Sex- and size-dependent impacts of tire wear particles and zinc oxide nanoparticles on adult zebrafish: integrated evidence from physiology, gut microbiota networks, and hepatic transcriptomics.

The co-occurrence of tire wear particles (TWPs) and engineered nanoparticles in aquatic environments raises concerns about their combined impacts on freshwater biota. Here, we assessed sex- and size-dependent responses of adult zebrafish following 15-day exposure to control conditions, ZnO-NPs (760 μg/L), large TWPs (LTWPs, 250-380 μm; 10 mg/L), small TWPs (STWPs, <120 μm; 10 mg/L), and co-exposure treatments combining ZnO-NPs with either LTWPs or STWPs. Across endpoints, females were more sensitive than males, showing broader reductions in growth-related and organ-somatic indices. Small TWPs generally induced broader and stronger adverse effects than larger TWPs, and combined exposure to TWPs and ZnO-NPs was associated with stronger physiological and oxidative-stress responses in selected endpoints, particularly in females. Gut microbiota analyses revealed sex-dependent community restructuring, with the female co-exposure group showing a more fragmented interaction network. Female hepatic transcriptomics revealed a graded molecular response across the selected exposure scenarios, characterized by a shared stress-response core together with exposure-specific signatures related to innate immune regulation, apoptosis, proteostasis, and mitochondrial bioenergetic remodeling. WGCNA of the female hepatic transcriptome identified an immune-associated hepatic module that covaried with exposure-responsive gut bacterial genera, which were further associated with antioxidant responses and reduced body or liver weight, supporting coordinated, correlation-based multi-organ and microbiota-associated signatures under particulate stress. Collectively, these findings highlight the importance of particle size, co-exposure context, and sex-specific susceptibility in shaping the toxicity of traffic-derived particulate contaminants in freshwater organisms.