Accumulation of paralytic shellfish poison (PSP) and biotransformation of its components in oysters, Crassostrea gigas, fed with the toxic dinoflagellate Alexandrium tamarense

As a part of our studies on the mechanism of uptake of paralytic shellfish poison (PSP) and the kinetics of its accumulation in bivalves, oysters Crassostrea gigas were experimentally contaminated with PSP by being fed with the toxic dinoflagellate Alexandrium tamarense for 2, 4, 6, 8 and 10 days. Temporal variations in the PSP contents and their profiles in oysters during the feeding experiment were monitored by high-performance liquid chromatography (HPLC) and the toxin profile of the oysters was compared with that of A. tamarense. Toxins excreted from the infested oysters into the seawater for 2 and 10 days were recovered and analyzed by HPLC. PSP toxicity rapidly appeared in the tissues of oysters and their toxicity levels reached 0.6 (0.3), 2.2 (1.1), 1.0 (0.5), 3.4 (1.6) and 1.1 (0.5) MU/g (nmol/g) shucked meat at 2, 4, 6, 8 and 10 days, respectively. The accumulation rates of toxin, calculated from the total amount (nmol) of toxins expressed by the total cell number fed during the exposure period and the toxicity of the oysters, were 14.1, 18.7, 5.1, 14.9 and 3.2% for 2, 4, 6, 8 and 10 days. During feeding experiments, the toxin profile of oysters changed substantially, showing marked differences from the proportions found in the toxigenic dinoflagellate used as food. The toxin components in this strain existed almost exclusively as beta-epimers, which accounted for 66.3 mol% of the total. This contrasts with the case of the oysters, where the beta-epimers represented 24.8, 29.8, 25.1, 27.3 and 25.2 mol% of the total at 2, 4, 6, 8 and 10 days, respectively. The amount of gonyautoxin-1 (GTX1) accumulated in oysters increased linearly and slowly for 8 days and the maximum content of GTX1 reached 51.3 mol%. The composition of GTX group compounds recovered from the seawater in which the oysters had been reared was a little different from that within the oyster tissues.     

Occurrence of paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamarense in Hiroshima Bay, Hiroshima Prefecture, Japan, during 1993-2004 and its PSP profiles

To assess levels of shellfish intoxication by the paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamarense, potential health risks to human shellfish consumers and the possible need for regulatory intervention, yearly variations of maximum cell density of this species were examined from 1993 to 2004 in Kure Bay and Kaita Bay, which are located within Hiroshima Bay, Hiroshima Prefecture, Japan. The seawater temperature was determined concomitantly. In Kure Bay, maximum concentrations of 1,400 and 1,300 cells/mL at 0 and 5 m depths were observed on 21 and 24 April 1997. In Kaita Bay, remarkably high concentrations above 1,000 cells/mL of A. tamarense were observed in two out of three years investigated. These facts suggest that the environment in both bays is favorable for the propagation of A. tamarense. The temperature range at which the natural population of A. tamarense blooms was generally from 12 to 16 degrees C. Four strains (ATKR-94, -95, -97 and -01) from Kure Bay and one strain (ATKT-97) from Kaita Bay were established. The strain ATKR-94, cultured in modified SW-2 medium at 15 degrees C for 15 days, showed a specific toxicity of 33.8 x 10(-6) MU/cell. The toxins in all five strains exist almost exclusively as beta-epimers (C2 (PX2 or GTX8), GTX3, dcGTX3 and GTX4), which accounted for 54.9 to 73.0 mol% of the total. The corresponding a-epimers (C1 (PX1 or epi-GTX8), GTX2, dcGTX2 and GTX1) accounted for 6.0 to 28.9 mol%. The toxin profiles of ATKR-97 and ATKT-97 were characterized by unusually high proportions of low-potency sulfocarbamoyl toxin, which comprised 62.4 and 68.2 mol%, respectively, of total toxins. In the toxic bivalves, the low-toxicity sulfocarbamoyl components, major components of A. tamarense, were present in amounts of only a few percent, suggesting that in vivo conversion of PSP occurs after ingestion. A comparison of the toxin profiles of the causative dinoflagellate and contaminated bivalves showed that PSP components exist in the bivalves in the form of alpha-epimers, presumably owing to accumulation or storage of the toxins.     

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.     

