| With the rising economy and the improvement of people's living standards,more and more pharmaceutical and personal care products(PPCPs)are used in our daily life.After intake of these PPCPs,studies suggest that human body will excrete 50-90% of them into sewage.The common wastewater treatment system can only decompose 50% of such compounds,the residues will be discharged into the environment.Because of some unique toxic effects caused by their drug mechanism,PPCPs pollution in the environment is getting increasing attention.The Yangtze Estuary is adjacent to the City of Shanghai,the most densely populated and developed area in China.The treated sewage of Shanghai was mainly discharged into the coastal area of the Yangtze River Estuary,thus causing a constant input of PPCPs into this area.Among these PPCPs pollutants,some commonly used pharmaceuticals have relatively high environmental concentrations.These substances could cause potential environmental risk,thus a toxicological investigation on them is necessary.In this study,five commonly used pharmaceuticals: tamoxifen(TAM),bisphenols A(BPA),ibuprofen(IBP),diclofenac(DCF)and paracetamol(PRCT)were selected as targets based on previous pollution reports.During the laboratory study,short-term exposure experiments using zebrafish embryo/larval were selected to assess the toxicity of these five PPCPs.Morphological,behavioral and gene expression changes caused by PPCPs exposure were investigated.Several toxicity indicators were then selected based on the results obtained.Their potential for characterizing the relative environment pollution was also discussed.Finally,this research conducted exposure experiments using the environmental water sample collected in the coastal area of the Yangtze Estuary.The effectiveness of pollution assessment using exposure experiments and biomarkers discussed above was evaluated,and the current pollution status of sampling sits was discussed.During the morphological study,this research observed some teratogenic effects caused by 500 ?g/L TAM exposure.The total body length and heart rate of 96 hpf zebrafish larval was reduced,and mild caudal fin damage as well as dull skin was observed.Genes related to epidermal growth and recovery,krt17 and egf,were also found to be up-regulated by TAM exposure,suggesting the caudal fin damage and dull skin effect was caused by epidermal damage induced by TAM.During the behavioral study,this research observed motor behavioral changes caused by IBP and DCF exposure.The spontaneous movement of 28 hpf zebrafish embryo was decreased in the presence of IBP(-25%)and DCF(-28%).During the embryo hatching process,a significant reduction in hatch rate at 55 hpf was caused by both IBP(-63%)and DCF(-58%)exposure.As for 120 hpf larval,high concentration of IBP significantly reduced the free swimming distance,duration and speed under dark condition by 41%,29% and 30%,respectively.High concentration of DCF also reduced the swimming distance as well as active duration by 17% and 13% under light stimulation.Among neuron related genes tested,the expression of neurog1 and neurod1 was altered by IBP and DCF exposure.These findings indicated that IBP and DCF significantly affected embryo locomotivity and were potentially neurotoxic.The expression of some endocrine related genes in 96 hpf zebrafish larval can be altered by TAM and BPA.Exposure to TAM caused up-regulation of esr1,cyp19a1 b,ar,hsd3b2 and ugt1a1 expression to 224%,2024%,153%,182% and 154% compared to the controls.However the expression of vtg1 was down-regulated to 71% by TAM.These results are in accordance with the identity of the TAM as a selective estrogen receptor modulator.Exposure to BPA also caused up-regulation of esr1,cyp19a1 b,ar,vtg1 and ugt1a1 expression to 188%,1554%,134%,141% and 205% compared to the controls.These results support the role of BPA as an environmental estrogen.Since TAM induced the expression of cyp19a1 b by up to 20 times,which is a key gene that controls estrogen synthesis;thus TAM may mainly act as an environmental estrogen during zebrafish early development.All five target PPCPs induced the expression of some genes related to zebrafish metabolic system.The findings suggested the pressure of PPCPs pollution to the metabolic and antioxidant system of zebrafish embryo/larval.The expression of phase I metabolic related gene cyp3a65 was up-regulated by all five PPCPs(up to 336%),suggesting its common role in zebrafish PPCPs metabolism.The expression of antioxidant system related genes gpx1 a and gsr was also induced by all target compounds by up to 184% and 147%,suggesting their important roles in zebrafish oxidation resistance.Other genes such as cyp1 a,gstp1 and cat were also induced in multiple PPCPs experiments(up to 160%,140% and 142%),suggesting the common presence of these genes in PPCPs metabolism and detoxification process.The results of these exposure experiments suggested that morphological indices such as body length,heart rate and malformation;behavioral indices such as spontaneous movement and hatch rate;gene expression such as esr1?cyp19a1b?ar ?vtg1?cyp1a?cyp3a65?gpx1a?gstp1?gsr?gss?sod1 and cat,are all potential biomarkers of environmental PPCPs pollution.During the exposure experiments using environmental water samples,all selected biomarkers were affected by polluted water samples from different sites.These results suggested that environmental pollution of low concentration can be assessed by exposure experiments using zebrafish embryo combined with the detection of these biomarkers.As the final part of this research,the pollution status in the coastal waters of the Yangtze Estuary was investigated based on the methods and biomarkers developed.The water samples were collected from selected sites along the Yangtze Estuary(LHK,SDK,WSK).During the morphological study,water from SDK caused 35.83% death rate among zebrafish embryo/larval tested,development delay was also observed.During behavioral study,water from LHK and SDK increased the spontaneous movement(113% and 121%)and caused significant early hatch effect(10 h and 14 h earlier than controls).Study on endocrine related genes reveal complex environmental hormone effects of all water sample.Water from LHK induced the expression of cyp19a1 b and ar(133% and 143%).Water form SDK induced the expression of esr1,cyp19a1 b and vtg1(183%,120% and 143%),but the expression of vtg1 was suppressed(80%).Water from WSK induced the expression of esr1 and ar(135% and 123%).Study on metabolic related genes suggested interferes with the metabolic and antioxidant processes caused by LHK and SDK water exposure.Water from LHK induced the expression of cyp1a(242%),yet reduced the expression of cyp3a65,gpx1 a and sod1(53%,77% and 84%).Water form SDK caused significant suppression of cyp3a65,gpx1 a,gstp1,gsr,gss,sod1 and cat(10%,63%,77%,85%,83%,64% and 44%).All these results suggested notable environmental risks in the coastal surface water of LHK,SDK and WSK sites,which in term indicated the pollution status of Yangtze Estuary is severe. |
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