海洋生态与环境科学重点实验室知识库

双酚类物质（Bisphenols BPs）是一类具有两个酚基功能团的持久性有机污 染物，因其能够提高聚合物的透明度、耐磨耗性和硬度被广泛应用于人工合成 环氧树脂等聚合物工业原材料的生产当中。由于在生产过程中的大量使用以及 工业废水处理的不充分，使双酚类物质流入到了自然环境中。有研究表明，双 酚类物质在我国河口和近海海域分布广泛，且主要积累在沉积物中。黄渤海近 海海域是我国北方重要的水产养殖海域，双酚类物质的广泛分布可能会对该海 域的重要经济种群的生存生长造成不利影响，而目前现有的研究中缺少针对黄 渤海近海海域中双酚类物质的监测和评估。刺参（Apostichopus japonicus）是黄 渤海近海海域重要的海洋经济物种，同时是典型的海洋底栖动物，主要以海洋 沉积物中的动植物碎屑和藻类为食。因此，双酚类物质对近海海域养殖的刺参 具有潜在的不利影响，而目前针对双酚类物质对刺参的生态毒性及评估的研究 较少。基于上述原因，本研究针对黄渤海近海海域中双酚类物质的分布和浓度 水平开展调查，明确了黄渤海近海海域中主要的双酚类物质种类、分布特征和 浓度水平。同时以调查海域主要的双酚类物质作为胁迫因子，研究了双酚类物 质干扰刺参正常代谢生理过程背后的调控机制。此外，基于自然环境双酚类物 质背景调查结果，以环境相关浓度作为胁迫浓度值，研究了环境相关浓度下双 酚类物质对刺参生存生长的影响，并通过计算整合生物标志物指数，评估了当 前环境背景值下双酚类物质对刺参的毒性效应大小，为养殖水域环境质量检测 和评估提供数据支撑与理论依据。本研究取得的主要实验结果如下： （1）黄渤海近海典型养殖海域双酚类物质环境浓度背景调查 围绕黄渤海近海的典型养殖海域选取五个样点（大连、唐山、烟台、威海 和青岛）开展污染情况调查，结果显示，该海域主要存在的双酚类物质为六氟 双酚 A（Bisphenol AF BPAF）。双酚类物质浓度水平存在显著的地区间差异， 与水层的深度并未呈现明显的相关性，且双酚类物质主要富集在沉积物当中， 其中烟台沉积物样品中 BPAF 的浓度最高，检测浓度范围为 4.040~15.320 μg/kg。 （2）双酚类物质胁迫下刺参的代谢调控变化 通过对刺参肠道代谢通路进行分析，结果表明，双酚类物质胁迫会对刺参双酚类物质对刺参的代谢和生理毒性效应及风险评估 II 肠道组织的细胞膜造成破坏，KEGG 差异代谢通路富集分析结果显示刺参肠道 组织中的甘油磷脂代谢通路上调。此外，双酚类物质胁迫会干扰刺参肠道组织 中的碳水化合物代谢和氨基酸代谢。 （3）环境相关浓度下双酚类物质对刺参的生理响应特征评估 环境相关浓度双酚类物质胁迫 28 天后，刺参的生存生长并未受到显著的影 响。呼吸树是水体暴露于双酚类物质的重要接触部位，通过对刺参呼吸树进行 组织切片学观察，结果显示，经过双酚类物质胁迫后，刺参呼吸树上皮组织间 出现融合，呼吸树组织中央腔空间增大的现象。最后通过计算整合生物标志物 指数，结果显示双酚类物质混合胁迫对刺参的毒性效应大于单一双酚类物质的 胁迫，其中 BPAF 对刺参的毒性效应大于双酚 A（Bisphenol A BPA）。 综上所述，本研究结果表明，当前黄渤海近海典型养殖海域中主要的双酚 类物质为 BPAF，且其在海洋沉积物中的浓度显著高于海水中的浓度。双酚类 物质胁迫会导致刺参体内脂质代谢发生紊乱，其中双酚类物质会破坏组织细胞 膜，从而导致刺参体内甘油磷脂代谢上调。通过对当前环境相关浓度双酚类物 质对刺参的毒性效应进行评估，结果显示环境相关浓度下 BPAF 和 BPA 对刺参 的生存生长无显著的不利影响。

