广东东部沿海麻痹性贝类毒素成分特征分析

为了弄清广东东部沿海贝类中麻痹性贝类毒素(Paralytic shellfish poisoning,PSP)的主要成分及其藻类来源,运用液相色谱—柱后衍生荧光检测技术,对2005-2006年间在粤东近岸重点养殖区采集的主要经济贝类进行了麻痹性贝类毒素成分分析。分析结果表明,在染毒的贝类样品中共检出PSP的12种成分,各成分的检出率时空分布特征有所不同,其中N-磺酸氨甲酰基类毒素C1和氨基甲酸酯类毒素GTX1的检出率最高,均达78%以上;其次为GTX4,达71%。在毒素含量方面,C1普遍较低,春季含量较其他季节高,而在春秋两季贝类中,GTX1和GTX4含量普遍较高。总体上,麻痹性贝毒在粤东海域的分布具有明显的地域性、季节性和贝类种间差异。地域上以柘林湾污染较为严重,毒素成分最多,且含量较高;季节上春季为染毒高峰期,秋季次之;主要染毒贝类为牡蛎(Crassostrea spp.)、结蚶(Tegillarca nodifera)、泥蚶(Tegillarca granosa)和蚶(Scapharca spp.),柘林湾2005年9月14日的结蚶毒性最高,其毒性当量值达到370μgSTX/100g。与其他贝类相比,蚶类对高毒力的GTX毒素有更强的积累能力。     

天津市售主要经济贝类中麻痹性贝类毒素污染现状及特征分析

使用液相色谱串联质谱法(LC-MS/MS)检测9种天津市售主要经济贝类中13种麻痹性贝类毒素(paralytic shellfish poisoning,PSP)的含量,分析PSP的污染现状和成分特征。结果显示,共计6个品种(紫贻贝、虾夷扇贝、牡蛎、毛蚶、脉红螺、菲律宾蛤仔)检出PSP。其中虾夷扇贝、紫贻贝、毛蚶3个品种的检出率较高且含量较高,检出率分别为66.7%、58.3%、58.3%。PSP中检出率较高的毒素包括N-磺酰胺甲酰基类毒素C1、C2和膝沟藻毒素GTX1、GTX2、GTX3,毒力占比主要以膝沟藻毒素GTX1-4为主,毛蚶蓄积STX毒素的能力强于其他贝类。全年的PSP含量季节变化为夏季最高,冬季最低。PSP主要存在于贝类的消化腺内,全年不同月份的PSP含量均在安全食用范围内。     

渤海海域唐山贝类养殖区腹泻性和麻痹性贝类毒素的监测与风险评估

为监测渤海海域唐山贝类养殖区贝类毒素的污染情况，防止食用贝类中毒事件发生，于2019年10月—2020年9月间，每月持续在渤海海域唐山贝类养殖区采集四角蛤(Mactra veneriformis)、菲律宾蛤仔(Ruditapes philippinarum)、脉红螺(Rapana venosa)、牡蛎(Crassostrea gigas)、青蛤(Cyclina sinensis)、文蛤(Meretrix meretrix)和硬壳蛤(Mercenaria mercenaria) 7种经济贝类样品，采用高效液相色谱–串联质谱(HPLC-MS/MS)法测试了5种腹泻性贝类毒素(diarrhetic shellfish poisoning, DSP)和14种麻痹性贝类毒素(paralytic shellfish poisoning, PSP)。结果显示，在7种经济贝类样品中均未检出DSP。检出的PSP成分包括石房蛤毒素(Saxitoxin, STX)、膝沟藻毒素1 (Gonyautoxin 1, GTX 1)、膝沟藻毒素2 (Gonyautoxin 2, GTX 2)和脱氨甲酰基膝沟藻毒素3 (Ddecarbamoy l gonyautoxin 3, dcGTX 3)，其中，GTX 1含量最高且最高值为537.95 μg/kg。不同季节贝类毒素蓄积含量有一定差异，PSP主要集中在4月检出。菲律宾蛤仔、牡蛎、文蛤和硬壳蛤中PSP的检出率分别为11.76%、47.06%、5.90%和8.82%，其他贝类均未检出。PSP总量均低于欧盟及中国的食用安全限量标准800 μg STXeq/kg。应用风险熵值法和点评估法进行食用安全风险评估，显示风险熵值和暴露风险指数均在安全范围内，结果表明，渤海海域唐山贝类养殖区7种经济贝类不存在食用安全风险。     

