Enantioselectivity in degradation and ecological risk of the chiral pesticide ethiprole

Intensive agricultural activities have caused land degradation due to soil pollution, particularly by pesticides. However, the degradation, metabolism, and toxicity of chiral pesticides by soil microorganisms are often enantioselective. This study aimed to determine the effect of chirality on the degradation of the enantiomers of ethiprole in soil and their impact on soil microbial communities. (R)‐ethiprole underwent directional chiral conversion to the (S)‐enantiomer in a paddy soil microcosm, leading to elevated concentrations of (S)‐ethiprole. Initially, the bacterial operational taxonomic units significantly decreased after 3 days of incubation with rac‐ethiprole, (R)‐ethiprole, and (S)‐ethiprole but gradually increased in the later stage. Principal coordinate analysis revealed that the bacterial community structure was enantioselectively affected by the ethiprole enantiomers. Within 3 days, both rac‐ethiprole and (R)‐ethiprole reshaped the original stochastic microbial community into a deterministic community (variable selection). Thus, we propose that the enantioselective behavior and ecotoxicology of chiral pesticides need to be considered, especially because there are numerous chiral pesticides currently in use within agricultural management. The comprehensive understanding of the ecological risk of chiral pesticide enantiomers is vital to the process of improving sustainable production and environmental health in agricultural ecosystems.

caused by source pollution in cultivated lands and to some extent by nonpoint source pollution due to long-range atmospheric deposition (Epple, Maguhn, Spitzauer, & Kettrup, 2002;Potter & Coffin, 2017). In recent decades, pesticide use in cultivated land grew due to an increase in global food demand. In 2017, approximately 1.8 × 10 6 and 3.9 × 10 5 tons of pesticides were applied in China and the United States, respectively (de Albuquerque, Carrão, Habenschus, & de Oliveira, 2018). However, it is estimated that the amount of pesticides reaching the target pests is less than 0.1% of the pesticide applied and the rest enters the environment (Pimentel, 1995). Therefore, pesticide pollution may adversely affect the structure and function of soil microbial communities in agricultural fields. Soil microbes play a critical role in global element cycling, as well as mediating transformation of organic matter through those processes, including the degradation of xenobiotics and mineralization of plant nutrients. (Bowles, Acosta-Martínez, Calderón, & Jackson, 2014;Zhao et al., 2016). Hence, the need for systematic evaluation of pesticides on the effects of microbial communities in various soil types.
The enantioselective behavior of chiral pesticides is closely related to soil properties, including pH, organic matter content, and incubation conditions (Buerge, Poiger, Müller, & Buser, 2003, 2006Frková et al., 2016; X. Wang et al., 2013). For example, (S)-methamidophos was preferentially degraded in soil obtained from Zhengzhou but was enriched in soils from Changchun and Nanchang (X. Wang et al., 2013). Furthermore, inversion of one enantiomer to its antipode has been observed in some soil samples probably due to microbial activity (Han, Kitagawa, Wzorek, Klika, & Soloshonok, 2018); for example, (R)-and (S)-malathion interconverted (Sun et al., 2012) and (S)-haloxyfop converted to (R)haloxyfop (Poiger, Müller, Buser, & Buerge, 2015). Hence, it is essential to understand the role of microbial communities in the stereoselective environmental behavior of chiral pesticides in different soils. However, previous studies showed that intensive agricultural activities and increased pesticide cause a reduction in microbial diversity and populations (Fang, Lian, Wang, Cai, & Yu, 2015). However, the effect of chiral pesticides on soil microbiome (especially with regard to microbial biomass and activity) is often overlooked due to complexities of microbe-soil and chiral interactions. Therefore, there is a need to determine the effect of pesticide chirality on bacterial communities as it affects the function and structure of the agricultural ecosystem.
Ethiprole is a broad spectrum phenylpyrazole insecticide with an asymmetric sulfur atom, existing as (R)-and (S)-enantiomers and applied as a racemic mixture. It exerts its toxicological effects by blocking the passage of chloride ions through GABA (γ-aminobutyric acid) receptors in the central nervous system and is effective against pests in paddy soils (Moffat, 1993). There is a lack of systematic studies on the stereoselective degradation of ethiprole and its impact on soil microbial communities. In this study, the mechanisms of enantioselective behavior of ethiprole in five latitudinal paddy rice soils with different organic matter content and pH values were investigated. High-throughput sequencing technologies were used to determine microbial community changes (here, focus on bacterial community) following exposure to chiral ethiprole and its enantiomers.
This study aims to establish the correlations between enantioselective degradation of ethiprole and microbial community. Those results have significant implications for the prediction of potential risks of chiral pesticides to agricultural ecosystems and public health.

