Dietary Glyphosate Exposure Disrupts Hepatic and Reproductive Function in Female Zebrafish at Regulatory Safe Levels

Glyphosate (GLY), the active ingredient in widely used herbicides, was long considered specific to plants and bacteria, yet mounting evidence shows it can impair endocrine and reproductive functions in animals. Given its widespread use and environmental persistence, assessing its effects at regulatory-approved doses is critical. Here, adult female zebrafish (Danio rerio) were exposed for 21 days to different concentrations of dietary GLY at 0.5 mg/kg body weight/day (GLY0.5, acceptable daily intake, ADI), 5 mg/kg/day (GLY5), and 50 mg/kg/day (GLY50, no-observed-adverse-effect level, NOAEL). Our findings show that dietary GLY induces dose-dependent perturbations along the hepato-gonadal axis. At the highest dose, chronic stress responses were evident through elevated cortisol and cortisone, accompanied by hepatic glycogen accumulation and ferroptotic stress. Although follicle histology appeared normal, alterations in several genes involved in oocyte maturation and estrogen receptor signaling translated into reduced fertilization, revealing compromised gamete quality rather than overt follicular development abnormality. Likewise, the lowest dose triggered modifications in genes crucial for oogenesis without altering the follicle development, although in this case, potential compensatory mechanisms could have led to enhanced fertilization. GLY5 did not alter the number of fertilized eggs but significantly increased embryo mortality. Overall, dietary GLY disrupted hepatic metabolism, endocrine signaling, and reproduction in a non-monotonic manner, even at levels considered safe by EFSA. These findings highlight the need to reevaluate current safety thresholds with attention to female-specific reproductive risks.