Probing the Stereochemistry of Successive Sulfoxidation of the

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Probing the Stereochemistry of Successive Sulfoxidation of the Insecticide Fenamiphos in Soils Xiyun Cai,* Weina Xiong, Tingting Xia, and Jingwen Chen Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China S Supporting Information *

ABSTRACT: Successive sulfoxidation is widely recognized as a general characteristic of the metabolism of chiral or prochiral thioethers, producing sulfoxides, and sulfones. However, information related to the stereochemistry of this process in soils is rare. In this study, the biotic transformation of the insecticide fenamiphos (a model thioether) was followed over two months in three soils, through separate incubations with fenamiphos parent, the sulfoxide intermediate (FSO), the sulfone intermediate (FSO2), and their respective stereoisomers. The results showed that the successive sulfoxidation involved oxidation of fenamiphos to FSO and subsequently to FSO2 as well as diastereomerization/enantiomerization of FSO, all of which were primarily biotic and stereoselective. The concomitant hydrolysis of fenamiphos, FSO, and FSO2 to phenols that occurred at lower rates was biotically favorable, but not stereoselective. The stereochemistry of this successive sulfoxidation transferred principally through two parallel systems, R(+)-fenamiphos → SRPR(+)-/SSPR(−)-FSO → R(+)-FSO2 and S(−)-fenamiphos → SRPS(+)-/SSPS(−)-FSO → S(−)-FSO2, between which unidirectional intersystem crossing occurred at FSO via isomeric conversions and created a system of S(−)-fenamiphos → SRPR(+)-/SSPR(−)-FSO → R(+)-FSO2. This pattern accounts for the enrichment of the intermediates SSPR(−)-/SSPS(−)-FSO and R(+)-FSO2 that are toxicologically close to the highly toxic S(−)-fenamiphos, associated with soil application of fenamiphos. Selective formation/depletion of these intermediate stereoisomers leads to dramatic variations in the ecotoxicological effects of the thioether insecticide.

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INTRODUCTION Sulfur has an electron configuration of 3s23p4 and presents 10 different oxidation states (i.e., −2 to +6). It fulfills many essential biological functions by constituting key biomolecules involved in in vivo redox reactions.1,2 Sulfur biochemistry has attracted extensive attention in attempts to design sulfur-containing fine chemicals, such as pesticides (fenamiphos, fipronil, etc.) and drugs (omeprazole, albendazole, etc.). These chemicals commonly undergo in vivo sulfoxidation (transformation of thioethers to sulfoxides or sulfoxides to sulfones), which in turn alter reactivity, translocation, and thus toxicity of the parents.2−5 Sulfoxides (R1R2SO), which are of particular biological importance, may be chiral with an asymmetric sulfur atom (R1 ≠ R2). Chiral sulfoxides often undergo selective formation/ depletion mediated by cytochrome P450 and flavin monooxygenase, leading to enantiomeric enrichment.2,6−8 The significance of chirality is widely recognized in the environmental sciences, especially for organic pollutants possessing asymmetric carbon atoms or axes.9−14 The stereoisomers of these pollutants commonly have distinct biological effects (e.g., toxicity and carcinogenicity).9,10 In most cases, they also differ in biotic degradation, resulting in varying stereoisomeric compositions of chiral pollutants commonly used in the racemate form. Enantiomeric selectivity of the toxicological and © 2014 American Chemical Society

degradation effects leads to variations in the environmental risks of chiral pollutants.9−11 This is the case for chiral sulfoxides (the primary metabolites of thioethers), which have been documented to have stereoselective in vivo metabolism and toxicity2,15−17 and to undergo stereoselective biodegradation in soils and sediments.18−21 However, less attention has been given to the understanding of the transformation of chiral or prochiral thioether chemicals (the precursors of sulfoxides) in soils, especially from the viewpoint of stereochemistry, although they make up the largest portions of sulfur-containing fine chemicals. Chemicals in this category, increasingly released into the environment, commonly undergo successive oxidation to produce sulfoxide and sulfone intermediates in animals, plants, and soil microorganisms.4,16,17 This successive sulfoxidation is widely recognized as a general characteristic of metabolism of thiothers.2,16 It is noteworthy that hazardous transformation products are often accumulated with the active moieties intact or more potent structures formed.22−24 The thioether insecticide fenamiphos, for example (Supporting Received: Revised: Accepted: Published: 11277

June 10, 2014 August 25, 2014 August 27, 2014 August 27, 2014 dx.doi.org/10.1021/es502834v | Environ. Sci. Technol. 2014, 48, 11277−11285

