Environmental Behavior of the Chiral Triazole Fungicide

Feb 16, 2012 - fungicide of the triazole class with one chiral center. In the present study, the .... and a garden red soil from Changsha City in Sout...
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Environmental Behavior of the Chiral Triazole Fungicide Fenbuconazole and Its Chiral Metabolites: Enantioselective Transformation and Degradation in Soils Yuanbo Li,† Fengshou Dong,† Xingang Liu, Jun Xu, Jing Li, Zhiqiang Kong, Xiu Chen, and Yongquan Zheng* Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Beijing 100193, People's Republic of China ABSTRACT: Fenbuconazole is a widely used systemic agricultural fungicide of the triazole class with one chiral center. In the present study, the enantioselective degradation of fenbuconazole and its chiral metabolites, RH-9129 and RH-9130, in two soils under aerobic and anaerobic conditions were investigated using a chiral OD-RH column on a reversed-phase liquid chromatography−tandem mass spectrometry system. Under aerobic or anaerobic conditions, the results showed the occurrence of enantioselectivity with (−)-fenbuconazole preferentially degraded in both soils. Further enantioselective analysis of converted products showed that the concentrations of four RH-9129 and RH-9130 stereoisomers were different from each other under both aerobic and anaerobic conditions. The four stereoisomer concentrations followed the order (−)-RH-9129 > (+)-RH-9129 > (−)-RH-9130 > (+)-RH-9130 in Langfang alkaline soil. However, in the case of Changsha acidic soil, different RH-9129 and RH9130 stereoisomer patterns were produced in the order (−)-RH-9129 > (+)-RH-9129 > (+)-RH-9130 > (−)-RH-9130. The (−)-RH-9129 stereoisomer had the highest concentration formed by transformation of fenbuconazole in both soils. The degradation of RH-9129 and RH-9130 in the two soils is also stereoselective under both aerobic and anaerobic conditions, the results indicating that the (+)-RH-9130 enantiomer degraded faster than the (−)-RH-9130 enantiomer and the (+)-RH-9129 enantiomer degraded faster than the (−)-RH-9129 enantiomer. In addition, the (−)-RH-9129 isomer exhibited the slowest degradation rate in both soils. This study provides the first experimental evidence of stereoselective degradation and transformation of fenbuconazole as well as its chiral metabolites in the environment.

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inhibitor of human aromatase activity.3 Previous studies also demonstrated that fenbuconazole is a phenobarbital-type inducer of mouse liver adenomas.4 Such data reflect both the biological and tumor response for fenbuconazole and have been suggested for consideration when evaluating cancer risks.4,5 The two metabolites RH-9129 and RH-9130 are being further assessed for potential groundwater contamination in accordance with the current EU guideline.1 Moreover, fenbuconazole is commonly commercialized worldwide as a racemate product and is released into the environment as a 1:1 mixture of its two enantiomers. Nearly all of the triazole fungicides are chiral, which is an important feature in evaluating their environmental behavior and toxicity. It is well-known that enantiomers from the same compound have identical physicochemical properties and abiotic degradation rates in an achiral environment,6 whereas their individual toxicities, biological activities, effects on

INTRODUCTION Fenbuconazole (Figure 1), (R,S)-4-(4-chlorophenyl)-2-phenyl2-(1H-1,2,4-triazol-1-ylmethyl)butyronitrile, is a 1,2,4-triazole fungicide used for the control of leaf spot, yellow and brown rust, powdery mildew, and net blotch on various agricultural and horticultural crops. It is a systemic foliar fungicide that acts by inhibiting sterol biosynthesis. Fenbuconazole is mainly biodegraded in soil, plants, and animals via oxidation of the carbon next to the chlorinated phenyl ring, which forms the benzylic alcohol intermediate and the subsequent lactone metabolites RH-9129 and RH-9130.1 Fenbuconazole has a single chiral center and consists of two enantiomers. Since there are two unsymmetrical substituted carbon atoms in the lactone metabolite, it exists as four stereoisomers (Figure 1). These exist as two diastereomers, termed RH-9129 and RH-9130, each consisting of a pair of enantiomers. Since fenbuconazole is used as an agricultural fungicide, there is concern regarding potential human exposure, as well as wildlife exposure, from its residues in the environment. This fungicide has been associated with an increase in the incidence of liver adenomas in female mice following long-term dietary exposure.2 In addition, fenbuconazole was identified as a new © 2012 American Chemical Society

Received: Revised: Accepted: Published: 2675

September 21, 2011 January 23, 2012 January 30, 2012 February 16, 2012 dx.doi.org/10.1021/es203320x | Environ. Sci. Technol. 2012, 46, 2675−2683

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Figure 1. Chemical structures of fenbuconazole, RH-9129, and RH-9130 (an asterisk indicates a chiral center).

