【作者】 王明华；

【导师】 王桂忠；

【作者基本信息】 厦门大学 ， 海洋生物学， 2007， 博士

【摘要】 重金属污染已成为一个全球性的环境问题,因此环境监测是修复和治理环境污染的一个必需手段,虽然常规的化学和物理等监测手段是环境监测必不可少的工具,然而由于该手段不能提供生物信息,继而不能满足当今环境监测的目标。由于可提供环境污染的生物信息,生物标志物(Biomarker)在现代化环境监测中占有非常重要地位,所以在环境监测中大规模应用生物标志物的问题有待于深入研究。本文选择桡足类为研究对象,进行重金属(镉或镍)对日本虎斑猛水蚤(Tigriopus japonicus)、双齿许水蚤(Schmackeria dubia)、中华哲水蚤(Calanussinicus)和婆罗异剑水蚤(Apocyclops borneoensis)急性毒性的测定;同时采用生化测定方法,研究为期12 d的不同浓度重金属(镉或镍)暴露对日本虎斑猛水蚤和双齿许水蚤体内多种生化指标{超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)、谷胱甘肽硫转移酶(GST)、乙酰胆碱酯酶(AChE)、还原型谷胱甘肽(GSH)、丙二醛(MDA)与还原型谷胱甘肽和氧化型谷胱甘肽的比值(GSH/GSSG)}的影响,以探讨镉或镍对桡足类的毒害效应以及动物的应激机制,同时筛选出重金属污染的生物标志物;本文也进行重金属(镉或镍)不同暴露时间(1、4、7和12 d)对日本虎斑猛水蚤体内金属硫蛋白(MT)水平的影响,以探讨该种桡足类体内MT在重金属胁迫下的诱导模式和应激机制;本文还选择重金属暴露12 d后的双齿许水蚤为研究对象,研究不同浓度重金属对该种桡足类肌肉组织和卵巢超微结构的影响,以探讨重金属对动物的毒性效应。同时,本文定量分析桡足类(日本虎斑猛水蚤、双齿许水蚤和婆罗异剑水蚤)生殖力在重金属胁迫下发生的变化,阐述重金属污染下桡足类种群变化的应对策略。最后,采用放射性同位素示踪法,研究不同氮水平和氮源对东海原甲藻(Prorocentrum donghaiense)吸收镍与该金属在藻类亚细胞和各生化组分中分布的影响,即探讨环境营养盐水平和来源对藻类吸收镍的影响机制;同时也研究氮或磷添加对镍在东海原甲藻和中肋骨条藻(Skeletonema costatum)胞内吸收和累积的影响;并将其分别投喂中华哲水蚤和真刺唇角水蚤(Labidocera euchaeta)后,探讨镍在桡足类体内的吸收和生理周转,即探讨不同营养盐浓度对镍在海洋食物链上传递的影响。主要研究结果如下:1.镉对桡足类的24和48 h半致死浓度均小于镍对应的半致死浓度,因此桡足类对镉的敏感性大于镍,即镉更具有毒性;在镍对各种桡足类24和48 h半致死浓度的比较中,日本虎斑猛水蚤最大,中华哲水蚤和双齿许水蚤最小(两者相差不大),而在镉对各种桡足类24和48 h半致死浓度的比较中,婆罗异剑水蚤最大,日本虎斑猛水蚤次之,中华哲水蚤最小,因此重金属对桡足类的毒性与金属类别和生物种类均相关。2.不同浓度的镉或镍均引起桡足类体内各种生化指标(SOD、GPx、GST、GSH和GSH/GSSG)的显著变化,该类指标的变化与重金属暴露剂量和时间均相关,因此提示该系列指标可作为重金属污染的生物标志物;特别是GSH.在12 d的重金属暴露过程中,GSH总是最先受到影响的,而且基本上处于抑制状态,因此该指标有望成为桡足类体内指示重金属污染的良好标志物。同时,镉和镍对桡足类均有神经毒性效应,因为不同浓度重金属均诱导桡足类体内AChE活性的显著变化,但重金属更多是通过AChE活性的提高而呈神经毒性。经过12 d的镉或镍暴露后,桡足类体内MDA含量更多地与该金属处理无相关性,因此该指标作为生物体氧化损伤程度的标志物时,需要慎重,应该辅以其它生物标志物才能更全面地反应生物体的氧化损伤水平。3.在12 d的暴露过程中,镉或镍既可诱导日本虎斑猛水蚤MT的合成,也可表现为抑制效应,即MT的变化与暴露剂量和时间均有相关性。本研究通过双齿许水蚤肌肉组织和卵巢超微结构的观察表明,在12 d的暴露过程中,镉和镍已对该种桡足类造成氧化损伤,因为肌纤维受到氧化损伤的迫害,同时生殖细胞发生凋亡。4.不同浓度镉或镍均可影响桡足类的生殖力,而且其影响与金属类别和生物种类均相关,特别是不同浓度镉或镍均显著抑制日本虎斑猛水蚤的生殖力,说明重金属可影响日本虎斑猛水蚤的种群繁衍。5.通过同位素示踪法研究表明,东海原甲藻培养在高浓度和低浓度镍下,不同氮水平和氮源均可显著影响其对镍的吸收,该吸收随着硝酸氮的升高而增加,同时以尿素为氮源时可显著促进藻类对该金属的吸收。当东海原甲藻吸收镍后,不同氮水平和氮源均可影响镍在藻类亚细胞中的分布,如细胞壁和可溶性物质中镍的百分含量随着硝酸氮浓度的升高而降低,细胞器中镍的百分含量随着硝酸氮浓度的升高而增加,而且尿素可提高镍在细胞壁中的分配率:同时,绝大部分镍(＞70%)分布在细胞的可溶性物质中。不同氮水平和氮源也可影响镍在藻类各生化组分中的分布,如碳水化合物中镍的含量随着硝酸氮浓度的升高而显著降低,蛋白质中镍的含量却随着硝酸氮浓度的升高而显著增加,尿素可显著提高镍在蛋白质中的分布然却降低该金属在碳水化合物中的百分含量,脂类中镍的含量与不同氮水平和氮源均无显著相关;镍主要分布在蛋白质中(＞50%)。6.经过24 h暴露后,氮或磷均显著影响东海原甲藻和中肋骨条藻对镍的吸收,且高浓度氮或磷均能促进镍在这两种藻类细胞内的吸收和累积。中华哲水蚤和真刺唇角水蚤对镍的吸收随着藻类培养液中营养盐(氮或磷)浓度的增加而显著升高;然而镍在中华哲水蚤或真刺唇角水蚤体内的生理周转率却不受氮或磷浓度的影响,似乎更受动物个体的生理状态的影响,如摄食不同磷浓度培养下的硅藻镍后,食物镍在中华哲水蚤体内的生理周转率均是真刺唇角水蚤的2倍左右。桡足类对镍的吸收率与藻类细胞内镍含量呈显著正相关,证明了“桡足类只吸收饵料细胞内‘水相’金属库中的金属”的假说。由此可见,近海富营养化可促进浮游植物对镍的吸收,进而影响该金属在浮游食物链上的传递。更多还原

【Abstract】 Heavy metal pollution is a global environmental problem. Thus, environmental monitoring is essential to restore and resolve environmental pollution. Although chemical and physical methods are indispensable to environmental monitoring, they can not provide biological information, hence never satisfying the goal of modern environmental-monitoring. Due to being capable of exhibiting biological information, biomarker playes a very important role in modern environmental-monitoring. However, it is a further challenge to put it into the practice of monitoring cosmically. In the present study, using copepod as the object, we examined the acute toxicity of Cd or Ni to four species of copepods （Tigriopus japonicus, Schmackeria dubia, Calanus sinicus and Apocyclops borneoensis）. Meanwhile, the