Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications

A number of recent acute human intoxications in Europe from the consumption of Irish mussels have been attributed to the presence of a new class of toxins named azaspiracids. The study demonstrates that azaspiracids behave differently from other polyether toxins, and this accounts for most false-negative results in the mouse bioassay employed by regulatory agencies to detect azaspiracids. Typically, polyether toxins are concentrated in the digestive glands of shellfish, but this is not always the situation with azaspiracids. Liquid chromatography-mass spectrometry (LC-MS), especially multiple tandem MS methods, have been applied to demonstrate that azaspiracid (AZA1) and its methyl- and demethyl- analogues, AZA2 and AZA3 respectively, are distributed throughout shellfish tissues. Using conventional mouse bioassay protocols, only 0-40% of the total azaspiracid content of shellfish was used in the assay, which could directly account for false-negative results. It was also observed that the toxin profiles differed significantly in various mussel tissues with AZA1 as the predominant toxin in the digestive glands and AZA3 predominant in the remaining tissues.     

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.     

麻痹性贝类毒素常规检测分析方法比较与 研究进展

麻痹性贝类毒素作为贝类产品中一种毒性最强、分布广泛的毒素,不仅严重威胁人们的身体健康,而且会造成相当大的经济损失。因此其监测检测方法的研究与改进一直是人们的研究热点。本文分析评述了麻痹性贝类毒素的三种常规检测分析方法的优缺点以及最新研究进展,并探讨了小白鼠生物法、免疫测定法和色谱联用技术作为主要的检测方法由于原理不同,结合不同的研究需求其应用的领域。其中,小白鼠生物测定法虽然概括毒性有效,但是其灵敏度低、误差大、并且需要大量活体动物而逐渐被色谱技术和免疫测定法所取代,此外,神经细胞分析法、毛细管电泳技术和表面等离子体共振传感器技术等方法也逐渐得到应用。不管怎样,这些方法由于需要专业人员、成本高等问题仍需进一步完善。     

贝类毒素检测中两种筛选方法的比较研究

目的 采用传统小鼠生物法(MBA)和酶联免疫吸附法(ELISA)对贝类样品中四类毒素进行检测, 为不同要求下建立或选择准确的贝类毒素检测快速筛选方法提供参考。方法 分别采用MBA和ELISA检测腹泻性贝毒(DSP)和麻痹性贝毒 (PSP), 并采用ELISA检测记忆缺损性贝毒(ASP) 和神经性贝毒(NSP)。结果 对2009~2011年8种67份贝类样品进行检测, 结果表明: 两种测试方法在实际应用中对DSP、PSP检测结果不存在差异, 检测结果有很好的吻合性。使用ELISA法对自制ASP、NSP模拟阳性样品进行检测, 均测得ASP、NSP, 检测结果满意。结论 两种筛选方法在贝类毒素检测中均有其应用空间。实验室可根据不同情况选择合适的检测方法。     

海产品中麻痹性贝类毒素快速检测技术研究进展

麻痹性贝类毒素是我国海洋赤潮中最常见的贝类毒素之一,分布最广,危害最大,事故发生率也最高,对人类健康构成了严重威胁,加强对该类毒素的检测监控成为保障海产品安全的重要措施。传统的检测方法主要有小鼠生物检测法、液相色谱法、液相色谱-串联质谱法和酶联免疫法,这些方法均有各自的优势,但在实际应用中还缺少用于现场检测的快速筛查技术。因此,开发快速、灵敏、准确、低成本的麻痹性贝类毒素检测技术具有重要的应用价值。本文主要介绍了麻痹性贝类毒素目前开发出来的快速检测方法,主要包括免疫层析技术和生物传感器技术,对各方法的特点迚行分析。最后对未来麻痹性贝类毒素快速检测技术在实际应用中面临的主要问题迚行了评述,幵对发展趋势迚行了展望。     

