Assessing the Combined Toxicity of Silver and Copper Nanoparticles in Rainbow Trout (Oncorhynchus mykiss) Fingerlings.

The growing use of silver (Ag) and copper (Cu) nanoparticles (NPs) for their antimicrobial properties has raised environmental health concerns due to their coexistence in aquatic ecosystems. This study assessed the combined physiological and molecular toxicity of AgNPs and CuNPs in rainbow trout (Oncorhynchus mykiss) exposed to sub-lethal concentrations of the NP mixture for 21 days. Fish were exposed to varying concentrations of co-exposure of AgNPs and CuNPs (T1 group 0.2 AgNPs + 0.2 mg/L CuNPs, T2 group 0.8 AgNPs + 0.6 mg/L CuNPs, and T3 group 1.4 AgNPs + 1.0 mg/L CuNPs). Behavioral alterations were evident, accompanied by a significant (p < 0.05) reduction in hemoglobin, red blood cell count, and hematocrit levels, while white blood cell counts increased, indicating immune activation. Serum biochemical analyses revealed metabolic disturbances linked to oxidative stress and physiological imbalance. Enzymatic activities in gills and liver showed a dynamic response, with elevated catalase (CAT) and superoxide dismutase (SOD) levels at T2 and T3 after 14 days, followed by a decline by day 21. Glutathione S-transferase (GST) activity increased in gills at T2 and T3 after 7 days and in the liver at T3 after 14 days, while lipid peroxidation (LPO) significantly increased in gills at T3 after 7 days and in the liver at T2 and T3 after 14 days. Molecular analysis confirmed upregulation of oxidative stress genes (SOD1, CAT) and inflammatory markers (HSP70, IL- 1β). Histopathological examination revealed gill damage, including lamellar fusion and hyperplasia, and liver degeneration, such as hepatocyte vacuolation and necrosis, with the most severe effects observed at T3. These findings highlight dose-dependent toxicity and oxidative damage caused by the AgNPs-CuONPs mixture, emphasizing its potential physiological and molecular impacts on aquatic organisms.