response of the copepods （T. japonicus and S. dubia） to Cd or Ni additions was investigated under laboratory-controlled conditions in the 12 d exposure at the biochemical level （SOD, GPx, GST, AChE, GSH, MDA and GSH/GSSG）, so as to discuss metal toxicity and the associated response of copepod, along with sieving out suitable biomarkers to metal pollution. We also investigated the response of MT levels in the copepod T. japonicus to different exposure duration （1, 4, 7 and 12 d） under different metal treatments （Cd or Ni）, in order to expatiate the induction mode and mechanism of MT in the copepod under metal stress. Using S. dubia as the object, which had experienced the metal （Cd or Ni） exposure for 12 d, we also studied the influence of different metal treatments on the ultrastructure of muscle and ovary in the copepod, so as to reflect the metal toxicity. Additionally, the influence of metal additions on the reproductive ability of the copepods （T. japonicus, S. dubia and A. borneoensis） was quantified, in order to expound the response of copepod population to metal stress. Finally, We examined the influence of different nitrogen levels and sources on Ni uptake by Prorocentrum donghaiense and its distribution in cellular substructure and biochemical components of algae using iotope approach, to discuss the possible effect mechanism of the ambient nutrient level and source to Ni uptake by algae. Meanwhile, we also investigated the effect of macronutrient （nitrate or phosphate） additions to Ni uptake by phytoplankton （P. donghaiense and Skeletonema costatum） and its subsequent transfer to marine copepods （C. sinicus and Labidocera euchaeta）, i.e., to discuss the influence of different nutrient levels on the transfer of Ni along the planktonic food-chain. The results of the present sturdy are described as follows:1. By contrast to Ni, the 24 and 48 h-LC₅₀ values of Cd to the copepods are smaller, hence the copepods showing more susceptivity to Cd than Ni. In the comparison concerning the 24 and 48 h-LC₅₀ values of Ni to different copepods, T. japonicus displayed the biggest value, and C. sinicus showed little difference from S. dubia, both of which showing a smaller value. However, in the Cd comparison, A. borneoensis held the biggest value, T. japonicus stayed at the second position, and C. sinicus had the smallest value. Therefore, the metal toxicity to copepod was correlated with metal classes and organism species.2. Different metal （Cd or Ni） treatments significantly influenced the variant biochemical indexes （SOD, GPx, GST, GSH, MDA and GSH/GSSG）, with the change of these indexes showing a relation with metal concentration and duration, implying that these biochemical parameters could be biomarkers of metal pollution, especially for GSH. Regardless of different metal treatments and durations, GSH always firstly responsed, and almost suffered the inhibition-effect, suggesting that this parameter could be a good biomarker in copepod to metal pollution. Meanwhile, both Cd and Ni exhibited the neurotoxicity to copepod, because different metal additions markedly influenced the copepod AChE activity. However, the metals showed their neurotoxcity, more by enhancing the activity of AChE in copepod. Nevertheless, after 12 d exposure of Cd or Ni, at more extent, MDA level in copepods are little correlated with metal