Effects Of Environmental Concentrations Of Oxytetracyeline And Polystyrene Microplastics On Growth And Intestinal Health Of Zebrafish

Oxytetracycline(OTC)is a broad-spectrum antibacterial drug that is widely used in human and animal disease control,animal husbandry and aquaculture,as a result,it is frequently detected in water environment.The distribution of OTC in water is also influenced by other pollutants.Polystyrene microplastics(PS-MPs)have been shown to adsorb a variety of antibiotics,including OTC,resulting in combined contamination effect.However,there are few studies on the toxic effects of combined exposure to OTC and PS-MPs on aquatic organisms.In this study,zebrafish were used as experimental animals to investigate the effects on zebrafish after exposure to environmental concentrations of OTC(500 ng/L and 5000 ng/L)alone or in combination with environmental concentrations of PS-MPs(200 μg/L)for 90 days.We investigated changes in digestive enzymes(trypsin(TRS),amylase(AMS),lipase(LPS)),gut health(serotonin(5-HT)content,tight junction protein TJP2 content,interleukin-1β(IL-1β)content)and intestinal flora to elucidate the possible reason causing growth and developmental effects.The results of the study are as follows:1.The results of the chemical analysis indicated that 200 μg/L of 5 μm PS-MPs accumulated in the intestine of zebrafish and that high concentrations(5000 ng/L)of OTC in combination with PS-MPs increased the accumulation of PS-MPs in the intestine.2.Toxicological endpoints showed that environmental concentrations of OTC(500ng/L,5000 ng/L)significantly inhibited body length and body weight of male and female zebrafish;PS-MPs significantly inhibited body weight of male zebrafish;Combined exposure of OTC and PS-MPs significantly inhibited the growth and development of zebrafish,including significant reduction of body length and body weight,and significant reduction of HSI and BSI,Compared with OTC exposure alone,the combined exposure of OTC and PS-MPs resulted in significant decreases of body length and BSI in male zebrafish and no significant changes in female zebrafish;Compared with PS-MPs alone,the high concentration of compound exposure resulted in a significant decrease in body length,body weight,BSI and HSI of zebrafish;the results indicated that OTC and PS-MPs showed a synergistic effect on the growth inhibition of zebrafish,with males being more affected.3.Histopathological results showed that environmental concentrations of OTC caused intestinal structural damage of female and male zebrafish,as evidenced by a significant decrease in the height of the villi and the number of goblet cells.Environmental concentrations of PS-MPs caused a significant decrease in villus height of female and male zebrafish and a significant decrease in goblet cells of male zebrafish,exhibiting intestinal damage effect.The combined exposure of OTC and PS-MPs exacerbated the intestinal tissue damage induced by OTC or PS-MPs alone,this was demonstrated by a significant reduction of intestinal villus height and intestinal somatic index(ISI)compared to OTC exposure alone,and a significant reduction of ISI compared to PS-MPs alone,showing a synergistic toxic effect.Intestinal physiological indicators showed that 5000 ng/L OTC significantly altered intestinal TJP2 and 5-HT levels and affected intestinal health in zebrafish;combined exposure to OTC and PS-MPs significantly altered intestinal TJP2 and 5-HT levels and had a negative but not synergistic effect on intestinal health.4.Analysis of the activities of digestive enzymes closely related to growth(TRS,AMS and LPS)showed that 5000 ng/L OTC led to a significant reduction in digestive enzyme activity of zebrafish;PS-MPs significantly reduced digestive enzyme activity of male zebrafish but had no significant effect on female zebrafish,which may be one of the main reasons for the sex-dependent inhibition of growth in male zebrafish by PS-MPs.The combination of OTC and PS-MPs further significantly inhibited the digestive enzyme activities of male and female zebrafish,showing synergistic toxic effects,which is consistent with the synergistic inhibition of growth and development in the combined group,these results suggest that OTC and PS-MPs may inhibit fish growth through synergistically inhibiting the activity of TRS AMS LPS.5.The results of the intestinal microbiological analysis showed that OTC(500 ng/L and 5000 ng/L)exposure alone resulted in a disruption of the zebrafish gut flora,as evidenced by a significant increase in the relative abundance of the proteobacteria in male and female zebrafish and a significant decrease of actinobacteria in female zebrafish;at the genus level,the relative abundance of pseudomonas was significantly increased in female zebrafish,while the relative abundance of oceanicaulis in male zebrafish was significantly decreased.PS-MPs exposure alone resulted in a significant increase in the relative abundance of actinobacteria in zebrafish and a significant decrease in the relative abundance of actinobacteria in male zebrafish.Compared to PS-MPs and OTC alone,the combined exposure exacerbated intestinal flora disorders,as evidenced by the relative abundance of proteobacteria and pseudomonas were significantly increaseand in female zebrafish and the relative abundance of actinobacteria and oceanicaulis were significantly decrease in male zebrafish.Further correlation analysis showed a significant negative correlation between proteobacteria and body length and body weight in female zebrafish,and a significant positive correlation between oceanicaulis and body weight in male zebrafish.This suggests that a significant increase in the pathogenic genus pseudomonas may be associated with growth inhibition in female zebrafish,and a significant decrease in the detoxification-related genus oceanicaulis may be associated with growth inhibition in male zebrafish.In conclusion,Our results show that exposure to environmental concentrations of both OTC and PS-MPs alone resulted in significant inhibition of growth and development in zebrafish,and that the combination of OTC and PS-MPs exacerbated the growth inhibitory effects of OTC and PS-MPs exposure alone,showing significant synergistic toxic effects.Further studies have shown that exposure to both OTC and PS-MPs alone resulted in intestinal injury and affected physiological indicators related to intestinal health,and inhibits growth and development mainly by inhibiting the activity of digestive enzymes,and that the combination of OTC and PS-MPs showed significant synergistic effects.Analysis of the gut microbial community structure showed that environmental concentrations of OTC and PS-MPs alone and in combination caused disturbances in the gut flora.Correlation analysis showed that a significant increase in the pathogenic genus pseudomonas may be associated with growth inhibition in female zebrafish,and a significant decrease in the detoxification-related genus oceanicaulis may be associated with growth inhibition in male zebrafish.