Environmental Science & Technology

Article

Separate incubations with the racemates and stereoisomers were performed in the three soils. Briefly, a stock solution of each chemical in methanol was spiked into the air-dried soils. The fortified soils were allowed to settle overnight in a fume cupboard to evaporate the solvent. The recovered soils of 9-fold dry weight were blended and homogenized by sieving and spraying with deionized water, yielding a fortification level of 5.0 mg/kg for each chemical. This level roughly corresponds to the median of the recommended application rates, typically 1−10 lbs fenamiphos/acre (0.9−9.0 mg/kg). The as-treated soils (10.0 g dry weight) were divided into 50 mL glass flasks covered with airpermeable, sterile cellulose plugs. All soil samples were then incubated for approximately 9 weeks. At certain time intervals, the flasks were withdrawn in triplicate for further extraction analysis. In parallel, other portions of the air-dried soils were autoclaved at 125 °C for 2 h and fortified by the tested chemicals, acting as control samples. The water content of all the samples was periodically checked by weighing, and water losses were offset by spraying with deionized water. Extraction Method. The extraction of fenamiphos and its transformation products was performed using the modified QuEChERS method.34 In detail, 10-g samples (dry weight) were transferred into 50 mL Teflon centrifuge tubes and mixed with 4 mL of deionized water and 10 mL of acetonitrile (containing 0.5% acetic acid) by vigorously shaking for 30 s and mechanically vortexing for 60 s. Next, 1.0 g NaCl and 4.0 g anhydrous MgSO4 were added. The tubes were immediately capped, followed by mechanical vortexing for 3 min and centrifugation for 10 min at 4000 r/min (2970 g). Aliquots of 8.0 mL supernatant were transferred into 10 mL centrifuge tubes containing 1.0 g anhydrous MgSO4 and 100 mg PSA (Primary Secondary Amine, 40−60 μm). The samples were again vortexed and centrifuged. Five-milliliter aliquots of the clear supernatant were withdrawn and evaporated to dryness under a gentle stream of nitrogen gas. A total of 1.5 mL n-Hexane was added in three 0.50 mL aliquots to redissolve the residue. The reconstituted solutions were purified by a 0.22-μm Teflon syringe filter and evaporated to dryness. The final residue was redissolved in 0.50 mL n-hexane for stereoselective and nonstereoselective quantification using HPLC systems equipped with a Daicel Chiralpak AD-H column and a Dalian Elite YWG C18 column, respectively (SI Text S2). This extraction yielded average percent recoveries of 96.3% for fenamiphos (0.05−5.00 mg/kg, n = 9) and its enantiomers (0.025−2.50 mg/kg, n = 9), 83.8% for FSO2 (0.05−5.00 mg/kg, n = 9) and its enantiomers (0.025−2.50 mg/ kg, n = 9), and 84.4% for FSO (0.10−5.00 mg/kg, n = 9), and its stereoisomers (0.025−1.25 mg/kg, n = 9). The data were corrected accordingly. Zebrafish Embryo Toxicity Assay and Microscopic Observation. Adult AB-strain zebrafish (Danio rerio) were cultured in our laboratory. Fertilized eggs (embryos) were collected from spawning adults with a female/male ratio of 1:235 and used for the 72-h embryo toxicity assay according to the OECD guideline for fish embryo toxicity test.36 In brief, 2.0 mL of test solutions was added to each cell of 24-well microtiter plates, and five embryos were placed into each cell. The tested chemicals were applied at experimental concentrations of 0, 0.10, 0.20, 0.50, 1.00, and 2.00 mg/L. The microtiter plates were then covered with lids and incubated at 28 ± 1 °C in a light:dark cycle of 14/10 h. The test solutions were reproduced daily by freshly prepared solutions. Development of embryos from the blastula to early larval states was observed every 12 h using an optical microscope. Embryo mortality, crooked body, pericardial edema,

