The Presence of Pseudo-nitzschia australis in North Atlantic Aquaculture Sites, Implications for Monitoring Amnesic Shellfish Toxins.

The farming of shellfish plays an important role in providing sustainable economic growth in coastal, rural communities in Scotland and acts as an anchor industry, supporting a range of ancillary jobs in the processing, distribution and exporting industries. The Scottish Government is encouraging shellfish farmers to double their economic contribution by 2030. These farmers face numerous challenges to reach this goal, among which is the problem caused by toxin-producing microplankton that can contaminate their shellfish, leading to harvesting site closure and the recall of product. Food Standards Scotland, a non-ministerial department of the Scottish Government, carries out a monitoring programme for both the toxin-producing microplankton and the toxins in shellfish flesh, with farms being closed when official thresholds for any toxin are breached. The farm remains closed until testing for the problematic toxin alone, often diarrhetic shellfish toxin (DST), shows the site to have dropped below the regulatory threshold. While this programme has proved to be robust, questions remain regarding the other toxins that may be present at a closed site. In this study, we tested archival material collected during site closures but only tested for DSTs as part of the official control monitoring. We found the presence of amnesic shellfish toxin (AST) in low concentrations in the majority of sites tested. In one case, the level of AST breached the official threshold. This finding has implications for AST monitoring programmes around Europe.

Combining Nanopore Sequencing with Recombinase Polymerase Amplification Enables Identification of Dinoflagellates from the Alexandrium Genus, Providing a Rapid, Field Deployable Tool.

The armoured dinoflagellate Alexandrium 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 Alexandrium 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 Alexandrium 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 Alexandrium genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different Alexandrium species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of A. minutum in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the A. catenella, A. minutum, A. tamutum, A. tamarense, A. pacificum, and A. ostenfeldii 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 A. catenella species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 (p ≤ 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 Alexandrium 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.

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.

Performance Characteristics of Refined LC-FLD and HILIC-MS/MS Methods for the Determination of Paralytic Shellfish Toxins in Shrimp, Whelk, and Crab.

BACKGROUND: Paralytic Shellfish Poison (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. OBJECTIVE: 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. METHODS: Matrices were shrimp (Crangon crangon), common whelk (Buccinum undatum), and edible crab (Cancer pagurus). The two methods assessed were the pre-column oxidation high-performance liquid chromatography-fluorescence detection AOAC 2005.06 Official Method of analysis and an internationally validated hydrophilic interaction chromatography-tandem mass spectrometry method. Brown and white crab meat were assessed separately. RESULTS: 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 mollusks. Acceptability of the majority of toxin/matrix combinations was evidenced through comparison of method performance characteristics against specific performance criteria, including Horwitz ratio values. CONCLUSIONS: Both PSP toxin detection methods were found to provide acceptable performance for the monitoring of shrimp, whelk, and crab species. HIGHLIGHTS: Two PSP toxin detection methods have been single-laboratory validated successfully for three non-bivalve shellfish species.

Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America.

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.

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.

Andean drought and glacial retreat tied to Greenland warming during the last glacial period.

Abrupt warming events recorded in Greenland ice cores known as Dansgaard-Oeschger (DO) interstadials are linked to changes in tropical circulation during the last glacial cycle. Corresponding variations in South American summer monsoon (SASM) strength are documented, most commonly, in isotopic records from speleothems, but less is known about how these changes affected precipitation and Andean glacier mass balance. Here we present a sediment record spanning the last ~50 ka from Lake Junín (Peru) in the tropical Andes that has sufficient chronologic precision to document abrupt climatic events on a centennial-millennial time scale. DO events involved the near-complete disappearance of glaciers below 4700 masl in the eastern Andean cordillera and major reductions in the level of Peru's second largest lake. Our results reveal the magnitude of the hydroclimatic disruptions in the highest reaches of the Amazon Basin that were caused by a weakening of the SASM during abrupt arctic warming. Accentuated warming in the Arctic could lead to significant reductions in the precipitation-evaporation balance of the southern tropical Andes with deleterious effects on this densely populated region of South America.