Bisphenols are persistent organic pollutants (POPs) with two phenolic functional groups, which are widely used in the production of raw materials for the polymer industry, such as synthetic epoxy resins, for their ability to improve polymer transparency, abrasion resistance and hardness. Due to the large amount of use in the production and inadequate treatment of industrial wastewater, bisphenols are discharged into the natural environment. There are studies have shown that bisphenols are widely distributed in China's estuaries and offshore waters, and are mainly accumulated in the sediments. The offshore area of Yellow and Bohai sea is an important aquaculture area in the north of China, and bisphenols may affect the survival and growth of economically important populations in this area. There is a lack of studies on investigating the toxicology and risk assessment of bisphenols in the coastal area of the Yellow and Bohai sea. Sea cucumber (Apostichopus japonicus) is an economically important marine species, and is also a typical marine benthic animal that feeds on plant and animal debris as well as algae in sediments. Therefore, bisphenol have potential adverse effects on the cultivation of sea cucumber in the coastal area of Yellow and Bohai sea, and there are limited studies on the ecotoxicity and assessment of bisphenols on sea cucumber. This study investigated the distribution and concentration of bisphenols in the coastal area of Yellow and Bohai sea, and unveiled the main bisphenol pollutant species, distribution characteristics and concentration in this area. The main bisphenol pollutants in the investigated sea area will be used as stress factors, and the sea cucumber was used as an experimental subject to study the regulatory mechanisms under the stress of bisphenols with the normal metabolic and physiological processes of the sea cucumber. In addition, based on the results of the field investigation, the effects of bisphenols on the survival and growth rate of the sea cucumber were investigated. The toxicology of bisphenol on sea cucumber under the current environmental relevant level were evaluated by calculating the integrated biomarker index, which provided data support and theoretical basis for the development of aquaculture envi-Toxicology and risk assessment of bisphenols on sea cucumber Apostichopus japonicus IV ronment quality standards and the monitoring of the environmental quality of sea cucumber culture. The main experimental results of this study are as follows: （1） Investigation of the occurrence of bisphenol along the coastal area of Yellow and Bohai sea Five sample sites (Dalian, Tangshan, Yantai, Weihai and Qingdao) were selected to investigate the occurrence of bisphenols, and the results showed that the main bisphenol in the seawater was Bisphenol AF （BPAF）. There were significant regional differences in the concentration levels of BPAF, with no obvious correlation with the depth of the water layer, and the BPAF were mainly enriched in the sediment, with the highest concentration of BPAF in the sediment samples from Yantai, ranging from 4.040 to 15.320 μg/kg. （2） Metabolic regulation mechanism of sea cucumber under the stress of bisphenol Through the analysis of the intestinal metabolic pathways of sea cucumber, the results showed that bisphenol contaminant stress would damage the cell membrane of sea cucumber intestinal tissues, and the results of differential metabolic pathway enrichment analysis showed that the glycerophospholipid metabolic pathway was upregulated in the sea cucumber intestinal tissues. In addition, bisphenol contaminant stress resulted in disruption of carbohydrate metabolism and amino acid metabolism in the intestine of the sea cucumber. （3） Evaluation of the toxicity of bisphenols on the survival and growth of sea cucumber at environmentally relevant concentrations Survival and growth of sea cucumber were not significantly affected after 28 days of stress with environmentally relevant concentrations of bisphenol pollutants. The respiratory tree is an important contact site of the water body exposed to bisphenol pollutants, and the histological sectioning of the respiratory tree of the sea cucumber showed that there was a fusion between the epithelial tissues of the respiratory tree of the sea cucumber and an increase in the space of the respiratory tree's inner lumen after the exposure of bisphenols. Finally, by calculating the integration biomarker index, the results showed that the toxic effect of the mixed bisphenol pollu-Abstract V tant stress on the sea cucumber was greater than that of the single bisphenol pollutant stress, in which the toxic effect of BPAF on the sea cucumber was greater than that of BPA. In summary, the results of this study showed that the main bisphenol contaminant in the offshore waters of the Yellow and Bohai Sea is BPAF, and its concentration in sediments is significantly higher than that in the seawater. Bisphenol can cause lipid metabolism disorders in sea cucumbers, in which the BPAF can damage tissue cell membranes, leading to the up-regulation of glycerophospholipid metabolism. The toxic effects of BPA and BPAF on the sea cucumbers were evaluated at the environmentally relevant concentrations, and the results showed that BPAF and BPA had no significant adverse effects on the survival and growth of the sea cucumbers at the current environmentally relevant level.