Information, SI, Figure S1), and its primary transformation products (sulfoxide (FSO) and sulfone (FSO 2 )) have nematicidal properties and are labeled as moderately to highly toxic substances. They are all included in the risk assessment of this insecticide, as indicated by fenamiphos total toxic residues (TTR, a sum of fenamiphos parent plus FSO and FSO2).25,26 Although information related to the stereochemistry of the successive sulfoxidation of thioethers is unavailable, data reported from direct monitoring and laboratory microcosm studies have indicated the selective depletion of other chiral pollutants and formation of chiral transformation products.10,11,14 Examples of these compounds include PCBs,13 the fungicides triadimefon, metalaxyl and fenbuconazole,12,27,28 herbicides diclofop and beflubutamid,14,29 insecticides acephate and paichongding,30,31 and plant growth regulators paclobutrazol and uniconazole.32 This information improves the understanding of the relative importance of biotic transformation of chiral pollutants in natural environments, and provides insights into potential risk alterations due to the formation/depletion of hazardous intermediate stereoisomers.10−12 The primary goal of this study was to probe the stereochemistry of the successive sulfoxidation of the insecticide fenamiphos (a model thioether) in soils and associated variances in risk assessments of this insecticide. Separate incubations with fenamiphos, FSO, FSO2, and their respective stereoisomers were performed in three aerobic soils to reveal the stereoselective transformation of the insecticide and formation/depletion of its intermediates. The same soils were autoclaved as controls. Acute zebrafish embryo toxicity assays and microscopic observations were conducted to assess developmental effects of the racemates and stereoisomers. The racemate- and stereoisomer-based hazard quotients were calculated to compare potential risks of fenamiphos toward zebrafish embryos.

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EXPERIMENTAL SECTION

Chemicals and Soils. Fenamiphos parent, FSO and FSO2 (chemical purity ≥99%) were purchased from Dr. Erenstorfer Gmbh (Augsburg, Germany). Fenamiphos and FSO2 have an asymmetric phosphorus atom and each consists of a pair of enantiomers, whereas FSO consists of four stereoisomers (two pairs of enantiomers) due to its two asymmetric atoms (phosphorus (P) and sulfur (S)). The stereoisomers of these chemicals (chemical purity ≥99% and optical purity ≥95%) were prepared using a chiral semipreparative HPLC system (SI Text S1). Phenol intermediates were prepared by the hydrolysis of these chemicals that respectively were incubated in 0.10-mol/L NaOH at 50 °C for 12 h.33 All organic solvents (HPLC grade) were purchased from Tedia (Fairfield, OH, U.S.A.). Three surface soils were collected at depths of 0−15 cm from geographically different agricultural regions of China (SI Table S1). The soils were air-dried at room temperature, passed through a 2 mm sieve, and stored in the dark at approximately 4 °C. Blank analysis revealed fenamiphos, FSO and FSO2 not present in the soils, with detection limits of 10.0 μg/kg (fenamiphos and FSO2) and 25.0 μg/kg (FSO). Incubation Experiments. Prior to fortification with the racemate or stereoisomers, the air-dried soils were adjusted to 60% of the maximum water holding capacity by sieving and spraying with deionized water. The moist soils were transferred into an artificial climate incubator and incubated in the dark at 20 ± 1 °C for 4 weeks to recover soil microbial activities (SI Figure S2). 11278

dx.doi.org/10.1021/es502834v | Environ. Sci. Technol. 2014, 48, 11277−11285


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Figure 1. Depletion of the fortified rac-fenamiphos in the nonautoclaved soils and selective formation/depletion of sulfoxide (FSO) and sulfone (FSO2) stereoisomers. (a) Profiles of the racemates of fenamiphos, FSO, FSO2 and total toxic residues (TTR, a sum of fenamiphos parent plus FSO and FSO2) in the soils; and, (b) Profiles of stereoisomers of fenamiphos, FSO and FSO2 in the soils.

day 11, accompanied by a remarkable loss of the parent (ca. 75%). In the autoclaved soil, however, only 26% of the insecticide was depleted; the formation percentage of FSO was 12%, and FSO2 was below the detection limit. Similar results were observed for the other two experimental soils. These differences demonstrated that fenamiphos sulfoxidation was the primary transformation process and was biotic. However, the concomitant hydrolysis of fenamiphos to phenol underwent at lower rates and seemed to be abiotic in nature, as indicated by similar formation percentages of the product in the autoclaved and nonautoclaved soils. The separate incubations with FSO and FSO2 demonstrated that, similar to the insecticide, FSO underwent both oxidation and hydrolysis, whereas FSO2 was depleted primarily by the hydrolysis to FSO2 phenol. Higher fractions of the two phenol intermediates were observed in the nonautoclaved soils, indicating that the hydrolysis of FSO and FSO2 is biologically favorable. The three phenols were all further depleted in the soils over incubation time, which is consistent with previous studies33,38−40 showing that they are rapidly degraded to final oxidation products (i.e., CO2 and H2O); for this reason, the complete quantification of these phenols was not included in this study. In the nonautoclaved soils, fenamiphos degraded readily with no lag periods, yielding half-lives of 5−13 days (Figure 1a, SI Table S2). FSO was rapidly formed, and the maximum concentrations were reached after approximately 9 days of incubation, after which the level slowly declined or remained constant. FSO2, a further sulfoxidation product, was simultaneously detected at low levels and accumulated with incubation time. The formation and depletion of the intermediates from the parent were quantified by the coefficient χP (eq 1) that ranged from 0 to 1.0 and accounted for the stoichiometry of the parent’s depletion in producing an intermediate.41