| Overview
The details of the experimental approach, including ethiprole-spiking procedures, extraction in soil, and chiral analysis, can be found in Q. Zhang et al. (2016). Briefly, the incubation experiment was conducted over a 180-day period in soil microcosms. Five types of soil samples (top 10-cm layer) were collected from different latitudinal in the main paddy rice sites in China (Ji Lin, Nan Jin, Jiang Xi, Guang Dong, and Hai Nan). The samples were air dried, sieved (2-mm mesh), and stored at 4°C. The properties of the five types of soils were analyzed using established methods (Xiong et al., 2017), and soil properties were described in Table S1. No ethiprole enantiomers were detected in any of the soil samples. Sterilized soils were used in the soil microcosms experiment after spiking with rac-ethiprole, (R)-ethiprole, or (S)-ethiprole in triplicate (Table S2). For each soil microcosm, 20.0-g soil (dry weight) was spiked with 0.1 ml of acetone containing either 800.0 μg of racemic ethiprole or 400.0 μg of individual enantiomers in a 500-ml conical flask. The samples were thoroughly stirred for 10 min. It was ensured that the acetone was adequately evaporated to avoid adverse effects on microbiological activity. An additional 380.0-g nonsterilized soil was added and mixed thoroughly, yielding a final concentration of racemic ethiprole or individual enantiomers of 2.0 or 1.0 μg g −1 , respectively. All soils were maintained at a 20.0% moisture content and incubated at 25°C in the dark to facilitate activation of soil microorganisms. Soil incubations containing only deionized water as control were performed to further understand the impact of chiral ethiprole on soil microbial diversity. At selected time intervals (2 hr, 1, 3, 7, 14, 21, 30, 45, 60, 90, 120, 150, 180 days), triplicate 20.0-g soil aliquots from each treatment were randomly removed and were immediately transferred to a −80°C freezer.

| Enantioselective degradation
Equivalent 10.0-g soil samples were extracted with 5.0 ml of distilled water and 50.0 ml of acetonitrile in 100-ml centrifuge tubes. Each tube was shaken vigorously for 4 min before undergoing sonication extraction for 15 min. The water was removed with the addition of 4.0 g of anhydrous magnesium sulfate (MgSO 4 ) and 2.0 g of sodium chloride (NaCl). The chemicals and materials used for extraction are described in Method S1. The mixture was vortexed vigorously and then centrifuged at 4,000 rpm for 3 min. The supernatant acetonitrile (25 ml) was collected and completely evaporated. The clean-up method of these soil extracts was performed according to an existing research protocol using a Florisil solid phase extraction column (Q. Zhang et al., 2016;. The ethiprole residue was then redissolved in 1 ml of methanol and water solution (65:35, v/v). After filtration through a 0.22-μm filter membrane, the soil extracts were analyzed on a chiral Lux Cellulose-2 by Agilent 1200 HPLC system (Agilent, USA) using a mixture of methanol and water (65:35, v/v) as the mobile phase at a flow rate of 0.7 ml min −1 . The UV detection was performed at 225 nm with a chiral column temperature at 35°C. The recoveries and limit of detection are shown in Method S2. Stereoselectivity was determined using the enantiomeric fraction (EF) and the enantiomeric ratio (ER), as well as over time using Δk (Method S3).
The degradation and chiral conversion (S → R) rate constants were determined based on first-order processes defined by the following equations (Poiger et al., 2015):