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EXPERIMENTAL SECTION Chemicals and Soils. Racemic fenbuconazole, RH-9129, and RH-9130 standards were obtained from Rohm and Haas Co., Ltd. (Philadelphia, PA) with purities of 99.9%, 99.8%, and 99.3%, respectively. Solutions of each analyte were analyzed by LC−MS/MS using multiple-reaction monitoring (MRM) (see the section “Entantioselective LC−MS/MS Analysis” below) to check for purity, which nominally matched the given purity. HPLC-grade acetonitrile (ACN) and ammonium acetate were purchased from Sigma-Aldrich (Steinheim, Germany) and Tedia (Fairfield, OH), respectively. Sodium chloride (NaCl), anhydrous magnesium sulfate (MgSO4), and ACN were of analytical grade from Beihua Fine-chemicals Co. (Beijing, People's Republic of China (PRC)). Ultrapure water was obtained from a Milli-Q system (Bedford, MA). Octadecylsilane (C18, 40 μm) sorbents were obtained from Agela Technologies Inc. (Tianjin, PRC). The mobile phase solvents were distilled and filtered through a 0.22 μm pore size filter membrane (Tengda, Tianjin, PRC) before use. Soil sources and collection data are as follows: Two soils representing different physicochemical properties and climatic environments were collected from two distinct sites in China. An agricultural yellow soil from Langfang City in North China and a garden red soil from Changsha City in South China were used in this study. The soils were collected from the 0−15 cm soil profile with a hand auger, transferred into a single container, and mixed thoroughly. The soil samples were then air-dried, passed through a 2 mm sieve, and stored in the dark until use within a few days. Blank determination of the soils prior to fortification revealed no fenbuconazole, RH-9129, and RH-9130 or their enantiomers (detection limit 2.5 μg/kg) present. Physicochemical characteristics of the two soils were as follows: (Langfang soil) pH 8.02; organic matter, 0.97%; clay, 11.45%; sand, 67.31%; silt, 21.24%. (Changsha soil) pH 4.69; organic matter, 2.43%; clay, 44.75%; sand, 16.77%; silt, 38.48%. Soil Incubation under Aerobic Conditions. Incubation experiments were carried out with the racemic compounds using 250 mL Erlenmeyer flasks covered with sterile cotton plugs. To avoid potential effects of solvents on the microbiological activity of the soils, the following fortification methods were adopted.28 A portion of the sediment (20 g) was first spiked with 100 μL of stock solution in acetone containing approximately 1000 μg of fenbuconazole and the resulting mixture stirred for 5 min. The spiked soils were then

nontarget organisms, and environmental biodegradation fates have been shown to differ.6−10 Thus, enantioselectivity plays an important role in the environmental fate and ecological risks of a chiral compound,11 as many environmental processes are enantioselective.10,12 One result is that the enantiomeric composition may be changed by enantioselective degradation over time. In some cases, only one enantiomer is being degraded, while the other enantiomer is being accumulated in the environment. For example, previous studies have shown that enantiomers of α-HCH, mecoprop, metalaxyl, and fipronil behave significantly differently during biodegradation and bioaccumulation in the environment.13−22 Furthermore, many chiral pesticides can be metabolized enantioselectively, and the transformation products from environmental dissipation may also be chiral and exhibit significant enantioselectivity in their chemical and toxicological properties.23−25 To date, however, enantioselectivity has not been studied for the environmental fates of fenbuconazole and its chiral metabolites. It should be kept in mind that the six stereoisomers of fenbuconazole and its two metabolites are independent entities with respect to many of their biological properties. Each isomer may differ in toxicity to a variety of species and may be transformed by microbes at different rates. Thus, information on the stereoselective degradation and environmental behavior of parent fenbuconazole as well as metabolites RH-9129 and RH-9130 will help us improve our understanding of the risk of this chiral fungicide to human and ecological health. In recent years, chiral liquid chromatography−tandem mass spectrometry (LC−MS/MS) has been highly favored for drug metabolism and pharmacokinetic studies of stereoisomers because of its high sensitivity and specificity,26 but few applications of this method in chiral pesticide studies have been conducted.27 In this study, we demonstrate a new robust analytical technique to determine the six stereoisomers of fenbuconazole and its metabolites simultaneously using chiral LC−MS/MS in the electrospray ionization (ESI) positive mode. The primary objective of this study was to determine if stereoselectivity occurred in the degradation and transformation processes of fenbuconazole as well as its metabolites in two soils. In addition, we investigated these processes in aerobic as well as anaerobic soils. Results of this research on stereoselectivity in the degradation and transformation of these stereoisomers may have implications for better environmental and human risk assessment of chiral triazole fungicides. 2676

