Chiral analysis and semi-preparative separation of metconazole stereoisomers by supercritical fluid chromatography and cytotoxicity assessment in vitro.

Metconazole is one of the widely-used chiral triazole fungicides in controlling wheat leaf rust, powdery mildew, Fusarium head blight with high efficacy, and so forth. In the current work, the effects of chiral stationary phases, alcoholic modifiers, and column temperature on the chiral separation of metconazole were discussed in detail. Amylose tris(3,5-dimethylphenylcarbamate)-coated chiral stationary phase exhibited much stronger chiral recognition ability toward metconazole stereoisomers in the CO2 /ethanol mixture as compared to the others. Then, a two-step semi-preparative separation of metconazole was performed through supercritical fluid chromatography and high-performance liquid chromatography, and the enantiomeric excess values of four stereoisomers were achieved over 98%. Moreover, the enantioselective cytotoxicity of cis-metconazole against HepG2 cells has been investigated, and the order of the cell proliferation toxicity against HepG2 cells was (1R, 5S)-metconazole > (1S, 5R)-metconazole > the mixture. Briefly, this study would provide valuable information in the preparative separation of optically pure metconazole products through chromatographic techniques and their environmental risk assessment.

Metconazole inhibits fungal growth and toxin production in major Fusarium species that cause rice panicle blight.

Rice panicle blight (RPB) caused by various Fusarium spp. is an emerging disease in the major rice-growing regions of China. Epidemics of this disease cause significant yield loss and reduce grain quality by contaminating panicles with different Fusarium toxins. However, there is currently no registered fungicide for the control of RPB in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against several Fusarium spp. that cause wheat head blight, wheat crown rot and maize ear rot. In this study, we investigated the specific activity of metconazole against six Fusarium spp. that cause RPB. Metconazole significantly inhibited mycelial growth, conidium formation, germination, germ tube elongation and major toxin production in Fusarium strains collected from major rice-growing regions in China, as well as disrupting cell membrane function by inhibiting ergosterol biosynthesis. Greenhouse experiments indicated a significant reduction in blight occurrence and toxin accumulation in rice panicles treated with metconazole. Overall, our study demonstrated the potential of metconazole for managing RPB and toxin contamination, as well as providing insight into its bioactivities and modes of action of metconazole against distinct Fusarium spp.

In Vitro and Ex Vivo Antifungal Activities of Metconazole against the Rice Blast Fungus Pyricularia oryzae.

Rice blast, caused by the filamentous fungus Pyricularia oryzae, has long been one of the major threats to almost all rice-growing areas worldwide. Metconazole, 5-(4-chlorobenzyl)-2, 2-dimethyl-1-(1H-1, 2, 4-triazol-1-ylmethyl) cyclopentanol, is a lipophilic, highly active triazole fungicide that has been applied in the control of various fungal pathogens of crops (cereals, barley, wheat), such as the Fusarium and Alternaria species. However, the antifungal activity of metconazole against P. oryzae is unknown. In this study, metconazole exhibited broad spectrum antifungal activities against seven P. oryzae strains collected from rice paddy fields and the wild type strain P131. Scanning electron microscopic analysis and fluorescein diacetate staining assays revealed that metconazole treatment damaged the cell wall integrity, cell membrane permeability and even cell viability of P. oryzae, resulting in deformed and shrunken hyphae. The supplementation of metconazole in vitro increased fungal sensitivity to different stresses, such as sodium dodecyl sulfate, congo red, sodium chloride, sorbitol and oxidative stress (H2O2). Metconazole could inhibit key virulence processes of P. oryzae, including conidial germination, germ tube elongation and appressorium formation. Furthermore, this chemical prevented P. oryzae from infecting barley epidermal cells by disturbing appressorium penetration and subsequent invasive hyphae development. Pathogenicity assays indicated a reduction of over 75% in the length of blast lesions in both barley and rice leaves when 10 µg/mL of metconazole was applied. This study provides evidence to understand the antifungal effects of metconazole against P. oryzae and demonstrates its potential in rice blast management.

