The Toxicity Effect And Mechanism Of Difenoconazole On Zebrafish(Danio Rerio)

With the increasing awareness of the negative effect of pesticides, environmental risk assessment has become an important foundation of the scientific application of pesticides. As an important biological ecosystem, fish is also one of the main foods of human. The toxicity evaluation of pesticides on fish is the focus of pesticide environmental risk assessment.Difenoconazole is a representative triazole fungicide and is widely used for the control of diseases caused by all kinds of pathogenic fungi on various crops. In China, difenoconazole has been used as the main pesticide to combat rice diseases for many years. It is therefore important to carry out further environmental toxicological studies on difenoconazole to determine the risk it poses to aquatic organisms.In this study, to investigat the action mechanism of difenoconazole on different life stages zebrafish, experimental work that is related in morphological, biochemical and molecular biological is conducted:1The acute toxicity of difenoconazole on zebrafishThe96h-LC5o of difenoconazole to zebrafish adult, embryo and larvae was1.45,2.34and1.17mg/L respectively. The sensitivity order of three life stages to difenoconazole is larvae> adult> embryo.2The developmental toxicity of difenoconazole on zebrafish embryosZebrafish embryos were exposed to0.5,1.0,1.5,2.0,2.5,3.0mg/L difenconazole for6days and the embryonic mophorlogical indicators were tested. The results indicated that a large suite of symptoms was induced in embryonic development by different dosages of difenoconazole, including hatching inhibition, abnormal spontaneous movement, slow heart rate, growth regression and morphological deformities. To further explore its active mechanism, another4days embryonic test was conducted. Embryos were exposed to0.5and2.0mg/L groups and the expression level of11genes that related in embryonic development was measured via quantative PCR. The results showed that the expression of IGF-1, GH, BMP2and BMP4was significantly decreased, while the expression of HE I and CYP26A1was significantly upregulated.3The oxidative stress caused by difenoconazole on zebrafishAdults and embryos were separately exposed to0.01,0.5and1.0mg/L difenoconazole. The activity of three antioxidases and their encoding genes transcription in adult liver and embryo was tested. In addition, the MDA content was measured to assess the oxidative damage in zebrafish. Results showed that the activity of three antixidases was decreased in both adult liver and embryo which is accompanied by the down-regulation of the three encoding genes. The inhibition of oxidative parameters in adult liver is stronger than embryo. According to the MDA assay, significant lipid peroxidation occurred in adult zebrafish liver, while no lipid peroxidation was observed in adult zebrafish embryos.4The effect of difenoconazole on lipid level of zebrafishAdults and embryos were separately exposed to0.01,0.5and1.0mg/L difenoconazole. The TCHO and triglyceride level was tested at4and8dpe. The transcription of6genes related in lipid synthesis and metabolism was measured by q-PCR. Biochemical results showed no significant change in TCHO and triglyceride level was observed after exposure. Quantitative PCR results showed that difenoconazole could alter the expression of lipid genes in both embryos and adults. For embryos, the expression of CYP51and HMGCRa increased and expression of FAS, ACC1, SREBF and PPARal decreased; while all of these six genes showed a significant reduction in adult liver.5Sex specific response in cholesterol level in zebrafish to difenoconazole.Adult zebrafish were exposed to environmental related dosage (0.1,10and500mg/L) difenoconazole. The body weight and hepatic total cholesterol (TCHO) level was tested at7,15and30days post exposure (dpe). The expressions of eight cholesterol synthesis genes and one cholesterol metabolism gene were assessed via Quantitative PCR method. The significant decrease of TCHO level in male zebrafish liver was observed at15and30dpe, which was accompanied by apparent hepatic cholesterol-genesis genes expression decline. In comparison with males, female zebrafish showed different transcription modification of tested genes, and the cholesterol content maintain normal level during the whole exposure.