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UPLC sample manager was used for the separation of analytes. A Chiralcel OD-RH column (Daicel Chemical Industries Ltd., Japan, 150 mm × 4.6 mm i.d., 5 μm particle size) packed with cellulose tris((3,5-dimethylphenyl)carbamate) was used for the enantiomer separation of fenbuconazole and its metabolites. A simultaneous separation of these compounds was carried out isocratically using solvent A (HPLC-grade ACN) and solvent B (2 mM ammonium acetate in ultrapure water) in a 60:40 (v/v) ratio and a 0.5 mL/min flow rate for 30 min. The sample injection volume was 10 μL. The column was kept at 25 °C, and the temperature in the sample manager was kept at 4 °C. A triple-quadrupole dtection (TQD) mass spectrometer (Waters Corp., Milford, Massachusetts) equipped with an ESI source was used to quantify fenbuconazole and its metabolites. The analyses were performed in the positive mode with a 3.0 kV capillary voltage, 120 °C source temperature, and 350 °C desolvation temperature. A 50 L/h cone gas flow and 500 L/h desolvation gas flow were used. The nebulizer gas was 99.95% nitrogen, and the collision gas was 99.99% argon with a pressure of 2 × 10−3 mbar in the T-wave cell. The Masslynx NT v.4.1 (Waters) software was used to collect and analyze the data obtained. MS analyses were performed in the MRM mode, measuring the fragmentation of the protonated pseudomolecular ions of fenbuconazole and its metabolites. MS/MS detection was performed in the positive ionization mode, and the monitoring conditions were optimized for fenbuconazole and its metabolites. After investigation of several dwell times in the 20−100 ms range, a dwell time of 20 ms per ion pair was used to maintain the high sensitivity of the analysis, and a number of data points across the chromatographic peak were required. Typical conditions were as follows: For fenbuconazole, transitions m/z 337 → 70 and m/z 337 → 125 were used for quantification and confirmation when the collision energies were set at 22 and 30 V, respectively. For metabolites, transitions m/z 354 → 70 and m/z 354 → 125 were used for quantification and confirmation when the collision energies were set at 23 and 32 V, respectively. The optimized cone voltages of fenbuconazole and its metabolites were both 30 V. In the present work, the elution orders of fenbuconazole, RH9130, and RH-9129 enantiomers were determined by measuring the optical rotation of each enantiomer using reversed-phase LC coupled with an online OR-2090 detector (Jasco, Japan), which performed using similar separation conditions with the UV detection at 223 nm. Under the described conditions above, six separated peaks with retention times of (+)-fenbuconazole, (−)-fenbuconazole, (+)-RH-9130, (−)-RH-9130, (−)-RH-9129, and (+)-RH-9129 of approximately 12.55, 15.50, 12.57, 14.76, 15.68, and 25.62 min, respectively, were obtained. These settings were utilized for all subsequent studies. A series of standard working solutions (0.02, 0.05, 0.1, 0.25, 0.5, 1, and 2.5 mg/L) of racemic fenbuconazole and its metabolites for the linearity of the six enantiomers were prepared from the stock solution by serial dilution in pure ACN. Correspondingly, matrix-matched standard solutions were obtained at the same concentrations by adding blank soil sample extracts to each serially diluted standard solution. Calibration curves were generated by plotting LC−MS/MS peak area vs the concentration of the enantiomers. Satisfactory linearities in the range of 0.02−2.5 mg/L fenbuconazole or the two metabolite (RH-9129 and RH-9130) enantiomers (n = 7) were obtained when the correlation coefficients (R2) were higher than 0.9991 in all cases. Blank analysis was performed to