Exploring the repairing mechanisms of reduced graphene oxide (rGo) on the dysregulation of Xenopus Laevis larva hypothalamus-pituitary-thyroid (HPT) axis caused by chiral triazole fungicide metconazole.

Replacing chair fungicide racemate marketed product by its enantiomer with high activity and low environmental risk for application is a more environmentally friendly methods to control crop diseases. Moreover, carbon-based nanomaterials, with the desirable chemical and mechanical properties, exhibits latent reduce fungicide toxicity capability, while the mechanism is still poorly understood. Therefore, the present study characterized the toxicity of rac-metconazole (Mez; (1RS,5RS;1RS,5SR)-5-(4-chlorobenzyl)-2,2-dimethyl-1-(1H)) and its two cis-enantiomers as well as the repairing effect of reduced graphene oxide (rGo) on Xenopus Laevis larva by examining growth appearance indexes, Mez bioaccumulation, and hypothalamus-pituitary-thyroid (HPT) axis related hormone contents and gene expression after 14 and 28 days exposure. Compared with two cis-Mez, rac-Mez was preferentially bioaccumulated in tadpoles, and rac-Mez treatment showed a higher toxicity effect on tadpole including growth stage and body weight inhibition by dysregulating tadpole thyroid stimulating hormone (TSH) and thyroid hormone (TH) contents and related gene expression. Enantioselectivity was observed in two cis-Mez treatments. Compared with R,S-Mez, S,R-Mez treatment showed more severe damage on tadpole HPT axis related physiological and biochemical processes. rGo could effectively decrease the toxicity of Mez, especially shown the capacity of repairing the hormone dysregulation caused by R,S-Mez treatment. Moreover, the addition of rGo can decrease the bioaccumulation of Mez in tadpoles. Therefore, R,S-Mez is less toxic to Xenopus Laevis larva growth, and its toxicity could be effectively repaired by the addition of rGO.

Antifungal activities of metconazole against the emerging wheat pathogen Fusarium pseudograminearum.

Fusarium crown rot of wheat is a serious fungal disease that occurs worldwide. The disease has been emerging in the major wheat-growing areas in China since 2010. Fusarium pseudogramineaum is the predominant causative pathogen of crown rot of wheat in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against Fusarium spp., but little is known about its specific activity against F. pseudogramineaum. Metconazole exhibited strong antifungal activities against all thirty-nine F. pseudogramineaum strains collected from the major wheat-growing areas in China. Metconazole inhibited mycelial growth and conidial germ tube elongation of F. pseudograminearum. Metconazole treatment significantly reduced the production of major toxins and the expression levels of toxin biosynthesis genes. Genome-wide transcriptional profiling of F. pseudograminearum in response to metconazole indicated that the expression of genes involved in ergosterol biosynthesis, including fungicide target genes (cyp51 genes), was significantly induced by metconazole. Nine ATP-binding cassette (ABC) transporter-encoding genes were significantly expressed in response to metconazole treatment. Reduced ergosterol production and antioxidant enzyme activities were observed after metconazole treatment. Greenhouse experiments indicated a significant reduction in crown rot occurrence in wheat after seed treatment with metconazole. This study evaluated the potential of metconazole to manage wheat crown rot and provides information to understand its antifungal activities and mechanism of action against F. pseudograminearum.

Enantioselective effects of the fungicide metconazole on photosynthetic activity in Microcystis flos-aquae.

