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Tetrodotoxin (TTX) is a potent marine neurotoxin found in several phylogenetically diverse organisms, some of which are sought as seafood. Since 2015, TTX has been reported in bivalve shellfish from several estuarine locations along the Mediterranean and European Atlantic coasts, posing an emerging food safety concern. Although reports on spatial and temporal distribution have increased in recent years, processes leading to TTX accumulation in European bivalves are yet to be described. Here, we explored the hypothesis that the ribbon worm species Cephalothrix simula , known to contain high levels of TTX, could play a role in the trophic transfer of the toxin into shellfish. During a field study at a single location in southern England, we confirmed C. simula DNA in seawater adjacent to trestle-farmed Pacific oysters Magallana gigas (formerly Crassostrea gigas ) with a history of TTX occurrence. C. simula DNA in seawater was significantly higher in June and July during the active phase of toxin accumulation compared to periods of either no or continually decreasing TTX concentrations in M. gigas . In addition, C. simula DNA was detected in oyster digestive glands collected on 15 June 2021, the day with the highest recorded C. simula DNA abundance in seawater. These findings show evidence of a relationship between C. simula and TTX occurrence, providing support for the hypothesis that bivalves may acquire TTX through filter-feeding on microscopic life forms of C. simula present in the water column at particular periods each year. Although further evidence is needed to confirm such feeding activity, this study significantly contributes to discussions about the biological source of TTX in European bivalve shellfish.

Tetrodotoxin (TTX), a potent neurotoxin mostly associated with pufferfish poisoning, is also found in bivalve shellfish. Recent studies into this emerging food safety threat reported TTX in a few, mainly estuarine, shellfish production areas in some European countries, including the United Kingdom. A pattern in occurrences has started to emerge, however the role of temperature on TTX has not been investigated in detail. Therefore, we conducted a large systematic TTX screening study, encompassing over 3500 bivalve samples collected throughout 2016 from 155 shellfish monitoring sites along the coast of Great Britain. Overall, we found that only 1.1 % of tested samples contained TTX above the reporting limit of 2 μg/kg whole shellfish flesh and these samples all originated from ten shellfish production sites in southern England. Subsequent continuous monitoring of selected areas over a five-year period showed a potential seasonal TTX accumulation in bivalves, starting in June when water temperatures reached around 15 °C. For the first time, satellite-derived data were also applied to investigate temperature differences between sites with and without confirmed presence of TTX in 2016. Although average annual temperatures were similar in both groups, daily mean values were higher in summer and lower in winter at sites where TTX was found. Here, temperature also increased significantly faster during late spring and early summer, the critical period for TTX. Our study supports the hypothesis that temperature is one of the key triggers of events leading to TTX accumulation in European bivalves. However, other factors are also likely to play an important role, including the presence or absence of a de novo biological source, which remains elusive., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-column oxidation, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay were assessed. For the majority of samples, qualitative results compared well between methods. Good statistical correlations were demonstrated between the majority of quantitative results, with a notably excellent correlation between the current EU reference method using pre-column oxidation LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins, although the findings of high numbers of false-positive results and two false negatives highlighted that some caution is still needed when interpreting results. This study demonstrated that effective replacement methods are available for countries that no longer wish to use the MBA, but highlighted the importance of comparing toxin data from the replacement method using local shellfish species of concern before implementing new methods in official control testing programs.

Bivalve molluscs represent an important food source within the Philippines, but the health of seafood consumers is compromised through the accumulation of harmful algal toxins in edible shellfish tissues. In order to assess the dynamics of toxin risk in shellfish, this study investigated the uptake, depuration, assimilation, and analogue changes of paralytic shellfish toxins in Perna viridis . Tank experiments were conducted where mussels were fed with the toxic dinoflagellate Alexandrium minutum . Water and shellfish were sampled over a six day period to determine toxin concentrations in the shellfish meat and water, as well as algal cell densities. The maximum summed toxin concentration determined was 367 µg STX eq./100 g shellfish tissue, more than six times higher than the regulatory action limit in the Philippines. Several uptake and depuration cycles were observed during the study, with the first observed within the first 24 h coinciding with high algal cell densities. Toxin burdens were assessed within different parts of the shellfish tissue, with the highest levels quantified in the mantle during the first 18 h period but shifting towards the gut thereafter. A comparison of toxin profile data evidenced the conversion of GTX1,4 in the source algae to the less potent GTX2,3 in the shellfish tissue. Overall, the study illustrated the temporal variability in Perna viridis toxin concentrations during a modelled algal bloom event, and the accumulation of toxin from the water even after toxic algae were removed.