allowed to air-dry for 10 min, before the remaining soil (180 g) was added, and the resulting combination was mixed thoroughly for another 15 min, yielding a fortification level of 2.5 μg/g (experiments SC1 and SL1; SC and SL stand for Changsha and Langfang soils, respectively). The water content of the soil samples was adjusted to about 60% of the field holding capacity (w/w) by adding deionized water. The soils were incubated at 25 ± 1 °C in the dark for up to 190 d. The samples were checked regularly for water content by weighing and were frequently mixed by hand for aeration. Similar experiments were carried out with the two metabolites RH9129 and RH-9130 (experiments SC2 and SL2). During the incubation, aliquots of 10 g of soil (based on dry weight) were removed from each treatment at different time intervals and immediately transferred into a freezer (−20 °C) until analysis. Triplicate samples were taken immediately after fortification and mixing (t = 0) to determine the recovery and reproducibility of extraction in the soils (see below). Three replicate samples were taken periodically thereafter. Soil Incubation under Anaerobic Conditions. Incubation experiments were carried out in 50 mL glass bottles with a Teflon septum screw cap. Portions of 20 g of air-dry soils were filled into separate bottles (48 bottles, 8 sampling points, triplicates of each of the 2 soil samples for 1 sampling point) in a glovebox under a high-purity N2 atmosphere. A 20 mL volume of deionized water, previously purged with N2 to remove O2, was added to each bottle. To favor anaerobic conditions, the soils were preincubated during 1 month prior to addition of fenbuconazole. The soil samples were fortified by adding 20 μL of an acetone solution of fenbuconazole with a 50 μL syringe through the septum (spiked level 2.5 μg/g dry soil, experiments SC3 and SL3). The soils were incubated at 25 ± 1 °C in the dark for up to 190 d. Similar experiments were carried out with the two metabolites RH-9129 and RH-9130 (experiments SC4 and SL4). To check the redox conditions, a piece of oxygen indicator (made by MGC and consisting mainly of resazurin) was added to each bottle. The color of the oxygen indicator was red upon anaerobic conditions; on the contrary, it was blue when there was only a small amount of oxygen in the bottle. The color of the oxygen indicator stayed red during these anaerobic incubations. Extraction Method. Samples were first thawed at room temperature. The extraction of fenbuconazole, RH-9129, and RH-9130 from soil was carried out by the QuEChERS (acronym for quick, easy, cheap, effective, rugged, and safe) method. In brief, 10 g samples (dry weight basis) were weighed in 50 mL Teflon centrifuge tubes, then 5 mL of water and 10 mL of ACN were added, and the mixtures were vigorously shaken for 30 min at 25 °C in a water bath shaker. Subsequently, 4 g of MgSO4 and 1 g of NaCl were added. The tubes were capped, immediately vortexed vigorously for 3 min, and then centrifuged for 5 min at a relative centrifugal force (RCF) of 2599g. Afterward, 1.5 mL of the ACN (upper) layer was transferred into a single-use 2 mL centrifuge tube containing 150 mg of anhydrous MgSO4 and 50 mg of C18. The samples were again vortexed for 1 min and centrifuged at RCF = 2077g for 5 min. The resulting supernate was then filtered using a 0.22 μm Nylon syringe filter for chromatographic injection. Enantioselective LC−MS/MS Analysis. A Waters ACQUITY UPLC system (Milford, MA) consisting of the ACQUITY UPLC binary solvent manager and the ACQUITY 2677

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Figure 2. Typical enantioselective LC−MS/MS MRM chromatograms of fenbuconazole, RH-9129, and RH-9130 from the incubation of fenbuconazole in Changsha soil (experiment SC1) after (A) 0, (B) 90, and (C) 150 d. (D)− (E), and (F) show the chromatograms of the incubation of fenbuconazole in Langfang soil (experiment SL1) after 0, 90, and 150 d, respectively.

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check interference from the matrix. The matrix effect was evaluated for the enantiomers in soil samples, and eventually the external matrix-matched standards were utilized for quantification to obtain more realistic results in this work. Quantification was by comparison of stereoisomer peak areas to a matrix-matched standard of similar concentration analyzed the same day. In addition to analysis of triplicate samples, quality control included chiral LC−MS/MS analysis of at least 1 standard for each 10 samples analyzed. Standard concentrations were verified by reference to standard curves of fenbuconazole or the two metabolites. Recoveries of fenbuconazole and its metabolites were determined immediately after fortification. Preliminary experiments showed that the recovery of the above procedure was >84% for all six enantiomers. The limits of quantification (LOQ; signal-to-noise ratio of 10) for these compounds were estimated to be 8 μg/kg on the basis of an acceptable RSD of 11%. Correspondingly, the limits of detection (LOD; signal-to-noise ratio of 3) for all compounds were 2.5 μg/kg.

RESULTS AND DISCUSSION

Simultaneous Enantioselective Separation of Fenbuconazole, RH-9129, and RH-9130 by LC−MS/MS. LC− MS/MS (MRM) chromatograms of fenbuconazole, RH-9129, and RH-9130, using the Chiralcel OD-RH chiral column, are shown in Figure 2. Under the conditions described above, baseline separation is achieved for the two fenbuconazole enantiomers, which are separated by approximately 2 min. The two diastereomers RH-9129 and RH-9130 consist of four stereoisomers, with the second and third peaks almost baseline separated, while the other stereoisomers are separated by a minimum of 2 min. As Figure 2B−C shows, the enantiomers of fenbuconazole and RH-9130 shared similar retention times on the same chiral column, thereby making their chromatographic separation impossible in general. Nevertheless, fenbuconazole and its metabolites have different molecular masses and can be selected in different mass channels. Thus, the fenbuconazole enantiomers were clearly distinguished from the RH-9130 enantiomers under mass spectrometric conditions. Thanks to the technique of LC combined with tandem MS (MRM), the 2678

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Table 1. First-Order Constants (K), Half-Lives (t1/2), Correlation Coefficients (R2), and Final EF Values for the Degradation of Fenbuconazole and Its Metabolites (RH-9129 and RH-9130) in Langfang Alkaline Soil (pH 8.02) experiment SL1

a

SL2a

incubated compound fenbuconazole RH-9129 and RH-9130

SL3b

fenbuconazole

SL4b

RH-9129 and RH-9130

enantiomer (+)-fenbuconazole (−)-fenbuconazole (+)-RH-9130 (−)-RH-9130 (−)-RH-9129 (+)-RH-9129 (+)-fenbuconazole (−)-fenbuconazole (+)-RH-9130 (−)-RH-9130 (−)-RH-9129 (+)-RH-9129

k × 102 (d ‑1)

t1/2 (d)

