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.

商业化的苯甲酰胺类杀菌剂用于作物，以控制卵菌类真菌引起的损害，并在水产养殖中用作兽药。这些杀真菌剂的作用机理是通过与β-微管蛋白结合诱导有丝分裂停滞，从而抑制微管蛋白聚合。但是，苯并咪唑类杀真菌剂在鱼类中的毒性数据很少。为了弥补这一知识差距，我们进行了斑马鱼胚胎毒性测试，以评估暴露于唑酰胺（0、0.5、1.0、2.5、5.0和10μM唑酰胺）后的畸形，存活和亚致死反应。我们假设由于斑马鱼抑制β-微管蛋白聚合的机制，骨骼畸形将在斑马鱼中普遍存在。Zoxamide对斑马鱼的胚胎和幼虫具有相对毒性，并且在受精后（dpf）2天，暴露量为10μM时，存活率降低了约50％，而在6 dpf下随着时间的推移，2.5μM暴露量使存活率降低了约20％。在斑马鱼中，以> 2.5μM的乙酰胺使3 dpf的孵化率也降低/延迟。在暴露于> 5μM的幼虫中观察到心包水肿，体长缩短和脊柱弯曲。使用XFe24通量分析仪评估了约30 hpf胚胎（24小时暴露）中的线粒体生物能学，回归分析表明基础呼吸作用与佐唑酰胺浓度呈负相关。超氧化物歧化酶1和胱天蛋白酶3 mRNA水平在暴露于2.5μM唑酰胺的6 dpf幼虫中均降低，但在0.5或1μM唑酰胺中表达均未改变。连续6天暴露于Zoxamide会降低2.5μM的幼虫活性；相反，在5μM下暴露24小时（5-6 dpf）会引起机体过度活跃，提示对鱼类行为的剂量和时间依赖性。基于亚致死终点，我们对抑制微管蛋白聚合的化学药品的不利结局进行了概念化。