First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum

During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 103 cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumersThe research leading to USC results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. In addition from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 315285 CIGUATOOLS and 312184 PHARMASEA. Inés Rodriguez is supported by a fellowship from Subprograma de Formación de Personal Investigador (AGL2012-40185-CO2-01), Spain. In depth investigation of the toxic episodes leading to the results and publication of the present work was undertaken by the Greek National Reference Laboratory of Marine Biotoxins (NRLMB) to fulfill the requirements of EU Regulation 178/2002/EC (Articles 6 and 7) regarding risk analysis and communication and scientific information needed for risk assessment and EU Regulation 882/2004/EC (article 7) with regard to transparency and information to the public. Collaboration of all the staff of the NRLMB is greatly appreciated. Thanks are also expressed to all the Greek regional veterinary services for their contribution to the shellfish samplings and for provision of the seawater analyses results for the presence of potentially toxic microalgae. The use of cell counts’ data within the period 2006–2009 and 2012 regarding P. minimum presence in seawater, derived from the Greek “National Programme for Monitoring of Bivalve Molluscs’ Production Areas for the presence of Marine Biotoxins” and conducted by the Laboratory Unit of Toxic Marine Microalgae (LUTMM), Department of Biology, Aristotle University of Thessaloniki (scientific coordinator: G. Nikolaidis (until February 2010) and M. Arsenakis (March 2010–to date)), as well as the restrictions of LUTMM regarding the use of data produced within 2013–2015 due to contract terms and ISO 17025 requirements are acknowledgedS

Palytoxin and Analogs: Biological and Ecological Effects

Palytoxin (PTX) is a potent marine toxin that was originally found in soft corals from tropical areas of the Pacific Ocean. Soon after, its occurrence was observed in numerous other marine organisms from the same ecological region. More recently, several analogs of PTX were discovered, remarkably all from species of the dinoflagellate genus Ostreopsis. Since these dinoflagellates are also found in other tropical and even in temperate regions, the formerly unsuspected broad distribution of these toxins was revealed. Toxicological studies with these compounds shows repeatedly low LD50 values in different mammals, revealing an acute toxic effect on several organs, as demonstrated by different routes of exposure. Bioassays tested for some marine invertebrates and evidences from environmental populations exposed to the toxins also give indications of the high impact that these compounds may have on natural food webs. The recognition of its wide distribution coupled with the poisoning effects that these toxins can have on animals and especially on humans have concerned the scientific community. In this paper, we review the current knowledge on the effects of PTX and its analogs on different organisms, exposing the impact that these toxins may have in coastal ecosystems

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INaInés Rodríguez is supported by a fellowship from Subprograma de Formación de Personal Investigador MINECO (AGL2012-40185-CO2-01), Spain. The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA.S

Development of a novel smoke-flavoured salmon product by sodium replacement using water vapour permeable bags

This is the peer reviewed version of the following article: Rizo Parraga, Arancha Maria, Fuentes López, Ana, Barat Baviera, José Manuel, Fernández Segovia, Isabel. (2018). Development of a novel smoke-flavoured salmon product by sodium replacement using water vapour permeable bags.Journal of the Science of Food and Agriculture, 98, 7, 2721-2728. DOI: 10.1002/jsfa.8767, which has been published in final form at http://doi.org/ 10.1002/jsfa.8767. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] BACKGROUND: Food manufacturers need to reduce sodium contents to meet consumer and public health demands. In this study the use of sodium-free (SF) salt and KCl to develop a novel smoke-flavoured salmon product with reduced sodium content was evaluated. Fifty percent of NaCl was replaced with 50% of SF salt or 50% KCl in the salmon smoke-flavouring process carried out using water vapour permeable bags. RESULTS: Triangle tests showed that samples with either SF salt or KCl were statistically similar to the control samples (100% NaCl). Since no sensorial advantage in using SF salt was found compared with KCl and given the lower price of KCl, the KCl-NaCl samples were selected for the next phase. The changes of physicochemical and microbial parameters in smoke-flavoured salmon during 42 days showed that partial replacement of NaCl with KCl did not significantly affect the quality and shelf-life of smoke-flavoured salmon, which was over 42 days. CONCLUSION: Smoke-flavoured salmon with 37% sodium reduction was developed without affecting sensory features and shelf-life. This is an interesting option for reducing sodium content in such products to help meet the needs set by both health authorities and consumers.We gratefully acknowledge the support of Tub-Ex Aps (Taars, Denmark) for suppling the water vapour permeable bags and for providing all of the necessary technical information. Arantxa Rizo thanks the Universitat Politecnica de Valencia for the FPI grant.Rizo Parraga, AM.; Fuentes López, A.; Barat Baviera, JM.; Fernández Segovia, I. (2018). Development of a novel smoke-flavoured salmon product by sodium replacement using water vapour permeable bags. Journal of the Science of Food and Agriculture. 98(7):2721-2728. https://doi.org/10.1002/jsfa.8767S2721272898

Effects of Marine Toxins on the Reproduction and Early Stages Development of Aquatic Organisms

