Developmental toxicity and thyroid hormone receptor antagonism of chlorothalonil in zebrafish: insights from transcriptomics and in vitro assays.

Chlorothalonil is a widely used organochlorine fungicide frequently detected in aquatic ecosystems, yet its mechanisms of developmental toxicity in fish remain incompletely understood. This study employed an integrated phenotype-to-mechanism approach in zebrafish embryos to assess the acute and developmental toxicity of chlorothalonil and elucidate potential molecular pathways. The 96-hour median lethal concentration (LC₅₀) was determined to be 89.5 μg/L. Exposure to concentrations below the acute lethal threshold (20-80 μg/L) resulted in significant developmental impairments, including reduced heart rate and body length, along with increased incidences of pericardial edema, tail malformation, and scoliosis. Transcriptomic analysis of embryos exposed to 50 μg/L chlorothalonil at 24 h post-fertilization revealed coordinated upregulation of xenobiotic metabolism genes and dysregulation of genes essential for cardiac and skeletal development, notably the thyroid hormone receptor gene thrab. Subsequent functional analysis using a zebrafish-specific thyroid hormone receptor (TR) yeast two-hybrid assay demonstrated that chlorothalonil acts as a direct antagonist of both TRα and TRβ, dose-dependently inhibiting triiodothyronine (T3)-induced receptor activation. These findings establish disruption of thyroid hormone signaling as a key mechanistic pathway for chlorothalonil-induced developmental malformations, enhancing the mechanistic understanding of its risk to early-stage development of teleosts.