Insights into the combined effects of environmental concentration of difenoconazole and tebuconazole on zebrafish early life stage.

Limited literature had focused on the combined effect of triazole fungicides on aquatic organisms at environmental concentrations. In this research, difenoconazole (DIF) and tebuconazole (TEB) mixture exhibited additive effect on the acute toxicity to zebrafish embryos. The transcriptomics and metabolomics demonstrated DIF and TEB mixtures at aquatic life benchmark and environmental concentration simultaneously influenced the lipid metabolism, arachidonic acid metabolism, steroid hormone biosynthesis and tryptophan metabolism, but showed diverse response patterns mediating the combined effects on zebrafish embryos after 120 h exposure. The DIF and TEB mixture at aquatic life benchmark caused combined effect on yolk sac resorption and metabolites, was less than the additive effect of individual DIF and TEB. It was found environmental concentration of DIF and TEB caused much lower levels of IFN and IL6, induced higher levels of PGE, l-kynurenine and formylanthranilate in zebrafish larvae, and their binary mixture caused synergistic effect on the accumulation of metabolites in metabolic pathways, which might cause more negative effect and risk on growth in zebrafish later life stages. Results further demonstrated that adding arachidonic acid (AA) increased the transcripts of Pla, Ptgs1, Cyp19a and Cxcl8b, allayed the accumulation of PLA and 17β-E, and induced more PGF2α, IFN and IL6 levels in zebrafish larvae, indicated AA metabolism might play important regulatory roles on hormone synthesis and immune response caused by DIF and TEB mixtures. Current results indicated the risk assessment of mixtures based on single concentration may not precisely estimate the environmental risk and health effect, it is crucially important to consider the multi-concentration combinations, and more attention should be paid to the environmental concentration.