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.

The Application of Nanopore Sequencing Technology to the Study of Dinoflagellates: A Proof of Concept Study for Rapid Sequence-Based Discrimination of Potentially Harmful Algae.

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 Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium 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.

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.

Development of a TaqMan qPCR assay for detection of Alexandrium spp and application to harmful algal bloom monitoring.

The Genus Alexandrium is a widespread dinoflagellate marine phytoplankton that is the primary causative organism causing Paralytic Shellfish Poisoning (PSP) intoxications in European waters. EU food safety directives specify that EU Member States must implement a routine monitoring programme to mitigate risks associated with bio-accumulation of biotoxins by bivalve shellfish, such as those produced by Alexandrium. This strategic drive comprises of both direct testing of bivalve flesh for the presence of regulated toxins and an early warning phytoplankton monitoring programme. In the UK the flesh testing moved away from animal bio-assays to analytical chemistry techniques, whereas phytoplankton monitoring methods have seen little technological advancement since implementation. Methods currently utilize light microscopy and manual enumeration of different algal species. These methods although proven are time consuming, reliant on highly trained staff, have high limits of detection (LOD) with low specificity, unable to reliably identify Alexandrium to species level. The implications of these limitations of the techniques mean that in the case of Alexandrium the LOD is also the action limit and as such it is easy to miss positive samples affecting the efficacy of any early warning strategy. This study outlines the development, preliminary method characterisation, validation and trial implementation of an alternative early warning technique, utilizing quantitative PCR to identify water samples containing Alexandrium cells. The approach outlined in this document, showed an improved correlation with flesh toxicity, improved sensitivity, improved throughput compared to traditional light microscopy methods and there was also good correlation with higher cell abundance samples when compared to the light microscopy results. The application of this approach to routine water samples was explored and was found to demonstrate potential as a corroborative method for use during flesh intoxication episodes. This study offers potential for future improvements in the accuracy and sensitivity of phytoplankton monitoring whilst ensuring continuity of public safety, providing cost savings and offering new research opportunities.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

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 Rapid Method for the Analysis of Paralytic Shellfish Toxins Utilizing Standard Pressure HPLC: Refinement of AOAC 2005.06.

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.

South Greenland ice-sheet collapse during Marine Isotope Stage 11.

Varying levels of boreal summer insolation and associated Earth system feedbacks led to differing climate and ice-sheet states during late-Quaternary interglaciations. In particular, Marine Isotope Stage (MIS) 11 was an exceptionally long interglaciation and potentially had a global mean sea level 6 to 13 metres above the present level around 410,000 to 400,000 years ago, implying substantial mass loss from the Greenland ice sheet (GIS). There are, however, no model simulations and only limited proxy data to constrain the magnitude of the GIS response to climate change during this 'super interglacial', thus confounding efforts to assess climate/ice-sheet threshold behaviour and associated sea-level rise. Here we show that the south GIS was drastically smaller during MIS 11 than it is now, with only a small residual ice dome over southernmost Greenland. We use the strontium-neodymium-lead isotopic composition of proglacial sediment discharged from south Greenland to constrain the provenance of terrigenous silt deposited on the Eirik Drift, a sedimentary deposit off the south Greenland margin. We identify a major reduction in sediment input derived from south Greenland's Precambrian bedrock terranes, probably reflecting the cessation of subglacial erosion and sediment transport as a result of near-complete deglaciation of south Greenland. Comparison with ice-sheet configurations from numerical models suggests that the GIS lost about 4.5 to 6 metres of sea-level-equivalent volume during MIS 11. This is evidence for late-Quaternary GIS collapse after it crossed a climate/ice-sheet stability threshold that may have been no more than several degrees above pre-industrial temperatures.

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.

A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins.

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.

