Studies On Bioaccumulation Behavior And Toxic Effects Of Three Chiral Triazole Fungicides In Zebrafish (Danio Rerio)

Little is known about the bioaccumulation and toxicity of chiral triazole fungicides,in spite of the fact that they are indispensable parts for a comprehensive assessment of their environmental fate and toxic effects in organisms of freshwater ecosystems.In this paper,the bioaccumulation behavior of hexaconazole,epoxiconazole and diniconazole as well as their toxic effects in zebrafish(Danio rerio)were investigated.The metabolomics strategy as well as histopathological examination was also carried out to study the toxic effects of hexaconazole enantiomers and epoxiconazole enantiomers on zebrafish.Significant enantioselective bioaccumulation was demonstrated when exposed to hexaconazole of 100 and 200 ?g L-1 for 21 days,finding that the(-)-enantiomer tended to accumulate in zebrafish more easily than(+)-enantiomer.Activities of antioxidant enzymes(SOD,CAT and GPx)and GSH content were all significantly decreased when zebrafish were exposed to 50 and 200 ?g L-1 hexaconazole for 21 days.A series of genes of the apoptosis pathway were examined in groups treated with 50 and 200 ?g L-1 hexaconazole for 21 days using real-time PCR.Significant up-regulation of p53,Puma,Apaf-1,caspase-3 and caspase-9 expression and down-regulation of Bcl-2/Bax expression ratio were proved.The overall results indicated that waterborne hexaconazole was able to produce oxidative stress and induce apoptosis through the involvement of caspases in adult zebrafish.Significant enantioselectivity was observed with(+)-epoxiconazole enrichment in the bioaccumulation process when exposed to epoxiconazole of 100 and 1000 ?g L-1 for 21 days.1H NMR-based metabolomics analysis showed that 100 and 1000 ?g L-1 epoxiconazole exposure for 21 days resulted in two similar but not identical patterns for the change of endogenous metabolites related to energy,lipid and amino acid metabolism.The expression of genes associated with mitochondrial respiratory chain,ATP synthesis and fatty acid p-oxidation were further measured to explore the reason for the disturbed energy metabolism,finding epoxiconazole had an inhibition effect on the genes expression of the above ways.No enantioselectivity was observed in the bioaccumulation process of both 70 and 300 ?g L-1 diniconazole exposure groups for 21 days.The 1H NMR-based metabolomics analysis revealed that 70?g L-1 diniconazole exposure for 21 days increased valine,leucine,isoleucine,alanine,lactate and choline,accompanied by decreased glucose,creatine and taurine,in comparison to the control.In the exposure group of 300 ?g L-1 diniconazole for 21 days,only down-regulated glucose and creatine were observed.The above results indicated that diniconazole disturbed energy metabolism,amino acid metabolism and lipid metabolism.Histological examination showed that the swell and vacuolization in the liver,as well as the reduction of spermatids in the low and high dose diniconazole exposure groups for 21 days,while no changes in brains were observed.Zebrafish were separately exposed to 100 and 1000 ?g L-1 hexaconazole and epoxiconazole enantiomers for 21 d.1H NMR-based metabolomics analysis showed that the exposure of low and high dose of hexaconazole enantiomers altered energy metabolism,lipid metabolism and amino acid metabolism of zebrafish,with the different metabolic profiles resulted from the same dose of(+)-hexaconazole and(-)-hexaconazole.Similar to hexaconazole enantiomers,the metabolic profiles,including the changes related to energy metabolism,lipid metabolism and amino acid metabolism,were demonstrated in low and high dose epoxiconazole enantiomers treatment groups.There are differences in the metabolic profiles of zebrafish between exposed to(+)-epoxiconazole and(-)-epoxiconazole of the same dose.The results of histological examination revealed that the exposure of both enantiomers for hexaconazole and epoxiconazole resulted in the similar histopathological changes.The exposure of hexaconazole and epoxiconazole enantiomers at low and high dose resulted the vacuolization and swell in the liver of the female and male zebrafish.Compared to female zebrafish,more liver damage were found in male zebrafish in the hexaconazole enantiomers exposure groups.The reduction of spermatids were observed in hexaconazole and epoxiconazole enantiomers treatment groups of both doses.Hexaconazole enantiomers exposure of low and high dose resulted the increase in the number of mature eggs,while such effect was not observed in epoxiconazole enantiomers exposure groups.Hexaconazole and epoxiconazole enantiomers exposure resulted in no changes in brains of female and male zebrafish.There are similarities between hexaconazole enantiomers and epoxiconazole enantiomers on their effects on the metabolic profile and histological damages of zebrafish.