The Ecotoxicological Effects And Mechanism Of Representative Contaminants On Adult Chinese Mitten Crab (Eriocheir Sinensis)

Along with socioeconomic development,large quantities of organic compounds are widely produced and used in industry,agriculture,livestock and other fields.The unique geographical environment of the Yangtze Estuary provides an ideal place for the habitat,breeding and growth of various fishes,shrimps and crabs.In addition,the Yangtze Estuary is the inevitable passage of many species in anadromous and catadromous migration,as well as the cradle and provenance base of the Chinese mitten crab(Eriocheir sinensis)from Yangtze water system.However,intense human activities in the area result in large-scale uses and emissions of chemicals and pharmaceuticals causing damages to the surrounding environment,and their environmental risks have received great attention.Therefore,in this study,Chinese mitten crab was selected as bio-indicator,representative pollutants erythromycin(ETM),methoxychlor(MXY)and cadmium(Cd)were chosen as the target compounds,to investigate the ecotoxicological effects on Chinese mitten crab and mechanisms of these pollutants in laboratory experiments.Meanwhile,sulfadiazine(SD)was chosen to contrast the bioaccumulation in tissues and the impacts on enzymes of SD and ETM,providing theoretical basis for the interactions of different contaminants with aquatic organisms.On this basis,the bioaccumulation effects and the influences on enzymes of four SD concentrations were examined to provide theoretical basis for accessing the environmental risk of contaminants in the Yangtze Estuary area.Key results are listed as follows: 1.The combined ecotoxicity of ETM,MXY and Cd on Chinese mitten crab 1.1 The bioaccumulation effect of ETM,MXY and Cd in tissues of Chinese mitten crabBased on modified ultrasonic extraction and purification method,accelerated solvent extraction method and mixed acid digestion method,the residues of ETM,MXY and Cd in four different tissues of Chinese mitten crab were determined following separate and combined exposure from ETM,MXY and Cd(each at 1 mg/L)for 21 days.After exposure,the highest residues of ETM were found in gill(3054 ng/g)followed by hepatopancreas(392 ng/g);the highest residues of MXY were in gill(294 ng/g)followed by ovary(61 ng/g)and hepatopancreas(35 ng/g);while the highest residues of Cd were in gill(1154 ?g/g)followed by hepatopancreas(461 ?g/g).Using Pearson correlation coefficient analysis,MXY and Cd improved the bioaccumulation of ETM in hepatopancreas,gill,muscle and ovary tissues;ETM enhanced the bioaccumulation of MXY in hepatopancreas and ovary;and Cd increased the bioaccumulation of MXY in hepatopancreas,gill and ovary.This suggested obvious bioaccumulation effect of pollutants in Chinese mitten crab at thelevel of 1 mg/L,and possible interactions of pollutans during bioaccumulation.1.2 The impacts of ETM,MXY and Cd on histomorphologyBasing on the bioaccumulation of ETM,MXY and Cd in Chinese mitten crab,the histomorphology observation of Chinese mitten crab was further performed using paraffin sections and ultrathin sections.The results showed obvious vacuoles,pyknosis and debris of cell nuclei in hepatic ducts;vacuoles,broken cellular structure,disintegrated epithelial cell layer and even draining chromatin were observed in gill arches;in ovary tissue,thinning cell walls of egg,irregularly cell and debased density of cytoplasm were observed.These damages became worse with the prolonged exposure time.After exposure to cadmium,the ultrastructure of four tissues was observed by transmission electron micrographs.Broken microvilli,decreased mitochondria,and vacuoles in hepatopancreas were observed;decreased and even disappeared infoldings of microvilli,swelled and broken mitochondria were observed in gill;in ovary tissue,the mucosa of egg cell became thickened,and the gap occurred between the endothelium of egg capsules and the matrix;and increased interspace of myofibrils,swelled and disintegrating mitochondria were seen in muscle tissue.This revealed that three pollutants can cause obvious damages in Chinese mitten crab at the level of 1 mg/L.1.3 The changes of ACHE,GST,MT and SOD activities in hepatopancreas and gill exposed by ETM,MXY and CdTo investigate possible toxicity mechanism on Chinese mitten crab at a cellular level,the activities of four key metabolic enzymes were determined in hepatopancreas and gill after exposure to ETM,MXY and Cd,including acetylcholin esterase(ACHE),glutathione S-transferase(GST),metallothionein(MT)and superoxide