近海水域贝类麻痹性毒素的研究进展

麻痹性贝类毒素的研究已成为国内外专家研究的热点。水科院南海水产研究所对近海水域中贝类麻痹性毒素进行检测研究。经过实际检测表明: 1、珠江口海域四种贝类PSP为未检出(<1.75-9.68Mu/g内);大鹏湾海域10种贝类PSP含量未检出;大亚湾海域13种贝类PSP含量为未检出; 2、大亚湾海域的华贵栉孔扇贝和翡翠贻贝     

珠江口及邻近海域贝类麻痹性毒素调查

于1990－1999年对珠江口、大鹏湾和大亚湾3个海域14种主要经济贝类进行麻痹性毒素（PSP）的毒性水平检测并比较其季节性变化和种间差异,采用麻痹性毒素小白鼠生物测定法测定PSP毒性水平,并以联合国粮农组织推荐的贝类安全食用标准进行分析评价。结果表明,珠江口海域的僧牡蛎轻微染毒（PSP）,大鹏湾海域的华贵栉孔扇贝和翡翠贻贝不同程度染毒,大亚湾海域的华贵栉孔扇贝,翡翠贻贝,马氏珠母贝棕带仙女蛤具潜在染毒危险。     

对天津市售贝类麻痹性贝类毒素的调查研究

对2011年2月至2012年1月天津市售双壳经济贝类麻痹性贝毒(PSP)污染状况进行为期一年的抽样,调查其食用安全性。分别采用小白鼠生物法和高效液相色谱法(HPLC)测定毒素含量及分析毒素组成。在所抽查的10种,103个贝类样品中,有3种16个样品呈阳性,虾夷扇贝全年均检测出PSP阳性,其中6-8月毒力超过食用标准,毛蚶在5、6、7月结果呈阳性,波纹巴非蛤在6月中有染毒现象,毒力范围分布在49⁵30Mu/100g之间。对阳性样品进行HPLC检测出6种麻痹性贝毒成分,其中低毒力的C1、C2为毒素主要成分,高毒力的STX、GTX1检出率低,中毒力的GTX2、GTX3均有检出。麻痹性贝毒在天津市场范围内检出率及含量整体水平不高,但应提示有关部门应在春季预防,在夏季加强对食用贝类的监测和宣传,保证市民健康安全。     

麻痹性贝类毒素GTX1&4和GTX2&3标准样品的制备技术

贝类毒素标准样品的制备是保障贝类质量安全的基础,本研究以有毒贻贝(Mytilus edulis)为原料,探究麻痹性贝类毒素标准样品的制备技术。以1%乙酸溶液提取贻贝中的麻痹性贝类毒素,采用羟丙基葡聚糖凝胶柱(Sephardex LH20)净化后收集目标馏分,再经高效液相色谱以TSK-GEL Hillic Amide-80色谱柱(4.6 mm×250.0 mm,5.0μm)分离、纯化,根据麻痹性贝类毒素的保留时间收集色谱峰流出液,连续制备合并馏分,40℃减压蒸馏浓缩近干,用75%乙腈水(含0.25%甲酸)溶解,即制得毒素标准样品溶液。分别采用四极杆/离子阱复合质谱与标准物质的多级质谱信息进行匹配,以高分辨质谱获得的精确质量数进行定性确证,样品溶液所含的麻痹性贝类毒素为4种膝沟藻毒素,分别为膝沟藻毒素-1-4 (Gonyautxins-1-4, GTX14)和膝沟藻毒素-2-3(Gonyautxins-2-3, GTX23)。均匀性实验中,经F检验表明在95%的置信区间范围内样品均匀性良好。稳定性考察实验中,标准样品在4℃和-18℃两种环境条件下储存6个月稳定性较好。标准样品经8家实验室联合定值,并评定了定值结果的不确定度。本研究制得的麻痹性贝类毒素标准样品为GTX 1(3 663μg/L)GTX4(1 147μg/L)和GTX 2(1 884μg/L)GTX3(797μg/L)的混合溶液,溶剂为75%乙腈水(含0.25%甲酸),为国内贝类毒素标准样品研发提供了技术支撑。[中国渔业质量与标准,2019,9(2):62-70]     

