【作者】 何志强；

【导师】 郑永华；

【作者基本信息】 西南大学 ， 水产养殖， 2008， 硕士

【摘要】 重金属作为一类主要的污染物其对鱼类的毒害作用，已日益受到人们的关注。目前人们对许多外源性化学物的单一毒性已有较多的了解，但对于环境中存在的多种污染物的联合毒性作用及其机理则认识的较少。本试验采用体长为4．5—5．6cm的中华倒刺鲃作为受试对象，以重金属铜、锌、汞作为污染物来研究其对中华倒刺钯的生物毒性效应，包括单因子急性毒性试验、联合急性毒性试验和亚急性毒性试验，其中亚急性毒性试验则是研究污染物在较长的时间内对鱼体体内酶类和DNA的影响。试验结果表明：1．单因子急性毒性试验采用静水染毒法，得出重金属铜、锌和汞对中华倒刺鲃的96h半致死浓度分别为0．534mg/L、10．13mg/L和0．141mg/L，安全浓度分别为0．053mg/L、1．013mg/L和0．014mg/L，其毒性大小为：Hg²⁺>Cu²⁺>Zn²⁺。并且其半致死浓度随着受试时间的增长而呈现下降的趋势。2．联合急性毒性试验在单一急性毒性试验的基础上，进行Cu²⁺—Hg²⁺、Cu²⁺—Zn²⁺、Hg²⁺—Zn²⁺三个组合的联合急性毒性试验。用96hLC₅₀（半致死浓度）作为1个毒性单位，本试验设6个浓度梯度且各处理组的毒性总和都为1。试验结果表明，Cu²⁺—Zn²⁺、Zn²⁺—Hg²⁺对的联合毒性效应均为增毒作用，而Cu²⁺—Hg²⁺中华倒刺鲃的毒性效应在Cu²⁺：Hg²⁺为0．2：0．8时为拮抗作用，在比值为0．4：0．6时为相加作用，随着Cu²⁺比例的增加其毒性效应表现为增毒作用。3．亚急性毒性试验取96h半致死浓度的5％、10％和20％设立3个染毒组和1个对照组。试验共进行28d，采用静态置换法每3d更新溶液50％，每天投喂无污染配合饲料一次，分别于7d、14d、21d和28d采集样本。试验结果表明：Cu²⁺、Zn²⁺和Hg²⁺对中华倒刺鲃鳃组织GPT和GOT活力影响呈现抑制作用，在28d时，鳃组织GPT活力出现最小值，其中2．026 mg/L Zn²⁺组抑制率最大达到57．45％，其对中华倒刺钯鳃组织GPT活力的抑制作用大小为：Cu²⁺<Hg²⁺<Zn²⁺。而在等毒性条件下，Hg²⁺较Cu²⁺和Zn²⁺对GOT的活力有更强的抑制作用，0．028 mg／LHg²⁺组在28d时对鳃组织GOT酶活力的抑制率达到最大值62．68％。随着处理组重金属离子浓度的增大，对ACP和AKP活力的抑制作用也随之增强，但Cu²⁺、Zn²⁺和Hg²⁺对鳃组织AKP活力的抑制作用在暴露时间上并未呈现较大的变化，反而AKP活力于21d时还均有不同程度的上升。同时各个处理组在随着暴露时间的增加对ACP活力也没有出现较大的差异，这可能是AKP酶和ACP酶较为敏感，在重金属暴露处理的前期（<7d）其活力就受到了抑制。虽然对SOD酶活力也存在抑制作用，但抑制作用不大，并未达到显著水平，可以看出，Cu²⁺、Zn²⁺和Hg²⁺对鳃组织的SOD酶活力没有明显的影响。随着处理时间延长，对中华倒刺鲃肝胰脏的DNA损伤程度呈现上升趋势，染毒7d后，Zn²⁺和Cu²⁺处理组对DNA的损伤以3级损伤为主，损伤率达到100％，染毒21d以后其差异不明显：2．026mg/L Zn²⁺组对中华倒刺鲃的DNA损伤最大，0．007mg／L Hg²⁺组为最小。其对DNA损伤的大小顺序：Zn²⁺>Cu²⁺>Hg²⁺。通过试验发现，用SCGE技术检测重金属对中华倒刺鲃肝胰脏细胞DNA损伤具有灵敏、快速、直接、可靠等优点，可以检测出细胞的DNA断裂，很好地反映出低剂量的遗传毒性，可作为检测早期生物学毒性效应的生物标志物。更多还原

