【作者】 郑晓晶；

【导师】 张育辉；

【作者基本信息】 陕西师范大学 ， 动物学， 2006， 硕士

【摘要】 随着工农业生产的快速发展,环境化学污染日益严重。烷基酚聚氧乙烯醚（alkylphenol polyethenoxy ethers, APEs）类物质,是一类在工业生产和日常生活中广泛应用有机化合物,其降解产物壬基酚（nonylphenol NP）和辛基酚（octylphenol OP）具有环境雌激素活性,对动物的个体生存和种系繁衍构成了极大的威胁。本研究以壬基酚水体染毒的方式,对中国林蛙蝌蚪分别进行急性和慢性染毒实验,旨在揭示壬基酚对中国林蛙蝌蚪的急性毒性、对生长发育的影响、对性腺分化的效应及其机制。实验的主要结果及结论如下: 1.在水温21±1℃条件下,通过急性毒性实验,测得中国林蛙蝌蚪暴露在NP中24、48、72、96h的蝌蚪死亡几率与NP浓度对数的回归方程分别为y=8.4087x+10.202、y=9.5104x+11.745、y=10.284x+12.498、y=10.619x+13.095;半数致死浓度(LC₅₀)分别为0.24、0.20、0.19、0.17mg·L^-1。安全浓度（SC）为0.017mg·L^-1;96h的零致死浓度（LC₀）为0.14mg·L^-1。 2.将26期林蛙蝌蚪暴露在100,60,30,10μg·L^-1NP中连续养殖直至完全变态,并以3、0.3μg·L^-1雌二醇（E2）作为阳性对照,分别统计林蛙蝌蚪暴露于NP和E₂中20天、40天以及完成变态后幼蛙的体重、全长以及从26期到完全变态所需的时间。结果表明,NP不影响林蛙蝌蚪的死亡率。100、10μg·L^-1NP与3μg·L^-1E₂的效应相似,可降低蝌蚪完全变态后幼蛙体重,滞后蝌蚪变态时间,但对死亡率没有影响。说明在100μg·L^-1以下,NP不是通过直接损伤机体、而是通过干扰内分泌活动影响林蛙幼体生长发育。 3.通过统计比较野生蝌蚪染毒NP的幼蛙和母体染毒NP后子代幼蛙的性别比例,研究NP对性腺分化的影响,结果表明:（1）NP浓度在100μg·L^-1和30μg·L^-1之间时,将诱导野生林蛙蝌蚪的雌性比例上升。并且,雌性比例与NP浓度呈正相关,说明NP可导致雄性野生蝌蚪雌性化。30μg·L^-1NP与0.3μg·L^-1E2效应相似,表明NP的雌激素效应浓度是E2的100倍。（2）NP仅通过雌性亲代染毒对其子代性腺分化产生隔代效应,而不通过雄性亲代产生隔代效应。（3）通过对母体染毒的子代蝌蚪再次染毒,幼蛙雌性比例可进一步上升,雌性比例最高可达到85.2%,比野生雌性比例高出40%。这些结果说明,水域中非致死剂量的NP会影响到两栖动物幼体性腺的正常发育,进而影响雌雄比例失衡、繁殖活动障碍、以至于种群数量下降。 4.采用免疫组织化学方法检测NP对野生林蛙蝌蚪变态后幼蛙性腺中睾酮更多还原

【Abstract】 With the rapid development of industry and agriculture, environmental contamination became seriously. A well known group of organic compound is the alkylphenol polyethoxylates （APEs）, which are widely used in industry and daily life. APEs undergo a biodegradation process to give derivatives such as nonylphenol （NP） and octylphenol （OP）, which have estrogenic effects. It brought an extreme threat on survival and reproduction of vertebrates. In this study, Rana chensinensis tadpoles were exposed to NP in acute toxicity tests and chronic aqueous exposure tests, and acute toxicity, development and sexual differentiation were examined after that period to evaluate the effects and the mechanisms of estrogenic effects of NP. The main results and conclusions as followings:1. A standard method was used to evaluate the acute toxicity of nonylphenol on Rana chensinensis tadpoles at the water temperature of 21±1℃.The results showed that the regress equation between the death probability of Rana chensinensis tadpoles and the concentration logarithm of NP were y=8.4087x+10.202, y=9.5104x+11.745, y=10.284x+12.498 and y=10.619x+13.095 respectively for 24, 48, 72, 96h. The mean LC₅₀ values were 0.24、 0.20、 0.19 and 0.17 mg·L^-1 respectively. The mean SC（safe concentration） values was 0.017 mg/l, The mean 96h LCo values was 0.14 mg · L^-1.2. Rana chensinensis tadpoles were continuously exposed to 100, 60, 30, 10μg ·L ^-1 NP and 3、 0. 3μg·L^-1 E2 （positive control）. Exposure occurred throughout the stage 26 to stage 66, and weight, total length were examined at day20, day40, complete metamorphosis respectivel.Time of metamorphosis were examined at period of metamorphosis. The results showed that NP did not affect mortality of Rana chensinensis tadpoles. 100 and 10μg ·L^-1 NP had similar effects induced by 3μg·L^-1 E₂, reduced the weight of froglets （metamorphs） and delayed the time of metamorphosis. It suggested that NP did not directly damage the organism but disrupted the endocrine system to affect the development of Rana chensinensis tadpoles under 100μg·L^-1 NP.3. The aim of this study was to evaluate both estrogenic effects in directly NP-exposed wild Rana chensinensis tadpoles and possible transgeneration effects in the offspring of exposed mature Rana chensinensis. Analysis of post-metamorphic sex ratio,indicated as followings: （1） wild tadpoles were exposed to NP, beween lOOjag ? L ’ and 3Ofjg ? L"1, female ratio was elevated. A tendency for a dose-dependent increase in the female ratio was observed in the NP expose groups. It suggested NP induced feminization in wild Rana chensinensis tadpoles. The necessary concentration of NP for inducing feminization was at least 102 times higher than that of E2. （2） Only female mature exposed to NP would had transgeneration effects on the offspring （3） Female progeny were exoposed to NP. Their maximal female ratio was 85. 2%, towered above the wild female ratio about 40%. This study demonstrated sub-lethiferous NP concentrations in water affected gonadal development and sexual differentiation in amphibian, and induce sex ratio deviating and reproductive obstacle. It resulted in population delines in amphibian species.4. To understand the effects of NP on the level of immunoreation of testosterone （T）, aromatase （ARO）, androgen receptor （AR） and estrogen receptor （ER） by immunocytochemisry in gonad in froglets of NP-exposed wild tadpoles. It indicated as followings: （1） The level of immunoreactive T was reduced in the gonad. （2） NP was in agreement with the effects of E2. An increase in the level of immunoreactive ARO was found in the gonad. （3） NP had similar effects induced by E2, reduced the level of immunoreactiveAR in the gonad. （4） The level of immunoreactiveER was just reduced in the testis of froglets of wild tadpoles were exposed to lOOug ? L"1 NP. （5） There was a similar effect on the level of immunoreactiveT, ARO, AR, ER in testis and ovary of froglets of NP-exposed wild tadpoles. Our results further suggested that NP increased the activity of ARO, and converted testosterone to estrogen.lt induced an increase in the E2 relative content in gonad. E2 inhibited the expression of AR, and inhibited androgen binding of AR, At last, NP induced feminization in gonad of froglets from NP-exposed wild tadpoles.更多还原