R2

EFd

0.49 0.54 1.30 0.86 0.53 0.95 0.32 0.35 0.67 0.55 0.48 0.57

141.5 ± 9.4 128.4c ± 7.8 53.4c ± 2.2 80.6c ± 6.2 130.8c ± 8.9 73.0c ± 3.5 216.6 ± 14..5 198.1 ± 10.6 103.5c ± 5.6 126c ± 6.3 144.4c ± 10.3 121.6c ± 7.1

0.96 0.95 0.98 0.97 0.93 0.96 0.92 0.96 0.95 0.99 0.97 0.94

0.53

c

0.31 0.35 0.51 0.43 0.44

a

Incubation experiment under aerobic conditions. bIncubation experiment under anaerobic conditions. cSignificantly different from each other, P < 0.05 (Student’s paired t test). dEF values of each enantiomer pair at the end of incubation (190 d).

Table 2. First-Order Constants (K), Half-Lives (t1/2), Correlation Coefficients (R2), and Final EF Values for the Degradation of Fenbuconazole and Its Metabolites (RH-9129 and RH-9130) in Changsha Acidic Soil (pH 4.69) experiment SC1

a

SC2a

incubated compound fenbuconazole RH-9129 and RH-9130

SC3b

fenbuconazole

SC4b

RH-9129 and RH-9130

enantiomer (+)-fenbuconazole (−)-fenbuconazole (+)-RH-9130 (−)-RH-9130 (−)-RH-9129 (+)-RH-9129 (+)-fenbuconazole (−)-fenbuconazole (+)-RH-9130 (−)-RH-9130 (−)-RH-9129 (+)-RH-9129

k × 102 (d ‑1)

t1/2 (d)

R2

EFd

0.38 0.50 0.88 0.54 0.51 0.69 0.23 0.29 0.41 0.33 0.31 0.39

182.5 ± 10.5 138.6c ± 6.2 78.8c ± 3.1 126.0c ± 8.5 135.9c ± 8.1 100.5c ± 6.6 301.3c ± 18.6 239.1c ± 12.8 169.1 ± 11.3 210.4 ± 10.4 223.6c ± 13.1 177.8c ± 7.9

0.91 0.95 0.99 0.93 0.94 0.91 0.96 0.95 0.98 0.97 0.92 0.94

0.55

c

0.34 0.43 0.53 0.45 0.47

a Incubation experiment under aerobic conditions. bIncubation experiment under anaerobic conditions. cSignificantly different from each other, P < 0.05 (Student’s paired t test). dEF values of each enantiomer pair at the end of incubation (190 d).

simultaneous quantification of all six enantiomers was possible in a signal run. The enantiomeric fraction (EF)29 was used as a measure of the enantioselectivity of a pair of enantiomers, and EF is defined by the following equation: EF = ( +)/( +) + ( −)

the corresponding EF values, the concentrations of individual enantiomers in soil were then calculated. Enantioselective Degradation of Fenbuconazole in Soils under Aerobic and Anaerobic Conditions. Fenbuconazole was found to be persistent in the two soils. Under aerobic conditions, fenbuconazole was degraded slowly in Langfang alkaline soil, and approximately 63.8% of the initially spiked fenbuconazole disappeared after 190 d of incubation (experiment SL1). At the same time, the EF value, defined as the concentration ratio of the first eluted (+)-fenbuconazole to the total fenbuconazole concentration (sum of the (+)- and (−)-fenbuconazole concentrations), changed gradually from the initial 0.49 to 0.53 after 190 d, suggesting slight enantioselectivity with the second eluted (−)-fenbuconazole preferentially degraded. For Changsha acidic soil, the degradation was slower with only approximately 56.5% of the fenbuconazole dissipated after 190 d of incubation (experiment SC1). The EF increased from 0.49 to 0.55 over the 190 d period, indicating a relatively higher enantioselectivity in this soil. Similarly, (−)-fenbuconazole was dissipated faster than (+)-fenbuconazole in Changsha soil. In Figure 2A−C, enantioselective LC−MS/MS MRM chromatograms show the elution of fenbuconazole and concurrent formation of its metabolites (RH-9129 and RH-9130) from samples of experiment SC1 (incubation with fenbuconazole) after 0, 90,

