Dogan_2026_Toxicol.Appl.Pharmacol__117967

Aquatic ecosystems are increasingly exposed to contaminant mixtures rather than single compounds, creating complex interactions that remain poorly understood in ecotoxicology. Agricultural pesticides and engineered nanoparticles are among the most pervasive stressors, often detected together in surface waters due to intensive farming practices and widespread industrial use. Spirotetramat (a systemic tetramic acid insecticide), methoxyfenozide (an ecdysone agonist), and zinc oxide nanoparticles (ZnO-NPs) were focused on as representative co-occurring contaminants with potential joint effects on aquatic organisms. To investigate their combined impacts, adult zebrafish (Danio rerio) were exposed for 21 days to single, binary, and ternary mixtures under sublethal conditions, and multibiomarker profiling was applied to integrate neurotoxicity, oxidative stress, endocrine disruption, and apoptosis. Sustained acetylcholinesterase inhibition indicated persistent neurotoxic stress, while GST and CAT induction, biphasic GPx regulation, and elevated TBARS, protein carbonyls, and 8-OHdG revealed overwhelmed antioxidant defenses and cumulative oxidative damage. Vitellogenin induction, particularly in males, provided clear evidence of endocrine disruption, and caspase-3 activation highlighted apoptotic stress compromising organismal resilience. Integrated biomarker response scores identified ternary mixtures as most disruptive, while joint toxicity analysis revealed synergistic interactions beyond additive expectations. Collectively, these findings provide mechanistic evidence that pesticide-nanoparticle mixtures intensify neurotoxicity, oxidative stress, endocrine disruption, and apoptosis in zebrafish. Our results underscore that mixture-based biomarker integration is critical for capturing ecologically relevant outcomes and advancing risk assessment of emerging contaminant combinations.