【作者】 徐永江；

【导师】 于志刚； 柳学周； 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2005， 硕士

【摘要】 利用半滑舌鳎（Cynoglossus semilaevis Günther）—一种中国近海大型的底栖经济类鲽形目鱼类—的胚胎为试验材料,研究了Cu、Hg、Zn、Cd、Pb五种重金属对胚胎发育的毒性效应,结果显示:0.01mg/L、0.08mg/L、0.1mg/L Cu²⁺处理时胚胎孵化率较低,与对照组差异显著（P<0.05）;其它几种重金属在不同浓度水平上对半滑舌鳎胚胎的孵化率影响相对较小,但初孵仔鱼的畸形率较高,与对照组差异显著;除0.125mg/L、0.25mg/L、0.5mg/LCd²⁺会促进胚胎发育速率外,其余四种重金属都会在不同程度上减慢胚胎孵化速率（孵化时间延长2～7h）,高浓度的pb²⁺和Zn²⁺能延长胚胎的破膜时间。重金属离子在胚胎发育过程中引起了各种各样的畸形现象,如眼睛残缺、胚胎异常死亡、胚体解体模糊、胚胎尾部弯曲;初孵仔鱼不能破膜而出,初孵仔鱼脊椎弯曲成“S”形、“V”形、“L”形等。从孵化率、仔鱼畸形率和胚胎畸形程度等综合指标看,这五种重金属对胚胎发育的毒性大小依次为:Cu>Hg>Cd>Zn>Pb。研究了Hg、Cu、Cd、Pb、Zn等五种重金属对半滑舌鳎初孵仔鱼、10～20d仔鱼和20～30d稚鱼急性（96h）毒性影响,并计算了几种重金属对半滑舌鳎仔稚鱼的半数致死浓度(LC₅₀)和安全浓度。仔稚鱼对高浓度的金属比较敏感且在触毒后较短时间内就死亡,仔稚鱼在重金属溶液中表现出脊柱弯曲成“L”形和“S”型等畸形现象。 实验室条件下,1龄半滑舌鳎（Cynoglossus semilaevis Günther）暴露在浓度为0.1×10^-6mg/L的pb²⁺、Zn²⁺、Cd²⁺、Cu²⁺溶液中,随暴露时间的延长,半滑舌鳎的游泳行为发生一定的变化:Cd²⁺和Cu²⁺处理组的鱼行动缓慢,应激性很差,偶尔还出现不协调的颤抖性游泳行为,试验结束时几乎不摄食:pb²⁺和Zn²⁺处理组的鱼游动积极,与实验前无大差别。利用常规组织切片法检查pb²⁺、Zn²⁺、Cd²⁺、Cu²⁺对半滑舌鳎鳃、肝脏、肾脏、脾脏、脑的组织损伤情况,结果显示:Cd²⁺和Cu²⁺对鳃、肝脏和肾脏的损伤最为严重,引起了次鳃小片结构的崩溃;肝脏细胞脂肪颗粒的变形和大量堆积,造血组织坏死;肾小球结构溃解和管细胞肥大。pb²⁺和Zn²⁺处理组鱼次鳃小片基部上皮呈空泡状,粘液细胞数量减少,氯细胞肥大增生:肝脏脂肪颗粒堆积,肝细胞肥大增生;肾脏血细胞坏死,肾小管形态溃解和管细胞增生。对脾脏和脑的损伤未观察到。将行为学变化和生理损伤结合可以对更多还原