Enantiomers of chiral fungicides usually display different toxic effects on nontarget organisms in the surrounding environment, although there are rare reports on the enantioselective toxicity of metconazole (MEZ) to aquatic organisms, such as Microcystis flos-aquae (M. flos-aquae). To explore the enantioselective toxicity of MEZ in algae, the impact of various concentrations (0.001, 0.003, 0.01, 0.03 and 0.1 mg/L) of MEZ on M. flos-aquae over 8 days was investigated. Significant differences were observed between the four enantiomers in chlorophyll a (Chl a) contents, carotenoids, photochemical efficiency (Fv/Fm), rapid light-response curves (RLCs), utilization efficiency of light energy (α) and protein contents during treatment time. MEZ can enantioselectively stimulate the chlorophyll fluorescence parameters (RLCs, Fv/Fm and α) and carotenoid and Chl a contents of M. flos-aquae, especially at low concentrations (0.001 or 0.003 mg/L). At high concentrations of 0.03 or 0.1 mg/L, the chlorophyll fluorescence parameters (RLCs, Fv/Fm and α), protein and Chl a contents of M. flos-aquae exposed to cis-enantiomers were lower than those of M. flos-aquae exposed to trans-enantiomers. These observations indicated that the enantiomers of MEZ pose different toxicities to M. flos-aquae, with the cis-enantiomers more toxic than the trans-enantiomers. These results are beneficial for understanding the enantioselective effects of MEZ enantiomers on nontarget organisms and helpful for evaluating their eco-environment risk.

Molecular and biological characteristics of laboratory metconazole-resistant mutants in Fusarium graminearum.

The Fusarium graminearum species complex (FGSC), the causal agents of Fusarium head blight (FHB) in wheat, has the different geographically distributed species. Our previous study suggested that a DMI fungicide metconazole exhibits a strong fungicidal activity in mycelial growth of Chinese FHB pathogens and metconazole is currently a most effective compound of commercial fungicides for controlling FHB in China. In the current study, metconazole-resistant F. graminearum mutants were induced by chemical taming and their molecular and biological characteristics were determined. Compared to the corresponding parental strains, three mutation genotypes (two single mutations G443S and D243N, and a combined mutation E103Q&V157 L) were observed in the FgCYP51A of metconazole-resistant mutants. In addition to FgCYP51A mutation, all the mutants had no change on sequences of FgCYP51B and FgCYP51C and promotor sequences of FgCYP51s, but expression patterns of FgCYP51s were different. Compared to the corresponding parental strains, overexpression of FgCYP51A, FgCYP51B and FgCYP51C was observed in the mutant conferring D243N mutation, overexpression of FgCYP51A and FgCYP51B was observed in the mutant conferring E103Q&V157L mutations, and overexpression of FgCYP51A was observed in the mutant conferring G443S mutation. Biological fitness of the mutants conferring D243N mutation or E103Q&V157 L mutations significantly decreased in comparison to the corresponding parental strains, suggesting a fitness penalty. The mutants conferring G443S mutation had no change in biological fitness as compared with the parental strain, indicating that the G443S mutation may emerge in field resistant populations of F. graminearum in the future. In addition, a positive cross resistance between metconazole and other tested DMI fungicides was observed in the mutants conferring D243N mutation or E103Q&V157L mutations, but no cross resistance between metconazole and ipconazole or prochloraz was observed in the mutants conferring G443S mutation. Therefore, we concluded that the mutation genotype of FgCYP51A may cause the differences of biological fitness, cross-resistance and FgCYP51s overexpression patterns. Such information will increase our understanding of resistance mechanism of F. graminearum to DMIs and could provide new reference data for the management of FHB.

Stereoselective study on chiral fungicide metconazole in four kinds of fruits: Absolute configuration, SFC-MS/MS enantioseparation, degradation and risk assessment.

