Oxidative stress response biomarkers in gills and liver of bullfrog tadpoles semi-chronically exposed to combined microplastics and titanium dioxide nanoparticles

Abstract Amphibians are among the most threatened groups of vertebrates worldwide, and the increasing release of emerging contaminants such as microplastics (MPs) and nanoparticles (NPs) poses a growing ecological risk to these organisms. This study evaluated the semi-chronic effects of polyethylene microplastics (60 mg/L) (PE-MPs) and titanium dioxide nanoparticles (10 µg/L) (TiO 2 NPs), individually and in combination (Mix), on oxidative stress and detoxification biomarkers in the gills and liver of bullfrog tadpoles ( Aquarana catesbeiana , in Gosner stages 25–26). Tadpoles were exposed for 15 days, and biochemical parameters (ethoxyresorufin-O-deethylase – EROD, glutathione-s-transferase - GST, superoxide dismutase - SOD, catalase - CAT, lipid peroxidation - LPO, protein carbonyls - PCO, and glucose levels) were quantified. In the gills, the Mix group significantly increased SOD and CAT activities, indicating sustained antioxidant responses associated with the formation of reactive oxygen species (ROS). In the liver, GST activity was elevated only in the Mix group, while LPO levels increased in all contaminant-exposed groups, demonstrating oxidative damage to membranes. Conversely, hepatic glucose concentrations decreased exclusively in the MP group, suggesting disturbances in energy metabolism potentially linked to contaminant bioaccumulation. Principal Coordinates Analysis (PCoA) and Integrated Biomarker Response indices (IBRv2) revealed organ-specific and treatment-dependent effects, with more pronounced biochemical alterations in the liver and stronger integrated responses in the MPs and Mix treatments. Altogether, these results demonstrate that MPs and NPs induce distinct oxidative and metabolic dysfunctions in tadpoles, with synergistic effects emerging under combined exposure. These findings highlight the importance of assessing mixtures of emerging contaminants and underscore the vulnerability of amphibian larvae to environmentally realistic pollution scenarios.