Enantioselective Toxic Effects Of Mefentrifluconazole And Its Enantiomers On Different Life Stages Of Zebrafish (Danio Rerio)

At present,agricultural production is heavily dependent on the use of pesticides.While pesticides have brought us many benefits such as ensuring food security and promoting social development,environmental problems and human health problems caused by pesticides have also been increasingly exposed.Mefentrifluconazole is a new type of the chiral triazole fungicide.Due to its unique isopropanol structure,mefentrifluconazole has excellent anti-bacterial activity and is used to prevent corn,grain,soybean,rice and other crops from damage of diseases.In addition,mefentrifluconazole has a chiral carbon,so its enantiomers may exhibit different biological activities,ecotoxicological and environmental behaviors in the environment.However,the toxicological effects of mefentrifluconazole on aquatic organism are scarce,especially at the enantiomer level.Therefore,in the present study,zebrafish were used as a model animal to explore the enantioselective toxic effects and mechanism of mefentrifluconazole on zebrafish at different life stages by combining the means of morphology,behavior,metabolomics and molecular biology.The main experimental contents and results are shown as follows:(1)Zebrafish embryos were exposed to different concentrations of rac-mfz,(-)-mfz,(+)-mfz.The acute toxicity and developmental toxicity of mefentrifluconazole to zebrafish embryos and the difference in toxicity between enantiomers were investigated.The acute toxicity assay results revealed that the 96h-LC₅₀ values of rac-mfz,(-)-mfz,(+)-mfz to zebrafish embryos were 1.101,1.552and 0.753 mg/L,respectively;the 96 h-LC₅₀ values of rac-mfz,(-)-mfz,(+)-mfz to zebrafish larvae were 0.753,1.187 and 0.553 mg/L,respectively;and the 96h-LC₅₀value of rac-mfz to adult zebrafish was 1.150 mg/L.In addition,the results showed that rac-mfz,(-)-mfz,(+)-mfz exposure caused adverse effects on zebrafish embryos,such as abnormal spontaneous movement,slow heartbeat,hatching inhibition,pericardial edema and yolk sac edema.Interestingly,our results clearly showed that rac-mfz and(+)-mfz was more toxic to zebrafish than(-)-mfz at the same concentration,whether it was acute toxicity or developmental toxicity.(2)Zebrafish larvae were exposed to rac-mfz/(-)-mfz/(+)-mfz at different concentrations(0.01,0.1 and 0.5 mg/L),and the enantioselective effects of mefentrifluconazole on the swimming behavior,neurotoxicity and cardiotoxicity of zebrafish in the early stage were investigated.The results showed that the active duration and the swimming speed of zebrafish larvae was significantly reduced compared to the contronl groupsin the high concentration of(0.5 mg/L)(+)-mfz exposure treatment group.Meanwhile,we also found that mefentrifluconazole and its enantiomers also changed the expression of neurotoxicity and cardiotoxicity-related genes in zebrafish larvae.Compared with the control group,the m RNA levels of the high concentration(0.5 mg/L)of rac-mfz and(+)-mfz exposure groups were significantly changed(p<0.05),while(-)-mfz exposure group were no significant changes,which proved that rac-mfz and(+)-mfz were more toxic than(-)-mfz.(3)Adult zebrafish were exposed to different concentrations of rac-mfz/(-)-mfz/(+)-mfz(0.01 and 0.1 mg/L)for 28 days.After the exposure,liver,heart and brain tissues of female and male zebrafish were collected.The enantioselective toxic effects of mefentrifluconazole on different tissues of adult zebrafish were investigated by means of metabonomics and quantitative real-time PCR.The results indicated that exposure to rac-mfz and(+)-mfz resulted in more significant changes in metabolites than exposure to(-)-mfz.In addition,exposure to mefentrifluconazole and its enantiomers significantly changed the expression of hepatotoxicity,neurotoxicity and cardiotoxicity-related genes in zebrafish,compared with control treated groups.The expression of lipid metabolism,apoptosis and CYP-related genes in the liver of female zebrafish in rac-mfz and(+)-mfz treatment groups were 1.61-108.92 times and 2.37-551.34 times higher than those in the(-)-mfz treatment group,respectively.The changes of m RNA expression levels of neurotoxicity and cardiotoxicity-related genes in rac-mfz and(-)-mfz treatment group were more than in(-)-mfz.Moreover,sex-specific differences were detected in zebrafish liver toxicity and cardiotoxicity.