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There is growing concern that sprayed neonicotinoid pesticides (neonics) persist in mixed forms in the environmental soil and water systems, and these concerns stem from reports of increase in both the detection frequency and concentration of these pollutants. To confirm the toxic effects of neonics, we conducted toxicity tests on two neonics, clothianidin (CLO) and imidacloprid (IMD), in embryos of zebrafish. Toxicity tests were performed with two different types of mixtures: potential mixture compounds and realistic mixture compounds. Potential mixtures of CLO and IMD exhibited synergistic effects, in a dose-dependent manner, in zebrafish embryonic toxicity. Realistic mixture toxicity tests that are reflecting the toxic effects of mixture in the aquatic environment were conducted with zebrafish embryos. The toxicity of the CLO and IMD mixture at environmentally-relevant concentrations was confirmed by the alteration of the transcriptional levels of target genes, such as cell damage linked to oxidative stress response and thyroid hormone synthesis related to zebrafish embryonic development. Consequently, the findings of this study can be considered a strategy for examining mixture toxicity in the range of detected environmental concentrations. In particular, our results will be useful in explaining the mode of toxic action of chemical mixtures following short-term exposure. Finally, the toxicity information of CLO and IMD mixtures will be applied for the agricultural environment, as a part of chemical regulation guideline for the use and production of pesticides.

This study aimed to examine the impact of chronic (30 days) exposure to polystyrene microplastics (PS-MPs) of different sizes (50 nm and 2 µm) and at different concentrations (0.5 μg/L and 100 mg/L) to marine copepod Tigriopus japonicus. Polystyrene microplastics affected survival rates in size- and concentration-dependent manners. The LC50s values of 50 nm and 2 µm PS-MPs were 0.10 mg/L and 3.92 mg/L, respectively. The developmental time was delayed by 50 nm PS-MPs, and Usp expression was downregulated. Reproduction was negatively affected by 2 µm PS-MPs even at environmentally relevant concentrations; however, the expression of Vtg was not altered. The production rates of reactive oxygen species and nitric oxide also increased after exposure to PS-MPs; but this effect was independent of particle size. The expression levels of Cat and Tnf, genes related to oxidative stress and inflammation, respectively, were upregulated by exposure to PS-MPs, independently of particle size. Meanwhile, the level of oxidative stress in T. japonicus was not significantly affected by PS-MPs at environmentally relevant concentrations. This study suggests that nano-sized PS-MPs are not always more toxic than micro-sized PS-MPs, and that oxidative stress is a key factor in determining the toxic effect on T. japonicus at high concentrations.