Metconazole is a novel chiral fungicide with two chiral carbon atoms, but the research on its stereoselective behavior is limited. Therefore, the stereoselective behaviors of metconazole in four fruits, including grape, peach, pear and jujube, were summarized in this study. After determining the absolute configuration of metconazole stereoisomers, a chiral separation method through supercritical fluid chromatography/tandem triple quadrupole mass spectrometry was first developed, which combined an improved QuEChERS method obtained the recoveries of 71.6-113 % with RSD ≤ 19.8 %. The LOD and LOQ were 4.30-95.9 and 10.5-143.2 ng/kg, respectively. Different stereoselective and diastereoselective behaviors were observed in four fruits. Dietary risk assessments of rac-metconazole were performed in populations with different ages and genders. Both acute (RQa, 0.0124-0.140 %) and chronic (HQ, 0.0234-0.0794 %) intake risks were acceptable. The results of this study would contribute to more complete risk assessments of metconazole and provide data for chiral studies.

Stereo-selective cardiac toxicity induced by metconazole via oxidative stress and the wnt/ß-catenin signaling pathway in zebrafish embryos.

Metconazole (MEZ), a chiral triazole fungicide, produces enantioselective adverse effects in non-target organisms. Among MEZ's isomers, cis-MEZ displays robust antimicrobial properties. Evaluating MEZ and cis-MEZ's toxicity may mitigate fungicide usage and safeguard non-target organisms. Our study evaluated the toxicity of MEZ and its cis-isomers at concentrations of 0.02, 0.2, 2, and 4 mg L-1. We report stereoselectivity and severe cardiovascular defects in zebrafish, including pericardial oedema, decreased heart rate, increased sinus venous and bulbous arteries distances, intersegmental vessel defects, and altered cardiovascular development genes (hand2, gata4, nkx2.5, tbx5, vmhc, amhc, dll4, vegfaa, and vegfc). Further, MEZ significantly increased oxidative stress and apoptosis in zebrafish, primarily in the cardiac region. Isoquercetin, an antioxidant found in plants, partially mitigates MEZ-induced cardiac defects. Furthermore, MEZ upregulated the Wnt/ß-catenin pathway genes (wnt3, ß-catenin, axin2, and gsk-3ß) and ß-catenin protein expression. Inhibitor of Wnt Response-1 (IWR-1) rescued MEZ-induced cardiotoxicity. Our findings highlight oxidative stress, altered cardiovascular development genes, and upregulated Wnt/ß-catenin signaling as contributors to cardiovascular toxicity in response to MEZ and cis-MEZ treatments. Importantly, 1R,5S-MEZ exhibited greater cardiotoxicity than 1S,5R-MEZ. Thus, our study provides a comprehensive understanding of cis-MEZ's cardiovascular toxicity in aquatic life.

Stereoselective cardiotoxic effects of metconazole on zebrafish (Danio rerio) based on AGE-RAGE signalling pathway.

Metconazole (MEZ) is a novel chiral triazole fungicide that is widely used to prevent and control soil-borne fungal pathogens and other fungal diseases. However, it has a long half-life in aquatic environments and thus poses potential environmental risks. This study evaluates the acute and stereoselective cardiotoxicity of MEZ in zebrafish (Danio rerio) embryos. In addition, transcriptomics, real-time quantitative PCR, enzyme activity determination, and molecular docking are performed to evaluate the molecular mechanisms underlying the cardiotoxicity of MEZ in zebrafish. MEZ decreases the heart rate while increasing the pericardial oedema rate; additionally, it induces stereoselective cardiotoxicity. 1S,5S-MEZ exhibits stronger cardiotoxicity than 1R,5R-MEZ. Furthermore, MEZ increases the expression of Ahr-associated genes and the transcription factors il6st, il1b, and AP-1. Heart development-related genes, including fbn2b, rbm24b, and tbx20 are differentially expressed. MEZ administration alters the activities of catalase, peroxidase, and glutathione-S-transferase in zebrafish larvae. Molecular docking indicates that 1R,5R-MEZ binds more strongly to the inhibitor-binding sites of p38 in the AGE-RAGE signalling pathway than to other MEZ enantiomers. Studies conducted in vivo and in silico have established the enantioselective cardiotoxicity of MEZ and its underlying mechanisms, highlighting the need to evaluate the environmental risk of chiral MEZ in aquatic organisms at the enantiomeric level.

