Marine Biotoxins in Whole and Processed Scallops from the Argentine Sea.

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.

The Common Sunstar Crossaster papposus-A Neurotoxic Starfish.

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 (Crossaster papposus) 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 (n > 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.

Paralytic shellfish toxins and associated toxin profiles in bivalve mollusc shellfish from Argentina.

Paralytic Shellfish Poisoning is a potentially fatal syndrome, resulting from the filter-feeding activities of marine molluscs accumulating harmful neurotoxins naturally occurring in microalgae. Outbreaks are well recognised throughout most regions of the world, but with the highest levels of toxicity to date recorded in mussels from Argentina. Whilst toxicity has been documented for selected outbreaks over the years, testing has been conducted using a mouse bioassay. Consequently there is a need to establish baseline data utilising modern chemical detection methods, which also facilitate the quantification of individual toxin analogues, giving useful data on toxin profiles as well as total sample toxicity. In this study, 151 shellfish samples harvested from the marine waters of Argentina between 1980 and 2012 were subjected to analysis by liquid chromatography with fluorescence detection, since Jan 2019 the European Union reference method for PSP determination. Total PST concentrations were found to vary enormously throughout the coastline of Argentina, with higher levels of toxins found in the central regions of Rio Negro and Chubut. Toxin profiles in terms of molar percentage of total concentrations were dominated by the gonyautoxins GTX1&4 and GTX2&3, followed by C1&2, STX and dcGTX2&3, with minor levels of other analogues previously not reported in the country. Profiles were found to vary significantly, with statistical clusters of profile types associated with a wide range of factors, including species, spatial and temporal differences, as well as likely source microalgae species and potential toxin transformation pathways. Overall application of the chemical detection method has confirmed both the significant risk to shellfish consumers in Argentina with periodic outbreaks of extremely high toxin levels and a large variability in toxin profiles relating in part to previously reported variabilities in microalgal toxin content. The study has demonstrated the potential for the method to systematically study the relationships between toxicity, toxin profile, source phytoplankton and other environmental factors.

Multiple New Paralytic Shellfish Toxin Vectors in Offshore North Sea Benthos, a Deep Secret Exposed.

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 Crossaster papposus. 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 (Alcyonidium diaphanum), the first known detection in the phylum bryozoan, as well as eleven other new vectors (>50 µg STX eq./kg), namely the opisthobranch Scaphander lignarius, the starfish Anseropoda placenta, Asterias rubens, Luidia ciliaris, Astropecten irregularis and Stichastrella rosea, the brittlestar Ophiura ophiura, the crustaceans Atelecyclus rotundatus and Munida rugosa, the sea mouse Aphrodita aculeata, and the sea urchin Psammechinus miliaris. The two species that showed consistently high PST concentrations were C. papposus and A. diaphanum. 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.

Outbreak of diarrhetic shellfish poisoning associated with consumption of mussels, United Kingdom, May to June 2019.

We report on six cases of diarrhetic shellfish poisoning following consumption of mussels harvested in the United Kingdom. Dinophysis spp. in the water column was found to have increased rapidly at the production site resulting in high levels of okadaic acid-group lipophilic toxins in the flesh of consumed mussels. Clinicians and public health professionals should remain aware of algal-derived toxins being a potential cause of illness following seafood consumption.

Variability of Amnesic Shellfish Toxin and Pseudo-nitzschia occurrence in bivalve molluscs and water samples-Analysis of ten years of the official control monitoring programme.