treatments. Therefore, it is cautious to choose this parameter as biomarker of oxidative injury, and other biomarkers should be involved, so as to reflect the extent of oxidative injury more comprehensively.3. During the 12 d exposure, heavy metal （Cd or Ni） not only induced the synthesis of MT in the copepod T. japonicus, but also displayed the inhibition-effect, i.e., the change of MT levels in the copepod was correlated with metal concentration and duration. According to the ultrastructure in the copepod S. dubia, heavy metal （Cd and Ni） had already brought the oxidative injury to the animal, due to the muscle being oxidatively injuried and the germ cell suffering from apoptosis.4. Different metal additions influenced the reproductive ability of the copepods, and the effect was associated with metal classes and animal species. Especially, different Cd or Ni significantly inhibited the reproductive capability of T. japonicus, illuminating that the metal effect had reflected at the population level of this copepod.5. Under both high- or low-Ni treatments, different nitrogen levels and sources both significantly influenced Ni uptake by P. donghaiense, with its uptake increasing with an increase of nitrate concentration and being enhanced markedly using urea as the nitrogen source. After being uptaken by P. donghaiens, Ni distribution in cellular substructure was also affected by different nitrogen levels and sources, e.g., Ni content in cell wall and soluble substance decreased, but the content in organelle increased, with an increase of nitrate level, and furthermore urea significantly facilitated the metal distribution in cell wall. Meanwhile, most of Ni distributed in soluble substance （> 70 %）. Similarly, the nitrogen concentration and source also affected the Ni distribution in different biochemical components. Ni content in carbohydrate decreased, but the protein-Ni level increased, with an increase of nitrate level. Additionally, urea markedly promoted Ni distribution in proteins, but decreased its content in carbohydrate. However, the lipid-Ni content showed little correlation with different nitrogen levels and sources. Additionally, Ni mainly distributed in proteins （> 50 %）.6. Ni uptake by phytoplankton after 24 h of exposure was markedly dependent on nutrient conditions, with a higher nutrient quota facilitating Ni accumulation in the algae. Trophic transfer was quantified by measurements of the Ni assimilation efficiency in C. sinicus and L. euchaeta, feeding on the algae under different nutrient treatments. Ni assimilation efficiency generally increased with an increase of nutrient concentration in the algae. However, ambient nutritional conditions had little effect on the physiological turnover rate constant of Ni by copepods, which was more affected by the physiological status of copepod. For example, feeding on the diatom-Ni under P treatments, in comparison to L. euchaeta, the physiological turnover rate constant of Ni in C. sinicus increased approximately 2 times. A significant positive correlation was found between the Ni assimilation efficiencies of the copepods and the % intracellular Ni in the algal cells, testifying the hypothesis "copepod only assimilates metal in the ’cytoplasmic’ pool. Thus, nutrient-enrichment may lead to an increase in Ni uptake and transfer in the marine plankton.更多还原