Paralytic Shellfish Toxins In Mussels Research Articles

Determination of hydrophilic marine biotoxins in bivalve molluscs by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

This work describes a method for the determination of paralytic shellfish toxins in mussels (M. galloprovincialis), oysters (Crassostrea gigas), and clams (Ruditapes decussatus) samples. The acetic acid extraction method is used for simultaneous analysis of Paralytic Shellfish Poisoning (PSP) and the separation of these compounds was achieved on ethylene bridged hybrid (BEH) Amide column with gradient mobile phase composed of acetonitrile (MeCN) and water (as eluent A and eluent B) both containing 2 mM ammonium formate and 50 mM formic acid. The method specificity, linearity, sensitivity, precision, and accuracy were successfully evaluated towards the detection of 13 hydrophilic marine biotoxins analyzed together (single run) in different sample matrixes such as mussels. oysters. and clams. Calibration curves were found to be linear (R2 ≥ 0.98). average Recovery% of 98.5–99.9%. limits of detection (LODs): 0.16–1.92 µg·kg−1; limits of quantitation (LOQs): 0.54–6.41 µg·kg−1) and the relative standard deviation (RSD%) of the precision was ≤ 2.8%. The method was applied for the determination of PSP in shellfish collected from several Tunisian locations. All LC-MS/MS results of analyzed molluscs collected from different Tunisia locations, reveal no PSP toxin group contamination and that the amount found does not exceeding the threshold of 800 µg STXequ·kg−1.

Detection of paralytic shellfish toxins by near-infrared spectroscopy based on a near-Bayesian SVM classifier with unequal misclassification costs.

Paralytic shellfish poisoning caused by human consumption of shellfish fed on toxic algae is a public health hazard. It is essential to implement shellfish monitoring programs to minimize the possibility of shellfish contaminated by paralytic shellfish toxins (PST) reaching the marketplace. This paper proposes a rapid detection method for PST in mussels using near-infrared spectroscopy (NIRS) technology. Spectral data in the wavelength range of 950-1700 nm for PST-contaminated and non-contaminated mussel samples were used to build the detection model. Near-Bayesian support vector machines (NBSVM) with unequal misclassification costs (u-NBSVM) were applied to solve a classification problem arising from the fact that the quantity of non-contaminated mussels was far less than that of PST-contaminated mussels in practice. The u-NBSVM model performed adequately on imbalanced datasets by combining unequal misclassification costs and decision boundary shifts. The detection performance of the u-NBSVM did not decline as the number of PST samples decreased due to adjustments to the misclassification costs. When the number of PST samples was 20, the G-mean and accuracy reached 0.9898 and 0.9944, respectively. Compared with the traditional support vector machines (SVMs) and the NBSVM, the u-NBSVM model achieved better detection performance. The results of this study indicate that NIRS technology combined with the u-NBSVM model can be used for rapid and non-destructive PST detection in mussels. © 2023 Society of Chemical Industry.

LC-HRMS Profiling of Paralytic Shellfish Toxins in Mytilus galloprovincialis after a Gymnodinium catenatum Bloom

Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels' whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs' profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity.

Recent findings of paralytic shellfish toxins linked to the genus Alexandrium Halim in Mediterranean mollusc production areas

Paralytic shellfish poisoning is a human intoxication syndrome associated with the consumption of seafood that has been contaminated with paralytic shellfish toxins (PSTs), a group of natural neurotoxic alkaloids produced by marine dinoflagellates, including some Alexandrium species. This study presents findings of PSTs in mussels (Mytilus galloprovincialis) during 2018–2019 in several mollusc production areas of Sardinia (Italy, western Mediterranean). Investigations of the presence and abundance of PST-producing microalgal species in marine water and of the toxins associated with shellfish were carried out concomitantly. Overall, the results suggested a spatio-temporal expansion of Alexandriumpacificum and Alexandriumminutum in recent years, with an increasing number of PSTs present in molluscs and increased occurrences of toxicity cases. Liquid chromatography with fluorescence detection determined the toxin profile to be composed primarily of the carbamate gonyautoxin-5 and N-sulphocarbamoyltoxins 1 and 2. The study highlights the potential high risk to consumers of poisoning by bivalve molluscs bred in Sardinia, where shellfish production is a very important industrial sector. For this reason, routine monitoring is strongly recommended in order to mitigate any harm to human health as well as negative socio-economic consequences.

Accumulation and Elimination Dynamics of the Hydroxybenzoate Saxitoxin Analogues in Mussels Mytilus galloprovincialis Exposed to the Toxic Marine Dinoflagellate Gymnodinium catenatum.

