Single And Combined Toxicity Of Beta-Cypermethrin And Chlorpyrifos To Zebrafish Embryo

Environmental pollution caused by the widely used organophosphate and pyrethroid insecticides in agriculture and everyday life has led to a potential hazard to the survival of aquatic organisms. In addition, pesticides can bioaccumulate in aquatic organisms through the food chains, ultimately posing a threat to human health. Effects of pesticides on aquatic organisms and the water environment has become the focus of increasing concern. In the present study, zebrafish was used as a model to investigate the individual and joint toxicity and toxicological mechanisms of beta-cypermethrin(BCP)and chlorpyrifos(CPF).In this research, the potential developmental toxicity, as well as the combined effects of the pesticides was evaluated by morphological observation. In the acute toxicity test, three hours post-fertilization(hpf)zebrafish embryos were exposed to a concentration gradient of single pesticide solution for 96 h, and malformation rate, hatchability and mortality were observed and recorded. The median lethal concentrations(LC50) of chlorpyrifos and beta-cypermethrin were calculated to be 1.18 mg/L and 0.029mg/L, respectively. The deformity rate of zebrafish embryos was high in higher concentrations of chlorpyrifos and beta-cypermethrin exposure groups, and both of the pesticides had an impact on the embryonic hatching. The toxicity of binary mixture(MIX) composed by 1∶1 toxic unit of chlorpyrifos and beta-cypermethrin was determined by the acute toxicity test. The median lethal concentration of binary mixture was calculated to be 0.38 mg/L. The concentration addition(CA), independent action(IA), Marking additive index and combination index were applied to analyze their mode of action. The results indicated that these two pesticides acted together in a synergistic manner.To assess the oxidative stress of chlorpyrifos, beta-cypermethrin and their binary mixture on zebrafish embryos, the expression of oxidative stress-related genes(Mn–sod、Cu/Zn–sod、cat、gstp2、nrf2、keap1、cox1、 ucp2 and bcl2) was determined by real time PCR, and activities of antioxidant enzymes(SOD,CAT, GST) and malondialdehyde(MDA) content were detected. When exposed for 72 h, activities of these antioxidant enzymes and the mRNA levels of most of genes significantly increased in the chlorpyrifos, beta-cypermethrin and their binary mixture exposed groups, except CAT and its encoding gene(cat) whose expression level decreased in the binary mixture exposed group. When exposed for 96 h, some of the genes were down-regulated in the chlorpyrifos and binary mixture groups. MDA content significantly increased in all exposed groups, indicating that single and joint exposure of these two pesticides could cause oxidative damage in embyros.The transcriptomic assessment was designed to further explore the individual and joint toxicity mechanism of these two pesticides. The transcriptome of 96 h-exposed zebrafish embryos was sequenced by RNA-Seq and the results were verified by the droplet digital PCR. The results suggested that there were 356 differentially expressed genes between the chlorpyrifos treatment group and the control group; 2198 differentially expressed genes between the beta-cypermethrin treatment group and the control group; and 2358 differentially expressed genes between the binary mixture treatment groupand the control group. The results of KEGG enrichment analysis of differentially expressed genes indicated that two pathways(ECM-receptor interaction and DNA replication) were significantly enriched in CPF vs Control group; two pathways(phototransduction and adherens junction) were significantly enriched in BCP vs Control group; five pathways were significantly enriched in MIX vs Control group, including focal adhesion, cell cycle, DNA replication, phototransduction and ECMreceptor interaction. The results of histological section showed that the liver lesions of juvenile zebrafish occurred in the treatment groups. In addition, single and combined exposure of beta-cypermethrin and chlorpyrifos could also induce severe apoptosis in eye, brain, liver, gut and tail.The single and combined toxicity of chlorpyrifos and cypermethrin was analyzed in this study. Our findings could help clarify the toxicity of these two pesticides on early life-stages of zebrafish, and could also provide information for the safety assessment and risk management.