Accumulation and elimination profiles of paralytic shellfish poison in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Gymnodinium catenatum

The paralytic shellfish poison (PSP)-producing dinoflagellate Gymnodinium catenatum (Gc) was fed to the short-necked clam Tapes japonica, and the accumulation, transformation and elimination profiles of PSP were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested most of the Gc cells (4 x 10(6) cells) supplied as a bolus at the beginning of the experiment, and accumulated a maximal amount of toxin (181 nmol/10 clams) after 12 hr. The rate of toxin accumulation at that time was 16%, which rapidly decreased thereafter. During the rearing period, a variation in toxin composition, derived presumably from the transformation of toxin analogues in the clams, was observed, including a reversal of the ratio of C2 to C1, and the appearance of carbamate (gonyautoxin (GTX) 2, 3) and decarbamoyl (dc) derivatives (decarbamoylsaxitoxin (dcSTX) and dcGTX2, 3), which were undetectable in Gc cells. The total amount of toxin contained in clams and residue (remaining Gc cells and/or excrement in the rearing tank) gradually declined, and only about 1% of the supplied toxin was detected at the end of the experiment.     

Analysis of paralytic shellfish toxins and their metabolites in shellfish from the North Yellow Sea of China

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.     

Analysis of paralytic shellfish toxins and their metabolites in shellfish from the North Yellow Sea of China

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.     

2018—2020年河北省市售贝类中麻痹性贝类毒素污染状况调查分析

目的了解2018—2020年河北省市售贝类中麻痹性贝类毒素(paralytic shellfish poison,PSP)污染状况。方法 2018年8月—2020年5月间,对河北省市售的7种双壳贝类,共508份进行检测分析。样品经0.5%乙酸水提取,石墨化碳黑固相萃取柱净化,采用高效液相色谱-串联质谱法进行检测。结果 508份样品,PSP阳性样品24份,检出率为4.7%, 15份样品超过世界卫生组织规定安全限量,超标率为3.0%。检出贝类为贻贝、毛蚶、杂色蛤、扇贝, PSP含量范围分别为217.0~13001.8μg石房蛤毒素当量(saxitoxin equivalent, STXeq/kg)、217.0~4893.2μg STXeq/kg、217.0~503.6μg STXeq/kg、217.0~11024.5μg STXeq/kg;超标贝类为贻贝、毛蚶、扇贝。贝类中检出的PSP类型有GTX1、GTX4、GTX2、GTX3、neoSTX、STX。结论河北省市售贝类麻痹性贝类毒素暴露风险整体较低,秦皇岛地区贻贝等贝类产品在4、5月份较易受到PSP污染,应持续关注,加强早期监测预警。     

石房蛤毒素免疫亲和柱制备及柱效测试研究

目的阐明石房蛤毒素(saxitoxin, STX)免疫亲和柱对11种麻痹性贝类毒素的亲和作用。方法通过纯化的STX单克隆抗体与琼脂糖凝胶(sepharose 4B)制备STX免疫亲和柱,采用11种麻痹性贝类毒素(paralytic shellfish poisoning,PSP)标液进行过柱实验,优化上柱条件,采用液相色谱-质谱串联法进行毒素检测。结果STX免疫亲和柱对新石房蛤毒素(neosaxitoxin,neo-STX)、脱氨甲酰基新石房蛤毒素(decarbamoylneosaxitoxin dihydrochloride,dcneo-STX)、膝沟藻毒素1(gonyautoxin-1,GTX1)、膝沟藻毒素4(gonyautoxin-4, GTX4)基本没有亲和力作用;而对7种PSP的亲和力强弱顺序为:STXN-磺酰氨甲酰基类毒素5(gonyautoxin-5, GTX5)脱氨甲酰基石房蛤毒素(decarbamoylsaxitoxin dihydrochloride, dcSTX)膝沟藻毒素3(gonyautoxin-3,GTX3)脱氨甲酰基膝沟藻毒素3(decarbamoylgonyautoxin-3,dcGTX3)脱氨甲酰基膝沟藻毒素2(decarbamoylgonyautoxin-2, dcGTX2)膝沟藻毒素2(gonyautoxin-2, GTX2),其中对STX、GTX5、dcSTX、GTX3的回收率为61.2%～99.0%。结论该STX免疫亲和柱对STX、GTX5、dcSTX、GTX3有较好的吸附效果,能够满足样品检测前处理的要求,为水产品中麻痹性贝类毒素的提取方法研究提供了新技术的参考依据。     

