Monthly Variation of Tetrodotoxin Levels in Pufferfish (Lagocephalus sceleratus) Caught from Antalya Bay, Mediterranean Sea.

The silver-cheeked toadfish (Lagocephalus sceleratus), an invasive alien pufferfish species that has rapidly settled throughout the Mediterranean region, poses significant threats not only to native marine species and fisheries but also to public health due to the tetrodotoxin (TTX) they harbor. In this study, TTX concentrations in L. sceleratus from Antalya Bay in the Northeastern Mediterranean Sea were investigated using Q-TOF-LC-MS on a monthly basis over a one-year period. Pufferfish were caught by angling from May 2018 to April 2019. The TTX levels in three different tissues (gonads, liver, and muscle) of 110 pufferfish in total were determined in both male and female individuals caught for 11 months. The highest TTX mean levels generally occurred in the gonads and the lowest in the muscle samples. As regards the maximum TTX contents, the highest concentrations determined were 68.2, 34.2, and 7.8 µg/g in the gonad, liver, and muscle tissues, respectively. The highest levels were generally observed in late autumn to winter (especially in November and December) in all tissues from both genders. Female individuals were generally found to be more toxic than male individuals. The TTX levels found confirm that the consumption of L. sceleratus from Antalya Bay remains dangerous throughout the year, and thus L. sceleratus constantly constitutes an important risk source for public health.

An Updated Review of Tetrodotoxin and Its Peculiarities.

Tetrodotoxin (TTX) is a crystalline, weakly basic, colorless organic substance and is one of the most potent marine toxins known. Although TTX was first isolated from pufferfish, it has been found in numerous other marine organisms and a few terrestrial species. Moreover, tetrodotoxication is still an important health problem today, as TTX has no known antidote. TTX poisonings were most commonly reported from Japan, Thailand, and China, but today the risk of TTX poisoning is spreading around the world. Recent studies have shown that TTX-containing fish are being found in other regions of the Pacific and in the Indian Ocean, as well as the Mediterranean Sea. This review aims to summarize pertinent information available to date on the structure, origin, distribution, mechanism of action of TTX and analytical methods used for the detection of TTX, as well as on TTX-containing organisms, symptoms of TTX poisoning, and incidence worldwide.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay.

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

Quantitative determination of the okadaic acid toxins group by a colorimetric phosphatase inhibition assay: interlaboratory study.

An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

Digital Technologies and Open Data Sources in Marine Biotoxins' Risk Analysis: The Case of Ciguatera Fish Poisoning.

Currently, digital technologies influence information dissemination in all business sectors, with great emphasis put on exploitation strategies. Public administrations often use information systems and establish open data repositories, primarily supporting their operation but also serving as data providers, facilitating decision-making. As such, risk analysis in the public health sector, including food safety authorities, often relies on digital technologies and open data sources. Global food safety challenges include marine biotoxins (MBs), being contaminants whose mitigation largely depends on risk analysis. Ciguatera Fish Poisoning (CFP), in particular, is a MB-related seafood intoxication attributed to the consumption of fish species that are prone to accumulate ciguatoxins. Historically, CFP occurred endemically in tropical/subtropical areas, but has gradually emerged in temperate regions, including European waters, necessitating official policy adoption to manage the potential risks. Researchers and policy-makers highlight scientific data inadequacy, under-reporting of outbreaks and information source fragmentation as major obstacles in developing CFP mitigation strategies. Although digital technologies and open data sources provide exploitable scientific information for MB risk analysis, their utilization in counteracting CFP-related hazards has not been addressed to date. This work thus attempts to answer the question, "What is the current extent of digital technologies' and open data sources' utilization within risk analysis tasks in the MBs field, particularly on CFP?", by conducting a systematic literature review of the available scientific and grey literature. Results indicate that the use of digital technologies and open data sources in CFP is not negligible. However, certain gaps are identified regarding discrepancies in terminology, source fragmentation and a redundancy and downplay of social media utilization, in turn constituting a future research agenda for this under-researched topic.

Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection.

An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006-2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 microg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells.

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.

Public Health Risks Associated with Tetrodotoxin and Its Analogues in European Waters: Recent Advances after The EFSA Scientific Opinion.

