【作者】 曹亮；

【导师】 窦硕增；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生态学， 2010， 博士

【摘要】 海洋污染是全球关注的热点问题之一。每年有大量的重金属通过各种人类活动进入到海洋环境中,严重影响海洋鱼类的繁殖、生长、发育和存活。大量研究表明鱼类的早期生活阶段比成鱼对重金属污染物更敏感,重金属暴露可导致鱼类孵化率下降、孵化时间延长、畸形率和死亡率增加、生长减慢等不利后果。长期污染使鱼类的资源补充失败、资源衰退或枯竭。此外,重金属在鱼类体内的积累,也对人类的食物安全和生命健康造成了巨大的危害。因此深入研究重金属对鱼类的毒理作用和致毒机理,并建立能够对重金属污染物的毒性水平进行早期预警和生态风险评估的生物监测体系对实现渔业资源的可持续发展和保证人类的食物安全具有十分重要的意义。本论文选取我国海洋水体中两种常见的重金属—铜（Cu）和镉（Cd）,分别研究了Cu和Cd对褐牙鲆（Paralichthys olivaceus）早期生活阶段（胚胎、仔鱼和稚鱼）的毒理作用。主要研究结果如下:1.急性毒理效应研究:Cu对褐牙鲆胚胎24 h半致死浓度(LC₅₀)和48 h LC₅₀分别为0.21 mg/L和0.11 mg/L;对仔鱼的48 h, 72 h, 96 h LC₅₀分别为0.46 mg/L,0.21 mg/L和0.12 mg/L。Cd对褐牙鲆胚胎24 h LC₅₀和48 h LC₅₀分别为7.98 mg/L和4.65 mg/L;对仔鱼的48 h, 72 h, 96 h LC₅₀分别为13.64 mg/L,7.41 mg/L和4.17 mg/L。结果表明: Cu比Cd对褐牙鲆的致死毒性更强;褐牙鲆胚胎比仔鱼对Cu、Cd暴露更敏感。2.胚胎-仔鱼亚急性毒理效应研究:受精后3小时（3 hours post fertilization, hpf）的褐牙鲆胚胎分别暴露于亚致死浓度的Cu（0-0.12 mg/L）和Cd（0-2.4 mg/L）溶液中直至仔鱼开口（130 hpf）。Cu和Cd的暴露对褐牙鲆胚胎-仔鱼的发育、生长和存活都产生了明显的毒理影响。≥0.06 mg/L的Cu以及≥0.8 mg/L的Cd暴露均使褐牙鲆的孵化率下降、孵化时间延迟、死亡率和仔鱼畸形率增加等。Cu和Cd均没有对褐牙鲆胚胎（42 hpf）的心率产生影响,然而高浓度的Cu（≥0.10 mg/L）和Cd（≥1.2 mg/L）显著抑制了130 hpf仔鱼的心率。≥0.10 mg/L的Cu和≥1.2 mg/L的Cd浓度组中仔鱼的全长均比对照组显著减少,而≥0.08 mg/L的Cu和≥0.8 mg/L的Cd显著降低了仔鱼对卵黄囊的吸收速率。各指标对Cu的敏感性为:累积死亡率>累积孵化率>仔鱼畸形率>生长指标;对Cd的敏感性为:仔鱼畸形率>累积死亡率>累积孵化率>生长指标。3.慢性毒理效应研究:实验室内模拟重金属对褐牙鲆整个早期生活史的毒理效应。褐牙鲆从胚胎开始分别在Cu（0-32μg/L）和Cd（0-48μg/L）溶液中持续暴露80天,研究三个阶段的褐牙鲆（变态期仔鱼、着底期仔鱼、稚鱼）对重金属的毒理响应。在变态期, Cu造成鱼体内还原型谷胱甘肽（GSH）含量降低,超氧化物岐化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽-S-硫转移酶（GST）活性下降,导致机体脂质过氧化产物丙二醛（MDA）含量显著升高;而Cd造成SOD和CAT酶活性下降, GSH含量增加,以及MDA含量升高。在着底期,Cu没有对仔鱼的抗氧化系统和脂质过氧化水平造成显著影响;而Cd造成CAT和GST酶活性下降,并且使GSH含量显著增加。在稚鱼期,Cu的暴露导致体内MDA含量显著升高,并使GSH含量以及SOD和GST酶活性显著增加;而Cd也造成稚鱼体内MDA含量显著升高,并使GST活性降低以及SOD活性增加。试验结束时32μg/LCu处理组褐牙鲆的体重显著低于对照组;24μg/L和48μg/L Cd处理组褐牙鲆的全长和体重均显著低于对照组。褐牙鲆体内的重金属蓄积量与暴露浓度呈明显的浓度依赖关系。4.稚鱼短期亚慢性毒理效应研究:褐牙鲆稚鱼暴露于不同梯度的Cd溶液（0-8 mg/L）28天后,4 mg Cd/L和8 mg Cd/L浓度组中褐牙鲆的生长受到了显著抑制。Cd在褐牙鲆组织中的蓄积呈浓度依赖性和组织特异性,Cd在四种组织中的蓄积量顺序为:肝脏>肾脏>鳃>肌肉。Cd抑制了褐牙鲆鳃组织中SOD,谷胱甘肽过氧化物酶（GPx）和GST的活性,降低了GSH的含量,导致鳃中MDA含量显著增加; Cd的暴露造成肝脏组织中GPx和GST的活性以及GSH含量下降,SOD活性增强,导致MDA含量上升;而肾脏组织中的GST和GPx活性显著提高, SOD活性显著降低,但是MDA含量并没有显著变化。这说明脂质过氧化作用因组织而异,其中过氧化损伤最严重的组织是鳃,其次是肝脏,而肾脏受到的损伤最轻。综上所述,本论文通过研究Cu、Cd对褐牙鲆不同生活阶段的生态毒理效应,阐明了重金属对褐牙鲆早期生活阶段生长和发育的毒理影响和致毒机理;探讨了重金属在褐牙鲆体内的积累规律;筛选氧化应激参数（包括抗氧化剂如SOD, CAT, GSH, GST, GPx和脂质过氧化水平）和胚胎-仔鱼发育过程中的敏感指标（如孵化率、畸形率和死亡率）作为生物指示物,初步构建了海洋重金属污染的生物监测体系,在生态风险评价中指示海洋中重金属的污染程度,并为海洋环境污染提供早期预警。更多还原