Feasibility studies into the production of gamma-irradiated oyster tissue reference materials for paralytic shellfish poisoning toxins.

A study was conducted to assess the feasibility for the production of sterile, stable and homogenous shellfish reference materials containing known concentrations of paralytic shellfish poisoning (PSP) toxins. Pacific oysters were contaminated with toxins following mass culturing of toxic algae and shellfish feeding experiments. Live oysters were shucked and tissues homogenised, before measuring into multiple aliquots, with one batch subjected to gamma irradiation treatment and the other remaining untreated. The homogeneity of both batches of samples was assessed using a pre-column oxidation liquid chromatography with fluorescence detection (Pre-COX LC-FLD) method and shown to be within the limits of normal within-batch repeatability. A twelve-month stability experiment was conducted for both untreated and gamma irradiated batches, specifically examining the effects of long term storage at -20 °C, +4 °C and +40 °C. Results indicated mostly good stability of PSP toxins in both materials when stored frozen at -20 °C, but with the instability of GTX2&3 concentrations in the untreated tissues eliminated in the irradiated tissues. Analysis using a post-column oxidation (PCOX) LC-FLD method also showed epimerisation in both GTX1&4 and GTX2&3 epimeric pairs in untreated samples after only 6 months frozen storage. This issue was not present in the tissues irradiated before long term storage. Biological activity testing confirmed the absence of bacteria in the irradiated samples throughout the 12 month study period. With such results there was clear evidence for the potential of increasing the scale of the mass culturing and shellfish feeding for the production of large batches of tissue suitable for the preparation of a certified matrix reference material. Overall results demonstrated the feasibility for production of oyster reference materials for PSTs, with evidence for prolonged stability following gamma irradiation treatment and storage at -20 °C.

Transformation of paralytic shellfish poisoning toxins in UK surf clams (Spisula solida) for targeted production of reference materials.

The periodic occurrence of Paralytic Shellfish Poisoning (PSP) toxins in UK surf clams and the recent move away from biological assays for PSP testing resulted in the need to determine method performance characteristics for the replacement analytical method in this species. With the requirement for laboratory reference materials to aid this validation together with known issues relating to toxin transformation in live clams and homogenised tissue, there was the need to assess the toxin transformation characteristics of PSP toxins in surf clam tissue. Initial work examined the rates of toxin transformation in UK surf clam tissue incubated with toxin standards, showing rapid transformation of N-sulfocarbamoyl toxins with slower transformation of carbamate toxins. Full transformational pathways were determined using a combination of three different analytical methods and confirmed the major expected transformations involving decarbamoylation, with some evidence for additional reaction pathways. Results obtained from the analysis of surf clam and oyster tissues incubated with varying concentrations of toxic Alexandrium algae highlighted expected transformation reactions, although significant differences were observed in the extent of the transformations amongst the range of toxins studied, with less efficient transformation of N-hydroxylated toxins as compared with other carbamate and N-sulfocarbamoyl toxins. Analysis of PSP-toxic incurred oyster, scallop and mussel tissues incubated with variable proportions of surf clam tissue showed large differences in the extent of the transformations. Total conversion of N-sulfocarbamoyl toxins was confirmed at low relative proportions of surf clam tissue in all three species, whereas transformation of carbamate toxins was found to occur only in the presence of higher proportions of surf clam tissue in oysters and mussels in comparison with scallops. Results enabled the production of three laboratory reference materials prepared following incubation of incurred homogenates with optimum proportions of surf clam tissue, resulting in materials containing a large number of PSP toxins. Stability experiments provided good preliminary evidence for the stability of these targeted materials under storage conditions. The work therefore provides both additional information relating to the transformational activity in UK surf clams and highlights a good potential method for the targeted production of reference materials which include a wider range of toxins than normally present in naturally incurred shellfish.

Rapid liquid chromatography for paralytic shellfish toxin analysis using superficially porous chromatography with AOAC Official Method 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.