| Bioinformatics and statistical analyses
After removing the adaptors and primer sequences, we assigned the raw sequences to each sample based on the unique barcode provided by QIIME (Caporaso et al., 2010). The split sequences of each sample were merged with FLASH V1.2.7 (Magoč & Salzberg, 2011). The retained sequences of each sample were processed following the established UPARSE pipeline (Edgar, 2013). In addition, the lower quality score (<0.5) and short length sequences (<200 bp) were removed.  (Shannon et al., 2003). Moreover, the abundanceweighted β-mean nearest taxon distance (βMNTD) was used to determine and quantify community phylogenetic turnover. The β-nearest taxon index (βNTI) was further used to quantify the magnitude and direction of a deviation between an observed βMNTD value and the null βMNTD distribution (Dini-Andreote, Stegen, van Elsas, & Salles, 2015). A |βNTI| > 2 indicates significant deviation following the dominance of deterministic processes, which includes homogeneous (βNTI < −2) and variable selection (βNTI > 2). When the |βNTI| < 2,

| Statistical analysis
Spearman's rank correlation coefficients between the indicator species the first stage being 2.9 times slower than the second stage. The soil microorganisms in Jiang Xi probably required a period of adjustment before the metabolism of ethiprole commenced; hence, the observed initial time lag. A short lag period suggests that the observed enantioselective degradation was probably mediated by soil microorganisms (Lao & Gan, 2012). It is possible that the long-term presence of ethiprole induces the corresponding microbial populations and enantiomer-specific enzymes (Dong et al., 2012;Yang & Ji, 2015). Therefore, the properties of soil and associated microbial communities have a substantial impact on the enantiomeric composition of chiral ethiprole in the soil.

| Directional chiral conversion of ethiprole in soils
For the racemic ethiprole incubation experiments, the final concentrations of (S)-ethiprole were shown to be higher than the spiked concentration ( Figure S1) The results suggest that there was a unidirectional chiral inversion of (R)-ethiprole to (S)-ethiprole. Ethiprole is a chiral sulfoxide (R 1 S(O)R 2 (R 1 ≠R 2 )); however, previous studies have shown that thermal, photochemical, or chemical processes can cause chiral inversion in chiral sulfoxides through processes such as pyramid atomic inversion (Jenks, Matsunaga, & Gordon, 1996;Marom, Biedermann, & Agranat, 2007). Furthermore, a study on flosequinan, a chiral sulfoxide quinolone vasodilator, demonstrated that bidirectional chiral inversion is probably mediated by microbes (Eiji, Tsuyoshi, Takashi, Masaaki, & Tetsuya, 1994). The mechanism of chiral inversion involved stereoselective reduction of (R)-or (S)-flosequinan by gut microbes to form a sulfide, which was subsequently oxidized to produce the antipode (Eiji et al., 1994). Therefore, because the soil microcosm was kept in darkness and the temperature was similar for all treatments, chiral inversion of (R)-ethiprole was probably caused by an interaction with chemical constituents of the different soil samples or microbial activity. Furthermore, chiral inversion was not observed in the sterilized soil microcosms.

| Correlation between the enantioselective behavior of ethiprole and soil properties
The correlation between the enantioselective behavior of ethiprole and soil properties was statistically analyzed based on five different regions of soil amended with rac-ethiprole. When the EF of the last  probably proliferated in high carbon and nitrogen soils. Previous studies suggested organic matter promotes pesticide degradation by providing carbon sources for the soil microbial growth (Qi et al., 2016;. However, high organic matter content may decrease the degradation rate because more pesticides will adsorb to the soil organic matter and become less available for contacting with soil microbes (Qi et al., 2016). Because stereoselectivity was not observed in sterilized treatments, chiral inversion in soils with high organic matter was probably caused by the increase in microbial activity and growth. Therefore, the enantioselectivity of ethiprole has a close relationship with the presence of soil the nutrients.

