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Comparative Lipid Peroxidation and Apoptosis in Embryo-Larval Zebrafish Exposed to Three Azole Fungicides, Tebuconazole, Propiconazole, and Myclobutanil, at Environmentally Relevant Concentrations.

Azole fungicides have entered the aquatic environment through agricultural and residential runoff. In the present study, we compared the off-target toxicity of tebuconazole, propiconazole, and myclobutanil using embryo-larval zebrafish as a model. The aim of this study was to investigate a relative toxicity of tebuconazole, propiconazole, and myclobutanil using multiple level endpoints such as behavioral endpoints and enzymatic and molecular biomarkers associated with their mode of action. Zebrafish embryos were exposed to azoles at environmentally relevant and high concentrations, 0.3, 1.0 and 1000 µg/L, starting at 5 hours post- fertilization (hpf) up to 48 hpf, as well as 5 days post fertilization (dpf). Relative mRNA expressions of CYP51, GST, Casp9, p53, and BAX were measured to assess toxicity due to fungicides at mRNA level, whereas caspase 3/7 (apoptosis) and MDA (lipid peroxidation) levels were measured at enzymatic level. Furthermore, mitochondrial dysfunction was measure through the Mito Stress test using the Seahorse XFe24 at 48 hpf. Additionally, light: dark movement behavior was monitored at 5 dpf using Danio Vision® to understand adverse effects at the organismal level. There was no significant difference in the light: dark behavior with exposure to azoles as compared to controls. The molecular biomarkers indicated that propiconazole and myclobutanil induced lipid peroxidation, oxidative stress, and potentially apoptosis at environmentally relevant concentrations (0.3 and 1 µg/L). The results from the mitochondrial respiration assay indicated a slight decrease in spare respiratory capacity with an acute exposure (48 hpf) to all three azoles at 1000 µg/L. Based on our results, propiconazole and myclobutanil are acutely toxic as compared to tebuconazole in aquatic organisms at environmentally relevant concentrations. This article is protected by copyright. All rights reserved.

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