Paralytic shellfish toxins (PST) and tetrodotoxin (TTX) are naturally-occurring toxins that may contaminate the food chain, inducing similar neurological symptoms in humans. They are co-extracted under the same conditions and thus their combined detection is desirable. Whilst PST are regulated and officially monitored in Europe, more data on TTX occurrence in bivalves and gastropods are needed before meaningful regulations can be established. In this study, we used three separate analytical methods - pre-column oxidation with liquid chromatography and fluorescence detection, ultrahigh performance hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (MS/MS) and HILIC high resolution (HR) MS/MS - to investigate the presence of PST and TTX in seawater and shellfish (mussels, clams) collected in spring summer 2015 to 2017 in the Mediterranean Sea. Samples were collected at 10 sites in the Syracuse Bay (Sicily, Italy) in concomitance with a mixed bloom of Alexandrium minutum and A. pacificum. A very high PST contamination in mussels emerged, unprecedentedly found in Italy, with maximum total concentration of 10851 μg saxitoxin equivalents per kg of shellfish tissue measured in 2016. In addition, for the first time TTX was detected in Italy in most of the analysed samples in the range 0.8-6.4 μg TTX eq/kg. The recurring blooms of PST-producing species over the 3-year period, the high PST levels and the first finding of TTX in mussels from the Syracuse bay, suggest that monitoring programmes of PST and TTX in seafood should be activated in this geographical area., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A simple, rapid UHPLC-MS/MS method has been developed and optimised for the quantitation of microcystins and nodularin in wide variety of sample matrices. Microcystin analogues targeted were MC-LR, MC-RR, MC-LA, MC-LY, MC-LF, LC-LW, MC-YR, MC-WR, [Asp3] MC-LR, [Dha7] MC-LR, MC-HilR and MC-HtyR. Optimisation studies were conducted to develop a simple, quick and efficient extraction protocol without the need for complex pre-analysis concentration procedures, together with a rapid sub 5min chromatographic separation of toxins in shellfish and algal supplement tablet powders, as well as water and cyanobacterial bloom samples. Validation studies were undertaken on each matrix-analyte combination to the full method performance characteristics following international guidelines. The method was found to be specific and linear over the full calibration range. Method sensitivity in terms of limits of detection, quantitation and reporting were found to be significantly improved in comparison to LC-UV methods and applicable to the analysis of each of the four matrices. Overall, acceptable recoveries were determined for each of the matrices studied, with associated precision and within-laboratory reproducibility well within expected guidance limits. Results from the formalised ruggedness analysis of all available cyanotoxins, showed that the method was robust for all parameters investigated. The results presented here show that the optimised LC-MS/MS method for cyanotoxins is fit for the purpose of detection and quantitation of a range of microcystins and nodularin in shellfish, algal supplement tablet powder, water and cyanobacteria. The method provides a valuable early warning tool for the rapid, routine extraction and analysis of natural waters, cyanobacterial blooms, algal powders, food supplements and shellfish tissues, enabling monitoring labs to supplement traditional microscopy techniques and report toxicity results within a short timeframe of sample receipt. The new method, now accredited to ISO17025 standard, is simple, quick, applicable to multiple matrices and is highly suitable for use as a routine, high-throughout, fast turnaround regulatory monitoring tool., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The south west coast of India has been showing a steady increase in shellfish cultivation both for local consumption and fishery export, over recent years. Perna viridis and Crassostrea madrasensis are two species of bivalve molluscs which grow in some selected regions of southern Karnataka, close to the city of Mangalore. In the early 1980s, shellfish consumers in the region were affected by intoxication from Paralytic Shellfish Poison present in local bivalves (clams and oysters) resulting in hospitalisation of many, including one fatality. Since then, there have been no further reports of serious shellfish intoxication and there is little awareness of the risks from natural toxins and no routine monitoring programme in place to protect shellfish consumers. This study presents the findings from the first ever systematic assessment of the presence of marine toxins in mussels and oysters grown in four different shellfish harvesting areas in the region. Shellfish were collected and subjected to analysis for ASP, PSP and lipophilic toxins, as well as a suite of non-EU regulated toxins such as tetrodotoxin and selected cyclic imines. Results revealed the presence of low levels of PSP toxins in oysters throughout the study period. Overall, total toxicities reached a maximum of 10% of the EU regulatory limit of 800 μg STX eq/kg. Toxin profiles were similar to those reported from the 1980 outbreak. No evidence was found for significant levels of ASP and lipophilic toxins, although some cyclic imines were detected, including gymnodimine. The results indicated that the risk to shellfish consumers during this specific study period would have been low. However, with historical evidence for extremely high levels of PSP toxins in molluscs, there is a strong need for routine surveillance of shellfish production areas for marine toxins, in order to mitigate against human health impacts resulting from unexpected harmful algal blooms, with potentially devastating socio-economic consequences., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Superficially porous column technologies have previously been shown to provide faster chromatographic analysis of toxin oxidation products when analyzing shellfish for paralytic shellfish toxins. While sub 3 μm fused core columns have facilitated enhanced method performance, including significantly lower analysis times and lower LOD, they were also found to last for only a few hundred injections before pressure increases rendered them unusable with standard HPLC. Recently 5 μm superficially porous columns have become commercially available. In this study, a 5 μm fused core column was used to develop a fast chromatographic method for the analysis of paralytic shellfish toxins, with performance characteristics and column lifetime being assessed. The 5 μm column was found to be able to perform approximately 3000 injections without significant increases in back pressure or reduction in performance. Data generated using the column were found to be equivalent to that determined using current HPLC column technologies for both screening and quantitation methods. Furthermore, an increase in sensitivity for all toxins tested under the routine monitoring program for British waters was observed and the overall run time of the analysis halved. Overall, the 5 μm fused core column provided a significant increase in sample throughput, a reduction in mobile phase consumption, and an increase in method sensitivity.

