Jorge Diogène

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.

Update on Methodologies Available for Ciguatoxin Determination: Perspectives to Confront the Onset of Ciguatera Fish Poisoning in Europe

Ciguatera fish poisoning (CFP) occurs mainly when humans ingest finfish contaminated with ciguatoxins (CTXs). The complexity and variability of such toxins have made it difficult to develop reliable methods to routinely monitor CFP with specificity and sensitivity. This review aims to describe the methodologies available for CTX detection, including those based on the toxicological, biochemical, chemical, and pharmaceutical properties of CTXs. Selecting any of these methodological approaches for routine monitoring of ciguatera may be dependent upon the applicability of the method. However, identifying a reference validation method for CTXs is a critical and urgent issue, and is dependent upon the availability of certified CTX standards and the coordinated action of laboratories. Reports of CFP cases in European hospitals have been described in several countries, and are mostly due to travel to CFP endemic areas. Additionally, the recent detection of the CTX-producing tropical genus Gambierdiscus in the eastern Atlantic Ocean of the northern hemisphere and in the Mediterranean Sea, as well as the confirmation of CFP in the Canary Islands and possibly in Madeira, constitute other reasons to study the onset of CFP in Europe [1]. The question of the possible contribution of climate change to the distribution of toxin-producing microalgae and ciguateric fish is raised. The impact of ciguatera onset on European Union (EU) policies will be discussed with respect to EU regulations on marine toxins in seafood. Critical analysis and availability of methodologies for CTX determination is required for a rapid response to suspected CFP cases and to conduct sound CFP risk analysis.

Comparative study of the use of neuroblastoma cells (Neuro-2a) and neuroblastoma × glioma hybrid cells (NG108-15) for the toxic effect quantification of marine toxins

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish.

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.

Resting cysts of the toxigenic dinoflagellate genus Alexandrium in recent sediments from the Western Mediterranean coast, including the first description of cysts of A. kutnerae and A. peruvianum

Cyst studies carried out in 2002–2003 on Mediterranean Sea sediment from seven different sites along the Catalan and Balearic coasts (Western Mediterranean) revealed a higher diversity of Alexandrium species in the region than was previously known. The cysts of eight species of the toxigenic, marine dinoflagellate genus Alexandrium are described, and some, such as A. kutnerae, A. margalefi, A. peruvianum and A. pseudogoniaulax, are reported from the area for the first time. This is also the first record of resting cysts of A. taylori in Mediterranean sediment, and the first description known to date of resting cysts for A. kutnerae and A. peruvianum. All the cysts were characterized by a smooth wall except for the paratabulated cyst of A. pseudogoniaulax, and most of them had a prominent yellow/orange accumulation body. Nevertheless, we have also detected an unparatabulated cyst of A. pseudogoniaulax, both from the sediment and in cultures. The cyst of A. kutnerae had a roughly cylindrical shape with rounded ends which makes it impossible to distinguish from resting cysts of A. tamarense and A. catenella, while the flattened round cysts of A. peruvianum were very similar to those of A. taylori. The cyst concentration data revealed A. catenella and A. minutum to be the most abundant cysts in the region, and they were detected in semi-enclosed waters, such as harbours. This highlights the importance of water exchange in the accumulation of cyst beds of these species, which has already been reported in the region by other authors for A. minutum. This paper contributes to the biogeographic distribution of some Alexandrium species, such as A. kutneare, A. margalefi, A. peruvianum and A. pseudogoniaulax, which have been reported only infrequently in a global context.

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea

ABSTRACT The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.

Determination of dissolved domoic acid in seawater with reversed-phase extraction disks and rapid resolution liquid chromatography tandem mass spectrometry with head-column trapping

Domoic acid (DA) is the principal neurotoxin responsible for amnesic shellfish poisoning (ASP) and is produced, among other species, by marine diatoms of the genus Pseudo-nitzschia. In this work, a method for the determination of dissolved DA and its isomers present in seawater has been developed, based on a solid-phase extraction (SPE) disks followed by rapid resolution liquid chromatography coupled with tandem mass spectrometry. SPE provided sample desalting and 20-fold concentration of dissolved DA, while complete resolution between DA and its isomers was achieved in less than 3 min with rapid resolution chromatography thus providing high sample throughput. Additionally, a simple on-column chromatographic procedure allowed head-column trapping of DA providing 15-fold higher sensitivity. The conditions developed in this work have shown appropriate quality parameters in a within-laboratory validation. The detection limit was 0.02 ng mL(-1) for the whole method, while trueness ranged between 92.1% and 110.6% recovery and precision between 8.4% and 19.0% relative standard deviation. Expanded uncertainty measured was 1.92, 0.23 and 0.03 for 10.0, 1.0 and 0.1 ng mL(-1) DA concentrations, respectively, which demonstrated the accuracy of this method for confirmation and quantification of DA present at very low concentration levels in seawater.

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.

Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands

The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml−1 according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC20). The LOQ was estimated as 0.0096 ng CTX1B eq. g TE−1 with 23–31% variability between experiments. These values were deemed sufficient even though quantification given at the IC50 (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17–19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g−1 of TE ranging from 0.058 (±0.012) to 6.23 (±0.713) ng CTX1B eq. g TE−1. The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

Alternative methods for the detection of emerging marine toxins: biosensors, biochemical assays and cell-based assays.

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs.