Prevalence and persistence of gymnodimines in clams from the Gulf of Gabes (Tunisia) studied by mouse bioassay and LC-MS/MS

In this work we studied the toxicity in clams from the Gulf of Gabes, Tunisia (Southern Mediterranean). Samples from two stations (M2 and S6) were collected monthly from January 2009 to September 2010, and analyzed by the official control method of mousse bioassay (MBA) for lipophilic toxins. All samples were also analyzed with the LC-MS/MS method for the determination of lipophilic toxins, namely: okadaic acid group, pectenotoxins, yessotoxins and azaspiracids, spirolides and gymnodimines (GYMs). The results showed prevalence of GYMs since it was the only toxin group identified in these samples with a maximum of 2,136 μg GYM -A kg-1 (February 2009 at M2). Furthermore, GYMs showed persistence in the area, with only one blank sample below the limit of detection. Interestingly, this blank sample was found in June 2009 after an important toxic episode which supports the recent findings regarding the high detoxification capability of clams, much faster than that reported for oysters. In comparison, good agreement was found among MBA, the LD50 value of 80-100 μg kg-1 reported for GYM- A, and quantitative results provided by LC-MS/MS. On the contrary to that previously reported for Tunisian clams, we unambiguously identified and quantified by LC-MS/MS the isomers GYM- B/C in most samples. Phytoplankton identification and enumeration of Karenia selliformis usually showed higher densities at site M2 than S6 as expected bearing in mind toxin results, although additional results would be required to improve the correlation between K. selliformis densities and quantitative results of toxins. The prevalence and persistence of GYMs in this area at high levels strongly encourages the evaluation of the chronic toxic effects of GYMs. This is especially important taking into account that relatively large quantities of GYMs can be released into the market due to the replacement of the official control method from mouse bioassay to the LC-MS/MS for lipophilic toxins (Regulation (EU) No 15/2011), and the lack of Regulation for this group of toxins.

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

We performed a comprehensive study to assess the fit for purpose of four chromatographic conditions for the determination of six groups of marine lipophilic toxins (okadaic acid and dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, gymnodimine and spirolides) by LC-MS/MS to select the most suitable conditions as stated by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). For every case, the elution gradient has been optimized to achieve a total run-time cycle of 12 min. We performed a single-laboratory validation for the analysis of three relevant matrices for the seafood aquaculture industry (mussels, pacific oysters and clams), and for sea urchins for which no data about lipophilic toxins have been reported before. Moreover, we have compared the method performance under alkaline conditions using two quantification strategies: the external standard calibration (EXS) and the matrix-matched standard calibration (MMS). Alkaline conditions were the only scenario that allowed detection windows with polarity switching in a 3200 QTrap mass spectrometer, thus the analysis of all toxins can be accomplished in a single run, increasing sample throughput. The limits of quantification under alkaline conditions met the validation requirements established by the EURLMB for all toxins and matrices, while the remaining conditions failed in some cases. The accuracy of the method and the matrix effects where generally dependent on the mobile phases and the seafood species. The MMS had a moderate positive impact on method accuracy for crude extracts, but it showed poor trueness for seafood species other than mussels when analyzing hydrolyzed extracts. Alkaline conditions with EXS and recovery correction for OA were selected as the most proper conditions in the context of our laboratory. This comparative study can help other laboratories to choose the best conditions for the implementation of LC-MS/MS according to their own necessities.

Protein phosphatase inhibition assays for okadaic acid detection in shellfish: matrix effects, applicability and comparison with LC-MS/MS analysis

The applicability of the protein phosphatase inhibition assay (PPIA) to the determination of okadaic acid (OA) and its acyl derivatives in shellfish samples has been investigated, using a recombinant PP2A and a commercial one. Mediterranean mussel, wedge clam, Pacific oyster and flat oyster have been chosen as model species. Shellfish matrix loading limits for the PPIA have been established, according to the shellfish species and the enzyme source. A synergistic inhibitory effect has been observed in the presence of OA and shellfish matrix, which has been overcome by the application of a correction factor (0.48). Finally, Mediterranean mussel samples obtained from Rı´a de Arousa during a DSP closure associated to Dinophysis acuminata, determined as positive by the mouse bioassay, have been analysed with the PPIAs. The OA equivalent contents provided by the PPIAs correlate satisfactorily with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Levels of PSP toxins in bivalves exposed to natural blooms of Alexandrium minutum in Catalan harbours

The development, validation, comparison and evaluation of analytical methods for marine toxins rely on the availability of toxic material. Within the project JACUMAR PSP, our interest is mainly focused on autochthonous bivalve species with the toxic profile of Alexandrium minutum, since this is the principal species involved regionally in PSP outbreaks. Mussels and oysters were exposed during few days in the harbor of Vilanova i la Geltrú, to blooms reaching a maximum A. minutum concentration of 200,000 cells L-1 in 2008, and 40,000 and 800,000 cells L-1, in 2009. Mussels, oysters and clams were exposed to one bloom of 22,000 cells L-1 in the harbor of Cambrils in 2009. In all situations higher toxic levels analyzed by HPLC-FD with postcolumn oxidation were observed in mussels (i.e. 1,200-2,500 μg eq. STX kg-1) than in oysters (i.e. 60-800 μg eq. STX kg-1) exposed to the same bloom. Blooms with higher concentrations of A. minutum did not correspond to higher levels of PSP toxins in bivalves. These differences may be explained by differences in A. minutum population dynamics, toxin production or in the physiological state or behaviour of shellfish. These results confirm that mussels concentrate more PSP toxins from A. minutum than oysters and clams.