Accurate analysis of paralytic shellfish toxins (PSTs) in shellfish is important to protect seafood safety and human health. In this study, the performance of different extraction protocols for PSTs from scallop tissues is compared and discussed, including regular extraction solvents hydrochloric acid (HCl) and acetic acid (AcOH) followed by heating and solid-phase extraction (SPE) purification, and a novel technique of matrix solid-phase dispersion (MSPD) without heating. The possible conversion of C1/2 and GTX2/3 standards after heating, and the stability of PSTs in wet scallop tissues stored at -20 °C for a 6-month period are also explored. Results showed that the MSPD technique could effectively mitigate matrix interference, but its recoveries of PSTs were significantly lower than those of the HCl and AcOH extraction methods followed by carbon SPE purification. The molar concentrations of M-toxins obtained by the MSPD method were generally lower than those analyzed by the HCl and AcOH extraction methods, which demonstrated a weak chemical conversion of C1/2 and GTX2/3 due to the heating process. Most of the PSTs were relatively stable in scallop tissues during 1-month storage at -20 °C, while the concentrations of PSTs in scallop tissues obviously changed after 6 months due to the degradation and transformation of PSTs during long-term storage at -20 °C. This work helps improve our understanding of the performance of different extraction methods and the stability of PSTs in scallop tissues stored at -20 °C., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The presence of phytoplankton responsible for the production of lipophilic marine biotoxins is well recognised throughout parts of South America. To date, the quantitation of lipophilic toxins in Argentinean shellfish has been limited to select and highly focussed geographical studies. This work reports the analysis for lipophilic marine biotoxins in shellfish harvested across five regions of Argentina between 1992 and 2012. LC-MS/MS analysis was used for the quantitation of all regulated lipophilic toxins. High concentrations of okadaic acid group toxins were quantified, with a clear dominance of the parent okadaic acid and more than 90% of the toxin present as esters. Results showed DSP toxins in shellfish from the Buenos Aires Province during 2006 and 2007, earlier than previously described. There was also strong evidence linking the presence of okadaic acid to human intoxications. Other lipophilic toxins detected were yessotoxin, pectenotoxin-2 and 13-desMeC spirolide. With evidence published recently for the presence of azaspiracid producers, this work reports the detection of low concentrations of azaspiracid-2 in shellfish. As such the data provides the first published evidence for yessotoxins and azaspiracids in Argentinean shellfish and further evidence for the continuing presence of lipophilic marine toxins in Argentinean waters., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Scotia Rapid Test for PSP is designed for qualitative identification of saxitoxins at levels in shellfish equivalent to the limit of detection of the biological reference method. However, issues with the method have been reported, including the low assay cross reactivity for some toxins, high numbers of false positive results and the subjective test interpretation. This study focussed on approaches to improve each of these issues. A refined test was found to improve GTX1&4 test sensitivity in samples containing high proportions of GTX1&4. The subjectivity of the test was successfully eliminated through use of an automated scanner, which enabled both the reliable identification of test results as well as the provision of a numerical result which could be utilised for more refined results interpretation. Finally the high proportion of false positive results in comparison with the LC-FLD was investigated, with a modified approach incorporating an additional extract dilution applied to a range of shellfish samples with different toxicities. Results showed highly variable limits of detection of the method and no significant reduction in false positive results when applying the additional dilution, which may be of concern to laboratories in receipt of high numbers of samples containing low concentrations of toxins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The need for homogenous reference materials stable for paralytic shellfish toxins is vital for the monitoring and quality assurance of these potent neurotoxins in shellfish. Two stabilisation techniques were investigated, heat treatment through autoclaving and the addition of preserving additives into the tissue matrix. Short and long-term stability experiments as well as homogeneity determination were conducted on materials prepared by both techniques in comparison with an untreated control using two LC-FLD methods. Both techniques improved the stability of the matrix and the PSP toxins present compared to the controls. A material was prepared using the combined techniques of heat treatment followed by spiking with additives and data is presented from this optimised reference material as used over a two year period in the Irish national monitoring program and in a development exercise as part of a proficiency testing scheme operated by QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) since 2011. The results were indicative of the long-term stability of the material as evidenced through consistent assigned values in the case of the proficiency testing scheme and a low relative standard deviation of 10.5% for total toxicity data generated over 24 months., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A single-laboratory validation study was conducted for the hydrophilic interaction-LC-MS/MS analysis of paralytic shellfish toxins (PSTs) in bivalve shellfish. The method was developed as an alternative to the precolumn oxidation AOAC 2005.06 and postcolumn oxidation AOAC 2011.02 LC with fluorescence detection methods, receptor binding assay AOAC 2011.27, as well as the mouse bioassay AOAC 959.08. PSTs assessed were saxitoxin, neosaxitoxin, deoxydecarbamoylsaxitoxin, decarbamoylsaxitoxin, decarbamoylneosaxitoxin, gonyautoxins 1-6, decarbamoylgonyautoxins 2-3, and N-sulfocarbamoyl gonyautoxins 2&3. The method also included the determination of decarbamoylgonyautoxins 1&4, N-sulfocarbamoyl gonyautoxins 1&4, and M toxins. Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops, and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly improved in comparison to current fluorescence-based detection methods, and the method was shown to be rugged. The method performed well in comparison to AOAC 2005.06, with evidence obtained from both comparative analysis of 1141 PST-contaminated samples and successful participation in proficiency testing schemes. The method is suitable for use in regulatory testing and will be submitted for an AOAC collaborative study.

