Toxicity Of Ambient Nitrite And Ammonia On Eriocheir Sinensis Juveniles And Nutritional Modulation Of Dietary VE

In this paper, the toxicity of ambient nitrite and ammonia on Eriocheir sinensis juveniles was studied and the detoxification mechanism of nitrite and ammonia by E. sinensis jueveniles was elucidated. Besides, the effect of dietary vitamin E supplementation on the ability of anti-stress was discussed. The results could provide more basic information for aquatic animal toxicology, nutritional immunology, and references for establishing the system of stress evaluation in crustacean, which could boost the sustainable development of crustacean aquaculture.1. LC₅₀ of nitrite and ammonia to E. sinensis and its detoxificationThe result of acute toxicity to E. sinensis juveniles showed that the 24h-, 48h-, 72h-, and 96h- LC₅₀ of ambient nitrite were 43.87-, 40.24-, 38.87- and 38.87-mg/l respectively, and 24h-, 48h-, 72h-, and 96h- LC₅₀ of ambient TAN (total ammonia nitrogen) were 251.68-, 217.61-, 156.05- and 119.67-mg/l respectively , and for the non-ionized ammonia, they were 2.75-, 2.37-, 1.70- and 1.31- mg/l, respectively. The safe concentration of ambient nitrite and ammonia were 3.8 mg/l and 12.0 mg/l. So, compared to the toxicity of ambient nitrite, E. sinensis juveniles was more tolerant to ambient ammonia.During nitrite exposure, the accumulation of nitrite in haemolymph increased with the concentration of ambient nitrite increasing, and the increased amount of haemolymph nitrite was much more than that of ambient nitrite. However, the level of ammonia in haemolymph was close to that of ambient ammonia, and it increased linearly with ambient ammonia increasing during ammonia exposure. The result showed that the accumulation of ambient nitrite in haemolymph was higher than that of ambient ammonia.The levels of glutamine, glutamate and urea were increased during nitrite exposure and ammonia exposure. With time elapsed, the levels of glutamine and urea in haemolymph and glutamate in hepatopancreas increased, while the level of glutamate in haemolymph decreased, which indicated that the extra ammonia and nitrite in tissues could be detoxified to glutamate or urea through the cycle of ornithine. Moreover, the extent of glutamine and glutamate in haemolymph induced by ambient nitrite was higher than that induced by ambient ammonia, which showedthat the detoxification of nitrite mainly depended on the formation of glutamate. In addition, partial nitrite could be transfered into nitrate.2. The acute toxicity of ambient nitrite and ammonia to E. sinensis juvenilesBased on the result of 96h- LC₅₀, five treatments of nitrite exposure (control, 10-, 20-, 30- and 40-mg/l) and five treatments of ammonia exposure (control, 20-, 40-, 60-and 80-mg/l) were designed and sampled at 3h, 6h, 24h and 48h. And the effects of acute nitrite exposure and acute ammonia exposure on the energy utilization, immunity, ion-regulation and expression of HSP70 in E. sinensis juveniles were discussed.Compared to the control, the level of oxyhemocynin in haemolymph decreased by acute nitrite exposure and ammonia exposure, and then lowered the oxygen-carrying capacity in haemolymph. So the energy was produced by anaerobic metabolic routes, which could lead to the decrease of glycogen level in hepatopancreas and elevation of glucose level in haemolymph and lactate level in haemolymph and hepatopancreas. At the same time, the decrease of triglycerides level in haemolymph indicated that lots of fat was used up to meet the demand of energy.The total hemocytes count (THC) and the activity of superoxide dismutase (SOD) in hepatopancrea were significantly increased after lower concentration of ambient nitrite and ammonia exposure in short time, which exhibited the phenomenon of "poison-intoxication-effect". With time elapsed, the intoxication phenomenon was diminished and THC and SOD activity gradually decreased. However the THC and SOD activity were decreased by higher concentrations of ambient nitrite and ammonia exposure, which could depress the ability of scavenging superoxide anion radicals and depressed the ability of immunity at some degree. Furthermore, the accumulation of malondialdehyde (MDA) in hepatopancrea was increased with ambient nitrite and ammonia increased and with time elapsed, which exaculated lipid peroxidation.With lipid peroxidated, the structure of cell membrane was damaged and then its permeability was altered. The concentrations of sodium, potassuim, chloride, strong ion difference (SID) and osmotic pressure were decreased by both nitrite and ammonia exposure. During nitrite exposure, chloride concentration and osmotic pressure were sensitive to the change of ambient nitrite, and there were notable differences in chloride concentration and osmotic pressure between different groups. However, sodium in haemolymph was sensitive to the change of ambient ammoniaand there was significant difference between different groups during ammonia exposure.Besides the physiological and chemical change caused by nitrite exposure and ammonia exposure mentioned above, there were still much more cellular changes. The experiment about effect of acute nitrite exposure on HSP70 (heat stress protein 70) expression in gills analyzed by Western-blotting showed that the expression of HSP70 peaked at 6h of 10- and 20-mg/l nitrite exposure and 3h of 30- and 40-mg/l, and the maximum