【作者】 唐超智；

【导师】 梁刚；

【作者基本信息】 陕西师范大学 ， 动物学， 2005， 硕士

【摘要】 三氯杀螨醇是现代农牧业生产中常用的有机氯杀虫剂之一,近些年来已有越来越多的证据表明,其在环境中的暴露对鱼类、爬行类、鸟类、哺乳类和人类有毒性和雌激素效应,而对两栖类的这些效应迄今尚未见实验性的报道。中华大蟾蜍（Bufo gargarizans）隶属两栖纲、无尾目、蟾蜍科,是我国重要的经济两栖动物,广泛分布于全国二十多个省区不同海拔的多种生境中,除冬眠和繁殖期在水中生活外,一般多在农田、果园、菜地、草丛、林下、居民点周围等潮湿地方栖息,其食性广、食量大,是农牧业区许多害虫的主要天敌之一。本文以中华大蟾蜍为研究对象,探讨了三氯杀螨醇对其幼体和成体的急性毒性和雌激素效应及其作用机制,旨在进一步积累三氯杀螨醇对两栖动物毒性和雌激素效应的实验证据,为野生动物资源的保护和人类食品的安全提供基础资料。实验用中华大蟾蜍成体购自西安市长安区斗门实验动物养殖厂,蝌蚪采集于西安市长安区杜曲镇。首先分别选取体重55-115g的健康成体80只、18-22日龄的蝌蚪300只进行急性毒性实验,再分别选取体重65-85g的健康成体28只、25日龄的蝌蚪160只进行雌激素效应实验。雌激素效应实验分别用5个和7个梯度浓度的三氯杀螨醇处理成体和蝌蚪,同时用任氏液和雌二醇分别作为空白对照组和雌激素对照组,主要通过组织学和免疫组织化学等方法,观察统计了三氯杀螨醇对中华大蟾蜍成体和蝌蚪性腺组织结构以及睾酮和雄激素受体表达的影响。从实验结果及其讨论分析中,得出以下结论。1.在15—25℃、染毒48h后,三氯杀螨醇对中华大蟾蜍成体的雄性和雌性的绝对致死浓度(LC₁₀₀)分别为300 mg.kg^-1。和400 mg.kg^-1,最大耐受浓度（LC₀）分别为100 mg.kg^-1和120 mg.kg^-1,半致死浓度(LC₅₀)分别为161.27±19.34 mgkg^-1和188.51±20.93mg.kg^-1。说明中华大蟾蜍雄性成体对三氯杀螨醇的耐受性比雌性差。2.在12-18℃和18-25℃、染毒48h后,三氯杀螨醇对中华大蟾蜍蝌蚪的LC₁₀₀分别为15.0 mg.1^-1和3.5 mg.1^-1,LC₀分别为5.Omg.1^-1和1.0 mg.1^-1,LC₅₀分别为8.64±0.97mg.1^-1和2.45±O.28mg.1^-1。说明在一定温度范围内,三氯杀螨醇对中华大蟾蜍蝌蚪的毒性随着温度升高而加重。3.一定浓度的三氯杀螨醇可导致部分中华大蟾蜍蝌蚪出现畸形和发育进程明显滞后:而且,O.016、O.04、0.08、0.16 mg.1^-1三氯杀螨醇处理组的雌性个体数量多,雄性个体数量少。4.一定浓度的三氯杀螨醇对中华大蟾蜍成体卵母细胞的发育有较强的抑制作用,可导致卵泡出现不同程度的闭锁,说明三氯杀螨醇可能对蟾蜍成体卵母细更多还原