Routine regulatory monitoring of paralytic shellfish toxins (PST) commonly employs oxidative derivitisation and complex liquid chromatography fluorescence detection methods (LC-FL). The pre-column oxidation LC-FL method is currently implemented in New Zealand and the United Kingdom. When using this method positive samples are fractionated and two different oxidations are required to confirm the identity and quantity of each PST analogue present. There is a need for alternative methods that are simpler, provide faster turnaround times and have improved detection limits. Hydrophilic interaction liquid chromatography (HILIC) HPLC-MS/MS analysis of PST has been used for research purposes, but high detection limits and substantial sample matrix issues have prevented it from becoming a viable alternative for routine monitoring purposes. We have developed a HILIC UPLC-MS/MS method for paralytic shellfish toxins with an optimised desalting clean-up procedure on inexpensive carbon solid phase extraction cartridges for reduction of matrix interferences. This represents a major technical breakthrough and allows sensitive, selective and rapid analysis of paralytic shellfish toxins from a variety of sample types, including many commercially produced bivalve molluscan shellfish species. Additionally, this analytical approach avoids the need for complex calculations to determine sample toxicity, as unlike other methods each PST analogue is able to be quantified as a single resolved peak. This article presents the method development and optimisation information. A thorough single laboratory validation study has subsequently been performed and this data will be presented elsewhere., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We report the first detection of tetrodotoxins (TTX) in European bivalve shellfish. We demonstrate that TTX is present within the temperate waters of the United Kingdom, along the English Channel, and can accumulate in filter-feeding molluscs. The toxin is heat-stable and thus it cannot be eliminated during cooking. While quantified concentrations were low in comparison to published minimum lethal doses for humans, the results demonstrate that the risk to shellfish consumers should not be discarded.