and yolk sac edema were recorded, and the 72-h cumulative magnitude was used to indicate toxic effects. Each exposure was tested in triplicate, and each chemical was tested in duplicate. Any dead embryos or larvae (no heartbeat observed) were removed immediately at each observation time. Hazard Quotient Assessment of Fenamiphos in Waters. The hazard quotient (HQ) of mixed pollutants37 toward zebrafish embryos is expected as

∑ HQ = i

MECi PNECi

where MECi is the measured environmental concentration of the ith component in an n-compound residue and PNECi is the predicted no-effect concentration of the ith component. The HQ was calculated using worst case assumptions. The MEC of fenamiphos, FSO and FSO2 was obtained from monitoring studies.25 The PNEC of these compounds was derived with a default assessment factor of 1000. Data Analysis. All the soil samples were weighed on a dryweight basis. Chemical concentration data were represented as the mean value (n = 3). Statistical analysis was determined by using one-way ANOVA in Origin 8.0 (Microcal Software, Inc., U.S.A.). Significant differences were defined at the 95% confidence interval without overlap.

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RESULTS AND DISSCUSSION Confirmation of Successive Sulfoxidation of Fenamiphos in Soils. Fenamiphos underwent oxidation and hydrolysis in the autoclaved and nonautoclaved soils, as indicated by the formation of oxidates (FSO and FSO2) and hydrolytic intermediates (phenols of both fenamiphos and the oxidates) (SI Figure S3). The degradation of the insecticide and formation of these intermediates (quantified as molar percentages of the fortified parent) was significantly inhibited in the autoclaved soils (SI Figure S4). The formation percentages were approximately 64% for FSO and 7% for FSO2 in the nonautoclaved XF soil on

d[Int]/dt = χP kP[P] − kInt[Int] 11279

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Figure 2. Chiral HPLC spectra of fenamiphos and its enantiomers in the nonautoclaved soils on day 11. Each soil was separately fortified by racfenamiphos and its enantiomers. The SD soil panel includes an expanded insert for the S(−)-fenamiphos plot. a, R(+)-fenamiphos; b, S(−)-fenamiphos; c, SR*PR(+)-FSO; d, SS*PR(−)-FSO; e, R(+)-FSO2; f, SR*PS(+)-FSO; g, S(−)-FSO2; and, h, SS*PS(−)-FSO.

Figure 3. Stereoisomeric conversion of fenamiphos sulfoxide (FSO) in the nonautoclaved soils on day 21. Each soil was separately fortified by FSO stereoisomers. c, SR*PR(+)-FSO; d, SS*PR(−)-FSO; e, R(+)-FSO2; f, SR*PS(+)-FSO; g, S(−)-FSO2; and, h, SS*PS(−)-FSO.

fractions (EF) of 0.45−0.48. In parallel, approximately 10% of FSO was detected with identical percentages of four stereoisomers, while FSO2 was not detected. The separately fortified enantiomers of fenamiphos did not undergo configuration inversion; each was transformed to its two equivalent diastereoisomers with the same configurations at the asymmetric phosphorus atom (e.g., formation of SRPR(+)- and SSPR(−)FSO from R(+)-fenamiphos). In the nonautoclaved soils, however, the losses of the racemate were enantioselective, as illustrated by the EF profiles (SI Figure S6), showing that the R(+)-fenamiphos commonly reacts faster than the antipode. Two new products, SRPR-(+)- and SSPR(−)-FSO, were detected at low levels in the S(−)-fenamiphos-fortified soils (Figure 2), neither of which occurred in the autoclaved soils (SI Figure S5). Although autoclaving may be not sufficient in many cases to completely eliminate microbial reactivity in soils, the above data indicate that the depletion of fenamiphos in soils is primarily biotic and enantioselective. Selective Formation/Depletion of Intermediate FSO. Figure 1b shows the fraction curves of the intermediate FSO stereoisomers in the rac-fenamiphos-fortified soils. The maximum fractions were reached at approximately 10−20 days. Interestingly, SSPS(−)- and SSPR(−)-FSO were always the two most abundant stereoisomers in the three soils, though all the stereoisomers varied temporally. In the SD soil, the fraction of SSPS(−)-FSO remained nearly constant (6.4−8.6%) throughout the incubation, while those of the other three stereoisomers declined, giving rise to the order of relative fractions: SSPS(−)- > SSPR(−)- > SPPS(+)- ≈ SPPR(+)-FSO. SSPR(−)-FSO was most abundant in the TL soil, followed by SSPS(−)-, SRPR(+)-

