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An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.

The application of ultra-performance rapid resolution LC on a 1.8 microm particle-size column coupled with tandem MS (RRLC-MS/MS) is described for the analysis of amnesic shellfish poisoning (ASP) toxins in shellfish. Complete resolution among domoic acid (DA) and the isomers was achieved in less than 3 min. The method was intralaboratory validated for direct analysis of crude extracts without further cleanup. It showed LODs ranging from 0.05 to 0.09 mg/kg and a working range that complied with the current regulatory level for DA of 20 mg/kg, and with the level of 4.5 mg/kg recently proposed by the European Food Safety Authority. Confirmatory capabilities were demonstrated according to the Commission Decision 2002/657/EC criteria. The results obtained by RRLC-MS/MS agreed with those provided by the reference LC-UV method, both intralaboratory for the analysis of blind samples (R2 = 0.9751) and interlaboratory through participation in the proficiency test for ASP toxins during 2009 (z-score = -0.962 and 0.177 for low- and high-contaminated samples, respectively). RRLC-MS/MS provided fast analysis and additional confirmatory capabilities for direct analysis of crude extracts while the performance and reliability of the results were maintained, even in very complex matrixes.

The contamination of different types of shellfish by okadaic acid (OA)-group toxin esters is an important problem that presents serious risk for human health. During previous investigations carried out in our laboratory by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), the occurrence of a high percentage of esters in relation to the total OA equivalents has been observed in several shellfish species. The determination of these kinds of toxins using LC/MS or other chemical methods requires a hydrolysis step in order to convert the sterified compounds into the parent toxins, OA, dinophysistoxins-1 (DTX-1) and dinophysistoxins-2 (DTX-2). Most of the hydrolysis procedures are based on an alkaline hydrolysis reaction. However, despite hydrolysis being a critical step within the analysis, it has not been studied in depth up to now. The present paper reports the results obtained after evaluating the hydrolysis process of an esterified form of OA by using a standard of 7-O-acyl ester with palmitoyl as the fatty acid (palOA). Investigations were focused on checking the effectiveness of the hydrolysis for palOA using methanol as solvent standard and matrices matched standards. From the results obtained, no matrix influence on the hydrolysis process was observed and the quantity of palOA converted into OA was always above 80%. The analyses of different Spanish shellfish samples showed percentages of palOA in relation to the total OA esters ranging from 27% to 90%, depending on the shellfish specie.

By the end of 2005, a toxic episode of phytoplankton origin in bivalve shellfish led to the closing down of several shellfish production areas in Galicia (northwestern region of Spain). During this time, different kinds of shellfish were collected and analysed by LC-MS/MS to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and spirolides. Samples were analysed before alkaline hydrolysis in order to investigate the presence of free OA and DTXs, AZAs, PTXs and spirolides, and after alkaline hydrolysis to detect OA and DTXs esters. All of the samples were found to be contaminated with OA and/or DTX-2, as well as esterified forms of these diarrhetic shellfish poison (DSP) toxins, at levels around and above European regulatory limit (160 microg of okadaic acid equivalents/kg). The analyses of mussels and razor clam also revealed the presence of 13-desmethyl spirolide C (SPX-1) at levels below 31 microg/kg. Likewise, in many of the samples different levels of pectenotoxin-2 secoacid (PTX-2sa) were detected. DSP toxin esters represent practically the 100% of the total OA equivalents for scallops, clams, razor clams and cockles.

