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.
•Recurring blooms of Alexandrium minutum and Alexandrium pacificum in Syracuse bay.•Toxin profile and content of seawater and shellfish collected in 2015, 2016, 2017.•Paralytic shellfish toxins contaminating mussels at levels up to 10851 μg STX eq/Kg.•First finding in Italy of tetrodotoxin in mussels at levels up to 6.4 μg TTX eq/Kg.•Comparison among PreCOX-HPLC-FLD, UHPLC-HILIC-MS/MS, and HPLC-HILIC-HRMS for PST.
PSP toxins have been reported in non-bivalve shellfish species, including crustaceans and gastropods. Routine surveillance of these species is currently conducted in parts of England. To date ...detection methods have not been validated for these matrices. Validation is required to ensure the test is fit for purpose, to give greater confidence in any results generated and ultimately facilitates accreditation.
The aim was to test and validate two independent PSP toxin detection methods previously validated for bivalve shellfish matrices, for applicability to commercial non-bivalve species of interest.
Matrices were shrimp (Crangon crangon), common whelk (Buccinum undatum) and edible crab (Cancer pagurus). The two methods assessed were the pre-column oxidation LC-FLD AOAC 2005.06 Official Method of analysis and an internationally validated HILIC-MS/MS method. Brown and white crab meat were assessed separately.
A refined extraction protocol was implemented with an increased solvent to sample ratio. The same extraction protocol was utilized for both methods, allowing both methods to be run simultaneously. Method sensitivity, recovery, repeatability, and method uncertainty were characterized in all matrix/toxin combinations. Overall, both methods performed similarly to that previously reported in bivalve molluscs. Acceptability of the majority of toxin/matrix combinations was evidenced through comparison of method performance characteristics against specific performance criteria, including Horwitz ratio values.
Both PSP toxin detection methods were found to provide acceptable performance for the monitoring of shrimp, whelk and crab species.
Two PSP toxin detection methods have been single-laboratory validated successfully for three non-bivalve shellfish species.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human ...health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus
. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on
the applicability to three additional genera of toxic algae, namely;
,
, and
was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations "one health" approach to marine monitoring.
The armoured dinoflagellate
can be found throughout many of the world's temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members ...produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant threat to animal and environmental health. Moreover, the consumption of bivalve molluscs contaminated with saxitoxin poses a threat to human health. The identification of
cells collected from sea water samples using light microscopy can provide early warnings of a toxic event, giving harvesters and competent authorities time to implement measures that safeguard consumers. However, this method cannot reliably resolve
to a species level and, therefore, is unable to differentiate between toxic and non-toxic variants. The assay outlined in this study uses a quick recombinase polymerase amplification and nanopore sequencing method to first target and amplify a 500 bp fragment of the ribosomal RNA large subunit and then sequence the amplicon so that individual species from the
genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different
species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of
in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the
,
,
,
,
, and
species from environmental samples, with just the alignment of the reads being sufficient to provide accurate, real-time species identification. By using sequencing data to qualify when the toxic
species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 (
≤ 0.05). Furthermore, a McNemar's paired test performed on qualitative data highlighted no statistical differences between samples confirmed positive or negative for toxic species of
by both phylogenetic analysis and real time alignment with the presence or absence of toxins in shellfish. The assay was designed to be deployed in the field for the purposes of in situ testing, which required the development of custom tools and state-of-the-art automation. The assay is rapid and resilient to matrix inhibition, making it suitable as a potential alternative detection method or a complementary one, especially when applying regulatory controls.