小鼠生物法检测麻痹性贝类毒素技术探讨及应用

为了确保贝类毒素监测结果的有效性和可信性,建立和规范快速准确的麻痹性贝类毒素(PSP)检测方法,本研究梳理了小鼠生物法(MBA)快速准确检测PSP的实验室规范要求、影响因素及操作要点。并运用MBA法,对浙江沿岸海域养殖区采集的贝类样品进行3个批次的PSP应急监测分析,结果表明,赤潮高发对贝类富集PSP有很大影响。此外,结合对浙江沿岸养殖区PSP的现状分析,提出了养殖贝类质量安全相关监管意见。     

麻痹性贝毒和腹泻性贝毒的性质及检测技术研究探讨

鱼、贝等海洋生物通过对赤潮毒素的富集作用产生贝毒,人误食了这些含有赤潮藻毒素的鱼类和贝类后则有可能中毒甚至死亡,麻痹性毒素(PSP)和腹泻性毒素(DSP)是其中重要的两种赤潮毒素,他们毒性大、作用强,严重威胁着人类以及海洋生物的生命安全。本文详细阐述了这两种贝毒的化学性质、致毒机理以及毒性,并对当前常用的几种检测技术进行了对比说明。     

10种麻痹性贝类毒素的固相萃取及液相色谱 串联质谱测定法

建立了10种麻痹性贝类毒素(PSP)的高效液相色谱-串联质谱检测方法。样品用0.5%甲酸溶液加热提取,提取液经乙酸乙酯、三氯甲烷和HLB固相萃取柱净化,乙腈去蛋白后,在正离子模式下以电喷雾串联质谱仪进行测定,TSK改性凝胶柱为色谱分离柱,5 mmol·L-1甲酸铵(含0.5%甲酸)-乙腈(含0.5%甲酸)为流动相,采用基质匹配的标准曲线进行定量,并以虾夷扇贝(Patinopecten yessoensis)和菲律宾蛤仔(Ruditapes philippinarum)为测试对象,对方法的有效性进行了评价。评价结果为:在50~600μg·kg-1添加水平下,10种麻痹性贝类毒素平均回收率为72.3%~91.1%(批内),相对标准偏差为3.9%~9.5%(批内)。STX、dc STX和neo STX定量限(S/N10)为100μg·kg-1,GTX1~GTX5、dc GTX2和dc GTX3定量限为50.0μg·kg-1。结果表明,该方法快速、准确,适合于贝类产品中麻痹性贝类毒素的测定。     

扇贝中麻痹性毒素的提取与分离纯化

为了获得麻痹性毒素标准品,以毒化扇贝为试验材料,对扇贝中的麻痹性毒素(PSP毒素)进行了提取和分离纯化。以酸性80%乙醇溶液反复提取麻痹性毒素,得到了总毒性为6170MU的毒素粗提液。毒素粗提液经过超滤、Bio-GelP-2凝胶柱层析和Bio-Rex70离子交换柱层析等二次层析柱分离纯化后得到以膝沟藻毒素群(GTXs)为主的纯化PSP毒素。HPLC分析结果显示,扇贝中主要含有膝沟藻毒素GTX3,GTX1、GTX2、GTX3、和GTX4的组成比例约为2∶4∶14∶1(以HPLC上的峰面积比例计)。纯化后的毒素可以作为标准物质用于GTX1-4的HPLC分析。     

Kinetics of diarrhetic shellfish poisoning toxins,okadaic acid,dinophysistoxin-1, pectenotoxin-6 and yessotoxin in scallops <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>

ABSTRACT:   Four toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-6 (PTX6), and yessotoxin (YTX), all associated with diarrhetic shellfish poisoning (DSP), were administered via syringe to Scallops Patinopecten yessoensis and their distribution in the hepatopancreas, adductor muscle, and combined other tissues (mantle, gill, gonad) was analyzed by liquid chromatography-mass spectrometry. Toxins exclusively remained in the hepatopancreas irrespective of the injection site, adductor muscle or hepatopancreas. When injected into hepatopancreas, OA, DTX1, and YTX were metabolized to 7- O -palmitoylOA, 7- O -palmitoylDTX1 and 45-hydroxyyessotoxin (45OH-YTX), respectively. Such metabolic changes were insignificant when toxins were injected into the adductor muscle. The residual ratio for each toxin in the hepatopancreas was less than 20%. Mortalities of scallops treated with PTX6 were lower than those treated with other toxins.     