An HPLC-FLD method, involving pre-chromatographic oxidation of the PSP toxins with hydrogen peroxide and periodate, has been AOAC validated through a collaborative trial and adopted as AOAC Official Method. This method could be a candidate for replacing the mouse bioassay (MBA) for the Official Control of PSP toxins at European level, once accepted by the legislation. An interlaboratory exercise has been organized by the CRLMB to evaluate its "fitness for purpose" for the Official Control of PSP toxins in the EU laboratories. Eighteen EU laboratories took part in the study and had to analyze six bivalve mollusc samples with several PSP toxic profiles. The performance of the participant laboratories in the application of this method was compared with that obtained at the collaborative trial. Information on problems/drawbacks encountered by participants in the application of this method was also sought. The HPLC validated method is only applicable for Official PSP Control for certain samples. This depends on sample PSP toxic profile. Results obtained for samples where only GTX2,3 and STX were present were satisfactory and in agreement with MBA results. Results obtained for a sample with a toxic profile dominated by GTX6 and suspected to contain also C1,2 and C3,4 were not satisfactory. GTX5 and dc-STX could be quantified, although the results achieved (total toxicity) were lower than those obtained by MBA. It can be also useful as a screening method, complementary to MBA, helping in the reduction of the animals used. However, the lack of several PSP standards, the fact that the method is not validated for all the PSP toxins, and several drawbacks found in its application are a handicap to fully implement it for Official PSP Control as a viable replacement for bioassay.

During the months of November and December 2005, a harmful algal bloom in the northwestern region of Spain caused the accumulation of different algal toxins in several types of shellfish. Many of the shellfish were contaminated at levels above European regulatory limit, presenting serious risks for human health. The analysis of mussels sampled in the affected areas to search for lipophilic toxins, using liquid chromatography tandem mass spectrometry (LC-MS/MS), showed the presence of free okadaic acid (OA) and dinophysistoxin-2 (DTX-2) as well as esters of these toxins. The results also revealed the presence of minor amounts of 13-desmethyl spirolide C (SPX-1) in the analysed samples, although this toxin has never been reported before in Spain. The combination of different MS modes of operation, just as enhanced MS (EMS) and MS(3) experiments, allowed to confirm the first occurrence of spirolides in Spanish shellfish.

Raw cow's milk collected from dairy farms in the province of Leon, Spain, was examined for aflatoxin M(1) (AFM(1)). The samples were analysed with a commercial competitive enzyme-linked immunosorbent assay (ELISA) kit and high-performance liquid chromatography (HPLC). The concentrations of AFM(1) in the milk extracts were initially estimated by ELISA, with recovery rates of 74.6-109% for artificially contaminated milk at levels of 10-80 ng x l(1). Samples found to contain more than 10 ng x l(1) were further quantified with HPLC. The mean recovery for this method was 89.3%. The quantification limit was 10 ng x l(1) for both ELISA and HPLC. Although AFM(1) was confirmed in only 3.3% of the samples, the concentrations in all these cases were lower than the maximum limit applicable to these products pursuant to European Union legislation. Both methods were validated with reference material certified by the Community Bureau of Reference.

In Portugal, high levels of diarrhetic shellfish toxins (DSTs) originating from Dinophysis spp. are accumulated in bivalves, mainly on the northwest coast (NWC). The occurrence of DSTs in spring and early summer is positively related to precipitation. A decrease in average precipitation after 2003 led to a reduction in DSTs after 2008. However, the accumulation of DSTs in the NWC increased in the period 2020–2022. The hydrological year of 2022 was marked by extreme drought. In cockles from the endemic area of Ria de Aveiro, 37.3% of the weeks tested presented samples above the regulatory limit (RL). The previous record was 31.9% in 2005, also an extremely dry year. The average percentage of weeks above the RL in severe drought years surpassed both the low and high precipitation years. In severe dry years, toxicity was dominated mainly by D. acuta toxins. While D. acuminata grows after the abundant river discharges in spring, D. acuta is capable of growing in summer during upwelling favourable conditions. In the last two decades, extensive forest wildfires were recurrent, but the area burnt reduced sharply after 2017 following tighter fire control measures. Low levels of DSTs or low percentage of weeks with cockles above the RL were related to high burnt areas in the previous year. The recent increase in 2020–2022 of DSTs, a planktonic biomarker in bivalves, was also coincident with the similar temporal increase in either the recruitment or the biomass of Sardina pilchardus, a planktivorous fish in decline after 2005/2006. Both low sardine recruitment and biomass were coincident with low precipitation or high burnt areas. Wildfires degrade coastal water quality, as seems reflected both in the cockle's DST and the sardine stock time series. [ABSTRACT FROM AUTHOR]