(1)

where (+) and (−) are the peak areas of the (+)- and (−)-enantiomers of fenbuconazole, RH-9129, and RH-9130, respectively (see Figure 2). The EF values can range from 0 to 1, with EF = 0.5 representing the racemic mixture. In this study, the EFs of fenbuconazole, RH-9129, and RH-9130 standards freshly spiked into soils at 0.1 and 1.25 μg/g levels were determined by enantioselective LC−MS/MS to be 0.49−0.51 with RSDs < 3% (n = 3) and agree well with those of authentic standards. Racemic standards were injected repeatedly to determine the reproducibility of EF measurements. For our standards, the EF of the fenbuconazole enantiomers was 0.494 ± 0.005 (n = 6), the EF of the RH-9130 enantiomers was 0.503 ± 0.008 (n = 6), and that of the RH-9130 enantiomers was 0.501 ± 0.007 (n = 6). RH-9129 and RH-9130 are two diastereomers, each of which is considered to be a racemic mixture for the purpose of this study. On the basis of the total concentrations of fenbuconazole, RH-9129, and RH-9130 and 2679

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Figure 3. Changes in the concentrations of the four RH-9129 and RH-9130 stereoisomers in the two soils: (A) experiment SC1 with fenbuconazole in Changsha soil, (B) experiment SL1 with fenbuconazole in Langfang soil.

bacterial consortia are active under aerobic and anaerobic conditions.32 However, the data from our studies showed that while enantioselective dissipation occurred for fenbuconazole, the direction of enantioselectivity did not shift under aerobic and anaerobic conditions. In other words, anaerobic conditions did not significantly alter the enantiomeric preference of the microbial community. Formation of RH-9129 and RH-9130 during Fenbuconazole Incubation Experiments. RH-9129 and RH-9130 were both detected during fenbuconazole dissipation in the two aerobic soils (Figure 2). Quantitative analysis showed the concentration of RH-9129 was higher than that of RH-9130 in both soils. Each of the two diastereomers RH-9129 and RH9130 consists of a pair of enantiomers. The soil concentrations of the four stereoisomers as a function of time were calculated and are plotted in Figure 3. Under aerobic conditions, the data show that the concentration of each stereoisomer of RH-9129 and RH-9130 increased steadily to a maximum and then decreased slowly to the end of the incubation in Langfang soil, indicating further degradation occurred for the stereoisomer. During the initial 3 d of incubation, the amounts of (+)-RH9130, (−)-RH-9130, (−)-RH-9129, and (+)-RH-9129 were approximately 4.5, 4.0, 5.0, and 4.3 μg/kg, respectively; after continued incubation, the concentrations of (+)-RH-9130, (−)-RH-9130, (−)-RH-9129, and (+)-RH-9129 reached a maximum of 61.6, 69.6, 132.4, and 104.7 μg/kg at day 120, respectively, and then appreciably decreased to 36.4, 52.5, 96.3, and 73.3 μg/kg at day 190, respectively. It is interesting that in Langfang alkaline soil (experiment SL1), the RH-9129 and RH9130 stereoisomer concentrations followed the order (−)-RH9129 > (+)-RH-9129 > (−)-RH-9130 > (+)-RH-9130 over the 190 d period (Figure 3B). It is apparent that the (−)-RH-9129 isomer was preferentially produced via the reduction reaction, whereas a smaller amount of (+)-RH-9130 was produced. In the case of Changsha acidic soil (experiment SC1), the highest concentrations of (+)-RH-9130, (−)-RH-9130, (−)-RH-9129, and (+)-RH-9129 were about 46.7, 39.1, 113.5, and 105.2 μg/ kg, respectively, occurring at the end of the incubation (day 190). In this soil, the disappearance of RH-9130 and RH-9129 stereoisomers was not observed. Since the incubation was only for 190 d, longer incubation times could have eventually shown the loss of these stereoisomers. Interestingly, we found that the stereoisomer concentration order was different from that of the Langfang soil, which was determined to be (−)-RH-9129 > (+)-RH-9129 > (+)-RH-9130 > (−)-RH-9130 at the end of incubation (Figure 3A). Again, the (−)-RH-9129 isomer was documented to be the highest amount of metabolite isomer. In