【Abstract】 Heavy metal is the principal pollutant in the river, people follow the bane action of fish closely day by day. People understand more knowledge about single toxicity of chemical substances at the present, but grasping the action and mechanism of multiple contamination’s joint toxicity is not enough. The experiment used Spinibarbus sinensis with 4.5-5.6cm stature as the trier, and researched the biotoxicity of the heavy metal to Spinibarbus sinensis, included single factor acute toxicity experiment, joint acute to xicity experiment and subacut acute toxicity experiment, subacute acute toxicity experiment researched the influence of pollutant to fish’s Enzymes and DNA. The results showed:1. Single factor acute toxicity experimentUsed method of static state, 96hours’ half lethal concentration of Copper, Zinc and Mercury on Spinibarbus sinensis was 0.534mg/L, 10.13 mg/L and 0.141 mg/L and safe concentration was 0.053 mg/L, 1.013 mg/L, 0.014 mg/L, So we could reach a conclusion that the magnitude of toxicity is Hg²⁺ >Cu²⁺>Zn²⁺. And the half lethal concentration presented the going down tendency along with the experiment’s time. 2. Joint acute toxicity experimentOn the basis of single factor acute toxicity experiment, joint acute toxicity experiment with three assembling of Cu²⁺-Hg²⁺, Cu²⁺-Zn²⁺, Hg²⁺-Zn²⁺ were carrying out. Regarding 96hLC₅₀ （96hours’ half lethal concentration） as a toxicity unit, the experiment set up 6 concentration gradient and everyone’s sum toxicity is 1. The results showed: the joint acute toxicity of Zn²⁺-Hg²⁺, Cu²⁺-Zn²⁺ were synergy, but the joint acute toxicity of Cu²⁺-Hg²⁺ was antagonism when Cu²⁺: Hg²⁺=0.2:0.8, the effect of Cu²⁺:Hg²⁺=0.4:0.6 was additive action, and the effect of toxicity were synergy with increasing the proportion of Cu²⁺.3. Subacute acute toxicity experiment5%, 10% and 20% of 96hours’ half lethal concentration were taken to be 3 experiment groups. The experiment carried out 28days, half of the experiment liquor could be replaced by new liquor every 3 days and fishes were be feeded once every day. The samples were gathered at 7^th, 14^th, 21^th and 28^thday. The result testified, the effect of Cu²⁺, Zn²⁺ and Hg²⁺ on the GPT and GOT enzyme activity of Spinibarbus sinensis gill was inhibition, the GPT enzyme activity of gill reached minimum value at 28^th day, in which the suppression ratio of 2.026 mg/L Zn²⁺ was maximum and reach 57.45%, so the inhibition sequence of heavy metal on GPT enzyme activity of gill was Cu²⁺<Hg²⁺<Zn²⁺. At the same toxicity, compare Hg²⁺ with Cu²⁺ and Zn²⁺ has stronger inhibition on the GOT enzyme activity, the suppression ratio of the gill GOT enzyme activity at 0.028 mg/L Hg²⁺ was maximum and reached 62.68%.With the level of the heavy metal ion increasing, the inhibition of the ACP and AKP enzyme activity become increased, but the inhibition of the AKP enzyme activity increased little, especially, the enzyme activity of AKP has different level of ascending at 21th day. And the ACP enzyme activity don’t become increasing by the experiment increasing either, this may be the AKP and ACP enzyme activity is more sensitive, which be to keep down at the preceeding of the experiment<7^thd). Heavy metal on the SOD enzyme activity is also inhibition, but the effect of increasing is small, can’t reach markedness level, so the Cu²⁺, Zn²⁺ and Hg²⁺ ion can’t reach the markedness effect on the SOD enzyme activity.The heavy metal ion on DNA damage appear the ascending trend, Zn²⁺ and Cu²⁺ on DNA damage almost reach third class and the damage ratio achieve 100% after 7^th day. The discrepancy is not markedness after 21th day, the max is 1.98 on 1.013mg/L Zn²⁺ at 21^th day. 2.026mg/L Zn²⁺ is maximal damage on heptopancreas DNA and 0.007mg/L Hg²⁺ is lowest, the sequence is Zn²⁺>Cu²⁺>Hg²⁺.Through experiment, we find out SCGE technique have sensitive, high-speed, immediante, reliable etc merits on heavy metal on the damage of Spinibarbus sinensis heptopancreas DNA, which can detect the DNA rupture and reflect inheritance toxicity by low dose, so regarding it as biomarker in inchoate biolog更多还原