Studies On The Toxic Mechanism Of Water-borne Cu²⁺ On Sesarma Dehaani

In our present study, the Sesarma dehaani was selected as experimental animals and the effects of water-borne copper on Sesarma dehaani have been conducted by use of morphologic, physiologic, biochemical, methods. Meanwhile, electric microscopic, biochemical enzymes and trace element analysis were also used for measurement. Our study aims at clarifying the mechanism of toxic effects of copper on Sesarma dehaani and supplying more reliable practical and theoretic data for preparation for Sesarma dehaani as the indicator organism to copper of the Yangtze estuary and coastal flat. Moreover, our results are bound to have both practical and theoretic significances for ecotoxicology of crustaceans. The main results in this thesis can be summarized as follows:1. Accumulation, distribution and toxic effects of copper sulfate on Sesarma dehaaniThe acute and sub-acute toxic effects of water-borne copper on Sesarma dehaani were conducted in our present study. All crabs survived in the control treatments after 96 h exposure to copper. And no mortality was found among the crabs exposed to 7.5mg/l copper for 96h. First acute toxicity was detected at copper concentrations of 10mg/l or higher after 24 h. 100% mortality was reached after 72 h at copper concentration of 32mg/l and all crabs exposed to 42mg/l copper died after 24 h. The 24-, 48-, 72- and 96-h LC50 of copper for Sesarma dehaani was 21.33, 16.59, 15.18 and 14.18mg/l, respectively. And the safety concentration for copper was 0.14 mg/l. The 2-week experiment was conducted to investigate the effects of different Cu²⁺( 0.00, 0.01, 0.10, 1.00, 5.00, 7.50mg/l ) in water on the accumulation, distribution in adult Sesarma dehaani. In control groups, Cu²⁺ concentrations in exoskeleton, gill and hepatopancreas were 0.0052, 0.0168 and 0.0351mg/g. With increasing of Cu²⁺ concentration in water, Cu²⁺ level increased significantly in all the tissues studied, especially the hepatopancreas. In the group exposed to 7.50mg/l Cu²⁺ level, the Cu²⁺ concentration in hepatopancreas was as 8.2 times as in control groups and was as 4.3 times and 2.3 times respectively as in the same Cu²⁺ level of exoskeleton and gill. Therefore, we concluded that hepatopancreas was the most important target organ in Sesarma dehaani, with the increasing Cu²⁺ concentration in water.2. Effects of water-borne copper on the microstructure and ultrastructure of Sesarma dehaaniEffects of different water-borne copper concentrations( 0.00,0.01,0.10,1.00, 5.00 , 7.50mg/l ) on microstructures of the gills and ultrastructures of hepatopancreas, in Sesarma dehaani were studied by Single Gradient Factor Experiments. The results showed as following:No significant differences were found in microstructures between the 0.01 mg/l copper treated group and the control group. However, when copper level increased, changes in the structure were observed and listed as following: the number of the haemocytes in the haemocoel of gill axis, thinningz of epithelial lamina of gill lamellae, swelling of lamellae, shrinkage of cuticle, augmentation of the space of haemocoel, Swelling of haemocytes and big vacuoles between cuticle and epithelium, necrosis of some epithelial cell, narrower sub-chitin space of gill lamellae were found in the gills.Similarly, changes in ultrastructure of the hepatopancreas were found in the groups treated with copper irons, and there is a significant structural damage in accordance when exposed to the high concentration of water-borne water. Compared with the control group, nucleus, basement membranes microvilli rough endoplasmic reticulum and mitochondira in hepatopancreas were seriously damaged: Nuclear membrane disintegrated; nuclear substance outflowed, heterochromatin was around nuclear membrane, breaking into many tablets, much rarer euchromatin and increased electron density; Swollen of basement membrane and separation of columnar cells from the basement membrane; Microvilli broke, fell and had a decreased number, Disintegrated of double membrane of mitochondrion, and vacuolization of mitochondrion; Ribosome on the RER fell, and became the smooth vesicles. The results indicate that exposure to high copper levels damages the ultrastructure of the hepatopancreas and furthermore affect the normal function of hepatopancreas in Sesarma dehaani. 