029 e−0.0696 t 6.7152 e−0.1148 t 2.9329 e−0.0621t 6.1897 e−0.0289 t 3.0604 e−0.0928 t 7.2715 e−0.0926 t 2.7473 e−0.0378 t 7.0256e−0.0201 t
R2
t1/2 (d)
0.9840 0.9803 0.9923 0.9931 0.9880 0.9845 0.9731 0.9691 0.9719 0.9861
10.1 8.36 9.96 6.04 11.2 24.0 7.47 7.49 18.3 34.5
ESa 0.095 0.245 −0.365 −0.001 −0.306
Enantioselectivity.
Table 3. Kinetic Data for Dissipation of the Two Enantiomers of Indoxacarb (1S/1R) in Soils soil no.
a
soil origins
alk-1
Haozhou
alk-2
Guiyang
neut-3
Jilin
ac-4
Hangzhou
ac-5
Haikou
enantiomer R-(−) S-(+) R-(−) S-(+) R-(−) S-(+) R-(−) S-(+) R-(−) S-(+)
regressive function C C C C C C C C C C
= = = = = = = = = =
−0.0630 t
4.9338 e 5.0758 e−0.0803t 4.8370 e−0.0677 t 4.6104 e−0.1030t 5.1290 e−0.0526 t 4.9289 e−0.0315t 5.3632e−0.0883t 5.4033 e−0.0874 t 4.7060 e−0.0308t 5.2248 e−0.0196t
R2
t1/2 (d)
0.9604 0.9658 0.9958 0.9876 0.9779 0.9855 0.9696 0.9706 0.8796 0.9292
11.0 8.63 10.2 6.72 13.2 22.0 7.85 7.93 22.5 35.4
ESa 0.121 0.207 −0.251 −0.005 −0.222
Enantioselectivity.
and S-indoxacarb, [α] 20 D = +69.4 (menthol) and mp 88 to 90 °C. Degradation of Indoxacarb Concentrate (2.33S/1R) in Soil. The degradation of the two enantiomers in the indoxacarb concentrate (2.33S/1R) followed first-order kinetics (Table 2 and Figure S1, Supporting Information), with R2 values of 0.9691 to 0.9931, which indicated acceptable linearity. Halflives (t1/2) in the two alkaline soils (alk-1 and alk-2) were about 10.1 and 9.96 days for R-(−)-indoxacarb and 8.36 and 6.04 days for S-(+)-indoxacarb. This finding suggests that the two enantiomers degraded selectively in weakly alkaline soil and that the (S)-(+)-indoxacarb was preferentially degraded. The corresponding ES values were 0.095 and 0.245. The half-lives (t1/2) of the two enantiomers were 11.2 and 24.0 d in neutral soil (neut-3). In soil neut-3, the degradation rates varied by a factor of 2, and the ES value of −0.365 indicates that (R)(−)-indoxacarb was preferentially degraded. Furthermore, the two enantiomers display different degradation trends in the two acid soils (ac-4 and ac-5). In soil ac-4, the half-lives (t1/2) of the two enantiomers were almost equal at 7.47 and 7.49 d. The ES value was −0.001; thus, the degradations of the two enantiomers was not selective. In soil ac-5, the half-lives (t1/2) of the two enantiomers were 18.3 and 34.5 d, with an ES value of −0.306. This result indicates that (R)-(−)-indoxacarb was preferentially degraded. Degradation of Indoxacarb Racemate (1S/1R) in Soil. The degradation of the two enantiomers in the indoxacarb racemate (1S/1R) followed first-order kinetics (Table 3 and Figure S2, Supporting Information), with R2 values of 0.8796 to 0.9958. In addition to a half-life of 35.4 d of S-(+)-indoxacarb in soil ac-5, the other half-lives were similar to the results studied by Li et al.16 and Sun et al.,20 which were less than 23 d. Table 3 presents the statistically significant differences in the degrada-
tion half-life of the two enantiomers in the two weakly alkaline soils (alk-1 and alk-2) at 2.37 and 3.48 d, respectively. The corresponding ES values were 0.121 and 0.207, thus suggesting that the two enantiomers degraded selectively in weakly alkaline soil and that the (S)-(+)-indoxacarb was preferentially degraded. It is also consistent with the findings with the concentrate. In the neutral soil (neut-3), the half-lives (t1/2) of the two enantiomers were 13.2 and 22.0 d. In soil neut-3, the ES value was −0.251, and (R)-(−)-indoxacarb was preferentially degraded. The degradation trends of the two enantiomers differed in acid soils ac-4 and ac-5. In soil ac-4, the half-lives (t1/2) of two enantiomers were almost equal at 7.93 and 7.85 d, and the ES value was −0.005. Thus, the degradations of the two enantiomers were not selective. In soil ac-5, the half-lives (t1/2) of two enantiomers were 22.5 and 35.4 d. The corresponding ES value was −0.222, thereby indicating that the (R)(−)-indoxacarb was preferentially degraded. Only one alkaline soil and one acid soil were selected for enantioselective degradation of indoxacarb racemate by Sun et al.20 Our result was consistent with the result reported by Sun et al. that S(+)-indoxacarb degraded faster than R-(−)-indoxacarb in alkaline soil. In one of the acid soils (ac-5), our result was consistent with the result reported by Sun et al. that R(−)-indoxacarb degraded faster than S-(+)-indoxacarb in acid soil. In the other acid soil (ac-4) in our study, the two enantiomers were nonselective. Comparison of Racemate (1S/1R) and Concentrate (2.33S/1R) in Terms of Selective Degradation. With the indoxacarb racemate, S-(+)-indoxacarb was preferentially degraded in the two alkaline soils (alk-1 and alk-2). Thus, the amount of S-(+)-indoxacarb residue was lower than that of R(−)-indoxacarb residue in the soil. As a result, the soil was enriched in R-(−)-indoxacarb. With the indoxacarb concen9069
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Figure 3. (a-1) Concentration−time curves and (a-2) chromatograms of indoxacarb concentrate (2.33S/1R) in soil alk-2 after 21d. (b-1) Concentration−time curves and (b-2) chromatograms of indoxacarb concentrate (2.33S/1R) in soil ac-5 after 51d. (c-1) Concentration−time curves and (c-2) chromatograms of indoxacarb racemate (1S/1R) in soil ac-4 after 21d.
