Biotoxic Effects Of Heavy Metals (Cu²⁺、Cr⁶⁺ And Cd²⁺) Combined In Water On Adult Zebrafish

Cu²⁺,Cr⁶⁺and Cd²⁺are common heavy metal contaminants in the aquatic environment and can cause physiological dysfunction in aquatic organisms.In the past decade,there have been more studies on the toxicity of single heavy metals to aquatic organisms,while there are fewer studies on the toxic effects of combined Cu²⁺,Cr⁶⁺and Cd²⁺on adult zebrafish.Therefore,in this paper,the combined toxic effects and toxicological mechanisms of Cu²⁺,Cr⁶⁺and Cd²⁺occurring in the coexistence of three metal ions were studied in zebrafish.In the single acute toxicity test,five concentration groups were set up for Cu²⁺,Cr⁶⁺and Cd²⁺,with three replicates in each group;in the combined acute toxicity test,the combined toxicity test of Cu²⁺,Cr⁶⁺and Cd²⁺was carried out in the ratio of 1:1,with four concentration groups of 1/4 times,1/2 times,1 times and 2 times 96 h-LC₅₀and a control group.In the chronic toxicity test,five concentration groups of Cu²⁺,Cr⁶⁺and Cd²⁺,1/40,1/30,1/20 and 1/10 96 h-LC₅₀were set up,with three replicates in each group and 25 fish in each replicate,and the trial was conducted for 8 weeks.The results of the study were as follows:1.The results of single acute toxicity test showed that the 96-h LC₅₀for Cu²⁺,Cr⁶⁺and Cd²⁺were 0.52 mg/L,18.83 mg/L and 184.38 mg/L for adult zebrafish,respectively.2.The results of the combined acute toxicity showed that the combined effects of Cu²⁺,Cr⁶⁺and Cd²⁺were antagonistic and then synergistic.3.Biotoxic effects of Cu²⁺,Cr⁶⁺and Cd²⁺on zebrafish3.1The results of antioxidant indexes showed that in liver tissue,superoxide dismutase（SOD）,glutathione peroxidase（GSH-PX）and catalase（CAT）activities decreased with increasing exposure concentrations of Cu²⁺,Cr⁶⁺and Cd²⁺,and malondialdehyde（MDA）content increased with increasing exposure concentrations;in gill tissue,the activities of CAT and GSH-PX in the 1/10 group were significantly lower in gill tissue.Among the expression of genes related to gill,liver and intestinal tissues,the relative expression of antioxidant-related genes（SOD,GSH-PX,CAT）showed a trend of up-regulation followed by down-regulation,and the relative expression in the 1/10 and 1/20 groups was lower than that in the control group（P<0.05）.3.2The results of immune indexes showed that the activities of immune-related enzymes（ACP,AKP,LZM）in liver tissues,showed a trend of increasing and then decreasing,while in intestinal tissues,there was a trend of significant increase compared to the control group（P<0.05）.The expression levels of interleukin-1β（IL-1β）,interleukin-8（IL-8）and tumor necrosis factor-α（TNF-α）were significantly upregulated in the expression of genes related to gill,liver and intestinal tissues,and the relative expressions of the highest concentrations（1/10 and 1/20）were both significantly different compared to the control group（P<0.05）.3.3The results of histopathological studies of liver,intestine and gill showed that different degrees of pathological damage were induced in liver,intestine and gill tissues under different Cu²⁺,Cr⁶⁺and Cd²⁺concentration exposures.The main manifestations were obvious central venous stasis,and peripheral nuclei and nuclear consolidation;gill tissue structure was relatively disorganized,with a rod shape as a whole;intestinal villi were severely broken,and mucosal epithelial cells were necrotic,accompanied by vacuolization of the muscular layer.3.416s revealed that the structure of intestinal flora of zebrafish exposed to different concentrations of Cu²⁺,Cr⁶⁺and Cd²⁺were significantly changed by heavy metals compared to the control group,with Proteobacteria,Firmicutes and Fusobacteriota The abundance of Proteobacteria,Firmicutes and Fusobacteriota in the 1/10,1/40 and 1/20 groups decreased significantly,while the abundance of Firmicutes increased significantly;the indices of Chao1,Shannon in the 1/10 group were significantly lower than those in the control group（P<0.05）.3.5The residues of Cu²⁺,Cr⁶⁺and Cd²⁺in muscle tissue showed that the residues of Cd²⁺and Cr⁶⁺in muscle tissue increased with increasing concentrations,and the residues of Cd²⁺and Cr⁶⁺in muscle tissue in the 1/40 group were 1.53 mg/kg and 2.57 mg/kg,respectively,after 8 weeks of zebrafish exposure,but the residues of Cu²⁺in muscle tissue decreased with increasing exposure However,the residues of Cu²⁺in muscle tissue decreased with the increase of exposure concentration.In conclusion,Cd²⁺and Cr⁶⁺had low toxicity and Cu²⁺had high toxicity to adult zebrafish.The relative abundance of Bacillus phylum and Thick-walled phylum in the intestinal tissues increased in the 1/10 and 1/20 groups,while the relative abundance of Actinomycetes and Aspergillus phylum decreased in the 1/10 group.The residues of Cd²⁺and Cr⁶⁺in muscle tissue were positively correlated with their concentrations,while the residues of Cu²⁺were negatively correlated with their concentrations.