腹泻性贝类毒素及检测技术研究进展

近些年来海洋污染越来越严重,赤潮频发直接导致海洋生物毒素对贝类产品大面积污染,这不仅直接影响海洋产品的品质和食用安全,更加阻碍了海洋养殖业的健康发展,对食品安全造成极大威胁。为了解决海洋产品的食品安全问题,保障人们生命健康,对海洋生物毒素分析检测的研究已越来越多。本文综述了腹泻性贝类毒素的主要特征、主要毒素成分及毒理效应,并对小鼠生物测试法、酶联免疫检测法、胶束电动色谱法、酶活力抑制法、毛细管电泳法、高效液相色谱以及高效液相色谱-质谱法等常用的检测方法作以综合性评价,并对其研究进展加以介绍。     

Azaspiracid poisoning, the food-borne illness associated with shellfish consumption

Azaspiracid poisoning (AZP) is a recently discovered toxic syndrome that was identified following severe gastrointestinal illness from the consumption of contaminated mussels (Mytilus edulis). The implicated toxins, azaspiracids, are polyethers with unprecedented structural features. Studies toward total toxin synthesis revealed that the initial published structures were incorrect and they have now been revised. These toxins accumulate in bivalve molluscs that feed on toxic microalgae of the genus Protoperidinium, previously considered to be toxicologically benign. Although first identified in shellfish from Ireland, azaspiracid contamination of several types of bivalve shellfish species has now been confirmed throughout the western coastline of Europe. Toxicological studies have indicated that azaspiracids can induce widespread organ damage in mice and that they are probably more dangerous than previously known classes of shellfish toxins. The exclusive reliance on live animal bioassays to monitor azaspiracids in shellfish failed to prevent human intoxications. This was a consequence of poor sensitivity of the assay and the fact that azaspiracids are not exclusively found in the shellfish digestive glands used for toxin testing. The strict regulatory control of azaspiracids in shellfish now requires frequent testing of shellfish using highly specific and sensitive methods involving liquid chromatography-mass spectrometry.     

Proficiency studies on the determination of paralytic shellfish poisoning toxins in shellfish.

Paralytic shellfish poisoning toxins are produced by dinoflagellates. Shellfish filtering these unicellular algae will accumulate the toxins and pose a health risk when consumed by man. In the European Union, paralytic shellfish poisoning toxins in bivalve molluscs are regulated at a maximum content of 80 microg/100 g (91/492/EEC). The current reference method in the European Union is the mouse bioassay, but alternative methods including the liquid chromatography methodology are preferred for ethical reasons. Analyses of suspected shellfish batches revealed, however, unacceptable differences in results reported by a small group of Dutch laboratories all using liquid chromatography methods with precolumn derivatization, followed by fluorescence detection. Therefore, a series of proficiency studies were undertaken among these laboratories. In the first three studies, participants were more or less allowed their own choice of method execution details. This approach yielded unsatisfactory results. A fourth study was then initiated in which a standardized method was mandatory. Two types of test material were used in the fourth study: lyophilized Cardium tuberculatum material containing saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX), and lyophilized mussel material containing dc-STX. The latter material was investigated in an interlaboratory study involving 15 participants and was considered as the reference material. Among the four laboratories, coefficients of variation (ANOVA) for C. tuberculatum material were 10% (n = 11) and 9% (n = 12) for STX and dc-STX, respectively, and for the reference material was 8% (n = 12) for dc-STX. The joint efforts showed that variability in analysis results between laboratories that all apply more or less the same method can be drastically improved if the methodology is rigorously standardized.     

贝类中麻痹性贝类毒素的蓄积及代谢研究进展

文章阐述了有害毒藻产生的麻痹性贝类毒素(Paralytic Shellfish Toxins,PSTs)具有的物种特异性,从PSTs的蓄积、转化、代谢及净化对麻痹性贝类毒素的相关研究进行了综述,并指出将暂养净化、生物吸附法、微生物代谢等净化方式进行优化或组合,对水产品进行高效脱毒是未来的研究方向。     

Screening for the presence of lipophilic marine biotoxins in shellfish samples using the neuro-2a bioassay