Tetrodotoxin (TTX) and its analogues are naturally occurring toxins responsible worldwide for human intoxication cases and fatalities, mainly associated with pufferfish consumption. In the last decade, TTXs were detected in marine bivalves and gastropods from European waters. As TTXs are not regulated or monitored at EU level, their unexpected occurrence in shellfish raised concerns as a food safety hazard and revealed the necessity of a thorough assessment on the public health risks associated with their presence. For this reason, the European Food Safety Authority (EFSA) was requested by the European Commission to provide a scientific opinion, finally adopted in March 2017, according to which a provisional concentration below 44 µg TTX equivalents/kg shellfish meat, based on a large portion size of 400 g, was considered not to result in adverse effects in humans. The EFSA expert panel, however, recognized a number of shortcomings and uncertainties related to the unavailability of sufficient scientific data and provided relevant recommendations for future research to overcome these data gaps identified in order to further refine the risk assessment on TTXs. The present review aims to summarize the knowledge obtained towards addressing these recommendations in the two years following publication of the EFSA opinion, at the same time highlighting the points requiring further investigation.

First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece).

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.

Effect of rosemary extract, chitosan and α-tocopherol on lipid oxidation and colour stability during frozen storage of beef burgers.

The effect of rosemary extract, chitosan and α-tocopherol, added individually or in combination, on lipid oxidation and colour stability of frozen (-18°C) beef burgers stored for 180 days was investigated. The burgers' lipid oxidation and appearance were evaluated through measurement of primary (conjugated dienes and peroxide value) and secondary (malondialdehyde) oxidation products, together with visual assessment and instrumental measurement of colour. Chitosan alone and in combination with either rosemary or α-tocopherol had the best antioxidative effect (P⩽0.05) compared to individual use of rosemary or α-tocopherol, while the best results were obtained with the combination of chitosan and rosemary. The differences of antioxidative effects, however, between individual use of rosemary or α-tocopherol as compared to the controls were also significant (P⩽0.05). Chitosan added individually or in combination with either rosemary or α-tocopherol had also a noteworthy effect on the burgers' appearance as it contributed to red colour retention for a much longer period (P⩽0.05) compared all other treatments and the controls. In conclusion, the best antioxidative effects were obtained with the combination of chitosan and rosemary extract.

Effect of rosemary extract, chitosan and α-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4°C.

The effect of rosemary extract, chitosan and α-tocopherol, added individually or in combination, on microbiological parameters and lipid oxidation of fresh pork sausages stored for 20days at 4°C was investigated. Microbiological determinations included the enumeration of Enterobacteriaceae, Pseudomonas spp., total viable count, yeasts and moulds and lactic acid bacteria, while pH values were also determined. Lipid oxidation was evaluated through measurement of primary (peroxide value) and secondary (malondialdehyde concentration) oxidation products. Chitosan addition resulted in significant (P⩽0.05) inhibition of microbial growth, while the lowest microbial counts were obtained in the samples containing both chitosan and rosemary, indicating a possible synergistic effect. Chitosan and its combinations with either α-tocopherol or rosemary, and especially the latter combination, showed also the most intense antioxidative effect (P⩽0.05), at least when compared to α-tocopherol alone or the controls, while use of rosemary extract alone had a comparable (P>0.05) antioxidative effect with chitosan and its combinations. Shelf life of samples containing chitosan was almost doubled compared to the remaining samples. In conclusion, the best antimicrobial and antioxidative effects were obtained from the combination of chitosan with the rosemary extract.

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 INa.

Relation of biogenic amines' formation with microbiological and sensory attributes in Lactobacillus-inoculated vacuum-packed rainbow trout (Oncorhynchus mykiss) fillets.

The biogenic amine (BA) content of vacuum-packed filleted rainbow trout (Oncorhynchus mykiss) inoculated or not with two different Lactobacillus strains, individually or in combination, was monitored during refrigerated storage for 20 days and related to respective bacteriological and sensory changes occurring during the same period. Eight amines, namely putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, histamine, spermine, and spermidine, were determined, whereas agmatine was not detected in any of the samples. In all cases, BA concentration was higher (P < or = 0.05) in the controls compared to all inoculated treatments, whereas the trend with regard to the bacterial populations (Enterobacteriaceae, pseudomonads, and H2S-producing bacteria) and the off-odor scores was similar. Inoculation with Lactobacillus sakei CECT 4808 showed the best preservative effect among inoculated treatments. Concentrations of putrescine and cadaverine, the main BAs formed, correlated well with both spoilage bacterial counts and off-odor scores and can be useful indicators of shelf life. Spermine and spermidine contents decreased during storage, while levels of the other determined BAs remained below 10 mg/kg even after sensory rejection.

