Biotoxic Effects Of Typical Sulfonamide Antibiotics In Aquatic Ecosystem On Adult Zebrafish

Antibiotics are not only used in the treatment and prevention of human diseases,but are also widely used in livestock and aquaculture industries to promote animal health and growth.However,the contamination of antibiotics in water has also become a topic of great concern in recent years,especially the contamination of sulfonamide antibiotics seriously affects the development and growth of aquatic organisms.In this paper,three typical sulfonamide antibiotics,including sulfadiazine(SD),sulfamethazine(SM2)and sulfamethoxazole(SMX),were first evaluated for their 96 h median lethality on zebrafish;The effects of SD and SMX on zebrafish growth were then investigated.The effects of SMX on antioxidant,immune,histopathological dynamic changes as well as intestinal microbiota in zebrafish will be finally evaluated to provide a scientific basis for developing aquatic ecological risk assessment of antibiotics.For acute toxicity testing,three antibiotics(SD,SM2 and SMX)were set up at four concentrations of 0(c),300(T300),600(T600)and 1200 mg/L(T1200)in three replicates each group.For chronic toxicity testing,SD and SMX were administered in five concentration groups of 0(C),3(T3),6(T6),12(T12)and 24 mg/L(T24),with five replicates per group for 8 weeks.The findings are as follows:1.Acute toxicity test results showed that the median lethal concentration(LC₅₀)at 96 h for SD and SMX on zebrafish was 4230.508 mg/L and 3091.003 mg/L with 95%confidence interval(CI)of 4230.508±485.979 mg/L and 3091.003±526.889 mg/L,respectively,and that at 96 h for SMX on zebrafish was greater than 1200 mg/L.2.The results of chronic toxicity test showed that different concentrations of SD and SMX did not significantly affect the growth of zebrafish(P>0.05).3.Biotoxic effects of SMX on zebrafish(1)The results of liver antioxidant enzyme activities showed a good linear relationship between the levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),catalase(CAT)and malondialdehyde(MDA)and different SMX concentrations.In the gill tissue,the SOD activity was first induced to rise and then inhibited to decrease as the concentration of SMX increased.The GSH-Px activity in the T24 group was significantly lower than that in the control group(P<0.05).There was a good linear relationship between CAT activity,MDA content and SMX concentration.(2)The relative gene expression in liver tissue results showed that the relative expression of SOD,GSH-Px,and CAT genes in the T24 group was significantly lower than that in the control group(P<0.05),and as the SMX concentration increased,the immune related gene expression of IL-1??IL-8 and TNF-?showed an upward trend.In the gill tissue,the gene expressions of SOD and CAT in T12 and T24 groups were lower than those in the control group,and the gene expression of GSH-Px in the T24 group was significantly lower than that in the control group(P<0.05).(3)The histopathological results of intestine and liver tissues showed that different degrees of pathological damage were induced in the intestine and liver tissues under different SMX concentration exposures,mainly,the villus height in the intestine was significantly reduced,the villus width was wider,the muscular layer thickness in the highest concentration group was significantly thinner,and the number of goblet cells was increased.At the same time,the liver tissue appears congestion,inflammatory cell infiltration,vacuolization,hepatocyte nuclear pyknosis and lysis.The damage of tissues was aggravated with the increase of SMX concentration.(4)The microbiome sequencing revealed that the gut microbiota structure of each SMX group was significantly changed compared with that of the control group,and the Proteobacteria,Fusobacteria,and Firmicutes were the predominant bacterial taxa in the zebrafish gut.The abundances of Proteobacteria and Firmicutes was significantly decreased in T6,T12 and T24groups,and those of Fusobacteria increased significantly.The Chao1,Shannon,Observed otus and Pielou-e indexes in T24 group were significantly lower than those of the control group(P<0.05).(5)The results of SMX residual in muscle tissue showed that the SMX residual improved with the increase of SMX concentration,and the residual of SMX in muscle tissue was 1.93?g/kg,3.64?g/kg in T3 and T24 groups,respectively.In summary,the SD,SM2,and SMX are considered to be hypotoxic to adult zebrafish.The SD and SMX at below 24 mg/L did not affect zebrafish growth.Under the stress of different SMX concentrations,it caused oxidative damage and immune system disorder in zebrafish liver and gill,which was the most significant in 12 and 24 mg/L groups.At the same time,it also caused varying degrees of pathological changes in intestinal and liver.With the increase of SMX concentration,the diversity and abundance of intestinal microbial community of zebrafish decreased significantly.