Stereoselective, Diastereoselective Dissipation and Risk Assessment of Chiral Metconazole in Soybean, Peanut, Cabbage, Celery, Tomato, and Soil.

The stereoselective behaviors and dietary risks of metconazole (MZE) in soil and five vegetables were investigated. The results showed that there was species-specific stereoselective and diastereoselective dissipation, and the half-lives ranged from 0.69 to 8.17 days. cis-(+)-1S,5R-MZE was preferentially dissipated in soybean pods, cabbages, celeries, and tomatoes, which was contrary to soybean plants and soil. trans-(+)-1R,5R-MZE was preferentially dissipated in peanut plants, peanut shells, celeries, and tomatoes, while trans-(-)-1S,5S-MZE was preferentially dissipated in soybean plants. cis-MZE was preferentially dissipated in the test vegetables and soil, except celery. The stereoisomeric excess changes were higher than 10%, indicating that the stereoselectivity and diastereoselectivity should be considered in the risk assessment of MZE in soybean plants, pods, and peanut plants. The acute and chronic dietary intake risks of rac-MZE for different groups of people were acceptable. The preferentially dissipated and high activity cis-(+)-1S,5R-MZE with lower toxicity might be suitable for application as monocase.

The triazole fungicide metconazole inhibits the homodimerization of human androgen receptors to suppress androgen-induced transcriptional activation.

We assessed the mechanism of human androgen receptor-mediated endocrine-disrupting effect by a triazole fungicide, metconazole in this study. The internationally validated stably transfected transactivation (STTA) in vitro assay, which was established for determination of a human androgen receptor (AR) agonist/antagonist by using 22Rv1/MMTV_GR-KO cell line, alongside an in vitro reporter-gene assay to confirm AR homodimerization was used. The STTA in vitro assay results showed that metconazole is a true AR antagonist. Furthermore, the results from the in vitro reporter-gene assay and western blotting showed that metconazole blocks the nuclear transfer of cytoplasmic AR proteins by suppressing the homodimerization of AR. These results suggest that metconazole can be considered to have an AR-mediated endocrine-disrupting effect. Additionally, the evidence from this study might help identify the endocrine-disrupting mechanism of triazole fungicides containing a phenyl ring.

Peer review of the pesticide risk assessment of the active substance metconazole.

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Belgium, and co-rapporteur Member State, the United Kingdom, for the pesticide active substance are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of metconazole as a fungicide on cereals and oilseed rape and as a plant growth regulator on oilseed rape. The reliable end points appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Enantioselective bioaccumulation, oxidative stress, and thyroid disruption assessment of cis-metconazole enantiomers in zebrafish (Danio rerio).

Chiral triazole pesticides may cause enantioselectively adverse effects to non-target organisms. In this work, we employed zebrafish as an aquatic organism model to explore stereoselective acute toxicity, bioaccumulation, oxidative stress, and thyroid disruption of cis-metconazole enantiomers. The median lethal concentration values of (1S, 5R)-metconazole, (1R, 5S)-metconazole, and the mixture of them against zebrafish were 4.01, 2.61 and 3.17 mg⋅L-1, respectively. (1R, 5S)-Metconazole was preferentially bioaccumulated in zebrafish than (1S, 5R)-metconazole, and the bioconcentration factor of (1R, 5S)-metconazole was 1.28-fold larger than that of (1S, 5R)-metconazole. Then, the activity order of catalase, superoxide dismutase, and glutathione-S transferase enzymes in zebrafish was expressed as (1S, 5R)-metconazole > the mixture > (1R, 5S)-metconazole, while the order of malondialdehyde content in zebrafish was (1R, 5S)-metconazole > the mixture > (1S, 5R)-metconazole. Moreover, cis-metconazole exhibited enantioselective regulation effects on the levels of triiodothyronine and thyroxine in zebrafish, and (1R, 5S)-metconazole possessed stronger thyroid disruption ability to zebrafish than the others. By virtue of molecular docking methodology, the binding affair and docking energy results supported that interactions between (1R, 5S)-metconazole and thyroid hormone receptors were much stronger than those between (1S, 5R)-metconazole and same receptors. This study of enantioselective evaluation of cis-metconazole in zebrafish can provide favorable information for risk assessments of chiral pesticides toward environment and health of aquatic organisms.