Marine organisms, and specially phytoplankton species, are able to produce a diverse array of toxic compounds that are not yet fully understood in terms of their main targets and biological function. Toxins such as saxitoxins, tetrodotoxin, palytoxin, nodularin, okadaic acid, domoic acid, may be produced in large amounts by dinoflagellates, cyanobacteria, bacteria and diatoms and accumulate in vectors that transfer the toxin along food chains. These may affect top predator organisms, including human populations, leading in some cases to death. Nevertheless, these toxins may also affect the reproduction of aquatic organisms that may be in contact with the toxins, either by decreasing the amount or quality of gametes or by affecting embryonic development. Adults of some species may be insensitive to toxins but early stages are more prone to intoxication because they lack effective enzymatic systems to detoxify the toxins and are more exposed to the toxins due to a higher metabolic growth rate. In this paper we review the current knowledge on the effects of some of the most common marine toxins on the reproduction and development of early stages of some organisms

First Evidence of Palytoxin and 42-Hydroxy-palytoxin in the Marine Cyanobacterium Trichodesmium

Marine pelagic diazotrophic cyanobacteria of the genus Trichodesmium (Oscillatoriales) are widespread throughout the tropics and subtropics, and are particularly common in the waters of New Caledonia. Blooms of Trichodesmium are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain several types of paralyzing toxins. The toxicity of water-soluble extracts of Trichodesmium spp. were analyzed by mouse bioassay and Neuroblastoma assay and their toxic compounds characterized using liquid chromatography coupled with tandem mass spectrometry techniques. Here, we report the first identification of palytoxin and one of its derivatives, 42-hydroxy-palytoxin, in field samples of Trichodesmium collected in the New Caledonian lagoon. The possible role played by Trichodesmium blooms in the development of clupeotoxism, this human intoxication following the ingestion of plankton-eating fish and classically associated with Ostreopsis blooms, is also discussed

Use of Biosensors as Alternatives to Current Regulatory Methods for Marine Biotoxins

Marine toxins are currently monitored by means of a bioassay that requires the use of many mice, which poses a technical and ethical problem in many countries. With the exception of domoic acid, there is a legal requirement for the presence of other toxins (yessotoxin, saxitoxin and analogs, okadaic acid and analogs, pectenotoxins and azaspiracids) in seafood to be controlled by bioassay, but other toxins, such as palytoxin, cyclic imines, ciguatera and tetrodotoxin are potentially present in European food and there are no legal requirements or technical approaches available to identify their presence. The need for alternative methods to the bioassay is clearly important, and biosensors have become in recent years a feasible alternative to animal sacrifice. This review will discuss the advantages and disadvantages of using biosensors as alternatives to animal assays for marine toxins, with particular focus on surface plasmon resonance (SPR) technology

Data quality in marine biotoxins’ risk assessment: Perceptions from data production to consumption

Marine biotoxins constitute one of the major hazards associated with seafood consumption. Risk assessments are essential for the effective management of problems arising from marine biotoxins occurrence, as they are a prerequisite for the establishment or periodic re-evaluation of marine biotoxins regulatory limits and for the adoption of appropriate risk management plans. Risk assessments are science-based data-intensive processes, and their successful outcomes are largely dependent on the quality of data used when they are carried out. In fact, data-related challenges are the most frequently reported issues rendering most marine biotoxins’ risk assessments conducted to date as inconclusive. Notably, data quality perceptions among the stakeholders involved in risk assessments may vary significantly, which may be a human factor influencing data quality. As such, the problem addressed in this thesis is the shortage of empirical information on how data quality is perceived by the different stakeholder roles involved in risk assessments relevant to marine biotoxins hazards. The focus of this thesis is thus to investigate the perceptions of diverse stakeholders within the information chain, namely data producers, collectors and consumers/users, regarding the quality of data used in risk assessments of marine biotoxins hazards, to provide a contribution directed towards data quality improvement. This was done by means of a survey, gathering data through interviewing a number of recognized marine biotoxins’ experts with documented experience in risk assessments. The research question of this study is: “What are the perceptions of data quality among diverse stakeholders along the information chain relevant to marine biotoxins’ risk assessments?” To answer the research question, the concept of data quality for marine biotoxins data destined for risk assessments was dissected into seven individual subtopics on which the perceptions of expert participants of all three roles were captured. The subtopics explored included: data quality challenges; changes in marine biotoxins data quality during the last decade; awareness on data quality legislation and standardization; importance of data quality dimensions, objectives and key performance indicators; importance of data quality-related feedback exchange between stakeholders of the relevant information chain; factors for successful adoption of harmonized standardized formats for marine biotoxins data collection; and suggestions for data quality improvement. The perceptions gathered per subtopic were analyzed using inductive thematic analysis, yielding a total of twelve themes, namely communication, compound, data/quality control, Information Technology or Data Collection Framework, legislation, method, organization, people, policy, risk assessment procedure, society/environment and toxicological aspects, with each subtopic containing items categorized within several of these themes. Certain differences were observed in the perceptions between participants of diverse data roles, in the sense that data producers and to a lesser extent data users mostly focused on themes relevant to analytical methodology, compound particularities, data and quality control, toxicological aspects and policies. On the other hand, data collectors’ views were more concentrated on items relevant to Information Technology or Data Collection Framework and organization. It is noted, however, that interpretation of these trends needs to consider that in many of the study participants different roles overlapped in the same person. This indicates that results should be cautiously generalized. Nevertheless, they could constitute a basis for further research to generate deeper knowledge in the field of data quality in risk assessments relevant to marine biotoxins and gain further insights on the differences in perceptions among data roles