Table 4. Kinetic Data for Dissipation of Pure S-Indoxacarb and Pure R-Indoxacarb in Soils soil no.
soil origins
alk-1
Haozhou
alk-2
Guiyang
neut-3
Jilin
ac-4
Hangzhou
ac-5
Haikou
enantiomer pure pure pure pure pure pure pure pure pure pure
regressive function C C C C C C C C C C
R-(−) S-(+) R-(−) S-(+) R-(−) S-(+) R-(−) S-(+) R-(−) S-(+)
= = = = = = = = = =
−0.0742t
8.6953 e 8.5341 e−0.0846t 7.6505 e−0.0806t 7.819e−0.1668t 8.7672 e−0.0853t 9.000e−0.0287t 9.4875e−0.0929t 9.5389e−0.0815t 9.8631e−0.0386t 10.4155e−0.0171t
R2
t1/2 (d)
0.9870 0.9930 0.9916 0.9859 0.9380 0.9480 0.9732 0.9664 0.9970 0.9836
9.34 8.19 8.60 4.16 8.13 24.2 7.46 8.50 18.0 40.5
−0.222 in the racemate, respectively, which showed the selective degradations of enantiomers varied more in the concentrate than in the racemate. Overall, the results indicate that the directions of selective degradation of the enantiomers in the concentrate are analogous to directions in the racemate for all five soil types. Degradation of Enantiopure Indoxacarb in Soil. The determination of samples of separate incubations with enantiopure R-(−)- and S-(+)-indoxacarb is necessary to understand the differences in their dissipation behaviors and detect potential enantiomerization of the compound. We therefore conducted separate incubations with enantiopure R(−)- and S-(+)-indoxacarb in all soils. As exhibited in Table 4, the half-lives (t1/2) of pure R-(−)-indoxacarb and pure S(+)-indoxacarb were 9.34 and 8.19 d in soil alk-1, and 8.60 and 4.16 d in soil alk-2, which was similar to the directions of enantioselective degradation in the concentrate and the racemate, namely, S-(+)-indoxacarb degraded faster than R(−)-indoxacarb. The half-lives (t1/2) of pure R-(−)-indoxacarb and pure S-(+)-indoxacarb were 8.13 and 24.2 d in soil neut-3, and 18.0 and 40.5 d in soil ac-5, respectively. The degradation rate of the pure R-(−)-indoxacarb was higher than that of pure S-(+)-indoxacarb, as with the directions of enantioselective degradation in the concentrate and the racemate in soils neut-3 and ac-5.
trate, the S-(+)-indoxacarb was also preferentially degraded in the two alkaline soils. Therefore, the difference between the R(−)- and S-(+)-residues in soil was reduced. As depicted in Figure 3a, the amount of S-(+)-indoxacarb was 2.33 times that of the R-(−)-indoxacarb at 0 d when the indoxacarb concentrate was added into soil alk-2. At 21 d, however, the amount of S-(+)-indoxacarb was only 0.90 times that of the R(−)-indoxacarb. In soils neut-3 and ac-5, the directions of selective degradation in indoxacarb racemate and concentrate were the same. In these soils, the R-(−) indoxacarb was preferentially degraded in both racemate and concentrate enantiomers. Consequently, the soil was enriched in S(+)-indoxacarb and S-(+)-residue was significantly more abundant than R-(−) residue, as shown in Figure 3b. In soil ac-4, the degradations of the two enantiomers in both racemate and concentrate were not selective, and the degradation rates of S-(+) and R-(−) were similar, as presented in Figure 3c. The ES values of the racemate and the concentrate varied greatly; these values indicated the degree of selective difference. In soil alk-1, the ES value was 0.095 in the concentrate, and it was 0.121 in the racemate, indicating the selective degradations of enantiomers differ more in the racemate than in the concentrate. In three of the soils (alk-2, neut-3, and ac-5), the ES values were 0.245, −0.365, and −0.306 in the concentrate, respectively, and were 0.207, −0.251, and 9070
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Table 5. Enantiomer Fraction (EF) of Indoxacarb upon Degradation in Sterilized Soil Samples (n = 3 or 2)a time (d) 0 5 10 20 40 60 93 a
EF (soil alk-1) 0.500 0.499 0.498 0.498 0.498 0.494 0.483
± ± ± ± ± ± ±
0.001 0.001 0.001 0.003 0.004 0.002 0.013
EF (soil alk-2) 0.500 0.500 0.500 0.497 0.496 0.494 0.494
± ± ± ± ± ± ±
EF (neut-3)
0.001 0.001 0.001 0.001 0.001 0.001 0.008
0.498 0.498 0.498 0.497 0.506 0.492 0.495
± ± ± ± ± ± ±
EF (soil ac-4)
0.002 0.002 0.002 0.003 0.014 0.004 0.001
0.500 0.496 0.499 0.499 0.499 0.496 0.499
± ± ± ± ± ± ±
0.001 0.005 0.001 0.001 0.001 0.001 0.001
EF (soil ac-5) 0.704 0.707 0.708 0.705 0.696 0.696 0.708
± ± ± ± ± ± ±
0.003 0.001 0.004 0.012 0.001 0.002 0.001
Values represent the mean ± SD (n = 3 or 2).
