Studies On The Toxicity Of Copper On Adult Onchidium Struma

The increase in human activities during the last few decades,especially rapid development of agriculture and industry,has resulted in a considerable increase in the presence of pollutants such as heavy metals in the estuaries,Heavy metals are now recognized to be among the most relevant contaminants in the marine environment and their concentrations are elevating in some coastal waters.Furthermore, water-borne pollutants including heavy metals are easy to be accumulated in the sediments of estuary considering of its special environment for deposition.And by mean of food chain,heavy metals could be significantly bioconcentrated, bioamplified and bioaccumulated into the tissues of aquatic organisms particularly benthic animals,and thus threatening the surviving and growth of benthic and intertidal animals living in the estuary.Since aquatic organisms living in polluted ecosystems often accumulate metals into their tissues,it has been argued widely that these organisms can be used as biomonitors indicating the bioavailability of contaminants and the degree of pollution.As located in the developed regions,wastewater coming from agricultural, industrial and municipal drainages pours into the middle and lower reaches of the Yangtze River and converges in the Yangtze River Estuary where pollutants are enriched gradually and lead to heavy pollutions.As emphasized in the Report on the State of the Fishery Eco-Environment in China for the Year 2005,the average Cu²⁺ level in this estuary reached to 0.0132 mg/L,which had exceeded evidently compared with the Water Quality Standard for Fisheries(GB 11607-89)(≤0.01 mg/L)and is still ongoing.As located in the Yangtze River Estuary,the largest estuary in China, Chongrning Island has been brought into Ecological Planning and Construction by Shanghai government.Therefore,ongoing copper pollution in Chongming Island has been of primary concern.As one typical kind of benthic organisms,marine gastropods have a wide geographical distribution,high abundance in the benthic areas,selective absorption of certain ions together with high sensitiveness towards water-borne pollutants,and thus being considered suitably as biomonitors.Furthermore,their responses to the most of stressors are potentially an excellent indicator of stress level and environmental quality in the intact animal.Marine gastropods,therefore,are used in the water quality studies to monitor heavy metals and other pollutants of water and sediments in the estuaries and other marine sites.As one of the most important micronutrient but essential elements for normal physiological functions in the mollusk,copper plays an indispensable role in a large number of enzymes,which is a cofactor for regulating the activity of copper-dependent enzymes and also an essential component required for synthesis of hemocyanin.The tissues of mollusk are able and easy to accumulate copper in accordance with the external copper.However,if excessively accumulated, toxicity of copper also leads to serious implication especially for those enzymes requiring copper as integral part for their structures and activities,and thus leading to dysfunctions as reflected at the molecular,cellular,tissular and individual level.As for our targeted marine gastropod,Onchidium struma,it is one of the most widespread mollusks across semitropical coast,especially throughout the Indo-Pacific estuaries and so is in the Yangtze River Estuary.In this study,Onchidium struma was selected as experimental animals and the effects of water-borne copper on Onchidium struma have been conducted using multiple methods such as morphologic, physiologic,biochemical and ecotoxicologic approaches.Meanwhile,electric microscopic,biochemical enzymes and trace element analysis were also adopted for determination so as to clarify the mechanism about toxic effects of Cu²⁺on adult Onchidium struma,Our study aims at providing valuable theoretic references towards the mechanism about metal toxicity on mollusks as well as practical value to determine water quality and estimate environmental pollution.The main results and conclusion in this thesis are as follows.1.Seasonal bioeoneentration of heavy metals in adult Onchidium struma from Yangtze River EstuaryThe seasonal concentration changes of selected heavy metal Cd,Cr,Cu,Fe,Mn, Pb and Zn in five tissues of marine gastropod Onchidium struma including hepatopancreas,albumen gland,vitelline gland,digenetic gland and muscle were studied in the Chongming Island,Changjiang Estuary on April 2007,July 2006, September 2006 and November 2006 respectively.The results demonstrated that the bioaccumulation factor of Cu(biomass/water)in all selected tissues was about 10⁴ magnitudes,Fe & Cd were 10³,Zn was 10²,and Mn,Pb and Cr were 10¹. Hepatopancreas was proven to be the dominant storage tissue of Cr,Cu,Mn and Zn, whereas Fe and Pb were mainly stored in muscle and digenetic gland,and Cd was stored in vitelline gland and albumen gland.Additionally,it was found that Cu,Fe, Mn and Zn were accumulated significantly by Onchidium struma(whole-body)in summer or autumn,and Cd,Cr & Pb increased slightly in spring and winter. Furthermore,the concentration of Cr was nearly 2-fold higher and Zn was 1.6-fold higher in the water comparing to the Water Quality Standard for Fisheries.With view of excessive amount of Pb,Cd and Cu according to seafood standard,the consumption of Onchidium struma might have the risk of health