dismutase(SOD).Combined with Pearson correlation analysis,it was found that the activities of ACHE,GST and SOD in hepatopancreas and gill tissues were all impacted by ETM,MXY and Cd,but different impacts caused by different compounds were observed.Among them,the activity of ACHE in hepatopancreas was elevated with the bioaccumulation of ETM;GST activities in hepatopancreas and gill tissues were significant correlated with MXY residues(P < 0.05);with the bioaccumulation of Cd,the activity of GST in hepatopancreas was decreased,and ACHE in gill tissue was increased.Therefore,hepatopancreas ACHE was considered as the potential candidate biomarker of toxicity by ETM,gill ACHE was considered as the potential candidate biomarker of toxicity by Cd,and GST was a potential candidate biomarker of MXY and Cd.2.The ecotoxicity of representative antibiotics ETM and SD on Chinese mitten crab 2.1 The bioaccumulation effects of ETM and SD in tissues of Chinese mitten crabETM was frequently detected in the aquatic environment and was shown in previous studies to bioaccumulate in Chinese mitten crab.So,sulfadiazine(SD),whose property was different with ETM,was chosen to investigate the bioaccumulation and toxicity of different kinds of compounds on Chinese mitten crab.From separate and joint exposure by ETM and SD(each 1 mg/L),the highest residues of ETM and SD in four tissues were detected after joint exposure,with the bioaccumulation concentrations decreasing as gill(867 ng/g)> hepatopancreas(116 ng/g)> muscle(13 ng/g)? ovary(13 ng/g)for ETM,and as gill(478 ng/g)> ovary(160 ng/g)> hepatopancreas(158 ng/g)> muscle(43 ng/g)for SD.Combining with Pearson correlation coefficient analysis,ETM could enhance the bioaccumulation of SD in gill and ovary tissues,and ETM might influence the bioaccumulation of SD in hepatopancreas;the presence of SD could promot ETM bioaccumulation in ovary tissue.2.2 The impacts of ETM and SD on activities of ACHE,GST and SODAdditionally,the activities of three key metabolism enzymes,ACHE,GST and SOD,were determined in hepatopancreas and gill after exposure by SD and ETM.The results showed SD and ETM obviously affected the activities of ACHE in hepatopancreas and gill tissues;and SD impacted GST and SOD in hepatopancreas and gill tissues;the combination of SD and ETM affected the three enzymes which was complex.Pearson correlation analysis between bioaccumulation concentration of SD and enzymes activities showed significant association between SD residues and ACHE in hepatopancreas(P < 0.05),asso between SD residues and GST activities in hepatopancreas and gill(P < 0.05).This showed obvious impacts on ACHE and GST in hepatopancreas and GST in gill by SD exposure,which could result in metabolism disorders of Chinese mitten crab.3.The ecotoxicity of low concentration SD on Chinese mitten 3.1 The distribution and level of SD in tissues of Chinese mitten crabDue to stronger hydrophilicity of SD,four different environmentally relevant concentrations of SD(50 ng/L,100 ng/L,500 ng/L and 1000 ng/L)were used to investigate the bioaccumulation effect on Chinese mitten crab.The results showed the SD residues in hepatopancreas,gill and muscle tissues were all less than 3 ng/g,suggesting limited bioaccumulation effect of low concentration SD on Chinese mitten crab.3.2 The impacts of low concentration SD on key metabolism enzymesThe activities of ACHE,alkaline phosphatase(AKP)and SOD were changed after SD exposure with different concentrations.The Pearson correlation analysis between enzymes activities and SD residues indicated that the activity of ACHE in gill tissue were negatively correlated with SD residues in 50 ng/L and 1000 ng/L groups(P < 0.05);in 100 ng/L group,AKP was significantly negatively correlated with SD residues(P < 0.01).These findings revealed that ACHE and AKP could be candidate biomarkers of toxicity effect from SD.This study focused on the ecological toxicity and mechanisms of antibiotics ETM and SD,pesticide MXY and persistent pollutant Cd on Chinese mitten crab,and detected obvious bioaccumulation effect and toxicity on Chinese mitten crab when contaminants were around 1 mg/L,suggesting serious toxicity on aquatic organisms.Various contaminants coexisted in the natural environment,which resulted in enlarged ecological toxicity because of their interactions.Therefore,this study provided strong evidence for controlling the pharmaceutical use in aquaculture,controlling the emissions of such contaminants into the environment,and assessing the environmental risk of such contaminants in the aquatic environment.