The Study Of Phenanthrene Toxicity On The Chinese Mitten-handed Crab, Eriocheir Sinensis

PAHs with carcinogenic, teratogenic and mutagenic, is a typical class of persistent organic pollutants. To study the toxicity effects of PAHs on aquatic animal and their toxicity mechanisms, in this study, phenanthrene, a typical PAH, was chosen as the research object, and Mitten-handed crab Eriocheir sinensis was chosen as experimental animals, which is a very important and delicious economic aquatic animal in our country. The research carried out at many levels, such as individual, physiological enzymes and molecules.1. Acute toxic effects of phenanthrene on Chinese mitten crab Eriocheir sinensisAn acute semi-static toxic test was carried out to expose E. sinensis to water-borne phenanthrene with different concentrations (1.00,1.63,2.65,4.31 and 7.00 mg·L-1) for 96 hours. Mortality and toxic reactions were used as effect criteria. Furthermore, the oxidative stress relative biomarkers (activities of CAT, SOD, GST and content of MDA in hepatopancreas and gills) were measured for E. sinensis exposed to four lower concentrations of phenanthrene (1.00,1.63,2.65 and 4.31 mg·L-1).The median lethal concentration (LC50) for 24,48 and 96 hour phenanthrene exposure was 16.7,2.96 and 2.14 mg·L-1, respectively, and the safe concentration (SC) of phenanthrene on E. sinensis was 0.214 mg·L-1. Phenanthrene significantly increased the activity of CAT in hepatopancreas and gills, and showed a significant dose-effect relationship in hepatopancreas. Phenanthrene, especially of high concentration, is highly toxic on E. sinensis, causing lipid peroxidative damage and even acute mortality. Meanwhile, E. sinensis can adapt to lower concentrations of phenanthrene by increasing the activities of SOD, CAT and GST. Because the CAT and GST activities of the gill and hepatopancreas are very sensitive to the presence of high-concentration phenanthrene, they can be used as high-sensitive biomarkers for phenanthrene polluted water.2. Subacute toxic effects of phenanthrene on the antioxidant enzymes of Eriocheir sinensisIn order to investigate the subacute toxic effects of phenanthrene on the lipid peroxidation status and the activity of antioxidant enzymes in Eriocheir sinensis, E. sinensis were exposed to water-borne phenanthrene with different concentrations (50, 100,150 and 200 μg·L-1) for different periods. During the exposure of phenanthrene, hepatopancreas and gills were separated for detecting the variation of CAT, SOD, GST, GPx activity and MDA content. The results show that in the subacute stress of phenanthrene, the activity of CAT, GPx, SOD, GST and MDA content in hepatopancreas and gills showed different sensitivity both in the same and different tissues. Namely, the activity of CAT, GST, GPx and SOD presented higher sensitivity in hepatopancreas than in gills, but the activity of GST in both hepatopancreas and gills are not sensitivity by compared with others.The activity of GPx SOD, CAT in hepatopancreas changed with time and the concentration of phenanthrene can be used as biomarkers for short-term exposure to organic pollutants. In addition, althoμgh the content of MDA increased significantly in the initial exposure period, but with the CAT, GPx, SOD and GST activity induced, the reactive oxygen species and free radicals generated by phenanthrene could be cleared effectively, then MDA content decreased, and finally stabilized.Even, when the content of MDA were high, the antioxidant capacity of E. sinensis have not been destoried by oxidative damage indicating that E. sinensis have a good ability to adapt to phenanthrene exposure which was below the safety concentration.3. Effects of phenanthrene on the expression and activity of detoxification enzymes of Eriocheir sinensisTo explore the effect of phenanthrene on the detoxifying enzymes gene expression and enzymes activity in E. sinensis, at first, this study cloned the P450 and a 816bp length of P450 fragment was gained in E. sinensis. BLAST analysis showed that the P450 fragment should be CYP4. To further explore the role of this gene in the biotransformation and detoxification of phenanthrene, expoused E. sinensis to the water borned 200 μg·L-1 phenanthrene after 28 days, half E. sinensis were exposed 21 days followed by a 7-day depuration period. The expression of CYP4 and GST in the hepatopancreas, as well as, the activity of metabolic enzymes EROD and GST in hepatopancreas, muscles and gills of E. sinensis was dected.The results showed that in the low concentrations of phenanthrene expouse, E. sinensis can upregulate the expression of CYP4 and GST which are associated with the biotransformation and detoxification of phenanthrene in E. sinensis, thus induced the activity of detoxification enzymes, EROD and GST, then effectively responded to the oxidative stress and cumulative toxicity caused by the phenanthrene. E. sinensis has a strong ability of detoxification phenanthrene.4. Accumulation and elimination of phenanthrene in Eriocheir sinensisTo investigate phenanthrene bioaccumulation and depuration in the hepatopancreas and muscle, oxidative stress in the hepatopancreas and gills of E. sinensis, and the relationship between the content of phenanthrene and the activity of enzymes, semi-static experiment was carried out. E. sinensis exposed to water-borne phenanthrene 200 μg·L-1 for different periods, some of E. sinensis after 21 days of exposure were transferred to clear water without phenanthrene for 7 days. Contend of phenanthrene in hepatopancreas and muscles, meanwhile, the content of MDA and the activity of SOD, CAT, GPx and GST in hepatopancreas and gills of E. sinensis were detected. As consequences phenanthrene expouse can induce GST, CAT, GPx, SOD activity in hepatopancreas and gills of E. sinensis, enhance oxidative defense ability, maintain oxidative damage in arecoverable degree. Both in hepatopancreas and gill, MDA level and GPx activity were significantly related, indicating there may be anti-oxidation pathway regulated by lipid oxidation presence in E. sinensis. At the same time, in gills, SOD activity and CAT activity, SOD activity and GPx activity were significantly correlated, indicating that the antioxidant pathways effected oxygen free radical not only found in vertebrate, but also found in invertebrates E. sinensis. In addition, in the muscle, the activity EROD and GST was very significant positive correlation, GST activity and the content of the phenanthrene was also significantly positively correlated, indicating that the detoxification pathways of exogenous organic discovered in vertebrates and involved in the activity of EROD and GST, also might exist in E. sinensis which is invertebrate. To some extent, the rising levels of phenanthrene enhanced phenanthrene biotransformation capabilities in E. sinensis.