In 2009, a refined HPLC method based on AOAC Official Method 2005.06 was developed and validated for the determination of paralytic shellfish poisoning (PSP) in mussels. A single-laboratory validation study of this method was undertaken here for the analysis of PSP toxins in oysters, cockles, clams, and razor clams. The method was characterized for selectivity, sensitivity, linearity, precision, repeatability, recovery, ruggedness, and uncertainty of measurement. Validation data were utilized to determine method performance characteristics for non-mussel bivalves for all commercially available certified reference toxins, extending the method to dcNEO and dcGTX2,3, where available. A period of parallel testing of oysters, cockles, and clams enabled a comparison of sample toxicities obtained using mouse bioassay (MBA) and HPLC methodologies, although only a very low number of PSP-positive samples were obtained through the United Kingdom official control monitoring program. Results from the MBA and HPLC methods were well-correlated for PSP-negative samples, but the low number of naturally contaminated PSP-positive samples has prevented any comparative statistical assessment of method performance for non-mussels between the two official methods. However, some evidence for potentially significant differences in total saxitoxin equivalents obtained by the two methods in some species has highlighted the need for further comparative testing in non-mussel samples to be conducted prior to implementation of the HPLC method in routine official control monitoring programs.

A single-laboratory validation study was undertaken for the analysis of paralytic shellfish poisoning (PSP) toxins in common mussels, extending AOAC Official Method 2005.06 to include the additional toxins dcNEO and dcGTX2,3. The method was refined to improve toxin oxidation product stability, analytical sensitivity of N-hydroxylated toxins, and throughput. Validation was performed to characterize the method for selectivity, sensitivity, linearity, precision, repeatability, recovery, ruggedness, and uncertainty. Parallel testing of naturally contaminated mussels enabled comparison of sample toxicities obtained using mouse bioassay (MBA) and high-performance liquid chromatographic (HPLC) methodologies. Performance characteristics of the method are reported for all commercially available certified reference toxins. Results from the MBA and HPLC methods were well correlated, and the analytical method has been instigated as the sole monitoring tool for UK official control surveillance of PSP toxins in common mussels.

Perna Viridis has become a common dish in daily life. Consumption of Perna Viridis, which is contaminated with diarrhetic shellfish poisoning (DSP) toxins can cause food poisoning in humans. Therefore, there is an urgent need for a rapid and accurate method to identify Perna Viridis contaminated with DSP toxins. In this study, a non-destructive method based on near-infrared spectroscopy (NIS) is proposed to detect DSP toxin-laden Perna Viridis. Spectral data of Perna Viridis were collected in the range of 950–1700 nm for DSP toxin-laden and non-laden (non-DSP) samples. The second derivative (D2) spectral preprocessing method was used to reduce the influence of noise and light scattering. A support vector machine (SVM) model based on Bayesian optimization (BO-SVM) was constructed as a classifier for the detection of the DSP toxin-laden Perna Viridis. The average accuracy for the D2-BO-SVM classifier in detecting DSP toxin-laden Perna Viridis in 50 random tests reached 99.97%. The recognition performance of the D2-BO-SVM model was analyzed to solve the problems of different ratios of training and test datasets, small sample datasets, and unbalanced class datasets in practical applications. The experimental results show that the D2-BO-SVM model was found to be superior to BO-SVM, D2-Standard SVM, and Standard SVM. These results suggest that the combination of NIS and artificial intelligence models has great potential for the detection of the DSP toxin-laden Perna Viridis. This study provides a new method for detecting the quality and safety of shellfish, which is of great practical importance for ensuring the safety of shellfish for consumers. [ABSTRACT FROM AUTHOR]

Harmful algal blooms is a widespread problem in aquatic ecosystems, in particular dinoflagellates that produce PSTs which are harmful to animal and human health. To explore the contamination status of PSTs in shellfish in the Southeastern China, a total of 2355 shellfish samples were analyzed by ultra high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of the 10 PSTs collected from the southeast coast of China from 2017 to 2021. From 2355 shellfish samples, 257 were detected (10.91%), with the highest value in samples of Perna viridis. Among the six source areas in China, the samples from Fujian recorded the highest detected rate (15.28%). PSTs were found in Fuzhou, Ningde, Quanzhou, Putian, Zhangzhou, and Xiamen, with Quanzhou and Fuzhou having the highest and lowest detection rates of 15.28% and 4.23%, respectively. Saxitoxin (STX), neosaxitoxin (neoSTX), gonyautoxin (GTX1, GTX2, GTX3, GTX4), N-sulfocarbamoyl toxin (GTX5), and decarbamoyl toxin (dcSTX, dcGTX2, dcGTX3) were detected, and GTX5 and dcGTX2 were dominant. In addition, the samples containing PSTs were mostly concentrated in May to August. The study confirms the risks of PSTs to shellfish consumers in the region. It will offer a great foundation for future monitoring of marine toxins and protecting the health of seafood consumers in China. This is the first detailed evaluation of PSTs occurrences and their profiles in shellfish from the Southeastern China over a period of multiple years. Highlights: 2355 mussels from China were analyzed by UPLC-MS/MS for PSTs in 2017–2021. The predominant PSTs were GTX5, neoSTX and dcGTX2. Arca granosa and Crassostyea gigas exhibited higher levels than other shellfish. Shellfish containing PSTs were mostly concentrated in May to August. Maximum detected level in shellfish was 2137.10 ug STXeq/kg. [ABSTRACT FROM AUTHOR]

