Aged nanoplastics exacerbate CPPD-induced ocular toxicity via the thyroxine pathway in zebrafish

The coexistence of naturally aged nanoplastics (NPs) and the antioxidant N-cyclohexyl-N'-phenyl-p-phenylenediamine (CPPD) in aquatic environments poses combined risks to ecosystems. This study evaluated the developmental and ocular toxicity of CPPD alone and in co-exposure with two types of aged polystyrene nanoplastics (PS) including ultraviolet-irradiated (PS-UV) and ozonated (PS-O₃) in embryonic zebrafish. Results showed that the presence of aged PS significantly exacerbated CPPD-induced developmental toxicity, manifested as synergistic inhibition of spontaneous movement, touch response, and heart rate, alongside increased malformation rates. Notably, co-exposure aggravated larval ocular abnormalities and visual behavior deficits (light sensitivity and phototaxis), accompanied by increased cell death and inflammation in the ocular region. The combined toxicity of PS-O₃ with CPPD was more potent than that of PS-UV. Mechanistic investigations revealed that the thyroid pathway inhibitor n-phenylthiourea (PTU), but not triiodothyronine (T₃), effectively alleviated ocular toxicities. While T₃ showed a partial response to CPPD alone, PTU treatment successfully rescued co-exposure-induced apoptosis, aberrant Olig2 and Huc expression, and Mbp suppression in the visual system. ELISA analysis confirmed that PTU restored ATP levels and thyroid hormones (T₃, T₄, TSH) reduced by co-exposure. qPCR showed that PTU attenuated cyp26a activation (a retinoic acid-metabolizing enzyme) and opn1sw1 suppression (a cone opsin gene). Further Mantel analyses revealed these biomarkers were highly correlated with most ocular development indexes, highlighting their potential as key indicators of early visual toxicity progression. These findings suggest that aged PS amplifies CPPD toxicity through synergistic disruption of oxidative stress, thyroxine axis function, and retinoid/phototransduction pathways, providing new insights for ecological risk assessment.