hazard.2.Toxic effects of copper on enzymes and metallothionein in adult Onchidium strumaThe acute and sub-acute toxic effects of water-borne copper on Onchidium struma were conducted.After 96h,no mortality was noted in controls(0.00mg/L)and 32.00mg/L copper treatment groups.All the marine gastropod exposed to 180.00 mg/L copper died after 96h,while partial death was spotted in other copper treatment groups.The 24-,48-,72- and 96-h LC₅₀(Lethal Concentration 50)of copper for Onchidium struma was 135.00,108.30,97.19 and 74.80 mg/L respectively.The Maximum Acceptable Toxicant Concentration(MATC)of Cu²⁺to adult Onchidium struma was 7.480±0.349 mg/L based on the body weight 8.53±0.30 g and length about 4.65±0.05 cm in this present study.The specific activities of antioxidative enzymes including Cu/Zn superoxide dismutase(Cu/Zn-SOD)and catalase(CAT),metabolic enzymes including acid phosphatase(ACP),alkaline phosphatase(AKP),glutarnic-oxaloacetic transaminase (GOT)and glutamic-pyruvic transaminase(GPT)in the hepatopancreas & muscle together with digestive enzymes including pepsin,amylase,typase,cellulose and lipase in the hepatopancreas were determined after 1-week exposure to copper(Cu²⁺) ranging from 1.35 to 4.20 mg/L.A progressive and negative concentration-dependent correlation was observed as for the Cu/Zn-SOD activity in the hepatopancreas, whereas CAT presented as a positive correlation just as ACP in the muscle.No matter which tissue it was,the threshold concentration of copper to GOT or GPT was 2.40 mg/L.These results suggest that Cu/Zn-SOD and CAT in Onchidium struma can be effective in indicating environmental copper stress with the priority of Cu/Zn-SOD to CAT,so is GPT to GOT and hepatopancreas to muscle.As for digestive enzymes, generally speaking,except for undetectable lipase,the alternation trends about the specific activities of pepsin and amylase were quite similar viz.initial decrease followed by subsequently rise and final drop,however that of typase and cellulose were parabola-shaped currents.Anyhow,some differences existed among different enzymes.A Cadmium-Saturation Method was adopted to determine the effects of copper (0.00,1.35,1.80,2.40,3.20 and 4.20 mg/L)on metallothionein(MT)synthesis in the hepatopancreas.As a whole,the content of MT increased as the concentration of copper.Compared with controls,significant difference in MT production occurred even under 1.35 mg/L Cu²⁺exposure with its largest increasing scale at 4.566-fold, which suggests that the synthesis of MT can be induced even under low level of copper.As Cu²⁺level raised,the MT content increase gradually.In the 4.20 mg/L Cu²⁺groups,the peak of MT content was observed,that is,2.928 mg/g(wet weight) showing that the MT volume was being saturated.Results of the present study indicate that even low copper exposure enables to induce the synthesis of MT in the hepatopancreas so as for detoxification to some degree.Meanwhile,the response of MT content can be applied for environmental determination.3.Effects of copper on the microstructure and ultrastructure of hepatopancreas in the adult Onchidium strumaEffects of different copper level(0.00,1.35,1.80,2.40,3.20 and 4.20 mg/L)on microstructure of the hepatopancreas in Onchidium struma were studied by Single Gradient Factor Experiments.No significant differences were found as for microstructure between the 1.35 mg/L copper treatment groups and the controls.As copper level increased,however,microstructure alternations were observed as follows. The epithelium cells in the hepatopancreas tubes were swollen,discomposed and shed down with the cavity between hepatopancreas tubes attenuated.The basement membranes were rumpled or even ruptured with vacuoles formed between the basement membrane and the simple columnar epithelia.The hemocytes clumped with the dissociated epithelium ruptured from basement membranes.The results indicated that exposure to escalated Cu²⁺levels can cause historical damages to the microstructure of the hepatopancreas.Effects of different copper level(0.00,1.35,1.80,2.40,3.20 and 4.20 mg/L)on ultrastructure of the hepatopancreas in Onchidium struma were also studied.The results indicated that as the increase of copper concentration,the degree of damages to ultrastructure of the hepatopancreas was also enlarged accordingly.The epithelium cells were misshapen,swollen and destroyed without any complete cellular membrane. And chromatin was heterogenizated bitterly with its density aggrandized.Meanwhile nuclear membrane was broken off with nuclear substance overflowed into cytoplasm. Furthermore,the nuclear substances were diluted and nucleus was misshapen.Results here indicate that exposure to escalated copper levels can cause damages to the ultrastructure of the hepatopancreas and furthermore affect the normal function of hepatopancreas in Onchidium struma.To sum up,faced with water-borne copper exposure,toxic effects on Onchidium struma were spotted firstly in the enzymes and large molecular as mentioned above and then at organical,individual and finally communitywide level.Our studies primarily clarified the bioconcentration characteristics of water-borne heavy metals in Onchidium struma as well as the influential effects of Cu²⁺on enzymes, metallothionein,tissues and organs.Furthermore,our study would provide valuable theoretic references towards the mechanism about metal toxicity on mollusks as well as practical value to determine water quality and estimate eco-environments.