我国近海藻毒素污染状况研究与毒素标准物质制备

	我国近海藻毒素污染状况研究与毒素标准物质制备
	柳阳
学位类型	博士
导师	于仁成研究员 ; 周名江研究员
	2017-05-25
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生态学
关键词	藻毒素浮游植物贝类高效液相色谱液-质联用标准物质
摘要	海洋中的一些微藻可以产生藻毒素，根据藻毒素极性的差异，可以将常见藻毒素分为水溶性藻毒素（如麻痹性贝类毒素等）和脂溶性藻毒素（如腹泻性贝类毒素等）两大类。这些毒素能够在滤食性海洋动物体内累积，给海产品食品安全带来潜在风险。以往调查结果表明，我国沿海养殖贝类沾染藻毒素的问题比较严重，多次发生食用染毒海产品导致的中毒事件。因此，亟待对我国近海藻毒素污染状况开展系统调查，加强对藻毒素的监测和研究工作。但我国目前尚无藻毒素标准物质，在一定程度上制约了相关监测和研究工作的开展。对此，本论文计划通过对我国近海重要海域藻毒素的分析，揭示藻毒素污染状况，同时推进藻毒素标准物质的研制和申报工作。针对我国近海藻毒素污染状况，本论文应用高效液相色谱-荧光检测（HPLC-FLD）和高效液相色谱-串联质谱（LC-MS/MS）等设备，分别建立了针对麻痹性贝类毒素和几类常见脂溶性藻毒素的分析检测方法。于2013年12月、2014年6月、9月和11月在渤海周边典型贝类养殖海域（烟台、莱州、汉沽、秦皇岛和葫芦岛）开展了四次调查，于2014年5-6月、9-10月和2015年8月在黄、东海海域开展了三次大面调查，于2015年2-9月在长江口及其邻近海域连续开展了多次重点调查，采集了浮游植物样品和贝类样品，并应用构建的毒素分析方法对样品进行了分析。此外，在实验室内开展了虾夷扇贝（Patinopecten yesoensis）暴露于有毒亚历山大藻（Alexandrium pacificum）的染毒实验。研究发现，我国近海各调查海域浮游植物样品中普遍存在麻痹性贝类毒素，其中基本以低毒性的N-磺酰氨甲酰基类毒素（N-sulfocarbamoyl-gonyautoxin2&3, C1&2）为主，但在渤海秦皇岛和莱山附近海域的浮游植物样品中，毒素组成却以高毒性的膝沟藻毒素（gonyautoxin1&4, GTX1&4）和脱氨甲酰基类毒素（decarbamoylgonyautoxins2&3, dcGTX2&3和decarbamoylsaxitoxin, dcSTX等）为主。基于麻痹性贝类毒素的组成特征，推断黄、东海海域（包括长江口及其邻近海域）麻痹性贝类毒素主要来源于塔玛亚历山大藻复合种（Alexandrium tamarense species complex），而渤海可能存在链状裸甲藻（Gymnodinium catenatum）等其它产麻痹性贝类毒素的有毒藻种。从毒素含量来看，长江口及其邻近海域的麻痹性贝类毒素含量（0-215 nmol m^-3）明显高于黄、东海其它海域（0-156 nmol m^-3），而渤海海域麻痹性贝类毒素含量相对较低（0-3.35 nmol m^-3）。对渤海周边养殖海域贝类中麻痹性贝类毒素的分析结果显示，部分贝类样品存在麻痹性贝类毒素超标（80 μg STX eq 100g^-1）现象，超标率为12%；贝类中麻痹性贝类毒素组成以高毒性的GTX1&4、GTX2&3（gonyautoxin 2&3）、NEO（neosaxitoxin）和STX（saxitoxin）为主，明显有别于浮游植物样品。室内扇贝染毒实验结果表明，当暴露于有毒亚历山大藻后，扇贝能够快速累积麻痹性贝类毒素，毒性水平在5-8天内超过食用安全标准，且毒素组成与有毒藻类存在差别，在扇贝各组织中肾脏是进行毒素转化的主要器官。调查表明，我国近海浮游植物样品中普遍存在脂溶性藻毒素，其中以PTX2（pectenotoxin2）、OA（okadaic acid）、DTX1（dinophysis toxin 1）、YTX（yessotoxin）和homo-YTX等毒素较为常见，而desMeC（13-desmethyl spirolide C）、GYM（gymnodimine）和AZA（azaspiracid）毒素也偶有检出。部分藻毒素可检出较多的同系物，如PTX2的同系物PTX11和PTX1, YTX的同系物trinor-YTX、45-OH-YTX、45-OH-homo-YTX、COOH-45-OH-YTX等。浮游植物样品中脂溶性藻毒素含量一般在6-9月份最高。在渤海周边养殖海域，贝类样品中也能够检测到脂溶性藻毒素，如OA、DTX1、PTX2、YTX、homo-YTX、AZA2、AZA3、GYM和desMec等。与其它贝类相比，扇贝、毛蚶和紫贻贝等更易沾染脂溶性藻毒素，贝类中脂溶性藻毒素含量同样在6-9月份较高，但远低于食用安全标准，因此渤海海域脂溶性藻毒素导致中毒事件的风险不大。通过对我国近海浮游植物和贝类样品中藻毒素的分析可以看出，麻痹性贝类毒素对贝类等海产品食品安全的影响远比脂溶性藻毒素严重，而N-磺酰氨甲酰基类毒素（C1&2）和膝沟藻类毒素（GTX1-4）是最为常见的麻痹性贝类毒素成分。对此，本研究针对C1&2、GTX2&3和GTX1&4等麻痹性贝类毒素开展了标准物质研制和申报工作。采用多柱多级分离方法，从室内培养的有毒亚历山大藻中分离纯化了C1&2、GTX1&4和GTX2&3等三组毒素样品，开展了毒素样品的均匀性和稳定性实验，并对毒素样品进行了定值和不确定度分析，向国家标准物质管理办公室申报了标准物质。总之，基于对我国近海的渤海、黄海和东海海域藻毒素的系统调查和分析，本论文基本查清了我国近海不同海域藻毒素的组成特征和污染状况，为针对性地开展藻毒素监测和研究工作提供了重要依据。在此基础上，开展了三组麻痹性贝类毒素样品的均匀性和稳定性研究，以及毒素样品的定值和不确定度分析，推进了毒素标准物质的申报工作，为藻毒素标准物质研制和毒素的监测、研究工作奠定了基础。