Paralytic shellfish poisoning (PSP) is a severe food-borne illness, caused by the ingestion of seafood containing paralytic shellfish toxins (PST), which are naturally produced by marine dinoflagellates and accumulate in shellfish during algae blooms. Novel PST, designated as hydroxybenzoate analogues (also known as GC toxins), was relatively recently discovered in Gymnodinium catenatum strains worldwide. However, to date, there have been no studies examining their accumulation in shellfish. In this study, mussels (Mytilus galloprovincialis) were exposed to G. catenatum for five days and then exposed to a non-toxic diet for 24 h, to investigate the toxin’s accumulation/elimination dynamics. As determined by UHPLC-HILIC-MS/MS, the hydroxybenzoate analogues, GC1 to GC6, comprised 41% of the algae toxin profile and only 9% in mussels. Elimination of GC toxins after 24 h was not evident. This study highlights that a relevant fraction of PST in mussels are not routinely analysed in monitoring programs and that there is a need to better understand the toxicological potential of the hydroxybenzoate analogues, in order to properly address the risk of G. catenatum blooms.

Single-Laboratory Validation of the Neogen Qualitative Lateral Flow Immunoassay for the Detection of Paralytic Shellfish Toxins in Mussels and Oysters.

Detection of paralytic shellfish toxins (PSTs) in bivalve shellfish by analytical methods is complicated and costly, requiring specific expertise and equipment. Following extensive blooms of Alexandrium tamarense Group 1 in Tasmania, Australia, an investigation was made into commercially available screening test kits suitable for use with the toxin profiles found in affected bivalves. The qualitative Neogen rapid test kit, with a modified protocol to convert gonyautoxins GTX1&4 and GTX2&3 into neosaxitoxin and saxitoxin (STX), respectively, with higher cross-reactivities, was the best fit-for-purpose. This validation study of the test kit and the modified protocol was undertaken following AOAC INTERNATIONAL guidelines for the validation of qualitative binary chemistry methods. The validation used four different PST profiles representing natural profiles found in Australia and in Europe: two in a mussel matrix and two in an oyster matrix. The test kit was shown to have appropriate selectivity of the toxin analogs commonly found in bivalve shellfish. The matrix and probability of detection (POD) study showed that the rapid test kit used with the modified protocol was able to consistently detect PST at the bivalve regulatory level of 0.8 mg STX⋅2HCl eq/kg, with a POD estimated via the binomial logistic regression of 1.0 at 0.8 mg STX⋅2HCl eq/kg in all tested profiles in both matrixes. The POD at 0.4 mg STX⋅2HCl eq/kg was 0.75 and 0.46 for the two toxin profiles in an oyster matrix and 0.96 and 1.0 for the two toxin profiles in a mussel matrix. No significant differences in the PODs of the PSTs at the regulatory level were found between production lots of the test kits. The results suggest the method is suitable to undergo a collaborative validation study.

A comparison of the physiological responses, behaviour and biotransformation of paralytic shellfish poisoning toxins in a surf-clam (Paphies donacina) and the green-lipped mussel (Perna canaliculus)

The accumulation of paralytic shellfish toxins (PSTs) in bivalves is species specific. We compared the physiological responses and the toxin profiles in tissues of the burrowing surf clam, Paphies donacina, and the green-lipped mussel, Perna canaliculus, exposed to the toxic dinoflagellate Alexandrium tamarense. Bivalves were supplied with the toxic algae for 10 days, then allowed a detoxification period of 8 days. Clearance rates of mussels and clams were similar when fed either with toxic A. tamarense or non-toxic A. margalefi. Byssus production in the mussel was inhibited and exhalent siphon activity in clams was erratic following exposure to A. tamarense. There were considerable differences in the toxic profile between the dinoflagellate A. tamarense, and tissues of the mussel and the surf clam, indicating that bioconversion of the PSTs had taken place. Toxin profiles of the tissues were both species and tissue specific. Following an 8-day detoxification period, total PSTs in mussels had fallen to safe concentrations below 50µg per 100g, whereas concentrations in clams remained high, with an average value greater than 600µg STX di-HCL equivalents per 100g. The results confirmed that mussels and clams are important monitoring organisms for toxic algal blooms and can be used to minimise the health risk of PSTs to humans.

Rapid Liquid Chromatography for Paralytic Shellfish Toxin Analysis Using Superficially Porous Chromatography with AOAC Official MethodSM 2005.06

The bioaccumulation of paralytic shellfish toxins in mussels, oysters, cockles, hard clams, razors, and king scallops is monitored in England, Scotland, and Wales by AOAC Official Method 2005.06 LC-with fluorescence detection (FLD). One of the commonly perceived disadvantages of using this method is the long turnaround time and low throughput in a busy laboratory environment. The chromatographic analysis of each sample typically utilizes a 15 min cycle time to achieve toxin oxidation product separation and column equilibration prior to subsequent analysis. A standard RP C18 analytical column, used successfully in recent years, achieves good separation with a long column lifetime. The analysis of a 40 sample qualitative screening batch takes approximately 18 h, including blanks, standards, and other QC samples. The availability of superficially porous column technology has offered the potential to reduce analysis time while retaining column performance on existing hardware. In this study, AOAC Official Method 2005.06 with LC-FLD was transferred to two different commercially available superficially porous columns, and the method performance characteristics were evaluated. Both columns separated all toxins adequately with cycle times less than half that of the existing method. Linearity for each toxin was acceptable up to two times the European maximum permitted limit of 800 microg di-HCl saxitoxin equivalent/kg flesh. LOD and LOQ values were substantially improved for the majority of toxins, with gonyautoxin 1&4 and neosaxitoxin showing up to a two- and fourfold improvement, respectively, depending on the column used. Quantification results obtained from parallel analysis of contaminated samples were acceptable on both columns. Comparative screen results gave a slight increase in the occurrence of contaminated samples, which was attributed to the improved detection limit for most toxins. Issues with rapidly increasing back pressure, however, were identified with both columns, with a limit of around 500 injections. This compares to the >3000 cycles routinely obtained with the standard RP-C18 HPLC columns currently in use. Overall, the gain achieved with these columns through shorter analysis time and improved analytical sensitivity is potentially of benefit in a high-throughput environment. For the routine high-throughput screening of shellfish samples, however, an improved column lifetime is desirable.