Development and application of an enzyme immunoassay based on a monoclonal antibody against gonyautoxin components of paralytic shellfish poisoning toxins

With a gonyautoxin 2/3 (GTX2/3)-specific monoclonal antibody (designated GT-13A) and a saxitoxin-horseradish peroxidase conjugate (STX-HRP), a direct competitive enzyme immunoassay (GTX-EIA) was established and its sensitivity to various toxin components was investigated. The concentrations resulting in 50% inhibition of the binding of STX-HRP to the solid-phase GT-13A antibody for GTX2/3, decarbamoyl-GTX2/3 (dc-GTX2/3), N-sulfocarbamoyl-GTX2/3 (C1/2), GTX1/4, STX, and neosaxitoxin (neoSTX) in GTX-EIA were found to be 0.28, 0.41, 0.52, 3.46, 4.06, and 89.37 ng/ml, respectively. When the minimum detection limit was assumed to be at a toxin concentration causing 30% inhibition of the binding of STX-HRP to the solid-phase GT-13A antibody, the detection limits for GTX2/3, dc-GTX2/3, C1/2, GTX1/4, STX, and neoSTX were found to be 0.15, 0.18, 0.19, 1.09, 1.50, and 22.93 ng/ml, respectively. These results indicate that all of the GTX components examined and STX are detectable at concentrations lower than the regulatory limit of 80 microg/100 g of shellfish tissue, even when a minimum dilution factor of 100 is applied to tissue extracts with the extraction procedure of the Association of Official Analytical Chemists. Therefore, GTX-EIA is thought to be a useful qualitative screening method for GTX components and STX in the mass monitoring of toxin-contaminated shellfish.     

A new analytical method for gonyautoxins based on postcolumn HPLC

A new ion-pairing high-performance liquid chromatography (HPLC) method on a C30 column with a volatile mobile phase was developed to separate the gonyautoxin group (GTXs) from contaminants, allowing the utilization of liquid chromatography/mass spectrometry (LC/MS) with higher performance. A mobile phase consisting of 5 mmol/L heptafluorobutyric acid and 2% acetonitrile in 10 mmol/L ammonium acetate was adopted for separation of GTXs because the C30 column strongly retains GTXs under acidic conditions. The newly adopted method could efficiently separate GTXs from contaminants, especially in the toxic short-necked clam, whereas the routine HPLC so far used has poor resolution to separate GTXs from unknown interfering substances. In our method, GTXs were eluted in the order of GTX5, GTX3, GTX4, GTX2 and GTX1 from the C30 column, and were successfully determined by sonic spray ionization mass spectrometry (SSI-MS) with high sensitivity. This method is characterized by the combination of HPLC using a fluorescence detection system for PSP, and SSI-MS for measurement of the mass number.     

上海市售贝类产品中麻痹性贝类毒素污染状况调查及其评价

目的 调查上海市售贝类产品中麻痹性贝类毒素污染状况。方法 2010年8月~2011年7月间, 在上海水产品批发市场进行5种贝类样品采集, 每月抽取样品24份, 全年共288份。采用生物法(SC/T 3023-2004)对其进行了麻痹性贝类毒素的检测, 其中虾夷扇贝的肠腺和肌肉(扇贝柱)进行分开测定。结果 缢蛏、菲律宾蛤仔、牡蛎、文蛤、虾夷扇贝肠腺和肌肉中麻痹性贝类毒素的含量范围分别为ND~121.5 MU/100 g、ND~113.4 MU/100 g、ND~177.7 MU/100 g、ND~124.6 MU/100 g、261.7~3363.5 MU/100 g和ND。全年麻痹性贝类毒素的平均含量分别在98.5±10.5 MU/100 g、78.6±9.3 MU/100 g、50.4±10.1 MU/100 g、40.6±14.8 MU/100 g、1242.2±974.3 MU/100 g和0。按照目前我国贝类产品主要出口国家和国际组织对麻痹性贝类毒素的限量要求进行评价, 仅仅是虾夷扇贝肠腺中麻痹性贝类毒素超标, 超标率为98%, 因此在食用扇贝时应去除其肠腺; 而其余贝类产品中麻痹性贝类毒素均在限量规定范围内。结论 上海市售贝类产品对食用的安全性不产生负面影响。     

Contamination of shellfish from Shanghai seafood markets with paralytic shellfish poisoning and diarrhetic shellfish poisoning toxins determined by mouse bioassay and HPLC

