Assessing the toxicity of the benzamide fungicide zoxamide in zebrafish (Danio rerio): Towards an adverse outcome pathway for beta-tubulin inhibitors.

Commercial benzamide fungicides are applied to crops to control damage caused by oomycete fungi and are used as veterinary pharmaceuticals in aquaculture. The mechanism of action of these fungicides is to induce mitotic arrest via binding to beta-tubulin, thus inhibiting tubulin polymerization. However, there are little toxicity data available for benzimidazole fungicides in fish. To address this knowledge gap, we conducted zebrafish embryo toxicity tests to assess deformities, survival, and sub-lethal responses following exposure to zoxamide (0, 0.5, 1.0, 2.5, 5.0 and 10 μM zoxamide). We hypothesized that skeletal deformities would be prevalent in zebrafish due to its mechanism of inhibiting beta-tubulin polymerization. Zoxamide was relatively toxic to zebrafish embryos and larvae, and survival was reduced ∼50 % at 2 days post fertilization (dpf) with 10 μM exposure and over time at 6 dpf, 2.5 μM exposure reduced survival by ∼20 %. Frequency of hatch was also reduced/delayed in zebrafish at 3 dpf with >2.5 μM zoxamide. Pericardial edema, body length shortening, and spine curvature were observed in larvae exposed to >5 μM. Mitochondrial bioenergetics were assessed in ∼30 hpf embryos (24-hour exposure) using an XFe24 Flux Analyzer and regression analysis revealed a negative relationship between basal respiration and zoxamide concentration. Superoxide dismutase 1 and caspase 3 mRNA levels were both decreased in 6 dpf larvae exposed to 2.5 μM zoxamide, but were not changed in expression at 0.5 nor 1 μM zoxamide. Continuous 6-day exposure to zoxamide reduced larval activity at 2.5 μM; conversely a 24-hour exposure (at 5-6 dpf) induced hyperactivity at 5 μM suggesting dose and time dependent effects on fish behavior. Based on sub-lethal endpoints, we conceptualize an adverse outcome pathway for chemicals that inhibit tubulin polymerization.