Liquid Chromatography Post-Column Oxidation (PCOX) Method for the Determination of Paralytic Shellfish Toxins in Mussels, Clams, Oysters, and Scallops: Collaborative Study

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfish toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specific analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoyl-gonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official Method 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official Method 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfish samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profiles of individual toxins. These concentrations are approximately equivalent to 1/2 maximum level (ML), ML, or 2xML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX diHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director's laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Official Method 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certified reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfish tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBAAOAC Official Method 959.08, and a detailed profile of the individual toxin present in the sample.

Liquid chromatographic post-column oxidation method for analysis of paralytic shellfish toxins in mussels, clams, scallops, and oysters: single-laboratory validation.

A single-laboratory validation study was conducted for the LC post-column oxidation analysis of paralytic shellfish toxins (PST): saxitoxin (STX); neosaxitoxin (NEO); gonyautoxins (GTX) 1-5; decarbamoyl gonyautoxins (dcGTX) 2 and 3; decarbamoyl saxitoxin (dcSTX); and N-sulfocarbamoyl-gonyautoxin-2 and 3 (C1 and C2) in mussels (Mytilus edulis), soft shell clams (Mya arenaria), scallops (Placopectin magellanicus), and oysters (Crassostrea virginicus). The instrumental technique was developed for the analysis of PST in shellfish as an alternative to the precolumn oxidation method, AOAC Official Method 2005.06, and a replacement for the current AOAC biological method 959.08. The method used reversed-phase LC with post-column oxidation and fluorescence detection. Test materials for method recovery were prepared by fortification of blank material with a cocktail of PST. Materials used to determine method repeatability and intermediate precision were prepared by blending blank material with naturally incurred material. The target total toxicity levels evaluated in the study were 0.40, 0.80, and 1.60 mg STX x diHCl equivalents per kilogram [(eq/kg) 1%, 1, and 2 times the regulatory limit]. Linearity, recovery, and within-laboratory precision parameters of the method were evaluated. Correlation coefficients of the calibration curves for all toxins studied were > 0.99. Total toxin recovery ranged from 94 to 106% at the three levels of interest. Repeatability and intermediate precision RSD ranged from 2 to 7% and 2 to 8%, respectively. The method LOD and LOQ (assuming the presence of all toxins) were determined to be equivalent to 0.18 and 0.39 mg STX x diHCl eq/kg. The method is intended for a regulatory framework and will be submitted for an AOAC collaborative study.

The uptake, distribution and elimination of paralytic shellfish toxins in mussels and fish exposed to toxic dinoflagellates

We exposed green-lipped mussels Perna viridis and black sea breams Acanthopagrus schlegeli to toxic dinoflagellates Alexandrium fundyense to evaluate the accumulation, distribution, transformation, and elimination of paralytic shellfish toxins (PSTs) in a controlled environmental condition. The mussels were fed A. fundyense for 7 days followed by 3 weeks of depuration, and the fish were fed toxic clams (pre-exposed to the dinoflagellates) for 5 days followed by 2 weeks of depuration. The toxin content and the compartmental distribution of PSTs were monitored throughout the experiments by high-performance liquid chromatography with post-column fluorescence derivatization (HPLC-FLD). This is the first report to assess the biokinetics of PSTs in marine fish under dietary exposure. The hepatopancreas in the mussels and the viscera in the fish accumulated most of the PSTs. Differential elimination of each toxin was observed in the mussels. The C2 toxins were eliminated rapidly in all organs; except in hepatopancreas, the more potent toxins such as GTX4, were eliminated slower during the depuration period. The relative proportions of various PSTs in the mussels changed over time, suggesting toxin-specific uptake and elimination rates, or biotransformation preferences between toxins. In the fish, the ratio of C1/C2 was 3.0 times ( p < 0.01) higher when compared to the clam tissues, indicating that conversion from C2 to C1 might have occurred when the toxin was transferred from the clams to the fish. In summary, species differences in uptake, distribution and elimination of PSTs were observed between mussels and fish, and this may influence trophic transfer of algal toxins in marine organisms.

A Fluorimetric Method Based on Changes in Membrane Potential for Screening Paralytic Shellfish Toxins in Mussels

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na + channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 μg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.