The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four postcolumn derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.     

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.     

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.     

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.     

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.     

Development and Use of A Proficiency Test Specimen for Paralytic Shellfish Poisoning

A proficiency test specimen was developed for evaluating laboratory precision in paralytic shellfish poisoning bioassays. Such a specimen is needed since much of health and economic consequence depends on an analyst's precision in measuring the toxin. Therefore, after testing clams, mussels, and mashed potato matrices, a test specimen composed of saxitoxin dihydrochloride and hydrated potato flakes was selected. Samples containing two toxin levels were sent to 14 collaborating laboratories. Analysis of results identified errors arising from poor dilution techniques. Recovery percentages and variances of mashed potato were comparable to those previously reported with shellfish matrices. Use of the potato matrix appears to offer cost and convenience advantages.     

Zur Problematik der selektiven Erfassung von PSP-Toxinen aus Muscheln

Zusammenfassung Die Messung der PSP-Belastung (paralytic shellfish poisoning) von Schalentieren erfolgt vor allem mit Hilfe des Maus-Biotestes. Um PSP-Toxine sowohl qualitativ als auch quantitativ besser bestimmen zu können, wurden chromatographische Verfahren mit Fluorescenzdetektion entwickelt. Diese HPLC-Methoden sowie die Kopplung HPLC/MS gelangten zum Einsatz, um in spanischen Muschelkonserven ein neben Saxitoxin vermutetes zweites PSP-Toxin nachzuweisen. Es zeigte sich, daß in den 1986 in der Bundesrepublik Deutschland wegen zu hoher PSP-Konzentrationen beanstandeten Muschelkonserven vor allem Decarbamoyl-Saxitoxin enthalten war.

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The problem of the selective determination of PSP-toxins in mussels

Summary 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.

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Herrn Professor Dr. A. Montag zum 60. Geburtstag gewidmet     

Do saxitoxin-like substances have a role in scombrotoxicosis?

Evidence is presented which establishes that mackerel fed in captivity can, by relay from contaminated shellfish via sand eels, accumulate paralytic shellfish poisons (PSP) in the edible flesh at a level (250 micrograms saxitoxin equivalents per kg) similar to that in the contaminated shellfish. Data from ELISAs performed independently in two laboratories show that commercial mackerel fillets which have been associated with incidents of scombrotoxicosis contained 0.02-1.30 micrograms saxitoxin equivalents per kg, concentrations some two to four orders of magnitude below that normally detectable by the mouse bioassay. The doses, expressed as saxitoxin equivalents, administered inadvertently during volunteer testing of such fillets ranged up to 0.5 ng/kg bw, at least four orders of magnitude less than the fatal oral dose for an adult. The doses associated with the rapid induction of nausea/vomiting and/or diarrhoea, 0.11-1.0 ng/kg bw, could not be distinguished from the doses which failed to produce such symptoms in susceptible volunteers (up to 0.5 ng/kg bw). Factors that might explain this lack of correlation between dose (saxitoxin equivalents) and volunteer response are discussed along with previously published reports of PSP relay through the food web. It is suggested that the relay of algal toxins, particularly PSP, but possibly in combination with diarrheic shellfish poisons, may be responsible for scombrotoxicosis.     

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。     

DEVELOPMENT OF A TROPICAL FISH ASSAY FOR SAXITOXIN

The tropical fish Notropis atherimoldes (0.2–0.4 g) was found to be highly sensitive to saxitoxin (STX) and well-suited for use in the biological estimation of saxitoxin. STX in distilled water at a concentration of 3.3 ng/mL resulted in a paralytic end-point being reached in 17.5 min. The assay is ideally suited for initially detecting the presence of paralytic shellfish toxins in bacterial culture supernates.     

Food safety implications of the distribution of azaspiracids in the tissue compartments of scallops ( Pecten maximus )

Azaspiracids, a new class of shellfish toxins, have been implicated in several recent incidents of human intoxications following the consumption of mussels ( Mytilus edulis ). A study was undertaken to examine the distribution of azaspiracid poisoning (AZP) toxins in scallops ( Pecten maximus ) and individual shellfish were dissected into five tissue fractions for the determination of toxin composition. Separation of the predominant azaspiracids, AZA1-3, was achieved using reversed-phase liquid chromatography with detection by positive electrospray multiple tandem mass spectrometry. The AZP toxin composition was determined in the adductor muscle (meat), gonad (roe), hepatopancreas (digestive glands), mantle and gill of scallops. Substantial differences in the AZP toxin levels between tissue compartments were observed and toxins were concentrated predominantly, about 85%, in the hepatopancreas. There was also a significant variation in the total toxin levels between individual scallops from the same sample batch and the RSD was 60% (n = 9). Interestingly, although all three AZP toxins were present in phytoplankton and mussels, AZA3 was not detected in the scallop samples examined. It was concluded that to improve food safety, only the adductor muscle and gonad of scallops should be permitted for sale to the public.     

