title: First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum.
abstract: 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.
authors.affiliation: Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. aristidis.vlamis@rai.usc.es.|National Reference Laboratory on Marine Biotoxins, Veterinary Centre of Thessaloniki, Ministry of Productive Reconstruction, Environment and Energy, 3A Limnou street, GR 54627 Thessaloniki, Greece. aristidis.vlamis@rai.usc.es.
authors.forename: Aristidis
authors.initials: A
authors.lastname: Vlamis
authors.affiliation: National Reference Laboratory on Marine Biotoxins, Veterinary Centre of Thessaloniki, Ministry of Productive Reconstruction, Environment and Energy, 3A Limnou street, GR 54627 Thessaloniki, Greece. biotoxin@otenet.gr.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Katikou
authors.affiliation: Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. ines.rodriguez@usc.es.
authors.forename: Ines
authors.initials: I
authors.lastname: Rodriguez
authors.affiliation: Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. veronica.rey@rai.usc.es.
authors.forename: Verónica
authors.initials: V
authors.lastname: Rey
authors.affiliation: Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. amparo.alfonso@usc.es.
authors.forename: Amparo
authors.initials: A
authors.lastname: Alfonso
authors.affiliation: National Reference Laboratory on Marine Biotoxins, Veterinary Centre of Thessaloniki, Ministry of Productive Reconstruction, Environment and Energy, 3A Limnou street, GR 54627 Thessaloniki, Greece. aopvet@gmail.com.
authors.forename: Angelos
authors.initials: A
authors.lastname: Papazachariou
authors.affiliation: National Reference Laboratory on Marine Biotoxins, Veterinary Centre of Thessaloniki, Ministry of Productive Reconstruction, Environment and Energy, 3A Limnou street, GR 54627 Thessaloniki, Greece. thetisz@otenet.gr.
authors.forename: Thetis
authors.initials: T
authors.lastname: Zacharaki
authors.affiliation: Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. anamaria.botana@usc.es.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Botana
authors.affiliation: Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. luis.botana@usc.es.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D000818:Animals; D049872:Bivalvia; D002853:Chromatography, Liquid; D004141:Dinoflagellida; D004784:Environmental Monitoring; D005506:Food Contamination / Q000032:analysis*; D041981:Gastrointestinal Tract / Q000737:chemistry; D006115:Greece; D008522:Mediterranean Sea; D051379:Mice; D012623:Seawater / Q000032:analysis; D012758:Shellfish; D053719:Tandem Mass Spectrometry; D013779:Tetrodotoxin / Q000032:analysis* / Q000633:toxicity; D014873:Water Pollutants / Q000032:analysis / Q000633:toxicity
chemical_list: D014873:Water Pollutants; D013779:Tetrodotoxin
keywords: Aegean Sea; Mediterranean Sea; Mytilus galloprovincialis; Prorocentrum minimum; UPLC-MS/MS; cultured mussels; emerging biotoxins; tetrodotoxin; toxic episode; venerupin shellfish toxin