【Abstract】 To help define the tolerance of marine environmental stress exerted by heavy metals, embryos, newly hatched larvae, 10-20days after hatching larvae,20-30 days after hatching larvae of Cynoglossus semilaevis Giinther^a kind of high-valued recreationally and commercially harvested flatfishes found along the coast line of China, which grows fast and is an ideal candidate for aquaculture development and stock enhancement programs--were incubated at different levels of copper, mercury, lead, zinc and cadmium solutions, respectively. Embryos incubated at 0.01 mg/L, 0.08 mg/L, 0.1mg/L copper solutions showed low hatching rate, which significantly different from the control （P<0.05）, other heavy metals didn’t affect the hatching rate of embryos notably, but the newly-hatched larvae in all heavy metals’ solutions exhibited significant difference from the control for the high deforming rate. 0.125 mg/L, 0.25 mg/L, 0.5 mg/L cadmium could boost the development speed of embryos, other metals retarded the hatching course by 2⁷ h. Malformation of embryos caused by the metals were observed during the experiment, which different from each other, such as monothphmia, cyclopia, embryo death, larvae have V-shaped, S-shaped, L-shaped vertebral abnormality, the larvae can’t hatched from the embryos, and so on. The 50% lethal concentration and safety concentration of these metals of 24 h,48 h,72 h and 96h for newly hatched larvae, 10-20 days after hatching larvae,20-30 days after hatching larvae are calculated to help establish water criterion and protect marine environment. Larvae were sensitive to metals at high concentrations, many died soon after entering the solution. The abnormal larvae affected by the metals showed the curled vertebra and inconsistent swimming behavior.1-year-old Cynoglossus semilaevis Gunther were exposed to 0.1×10^-6mg/L lead,zinc,cadmium,copper solutions for 29 days and 10 days respectively in laboratory.Behavioral alteration has been detected: jerky, uncoordinated, spontaneous and slow movement, boeotian irritability was observed for fish in cadmium and copper solutions as the experiment processed, but different in fish in lead and zinc solutions. Effects of lead,zinc,cadmium,copper on the morphology of gill, kidney, liver and brain were investigated using routine histological technique and light microscope analysis. Cadmium and copper caused more serious lesion: gill lamellae structure completely damaged, pillar cells, mucus cells and chloride cells were not seen; accumulation of numerous droplets of fat and necrosis of hemopoietic tissue in liver; tubular necrosis and hyperplastic tubular cells were seen in kidney. Lead and zinc caused balloonlike appearance in gill basi-lamellae epithelium, shortage in mucus cells and hypertrophy of chloride cells, accumulation of fat droplets in hepatic cells, necrosis of blood cells in kidney and damaged tubules and tubular cells with hyperplasia and hypertrophy. Lesion in spleen and brain were not detected. Combination of abnormal behavior and histological lesion might be an useful way to monitor the heavy metals pollution in marine environment and artificial rearing offish.Histological alterations and Na+-K+-ATPase activity changes in gill of Cynoglossus semilaevis Gunther induced by subchronic copper exposure at different levels were studied in laboratory by combinding light and transmission electron microscope techniques and biochemistry methods. Lesion caused by copper in gill included epithelial changes such as lifting, rupture（vacuols）, lamellar fusion, hyperplasia and cellular hypertrophy; mucus cells increased , rupture of pilar cells system, erythrocyte congestion, and so on. Hytrophy and hyperplasia of chloride cells and epithelial cells increased the thickness of the water-blood barrier, which indicated severe impairment of ion regulation and gas transfer of fish exposed to copper. The results of the study indicate that histology is a successful tool capable of revealing sensitively and selectively even the sublethal effects of heavy metals on the environment and aquatic biota. Na+-K+-ATPase activity decreased significantly as the copper concentration increased, showed significant difference from the control （P^ 0.05）. Fish exposed to 150 u.g/L exhibited behavioral alteration: uncoordinated and jerky swimming behavior, slow movement and reaction to exterior stimulus wasobserved after 12 days exposure. As the sensitivity and dose-depended relationship between the Naf-K+-ATPase activity and copper concentration in water, it has the potential to be a bio-indicator for copper pollution in aquaculture and marine environment.Routine histological methods and electron microscope technique were combined to investigate the lesion and the changes of several functional enzymes in liver of Cynoglossus semilaevis Giinther after subchronic copper exposure 15 days. The liver experienced variable changes caused by copper: hepatocytes decreased in diameter with abnormal or necrotic nucleus, cytoplasm showed vacuols and accumulation of lot of glycogen, ribosomes fell off the endoplastic reticulum into the cytoplasm, mitochondria enlarged with abnormal shape such as round cake and dumbbell, sinusoids and bile canaliculus damaged with decrease in quatity which indicated an turbulence in fat metabolism, lysosomes manifolded, and so on. Glutamate-oxaloacetate transaminase （GOT）, glutamate-pyruvate transaminase （GPT）, succinate dehydrogenase（SDH）, antioxidant enzymes catalase（CAT）, glutathione peroxidase（GSH-Px）, superoxide dismutase（SOD）, glutathione reductase（GR） activity changes were investigated in vivo in liver following 15 days of copper administration. The specific activities of antioxidant enzyme CAT , GSH-Px and GR was strongly reduced by copper exposure. Meanwhile SOD and GPT activity were strongly stimulated in contrast, whereas the GOT and SDH activity were stimulated notably by 37.5u.g/L copper exposure. As far as the 150 ug/L is concerned, the enzyme activity all decreased slightly. These data suggest that copper in fish hepatocytes altered cell membrane structure and concomitantly induced some perturbation in the liver function. As CAT, GSH-Px, GR activity decreased with the elevated copper concentration in water and the dose-depengded relation to copper concentration, these enzymes would be effective bio-indicators for copper pollution in marine and aquaculture environment.更多还原