The content and composition of paralytic shellfish toxins (PSTs) in Japanese basket clam (Corbicula japonica) and mussels (Mytilus galloprovincialis) from Osaka Bay, Japan, were investigated using a mouse bioassay (MBA) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), and the association between toxicity values of MBA and HILIC-MS/MS was verified based on research data. The overall toxicity in Japanese basket clam was lower than that in the mussel. The PSTs of Japanese basket clam and mussel consisted mainly of C1, C2, and gonyautoxins 1–4 (GTX1–4) taking toxins compositional differences as mol%. When multiplying the content of different toxins by the toxic equivalent factor (TEF), C2 and GTX1–4 accounted for more than 90% of total toxicity (MU TEF/g) based on the MU TEF score converted by TEF for the two species. The total content of C2 and GTX1–4 converted to toxicity was significantly correlated with the toxicity determined by MBA for the two species (r2 > 0.983). This study provides a suitable and ethical monitoring method to investigate toxicity in bivalves contaminated with A. tamarense by analysis of only predominant toxins, along with reducing use of MBA. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the occurrence of the most representative cyclic imines (CIs) gymnodimine (GYM), pinnatoxin G (PnTX-G), and 13-desmethyl SPX C (SPX1) in Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) (n = 416), European oysters (Ostrea edulis Linnaeus, 1758) (n = 104), Queen scallops (Aequipecten opercularis Linnaeus, 1758) (n = 52) and edible ascidians of the Microcosmus spp. (n = 104) originating from nine harvesting and breeding areas in the northern part of the Adriatic Sea using ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). All CI concentrations were far below the guidance level of 400 μg SPXs/kg proposed by the EU Reference Laboratory for Marine Toxins. In contrast to Queen scallops and ascidians, in Mediterranean mussels and European oysters CIs were found throughout the year. Our data reveal the differences between species predisposed for CIs accumulation, as well as seasonal and locational variations in CIs occurrence. [ABSTRACT FROM AUTHOR]

Individuals of Mytilus platensis were exposed to Alexandrium catenella to evaluate the accumulation and metabolization of paralytic shellfish toxins (PST) over a period of 25 days. Mussels were collected from the intertidal zone of Cerro Avanzado, Argentine Patagonia. After 16 days, the toxins in the tissues of mussels were detected by the methods of mouse bioassay and high performance liquid chromatography with fluorometric detection (HPLC-FDL). The accumulation kinetics of PST toxins in M. platensis fed with A. catenella fitted to a linear function, in which the accumulation rate was 31.2 µg STX eq kg−1 day−1. After 16 days, the PST toxin level in tissues of mussels reached 1178 µg STX eq kg−1 exceeding the safety limit for human consumption (800 µg STX eq kg−1 tissue), whereas the highest PST toxin level was reached at the end of the experimentation (1613 µg STX eq kg−1) at 25 days. Differences in the toxin profile of the dinoflagellates and the tissues of the mussels confirmed biotransformation of PST in the mussel digestive system. The toxin profile of M. platensis was dominated by the gonyautoxins GTX1 and GTX4, while the toxin profile of A. catenella was dominated by the N-sulfocarbamoyl toxin C2. To our knowledge, this is the first experimentation on a laboratory scale of PST toxins accumulation in M. platensis with a native strain of A. catenella of Argentine Patagonia. [ABSTRACT FROM AUTHOR]

