Study On The Combined Toxic Effects Of Dibutyl Phthalate And Three Nanomaterials On Zebrafish (Danio Rerio)

Dibutyl phthalate(DBP),as a typical phthalate plasticizer,has a high detection rate in theenvironment and has been included in the list of key pollutants in China.A large number of studies have shown that DBP has neurotoxicity,cardiotoxicity and endocrine disrupting effects on organisms.Man-made nanomaterials(MNMs)have large specific surface area and high surface activity,which are inevitably released into the environment due to production and use.Studies have shown that it can affect the migration,transformation and biological toxicity of other pollutants in the environment,thereby posing a more serious threat to the environment.However,the complex toxic effects and mechanisms of DBP and MNMs are still unclear.Therefore,this paper selects DBP and three representative nanomaterials:nano-titanium dioxide(n-TiO₂),multi-walled carbon nanotubes(MWCNTs),carboxylated multi-walled carbon nanotubes(MWCNTs-COOH)as research targets,using adsorption experiments,acute toxicity and developmental toxicity experiments,enzyme activity detection,real-time fluorescence quantitative PCR,acridine orange staining and metabolomics methods to explore the toxic effects and mechanisms of DBP single exposure and compound exposure with DBP and nanomaterials on zebrafish embryos/larvae.The results are as follows:(1)Adsorption effect and mechanism of three nanomaterials on DBP.The adsorption equilibrium time of DBP on the three nanomaterials was all within 24 h,and the adsorption amount of DBP on the three nanomaterials was MWCNTs>MWCNTs-COOH>n-TiO₂.The adsorption process of DBP on the three nanomaterials can well be fitted by the Freundlich adsorption isotherm model.There has no significant effect on the adsorption process in the range of p H 5.3-7.9,and the adsorption capacity of DBP on MWCNTs could be significantly increased by humic acid.The adsorption experiments of the three nanomaterials on DBP are endothermic spontaneous processes,and the higher the temperature,the more spontaneous the adsorption process.(2)The zebrafish embryos were exposed to DBP(0.1,0.5,1.0 mg/L),n-TiO₂(0.5 mg/L) and the combined exposure group of DBP with different concentrations and n-TiO₂,respectively.The acute toxicity of combined exposure group(96 h-LC₅₀:1.578 mg/L)is greater than DBP single exposure group(96 h-LC₅₀:2.188 mg/L).Both single DBP exposure and combined exposure can cause developmental toxicity such as abnormal voluntary movement,slow heart rate and delayed hatching of zebrafish embryos.The deformity rate was significantly increased(1.26-1.8 times,p<0.05),and the active duration of larvae was significantly decreased(11.6%-14%,p<0.05)after combined DBP and n-TiO₂ exposure.The effects of oxidative stress,neurodevelopment,and apoptosis-related genes after combined exposure were further evaluated.The results show that it could be significantly increased antioxidant enzyme activity(1.2-1.9-fold,p<0.05)by co-exposure DBP with n-TiO₂ and induce oxidative stress.DBP and n-TiO₂ co-exposure aggravated oxidative damage and cell death which are mediated by single DBP exposure through affecting the expression of genes related to antioxidant(down-regulated 27.5%-75.9%,p<0.05)and apoptosis(up-regulated 1.43-8.7-fold,p<0.05).The high concentration DBP combine exposure group significantly affect the neurodevelopment related gene expression,resulting in the neurodevelopment toxicity of zebrafish larvae.In addition,it will be exacerbated disturbances in amino acid metabolism and interfered with lipid metabolism compared with DBP single exposure in DBP and n-TiO₂ co-exposure treatment.Therefore,the combined exposure of DBP and n-TiO₂ may aggravate the development and neurotoxicity of zebrafish embryos/larvae through oxidative stress,inducing apoptosis,interfering with amino acid metabolism and lipid metabolism.(3)The zebrafish embryos were exposed to DBP(0.1,0.5,1.0 mg/L),MWCNTs/MWCNTs-COOH(0.5 mg/L)and different concentrations of DBP and MWCNTs/MWCNTs-COOH combined exposure groups,respectively.The acute toxicity of the co-exposure group(96 h-LC₅₀:1.540 mg/L,1.818 mg/L)is greater than that of the DBP single exposure group.Combined exposure caused developmental toxicity,including a significant increase in heart rate and malformation rates(pericardial edema,cardiac malformations).The results of enzyme activity detection and real-time fluorescence quantitative PCR show that antioxidant enzyme activity(1.2-1.6 times,p<0.05)and the expression of genes related to antioxidant,energy metabolism(down-regulated by 12.4%-56.6%,p<0.05),cardiac function development(down-regulated by 21.2%-98.5%,p<0.05)and apoptosis(up-regulated by 1.19-1.31-fold,p<0.05)can be affected by combined exposure,resulting in developmental and cardiotoxic effects on zebrafish embryos/larvae.For oxidative stress and cardiotoxicity,MWCNTs/MWCNTs-COOH aggravated the toxic effects of moderate concentrations of DBP on embryos,while MWCNTs/MWCNTs-COOH had no significant effect on the toxic effects of high concentrations of DBP.In addition,co-exposure can alleviate the disturbance of amino acid metabolism caused by single exposure to DBP.In conclusion,n-TiO₂/MWCNTs/MWCNTs-COOH may aggravate DBP-induced oxidative stress,neurotoxicity,apoptosis and cardiotoxicity in zebrafish by adsorbing DBP.It is necessary to pay more attention to the impact and harm of compound pollutants on organisms and the environment when evaluating the toxic effects of pollutants.