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).

Reconstrucció de les temperatures superficials marines dels últims dos milions d'anys al Corrent de Benguela utilitzant l'índex TEX86

Per tal de reconstruir i obtenir una evolució climàtica a partir de les temperatures superficials marines dels últims dos milions d’anys al Corrent de Benguela (costa oest sud-africana) s’han analitzat 60 mostres del testimoni amb ODP 175-1084. Per fer-ho, s’ha utilitzat la nova calibració de l’índex TEX86 (Kim et al., 2007) i s’han representat els resultats juntament amb els valors obtinguts amb altres índexs referents a les temperatures mitjanes anuals de l’aire (MAAT) i el grau d’aportació sedimentària d’origen continental als sediments marins (BIT). També s’han comparat amb els registres de temperatura d’altres estudis en la mateixa àrea obtinguts a partir de proxies diferents. Els resultats del TEX86 mostren certa concordança amb alguns dels valors obtinguts en altres estudis i proposen hipòtesis que relacionen de manera directa els tres índexs calculats.

Béns i serveis associats als ecosistemes de fanerògames marines i estimacions de pèrdues causades per la seva degradació

Les praderies de posidònia de la costa catalana són ecosistemes d’alta importància, ja que tenint en compte la seva poca extensió, ens aporten una gran multiplicitat de béns i serveis ecosistèmics. No obstant, els danys observats en els prats en les últimes dècades, tant pel que fa a la regressió en superfície com a la pèrdua d’integritat ecològica, ens indiquen que aquests ecosistemes s’estan enfrontant a una crisi causada principalment per les pressions de les activitats antròpiques a les zones costaneres. En aquest projecte es pretenen identificar tots els béns i serveis que ens proporcionen aquests herbeis, així com també dictaminar quina és la importància de cada un d’ells en el context de la costa catalana. D’altra banda, es pretenen avaluar els impactes i conseqüències de la seva degradació i també quantificar, de manera relativa, la pèrdua d’aquests béns i serveis a la costa catalana, partint de la base que, segons els resultats obtinguts, la pèrdua d’abundància en els prats ha estat d’aproximadament el 60% des d’un hipotètic estat “pre-industrial” fins l’actualitat.

Ús de Sensor Web Enablement per a gestió de dades marines i accés al servei d'observació de sensors a través de client Android

El present treball és un estudi de l'estat de Sensor Web Enablement (SWE) aplicat a l'àmbit marí d'investigació oceanogràfica. Per assolir aquest objectiu s'han avaluat les diferents solucions d'arquitectura de sistemes més adients a la problemàtica indicada i s'ha dissenyat un client de Sensor Observation Service (SOS) per plataforma mòbil Android que permeti consultar la descripció del sensor (SensorML) i conjunts d'observacions mitjançant estàndards oberts de SWE i peticions web (SOAP). Els dissenys anteriorment indicats han permès que quedés palès l'ampli ventall de possibilitats i avantatges que s'obtenen pel fet d'emprar SWE, ja que ens permet integrar fàcilment les dades proporcionades pels sensors i les pròpies descripcions dels sensors en diferents entorns, tal i com és el cas del disseny presentat en Android, a través d'estàndards oberts independents de les múltiples especificacions de cada fabricant, en un entorn, com és l'àmbit de la investigació marina on hi ha una gran heterogeneïtat de sensors i protocols.

Diversitat bacteriana a les esponges marines

L’objectiu principal del projecte era aprofundir en el coneixement de les malalties de les esponges, centrant-nos en la diversitat bacteriana que acullen. Als estius 2008 i 2009 va haver episodis d’elevades temperatures de l’aigua de mar que van suposar importants mortalitats massives d’esponges a la Mediterrània. Contra tot pronòstic, els estius del projecte, el 2010 i el 2011, no van resultar especialment calorosos i no hi va haver episodis de mortalitat. És un fet molt positiu per les comunitats bentòniques però algun dels objectius específics de la meva proposta no es va poder dur a terme com havien estat platejats inicialment. Disposàvem de diferents mostres d’esponges malaltes de l’espècie Ircinia fasciculata, sanes i aigua circumdant recollides l’estiu 2009 i fixades per DNA, per això s’han pogut identificar i caracteritzar els canvis en la comunitat microbiològica de les esponges malaltes respecte de les sanes. Hem constatat que més que una infecció per un o pocs patògens puntuals, com havien proposat alguns autors, hi ha un canvi dràstic en la comunitat microbiològica associada a les esponges. Les esponges malaltes, on dominen els bacteris heterotròfics, presenten una major diversitat bacteriana que les sanes, on dominen els autòtrofs. Tot i que no es va poder realitzar un nou mostreig d’individus malalts, i algun objectiu específic no es va poder desenvolupar, vaig aprofitar l’estada en un centre de referència en estudis de la diversitat microbiana, per ampliar el coneixement general de les comunitats bacterianes associades a esponges. Es van realitzar nous estudis en què vam testar si totes les esponges (bacteriosponges i no) presenten simbionts reals o si les no-bacteriosponges presenten un enriquiment de les comunitats de l’aigua de mar però no una flora específica. El resultat és que totes les esponges presenten associacions molt específiques malgrat hi ha fortes diferències entre les comunitats microbianes associades a bacteriosponges i a no-bacteriosponges.