Quantification of lipophilic toxins associated with diarrhetic shellfish poisoning in Japanese bivalves by liquid chromatography-mass spectrometry and comparison with mouse bioassay

ABSTRACT:   Quantification of lipophilic toxins in bivalves associated with diarrhetic shellfish poisoning was investigated by liquid chromatography–mass spectrometry (LC–MS). Using a C8-silica reversed phase column and a mobile phase of aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, okadaic acid, dinophysistoxin-1, 7- O -palmitoyldinophysistoxin-1, pectenotoxin-1, pectenotoxin-2, pectenotoxin-6, pectenotoxin-2 seco-acid, yessotoxin, and 45-hydroxyyessotoxin in bivalves were quantified by LC–MS in the negative mode. When the crude 90% methanol extracts were analyzed by LC–MS, there were no significant effects from bivalve matrices on the quantification of toxins. More than 200 bivalve samples collected from various production areas in Japan were analyzed by LC–MS. Pectenotoxin-6 and dinophysistoxin-1 were the dominant toxins in scallops and mussels, respectively. Yessotoxin and 45-hydroxyyessotoxin were also detected in both species. Comparison of the quantitative results obtained for these bivalve samples between LC–MS and mouse bioassay indicates that LC–MS is suitable for routine monitoring of lipophilic toxins in Japanese bivalves.     

Difference in the level of paralytic shellfish poisoning toxin accumulation between the crabs <Emphasis Type="Italic">Telmessus acutidens</Emphasis> and <Emphasis Type="Italic">Charybdis japonica</Emphasis> collected in Onahama,Fukushima Prefecture

ABSTRACT:     The difference in paralytic shellfish poisoning (PSP) toxin accumulation in the crabs Telmessus acutidens and Charybdis japonica was investigated at Onahama, Fukushima Prefecture, from 2002 to 2005. The level of toxin accumulation in the hepatopancreas of T. acutidens corresponded to that of mussels when examined on a yearly basis. In 2003, some crabs had a high toxicity of approximately 1000 MU, which compares to one-third of the human minimum lethal dose. Therefore T. acutidens should be monitored as a vector species of PSP toxin. The toxin profile of T. acutidens was also investigated. Because an increase in highly toxic species of PSP toxins with a decrease in low toxic species, such as N -sulfocarbamoyl-11-hydroxysulfate toxins, was not clearly observed between consecutive samples, toxin transformation in T. acutidens was considered to have a minimal impact on toxicity. PSP toxins were also detected in several specimens of C. japonica , but the highest toxicity was only 7.4 MU/g in the hepatopancreas. Because C. japonica is widely distributed in the coastal waters of Japan, investigation of toxin accumulation in the crab should be continued, including its investigation in different areas of water from Onahama, where the causative dinoflagellates of PSP occur in different conditions.     

Paralytic shellfish poison as an attractant for toxic snails

The attracting effect of paralytic shell poison (PSP) on eight toxic snail species (Polinices didyma, Natica lineata, N. vitellus, Zeuxis sufflatus, Niotha clathrata, Oliva miniacea, O. mustelina, O. hirasei) and two non-toxic species (Pomacea canaliculata, Satsuma bairdi) was investigated. Eight toxic species were reported to contain tetrodotoxin, and three out of these contained minor PSP. The minimum lethal dose of PSP in most toxic gastropods was estimated to be more than 150 MU PSP/20 g body weight, but in non-toxic gastropods was less than 15 MU PSP/20 g body weight. After the attracting test, all toxic gastropods showed significant positive linear relationship (Y=3.716X+0.363, r=0.8427) between comparative attracting variation and the toxicity reported. The relationship between PSP resistance ability and the toxicity also had a positive correlation (Y=0.91X+210.090, r=0.6803). However, the non-toxic species showed a negative response. This indicates that PSP is an attractant for toxic snails.     