This paper reports the results of investigations of shellfish toxin contamination of products obtained from Shanghai seafood markets. From May to October 2003, 66 samples were collected from several major seafood markets. Paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) toxins in shellfish samples were monitored primarily by a mouse bioassay, then analysed by HPLC for the chemical contents of the toxins. According to the mouse bioassay, eight samples were detected to be contaminated by PSP toxins and seven samples were contaminated by DSP toxins. Subsequent HPLC analysis indicated that the concentrations of the PSP toxins ranged from 0.2 to 1.9 µg/100 g tissues and the main components were gonyautoxins 2/3 (GTX2/3). As for DSP, okadaic acid was detected in three samples, and its concentration ranged from 3.2 to 17.5 µg/100 g tissues. Beside okadaic acid, its analogues, dinophysistoxins (DTX1), were found in one sample. According to the results, gastropod (Neverita didyma) and scallop (Argopecten irradians) were more likely contaminated with PSP and DSP toxins, and most of the contaminated samples were collected from Tongchuan and Fuxi markets. In addition, the contaminated samples were always found in May, June and July. Therefore, consumers should be cautious about eating the potential toxic shellfish during this specific period.     

Thermal Degradation of Partially Purified Paralytic Shellfish Poison Toxins at Different Times, Temperatures, and pH

ABSTRACT: Mixtures of purified and partially purified paralytic shellfish poisoning (PSP) toxins including C1/2 and B1 toxins, gonyautoxins 1-4 (GTX), neosaxitoxin (NEO), and saxitoxin (STX) were heated at different temperatures (90 to 130 °C), heating times (10 to 120 min), and pH (3 to 7) and analyzed by HPLC. C toxins declined rapidly at low pH, and GTX 1/4 toxins decreased at high temperatures and at high pH. GTX 2/3 increased initially at low pH and then declined with subsequent heating, whereas STX increased consistently at pH 3 to 4. The integrated total specific toxicity declined at high pH (6 to 7). The kinetics of thermal destruction were 1^st order, and the efficacy of thermal destruction was highly dependent on pH, with rapid thermal destruction of carbamate compounds at higher pH. D values of carbamate toxins decreased with increasing temperature at high pH. Heating at low pH resulted in conversion of least toxic compounds to highly toxic compounds.     

舟山海域麻痹性贝类毒素污染情况及其2种检测方法比较

目的检测舟山东极与嵊泗枸杞2个海域养殖贻贝中的麻痹性贝类毒素(paralytic shellfish poison,PSP),比较小鼠生物测定法与酶联免疫分析法(ELISA)的测定结果。方法采用小鼠生物测定法与酶联免疫吸附法检测贝类中的麻痹性贝类毒素,并将2者的检测结果进行比较分析。结果 2种检测方法检测的麻痹性贝类毒素含量结果基本一致。5月份东极岛海域的厚壳贻贝中检出PSP((500±3.2)MU/100 g),超标率为5%;嵊泗枸杞海域贝类PSP含量较低,未超出安全食用标准。2个海域的紫贻贝PSP含量均未超出安全食用标准。结论小鼠生物法与ELISA方法的评价结果基本一致,其检测出的PSP结果可以为摄入PSP风险评估提供数据支撑。由于ELISA方法的检测成本较高,因此可采用小鼠生物法进行麻痹性贝类毒素风险监测。     

Toxins in toxic Taiwanese crabs

Abstract

A total of 459 specimens covering 51 species in 9 families was collected from October 1992 to May 1996 in Taiwan. All specimens were assayed for the presence of tetrodotoxin (TTX) and paralytic shellfish poison (PSP). The specimens of five xanthid crabs Zosimus aeneus, Lophozozymus pictor, Ategatopsis germaini, Atergatis floridus, and De‐mania reynaudi were found to contain potent toxins. Among them, A. germaini showed the highest toxicity. The toxin profile of each toxic crab species was as follows: 82% TTX and 18% PSP in Z. aeneus, 89% TTX and 11% PSP in L. pictor, 3% TTX and 97% PSP inA germaini, 85% TTX and 15% in A. floridus, and 88% TTX and 12% PSP in D. reynaudi. PSP was mainly composed of gonyautoxins (GTXs) 1–4 in Z. aeneus, L. pictor, and A. floridus, but GTX 3 and hydroxysaxitoxin in A. germaini, and neosaxitoxin in D. reynaudi. The PSP‐producing dinoflagellate plankton Alexandrium minutum and TTX‐producing bacteria including Vibrio alginolyticus and Vibrio parahaemolyticus were isolated and considered as the sources of the toxins.     

