The Toxicokinetics And Toxic Mechanism Of PFOS Alternative OBS In Zebrafish(Danio Rerio)

Sodium p-perfluorous nonenoxybenzene sulfonate(OBS),as a new alternative to perfluorooctane sulfonate and its salts(PFOS),has been detected in various environmental matrices,wild organisms and even humans.This may cause potential risks to the ecological environment and human health.However,the current research on the toxic effects of the emerging compound OBS and its potential health effects on humans is still relatively scarce.In this study,zebrafish(Danio rerio)was used as a model animal to carry out the toxicokinetics experiments of OBS in larvae and adult zebrafish.The specific distribution of OBS in different tissues of adult zebrafish was studied,and the effects of environmental related concentrations of OBS exposure on the intestinal microflora structure of adult zebrafish were investigated.At the same time,the effect of OBS on zebrafish developmental toxicity and oxidative stress and its mechanism were discussed.The experimental results can provide a scientific basis for the risk prevention,control and management of OBS.The main conclusions are as follows:(1)72 h after fertilization,zebrafish larvae were exposed to 10,100?g/L OBS and 10?g/L PFOS for 48 h,and then transferred to clean water for 24 h.The results of toxicokinetic experiments showed that the concentrations of OBS and PFOS remained relatively stable in the exposure solution,and there was no significant decrease in the concentration of exposure solution due to the adsorption of OBS and PFOS on the surface of the exposure container.The uptake and depuration of OBS and PFOS in zebrafish larvae were in good agreement with the first-order kinetic model(R²>0.8).According to the toxicokinetic parameters,the absorption rate constants k_u were 2.41 L kg^-1 h^-1 and 1.94 L kg^-1 h^-1 in 10?g/L and 100?g/L OBS exposure groups,respectively,and 2.01 L kg^-1 h^-1 in 10?g/L PFOS exposure group;the half lives of OBS and PFOS were 69.7?85 h and 222.2 h,respectively.The BCF_kvalues of OBS ranged from 238 to 242.5,which was much lower than that of PFOS(644.2),indicating that OBS had a weaker bioaccumulation capacity than PFOS in zebrafish larvae.Adult zebrafish were exposed to the normal blank control group,10and 100?g/L OBS through the fully automatic drug delivery exposure system for 28days,then transferred to clean water for 28 days.The results showed that the mortality of low-dose and high-dose long-term OBS exposure was 1.67%and 6.67%,respectively.There was no significant difference in body length between the two groups,and there was no serious impact on the physiological status of adult zebrafish,which met the requirements of quality control in the process of exposure experiment.The purification rate of OBS in adult zebrafish accorded with the first-order kinetic equation.The concentration of 10?g/L and 100?g/L OBS in the whole zebrafish reached the maximum at about 21 days,and then remained in a relative equilibrium state.The BCF_k values of the 10?g/L and 100?g/L OBS exposure groups were 497.2and 302.45,and the half-lives were 10.78 d and 14.16 days,respectively.(2)The concentration of OBS in different tissues of adult zebrafish varied greatly,showing obvious tissue specificity.The order of enrichment ability of OBS in different tissues:blood>liver>intestine>gill>testis>brain>muscle.OBS mainly accumulated in the blood and liver with higher protein content,and has the lowest content in muscles with the lowest protein content.The absolute amount of OBS in muscle is the second highest,accounting for more than 30%of the total OBS in the zebrafish body.Although the concentration of OBS in the liver is relatively high,it only accounts for no more than 3%of the total OBS in the whole fish.Protein content and tissue type play an important role in the tissue distribution and bioaccumulation of OBS in zebrafish.(3)Normal zebrafish embryos 2 hours after fertilization were exposed to the solvent control,15,20,25 mg/L OBS and 15 mg/L PFOS.The results of 168 hour developmental toxicity experiments show that both OBS and PFOS exposure can cause acute damage to zebrafish embryos and significantly affect the growth and development process of embryos,including abnormal autonomous movement,inhibition of hatching rate,increased mortality,increased heart rate,and morphological changes,including yolk sac edema,pericardial edema,tail curvature and spinal curvature,etc.In addition,normal zebrafish embryos 2 hours after fertilization were exposed to the solvent control group,15,20,25 mg/L OBS and 15mg/L PFOS positive control group for 120 h.The real-time fluorescent quantitative method(q RT-PCR)was used to detect the changes in the expression of genes related to apoptosis in embryos.The expression of bcl-2 gene in zebrafish larvae was down regulated by exposure of OBS and PFOS to different degrees,and up-regulate Bax,Caspase-3 and Caspase-9,which increased the level of apoptosis in zebrafish larvae,inhibits anti-apoptotic factors,and led to apoptosis.(4)The effect of OBS exposure on the antioxidant system of zebrafish larvae and adult zebrafish showed that both low-dose(10?g/L)and high-dose(100?g/L)OBS could cause changes in the antioxidant system.Western blot experiments showed that the expression levels of Nrf2 and SOD protein increased significantly,and in a dose-dependent manner,indicating that OBS induced oxidative damage in the zebrafish larvae and livers of the adult zebrafish.It is speculated that oxidative stress is one of the important ways for OBS to produce toxic effects on aquatic organisms.(5)Adult zebrafish were exposed to 0,10?g/L PFOS,10 and 100?g/L OBS for21 days.Based on 16S r RNA high-throughput sequencing technology,the microbial diversity of zebrafish intestinal flora was analyzed.The results indicated that after long-term exposure to environmentally relevant concentrations of OBS and PFOS for21 days,the intestinal microflora of the adult zebrafish was dysbiosis.Among them,Proteobacteria,Fusobacteria and Actinobacteriata are the dominant phylum,and Plesiomonas,Shewanella and Aeromonas are the absolute dominant genus.However,the relative abundance of microorganisms in each exposure group differed at different taxonomic levels.Furthermore,38 significant differences were detected as active biomarkers in the exposure group by LEf Se multi-level species difference discriminant analysis.