expression of HSP70 was positively correlative to ambient nitrite concentration. Therefore, HSP70 could be expressed before the organism was seriously damaged and could be used as an early biomarker of physiological status evaluation.3. The chronic toxicity of ambient nitrite and ammonia to E. sinensis juvenilesIn order to study the effect of chronic toxicity of ambient nitrite and ammonia on the growth and immunity in E. sinensis juveniles and elucidate the mechanism of long time nitrite and ammonia exposure on E. sinensis juveniles, five nitrite treatments (control, 5-, 10-, 15- and 20-mg/l) and five ammonia treatments (control, 10-, 20-, 30-and 40-mg/l) were designed and the effect of 60d-exposure on the growth, immunity and histological structure and ultra-structure in hepatopancrea were discussed.During the first 10d-exposure of nitrite and ammonia, the survival number decreased with ambient nitrite and ammonia increased and with time elapsed. And the body weight gain was decreased but the feed efficiency was elevated after nitrite exposure and ammonia exposure.The acid phosphate (ACP) and alkaline phosphate (ALP) activity in hepatopancreas were significantly decreased when the ambient nitrite concentration was higher than 5 mg/l, and the same phenomenon occurred when the ambient ammonia concentration was higher than 20 mg/l, which indicated that the effect of nitrite exposure on ACP and ALP activity, even the ability of protein synthesis or immunity, was stronger than that of ammonia.After the crabs exposed to 20 mg/l nitrite and 40 mg/l ammonia for 60d, the ultrastructure of hepatopancreas showed the organelles with membranous structure in hepatopancrea cells were easily damaged, such as mitochondria and nucleus. Thenuclear membrane changed from spreading all over the place to distintegrated, and the penetration of the mitochondrial outer membrane and inner membrane was changed. The mitochondria showed swelling and vacuolating. Mitochndrial cristae partly distintegrated, then fully disintegrated. So the damage of the structural and functional integrality of the mostly organelles affected the normal physiological function of hepatopancreas cells, which was the main mechanism of poison caused by ambient nitrite and ammonia.4. The effect of dietary VE supplementation on the ability of anti-stress in E. sinensis juvenilesThis study was aimed at determining if the increase of body vitamin E content through dietary supplementation in E. sinensis juveniles could enhance the resistance to nitrite stress. Three experimental diets were formulated which were control (basal diet, 360 mg VE kg^-1), diet 2 (660 mg VE kg^-1) and diet 3 (960 mg VE kg^-1) with supplementation of 300- and 600- mg VE kg^-1 respectively.The specific growth rate (SGR) and feed efficiency (FE) were recorded at day 20, 40 and 60, and the results showed that SGR increased significantly but FE decreased significantly after the juveniles were fed Diet 2 for 20d, however, the growth was inhibited and FE was increased after fed Diet 3.After fed 60 days, half of the crabs per group were treated by 10 mg/l nitrite stress for 24 h. The results showed that after nitrite stress, SOD activity and MDA level in hepatopancreas and gills, ALP activity in gills, ACP activity in hepatopancreas significantly increased, however, the ALP activity in hepatopancreas and ACP activity in gills decreased. But the differences of SOD, ALP and ACP activity and MDA level before and after stress could be shortened after dietary VE suppletmented, and the effect of dietary VE supplementation was significant in diet 2. Haemolymph oxyhemocynin level in the control group was significantly decreased while the groups of dietary VE supplementation had no notable decreases after nitrite stress. Vitamin E levels in hepatopancreas and gills increased with dietary vitamin E supplementation increasing but there was a significant decrease after nitrite stress. These results suggest that nitrite stress depress immune function and oxygen affinity of hemocyanin in crabs while vitamin E supplementation could alleviate the adverse effects caused by nitrite stress.5. The optimal requirement of VE in E. sinensis juveniles under nitrite exposureIn order to know the requirement to dietary VE under environmental stress, four diets including 0-, 300-, 1000-, 2000- mg/kg VE respectively were formulated, and the effect of different dietary VE on the growth, immunity and anti-stress ability of E. sinensis under 10 mg/l nitrite exposure was discussed.The results showed that except diet 4, WG and SGR increased while FE decreased significantly with dietary VE increasing. The survival rate of crabs fed with diet 1 was the lowest, followed by those fed with diet 4, and there was no notable difference in crabs fed with diet 2 and diet 3. And antioxidant enzymes activity in hepatopancreas was significantly highest and MDA level in hepatopancreas was significantly lowest in the treatment of diet 3. After 6h air exposure, the extent of SOD activity and MDA level increased was the least in crabs fed with diet 3.Except diet 4, the VE content in hepatopancreas and gills significantly increased with dietary VE increasing. However, they were decreased after 6h air exposure, and they were highest in diet 3, followed by diet 4, and lowest in diet 1.Therefore, the optimal requirement of VE under 10mg/l nitrite exposure in E. sinensis was 1 000 mg/kg or so.