Histology and multi-omic profiling reveal the mixture toxicity of tebuconazole and difenoconazole in adult zebrafish.

The combination effects of triazole fungicides on aquatic organisms remain largely unknown. In current study, an integrated histological, transcriptome, metabonomics and microbiology was applied to investigate the mixture effects and risk of tebuconazole (TEB) and difenoconazole (DIF) co-exposure on zebrafish liver and gonad at aquatic life benchmark. TEB and DIF mixture showed additive effect on the acute toxicity to adult zebrafish, the combined toxicity on liver was less than the additive effect of individual TEB and DIF, and TEB and DIF mixture also reduced the toxic effects on gonad and intestinal microflora. Transcriptomics and metabolomics further showed TEB and DIF mixture could induce more differentially expressed genes (DEGs) to regulate the metabolic pathways involved in energy metabolism, steroid hormone biosynthesis, retinol metabolism and microbial metabolism, to balance the energy metabolism and supplies, and maintain the steroid hormone and RA level, further reduced the toxic effect on liver and gonad caused by TEB and DIF. Our results showed the different responses and patterns on transcriptional and metabolic profiles mediated in the diverse toxicity and combination effects of TEB and DIF. The present results provided a deep mechanistic understanding of the combined effects and mode of action of DIF and TEB mixture on aquatic organisms, suggesting the concept of additive effects might sufficiently protective when evaluated the combination effects and ecological risk of TEB and DIF at aquatic life benchmarks.