Ecotoxicological Effects Of Butyl Benzyl Phthalate On Zebrafish

Phthalates(PAEs)are now widely used as additives in the manufacture and processing of various plastic products,and their global production and use are growing rapidly.However,since PAEs are not bound to plastics by stable chemical bonds but by hydrogen bonds or van der Waals forces,they can easily escape from plastics and enter the surrounding environment during use,thus posing potential ecological hazards.Among them,butyl benzyl phthalate(BBP)is one of the widely used phthalate plasticizers among PAEs.As a persistent environmental pollutant,the water environment may be contaminated by BBP through various pathways in natural and urban environments,posing potential risks to aquatic organisms,therefore,it is particularly important to systematically investigate the ecotoxicological effects of BBP.To explore the ecotoxicity of BBP to aquatic organisms,this study used adult zebrafish as the model organism and BBP as the target contaminant,and set three environmentally relevant concentrations of 5 μg/L,50 μg/L and 500 μg/L,and used chronic exposure tests,the trends of superoxide dismutase(SOD)activity,catalase(CAT)activity,glutathione-S-transferase(GST),reactive oxygen species(ROS)content and malondialdehyde(MDA)content were measured in zebrafish gill tissues at four cycles(days 7,14,21 and 28)to evaluate the oxidative damage of BBP on zebrafish;Evaluation of the tissue damage of BBP on zebrafish by HE staining of intact tissues of the gills of zebrafish in the control and high concentration 500 μg/L treated groups;evaluation of DNA damage by BBP in zebrafish based on changes in 8-hydroxydeoxyguanosine(8-OHd G)activity in the gills of zebrafish;RT-q PCR was used to determine the changes in the expression of p53 and caspase-3 in zebrafish gill and apoptosis-related genes,and 18 rs rrn and mt-ndl in mitochondria-related genes to determine the effects of BBP on zebrafish gill apoptosis and mitochondrial function-related effects,and finally,molecular docking techniques were used to predict the mode of action of simulated BBP on zebrafish.The main findings were:(1)The effects of BBP on the activity changes of antioxidant enzyme system and lipid peroxidation in the gills of zebrafish were determined at different test cycles.The specific effects were that BBP induced an increase and then a decrease in the reactive oxygen species content in the gills of zebrafish,and the ROS content in the high concentration treatment group was still higher than that in the control group;the activities of antioxidant enzymes CAT and SOD were inhibited,and the activity of GST showed a trend of activation and then inhibition;BBP caused an overall trend of increasing the MDA content in the gills of zebrafish,and at 21 d and 28 d,the MDA content increased with the increase of BBP The MDA content increased with the increase of BBP concentration at 21 d and 28 d,with a certain dose-effect relationship(2)The effects of BBP on DNA damage in the gills of zebrafish were determined for different test cycles.Specifically,the 8-OHd G content in the gills of zebrafish increased significantly after the poisoning and had a dose-effect relationship in the middle of the test.(3)BBP caused tissue damage to the gills of zebrafish,and after 28 d of exposure to 500μg/L BBP,swelling and even necrosis of gill lamellae cells,slight adhesions at the base,and cavitation of gill filament vessels were observed by HE staining.(4)The binding of two important antioxidant enzymes,CAT and SOD,to BBP was investigated by molecular docking means,and the results showed that BBP was able to bind to the active sites of both proteins,which may have some influence on the protein morphology and structure,and thus on the normal performance of the related functions.(5)The effects of different experimental cycles of BBP on the relative expression of caspase-3,and p53 genes associated with apoptosis in the gills of zebrafish were determined.Specifically,caspase-3 gene showed activation-inhibition-activation,and the cycle of activation showed a dose-effect relationship;p53 gene was inhibited in the early stage of the test,and showed activation-inhibition-activation with the increase of BBP concentration in the late stage of the test.(6)The effects of BBP on the expression of 18 rs rrn and mt-ndl genes in zebrafish gill and mitochondrial DNA replication and transcription were investigated at different experimental periods.Specifically,the 18 rs rrn gene showed that it was activated in the early stage and repressed in the late stage,and showed a certain dose-effect relationship in the early stage of the test;the mt-ndl gene showed that it was activated and then repressed in the early stage and late stage of the test,and had a certain dose-effect relationship,and showed a repressed state in the middle of the test.