其他摘要	Some microalgae in the sea can produce phycotoxins, which can be divided into hydrophilic marine toxins and lipophilic algal toxins based on their polarity. Phycotoxins can be accumulated in filter-feeding marine animals, and pose potent threats to seafood consumers. Previous studies have shown that contamination of phycotoxins in shellfish is a serious issue along the coast of China, and many poisoning incidents caused by contaminated seafood were recorded. Therefore, it is urgent to carry out systematic investigation to figure out the contamination status of phycotoxins and to strengthen the monitoring and research efforts on phycotoxins. However, there are no ceritified reference materials of phycotoxins yet in China, which restricts the relevant monitoring and research work on phycotoxins. Therefore, this study aims to reveal the contamination status via analyzing phycotoxins in phytoplankton and shellfish samples collected from coastal waters of China, and promote the preparation and declaration of toxin reference materials. In order to reveal the contamination status of phycotoxins, methods of high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) were established to analyze paralytic shellfish toxins (PSTs) and lipophilic algal toxins, respectively. Besides, samples of phytoplankton and shellfish were collected during four surveys in representative mariculture zones (including Yantai, Laizhou, Hangu, Qinhuangdao and Huludao) around the Bohai Sea from December 2013 to November 2014, three surveys in the Yellow Sea and the East China from May 2014 to August 2015, and surveys in the Changjiang River estuary and its adjacent waters from February to September 2015. Phycotoxins in theses samples were analyzed with the established methods. Moreover, an experiment of PST accumulation and transformation in scallops Patinopecten yesoensis was conducted in the present study. Based on the analytical results, PSTs were commonly found in phytoplankton samples collected from coastal waters of China. PSTs in most of the phytoplankton samples were dominated by low-potency N-sulfonylcarbamyl toxins (C1&2). But in the phytoplankton samples from Qinhuangdao and Laishan, PSTs were dominated by highly potency carbamate toxins (GTX1−4) and decarbamoly toxins (dcGTX2&3 and dcSTX), respectively. Based on toxin profiles of PSTs, it is deduced that PSTs in the Yellow Sea and the East China Sea are mainly produced by Alexandrium tamarense species complex, while Gymnodinium catenatum and other toxic species may be responsible for PSTs in the Bohai Sea. Toxin content of PSTs in the Changjiang River estuary and its adjacent waters (0−215 nmol m^-3) was much higher than that in the Yellow Sea and East China Sea (0−156 nmol m^-3), while that in the Bohai Sea is the lowest (0−3.35 nmol m^-3). For the shellfish samples collected from the Bohai Sea, PST toxicity in 12% of the samples exceeded the food safety standard (80 µgSTXeq 100g^-1). The composition of PSTs in shellfish was dominated by high potency gonyautoxin 1&4 (GTX1&4), gonyautoxin 2&3 (GTX2&3), neosaxitoxin (NEO) and saxitoxin (STX), significantly different from toxin profiles of phytoplankton. According