Combined toxicity of imidacloprid,acetochlor, and tebuconazole to zebrafish ( Danio rerio ): acute toxicity and hepatotoxicity assessment

Compound pollution refers to two or more kinds of pollutants with different properties, a pollutant from different sources, or the simultaneous existence of two or more different types of pollutants in the same environment. In this study, we aimed to investigate the individual and combined toxicity of the insecticide imidacloprid (IMI), the herbicide acetochlor (ACT), and the fungicide tebuconazole (TBZ) to zebrafish. The acute toxicity test results showed that the 96-h LC50 values of IMI, ACT, and TBZ were 276.84 (259.62–294.35) mg active ingredient (a.i.) L−1, 1.52 (1.34–1.74) mg a.i. L−1, and 8.16 (7.7–8.6) mg a.i. L−1, respectively. The combinations of IMI, ACT, and TBZ with toxicity ratios of 1:2:2, 1:4:4, 2:4:1, and 4:1:4 displayed synergistic toxic effects on zebrafish, while the toxicity ratios of 1:1:1, 1:1:2, 2:1:2, 2:2:1, and 4:2:1 of IMI, ACT, and TBZ, respectively, exhibited antagonistic toxic effects on zebrafish. The following experiments were performed with a toxicity ratio of 1:4:4 (IMI:ACT:TBZ). The activities of four enzyme biomarkers related to oxidative stress in the liver, catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and malondialdehyde (MDA) content were evaluated in each exposure group on days 7, 14, 21, and 28. Compared with those of the control group, the activities of CAT, SOD, and GST and the MDA content were significantly altered at different time points in the individual and combined exposure groups. Additionally, the activities of CAT, SOD, and GST and the MDA content were significantly altered in the combined group compared with those of the individual group after 14 days or 21 days of exposure. Therefore, it was confirmed that combined toxicity studies are indispensable in risk assessment.