Toxicity, bioactivity of triazole fungicide metconazole and its effect on mycotoxin production by Fusarium verticillioides: New perspective from an enantiomeric level.

The chiral triazole fungicide metconazole has four stereoisomers, is a broad-spectrum fungicide and is widely used for controlling Fusarium head blight caused by Fusarium species. In this study, systemic assessments of metconazole stereoisomers were performed, including stereoselective toxicity toward the aquatic organism Daphnia magna, fungicidal activity and effects on fumonisin production by the pathogen Fusarium verticillioides (F. verticillioides) in relation to different conditions. The toxicity of metconazole was enantioselective, and there was a 2.1-2.9-fold difference. The activities of superoxide dismutase (SOD) and catalase (CAT) increased and decreased, respectively, after treatment with metconazole stereoisomers, and the differences were observed among the stereoisomers. Among the four stereoisomers, (1S,5R)-metconazole showed the highest fungicidal activity under all assayed conditions, and the differences ranged from 4.4 to 45.2 times. Moreover, metconazole stereoisomers can stereoselectively affect on fumonisin B1 production by F. verticillioides and abiotic factors, such as water activity and temperature, play an important role. Our study provides new insight into metconazole at the stereoisomeric level, including its toxicity, bioactivity, and effect on Fusarium species producing mycotoxins.

Systematic investigation of stereochemistry, stereoselective bioactivity, and antifungal mechanism of chiral triazole fungicide metconazole.

In this study, the stereochemistry, stereoselective fungicidal bioactivity, and antifungal mechanism of chiral triazole fungicide metconazole were investigated. The configurations of metconazole stereoisomers were determined to be (1R, 5R)-metconazole, (1R, 5S)-metconazole, (1S, 5S)-metconazole, and (1S, 5R)-metconazole through using electronic circular dichroism spectroscopy. The bioactivities of four stereoisomers and their stereoisomer mixture toward Fusarium graminearum Schw and Alternaria triticina were found to be in the following order: (1S, 5R)-metconazole > the stereoisomer mixture > (1S, 5S)-metconazole > (1R, 5R)-metconazole > (1R, 5S)-metconazole. In addition, the fungicidal activities of (1S, 5R)-metconazole against two tested pathogens was 13.9-23.4 times higher than those of (1R, 5S)-metconazole. Molecular docking methodology was applied to characterize the docking energy and distances between Cytochrome P450 CYP51B and the metconazole stereoisomers, and (1S, 5R)-metconazole showed the strongest binding energy and the shortest distance binding to CYP51B than the other three stereoisomers. Moreover, enantioselective metabolisms of (1S, 5R)-metconazole and (1R, 5S)-metconazole by Fusarium graminearum Schw were investigated through NMR-based metabolomics. The amounts of alanine, arginine, acetate, ethanol, and dimethylamine produced in the presence of (1R, 5S)-metconazole were significantly higher than corresponding amounts in the presence of (1S, 5R)-metconazole, whereas the amounts of glucose, glycerol, glutamate, methionine, and trimethylamine formed in the presence of (1R, 5S)-metconazole were much less than those in the presence of (1S, 5R)-metconazole. This systematic investigation of metconazole stereoisomers would provide a new perception of metconazole in stereoisomeric level, including bioactivities, metabolic behaviors and antifungal mechanism.