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。     

Effect of ozonation and γ-irradiation on post-harvest decontamination of mussels (Mytillus galloprovincialis) containing diarrhetic shellfish toxins

Contamination of shellfish with diarrhetic shellfish poisoning (DSP) toxins readily occurs during algal blooms. Such phenomena raise important public health concerns and thus comprise a constant challenge to shellfish farmers, the seafood industry and health services, considering the increasing occurrence of toxic episodes around the world. To avoid the detrimental effects of such episodes, research has focused on the use of various detoxification methodologies that should be rapid, efficient, easy to apply, and will not alter the quality and sensory properties of shellfish. In the present study, both ozonation (15 mg kg(-1) for 6 h) and γ-irradiation (6 kGy) were utilised in order to reduce the toxin content of contaminated shucked mussels, collected during the DSP episodes of 2007 and 2009 in Greece. DSP toxicity was monitored using the mouse bioassay (MBA) whilst the determination of toxin content of the okadaic acid (OA) group (both free and esterified forms) was carried out by LC/MS/MS analysis. Toxin reduction using γ-irradiation was in the range of 12-36%, 8-53% and 10-41% for free OA, OA esters and total OA, respectively. The appearance and texture of irradiated mussels deteriorated, pointing to a low potential for commercial use of this method. Ozonation of mussels resulted in toxin reduction in the range of 6-100%, 25-83% and 21-66% for free OA, OA esters and total OA, respectively. Reduction of OA content was substantially higher in homogenised mussel tissue compared with that of whole shucked mussels. In addition, differences detected with regard to quality parameters (TBA, sensory attributes) between ozonated and control mussels were not considerable. Even though varying percentage reductions in OA and its derivatives were achieved using ozonation under specific experimental conditions tested, it is postulated that upon optimisation ozonation may have the potential for post-harvest commercial DSP detoxification of shucked mussels.     

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.     

Lipophilic toxin profiles detected in farmed and benthic mussels populations from the most relevant production zones in Southern Chile

Lipophilic toxins associated with diarrhoeic toxins were found in Mytilus chilensis (Blue mussels) and Aulacomya ater (Ribbed mussels). These shellfish samples were collected from Chiloe Island, Southern Chile. The samples were tested by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After the analysis, four toxins were found: DTX-1, DTX-3, YTX and PTX. All toxins were identified by comparing their HPLC retention times with those of analytical standards and confirmed by LC-MS/MS. Dinophysistoxin-1 (DTX-1) and dinophysistoxin-3 (DTX-3) toxins were the major components within the mussel extracts. Nevertheless, the percentages of these toxins differed depending on the area they were collected from and/or the sampling date. The levels detected in Butacheuques Island for okadaic acid (OA) was 267 ± 3.5 μg OA eq kg(-1) (p < 0.05) and for DTX-3 was 183.4 ± 7.5 μg kg(-1) in ribbed mussels. Pectenotoxin (PTX) and yessotoxin (YTX) were the toxins detected in minor proportions in the toxic profile of the bivalves. The maximum concentration of YTX detected in ribbed mussels was 85.2 ± 2.8 μg kg(-1) in Mechuque Island, whereas the PTX-2 level in ribbed mussels was 82.0 ± 2.4 μg kg(-1) in Cailin Island. Analogues of YTX and PTX-2 were not detected in any of the analysed mussels, which did not support the supposed presence of isomers of toxins as a result of the enzymatic metabolism of bivalves. This study found evidence proving co-occurrence of lipophilic toxins - like PTX and YTX - with diarrhoeic toxin in samples collected in Southern Chile, which is, to date, the more complex mix of lipophilic toxins ever found in mussels samples from Southern Chile.     

Geographical and annual variation in lipophilic shellfish toxins from oysters and mussels along the south coast of Korea

