【作者】 朱丽岩；

【导师】 董双林；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2005， 博士

【摘要】 鉴于目前内分泌干扰物(EDCs)对海洋环境的危害以及海洋动物尤其是无脊椎动物在海洋生态系统中的重要性,本文以有机锡——三丁基氧化锡(TBTO)、多环芳烃——蒽、有机磷农药——久效磷和甲基对硫磷等作为内分泌干扰物,以青岛近海常见海洋动物——瓣鳃类软体动物菲律宾蛤仔(Ruditapes philippinarum)、螠虫类动物单环棘螠(Urechis unicinctus)、甲壳类动物海洋伪镖水蚤(Pseudodiaptomus marinus)、中华哲水蚤(Calanus sinicus)、美丽猛水蚤Nitocra sp.、脊尾白虾(Exopalaemon carinicauda)和日本(虫寻)(Charybdis japonica)以及海洋鱼类牙鲆(Paralichthys olivaceus)鳃的永久性体外培养细胞系FG-9307等——为实验对象,在个体、组织器官、细胞以及分子等多个层次上研究了海洋环境中内分泌干扰物对海洋动物活体和体外毒性效应,为建立快速、有效、实用的内分泌干扰物的活体和离体生物监控和测试技术提供了基础资料,同时也为研究环境激素对海洋生态系统的影响进行了有益的尝试。研究工作包括以下几个方面: 1.通过菲律宾蛤仔-水环境的三丁基氧化锡(TBTO)暴露模拟体系,研究菲律宾蛤仔的三种器官——消化腺(肝脏)、鳃和外套膜在不同TBTO(浓度:76.55ngSn/L)暴露时间下的富集作用、组织和细胞的毒性效应。还研究了TBTO作用下血细胞数量和结构、酚氧化酶产量和活性的改变和繁殖发育所受的影响。 选用气相色谱与电子俘获检测器(GC-ECD)联用的方法对菲律宾蛤仔体内TBTO含量进行了测定。结果显示,该方法快速、简便且定量准确;菲律宾蛤仔体内的TBTO含量随着时间的延长不断积累,富集能力存在器官上的差异,由强到弱依次为消化腺>鳃>外套膜。暴露5周时消化腺的富集因子(BCF)为3275,说明菲律宾蛤仔是一种富集能力强且较为敏感的实验无脊椎动物。 TBTO对蛤仔各器官超微结构的破坏随时间延长而加剧。长期暴露于低浓度TBTO的蛤仔消化腺细胞中最敏感的细胞器是内质网,其次为线粒体;与消化腺细胞不同,鳃细胞受损细胞器主要为细胞核,其次为内质网及线粒体;外套膜各细胞器的变化与鳃类似。实验结果表明,蛤仔体内器官尤其是消化腺中细胞及组更多还原

