Aged polystyrene nanoplastics and bisphenol A co-exposure impair mitochondrial respiration in juvenile horseshoe crab Tachypleus tridentatus.

Despite mounting evidence that environmental weathering alters the surface chemistry of nanoplastics (NPs), most ecotoxicological studies continue to use pristine particles, leaving the toxicological consequences of NP aging insufficiently resolved. Here, we systematically compared the toxicity of pristine and aged polystyrene NPs (PS-NPs), in combination with bisphenol A (BPA), on juvenile Chinese horseshoe crabs (Tachypleus tridentatus), an endangered IUCN Red List marine sentinel. Six treatments-control, BPA, pristine NPs, aged NPs, pristine NPs + BPA, and aged NPs + BPA-were administered over 21 days, with sampling at days 7 and 21. Biomarkers spanning surface chemistry, mitochondrial function, membrane integrity, respiratory chain activity, and oxidative stress were integrated using IBRv2, PCA, and Pearson correlation. UV aging markedly altered NP surfaces: zeta potential shifted from -5.6 mV to -49.9 mV, and the carbonyl index increased 12.8-fold. IBRv2 scores at day 7 ranked: aged NPs (96.7) > aged NPs + BPA (53.6) > pristine NPs + BPA (44.5) > pristine NPs (37.3) > BPA (21.2); at day 21, the ranking reorganized to aged NPs + BPA (88.7) > pristine NPs (57.3) > aged NPs (49.0) > pristine NPs + BPA (46.5) > BPA (45.0). Critically, aged NPs + BPA caused toxicity dominated by mitochondrial respiratory chain impairment (SDH declining to 22% of control; MDA reaching 206 nmol·mL-1 at day 21), whereas pristine NPs + BPA drove acute membrane lipid peroxidation-a divergence confirmed by PCA. Our findings demonstrate that NP aging shifts the mode, rather than merely the intensity, of combined toxicity with BPA, with implications for environmental risk assessment.