To better understand critical aspects of diarrhetic shellfish poisoning (DSP) occurrence in a chief producing region of bivalves in Korea, the geographical and annual variation of DSP toxins and other lipophilic toxins in mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas) were investigated by liquid chromatography-tandem mass spectrometry in an area on the south coast of Korea from 2007 to 2009. The total lipophilic shellfish toxin (LST) levels in bivalves showed geographical and annual variations. LSTs were detected mostly in the hepatopancreas of mussels from Jinhae Bay throughout the entire year, except in November and December of 2007, but were almost undetectable in all samples during the entire year in 2009. The peak DSP toxin (okadaic acid plus dinophysistoxin 1) levels in the hepatopancreas of mussels from Jinhae Bay and the Tongyeong region were 945.3 and 37.6 ng/g, respectively. The DSP toxin content was about 10 times higher in mussels than in oysters collected from the same region. The major toxins in bivalves were okadaic acid and dinophysistoxin 1; however, pectenotoxin 2 or yessotoxin was occasionally detected as a major component. The results of a quantitative analysis of phytoplankton showed that Dinophysis acuminata was the most probable source of the LSTs, with the exception of yessotoxin. When the highest DSP toxin level was measured (945.3 ng/g in the hepatopancreas of mussels from Jinhae Bay), the toxin concentration in whole mussel tissue was calculated to be 114.0 ng/g. The calculated highest DSP toxin level in whole oyster tissue from both regions was 15.0 ng/g. The calculated maximum toxicities in whole mussel and oyster tissues were lower than the regulatory limit (160 to 200 ng/g) in Korea, the European Union, and the United States. Korean oysters (242 samples) and mussels (214 samples) were thus deemed safe for consumption. But because such variation was detected in a relatively small area of the coast, it is possible that at some locations or during a specific period LST levels could exceed the standard and a few consumers could be at risk of experiencing DSP.     

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.     

Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use

The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg^?1 to 13,000 µg STX equiv. kg^?1 by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg^?1 on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg^?1) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.     

Analytical screening of marine algal toxins for seafood safety assessment in a protected Mediterranean shallow water environment

Microalgal species growing in marine and aquaculture environments can be responsible for harmful events because of their ability to produce potent natural toxins that can accumulate in edible mollusc species. Their consumption can cause severe illness and even be lethal. The European Union provides comprehensive regulations covering various general food safety aspects to manage the risk of contamination in shellfish farms. Many analytical methods have been proposed to evaluate algal toxins presence in the environment and in food products, for conducting surveillance studies of the main molluscs production sites and, where necessary, immediate monitoring of possible contamination of shellfish. In this work, a one-year analytical surveillance study was carried out to verify the possible presence of algal biotoxins in molluscs from a Mediterranean breeding area. Water and molluscs were sampled from a district of the North-East coast of Sicily, consisting of a unique brackish ecosystem of two lakes connected to each other and to the sea by narrow canals. Water samples were collected to investigate phytoplankton i by microscope analysis to assess the presence of potentially toxin-producing species, such as Pseudo-nitzschia spp, Alexandrium spp and Gonyaulax spinifera, although the presence of toxic phytoplankton has never reached alert levels. Mussels and clams samples were submitted to analysis of paralytic shellfish poisoning toxins, amnesic shellfish poisoning toxins and lipophilic toxins by liquid chromatography-based methods Only a few yessotoxins were detected, having concentrations always below the regulation limits. An existing liquid chromatography-tandem mass spectrometry-based multiresidue method for lipophilic biotoxins was adopted and extended to cover emerging biotoxins such as cyclic imines. The performance of the analytical method for Gymnodimine A and Spirolide 13-desMeC was assessed, obtaining respective quantitation limits of 20 and 10 µg kg^?1, a precision always lower than 13% and trueness in the 81–120% range. Method applicability was confirmed using certified materials and a naturally contaminated sample.     

Assessment of the quantitative determination of paralytic shellfish poisoning toxins by pre-column derivatization and elimination of interfering compounds by solid-phase extraction

Monitoring programmes for paralytic shellfish poisoning toxins in bivalve molluscs still rely heavily on the use of mouse bioassays (MBA) for consumer protection. A high-performance liquid chromatography (HPLC) methodology (Lawrence method) was implemented in 1996 in the Portuguese monitoring programme as a complementary means of analysis. Comparison between MBA and HPLC was done at the time only by a qualitative approach due to the scarce number of positive samples tested. More quantitative data were obtained recently when studying toxin profiles in Moroccan shellfish, and the correlation found between these two methodologies is reported here for the first time. Two different matrices were studied: blue mussel and the giant cockle Acanthocardia tuberculatum. A good linear correlation was obtained for both matrices. However, a second-degree polynomial best fitted the data at both low and high extremes of toxicity. According to the HPLC quantitative results, 13% of false-negatives could be obtained by MBA due to an underestimation of toxicity near the limit of detection of the MBA. Difficulties on relying solely on HPLC for consumer protection have been aroused with uncommon matrices, such as imported clams or crustaceans, due to the presence of high concentrations of interfering compounds. The solid-phase extraction step of the Lawrence method was implemented to eliminate an unknown compound that could be mistaken for saxitoxin, and an 80% reduction of another common unknown compound eluting close to decarbamoylsaxitoxin. The implementation of the HPLC methodology achieved so far allows a high degree of consumer protection without the need to resource to animal sacrifice.