As the official control laboratory for marine biotoxins within Great Britain, the Centre for Environment, Fisheries and Aquaculture Science, in conjunction with the Scottish Association for Marine Science, has amassed a decade's worth of data regarding the prevalence of the toxins associated with Amnesic Shellfish Poisoning within British waters. This monitoring involves quantitative HPLC-UV analysis of shellfish domoic acid concentration, the causative toxin for Amnesic Shellfish Poisoning, and water monitoring for Pseudo-nitzschia spp., the phytoplankton genus that produces domoic acid. The data obtained since 2008 indicate that whilst the occurrence of domoic acid in shellfish was generally below the maximum permitted limit of 20 mg/kg, there were a number of toxic episodes that breached this limit. The data showed an increase in the frequency of both domoic acid occurrence and toxic events, although there was considerable annual variability in intensity and geographical location of toxic episodes. A particularly notable increase in domoic acid occurrence in England was observed during 2014. Comparison of Scottish toxin data and Pseudo-nitzschia cell densities during this ten-year period revealed a complex relationship between the two measurements. Whilst the majority of events were associated with blooms, absolute cell densities of Pseudo-nitzschia did not correlate with domoic acid concentrations in shellfish tissue. This is believed to be partly due to the presence of a number of different Pseudo-nitzschia species in the water that can exhibit variable toxin production. These data highlight the requirement for tissue monitoring as part of an effective monitoring programme to protect the consumer, as well as the benefit of more detailed taxonomic discrimination of the Pseudo-nitzschia genus to allow greater accuracy in the prediction of shellfish toxicity.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: azaspiracids and yessotoxins.

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC-MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton. On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams. The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45-OH homo-YTX were detected throughout the monitored period. 45-OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45-OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs. This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: Okadaic acid, dinophysis toxins and pectenotoxins.

Official control biotoxin testing of bivalve molluscs from Great Britain has been conducted by Cefas for over a decade. Reflecting the changes in legislation, bioassays were gradually replaced by analytical methods, firstly for analysis of Paralytic shellfish toxins, followed by introduction of liquid chromatography tandem mass spectrometric (LCMS/MS) method for lipophilic toxins (LTs) in 2011. Twelve compounds, representing three main groups of regulated lipophilic toxins, as well as two non-regulated cyclic imines were examined in over 20,500 samples collected between July 2011 and December 2016. The toxins belonging to Okadaic acid (OA) group toxins were the most prevalent and were quantified in 23% of samples, predominantly from Scotland. The temporal pattern of OA group occurrences remained similar each year, peaking in summer months and tailing off during autumn and winter, however their abundance and magnitude varied between years significantly, with concentrations reaching up to 4993 µg OA eq./kg. Three toxin profiles were identified, reflecting the relative contribution of the two main toxins, OA and dinophysis toxin-2 (DTX2). Dinophysis toxin-1 (DTX1) was less common and was never detected in samples with high proportions of DTX2. Inter-annual changes in profiles were observed within certain regions, with the most notable being an increase of DTX2 occurrences in north-west Scotland and England in the last three years of monitoring. In addition, seasonal changes of profiles were identified when OA, the dominant toxin in early summer, was replaced by higher proportions of DTX2 in late summer and autumn. The profile distribution possibly reflected the availability of individual Dinophysis species as a food source for shellfish, however persistence of DTX2 during autumn and winter in mussels might have also been attributed to their physiology. Mussels were the only species with higher average proportions of non-esterified toxins, while Pacific oysters, cockles, surf clams, razors and queen scallops contained almost exclusively ester forms. In addition, a temporal change in proportion of OA and DTX2 free form was observed in mussels. Pectenotoxin-2 (PTX2) was quantified only on rare occasions.

A review of the global distribution of Alexandrium minutum (Dinophyceae) and comments on ecology and associated paralytic shellfish toxin profiles, with a focus on Northern Europe.

Alexandrium minutum is a globally distributed harmful algal bloom species with many strains that are known to produce paralytic shellfish toxins (PSTs) and consequently represent a concern to human and ecosystem health. This review highlights that A. minutum typically occurs in sheltered locations, with cell growth occurring during periods of stable water conditions. Sediment characteristics are important in the persistence of this species within a location, with fine sediments providing cyst deposits for ongoing inoculation to the water column. Toxic strains of A. minutum do not produce a consistent toxin profile, different populations produce a range of PSTs in differing quantities. Novel cluster analysis of published A. minutum toxin profiles indicates five PST profile clusters globally. Some clusters are grouped geographically (Northern Europe) while others are widely spread. Isolates from Taiwan have a range of toxin profile clusters and this area appears to have the most diverse set of PST producing A. minutum populations. These toxin profiles indicate that within the United Kingdom there are two populations of A. minutum grouping with strains from Northern France and Southern Ireland. There is a degree of interconnectivity in this region due to oceanic circulation and a high level of shipping and recreational boating. Further research into the interrelationships between the A. minutum populations in this global region would be of value.