where [Int] and [P] are the concentrations of the intermediate and its corresponding parent, respectively, and kInt and kP are their respective depletion rate constants. The results (SI Table S3) indicated that 48−99% of fenamiphos underwent oxidation to produce FSO, further showing the dominant role of sulfoxidation. The product FSO was depleted with rate constants of 0.12 d−1 (SD soil), 0.02 d−1 (TL soil), and 0.03 d−1 (XF soil), respectively, corresponding to the oxidation of ca. 33%, 99%, and 96% of this chemical to FSO2. The product FSO2 was further hydrolyzed in three soils with identical rate constants. Surprisingly, the fortified FSO that was a starting chemical had rate constants of 0.06−0.12 d−1 and was more degradable than its reaction intermediate form. This finding contradicts the wellknown slow decline of FSO to FSO2 in soils.33,38,40,42 Two possible explanations are provided for the pseudorecalcitrance of FSO in the intermediate form. One is that FSO depletion can be offset by its concurrent production from fenamiphos. The other is that FSO has low availability because the majority of the compound as a reaction product usually detected at low levels are adsorbed in the soil and cannot be utilized by microorganisms.42−44 Interestingly, the fortified FSO had the same order of degradability (indicated by rate constants in SI Table S2) as fenamiphos total toxic residues, supporting the hypothesis that FSO be the active species of the insecticide.26,33,38,40 Enatioselectivity in Sulfoxidation of Fenamiphos to FSO. Figure 2 and SI Figures S5 and S6 illustrate the selective degradation of rac-fenamiphos and its enantiomers separately fortified in the soils. In the autoclaved soils (SI Figure S5), approximately 41−53% of rac-fenamiphos remained intact on day 65 and was in a nearly racemic mixture, with enantiomeric 11280



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conversions into other stereoisomers, although it was most recalcitrant to degradation. SRPR(+)-FSO was lowest abundant in the rac-FSO-fortified soil, attributed to both its high degradability and the negligible formation of this stereoisomer from SSPR(−)-FSO. Product identification showed that SRPR(+)- and SSPR(−)-FSO underwent oxidation to R(+)FSO2 at higher rates than the other two stereoisomers to S(−)FSO2. R(+)-FSO2 was also detected in the soil fortified by SRPS(+)-FSO with a formation rate of ca. 11% (on day 9), as this FSO stereoisomer inversed into SSPR(−)-FSO that could generate R(+)-FSO2 (SI Figure S8). These two effects may cause the enrichment of R(+)-FSO2 (SI Figure S7). In comparison to the above two soils, the XF soil had a moderate organic matter content. In this soil, four stereoisomers of the fortified rac-FSO all slowly declined in concentration (SI Figure S7, Table S4). It is surprising that SRPR(+)- and SSPR(−)-FSO undergo dramatic bidirectional diastereomerization, with yielding rates of ca. 19% (on day 21) for the conversion of SRPR(+)- into SSPR(−)-FSO and 9% for the reverse. These values were comparable to the rates of sulfoxidation (i.e., 16% and 7%) of them to R(+)-FSO2. Other isomeric conversions occurred at low rates, e.g., 2.43% for the conversion of SSPS(−)into SRPS(+)-FSO and 0.52% for the conversion of SRPS(+)into SSPR(−)-FSO. The high fraction of SSPR(−)-FSO in the rac-FSO-fortified soil resulted from dramatic isomeric conversions of other stereoisomers into this one. However, SSPS(−)-FSO was also abundant, attributable to its recalcitrance to depletion. Similarly, the concomitant oxidation of SRPR(+)and SSPR(−)-FSO to R(+)-FSO2 was more pronounced than the reaction of the other two stereoisomers to S(−)-FSO2 (Figure 3, SI Figure S8), resulting in the enrichment of R(+)FSO2 in the soil. To our knowledge, this is the first evidence of isomeric conversions of sulfoxides in soils. In general, chiral sulfoxides are configurationally stable, attributable to a large energy barrier (35−43 kcal/mol) for configuration inversion at the asymmetric sulfur atom.2 Chemical racemization, however, may occur under certain conditions (e.g., high temperatures and acidic/alkaline solutions),2,8,45 and biotic racemization is exemplified by its occurrence in rats, sheep or cattle treated with certain sulfoxide drugs (i.e., flosequinan, pantoprazole, and albendazole sulfoxide).46−48 Proposed mechanisms for the chiral inversion of sulfoxides include pyramidal inversion via planar transition state formation,8 sequential sigmatropic rearrangements via sulfoxidesulfenate transformation,45,49,50 oxygen exchanges via formation of either a dication or a radical cation intermediate,45 and in vivo reversible metabolism between sulfoxides and thioethers/ sulfones.46,47 Here, any consideration of the above chemical mechanisms for the isomeric conversions of FSO in soils is precluded because the isolated stereoisomers of fenamiphos, FSO, and FSO2 remained intact in the stock solutions at room temperature for at least two years. The successive sulfoxidation of fenamiphos is found to be unidirectional in soils, indicating that reversible metabolism does not occur. Thus, these proposed mechanisms all fail to describe the isomeric conversions of FSO in soils, highlighting the need for further investigations. Selective Depletion of FSO2 Fortified in Soils. After the 65-day incubation, 38−59% of the fortified rac-FSO2 as a starting chemical was depleted in the nonautoclaved soils compared to losses of 14−22% in the autoclaved soils (SI Figure S9). The EF values (0.52−0.54) of residual FSO2 approximated the racemic one in all the soils, indicating a lack of stereoselectivity. As indicated by the intact configuration of each separately fortified