【Abstract】 Abstract: Dicofol is a kind of organochlorine insecticide, which is widely used by modern animal breeding and agriculture production. Recent years, more and more researches indicated that dicofol exposing in the environment would have toxicity and estrogen effects on Fishes, Reptiles, Aves, Mammals, and even humans. Yet there is no experimental reports about Amphibian. Bufo gargarizans, the species of Bufonidae in Anura, is an important economic animals in our country, and distributes in many kinds of living conditions with different altitude in more than 20 provinces of our country. They inhabit in the moist places such as farmlands, orchards, brushwood, vegetable fields, hurst or aroud the human resident. B. gargarizans with features of euryphage and hearty eater, is one of the principle natural enemies of many agriculture pests. With the experimental material of B. gargarizans, the paper investigated the acute toxicity and estrogen effects that dicofol have on the tadpoles and the adults of toad, meanwhile dicussed the mechanism of these biological effects, which would provide more evidences about the researches on the toxcity and estrogen effects of dicofol on the Amphibian, and give the basic materials of protecting the wild animal resources and human food safety.The adults of toad bought from doumen laboratory animal culture plant Xi’an city, the tadpoles collected from Duqu town of Chang’an County. Select 80 healthy adults weighing 55-115 g and 300 tadpoles aged 18-22 days for acute toxicity experiment;select 28 healthy adults weighing 65-85 g and 160 tadpoles aged 25 days for estrogen effects experiment. In estrogen effects experiment, the adults were dealt with 5 concentration gradient of dicofol and the tadpoles were dealt with 7 concentration gradient of dicofol respectively as disturbing team;and with physiological brine and estradiol as the two control team. With the methods of histology and immunohistology, the influence of dicofol effect on the structure of sexual gland was observed and the effect on the expression of testosterone and androgen receptors was detected. From the experiment results, discussion and analysis, the paper reaches the following conclusions:1. On the condition of 15-25℃ room temperature, injecting dicofol into the adult of toad. After 48 hours, the absolute lethal concentration(LC₁₀₀) of dicofol affected on the male and female of the adults of toad is respectively 300 mg.kg^-1 and 400 mg.kg^-1, the maximal tolerance concentration（LC₀） is respectively 100mg.kg^-1 and 120 mg.kg^-1,the medican lethal concentration（LC5o） is respectively 161.27+19.34 mg.kg’1 and 188.51+20.93 mg.kg"1. The result indicated that the resistance ability of the male adult toad to dicofol was poorer than that of the female.2. On the condition of 12-18 °C and 18-25°C, exposing in dicofol for 48 hours, the LCioo of dicofol effected on the tadpoles is respectively 15.0 mg.l"1 and 3.5 mg.l"1, the LCois respectively 6.0 mg.l"1 and 1.0 mg.l"1, the LC50 is respectively 8.64 + 0.97 mg.l"1 and 2.45 + 0.28 mg.l’1. The result showed that in certain a range of temperature, the toxicity of dicofol effected on the tadpoles would aggravated with the rising of the temperature.3. Certain a range of concentration of dicofol would lead to some tadpoles abnormalllity and lagged development. Furthermore, the tadpoles dealt with the concentration of 0.016, 0.04, 0.08, 0.16 mg.l*1, there were more females than males.4. Certain a range of concentration of dicofol had strong inhibitory effects on the development of oocytes in the female adults of B. gargarizans, which would lead different degree of follicle atresia. The result showed that dicofol may have toxicity effects on the development of oocytes in female adults. Furthermore, certain a range of concentration of dicofol also had inhibitory effects on the expression of testosterone and androgen receptors in oocytes, which we can concluded that the dicofol maybe have estrogen or antiandrogen effects.5. Certain a range of concentration of dicofol would make the structure of Seminiferous tubule, Spermatogonium, Leydig cells shrinked, and the quantity of sperm decreased. And that, the influence expanded with the increasing of the concentration. Moreover, dicofol had reduced the expression of testosterone and androgen receptors in the testis of male adults, and the reducing degree of testosterone expression was much more apparent than that of androgen receptors. The result indicated that Certain a range of concentration of dicofol would lead the hypofunction of testis in male adults, and the mechanism may be that dicofol had restrained the expression of testosterone.6. Certain a range of concentration of dicofol also had bad influence on the ovary of tadpoles of B. gargarizans, which would result in abnormal phenomenon such as the shrink of the oocytes nucleus, and disturb the normal process of oocytes development. Furthermore, dicofol also had inhibitory effects on the expression of testosterone and androgen receptors, and the reducing degree of testosterone expression was much more apparent than that of androgen receptors. The result showed that the main mechanism of the estrogen effect of dicofol was to directly restrain the expression of testosterone.7. Certain a range of concentration of dicofol had repressing influence on the structure of Seminiferous tubule and Leydig cells of tadpoles of B. gargarizans, and the influence expanded with the increasing of the concentration. Alike, dicofol reduced the expression of testosterone and androgen receptors, and the inhibitory degree of testosterone expression was much more apparently. The results can be concluded that the estrogen effects of dicofol achieved by the way of directly restraining the expression of testosterone.更多还原