different microorganisms affected by the collective soil environment. In conclusion, we investigated the enantioselective degradation of the indoxacarb concentrate (2.33S/1R) and racemate (1S/1R) in five types of soil using a chiral Chiralpak IA column under normal-phase conditions. We initially determined the absolute configuration of indoxacarb enantiomers based on single-crystal X-ray diffraction. We found that the directions of selective degradation of the enantiomers in the concentrate are analogous to directions in the racemate for all five soil types. Even in the five soils, the degradation of enantiopure R-(−)and S-(+)-indoxacarb was similar to the directions of enantioselective degradation in the concentrate and racemate. As our study shows, enantioselective degradation and soil properties such as pH level, organic substances, and soil texture were not linearly correlated.
These results differed from the degradation study of enantiopure indoxacarb by Sun et al. that showed conversion between R-(−)-indoxacarb and S-(+)-indoxacarb in soils.20 However, no enantiomerization was observed in our study. The current experiment results show that neither (S)-(+)-indoxacarb nor (R)-(−)-indoxacarb convert directly; both enantiomers are configurationally stable in the selected soils. Degradation of Indoxacarb Racemate (or Concentrate) in Soils under Sterile Conditions. No significant difference in enantioselectivity was found for indoxacarb racemate (or concentrate) degradation in five soils under sterile conditions, the degradation rates of S-(+)- and R(−)-indoxacarb were the same. The concentrations of the two enantiomers of indoxacarb in sterile soils were determined. The EF values of indoxacarb in sterile soils were approximately 0.50 (for the racemate) and 0.70 (for the concentrate) before, during, and after incubation (Table 5). In sterilized soils, indoxacarb enantiomers showed slow dissipation rates. The spiked concentrations dissipated 73%, 84%, 18%, 13%, and 24% in soils alk-1, alk-2, neut-3, ac-4, and ac-5, respectively, after 93 d of incubation. The results indicated that the biological process by microorganisms played important roles in the enantioselective degradation of indoxacrb, consistent with the other studies about many chiral chemicals in soils.20,25−28 The correlation between enantioselectivity and microbial biomass carbon was considered. However, we observed no clear linear relationship between the selective degradation of indoxacarb and soil microbial biomass. Incubation conditions in different types of soils from various locations may have affected the makeup of microorganisms and hence influenced the rate and direction of the observed enantioselectivity. Relationship between the Selective Degradation of Indoxacarb and Soil Properties. The selective degradation of indoxacarb concentrate (or racemate) varies across the five selected soils. In the two alkaline soils, with positive ES values, the degradation rate of the (S)-(+)-indoxacarb was faster than that of the (R)-(−)-indoxacarb. In two other soils, one acid and one neutral, with negative ES values, the degradation rate of the (R)-(−)-indoxacarb was faster than that of the (S)-(+)-indoxacarb. In the other acid soil, the degradation rates of enantiomers were similar and are not selective. Hence, its ES value was 0. The activity and types of soil microorganisms were affected by pH level, organic substances in soil, and soil texture. Therefore, the correlation between ES value and pH was considered, along with that between organic substances and the content of sand and clay and silt in soil. However, we observed no clear linear relationship between the selective degradation of indoxacarb and soil properties. Combined with the results of previous research of indoxacarb in soils,16,20 we believed the selectivity may vary with different soils, which contained
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ASSOCIATED CONTENT
S Supporting Information *
Crystallographic data and the recovery and precision data in five soils, and concentration of the indoxacarb concentrate (2.33S/1R), racemate (1S/1R), and enantiopure indoxacarb in five soils versus incubation time. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86 851 362-0521. Fax: +86 851 362-2211. E-mail:
[email protected]. Funding
We thank the National Natural Science Foundation of China (No. 21367007) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201203022) for financial support. Notes
The authors declare no competing financial interest.
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REFERENCES
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