The identification of the metabolites of chlorothalonil in zebrafish (Danio rerio) and their embryo toxicity and endocrine effects at environmentally relevant levels

Abstract

Chlorothalonil is a broad spectrum fungicide with high annual production and environmental contamination. Despite its high consumption, studies regarding the potential ecological risks of chlorothalonil, especially its metabolites, to aquatic organisms are still limited. In this study, we selected the zebrafish (Danio rerio) as the in vivo model and first identified the metabolite (4-hydroxychlorothalonil) of chlorothalonil in zebrafish by tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF). Then, the in vivo and in vitro models were applied to comprehensively estimate the embryo toxicity and potential endocrine effect of chlorothalonil and 4-hydroxychlorothalonil. The data from zebrafish embryo toxicity showed that the lowest observed effect concentrations of both chlorothalonil and 4-hydroxychlorothalonil were 50 μg/L after 96 h of exposure. The mortality rate of the 4-hydroxychlorothalonil was 2.6-fold higher than that of the parent compound at the concentration of 50 μg/L. Dual-luciferase reporter gene assays indicated that both chlorothalonil and 4-hydroxychlorothalonil exerted estrogen receptor α (ERα) agonist activity with REC₂₀ values of 2.4 × 10^-8 M and 3.6 × 10^-8 M, respectively. However, only 4-hydroxychlorothalonil exhibited both thyroid receptor β (TRβ) agonistic and antagonistic activities. Lastly, we employed molecular docking to predict the binding affinity of chlorothalonil and 4-hydroxychlorothalonil with ERα or TRβ. The results revealed that the potential endocrine effect of chlorothalonil and 4-hydroxychlorothaloni might be attributed to the different binding affinities with the receptors. In conclusion, our studies revealed that 4-hydroxychlorothalonil exhibited potent endocrine-disrupting effects compared to its parent compound, chlorothalonil. The results provided here remind us that the assessment of the potential ecological and health risks of the metabolites of fungicides in addition to their parent compounds should arouse great concerns.

Keywords: 4-hydroxychlorothalonil; Chlorothalonil; Developmental toxicity; Endocrine disrupting effects.