【Abstract】 Marine pollution is one of the major issues of global concern. Large quantities of heavy metals enter the aquatic environment via natural source and anthropogenic sources every year. Heavy metals at excessive amount in marine environments may adversely affect the growth, survival, and reproduction of marine fishes. Previous studies show that early life stages of fish are more sensitive to toxicity of heavy metals than adults, which may subsequently affect the recruitment and population wellness of the next cohorts. In addition, the accumulation of heavy metals in fish tissues could also seriously threaten the safety of fish products and thus consumer health. Therefore, the establishment of biological early warning systems for marine pollution is very important for the sustainable utilization of marine fishery resource and human health.Copper （Cu） and cadmium （Cd） are two common heavy metals in the Chinese coastal waters. This study investigated the toxic effects of Cu and Cd on flounder （Paralichthys olivaceus） in different early life stages （ELS: embryos, larvae, and juveniles）. The main results are as follows:（1） Acute toxicity tests: The 24- and 48-h LC₅₀ values of Cu for embryos were 0.21 and 0.11 mg/L, whereas the 48-, 72-, and 96-h LC₅₀ values for larvae were 0.46, 0.21, and 0.12 mg/L, respectively. The 24- and 48-h LC₅₀ values of Cd for embryos were 7.98 and 4.65 mg/L, whereas the 48-, 72-, and 96-h LC₅₀ values for larvae were13.64, 7.41, and 4.17 mg/L, respectively. These results suggest that Cu is much more toxic than Cd to the embryonic and larval survival; and embryos were more sensitive to these heavy metals than larvae.（2） Embryonic-larval toxicity tests: embryos were exposed, respectively, to different concentrations of Cu （0-0.12 mg/L） and Cd （0-2.4 mg/L） solutions from 3 to 130 hours post hatching （hpf） when larvae had opened mouth and were ready to initiate feeding. The results demonstrated that Cu and Cd had distinctly toxic effects on their embryonic-larval development and survival. Cu at≥0.06 mg/L and≥0.8 mg/L Cd concentrations caused low hatching success, delay in the time to hatching of embryos, reduction in yolk absorption rate of the larvae and high mortality and morphological malformations in the embryos and larvae; Neither Cu nor Cd significantly affect the heart rate of the embryos, but it significantly decreased the heart rate of the 130 hpf larvae when Cu concentration was≥0.10 mg/L and Cd concentration was≥1.2 mg/L; Total length of the larvae at the end of the tests was significantly reduced at≥0.10 mg/L Cu and≥1.2 mg/L Cd concentrations in comparison with those in the controls. The sensitivity sequence of toxicological endpoints for Cu was accumulative mortality > accumulative hatchability > morphological abnormality > growth, while that for Cd was: morphological abnormality > accumulative mortality > accumulative hatchability > growth.