and SSPS(−)-FSO; the fractions of all the stereoisomers declined during incubation. The order of the relative fractions of the stereoisomers in the XF soil was the same as in the SD soil, although different trends were observed for the four stereoisomers (i.e., an increase in SSPS(−)-FSO, initial stability and subsequent decline for SSPR(−)-FSO, initial decline and subsequent stability for SRPS(+)-FSO, and an overall decline for SRPR(+)-FSO). Consequently, this intermediate was enriched in SSPR(−)- and SSPS(−)-stereoisomers (SI Figure S6), accounting for the dramatic enantioselectivity for two pairs of enantiomers (i.e., SRPR(+)-FSO versus SSPS(−)-FSO and SSPR(−)-FSO versus SRPS(+)-FSO). The former declined while the latter increased. Dramatic stereoselectivity also occurred for other stereoisomer pairs. In contrast, FSO2 produced from FSO exhibited similar stereoselectivity profiles in the soils with the R(+)-enantiomer enrichment. Isomeric Conversions and Selective Depletion of the Fortified FSO. However, the relative fractions of stereoisomers of the fortified rac-FSO in three soils occurred in the same order irrespective of soil type and incubation time: SSPR(−)- ≥ SSPS(−)- > SRPS(+)- ≈ SRPR(+)-FSO (SI Figure S7). Obviously, the fortified FSO differred in patterns of depletion and selectivity from this chemical in the reaction intermediate form, as the latter underwent the parallel formation and depletion processes (SI Figure S6). Moreover, the separate incubations with the stereoisomers indicated that pure stereoisomers underwent configuration conversions and their patterns depended on soil type (Figure 3, SI Figure S8). In addition to SRPR(+)- and SSPR(−)-FSO, SSPS(+)- and SRPS(−)-FSO were produced in the three nonautoclaved soils fortified by S(−)fenamiphos, demonstrating that the conversions occur in the presence of microorganisms (Figure 2). Specifically, in the low-carbon SD soil, SRPR(+)-FSO was the most degradable stereoisomer, with a removal rate of 91% (on day 21), followed by SRPS(+)- (77%), SSPR(−)- (76%), and SSPS(−)-FSO (56%). This order of degradability may account for the profile of the relative fractions of these stereoisomers in the soil (SI Figure S7). The same order was observed for the formation rate of FSO2 from these stereoisomers, explaining the enrichment of R(+)-FSO2 in the soil (Figure 3, SI Figure S7). Furthermore, FSO underwent isomeric conversions at low rates, including the transformation of SSPS(−)- into SRPS(+)-FSO and formation of SRPR(+)-FSO from the other three stereoisomers (Figure 3). The transformation of SSPS(−)- into SRPS(+)-FSO and SRPS(+)-/SSPR(−)- into SRPR(+)-FSO was unidirectional diastereomerization, and the conversion of SSPS(−)- into SRPR(+)-FSO was unidirectional enantiomerization. This finding well explains the formation of SRPR(+)- and SSPR(−)-FSO in the S(−)-fenamiphos-fortified soil (Figure 2). In the carbon-rich TL soil, bidirectional diastereomerization between SRPR(+)- and SSPR(−)-FSO or SRPS(+)- and SSPS(−)-FSO occurred; the former with a conversion rate of approximately 13% (on day 21) was responsible for the formation of SRPR(+)- and SSPR(−)-FSO in the S(−)fenamiphos-fortified soil (Figure 2). SSPR(−)-FSO that itself had the second highest degradability was pseudopersistent in the rac-FSO-fortified soil, because of high rates of conversion of both SRPR(+)- and SRPS(+)-FSO into this stereoisomer (Figure 3). SSPS(−)-FSO appeared to be stable in the rac-FSO-fortified soil. Two explanations for this are proposed: SSPS(−)-FSO may actually be the least degradable in the soil, and the formation of this species from SRPS(+)-FSO may offset its loss. SRPS(+)FSO underwent rapid depletion primarily by configuration 11281