3. Effects of water-borne copper on enzymes activity and metallothionein synthesis in Sesarma dehaaniEffects of different water-borne copper (0.00, 0.01, 0.10, 1.00, 5.00 , 7.50mg/l) on the protein content as well as on the enzymes activities in hepatopancreas of Sesarma dehaani were studied by Single Gradient Factor Experiments. The results showed that: different concentrations of Cu²⁺ in water showed different inhibition on the pepsin, amylase, cellulose, and typase in hepatopancreas. High Cu²⁺ level all showed more inhibitive on the four digestive enzymes. Typase is the most sensitive among the four digestive enzymes. When exposed to 0.01 mg/L Cu²⁺ level, the inhibition rate had achieved to 32.86%. In addition, the inhibition on typase is the most notable. The average inhibition rate was 45.85%. Therefore, we concluded that the activities of its digestive enzymes can reflect the toxic effects of water-borne copper ion sensitively.Effects of different water-borne copper on major metabolic enzymes activities have been conducted in the hepatopancreas of Sesarma dehaani. The results showed that: with the increasing level of water-borne copper, the activity Cu-Zn SOD decreased first and then increased. And the activity of ACP increased first and then decreased, it reached its maximum activity and minimum activity at 0.1 mg/l copper level group and minimum activity at 7.50 mg/1 copper level group. The activity of AKP was very complicated. It increased first, then decreased, increased next and decreased finally. It reached its minimum activity at 7.50 mg/1 copper level group. The tendency of GPT activity and GOT activity were similar. They both decreased first and then increased and finally increased. The maximum activities of them were 1.52 U/mg and 3.80 U/mg respectively. The metabolic enzymes are much more sensitive than digestive enzymes. However, with the increasing concentration of water-borne copper, activities of the metabolic enzymes of the hepatopancreas of Sesarma dehaani were not completely inhibited more severely. It suggested that low concentration of copper may induce the synthesis of some metabolic enzymes , and high concentration of copper can inhibit the activities of the metabolic enzymes. Therefore, we can evaluate the copper pollution in the variation of the its metabolic enzymes of the hepatopancreas.The Cadmium-Saturation Method was used to investigate the effects of copper on MT synthesis in the hepatopancreas. After being exposured to increasing concentrations of copper for ten days, the content of MT in the hepatopancreas of Sesarma dehaani showed significant increases. When exposed to 0.01mg/l copper, the content of MT is 0.99±0.16mg/g ,which increased slightly compared to the control groups (0.88±0.12mg/g), but there was no statistically significant difference. The content of MT increased significantly to3.75±0.20, 4.89±0.12, 5.77±0.50 mg/g exposed to 0.10, 1.00, and 5.00mg/l copper, respectively. When exposed to 7.50mg/l copper, the content of MT add up to5.89±0.43 mg/g, which was the highest level in all the test groups and was as much as about 3 times higher than that in controls. But no statistically significant difference between 5.00 and 7.50mg/l copper. We can conclude that the synthesis of MT can be induced. The content of MT increased when exposured to the increasing level of copper, but never increased all the time with the elevated concentration of copper exposure. Metallothionein is sensitive to the water-borne copper. So the content of MT in the hepatopancreas can be as an index of detecting copper in environment.Results suggest that: the water-borne copper spread with the hemolyphy, distributed to all parts of body in Sesarma dehaani, and finally accumulated in the hepatopancreas. Therefore the hepatopancreas are the main copper bioaccumulation site. Moreover, the water-borne copper has an effect on enzymes and proteins which distributed all over the body with the spread of hemolymphy. The changes of enzyme activities and MT content can sensitively reflected the inhibition effect of copper, which suggested that these indexes can be used as biomonitor of copper pollution. So, we can take Sesarma dehaani as an indicator organism to copper of the Yangtze estuary and coastal flat.In summary, when Sesarma dehaani was treated with water-borne copper, toxic effects were observed first in enzymes and large molecular and then in the organs, individuals and in the community finally. Our studies primarily clarified the accumulation characteristic of water-borne copper in Sesarma dehaani, demonstrated effects of Cu²⁺ on enzymes as well as on tissues and organs. And what is more, it is our studies that firstly evaluated toxic mechanisms of Cu²⁺ in Sesarma dehaani, which are of great importance both in practice and in theories. Moreover, it supplied more data and relevant information for the preparation for the indicator organism to copper of the Yangtze estuary and coastal flat. In addition, it also supplied an integrate method for the study of other heavy metals.