第1章       绪论... 1

1.1 研究背景... 1

1.1.1双酚类物质概述... 2

1.1.2. 双酚类物质的生态毒性... 7

1.2 研究内容及意义... 11

1.2.1 科学问题... 11

1.2.2 研究内容... 11

1.2.3 技术路线... 12

1.2.4 研究意义... 12

1.3 本章小结... 12

第2章       黄渤海近海典型养殖海域双酚类物质污染水平背景调查    13

2.1 引言... 13

2.2 材料与方法... 14

2.2.1 实验试剂与仪器... 14

2.2.2 样品采集与处理... 14

2.2.3 液相色谱质谱联用仪（LC-MS）检测... 15

2.3 调查结果... 16

2.4 讨论... 17

2.5 本章小结... 18

第3章       双酚类物质胁迫下刺参的代谢调控变化... 19

3.1 引言... 19

3.2 材料与方法... 20

3.2.1 实验试剂与仪器... 20

3.2.2 实验设计与样品采集... 21

3.2.3 刺参肠道代谢物组学分析... 22

3.2.4 刺参体腔液免疫酶活性测定... 22

3.2.5 数据分析方法... 24

3.3 实验结果... 24

3.3.1 刺参肠道差异代谢物分析... 24

3.3.2 刺参肠道差异代谢通路KEGG富集分析... 27

3.3.3 刺参体腔液免疫酶活性... 31

3.4 讨论... 32

3.4.1 双酚类物质对刺参肠道生理过程的影响... 32

3.4.2 双酚类物质导致刺参产生氧化应激损伤... 34

3.5 本章小结... 35

第4章       环境背景值下双酚类物质对刺参的生理响应特征评估    37

4.1 引言... 37

4.2 材料与方法... 38

4.2.1 实验试剂与仪器... 38

4.2.2 刺参的采集与暂养... 39

4.2.3 刺参生长率的测定... 40

4.2.4 刺参呼吸树组织切片观察... 41

4.2.5 刺参肠道免疫酶活性测定... 41

4.2.6 整合生物标志物指数（IBR）... 42

4.2.7 数据分析方法... 42

4.3 实验结果... 42

4.3.1 双酚类物质慢性胁迫对刺参生长的影响... 42

4.3.2 刺参呼吸树组织变化... 46

4.3.3 抗氧化功能相关酶活性变化... 46

4.3.4 IBR指数... 48

4.4 讨论... 49

4.4.1 双酚类物质对刺参生存生长的影响... 49

4.4.2 双酚类物质对刺参生理的影响... 49

4.4.3 双酚类物质对刺参呼吸树组织结构的影响... 50

4.4.4 基于IBR指数评估环境相关浓度双酚类物质对刺参的毒性效应大小... 51

4.5 本章小结... 52

第5章       研究总结与展望... 53

5.1 研究总结... 53

5.2 主要创新点... 54

5.3 存在问题... 54

5.4 研究展望... 54

参考文献        55

致 谢        67

作者简历及攻读学位期间发表的学术论文与其他相关学术成果    68