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Figure 4. Proposed pattern of the stereochemistry of successive sulfoxidation of fenamiphos to FSO and then to FSO2 in the nonautoclaved soils. The sign of atoms is indicative of reaction pathways of intermediate stereoisomers from an optically active precursor.

Similar results have been observed for other chiral pollutants, demonstrating that the enantioselectivity is related to many factors (e.g., soil pH, organic matter, and redox characteristics).12,19,41 This index, for example, is a function of soil pH for certain compounds (e.g., cis-epoxiconazole, metalaxyl, and dichlorprop); however, for other compounds (e.g., metalaxyl acid, two diastereomers of cyproconazole, and Ethofumesate), no correlation is observed.27,41 The variation in enantio-/ stereoselectivity with soil type is commonly attributed to the diversity of soil microorganisms (related to soil type), and the relatively constant selectivity may be a consequence of similar communities of microorganisms in soils.9,12 Stereoselectivity in Developmental Effects toward Embryos. Fenamiphos and the two oxidates showed different stereoselectivity in morphological abnormalities and hatching rates, as indicated by their respective dose−response curves and LC50 values (SI Figure S10, Table S6). For the 2.0 mg/L case (SI Figure S11), both crooked body and yolk sac edema were observed for the rac-fenamiphos exposure, crooked body was observed for the R(+)-fenamiphos exposure, and both pericardial edema and yolk sac edema were observed for the antipode exposure. Crooked body occurred for both the rac- and R(+)FSO2 exposures, compared to only pericardial edema for the S(−)-FSO2 exposure. No abnormality was observed in the SRPS(+)-FSO exposure, while both pericardial edema and yolk sac edema for the SSPS(−)-FSO exposure and crooked body for the other three exposures were observed. A comparison of the LC50 values showed that the R(+)-enantiomers of fenamiphos and FSO2 had higher efficacies than the antipodes and that SSPR(−)- and SSPS(−)-FSO were more active than the other two stereoisomers (SI Figure S10, Table S6). For fenamiphos, similar toxicological selectivity has been reported in other biological assays (e.g., acute Daphnia toxicity, AChE inhibition, and PC12 cell line toxicity).51,52 Although no data related to the selective toxicity for FSO and FSO2 are available, similar metabolites of other organophosphorus insecticides (e.g., fenthion and profenofos) have been found to be enantioseletive and one to 2 orders of magnitude more inhibitory to AChE.16,53,54 These organophosphorus insecticides and metab-

