Application Of An Integrative Zebrafish Embryo Testing On The Toxicity Evaluation Of Environmental Mixtures

Numerous of chemicals that lack of toxic information will enter the environmental water with industrial,agricultural,domestic water and surface runoff for our living needs.Thus,chemicals in water should be complex and diverse.The long-term exposure to polluted water will pose a great threat to human and ecological health.Therefore,identifying the biological toxicity of chemical pollutants and environmental polluted water is of great significance for the assessment of human health and ecological risks.However,the traditional toxicity tests have many restricts which can not meet the needs of chemical toxicity test,low testing flux,long time consumption,high cost and animal ethical problems.Although zebrafish is an advanced vertebrate model that can be conducted with high throughput,the current zebrafish testing methods still have several deficiencies,such as a single toxicity end point(acute toxicity of individual level),low sensitivity(high exposure concentration)and so on,which can not accurately identify the toxic effects of environmental pollutants.With the development of omics technology,toxicogenomics can get molecular information effectively,which can be used for the identification and prediction of biological toxicity of single substance or mixture pollutants.In this stuy,an integrative zebrafish toxicology testing method,including zebrafish embryo high content imaging test(HCT),zebrafish photo motor response test(PMR)and reduced zebrafish transcriptome test(RZT),was established for identifying the toxicity of chemicals and environmental samples effectively.Firstly,we selected the well studied environmental pollutants(bisphenol A,propiconazole,triphenylphosphate and triclosan)to do the aggregate zebrafish toxicology test.The reliability,sensitivity and pollutant identification ability of the aggregate test technology were evaluated by comparing with the existing toxicity data.Secondly,this study selected four different environmental water bodies in the middle and lower reaches of the Yangtze River,including urban river waters,aquaculture waters,farming activities waters and chemical industry park wastewater treatment plants(WWTP).The toxicity of these four environmental samples were evaluated by zebrafish embryo toxicity test,combining with the results of water quality index and chemical analysis.Finally,the aggregate test technique is applied to the biological toxicity test part of the effect-directed analysis to identify the most toxic components of the environmental sample and try to use the biotoxicity information and the non-target chemical analysis to give the list of the key chemicals.The main results are summarized as follow:(1)The accuracy of three testing methods(HCT,PMR,RZT)was validated by comparing with previous study.HCT can get the phenotypical endpoints on the organism level,for example,mortality and organ malformation;PMR test can identify the chemicals that have neurotoxicity.However,these two test cannot distinguish the chemicals by mode of action.The aggregate zebrafish toxicology test can cover that shortage by integrating HCT,PMR and RZT.(2)The results of water quality index showed that all the water was contaminated by total nitrogen,and the WWTP samles had high chemicals concentration,as well as urban river was contaminated by ammonia nitrogen and total phosphorus,while the water influenced by agriculture had low dissolved oxygen concentration and the aquaculture water showed a better water quality.The HCT results demonstrated that WWTP waters were the most toxic ones,while PMR results elucidated that 9#(WWTP1)sample have teratogenic effect and neurotoxicity;36#(WWTP2)sample have lethal effect,teratogenic effect and neurotoxicity;38#(WWTP3)sample just have lethal effect and teratogenic effect.Meanwhile,RZT analysis showed that the sensitive response genes,such as rps11,prpf40a,mad211,snrpd1,raplaa,rps4x and so on,could be related to the development and endocrine biological pathway,including cell division,nervous system and organ development,cellular metabolism,signaling pathway and oocyte meiosis.In addition,the target chemical analyze detected numerous of neurotoxicants and other chemicals which can lead the zebrafish dystrophy.All the results elucidated that the aggregate test technique could distinguish the environmental samples and help to evaluate samples’ toxicity.(3)Using the aggregate zebrafish toxicology testing method to test the fractions of 36#sample(F1,F2,F3,F4)and 36#re sample(recombine these four fractions).HCT results meant that only the F2 sample could lead to pericardial cyst(LOEC = 1 Ox),other samples have no observed effects.PMR results showed that 36#re,F1 and F2 could cause the zebrafish larvae more active under a dark stimulating condition,with the LOEC are 5x,5x and 1x,respectively.RZT results showed that the molecular response of F2 was similar to 36#sample,and the toxicity potency of F2 was the highest,while the response pathway after F2 exposure was related to development,stress response,endocrine regulation and metabolic process.All of these results demonstrated that F2 was the most toxic fraction.The results of this study show that the integrative zebrafish embryo test has good accuracy and sensitivity,and could be used to identify environmental pollutants with different modes of action.Meanwhile,the integrative zebrafish embryo test can distinguish the environmental sample well and identify its biological toxicity.What’s more,combined with fractionation separation technology,this testing method can be used to trace the most toxic components,which will provide information for subsequent chemical analysis.The integrative zebrafish embryo testing technology can be applied to the environment toxicity identification and ecological risk assessment in view of its benefits.