The neuro-2a bioassay is considered as one of the most promising cell-based in vitro bioassays for the broad screening of seafood products for the presence of marine biotoxins. The neuro-2a assay has been shown to detect a wide array of toxins like paralytic shellfish poisons (PSPs), ciguatoxins, and also lipophilic marine biotoxins (LMBs). However, the neuro-2a assay is rarely used for routine testing of samples due to matrix effects that, for example, lead to false positives when testing for LMBs. As a result there are only limited data on validation and evaluation of its performance on real samples. In the present study, the standard extraction procedure for LMBs was adjusted by introducing an additional clean-up step with n-hexane. Recovery losses due to this extra step were less than 10%. This wash step was a crucial addition in order to eliminate false-positive outcomes due to matrix effects. Next, the applicability of this assay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhetic shellfish toxins/poisons (DSPs). For comparison, the samples were also analysed by LC-MS/MS. Standards of all regulated LMBs were tested, including analogues of some of these toxins. The neuro-2a cells showed good sensitivity towards all compounds. Extracts of 87 samples, both blank and contaminated with various toxins, were tested. The neuro-2a outcomes were in line with those of LC-MS/MS analysis and support the applicability of this assay for the screening of samples for LMBs. However, for use in a daily routine setting, the test might be further improved and we discuss several recommended modifications which should be considered before a full validation is carried out.     

Evidence of paralytic shellfish poisoning toxin in milkfish in South Taiwan

Natural phytoplankton blooms of the dinoflagellate Alexandrium minutum, milkfish (Chanos chanos) exposed to natural blooms, sediment and mangrove crab (Scylla serrata) were analysed for paralytic shellfish poisoning toxins by high-performance liquid chromatography. The toxin profiles of milkfish and mangrove crab were similar to that of A. minutum collected from blooming fishponds. In a laboratory A. minutum-blooming environment, the stomach and intestine of milkfish accumulated paralytic shellfish poisoning toxins during the exposure period. The non-visceral tissues were non-toxic. However, milkfish lost their entire body burden of toxin on the first day of transferring to a toxic algae-free environment. The result shows that milkfish concentrate paralytic shellfish poisoning toxins in digestive organs and did not retain toxins.     

Comparative Toxicity and Paralytic Shellfish Poisoning Toxin Profiles in the Mussel Mytilus galloprovincialis and the Oyster Crassostrea gigas Collected from a Mediterranean Lagoon in Tunisia: A Food Safety Concern

Edible shellfish Mytilus galloprovincialis and Crassostrea gigas have been investigated for the paralytic shellfish poisons using mouse bioassay and high performance liquid chromatography with fluorescence detection. Paralytic shellfish poisons toxins were detected in mussels and oysters from September 2007 to May 2008. The level of paralytic shellfish poisons toxins in mussels reached the maximum in November with 832.9 μg saxitoxin-eq/100 g tissue. In oysters, toxins were detected with a maximum of 11.2 μg saxitoxin-eq/100 g tissue. The toxin high performance liquid chromatography profiles in mussels and oysters revealed the dominance of gonyautoxin 5 and N-sulfocarbamoyl-gonyautoxin-2 and -3 (C1-2), whereas GTX1-4, saxitoxin, and neosaxitoxin were found at low amounts. Overall, levels of paralytic shellfish poisons toxins were 20–70 times greater in mussels than in oysters. This is the first report on the qualitative and quantitative paralytic shellfish poisons content of M. galloprovincialis and C. gigas from a shellfish farming lagoon in Tunisia.     

液相色谱-串联质谱法检测9种腹泻性贝毒毒素

目的建立腹泻性贝毒毒素9种代表性化合物:大田软海绵酸、鳍藻毒素、蛤毒素2、扇贝毒素及其衍生物、环亚胺米氏裸甲藻毒素、螺环内酯毒素1和原多甲藻酸的高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)检测方法。方法以80%甲醇水溶液对贝类组织中的毒素进行提取,再以亲水亲脂平衡柱(HLB)对提取液进行净化,最后以高效液相色谱-电喷雾串联质谱测定。结果 DSP毒素9种代表性化合物在各自浓度范围内线性良好,回收率为68.9%~94.2%,精密度为3.5%~9.5%。结论该方法具有灵敏度高、重现性好、操作简便、准确可靠等特点,适用于贝类中腹泻性贝毒毒素的测定。     

Distribution of diarrhetic shellfish poisoning toxins in consignments of blue mussel

Data describing the distribution of diarrhetic shellfish poisoning toxins in 13 consignments of Danish-produced blue mussels are reported. The content of diarrhetic shellfish poisoning toxins was measured by a liquid chromatography coupled with tandem mass spectrometry detection method, and mean levels in the 13 consignments varied from 58 to 243 μg kg^-1. The distributions of diarrhetic shellfish poisoning toxins in the consignments were relatively homogenous as the relative standard deviation of the content varied from 7 to 19%. The results are discussed in relation to food safety, the uncertainty of sampling and analysis, and the newly introduced European Union maximum levels of marine biotoxins in seafood products.     

