A Comprehensive Assessment of Pollutant Exposure (Cu²⁺, Cr⁶⁺ and Cd²⁺) in Adult Zebrafish

Copper (Cu²⁺), hexavalent chromium (Cr⁶⁺), and cadmium (Cd²⁺) are pervasive heavy metal pollutants in aquatic ecosystems, often coexisting and posing potential risks to aquatic organisms. While the individual toxicity of these metals is well documented, their combined effects at environmentally relevant concentrations remain poorly understood. This study investigated the chronic toxicity of a ternary mixture of Cu²⁺, Cr⁶⁺, and Cd²⁺ in adult zebrafish (Danio rerio) across multiple biological endpoints. In the chronic toxicity test, the toxicity of Cu²⁺, Cr⁶⁺, and Cd²⁺ was set up in five concentration groups, namely the control group, 1/40 (Cu²⁺ 0.013 mg/L, Cr⁶⁺ 4.610 mg/L, and Cd²⁺ 0.471 mg/L), 1/30 (Cu²⁺ 0.017 mg/L, Cr⁶⁺ 6.147 mg/L, and Cd²⁺ 0.628 mg/L), 1/20 (Cu²⁺ 0.026 mg/L, Cr⁶⁺ 9.220 mg/L, and Cd²⁺ 0.942 mg/L), and 1/10 (Cu²⁺ 0.052 mg/L, Cr⁶⁺ 18.440 mg/L, and Cd²⁺ 1.884 mg/L) of the 96 h-LC50 values. Each group had three replicates, with 25 fish in each replicate. Under the exposure of different concentrations of Cu²⁺, Cr^{6 +}, and Cd²⁺, the liver, intestine, and gill tissue induced different degrees of pathological damage, and caused oxidative damage and immune system disorder in liver and gill tissue. In the 1/10 and 1/20 concentration group, the relative abundance of Bacteroidetes and Firmicutes in intestinal tissue increased, while the relative abundance of Actinobacteriota and Proteobacteria decreased. The residual amounts of Cd²⁺ and Cr⁶⁺ in muscle tissue are positively correlated with their concentrations, while the residual amounts of Cu²⁺are negatively correlated with their concentrations.