BACKGROUND Aflatoxin M1 (AFM1) is a mycotoxin found in milk as a result of the ingestion of aflatoxin B1 (AFB1) by dairy cattle. Because of its carcinogenic properties, the control of its occurrence in dairy products is of great importance. We evaluated the occurrence and seasonality of AFM1 in milk from farms with subtropical and temperate climates, where significant milk production occurs. Forty samples of raw milk were collected from bulk tansk milk during the summer (five samples from a subtropical climate and 14 from a temperate climate) and winter (six samples from a subtropical climate and 15 from a temperate climate) months of 2017 and conducted an analysis using an enzyme‐linked immunosorbent assay (ELISA) (detection limit 5 ng L−1). Data from eight random samples were compared with respect to liquid chromatography with mass spectrometry detection and ELISA. A significant correlation was observed. RESULTS: The presence of AFM1 was detected in 87.50% (n = 35) of the samples analyzed (a mean of 16.66 ng L−1 and a median of 12.42 ng L−1 in positive samples); however, its concentration in all samples was below the maximum limit allowed by European (50 ng L−1) and Brazilian (500 ng L−1) legislations. There were no significant differences in the levels of AFM1 between the properties located in the two climate zones, in both summer and winter. The estimated daily intake of AFM1 based on the milk analyzed was 0.0107 ng kg−1 day−1 for adolescents, 0.0072 ng kg−1 day−1 for adults and 0.0098 ng kg−1 day−1 for the elderly. CONCLUSION: The present work demonstrated a low exposure to AFM1. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Marine biotoxins regularly occur along the coast, with several consequences for the environment as well as the food industry. Monitoring of these compounds in seawater is required to assure the safety of marine resources for human consumption, providing a means for forecasting shellfish contamination events. In this study, an analytical method was developed for the detection of ten lipophilic marine biotoxins in seawater: azaspiracids 1, 2, 3, 4 and 5, classified as azaspiracid shellfish poisoning toxins, and pectenotoxin 2, okadaic acid and the related dinophysistoxin 1, yessotoxin and homoyessotoxin, classified as diarrheic shellfish poisoning toxins. The method is based on the application of solid-liquid ultrasound-assisted extraction and solid-phase extraction, followed by high-performance liquid chromatography coupled with high-resolution mass spectrometry. The limits of detection of this method are in the range of nanograms per litre and picograms per litre for most of the compounds, and recoveries range from 20.5% to 97.2%. To validate the effectiveness of this method, 36 samples of surface water from open coastal areas and marinas located along the Catalan coast on the Mediterranean Sea were collected and analysed. Eighty-eight per cent of these samples exhibited okadaic acid in particulate and aqueous phases in concentrations ranging from 0.11 to 560 μg/g and from 2.1 to 1780 ng/L respectively. Samples from open coastal areas exhibited higher concentrations of okadaic acid in particulate material, whereas in samples collected in sportive ports, the particulate material exhibited lower levels than the aqueous phase. [ABSTRACT FROM AUTHOR]

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An analytical procedure for the determination of aflatoxin B in eggs was introduced and validated in laboratory 1. The method consisted of the extraction of aflatoxin B from a sample, purification of the extract with solvents, immunoaffinity column cleanup and the determination by liquid chromatography with post-column bromination and fluorescence detection at λ = 362 nm and λ = 425 nm. The method was transferred to laboratory 2, where it was modified and validated. The limit of detection (LOD) and limit of quantification (LOQ) obtained in laboratory 1 were 2 and 6 ng/kg, respectively, and 2 and 5 ng/kg in laboratory 2, respectively. The repeatability of measurements in laboratory 1, represented by differences between results of duplicate measurements, was 10 ng/kg at the contamination level of 50 ng/kg. At the same concentration level, the standard deviation ( s) and the relative standard deviation ( RSD) for the within-laboratory reproducibility were 5.5 ng/kg and 11 %, respectively, and the measurement uncertainty was ±10 ng/kg. The mean recovery was 70 %. In laboratory 2, the repeatability of measurements at the contamination level of 20 ng/kg, represented by the standard deviation ( s), repeatability ( r) and relative standard deviation ( RSD) was 4 ng/kg, 11 ng/kg and 20 %, respectively, and the recovery was 67 %. The results indicate that the procedures are suitable for the determination of aflatoxin B in eggs and can be implemented for the routine analysis. Using the procedure validated in laboratory 1, 25 samples from farms in Slovenia were analysed. In none of the analysed samples, aflatoxin B was detected. [ABSTRACT FROM AUTHOR]

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−1for 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. [ABSTRACT FROM PUBLISHER]

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g−1) exceeded largely the values for the other quantified compounds (0.09–58 ng g−1). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 µg STX equivalents kg−1. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200–800 µg STX equivalents kg−1 an increase was found between 44 and 28%. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]