Influence of body weight of mice on the susceptibility to okadaic acid,a diarrhetic shellfish poisoning toxin

The mouse bioassay (MBA) for diarrhetic shellfish poisoning (DSP) toxins has been widely used in many countries of the world. However, different body weight ranges of mice are designated to be used in the Japanese official method and European Union procedure. In this study we investigated whether and to what extent the body weights of the mice affect the susceptibility to DSP toxins. A lethal dose of okadaic acid, one of the representative DSP toxins, was injected intraperitoneally into mice of five different body weight range groups, from 14 to 24 g. The mice were observed until 24 h after injection. The lethality was 100% in the 14–15 and 16–17 g groups, 80% in the 19–20 g group, 50% in the 21–22 g group, and 40% in the 23–24 g group, with significant differences. Survival analysis indicated a relationship between body weights of mice and susceptibility to okadaic acid. These results would be quite useful not only for the MBA, but also to improve understanding of the biological responses to DSP toxins.     

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.     

Extraction recoveries and stability of diarrhetic shellfish poisoning (DSP) toxins in naturally contaminated samples

During the last few years the occurrence of a high percentage of esters of diarrhetic shellfish poisoning (DSP) toxins has been observed in shellfish from the Portuguese coast. Most of the commercial bivalves contain DSP toxins in ester forms, either acyl derivatives of okadaic acid (OA) or of dinophysistoxin-2 (DTX-2). The stability of these toxins in shellfish tissues and in raw methanol extracts was investigated in two different naturally contaminated species, mussel and carpet shell, over a 4-week period. The results for both species revealed that DSP toxins were more stable in tissue than in raw methanol extracts. Losses of DSP toxins were seen in the first 2 weeks and were more than 30%, but after that a period of stabilization was observed. The decrease was due probably from losses of esters of OA and DTX-2, the free toxins were stable over the period studied. The extraction most commonly used for chemical and biochemical assays relied on methanolic extraction with aqueous 80% methanol. In this work we have tested the extraction solvent on the extractability of DSP toxins from several naturally contaminated species. A single dispersive extraction with methanol, with solvent ratios of 70%, 80%, 90% and 100%, were tested. After alkaline hydrolysis of esterified toxins and clean-up with hexane and dichloromethane, the samples were analysed by liquid chromatography-mass spectrometry (LC-MS). The recovery of DSP toxins increased with increasing percentages of methanol up to 90%. A decrease in recovery with 100% methanol was observed probably due to problems during the liquid–liquid partitioning.     

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.     

Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay

This study compared five methods of measuring paralytic shellfish toxins (PSTs) including the long-used mouse lethality bioassay, a commercially available cell culture test (MIST ® Quantification kit), HPLC analysis, and two newly developed radioreceptor assays utilizing mammalian sodium channels and saxiphilin. Methods were challenged with toxic shellfish extracts prepared according to the AOAC official method. The best correlations between predicted toxicity values being 0.9 or better, were those between HPLC analysis when compared with both radioreceptor assays and the mouse lethality bioassay, as well as that between the saxiphilin and the sodium channel radioreceptor assays. In all cases, statistically significant correlations existed between the toxicity measurements of the same extracts. The ratios between some methods were not unitary as measured by the slopes of the regression lines used for correlation analyses. HPLC analysis predicted more toxicity than all of the bioassays. The saxiphilin assay underestimated toxicity relative to the mouse bioassay, the MIST ® kit determinations and the sodium channel assay. The sodium channel assay predicted there to be less toxicity than the mouse bioassay and the MIST ® kit. Of all of the techniques used, the MIST ® kit correlation with the mouse bioassay was nearest to one. Each method possesses different virtues and it may be that a multi-method approach would harness the benefits of each method for various aspects of a shellfish testing regime.     

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.