Co-exposure to microplastic and plastic additives causes development impairment in zebrafish embryos

•Microplastic mixtures cause developmental toxicity in zebrafish embryos.•Synergistic effects on phenotypic toxicity are shown in the binary mixtures.•Microplastic mixtures can cause cell damage by oxidative stress or embryonic malformation by inhibition of thyroid hormonal activation.•Toxic effects of microplastic mixtures depend on the mixed combination of components in the mixture. Since the run off of microplastic and plastic additives into the aquatic environment through the disposal of plastic products, we investigated the adverse effects of co-exposure to microplastics and plastic additives on zebrafish embryonic development. To elucidate the combined effects between microplastic mixtures composed of microplastics and plastic additives in zebrafish embryonic development, polystyrene (PS), bisphenol S (BPS), and mono-(2-ethylhexyl) phthalate (MEHP) were chosen as a target contaminant. Based on non-toxic concentration of each contaminant in zebrafish embryos, microplastic mixtures which is consisted of binary and ternary mixed forms were prepared. A strong phenotypic toxicity to zebrafish embryos was observed in the mixtures composed with non-toxic concentration of each contaminant. In particular, the mixture combination with ≤ EC10 values for BPS and MEHP showed a with a strong synergistic effect. Based on phenotypic toxicity to zebrafish embryos, change of transcription levels for target genes related to cell damage and thyroid hormone synthesis were analyzed in the ternary mixtures with low concentrations that were observed non-toxicity. Compared with the control group, cell damage genes linked to the oxidative stress response and thyroid hormone transcription factors were remarkably down-regulated in the ternary mixture-exposed groups, whereas the transcriptional levels of cyp1a1 and p53 were significantly up-regulated in the ternary mixture-exposed groups (P < 0.05). These results demonstrate that even at low concentrations, exposure to microplastic mixtures can cause embryonic damage and developmental malformations in zebrafish, depending on the mixed concentration-combination. Consequently, our findings will provide data to examine the action mode of zebrafish developmental toxicity caused by microplastic mixtures exposure composed with microplastics and plastic additives.