The lethal neurotoxins, paralytic shellfish toxins (PSTs), and tetrodotoxin (TTX) have recently been found in marine shellfish from many coastal states. Herein, we applied a sensitive and reliable ultra-performance hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS) method to determine 13 PSTs and TTX in marine shellfish using a porous carbon solid-phase extraction (SPE). This in-house validation study required the development of a novel chromatographic separation using a HILIC-Z column, which was necessary to retain highly polar compounds. Using acetonitrile as the organic phase and ammonium formate-formic acid buffer as the aqueous phase, the quantitative analysis was carried out with an external standard method in the multiple reaction monitoring modes using positive electrospray ionization. To reduce interference, 1% aqueous acetic acid extracts of the shellfish samples were cleaned up by ion-pair SPE using a porous graphitic carbon cartridge. The calibration curves for PSTs and TTX were linear (R2 > 0.995), and the sensitivity was good, with limits of detection (LODs) of 1.7–13.7 µg/kg, and limits of quantitation (LOQs) of 5.2–41.0 µg/kg. The recoveries were 76.5–95.5% with RSDs of 3.1–12.0%. Finally, We applied the method for the determination of PSTs and TTX in three batches of Nassarius showing excellent method accuracy against expected values. [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]

Besides human health risks, phycotoxins may cause physiological injuries on molluscan shellfish and, consequently, damages to marine ecosystems and global fisheries production. In this way, this review aimed to present an overview of HABs impacts on marine shellfish by evaluating the effects of cultivated molluscs exposure to microalgae and cyanobacteria that form blooms and/or synthesize toxins. More specifically, it was assessed the main molluscan shellfish responses to harmful algae, trophic transfer and dynamics of phycotoxins, and the risks for human health. Of the 2420 results obtained from literature search, 150 scientific publications were selected after thorough inspections for subject adherence. In total, 70 molluscan species and 37 taxa of harmful algae were assessed from retrieved scientific publications. A significant positive correlation was found between the marine production of molluscs and the number of available studies by molluscan category. Molluscan responses to HABs and phycotoxins were categorized and discussed in three sub-sections: effects on grazing and behavior, metabolic and physiological reactions, and fitness consequences. The main histopathological injuries and toxin concentrations in molluscan tissues were also compiled and discussed. Bivalves often accumulate more toxins than gastropods and cephalopods, occasionally exceeding recommended levels for safe consumption, representing a risk for human health. Harmful algae impact on molluscan shellfish are complex to trace and predict; however, considering the perspective of increase in the occurrence and intensity of HABs, the intensification of efforts to expand the knowledge about HABs impacts on marine molluscs is crucial to mitigate the damages on economy and human health. [ABSTRACT FROM AUTHOR]

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80–400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 105Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays. [ABSTRACT FROM AUTHOR]

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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]

Bivalve molluscs can acquire marine biotoxins by filter-feeding upon certain toxin-producing microalgae. The two most common syndromes observed in temperate coastal waters have been diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP). While DSP is a non-fatal gastrointestinal syndrome, PSP is a neurological syndrome which can lead to death by respiratory paralysis in high intoxication scenarios. In Portugal, the presence of both DSP and PSP toxins leads to recurrent seasonal bans of bivalve harvesting. On a few occasions, the bans were not placed in time, not properly disseminated to the public or were disregarded by recreational harvesters. Several cases of poisonings have been studied in collaboration between health authorities and the laboratory in charge of the biotoxin monitoring programme. Some of the outbreaks have even called the attention of the local media. In several of these recorded cases, a common trait has emerged throughout the years: bivalve harvest had often been done during very low tides attributed to either new or full moons. These tides expose intertidal bivalves more widely, increase harvesting time, and allow picking of larger-sized specimens. In some occasions, the consumers were extremely unfortunate because a noxious coincidence had occurred: larger-sized specimens were available but had attained the highest toxin content of the toxic season. This review alerts that despite costly monitoring programmes have been perfected through the years, human poisonings still take place due to the rapid increase in toxin levels and/or disrespect of harvest bans. [ABSTRACT FROM AUTHOR]