and 150 d, respectively. Correspondingly, Figure 2D−F presents the chromatograms of the incubation of fenbuconazole in Langfang soil (experiment SL1) after 0, 90, and 150 d, respectively. Assuming an exponential equation of first-order reaction, the kinetic data (degradation rate constant k and halflife T1/2) of the two enantiomers were calculated and are listed in Tables 1 and 2. The fit was good with R2 > 0.91 in most cases. For Langfang soils, the rate constants of (+)-fenbuconazole and (−)-fenbuconazole were 0.0049 and 0.0054 d−1, corresponding to half-lives of 141.5 and 128.4 d, respectively. For Changsha soils, the rate constants of (+)-fenbuconazole and (−)-fenbuconazole were 0.0038 and 0.0050 d−1 with halflives of 182.5 and 138.6 d, respectively. In the case of fenbuconazole incubated under anaerobic conditions (SC3 and SL3), dissipation of fenbuconazole was also enantioselective, the same as that in aerobic soils. Tables 1 and 2 present the half-lives and degradation rate constant values in the two soils under anaerobic conditions, and the results indicate that both fenbuconazole enantiomers were degraded more slowly in anaerobic conditions compared to aerobic conditions. The half-lives of (+)-fenbuconazole under aerobic conditions were 141.5 and 182.5 d for Langfang and Changsha soils, but increased to 216.6 and 301.3 d under anaerobic conditions, respectively. The half-lives of (−)-fenbuconazole under aerobic conditions were 128.4 and 138.6 d for Langfang and Changsha soils, increasing to 198.1 and 239.1 d under anaerobic conditions, respectively. After 190 d of treatment, the EF increased from 0.49 to 0.51 and 0.53 for Langfang and Changsha soils under anaerobic conditions, respectively. Both of the changed EF values of the two soils were lower than that in the aerobic conditions, suggesting a relatively lower enantioselectivity in anaerobic soils. Especially for Langfang anaerobic soil, the dissipation of fenbuconazole was not enantioselective during the incubation period in this work. In some other work, it was shown that the direction of enantioselectivity for some chiral pesticides may be changed with the incubation conditions. For example, metalaxyl was usually found to be environmentally enriched in the (S)enantiomer; however, a reversed enantioselectivity was observed in anaerobic degradation in sewage sludge, resulting in residues enriched in the (R)-enantiomer.30 Besides, (S)-cisbifenthrin degraded more rapidly than (R)-cis-bifenthrin in San Diego Creek sediment under anaerobic conditions, but the direction of enantioselectivity was reversed in aerobic soils, with (S)-cis-bifenthrin degrading more slowly than (R)-cis-bifenthrin.31 Those studies indicate that redox conditions affect the biotransformation of some chiral compounds, as different 2680

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Figure 4. Typical enantioselective LC−MS/MS chromatograms from the incubation of RH-9129 and RH-9130 in Langfang soil (experiment SL2) after (A) 0, (B) 60, and (C) 150 d. Panel D shows the chromatogram of blank soil.

anaerobic conditions; only a small amount of total fenbuconazole (∼46.8% or ∼36.4% for Langfang or Changsha soil, respectively) disappeared after 190 d of incubation. The RH-9129 and RH-9130 stereoisomers could be detected above our quantitation limit after day 60. The total concentration of RH-9129 reached a maximum at about 190 d in Langfang and Changsha soils under anaerobic conditions, approximately 62.3 and 57.8 μg/kg, respectively. These values were much lower than the maxima of approximately 237.6 and 220.7 μg/kg in the two aerobic soils. A similar result was also observed for the diastereomer RH-9130. Furthermore, it was observed that the four RH-9129 and RH-9130 stereoisomer concentration orders in the two anaerobic soils were consistent with those under aerobic conditions. It could be concluded that anaerobic conditions did not significantly alter the patterns of transformation. Degradation of the Metabolites RH-9129 and RH9130 in Soils under Aerobic and Anaerobic Conditions. In Langfang aerobic soil (experiment SL2), the residues of RH9129 and RH-9130 decreased readily with time elapsed and approximately 76.8% or 87.9% of the initially spiked RH-9129 or RH-9130 had degraded after 190 d of incubation. From Table 1 and Figure 4, we can see that the degradation of the two diastereomers in this soil was enantioselective. Apparently, the (+)-RH-9130 isomer dissipated fastest among the four RH9129 and RH-9130 stereoisomers, and there were also some differences in the degradation rates for the other three enantiomers (Figure 4A−C). The degradation rates of the

summary, when fenbuconazole was treated in different soils (experiments SL1 and SC1), each produced its own characteristic pattern of stereoisomer composition of the metabolites RH-9129 and RH-9130 (Figure 3). In experiment SL1, the concentration of the (−)-RH-9130 isomer was lower in the former part and higher in the latter part of the incubation than that of the (+)-RH-9130 isomer. In contrast, in experiment SC1, the data showed that the concentration of the (−)-RH9129 isomer was lower in the former part and higher in the latter part of the incubation than that of the (+)-RH-9129 isomer. The reason for the patterns in the two experiments is unknown; it is possibly due to different formation and dissipation rates of the isomers. The above data show that the different RH-9129 and RH-9130 stereoisomer patterns produced from fenbuconazole depend on the soil type. Interestingly, these observations are similar to previous investigations for another triazole fungicide. In earlier studies, Li et al. reported a stereoselective transformation of triadimefon to triadimenol in two soils.24 They found that the patterns of triadimenol A and triadimenol B stereoisomers produced from triadimefon varied with the soil type. Another similar observation has also been reported recently in studies of the chiral transformation of triadimefon to triadimenol when triadimefon was metabolized in three different soil types.23 Analysis of the two anaerobic soils (experiments SL3 and SC3) showed only low levels of RH-9129 and RH-9130 being formed during the incubation period. Likewise, as described above, fenbuconazole was degraded rather slowly under 2681

dx.doi.org/10.1021/es203320x | Environ. Sci. Technol. 2012, 46, 2675−2683

Environmental Science & Technology

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Figure 5. Enantiomer fraction (EF) vs time for loss of RH-9129 and RH-9130 from the two soils: (A) experiment SC2 with RH-9129 and RH-9130 in Changsha soil, (B) experiment SL2 with RH-9129 and RH-9130 in Langfang soil.

