Dioxin-like, non-dioxin like PCB and PCDD/F contamination in European eel (Anguilla anguilla) from the Loire estuarine continuum: spatial and biological variabilities

To characterize the eel contamination by dioxin-like (dl) and non dioxin-like (ndl) PCBs and PCDD/Fs, 62 eels from the Loire estuary (France) were analyzed. PCB contamination significantly increased from glass eel stage (3.7 ±1.9 and 15.2±4.2 ng.g-1 dw) to other life stages (for yellow eels: 62.8±34.4 and 381.8±181.8 ng.g-1 dw; for silver eels: 93.7±56.3 and 463.2±244.6 ng.g-1 dw respectively for dl and ndl PCB). An inter-site variability based on PCB levels and fingerprints was observed between the three studied sites. The glass eel pattern was mainly characterized by the less chlorinated PCBs contrarily to the other eels, underlying a different bioaccumulation pathway. Overall, eels from this estuary showed an intermediate contamination level compared to other international/national areas. However, more than 60% of studied silver eels displayed WHO2005 PCDD/F and dl-PCB TEQ values higher than the recommended level of 10 pg.g-1 ww. This statement indicates a potential exposure to PCBs through eel consumption, especially with silver individuals, and could potentially lead to damages for the eel population

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area

Chemical composition and antimicrobial activity of the essential oils from New Caledonian Citrus macroptera and Citrus hystrix

The essential oils from the leaves of Citrus macroptera and C. hystrix, collected in New Caledonia, have been analyzed by gas chromatography/mass spectrometry (GC/MS) and evaluated for their antimicrobial activity. A total of 35 and 38 constituents were identified, representing 99.1 and 89.0% of the essential oils. respectively. Both essential oils were rich in monoterpenes (96.1 and 87.0%, rest) with beta-pinene as major component (33.3 and 10.9%, resp.), and poor in limonene (2.4 and 4.7%. resp.). Other main components of C. macroptera oil were alpha-pinene (25.3%), p-cimene (17.6%), (E)-beta-ocimene (6.7%), and sabinene (4.8%). The essential oil of C hystrix was characterized by high contents of terpinen-4-ol (13.0%). alpha-terpincol (7.6%), 1,8-cineole (6.4%), and citronellol (6.0%). The antimicrobial activity was evaluated against five bacteria and five fungi strains. Both oils were inactive against bacteria. However. the C. macroptera leaf oil exhibited a pronounced activity against Trichophyton mentagrophytes var. interdigitale, with a minimal-inhibitory concentration (MC) of 12.5 mu g/ml

Detection of pacific ciguatoxins using liquid chromatography coupled to either low or high resolution mass spectrometry (LC-MS/MS)

Ciguatera Fish Poisoning (CFP) is primarily caused by consumption of tropical and sub-tropical fish contaminated by Ciguatoxins (CTXs). These lipid-soluble, polyether neurotoxins are produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa. While there is no regulatory level in Europe for CTXs, the European Food Safety Authority (EFSA) adopted the United States guidance level of 0.01 mu g P-CTX1B eq.kg(-1) of fish. This limit is extremely low and requires significant improvement in the detection of CTXs. In this study, we compared analytical protocols based on liquid chromatography coupled to tandem low or high resolution mass spectrometry (LC-LRMS or HRMS) to find the best conditions for sensitivity and/or selectivity. Different approaches such as LC conditions, ion choice and acquisition modes, were evaluated to detect the Pacific-ciguatoxins (P-CTXs) on a triple quadrupole (API4000 Qtrap, Sciex) or a quadrupole time of flight (QTOF 6550, Agilent Technologies) spectrometer. Moreover, matrix effects were calculated using matrix-matched calibration solutions of P-CTX1B and P-CFX3C prepared in purified fish extract. Subsequently, the method performance was assessed on naturally contaminated samples of seafood and phytoplankton. With LRMS, the ammoniated adduct ion used as a precursor ion showed an advantage for selectivity through confirmatory transitions, without affecting signal-to-noise ratios, and hence limits of detection (LODs). As also reported by some studies in the literature, methanol-based mobile phase gave better selectivity and sensitivity for the detection of P-CTXs. While the LOD for P-CTX1B and P-CTX3C met the EFSA recommendation level when using LRMS, the findings suggested careful evaluation of instrumental parameters for determination of CTXs. LODs were significantly higher for HRMS, which currently results in the need for a significantly higher sample intake. Nevertheless, HRMS allowed for the identification of artefacts and may allow for improved confirmation of the identity of P-CTXs analogues. Consequently, LRMS and HRMS are considered complementary to ensure adequate quantitation and identification of P-CTXs

Effect of Azadinium spinosum on the feeding behaviour and azaspiracid accumulation of Mytilus edulis

Azadinium spinosum, a small toxic dinoflagellate, was recently isolated and identified as a primary producer of azaspiracid toxins (AZAs). Previous experiments related to AZA accumulation in blue mussels upon direct feeding with A. spinosum revealed increased mussel mortality and had negative effects on the thickness of the digestive gland tubules. Therefore we conducted follow up experiments in order to study effects of A. spinosum on mussel feeding behaviour. Individual assessment of mussel feeding time activity (FTA), clearance rate (CR), filtration rate (TFR), absorption rate (AR), faeces and pseudofaeces production were carried out on mussel fed either toxic (A. spinosum) or non-toxic (Isochrisis aff. galbana (T-Iso)) diets. Furthermore, AZA accumulation and biotransformation in mussels were followed using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). A. spinosum had a significant effect on mussel feeding behaviour compared to T-Iso: CR was lower by a factor of 6, FTA by a factor of 5, TFR by a factor of 3 and AR even decreased to negative values for the last day of exposure. Even so, a rapid AZA accumulation was observed during the first hours of the trial; less than 6 h of feeding were required to reach AZA concentration in mussel above regulatory level. In consistence with physiological observations, AZA concentration of about 200 �g kg−1 did not increase further until the end of the study. AZA bioconversion was also found to be a fast process: after 3 h of exposure AZA17, -19 and AZA7-10 were already found, with a proportion of AZA17 equal to AZA2. These results show a negative effect of A. spinosum on blue mussel feeding activity and indicate a possible regulation of AZA uptake by decreasing filtration and increasing pseudofaeces production