From cells to tadpoles: mechanistic insights into ZnO-nanoparticle-induced oxidative stress and developmental toxicity in Bufo bufo.

Zinc oxide nanoparticles (ZnO-NPs) are widely used engineered nanomaterials increasingly present in freshwater ecosystems, yet their effects on amphibians remain poorly understood. Amphibians are ecologically important and highly sensitive to contaminants, making them valuable bioindicators. This study provides the first integrated assessment of ZnO-NP effects on the European toad (Bufo bufo, Gosner stages 26–27), combining biochemical, endocrine, and histological endpoints. Tadpoles were exposed for seven days to sub lethal ZnO-NP concentrations (0.1, 1.0, and 10 mg/L) under controlled laboratory conditions, then maintained in clean water until metamorphosis to evaluate survival, growth, developmental progression, and tissue integrity. Exposure induced ROS-mediated oxidative stress, evidenced by increased SOD and CAT activities and elevated malondialdehyde levels, while corticosterone elevation revealed systemic endocrine disruption. Histological analyses showed dose-dependent damage in gills, liver, eyes, and limb buds, with limb buds and gills exhibiting the highest sensitivity. These combined morphological and physiological impairments led to developmental delays, stage-specific mortality peaks, and arrested metamorphosis, highlighting critical windows of larval vulnerability. Our findings reveal a mechanistic pathway linking oxidative stress, endocrine disruption, and tissue damage and underscore the importance of integrating species-specific physiological and morphological responses in ecological risk assessments of nanoparticles.