| Indicator species by the treatment of chiral ethiprole
The indicator species of bacterial OTUs are used to assess the efficacy of chiral ethiprole in agricultural management (Hartmann, Frey, Mayer, Mader, & Widmer, 2015;Siddig, Ellison, Ochs, Villar, & Lau, 2016;Sun et al., 2016). Indicator species (OTUs) for the bacteria present in soils amended with different chiral enantiomers are shown in Figure 4. The control soil samples amended with water contain extremely abundant indicator species, and they mainly included Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The higher relative abundances of indicator species in (S)-ethiprole amended soil samples were OTU5,11,17,and 438,all (Table S4). The relative abundances of the indicator species decreased at higher concentrations of (R)-ethiprole in the soil.
Hence, the chiral conversion of (S)-ethiprole to its antipode influenced these bacterial OTUs. A soil microcosm study found that soil samples treated with (R)-imazethapyr had the lowest diversity compared whereas (S)-imazethapyr-treated samples appeared to be more diverse based on Shannon's and Simpson's diversity indices (Qian et al., 2015).
However, the presence of rac-ethiprole caused massive reductions of indicator species, with only OTU113 (Oryzihumus) and 464 (candidate division WPS-1) observed in paddy rice soil following ethiprole addition. Therefore, the ecological and toxicological impact of rac-ethiprole on bacterial communities will probably create a higher ecological risk in agriculture fields.

| Enantioselectivity in microbial assembly
The application of agricultural pesticides in farming systems is increasing the importance of deterministic selection in ecological succession.
The βNTI value in water treated and (S)-ethiprole treated soils showed that the stochastic processes (−2 < βNTI < +2) were dominant throughout the time series ( Figure 5). The βNTI values in soil incubated with rac-ethiprole and (R)-ethiprole revealed microbial assembly distribution progressively shifted with increasing incubation time, from initially being stochastic community assembly (−2 < βNTI < 2) to subsequently being a deterministic selection (variable selection; βNTI > 2) within 3 days. The soil incubation with (R)-ethiprole restored stochastic community assembly (−2 < βNTI < 2) from 7 to 60 days (Dini-Andreote et al., 2015). The soil incubation with rac-ethiprole restored stochastic community assembly (−2 < βNTI < 2) from 7 to 14 days and then transitioned to deterministic selection (homogeneous selection; βNTI < −2) after 30 days (Dini-Andreote et al., 2015). (R)-ethiprole and rac-ethiprole had a strong driving effect on bacterial community succession in the soil. Therefore, it appears that only one enantiomer may be expected to contribute significantly to the ecological effects for some chiral insecticides. This knowledge will contribute to a complete understanding of ecological effects caused by the application of chiral pesticides.

| CONCLUSION
The present outcomes indicate that the enantioselective behavior and ecological effects of the chiral phenylpyrazole insecticide ethiprole occurred concurrently in a simulated agricultural ecosystem. (R)ethiprole was readily converted to the (S) isomer in paddy soils resulting in (S)-ethiprole occupying a higher percentage of total ethiprole in agricultural fields. Enantioselectivity for ethiprole shows a close relationship with the presence of soil nutrients, (i.e., total organic carbon and nitrogen). Therefore, different enantioselectivity occurs for chiral pollutants depending on the application of various fertilizers, such as organic fertilizer, which may be effective in reducing the concentrations of highly toxic chiral enantiomers in agricultural systems (Mueller & Buser, 1995). Moreover, the selective conversion of chiral ethiprole proliferated within the groups Luteimonas, Comamonadaceae, and Xanthomonadaceae in bacteria.
This information is conducive to the development of chiral pharmaceuticals and pesticides with a short half-life, high bioactivity, and low ecological toxicity. Importantly, the chiral ethiprole enantiomers exert dramatically different influences on soil microbial community structures and ecological succession in agroecosystems (Hawkesworth & García Pérez, 2003). Therefore, we propose that the enantioselective behavior and resultant ecotoxicology of chiral pesticides need to be considered in pesticide registration before the application of a chiral pesticide in agricultural systems. Our results reveal a potential implication for environmental protection and ecosystem service. The comprehensive understanding of chiral pesticide fate is aimed at reducing the concentrations of high-risk chiral pesticide enantiomers and improving sustainable production in agroecosystems.

ACKNOWLEDGMENT
This study was supported by the National Key Research and Development Program of China (2016YFD0200207).