A literature review was conducted to determine the barriers and facilitators to shellfish cultivation using Northern Ireland (NI) as a case study. This review included 23 papers, published in 14 journals, indexed in the Web of Science database. The eligibility criteria included (i) peer‐reviewed articles or websites from key government or agency stakeholders in the aquaculture supply chain; (ii) had a primary focus on shellfish production; and (iii) made reference to the United Kingdom, Ireland or NI. Six key categories were identified as impacting the aquaculture sector: Economic, Environmental, Technical, Welfare, Political and Consumer factors. The most commonly cited codes within these categories included regulations, management decisions, environmental welfare, weather conditions, consumer needs and water quality. Within the scientific literature, the study designs were variable and NI‐specific data were limited. However, the recommendations were relatively consistent. More evidence‐based data collection is needed to better establish the status of the aquaculture industry in this region and to provide more effective management practices and recommendations. The information gained from this review allowed the identification and discussion of the challenges and opportunities for shellfish production worldwide; an insight into production in NI; and preliminary recommendations for improving this industry. As a result, the review offers important foundations and lessons for other aquaculture production units across the world to assess their industry. [ABSTRACT FROM AUTHOR]

There are two official PSP detection methods (mouse bioassay and HLPC-FLD) and a number of alternative methods. Ethical considerations have led to regulations being adopted in some countries that limit or prohibit the application of mouse bioassay. Analytical methodologies (e.g. HPLC-FLD or LC-MSMS) have the disadvantages of not being able to detect new toxins or analogues or reflecting the overall toxicity of the sample. In addition, they require highly trained personnel and expensive equipment, which are not always available. In this work, we have evaluated a method based on the Neuro-2a cell-based assay to detect substances that inhibit voltage-dependent sodium channels (Manger's method). We tested PSP standards and natural samples contaminated with PSP. Here we demonstrate that the adapted Manger's method is suitable for calculating Toxicity Equivalency Factors (TEF) for STX-analogues. The method was shown to be useful for screening contaminated natural samples in concentrations above the regulatory limit for these toxins (80 μg STX equivalents/100 g shellfish). We were able to detect PSP in 19 natural mollusc samples from South Chile despite the presence of other marine toxins. These preliminary results suggest that the method could be used as a first step in screening programmes. [ABSTRACT FROM AUTHOR]

A rapid screening method is described for the determination of paralytic shellfish toxins (PST), in fresh marine vectors (bivalves and gastropods), at levels ranging from 0.05 to 5.0 mg STX-eq kg−1. PST are extracted from marine vector homogenates with acetic acid according to the Pre-COX-LC-FLD method. At the same time, the obtained extract is oxidised simultaneously in hydrogen peroxide and periodate oxidate to determine PST, non-N-hydroxylated and N-hydroxylated toxins, respectively. Then, they are analysed using a microplate fluorometer (Ex: 335 nm/Em: 405 nm). All the samples were compared with the liquid chromatography post-column oxidation method. Recoveries of PST added to fresh and processed marine vectors averaged 93.9% with a coefficient of variation of 6.1%. Both methods showed a good linear regression (r2 = 0.97). The method shows good intra- and inter-day precisions with a relative coefficient of variation of ≈ 3.8% and 5.7%, respectively. The limit of quantification of the rapid screening fluorescence method was ≈ 0.082 mg STX-eq kg−1, with ≤5% false positives. The established rapid screening fluorescence methods offer highly effective and verifiable pre-analyses of PST contamination in marine vectors and can be used for routine screening of the PST in seafood before formal identification by confirmatory methods (Pre-COX LC-FLD method, Lawrence method). [ABSTRACT FROM AUTHOR]

Microcystins (MCs) pose a major threat to shellfish supplies, and humans that have been in contact with or consume contaminated products. To decrease health risks to the public, an efficient and accurate method for the synchronously determination of seven harmful MCs in shellfish samples was developed by employing a hydrophile lipophile balance/polydimethylsiloxane (HLB/PDMS)-coated stir bar sorptive extraction (SBSE) method coupled with high-performance liquid chromatogram and tandem mass spectrometry (HPLC-MS/MS). The most influential factors of SBSE, sample loading pH, extraction time, and desorption time, were optimized and found to be 5, 60 min, and 10 min, respectively. For the MS ionization, all the doubly charged ions of MCs were used as precursors instead of [M + H]+ for the determination of MCs in the multiple-reaction monitoring (MRM) mode. The method was validated in terms of linearity (R2 = 0.9953~0.9991), sensitivity (LOD ≤ 0.6 μg kg−1 and LOQ ≤ 2.0 μg kg−1), precision (RSD ≤ 12.7%), and recovery (51.7 to 72.3%). With this approach, three randomly chosen batches of Patinopecten yessoensis (P. yessoensis), Haliotis discus (H. discus), and Crassostrea virginica (C. virginica) from the local market were tested. MC-RR and MC-LR were detected to be MCs positive, which were probably induced by the diet of planktonic cyanobacteria. The successfully applied HLB/PDMS-coated SBSE and HPLC-MS/MS method exhibited great advantage in fast analysis of low concentrations of MCs in foods to ensure the safety of shellfish and its products.ᅟᅟ [ABSTRACT FROM AUTHOR]