In early 2018, a large easterly storm hit the East Anglian coast of the UK, colloquially known as the 'Beast from the East', which also resulted in mass strandings of benthic organisms. There were ...subsequent instances of dogs consuming such organisms, leading to illness and, in some cases, fatalities. Epidemiological investigations identified paralytic shellfish toxins (PSTs) as the cause, with toxins present in a range of species and concentrations exceeding 14,000 µg STX eq./kg in the sunstar
. This study sought to better elucidate the geographic spread of any toxicity and identify any key organisms of concern. During the summers of 2018 and 2019, various species of benthic invertebrates were collected from demersal trawl surveys conducted across a variety of locations in the North Sea. An analysis of the benthic epifauna using two independent PST testing methods identified a 'hot spot' of toxic organisms in the Southern Bight, with a mean toxicity of 449 µg STX eq./kg. PSTs were quantified in sea chervil (
), the first known detection in the phylum bryozoan, as well as eleven other new vectors (>50 µg STX eq./kg), namely the opisthobranch
, the starfish
,
and
, the brittlestar
, the crustaceans
and
, the sea mouse
, and the sea urchin
. The two species that showed consistently high PST concentrations were
and
. Two toxic profiles were identified, with one dominated by dcSTX (decarbamoylsaxitoxin) associated with the majority of samples across the whole sampling region. The second profile occurred only in North-Eastern England and consisted of mostly STX (Saxitoxin) and GTX2 (gonyautoxin 2). Consequently, this study highlights widespread and variable levels of PSTs in the marine benthos, together with the first evidence for toxicity in a large number of new species. These findings highlight impacts to 'One Health', with the unexpected sources of toxins potentially creating risks to animal, human and environmental health, with further work required to assess the severity and geographical/temporal extent of these impacts.
•Toxin profiles can be categorised statistically.•UK Alexandrium species produce statistically separable toxin profiles.•Source microalgal species affects the paralytic toxin profile of ...shellfish.•Chemotaxonomic analysis of toxin profiles can allow inference of causative species.•Chemotaxonomy can enhance monitoring programmes with little added expense.
Although phytoplankton is ubiquitous in the world's oceans some species can produce compounds that cause damaging effects in other organisms. These include the toxins responsible for paralytic shellfish poisoning, which, in UK waters, are produced by dinoflagellates from the Alexandrium genus. Within Great Britain (GB) a monitoring programme exists to detect this harmful genus as well as the Paralytic Shellfish Poisoning (PSP) toxins in the flesh of shellfish from classified production areas. The techniques used for toxin analysis allow for detailed analysis of the toxin profiles present in contaminated shellfish. It is possible to compare the toxin profiles of contaminated shellfish with the profiles from toxin producing algae and use this information to infer the causative microalgal species responsible for the contamination. This study sought to evaluate the potential for this process within the GB monitoring framework. Two species of toxic Alexandrium, A. catenella from Scotland and A. minutum from Southern England, were fed to mussels (Mytilus sp.) under controlled conditions. The toxin profile in mussels derived from feeding on each species independently, when mixed and when introduced sequentially was analysed and compared to the source algal cultures using K means cluster analysis. Toxin profiles in contaminated shellfish clustered with those of the causative algae and separately from one another during toxin accumulation and, where A. catenella was the sole toxin source, during depuration. During depuration after feeding with A. minutum and where mixed or sequential feeding was undertaken deviant toxin profiles were observed. Finally, data generated within this experimental study were compared to monitoring data from the GB official control programme. These data indicated that the causative algal species in sole source contaminations could be inferred from toxin profile analysis. This technique will be of benefit within monitoring programmes to enhance the value of data with minimal additional expense, where the toxin profiles of causative microalgae have been well described.
Microcystins and nodularins, produced naturally by certain species of cyanobacteria, have been found to accumulate in aquatic foodstuffs such as fish and shellfish, resulting in a risk to the health ...of the seafood consumer. Monitoring of toxins in such organisms for risk management purposes requires the availability of certified matrix reference materials to aid method development, validation and routine quality assurance. This study consequently targeted the preparation of a mussel tissue reference material incurred with a range of microcystin analogues and nodularins. Nine targeted analogues were incorporated into the material as confirmed through liquid chromatography with tandem mass spectrometry (LC-MS/MS), with an additional 15 analogues detected using LC coupled to non-targeted high resolution mass spectrometry (LC-HRMS). Toxins in the reference material and additional source tissues were quantified using LC-MS/MS, two different enzyme-linked immunosorbent assay (ELISA) methods and with an oxidative-cleavage method quantifying 3-methoxy-2-methyl-4-phenylbutyric acid (MMPB). Correlations between the concentrations quantified using the different methods were variable, likely relating to differences in assay cross-reactivities and differences in the abilities of each method to detect bound toxins. A consensus concentration of total soluble toxins determined from the four independent test methods was 2425 ± 575 µg/kg wet weight. A mean 43 ± 9% of bound toxins were present in addition to the freely extractable soluble form (57 ± 9%). The reference material produced was homogenous and stable when stored in the freezer for six months without any post-production stabilization applied. Consequently, a cyanotoxin shellfish reference material has been produced which demonstrates the feasibility of developing certified seafood matrix reference materials for a large range of cyanotoxins and could provide a valuable future resource for cyanotoxin risk monitoring, management and mitigation.