to the simulation experiment, scallops exposed to toxic Alexandrium could quickly accumulate PSTs, and the toxicity level exceeded the regulatory limit for PSTs within 5 days. Toxin composition of scallops was different from toxic algae, and the kidney was the major organ for toxin transformation in scallops. Lipophilic algal toxins were commonly found in phytoplankton samples from the coastal waters in China. Pectenotoxin2 (PTX2), okadaic acid (OA), dinophysis toxin 1 (DTX1), yessotoxin (YTX) and homo-YTX were major toxin components, and 13-desmethyl spirolide C (desMec), gymnodimine (GYM) and azaspiracids (AZAs) were occasionally detected. For some groups of phycotoxins, homologues of toxins could be detected in phytoplankton samples, such as PTX11 and PTX1 of PTX2, and trinor-YTX, 45-OH-YTX, 45-OH-homo-YTX, COOH-45-OH-YTX of YTX. The highgest content of lipophilic algal toxins in phytoplankton generally occurred during the period from June to September. In shellfish samples collected from the aquculture zones of the Bohai Sea, OA, DTX1, PTX2, YTX, homo-YTX, AZA2, AZA3, GYM and desMec were detected. Among the different species of shellfish, scallops, clams and mussels were more likely to accumulated lipophilic algal toxins. The content of lipophilic algal toxins in shellfish was also the highest from June to September. However, toxin content is much lower than the food safety standards, and the risk of seafood poisoning caused by lipophilic algal toxins is low. Based on the investigations, PST contamination in coastal waters of China is the most serious. Therefore, C1&2, GTX2&3 and GTX1&4 were chosed to prepare reference materials. C1&2, GTX1&4, GTX2&3 were extracted from cultured cells of Alexandrium, and purified with multiple columns. The purified toxins were assessed for homogeneity and stability, and determined for their values and uncertainties. The purified toxins were then declared for reference material to National Administrative Committee for CRM’s of China. In conclusion, based on the systematic investigations of phycotoxins, this study figures out the contamination status of phycotoxins in coastal waters of the Bohai Sea, Yellow Sea and East China Sea. Moreover, some PSTs were purified from toxic algae and then examined for homogeneity and stability. The purified toxins were determined for their values and uncertainties, and declared for reference materials. The results of the current study will promote the declaration of toxin reference materials, and lay a sound foundation for monitoring and research of phycotoxins in China.
学科领域	海洋调查与监测
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136647
专题	海洋生态与环境科学重点实验室
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
推荐引用方式 GB/T 7714	柳阳. 我国近海藻毒素污染状况研究与毒素标准物质制备[D]. 北京. 中国科学院大学,2017.

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