【Abstract】 The threat of endocrine disrupting chemicals (EDCs) to the marine environment are becoming more serious and marine animals especially invertebrates play the important role in marine ecosystem. In this paper, several kinds of usual marine animals in the offing of Qingdao, lamellibranch mollusc Ruditapes philippinarum, echiuran Urechis unicinctus, crustacean Pseudodiaptomus marinus, Calanus sinicus, Nitocra sp., Exopalaemon carinicauda, Charybdis japonica, as well as FG-9307 cell line derived from the gill of marine fishes Paralichthys olivaceus are used to investigate the effects of EDCs such as tributyltin oxide (TBTO), anthracene, monocrotophos and methylparathion. The toxic effects of EDCs in marine environment on marine animals in vivo and in vitro are studied on individual, organ, cell and molecule levels, and offer data for the establish of a fast, efficient and practical biological monitoring and testing technology in vivo and in vitro.1. The bioconcentration function of three organs in Ruditapes philippinarum, digestive gland (liver), gill, and mantle exposed to 76.55ngSn/L TBTO for different periods and the toxic effect in their organizes and cells are investigated. Gas Chromatography-Electron Capture Detection (GC-ECD) is used as detection and quantification method for TBTO in clam’s organs. The results show that GC-ECD is a fast, convenient and accuracy method. TBTO accumulates in clam’s tissues with the extension of exposure time, and the bioconcentration abilities are different in various organs. The order of bioconcentration abilities of various organs are digestive gland > gill>mantle. Bioconcentration factor (BCFs) of digestive gland after 5 weeks exposure is 3275, and it indicates that Ruditapes philippinarum is a kind of sensitive experimental invertebrate with better bioconcentration ability.Damages of TBTO on the structure of Ruditapes philippinarum increase with exposure time. In hepatocyte, the most sensitive organelle to TBTO of clam under long-term exposure is endoplasmic reticulum, and sub-sensitive one is mitochondria; the main damaged organelle in gill cells is nucleus, and then the endoplasmic reticulum and mitochondria; there are similar effects of TBTO on the mantle and gill of the subject. The results show that the changes of cells and organize’s structure in organs in vivo especially in digestive gland can reflect the period and degree of the lowdose TBTO pollution.The activity of digestive diverticula’s acid phosphatase, alkaline phosphatase and cytochrome oxidase decreased with the prolonging of exposure to TBTO, and can be judged from the fade of tissues, but there are few changes on principal active positions.After 48h exposure of Ruditapes philippinarum to TBTO, the amount ofhemocytes is reduced by 40.3%, of which the amounts of small granule cells reduced, large granule cells and clear cells are reduced by 46.46%, 30.77%, 1.08% respectively; the production of phenoloxidase is reduced by 34.48%, and the general enzymatic activity of phenoloxidase is reduced by 47.1%, and the unit enzymatic activity is reduced by 23.6%.The fertility rate of Ruditapes philippinarum adults exposed to 200ng/L TBTO for about 20 days is lower, the activity of sperms is affected, and the hatching rate of oosperms is 60%-70%; the development and metamorphosis of larvae are significant different between the treated group and the control group. The mortality of larvae in the treated group is 80.5% after the 16 days exposure.2. Effects of monocrotophos, anthracene and TBTO on the fertilization and the early development of Urechis unicinctus, as well as effects of monocrotophos on the activities of various enzyme are preliminary studied. Results show that the fertiligy rate of the sperms and ovums of Urechis unicinctus treated by 1.5 nl/L TBTO is significantly declined, and the effect of the treatment on sperms on fertiligy rate is more significant than which of the treatment on ovums. The ultrastructure of the membranaceous is significantly changed after exposed the sperms to TBTO. Each concentration of monocrotophos, anthracene and TBTO can all affect the fertilization and the early development of Urechis unicinctu, and the effects are more significant with the increase of the concentration. In addition, the values of 24h LC50 and 48h LC50 of monocrotophos, anthracene and TBTO to Urechis unicinctus trochophore and somite larvae are measured.Monocrotophos can seriously inhibit acetylcholinesterase(AChE), the activity of AChE in Urechis unicinctus exposed to very low concentration of monocrotophos is significantly depressed; the colored depth and sties of Cytochrome C oxidase in Urechis unicinctus exposed to low concentration of monocrotophos are increased; no significant effects of low concentration of monocrotophos (^ 150ng/L) on alkaline phosphatase in Urechis unicinctus are observed.3. Effects of TBTO and anthracene on survival, reproduction, paedomorphosis and the activity of phenoloxidase of crustaceans such as copepods are investigated.The 48hLC50 and 96hLC50 values of Pseudodiaptomus marinus are 1.23p.g/L and 0.57ng/L, and the metamorphosis rates of the first and second generation exposed to each concentration of TBTO are reduced. Sex ratio ($/c?) of the second generation exposed to 20ng/L are reduced. The body length exposed to each concentration of TBTO.Survival, egg production and the percentage of gravid females are all reduced at 60ng/L TBTO.The 72hLC50 of TBTO to Calanus sinicus is 1.26ug/L, and the egg production rates of animals exposed to 20ng/L and 200ng/LTBTO for 96h are reduced markedly.The filtration fate and grazing rate of copepod Nitocra sp. are reduced along with the elevation of concentration of TBTO. There is significant difference between 2ng/LTBTO exposed group and the control group (P<0.05), and the filtration fate and grazing rate of copepods are significantly reduced (P<0.01) when exposed to 2jag/LTBTO.After 48h exposure of Charybdis japonica to TBTO, the amount of hemocytes is reduced by 34.9%, of which the amounts of small granule cells reduced, large granule cells and clear cells are reduced by 40.1%, 4.8% and 31.3%, respectively. Compared TBTO treated group with the control group, the heterochromatin of nucleus in clear cells are more concentrate, it is seriously vacuolization in cytoplasm, and the amounts of free ribosomes and mitochondrions are relatively reduced. The heterochromatin of nucleus in small granule cells are increased; there are large vacuoles in cytoplasm, and the amount of mitochondrions are significantly reduced, the volumes of mitochondrions and the amount of cristae are both reduced; the amount of free ribosomes is significantly reduced. After 48h exposure of Charybdis japonica to TBTO, the production of phenol oxidase is reduced by 42.68%, and the general enzymatic activity of phenol oxidase is reduced by 62.7%, and the unit enzymatic activity is reduced by 35.1%.The 72h LC50 of anthracene to Exopalaemon carinicauda is 13. 30ug/L. The development of Exopalaemon carinicauda larvae exposed to anthracene at low concentration is affected, and with the increase of the concentration, the time of metamorphosis in each stage is prolonged, and the metamorphosis rates are reduced.4. Cytotoxic effects of methylparathion and TBTO on FG-9307 cell line derived from the gill of flounder Paralichthys olivaceus in vitro are measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay, cell protein assay, and transmission electron microscopic examination of cytoarchitacture. FG-9307 is more sensitive to methylparathion, and results of NR^ MTT and cell protein assay indicate that cytoactive is affected at 5ug/L, and the toxic effects of methylparathion is dose-dependent. The ultrastructures of methylparathion exposed cells are markedly altered, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear fragmentation, and increased levels of lysosomes and bleb.Results indicate that the dose of TBTO at O.lug/L could affect the cytoactive of this cell line with the three test systems, and the ultrastructures are markedly altered, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear membrane dissolution, and increased level of lysosomes. This renders FG cells one of several choices for rapidly evaluating the acute toxicities of methylparathion and TBTO.更多还原