Dynamic mixture toxicity of glufosinate-ammonium and CuO nanoparticles in zebrafish: oxidative, apoptotic, and endocrine responses.

Aquatic ecosystems face increasing pressure from the co‑occurrence of agrochemicals and engineered nanoparticles, posing significant toxicological risks. Glufosinate‑ammonium (GA) and copper oxide nanoparticles (CuO-NP) are environmentally persistent contaminants whose combined effects on fish physiology remain poorly understood. Here, adult zebrafish (Danio rerio) were exposed to graded concentrations of GA, CuO-NP, and their mixtures for 7, 14, and 21 days. A suite of biomarkers was assessed, including protein content, antioxidant enzyme activities, oxidative stress indicators, apoptosis markers, and vitellogenin. Early responses involved depletion of protein reserves and activation of antioxidant defenses, reflecting an initial attempt to preserve cellular homeostasis. With prolonged exposure, oxidative stress intensified, apoptosis became persistent, and protein reserves failed to recover, indicating progression toward sustained cellular stress. Vitellogenin induction was consistently observed in males, indicating sex‑specific endocrine disruption, while females remained largely unaffected. IBRv2 analysis reflected these dynamics, with transient reductions early and pronounced elevations at later stages. Joint effect evaluation revealed synergistic toxicity in GA + CuO-NP groups, where biomarker scores and IBR indices exceeded single exposures, amplifying both biochemical strain and endocrine imbalance. These findings demonstrate that mixture effects are dynamic and cannot be predicted from single‑agent exposures, underscoring the importance of incorporating contaminant interactions into ecological risk assessment.