Saxitoxins (STXs) are a family of potent neurotoxins produced naturally by certain species of phytoplankton and cyanobacteria which are extremely toxic to mammalian nervous systems. The accumulation ...of STXs in bivalve molluscs can significantly impact animal and human health. Recent work conducted in the North Sea highlighted the widespread presence of various saxitoxins in a range of benthic organisms, with the common sunstar (
) demonstrating high concentrations of saxitoxins. In this study, an extensive sampling program was undertaken across multiple seas surrounding the UK, with 146 starfish and 5 brittlestars of multiple species analysed for STXs. All the common sunstars analysed (
> 70) contained quantifiable levels of STXs, with the total concentrations ranging from 99 to 11,245 µg STX eq/kg. The common sunstars were statistically different in terms of toxin loading to all the other starfish species tested. Two distinct toxic profiles were observed in sunstars, a decarbomylsaxitoxin (dcSTX)-dominant profile which encompassed samples from most of the UK coast and an STX and gonyautoxin2 (GTX2) profile from the North Yorkshire coast of England. Compartmentalisation studies demonstrated that the female gonads exhibited the highest toxin concentrations of all the individual organs tested, with concentrations >40,000 µg STX eq/kg in one sample. All the sunstars, male or female, exhibited the presence of STXs in the skin, digestive glands and gonads. This study highlights that the common sunstar ubiquitously contains STXs, independent of the geographical location around the UK and often at concentrations many times higher than the current regulatory limits for STXs in molluscs; therefore, the common sunstar should be considered toxic hereafter.
Harmful algal blooms are an increasing worldwide threat to the seafood industry and human health as a consequence of the natural production of biotoxins that can accumulate in shellfish. In the ...Argentine Sea, this has been identified as an issue for the offshore fisheries of Patagonian scallops (Zygochlamys patagonica), leading to potentially harmful effects on consumers. Here we assess spatial and temporal patterns in marine biotoxin concentrations in Patagonian scallops harvested in Argentinian waters between 2012–2017, based on analyses for paralytic shellfish toxins, lipophilic toxins, and amnesic shellfish toxins. There was no evidence for concentrations of lipophilic or amnesic toxins above regulatory acceptance thresholds, with trace concentrations of pectenotoxin 2, azaspiracid 2 and okadaic acid group toxins confirmed. Conversely, paralytic shellfish toxins were quantified in some scallops. Gonyautoxins 1 and 2 dominated the unusual toxin profiles (91%) in terms of saxitoxin equivalents with maximum concentrations reaching 3985 µg STX eq/kg and with changes in profiles linked in part to seasonal changes. Total toxin concentrations were compared between samples of the adductor muscle and whole tissue, with results showing the absence of toxins in the adductor muscle confirming toxin accumulation in the digestive tracts of the scallops and the absence of a human health threat following the processing of scallop adductor meat. These findings highlight that paralytic shellfish toxins with an unusual toxin profile can occur in relatively high concentrations in whole Patagonian scallops in specific regions and during particular time periods, also showing that the processing of scallops on board factory ships to obtain frozen adductor muscle is an effective management process that minimizes the risk of poisonings from final products destined for human consumption.
Abstract
Background
Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish ...samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods.
Objective
The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods.
Methods
Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores.
Results
Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC–MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC–MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC–MS/MS.
Conclusion
The results from this study demonstrate that current LC–MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC–MS/MS protocol to further improve interlaboratory precision.
Highlights
Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
