Single and Combined Embryotoxic Effects of Metal Oxide nanoparticles (Aluminium Oxide (Al2O3) and Zinc Oxide (ZnO)) on the African catfish (Clarias gariepinus)

• Combined Al₂O₃+ZnO reduced hatching to 58% at 10 mg/L after 48 hpf. • Single treatments showed higher hatchability (Al₂O₃ 77%, ZnO 87%) than mixtures • Survival declined significantly from 24–48 hpf under combined exposure (p<0.001) • Combined exposure significantly reduced larval swimming speed and distance (p<0.05) • CAT, SOD and GPx activities were lowest in combined exposure after 48 hpf The widespread application of aluminium oxide (Al₂O₃) and zinc oxide (ZnO) nanoparticles (NPs) in industrial and consumer products has heightened concerns regarding their ecological risks, particularly in aquatic environments. This study investigated the in vivo embryotoxic effects of Al₂O₃ and ZnO NPs, individually and in combination, on Clarias gariepinus embryos/larvae. Fertilised embryos were exposed for 48 hours, following standard guidelines, to 0, 0.5, 1, 5, and 10 mg/L of single and combined nanoparticles. Endpoints evaluated included Zn and Al bioaccumulation, survival, hatchability, morphological abnormalities, swimming speed, enzymatic antioxidants (CAT, SOD, GPx), non-enzymatic antioxidants (GSH, total proteins), and lipid peroxidation (MDA). The nanoparticles (<50 nm; mixture size 27.65 ± 10.32 nm) formed stable dispersions and showed concentration-dependent accumulation, with significantly higher metal uptake under combined exposure (p < 0.05). Survival did not differ significantly from the control; however, hatching success declined at 10 mg/L to 77% (Al₂O₃), 87% (ZnO), and 58% (combined). Exposure also induced dose-dependent developmental abnormalities and impaired swimming performance, with a 79% reduction in swimming speed at 10 mg/L in the combined treatment. Marked oxidative stress was evident with CAT decreasing by 90%, 82%, and 77%; SOD by 56%, 32%, and 26%; and GPx by 96%, 68%, and 65% under combined, Al₂O₃, and ZnO treatments, respectively. Conversely, GSH, total protein, and MDA levels increased markedly, with the highest elevations in the combined group. These findings demonstrate synergistic toxicity of Al₂O₃ and ZnO mixtures and highlight the ecological relevance of assessing nanoparticle co-exposures in environmental risk assessment.