enantiomer, FSO2 did not undergo chiral inversion. Thus, the hydrolysis of FSO 2 was biotically facilitated, but not enantioselective. Obviously, the R(+)-enantiomer enrichment of FSO2 as a reaction intermediate in the degradation of fenamiphos and FSO was an outcome of the selective sulfoxidation of FSO stereoisomers yielding R(+)-FSO2 at higher rates as a whole (SI Figures S6 and S7). Stereochemistry of Successive Sulfoxidation of Fenamiphos in Soils. Fenamiphos underwent a variety of concomitant reactions in soils, including oxidation of the parent (fenamiphos) to the primary intermediate (FSO), further oxidation of FSO to FSO2, isomeric conversions of FSO, and hydrolysis of fenamiphos and the two oxidates to phenol intermediates (Figure 4). The former three reactions constituted the stereoselective successive sulfoxidation and were pertinent to the asymmetric sulfur atom, whereas the last one was nonstereoselective at the asymmetric phosphorus atom. During the successive sulfoxidation, the stereochemistry transferred principally through two parallel systems: R(+)-fenamiphos → SRPR(+)-/SSPR(−)-FSO → R(+)-FSO2 and S(−)-fenamiphos → SRPS(+)-/SSPS(−)-FSO → S(−)-FSO2. The stereochemistry also transferred by a secondary system of S(−)-fenamiphos → SRPR(+)-/SSPR(−)-FSO → R(+)-FSO2, because the isomeric conversions of FSO resulted in unidirectional intersystem crossing between the two systems, and hence S(−)-fenamiphos was transformed to R(+)-FSO2. Enantio- or stereoselectivity of degradation of chiral pollutants can be described by the ratio of the difference between degradation rates of two isomers to their sum.27,41 Interestingly, the enantioselectivity for fenamiphos decreased linearly with soil organic matter increasing (R2 = 0.89); the enantioselectivity for FSO2 remained nearly constant, while both the enantio- and stereoselectivity for FSO varied with soil characteristics (SI Table S5). The enantioselectivity for SSPR(−)- and SRPS(+)-FSO, one of the two enantiomer pairs of FSO, was nearly constant (−0.233 in the XF soil to −0.210 in the TL soil) across soil types, whereas, for other stereoisomer/enantiomer pairs of FSO, no statistically significant correlation was found between the enantio-/stereoselectivity and soil organic matter (or pH). 11282



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of these compounds, a more comprehensive understanding of the significance of stereochemistry is imperative for improving risk assessment and regulation.

olites (including fenamiphos and the oxidates) are commonly considered to be highly toxic, because they are potent AChE inhibitors toward organisms.16,25,54 Note that SSPR(−)- and SSPS(−)-FSO, both of which are most abundant in the soils with fenamiphos application, are toxicologically close to the highly toxic S(−)-fenamiphos, further supporting that FSO dominates insecticidal efficiency of the insecticide.26,33,38,40 Environmental Implications. Fenamiphos is a representative of currently used thioether insecticides. The stereoisomers of this insecticide and its primary transformation products were found to exhibit dramatic differences in their biotic transformation in soils and acute toxicity toward zebrafish embryos. As the parent and transformation products are all of toxic concern, the formation/depletion of intermediate stereoisomers may be an important issue for ecological risk assessment. This concern was highlighted by the monitoring data based on the racemic concentration showing that both detection frequency and concentration in waters follow the order FSO ≫ FSO2 > fenamiphos (SI Table S7).25,55,56 It is reasonable to expect from the stereochemistry of the successive sulfoxidation (Figure 4) that stereoisomeric compositions of these compounds deviate from the racemate. Then, the fraction of each stereoisomer was designed as the levels of 0−1.0 to calculate a hazard quotient (HQ). The HQ of fenamiphos total toxic residues at the stereoisomer level was 0.116−0.977 (California), 0.030−30.380 (Georgia), and 0.033−1.171 (Florida), as compared with their respective racemate-based values of 0.230−0.527, 0.069−13.025, and 0.085−0.646 (SI Table S7). Obviously, the inclusion of stereochemistry indeed leads to dramatic variations of the potential risks of the insecticide toward embryos (Figure 5), suggesting that the use of the racemate-based data, as currently practiced, may underestimate or overestimate its risks. More importantly, sulfur-related chemicals, being chiral or prochiral, commonly share the same mode of action and/or pattern of metabolism. The stereochemistry observed for fenamiphos and its transformation products may be extrapolated to other pesticides or drugs in its class. Given the widespread use

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S Supporting Information *

Additional text, tables, and figures as described in the text. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +86-411-8470 7844; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Basic Research Program of China (No. 2013CB430403), the National Natural Science Foundation of China (Nos. 41171382, 21477013, and 21137001), the Fok Ying Tung Education Foundation (No. 114042), the Program for New Century Excellent Talents in University (No. NCET-11-0047), the Fundamental Research Funds for the Central Universities, the open foundation of Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education (No. KL-PPEC-2013-03), and the program for Changjiang Scholars and Innovative Research Teams in University (No. IRT-13R05). The artificial climate incubator used was donated by the International Foundation for Science (No. F/4580-1).

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REFERENCES

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Figure 5. Comparisons of racemate- and stereoisomer-based hazard quotients of fenamiphos, FSO, FSO2, and total toxic residues (TTR, a sum of fenamiphos parent plus FSO and FSO2). 11283



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Probing the Stereochemistry of Successive Sulfoxidation of the

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