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 consumers.

Assessing the neurotoxic effects of palytoxin and ouabain, both Na⁺/K⁺-ATPase inhibitors, on the myelinated sciatic nerve fibres of the mouse: an ex vivo electrophysiological study.

Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na⁺/K⁺-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na⁺/K⁺-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibres. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT50) incubated in normal saline was 24.5 ± 0.40 h (n = 5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT50 of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n = 5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n = 5) and inhibitory concentration to 50% (IC50) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 µM (n = 5) and IC50 of 370.0 ± 18.00 µM (incubation 24.5 h). Finally, when the two compounds were combined--e.g. pre-incubation of the nerve fibre in 250.0 µM ouabain for 1 h and then exposure to 1.0 nM PlTX--ouabain offered minor a neuroprotection of 9.1-17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 µM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay--the standard ex vivo test for PlTX toxicity.

Occurrence of palytoxin-group toxins in seafood and future strategies to complement the present state of the art.

Palytoxin (PlTX) and palytoxin-like (PlTX-like) compounds in seafood have been raising scientific concern in the last years. The constant increase in record numbers of the causative dinoflagellates of the genus Ostreopsis together with the large spatial expansion of this genus has led to intensification of research towards optimization of methods for determination of PlTX presence and toxicity. In this context, identification of seafood species which could possibly contain PlTXs constitutes an important issue for public health protection. In the present paper, worldwide occurrence of PlTX-like compounds in seafood is reviewed, while potential future strategies are discussed. PlTX has been reported to be present in several species of fish, crustaceans, molluscs and echinoderms. In one occasion, PlTX has been identified in freshwater puffer fish whereas all other records of PlTXs refer to marine species and have been recorded in latitudes approximately between 43°N and 15°S. PlTX determination in seafood has relied on different methodologies (mainly LC-MS, mouse bioassay and hemolysis neutralization assay) that have evolved over time. Future recommendations include systematic screening of PlTX in those species and areas where PlTX has already been recorded implementing updated methodologies.

Determination of okadaic acid, dinophysistoxin-1 and related esters in Greek mussels using HPLC with fluorometric detection, LC-MS/MS and mouse bioassay.

An approach involving both chemical and biological methods was undertaken for the detection and quantification of the marine toxins okadaic acid (OA), dinophysistoxin-1 (DTX-1) and their respective esters in mussels from different sampling sites in Greece during the period 2006-2007. Samples were analyzed by means of a) high performance liquid chromatography with fluorometric detection (HPLC-FLD), using 9-athryldiazomethane (ADAM), as a pre-column derivatization reagent, b) liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and c) the mouse bioassay. Free OA and DTX-1 were determined by both HPLC-FLD and LC-MS/MS, while their respective esters were determined only by LC-MS/MS after alkaline hydrolysis of the samples. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the HPLC-FLD method were 0.015 microg/g HP and 0.050 microg/g HP, respectively, for OA. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the LC-MS/MS method were 0.045 microg/g HP and 0.135 microg/g HP, respectively, for OA. Comparison of results between the two analytical methods showed excellent agreement (100%), while both HPLC-FLD and LC-MS/MS methods showed an agreement of 97.1% compared to the mouse bioassay.

First report on toxicity assessment of the Lessepsian migrant pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European waters (Aegean Sea, Greece).

According to the current European Union legislative requirements (Regulation 853/2004/EC; Regulation 854/2004/EC, poisonous fish of the family Tetraodontidae and products derived from them must not be placed on the European markets. Following the increased publicity regarding the presence of the pufferfish species Lagocephalus sceleratus in Greek waters, this study was undertaken in order to confirm its toxicity and assess the risk of poisoning in case of accidental consumption. Acidic extracts from tissues of L. sceleratus specimens of different sizes were examined by means of the official mouse bioassay for tetrodotoxin, while some of the extracts were also tested for the presence of Paralytic Shellfish Poisoning (PSP) toxins with a commercial ELISA kit. Toxicity in mice, with symptomatology indicative of tetrodotoxin, was confirmed in a number of samples and indicated a correlation with fish size. Toxicity of certain tissues (liver, gonads, gastrointestinal tract) in larger individuals, expressed as microg/g tetrodotoxin equivalents, was largely above levels required to cause death in human adults. On the other hand, all tested extracts provided a positive reaction in the ELISA test for PSP toxins. This constitutes the first report for presence of toxicity in L. sceleratus caught in European coastal waters.