Effect Of Common Fungicide Fluosilazole On Zebrafish

Fluosilazole is a three-azole internal absorbability bactericide,which inhibits demethylation of sterols.Mainly for the prevention and treatment of Ascomycetes,basidiomycetes and Deuteromycetes fungi,and prevent the destruction of the biosynthesis of ergosterol,the membrane can not be formed,the bacteria are killed.Therefore,it is widely used in domestic and animal safety,and is highly effective against pathogens.The half life in soil is 12+ 2 days,which will make fluosilazole enter the water through soil and then concentration.There is no enough report on the toxicity of fluosilazole to aquatic organisms,and the assessment of the fluosilazoles on zebrafish(Danio rerio)embryos acute toxicity only reported in foreign countries.Zebrafish is a model organism recommended by OECD.This text will use zebrafish to evaluate the toxicity of fluosilazole.We hope to find out its action mechanism and provide suggestions for the rational use of fluosilazole.Developmental toxicity and the mode of action concerning apoptosis for fluosilazoles on zebrafish embryos were studied using the method of acridine orange staining and TUNEL assay,along with RT-qPCR.Developmental toxicity together with the whole mount and apoptosis at 24h for cyantranilipole on zebrafish embryos were investigated with acridine orange staining,TUNEL assay,Annexin V FITC/PI double-staining,JC-1 staining flow cytometry and RT-qPCR,meanwhile,studies on DNA damage,developmental toxicity and the genetic toxicity regulatory mechanism on zebrafish embryos were also applied using DAPI staining and single cell gel electrophoresis(SCGE).The results indicate that:1.The zebrafish embryos acute toxicity of was 4.95(3.315-14.29)mg/L-1.High concentration treatment group induced zebrafish embryos spontaneous movement ability,heart rate,hatching rate and increase pericardial cyst and tail deformity,and the induction ability increased with fluosilazole concentration increasing;2.Both the results of acridine orange staining and TUNEL assay indicate that the main site of flusilazole induced apoptosis in 24h cells of zebrafish embryos is in the heart and caudal spinal cord area,the main part of flusilazole induced apoptosis on 96,168h zebrafish embryo cells is in the heart,tail spinal cord and the neck region,the ability of cell apoptosis increases with the increase of flusilazole concentration.The RT-qPCR technique was used to investigate the apoptosis pathway of zebrafish embryos induced by fluosilazole,and the results showed that fluosilazole induced apoptosis through the apoptosis pathway mediated by p53 gene;3.Embryonic DNA damage of cyantranilipole was detected using comet assay and DAPI staining micronucleus at the stage of 24hpf,the result indicates that the micronucleus rate,the DNA content on the comet tail and the tail distance are all significantly increased when its concentration goes up,which signifies that there is a good dose-effect relation between cyantranilipole and the induction of the embryonic DNA damage;4.Sultan black B staining was used to detect the effect of fluosilazole on hematopoietic cells of 48 and 72h zebrafish embryos.The results indicted that with the increase of fluosilazole concentration,the hematopoietic cells of embryonic tail decreased;5.The influence of fluosilazole on the apoptosis related genes in zebrafish embryos was studied by RT-qPCR technology.The results suggest that fluosilazole may induce apoptosis in zebrafish embryo cells through mitochondrial apoptosis pathway and death mediated pathway.The main pathway is that fluosilazole induces DNA damage in zebrafish embryos and induces the up regulation of the p53 gene,thus activating the Bid gene in the cytoplasm,promoting the transfer of the Bax gene to the mitochondrial membrane to change the membrane permeability,causing the decrease of the mitochondrial membrane potential and activating the Caspase-9 precursor protein induced Caspase cascade reaction to induce apoptosis.The expression of Bax,Bid,p53,Caspase-8 and Caspase-9 increased and the expression of Bcl-2 was not significantly increased.6.By flusilazole on acute toxicity test of adult zebrafish,with flusilazole concentration increases,increased mortality of adult zebrafish,spinal cord hemorrhage increased,and caused irreversible tail bending;7.By flusilazole recovery test on chronic toxicity of adult zebrafish,flusilazole may cause damage to the zebrafish with the increase of the concentration of resistance,after a certain time,zebrafish can repair a certain amount of the damage of flusilazole.