Studies On The Toxic Effect Of Water-borne Hg²⁺ On Eriocheir Sinensis

Eriocheir sinensis is one of the most important cultured crustacean species in China and the annual profit was more thanï¿¥10 billion. Mercury is one of the most toxic heavy metal in environment. In the present study, E. sinensis was used as test animal to investigate the toxic mechanism of water-borne Hg²⁺. Morphological, physiological, biochemical methods and TEM, enzyme determinant and element analyzable methods were used in present study. The whole thesis consists five chapters.According to related references, the first chapter reviewed the studies on the toxic effects of water-borne heavy metals on crustaceans in recent years. Insufficiency of the study of the mechanisms of heavy metal on crustaceans has been pointed out and the technique route of the present study has also been expressed.The second chapter reported the toxic effects of water borne Hg²⁺ on the molting rate, the weight gain and the survival rate in juvenile E. sinensis and accumulation of mercury in different tissues of the crab. The 24, 48, 72 and 96 h LC₅₀S of Hg²⁺ on juveniles crabs were 0.6248, 0.5716, 0.5332 and 0.4423 mg/l, respectively. According to the sublethal experiment, the molting rate, weight gain and survival rate of juvenile crab decreased stably in accordance with the increase of Hg²⁺ level in the Hg²⁺ treated groups (0.01, 0.05, 0.10, 0.20, 0.30 mg/l). Mercury content in gills, hepatopancreas, and exoskeleton increased with Hg²⁺ in the water and among these three tissues, gills and hepatopancreas should be the main mercury bioaccumulation and the metabolism site for mercury in E. sinensis.In the third chapter, effects of different water borne Hg²⁺ concentrations (0.00, 0.01, 0.05, 0.10, 0.20 and 0.30 mg/l) on microstructures and ultrastructures of anterior and posterior gills, hepatopancreas in E. sinensis were studied by Single Gradient Factor Experiments. No significant differences were found in microstructures of both anterior and posterior gills between the 0.01 mg/l Hg²⁺ treated group and the controls. When Hg²⁺ level increased, pathological changes in the structure of the anterior and posterior gills were observed as irregularly thickened lamella, enlarged haemocoels, increased hemocytes and damaged or disassembled epithelia in the anterior gill. Obvious pathological changes in ultrastructure of the anterior and posterior gills were found even in the group exposed to 0.01 mg/l Hg²⁺ compared with the control group. Severe damage can be seen under TEM (Transmission Electron Microscope) such as lamellae hyperplasia, basement membrane infolded, microvillus decreased even disappeared, mitochondria were damaged and cristae partly disappeared; necrosis masked epithelial cell structures, empty cytosol, and epithelial cell vacuolated. Considerable damage also can be seen in posterior gills, which includes lamellae hyperplasia, basement membrane infolded, lack microvillus and apical membrane folds, mitochondria valuated; necrosis masked epithelial cell structures, empty cytosol, and epithelial cell vacuolated. Results indicated that exposure to elevated Hg²⁺ levels damages the microstructure and ultrastructure of the gills and furthermore affect the normal respiratory and ionoregulatory function of E. sinensis.Discernable microstructure difference were noted in crabs exposed to more than 0.05 mg/l Hg²⁺, which include basal lamina thickened and rippled from epithelium. In some area of tubule, the number of R cells increased and these increased cells huddled together. Cytoplasmic vacuolization, tumefied epithelial cells, deflation of the space of tubule lumen and melanin-like materials (M) deposited in the B cells and R cells could been noted in hepatopancreas of Hg²⁺ treated crabs. In crabs exposed to 0.30 mg/l Hg²⁺, tubule lost its organization, necrotic tubule (NT) in the hepatopancreas separated from the basal lamina, tissue debris deposited in the tubule lumen.Under TEM, the variation found in the ultrastructure of cells included disintegrated microvillus, damaged nuclei, disfigured and disintegrated endoplasmic reticulum, and excessive accumulated of glycogen in the endoplasmic reticulum. The mitochondrial membrane was distended in some locations and partially degenerated; rough endoplasmic reticule was broken down into minted pieces and distributed throughout the cells. Mitochondria lost their characteristic shape and disintegrated. The epithelial cell lost much of cytoplasm and become a big vacuole.By Single Gradient Factor Experiments effects of different water-borne Hg²⁺ on some important enzyme activities in gill, hepatopancreas and haemolymph and the metallothionein content in hepatopancreas of E. sinensis were studied in the fourth chapter.Firstly, antioxidant enzymes included superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and malondialdehyde (MDA) content in haemolymph and hepatopancreas of E. sinensis exposed to different Hg²⁺ concentrations were determined by biochemical methods. The results showed that SOD activity in both hepatopancreas and haemolymph increased in low Hg²⁺ concentration exposed groups while decreased in high Hg²⁺ groups. GPX activity in hepatopancreas of crabs exposed to 0.01 mg/l Hg²⁺ was significantly higher than controls (P＜0.05). In the other mercury treated groups GPX activities were significantly lower than controls. GPX activity in haemolymph of crabs exposed Hg²⁺ tended to decrease with waterborne Hg²⁺. The tendency of CAT activities in hepatopancreas and haemolymph of E. sinensis was similar in this report that decreased when waterborne Hg²⁺ increased. MDA is one of the most important substances from lipid peroxidation. In present study, MDA content in hepatopancreas and haemolymph of E. sinensis increased with Hg²⁺ that means the extent of lipid peroxidation grow on with Hg²⁺.Secondly, gill ATPase (Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase) and five digestive enzymes (tryptase, pepsin, amylase, cellulase and lipase) in hepatopancreas of E. sinensis were also determined. The result showed that both Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase decreased greatly with waterborne Hg²⁺. In crabs exposed to 0.30 mg/l Hg²⁺, Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were 1.78 and 1.89μmol Pi/ mg protein that values were only about 1/3 value appeared in control group. That means that gill ATPase is sensitive to waterborne Hg²⁺. Furthermore, the inhibition effect of Hg²⁺ in activities of some important digestive enzymes, e.g. the tryptase, pepsin, amylase and cellulase could be noted in the hepatopancreas in varied extents when increasing the level of water-bome Hgu²⁺. However, the activity of the lipase increased in all mercury treated groups and the maximum value appeared in 0.10 mg/l Hg²⁺ group. The phenomena above suggest that the different function of the gill, hepatopancreas of E. sinensis can be affect by Hg²⁺. Moreover, the variations on the activity of Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase in gills and tryptase, pepsin, amylase and cellulase enzymes in hepatopancreas can indicate sensitively the inhibition effect and toxicity of Hg²⁺ in E. sinensis.Effects of different water-borne Hg²⁺ activities of several major metabolic enzymes have been conducted in the hepatopancreas and haemolymph. With the increase of Hg²⁺ level Acid Phosphatase (ACP) and Alkaline Phosphatase(AKP) activity elevated at first and then suppressed in the hepatopancreas, and reached its highest 148.693 U/mg at 0.0 mg/l and 295.207 U/mg at 0.05 mg/l Hg²⁺ treated group. However, decrease of Glutamic Pyruvic Transaminase (GPT) and Glutamic Oxaloacetic Transaminase (GOT) activities can be noted in present study and the highest and lowest value in the controls and 0.30 mg/l Hg treated group. Things changed in haemolymph of E. sinensis exposed to different Hg²⁺ groups. GPT, GOT, ACP and AKP activities in haemolymph of crabs exposed to Hg²⁺ groups elevated in low Hg²⁺ groups and decreased in high Hg²⁺ groups. The highest values of GPT, GOT and AKP activities in haemolymph of E. sinensis occurred in 0.05 Hg²⁺ treated group and that value of ACP occurred in 0.01 mg/l Hg²⁺ groups.The Sephadex G-75 elution profiles of heat-stable protein with metal derived from mercury treated E. sinensis showed two clear protein peaks. According to the results of molecular weight determination, these two metal-associated protein peaks had native molecular weighs of about 20,000 and 11, 000. A Cadmium-Saturation Method was used to investigate the content of MT synthesis in the hepatopancreas by water-borne Hg²⁺. MT was induced even in 0.01 mg/l waterborne Hg²⁺. The maximum content of MT was found in the group exposed to 0.10 mg/l Hg²⁺.Based on all results mentioned above, the mechanism of water-borne Hg²⁺ toxic effects could be summarized as: water-borne Hg²⁺ is primarily absorbed by the gills and then transported through haemolymph. Gill and hepatopancreas are regarded as the major accumulation sites for this heavy metal. When Hg²⁺ was transported through haemolymph, it affects enzymes and the large molecular at first. If Hg²⁺ level have a continuous elevation, irreversible damage will be observed in the structure and function of the gills, hepatopancreas. Accordingly, normal physiological functions of the organs were affected, causing the decrease of molting rate and growth of E. sinensis.The study has both realistic and theoretic significances on further study of toxic mechanism of heavy metals on crustaceans, as well as provides useful information for effective water quality evaluation of aquaculture environment for these animals.