Dose-dependent acute toxicity of chitosan nanoparticles with dual assessment of multisystem toxicopathology and oxidative stress biomarkers in Nile tilapia

Nano-chitosan (nCS) is widely used in aquaculture for its antimicrobial and immunostimulatory properties, but its toxicological profile in fish remains poorly understood. This study assessed the dose-dependent acute toxic effects of nCS in Nile tilapia on oxidative stress biomarkers and multisystemic histopathology to clarify the mechanisms of systemic injury. Healthy fish (n = 80) were exposed to spherical nCS (8-20 nm (at concentrations of 0, 5, 10, and 20 mg/L for 96 h. Exposure to nCS caused dose-dependent toxicity. While no mortality was observed at concentrations up to 5 mg/L, the 20 mg/L dose induced significant behavioral changes and 50% mortality within 96 h. Biochemical analysis indicated systemic oxidative stress, with increased lipid peroxidation and depleted antioxidant defenses across the gills, kidney, liver, and muscle. Comprehensive histopathological examination of eight distinct tissues confirmed severe tissue damage, including branchial lamellar fusion, renal tubular necrosis, and neurotoxic effects in the brain, correlating with neurobehavioral distress. These findings indicate that acute exposure to nCS concentrations of ≥ 10 mg/L induces severe multisystemic necrosis, establishing a no-observed-adverse-effect-level (NOAEL) below 5 mg/L. The study identifies the gills and kidney as key nanotoxicity biomarkers, suggesting that strict application limits are essential to balance the therapeutic benefits and ecological risks of nCS in aquaculture.