The neuro-2a bioassay is considered as one of the most promising cell-basedin vitrobioassays 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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Shellfish is a major component of global seafood production. Specific items include shrimp, lobsters, oysters, mussels, scallops, clams, crabs, krill, crayfish, squid, cuttlefish, snails, abalone, and others. Shellfish, in general, contain appreciable quantities of digestible proteins, essential amino acids, bioactive peptides, long-chain polyunsaturated fatty acids, astaxanthin and other carotenoids, vitamin B12 and other vitamins, minerals, including copper, zinc, inorganic phosphate, sodium, potassium, selenium, iodine, and also other nutrients, which offer a variety of health benefits to the consumer. Although shellfish are generally safe for consumption, their exposure to diverse habitats, the filter feeding nature of shellfish such as oysters, clams, and mussels, and unhealthy farming and handling practices may occasionally entail health risks because of possible presence of various hazards. These hazards include pathogenic organisms, parasites, biotoxins, industrial and environmental pollutants, heavy metals, process-related additives such as antibiotics and bisulfite, and also presence of allergy-causing compounds in their bodies. Most of the hazards can be addressed by appropriate preventive measures at various stages of harvesting, farming, processing, storage, distribution, and consumption. Furthermore, consumer safety of shellfish and other seafood items is strictly monitored by international, governmental, and local public health organizations. This article highlights the nutritional value and health benefits of shellfish items and points out the various control measures to safeguard consumer safety with respect to the products. [ABSTRACT FROM AUTHOR]

Contamination of shellfish with lipophilic marine biotoxins (LMB), pectenotoxins (PTXs), yessotoxins (YTXs) and okadaic acid (OA) toxin groups in southern Chile is a constant challenge for the development of miticulture considering the high incidence of toxic episodes that tend to occur. This research is focused on using methodologies for assessing the decrease in toxins of natural resources in Chile with high value, without altering the organoleptic properties of the shellfish. The species were processed through steaming (1 min at 121°C) and subsequent canning (5 min at 121°C). Changes in the profiles of toxins and total toxicity levels of LMB in endemic bivalves and gastropods were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total reduction of toxicity (≈ 15%) was not related to the destruction of the toxin, but rather to the loss of LMB on removing the shells and packing media of canned products (***p < 0.001). Industrial processing of shellfish reduces LMB contents by up to 15% of the total initial contents, concomitant only with the interconversion of PTX-group toxins into PTX-2sa. In soft bottom-dwelling species with toxicities beyond the standard for safe human consumption (≥ 160 μg OA-eq kg–1), toxicity can be reduced to safe levels through industrial preparation procedures. [ABSTRACT FROM PUBLISHER]

Matrix reference materials are an essential component for the validation and quality control of analytical methodologies for the quantitation of marine biotoxins in shellfish. Given the potential advantages of reference materials in powder form, a study was conducted to assess the feasibility for the production of a freeze-dried oyster tissue reference material containing a range of important paralytic shellfish poisoning toxins. One bulk sample of a wet oyster tissue homogenate was generated following mass culturing of toxic Alexandrium and oyster feeding experiments. The bulk tissue was used to prepare untreated wet frozen aliquots with the remainder being freeze-dried and processed into appropriately-sized powder samples. A pre-column oxidation LC-FLD analysis was used to confirm the absence of any chromatographic artefacts resulting from the processing and to confirm acceptable homogeneity of the tissues. Excellent stability over both the short-term (1 month) and long-term (1 year) of the freeze-dried material was demonstrated as compared with the stability of the untreated wet tissue. A post-column oxidation LC-FLD method was used to confirm the absence of toxin epimerisation in freeze-dried tissues which were observed in the wet tissues. Overall the work showed the feasibility of an approach to produce a homogenous freeze-dried oyster matrix material with enhanced stability in comparison to the untreated wet tissue. The potential for use of the process for preparation of large scale production batches of a freeze-dried CRM for paralytic shellfish poisoning toxins has therefore been demonstrated. [ABSTRACT FROM AUTHOR]

refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]