麻痹性贝类毒素在栉孔扇贝体内的代谢轮廓

将栉孔扇贝(Chlamys farreri)直接暴露于产毒藻,通过比较各组织器官中毒素的蓄积及代谢转化特征,研究麻痹性贝类毒素(Paralytic Shellfish Toxins,PSTs)在栉孔扇贝体内危害形成的过程。结果显示,分布于中国的一株塔玛亚历山大藻(Alexandrium tamarense,AT5-3株),其生长及产毒性状稳定,产毒能力强,主要成分为Gonyautoxins-1-4(GTX14),单细胞产毒能力为7.95 fmol/cell;栉孔扇贝对该产毒藻具有较强摄食能力及PSTs蓄积能力,总体蓄积效率达到84.4%,最高蓄积浓度为1903μg STX eq./kg,不同组织蓄积能力由高到低依次为内脏团、性腺、外套膜、闭壳肌;内脏团对PSTs的代谢能力也最强,是该毒素在栉孔扇贝体内蓄积代谢的靶器官;此外,栉孔扇贝对PSTs表现出较强的生物转化能力,主要转化途径为:N-磺酰胺甲酰基类毒素(N-Sulfocarbamoylgonyautoxin-2,C1)→膝沟藻毒素(Gonyautxins-2,GTX2)/脱氨甲酰基类毒素(Decarbamoylgonyautoxins-2,dc GTX2),N-磺酰胺甲酰基类毒素(N-Sulfocarbamoylgonyautoxin-3,C2)→膝沟藻毒素(Gonyautxins-3,GTX3)。本研究中,栉孔扇贝对PSTs总体呈现出迅速蓄积和缓慢代谢的特点,同时,GTX14和NEO等高毒成分所占比例较高,造成扇贝中PSTs毒性和风险性均显著升高。本研究有助于科学评估PSTs危害的形成机制,为系统监控我国PSTs风险性提供科学支撑。     

冷冻时间对贝类大肠杆菌、细菌总数结果的影响

研究探讨了贝类以缢蛏为代表在室温16℃、湿度50%～70%条件下,不同冷冻时间对贝类大肠杆菌、细菌总数的影响。结果表明,随着存放时间延长,贝类菌落总数呈下降趋势,变化范围分别在1.0×102～1.0×103 CFU/g之间。而大肠杆菌数基本小于30MPN/100g,低于农业部无公害贝类标准。存储8d以后,大肠杆菌数量无明显变化,而细菌总数在6d以后呈下降趋势。可见贝类在-11～-18℃的温度下,大肠杆菌数存储8d无明显变化,细菌总数存储6d无明显变化。     

Paralytic shellfish poisoning toxin analysis of the genus <Emphasis Type="Italic">Alexandrium</Emphasis> (Dinophyceae) occurring in Korean coastal waters

ABSTRACT:   Paralytic shellfish poisoning (PSP) toxins produced by Alexandrium isolates from Korea were analyzed by high-pressure liquid chromography. Species designation of the regional isolates was determined by morphological criteria and ribotyping inferred from sequences of the 28S rDNA D1-D2 region. Toxin analysis performed at the exponential growth phase, revealed that the two strains of A. fraterculus were non-toxic, while the strains of A. tamarense and A. catenella were toxic. Toxic isolates DPC7 and DPC8 of A. catenella produced GTX1, 2, 3, 4, 5, dcGTX2, 3, C1, 2, neoSTX and STX with trace or non-detectable levels of C3 and C4, while isolates UL7, KDW981, SJW97043, SJW97046, KJC97111 and KJC97112 of A. tamarense produced GTX1, 2, 3, 4, dcGTX3, C1, 2, neoSTX with trace or non-detectable levels of C3, 4, dcSTX and STX, and no GTX5 and dcGTX2. The major toxins produced by A. catenella were C1 +2, and those of A. tamarense were C1 +2 and GTX4 in most of the isolates. A. tamarense strains other than SJW97046 produced a relatively high proportion of carbamate toxins, reflecting the high toxicity scores of shellfish intoxication in sampled coastal areas. Two representative toxic isolates, A. tamarense SJW97043 and A. catenella DPC7, were cultured for 30 days in batch mode and subjected to toxin analysis at 5-day intervals. Comparison of toxin productivity in terms of total toxin content, toxin components, and their variations with culture age revealed marked differences between the two strains.