Fatal Canine Intoxications Linked to the Presence of Saxitoxins in Stranded Marine Organisms Following Winter Storm Activity.

At the start of 2018, multiple incidents of dog illnesses were reported following consumption of marine species washed up onto the beaches of eastern England after winter storms. Over a two-week period, nine confirmed illnesses including two canine deaths were recorded. Symptoms in the affected dogs included sickness, loss of motor control, and muscle paralysis. Samples of flatfish, starfish, and crab from the beaches in the affected areas were analysed for a suite of naturally occurring marine neurotoxins of dinoflagellate origin. Toxins causing paralytic shellfish poisoning (PSP) were detected and quantified using two independent chemical testing methods in samples of all three marine types, with concentrations over 14,000 µg saxitoxin (STX) eq/kg found in one starfish sample. Further evidence for PSP intoxication of the dogs was obtained with the positive identification of PSP toxins in a vomited crab sample from one deceased dog and in gastrointestinal samples collected post mortem from a second affected dog. Together, this is the first report providing evidence of starfish being implicated in a PSP intoxication case and the first report of PSP in canines.

Analysis of Microcystins in Cyanobacterial Blooms from Freshwater Bodies in England.

Cyanobacterial blooms in freshwater bodies in England are currently monitored reactively, with samples containing more than 20,000 cells/mL of potentially toxin-producing species by light microscopy resulting in action by the water body owner. Whilst significantly reducing the risk of microcystin exposure, there is little data describing the levels of these toxins present in cyanobacterial blooms. This study focused on the quantitative LC-MS/MS analysis of microcystins in freshwater samples, collected across England during 2016 and found to contain potentially toxin-producing cyanobacteria. More than 50% of samples contained quantifiable concentrations of microcystins, with approximately 13% exceeding the WHO medium health threshold of 20 µg/L. Toxic samples were confirmed over a nine-month period, with a clear increase in toxins during late summer, but with no apparent geographical patterns. No statistical relationships were found between total toxin concentrations and environmental parameters. Complex toxin profiles were determined and profile clusters were unrelated to cyanobacterial species, although a dominance of MC-RR was determined in water samples from sites associated with lower rainfall. 100% of samples with toxins above the 20 µg/L limit contained cell densities above 20,000 cells/mL or cyanobacterial scum, showing the current regime is suitable for public health. Conversely, with only 18% of cell density threshold samples having total microcystins above 20 µg/L, there is the potential for reactive water closures to unnecessarily impact upon the socio-economics of the local population. In the future, routine analysis of bloom samples by LC-MS/MS would provide a beneficial confirmatory approach to the current microscopic assessment, aiding both public health and the needs of water users and industry.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Variability of paralytic shellfish toxin occurrence and profiles in bivalve molluscs from Great Britain from official control monitoring as determined by pre-column oxidation liquid chromatography and implications for applying immunochemical tests.

As the official control monitoring laboratory in Great Britain for the analysis of marine biotoxins in shellfish, Cefas have for the past five years conducted routine monitoring for paralytic shellfish poisoning toxins (PST) using a non-animal alternative method to the mouse bioassay reference method; a refined version of the AOAC 2005.06 pre-column oxidation liquid chromatography method. Application of this instrumental methodology has enabled the generation of data not only on the occurrence and magnitude of PST events, but also the quantitation and assessment of different PST profiles. Since implementation of the method in 2008, results have shown huge variabilities in the occurrence of PSTs, with large spatial and temporal variabilities around the coastline. Mean PST profiles were not found to correlate either with total PST content of the shellfish, the year of sampling or with a few notable exceptions, the shellfish species. Toxin profiles were found to fall into one of four distinct profile types, with one relating solely to the exclusive presence of decarbamoyl toxins in surf clams. The other profile types contained variable proportions of gonyautoxins, N-sulfocarbamoyl toxins, neosaxitoxin and saxitoxin. While some indications of geographical repeatability were noted, this was not observed for all profile types. Consequently, the application of rapid immunochemical testing methods to end product testing would need to be considered carefully given the large differences in PST congener cross-reactivities.