（3） Chronic toxicity tests: fish were exposed to waterborne Cu （0-32μg/L） and Cd （0-48μg/L） from embryonic to juvenile stages for 80 days, respectively. Activities of superoxide dismutase （SOD）, catalase （CAT）, glutathione S-transferase （GST）, and levels of reduced glutathione （GSH） and lipid peroxidation （LPO） were investigated at three developmental stages （metamorphosing larvae, settling larvae, and juvenile）. In metamorphosing larvae, Cu exposure caused decrease in GSH level and SOD, CAT, and GST activities, but increase in malondialchehyche （MDA） concentrations. Cd exposure caused decrease in SOD and CAT activities, but increase in the levels of GSH and MDA. Cu exposure did not have significant effects on setting larvae in any concentrations, but CAT and GST activities of settling larvae were inhibited and their GSH level was elevated at the high Cd concentrations. In juveniles, both Cu and Cd caused an increase in MDA concentrations. The level of GSH and the activities of SOD and GST were increased in high Cu treatments. However, SOD activity was increased but GST activity was decreased in high Cd treatments. At the end of the tests, flounder growth was reduced in both Cu and Cd treatments and the whole body metal accumulations were elevated with increasing concentrations.（4） Short-term subchronic toxicity tests with juveniles: following subchronic Cd （control, 2, 4, and 8 mg Cd/L） exposure for 28 days, fish growth was significantly reduced at≥4 mg/L Cd concentrations compared to the Cd-free controls. Accumulation of Cd in fish was dose-dependent and tissue-specific, with the greatest accumulation in the liver, followed by the kidney, gill, and muscle. Although the gill and liver mounted active antioxidant responses to cope with oxidative stress at≥4 mg/L Cd including a decrease in GSH level and GST and glutathione peroxidase （GPx） activities, the antioxidant response failed to prevent MDA production in these organs. In the kidney, increased GPx and GST activities and decreased SOD activity were observed in fish exposed to high Cd concentrations, but no significant differences were found in MDA levels for any of the exposure concentrations. The antioxidant defense system in the kidney may have effectively scavenged ROS and thereby succeed in preventing tissue-specific oxidative damages. The gill was most sensitive to oxidative damage, followed by the liver; the kidney was the least affected tissue.Overall, this present study investigated the toxic effects of heavy metals （Cu and Cd） on development, survival, growth, accumulation and antioxidative responses in different ELSs of flounder. Some sensitive endpoints such as biological parameters （i.e., hatchability, morphological abnormality, and mortality） and oxidative stress parameters （SOD, CAT, GSH, GST, GPx, and LPO） could be used as bioindicators of marine pollution in ecological risk assessment.更多还原