half-lives and the rate constants in the two soils under anaerobic conditions, and the results suggest that both RH9129 and RH-9130 were degraded more slowly under anaerobic conditions than under aerobic conditions. Individual rate constants of (+)-RH-9130, (−)-RH-9130, (−)-RH-9129, and (+)-RH-9129 in Changsha soil were 0.0041, 0.0033, 0.0031, and 0.0039 d−1 and corresponded to those in Langfang soil of 0.0067, 0.0055, 0.0048, and 0.0057 d−1, respectively. Again, (+)-RH-9130 was documented to be the consistently fastest dissipated isomer among the four stereoisomers in experiments SL4 and SC4, and the degradation rate order was consistent with that in the two aerobic soils. It is also notable that (−)-RH-9129 in this research was documented to have the highest concentration of the metabolites produced by fenbuconazole; however, it showed the slowest dissipation rate among the four stereoisomers of RH-9129 and RH-9130. As a consequence, the use of the racemic fenbuconazole product is expected to result in temporal increases in the concentration of the environmentally more persistent (−)-RH9129 stereoisomer relative to the other three stereoisomers. Stereoselectivity in these biodegradation and transformation processes is expected to result in ecotoxicological effects that cannot be predicted from our existing knowledge and must be considered in further risk assessment.

four stereoisomers hence followed the order (+)-RH-9130 > (+)-RH-9129 > (−)-RH-9130 > (−)-RH-9129. Individual rate constant (k) of (+)-RH-9130, (+)-RH-9129, (−)-RH-9130, and (−)-RH-9129 were 0.013, 0.0095, 0.0086, and 0.0053 d−1, respectively. By summing the concentrations of individual enantiomers, the rate constants for total RH-9129 and RH9130 were estimated to be 0.0070 and 0.01125 d−1 and corresponded to half-lives of 105.7 and 63.2 d, respectively, indicating the dissipation of RH-9130 in this soil was faster than that of RH-9129. For Changsha aerobic soil (experiment SC2), the degradation was slower with approximately 68.1% or 73.5% of RH9129 or RH-9130 eventually disappearing after 190 d of treatment. Also, (+)-RH-9130 was the fastest dissipated isomer among the four stereoisomers, and the order of the degradation rates for the four stereoisomers in Changsha acidic soil was in accordance with that in Langfang alkaline soil (Tables 1 and 2). This faster degradation of the (+)-RH-9130 stereoisomer in comparison to the other three stereoisomers indicates a preferential biotransformation of the (+)-RH-9130 isomer by the soil’s enzymatic systems and microbes when this isomer is applied as a racemic mixture. In addition, the rate constants of overall RH-9129 and RH-9130 in Changsha soil were estimated to be 0.0062 and 0.0071 d−1, respectively. Similarly, RH-9130 was degraded faster than RH-9129 in Changsha soil. The EF curves of RH-9129 and RH-9130 in aerobic conditions were calculated and are plotted in Figure 5. The EF values of RH-9129 and RH-9130 consistently decreased with time in experiments SL2 and SC2. After 190 d of incubation, the EF of RH-9130 decreased gradually from the initial 0.50 to 0.31 and 0.34 for Langfang and Changsha aerobic soils, respectively, indicating similar enantioselectivities in the two soils. Similarly, over the 190 d treatment period, the EF of RH-9129 steadily changed from the initial 0.50 to 0.35 and 0.43 for Langfang and Changsha soils, respectively, suggesting a higher enantioselectivity in Langfang alkaline soil. Meanwhile, the EFs of RH-9129 between Changsha soil and Langfang soil were all significantly different (P < 0.05, Student’s paired t test). These results indicate the same stereoselectivity in the degradation of RH-9129 and RH-9130 stereoisomers in the two aerobic soils, especially for the (+)-RH-9130 and (+)-RH9129 stereoisomers. The dissipation of RH-9129 and RH-9130 was also investigated under anaerobic conditions (experiments SL4 and SC4). The degradations of RH-9129 and RH-9130 were consistently enantioselective. Tables 1 and Table 2 show the

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +86-0162815908, fax: +86-01-62815908. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

This work was financially supported by the foundation established by the Agricultural Ministry of the People's Republic of China, the National Basic Research Program of China (The 973 Program, Grant 2009CB119000), the National Natural Science Foundation of China (Grants 31071706, 31000863, and 30900951), and the Public Service Sector Research and Development Project (Grants 200903054 and 200903033). 2682

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