河北省市售贝类中脂溶性毒素的调查研究

目的调查河北省市售贝类中脂溶性贝类毒素的污染状况。方法在2018年8月—2020年4月间,对河北省市售7大类贝类样品进行采集,共354份,采用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)进行脂溶性贝类毒素的检测。结果 354份样品中,阳性样品17份,检出率为4.8%,检出含量较低,检出品种有毛蚶、贻贝、扇贝、牡蛎,毒素组分有虾夷扇贝毒素(yessotoxin,YTX)、同源虾夷扇贝毒素(homo-YTX)、大田软海绵酸(okadaicacid,OA)、鳍藻毒素(dinophysistoxins, DTXs)(DTX1、DTX2)、蛤毒素2(pectenotoxin-2, PTX2)。结论河北省市售贝类中脂溶性贝类毒素暴露风险很低。     

Application of HPLC for the Determination of PSP Toxins in Shellfish

A high performance liquid chromatographic (HPLC) procedure for determination of the toxins associated with paralytic shellfish poisoning (PSP) is compared to the standard AOAC mouse bioassay method on 100 shellfish samples representing a variety of species. For those samples with toxin content below the detection limit of the bioassay (35 μg saxitoxin (STX)/100g) HPLC analysis indicated a similar low level with a range of <10 to 56 μg STX/100g (n = 60). A correlation coefficient of 0.92 was determined for the 40 samples exhibiting toxicity in the bioassay (i.e., >35 μg STX/100g). Among the advantages of the HPLC method over the bioassay are significantly better sensitivity, greater sample through-put, and ability to determine the levels of each individual PSP toxin.     

Occurrence of tetrodotoxin and paralytic shellfish poisons in a gastropod implicated in food poisoning in southern Taiwan

The toxicity of the gastropod Nassarius papillosus implicated in a food paralytic poisoning incident in Liuchiu Island, Taiwan, in October 2005 is reported. The symptoms of a victim (67 years old) were featured by general paresthesia, paralysis of phalanges and extremities, paralysis, coma, and aphasia. The remaining specimens of shell were assayed for toxicity. The range of specimen toxicity was found to be 63-474 mouse units (MU) per specimen for N. papillosus by a tetrodotoxin (TTX) bioassay. The mean (SD) toxicity of the digestive gland and other portions were 296 ± 120 and 382 ± 156 MU in N. papillosus. The toxin was partially purified from the acidic methanol extract of the gastropod by using a C18 solid-phase extraction column. The eluate was then filtered through a 3000 MW cut-off ultrafree microcentrifuge filter. It was shown that the toxin purified from gastropods analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry contained TTX 42-60 µg g^-1 (about 90%), whereas along with minor paralytic shellfish poisons (PSP) it was 3-6 µg g^-1 (about 10%).     

The problem of selective determination of PSP toxins in mussels

Levels of paralytic shellfish poisoning (PSP) toxins in shellfish are routinely determined by mouse bioassay. In order to improve the qualitative and quantitative determination of PSP toxins, chromatographic techniques with fluorescence detection have been developed. These HPLC methods and the HPLC/MS coupling were used to determine a second PSP toxin which was found, in addition to saxitoxin, in canned Spanish mussels. These canned mussels were rejected in 1986 by the German food control because PSP concentrations were too high. It has been shown that these samples contained mainly dc-saxitoxin.     

Semiautomated Method for the Analysis of PSP Toxins in Shellfish

The method uses an autoanalyzer continuous flow reaction system to oxidize toxin in standard acid extracts of shellfish, prepared for mouse bioassay, to derivatives which are detected by fluorescence. Oxidation is by periodic acid under alkaline (NH₄OH) conditions and is followed by acidification by acetic acid. Concentrations of 10 μg/100g toxin and above can be measured with good reproducibility and accuracy: coefficient of variation was 9.5% for samples with 60 μg/100g or greater. Correlation with the mouse bioassay was 0.82 for 204 samples (toxin from 0–2000 μg/100g). The method is proposed to screen shellfish samples for PSP toxins with only samples falling into the range 60–250 μg/100g being subject to the more tedious and expensive mouse bioassay.