Combined exposure to nano-TiO₂ and carbamazepine exacerbates reproductive toxicity in mussels Mytilus coruscus by disrupting steroid hormone homeostasis.

Titanium dioxide nanoparticles (nano-TiO₂) and carbamazepine (CBZ) are both emerging contaminants of concern in coastal environments. Current monitoring data indicate that nano-TiO₂ occur in nearshore seawater affected by land-based inputs, and CBZ is persistently detected in estuarine and nearshore waters; therefore, these contaminants may co-occur at a regional scale. In this study, the thick-shelled mussel (Mytilus coruscus) was used as a model organism. Nominal concentrations of nano-TiO₂ (100 μg/L) and CBZ (10 μg/L) were applied in single and combined exposure experiments over 14 days. Particle characterization, gonadal morphology, sex steroid hormone levels, and key gene expression analyses were integrated to evaluate reproductive hormone-disrupting effects. The results showed that nano-TiO₂ readily aggregated in seawater, and distinct nano-TiO₂ aggregates were observed on the surface of CBZ crystals in dried mixed samples. Phenotypically, exposure to either pollutant alone reduced the gonadal area and gonadosomatic index, showing significant sex-specific differences in the CBZ group. In contrast, combined exposure caused more pronounced reproductive tissue damage. Hormonally, exposure disrupted sex steroid homeostasis, decreasing progesterone, estrone, and estradiol (E2), accompanied by increased testosterone (T). These hormonal changes exhibited sex-dependent patterns across treatments, with a decrease in E2 and an increase in T being more pronounced in the combined-exposure group. Molecularly, exposures significantly downregulated SF-1 and Wnt signaling genes (β-catenin, Wnt4), while upregulating Wnt7b. Overall, single exposure to nominal concentrations of nano-TiO₂ or CBZ was sufficient to induce sex-specific reproductive damage in M. coruscus, whereas combined exposure caused more pronounced changes at the morphological, hormonal, and transcriptional levels.