Stereoselective Physiological Effects of Metconazole on Seed Germination and Seedling Growth of Wheat.

In addition to their fungicidal activity, many triazole fungicides function as plant regulators, which might impose adverse effects on the growth and development of crops. For chiral triazole fungicides, these effects can be alleviated by applying stereoisomers with high fungicidal and low regulator activities. This study investigated the stereoselectivity of four stereoisomers and the racemate of metconazole (2.5 g/100 kg seeds) on emergence and growth of seedlings (BBCH 01-14) in wheat. Wheat seedlings, coated with cis-1S,5R-metconazole, had a significantly lower seedling emergence ratio and shoot length than other metconazole treatments; however, the opposite effects were observed in the trans-1S,5S-metconazole treatment. With regard to the hormonal level, enzyme activity, and gene transcription of gibberellin (GA) and jasmonic acid (JA), cis-1S,5R-metconazole treatment inhibited GA biosynthesis while trans-1S,5S-metconazole treatment promoted GA biosynthesis. Moreover, cis-1S,5R-metconazole, trans-1S,5S-metconazole, trans-1R,5R-metconazole, and racemate treatments increased JA biosynthesis. The oxidative stress responses in trans-1R,5R-metconazole and racemate treatments were more intensive. Therefore, compared with the control, treatment with cis-1R,5S-metcoanzole exhibited minimal influence on wheat seedling growth. The results showed that the application of pure cis-1R,5S-metcoanzole (instead of the racemate) in agricultural management could decrease the risks associated with crop growth and developmental damage.

Stereoselective toxicity of metconazole to the antioxidant defenses and the photosynthesis system of Chlorella pyrenoidosa.

Metconazole (MEZ) is a broad-spectrum fungicide with four optical stereoisomers. Compared to traditional fungicides, it achieves better control effect at lower dosages. However, its toxicity to non-target organisms has rarely been investigated. This study investigated the stereoselective toxicity of metconazole to Chlorella pyrenoidosa (C. pyrenoidosa). The results indicate that the presence of the racemate and four stereoisomers of MEZ caused a sudden increase of reactive oxygen species (ROS). This in turn stimulated antioxidant defense, impaired photosynthesis and responses of subcellular structure, and eventually inhibited cell growth. The 96 h-EC50 of the racemate, cis-1R,5S-MEZ, cis-1S,5R-MEZ, trans-1S,5S-MEZ, and trans-1R,5R-MEZ were 0.058, 0.182, 0.129, 0.032, and 0.038 mg/L, respectively. Furtheromre, the generation of ROS, antioxidant response, and the loss of photosynthetic function in C. pyrenoidosa were all preferentially trans-1S,5S-MEZ induced. These results aid the understanding of the stereoselective effects of chiral pesticides on C. pyrenoidosa. Such stereoselective differences must be considered when assessing the risk of metconazole to environment.

Enantioselective determination of metconazole in multi matrices by high-performance liquid chromatography.

A reliable and effective HPLC analytical method has been developed to stereoselectively quantify metconazole in soil and flour matrices. Effects of polysaccharide chiral stationary phase, type and content of alcoholic modifier on separation of racemic metconazole have been discussed in detail. Resolution and quantitative determination of metconazole stereoisomers were performed by using an Enantiopak OD column, with the n-hexane-ethanol mixture (97:3, v/v) at the flow rate of 1.0mL/min. Then, extraction and cleanup procedures followed by the modified QuEChERS (quick, easy, cheap, effective, rugged and safe) method were used for metconazole racemate in soil and flour matrices. The residual analysis method was validated. Good linearity (R2 ≥ 0.9997) and recoveries (94.98-104.89%, RSD ≤ 2.0%) for four metconazole stereoisomers were obtained. In brief, this proposed method showed good accuracy and precision, which might be applied in enantioselective determination, residual quantitative analysis, and degradation of metconazole in food and environmental matrices.