PSP detoxification kinetics in the mussel Mytilus galloprovincialis. One- and two-compartment models and the effect of some environmental variables

Paralytic shellfish poisoning (PSP) toxins are accumulated by bivalves during toxic plankton blooms. In these bivalves the toxins are distributed into different body tissues which have varying affinities for them, and later these toxins are transferred by the bivalves to other trophic levels. After the disappearance of the toxic cells, shellfish remain toxic for a variable period of time, depending on the detoxification kinetics. We studied these kinetics in mussels Mytilus galloprovincialis previously exposed to a bloom of the PSP producing dinoflagellate Gymnodinium catenatum. The toxin profile observed in the mussels was very similar to that of G. catenatum, showing that toxin transformations (chemical or enzymatic) had little or no importance in this case. The detoxification rates at all the sampling points decreased progressively from ca 0.25 to 0 d-1 following an inverse hyperbolic-like curve. These rates were related to different degrees to the environmental factors studied (salinity, temperature, and light transmission as a measure of seston volume, and in vivo fluorescence as a measure of phytoplankton concentration) and to fresh body weight during each sampling period (estimated by multiple regression). In general, detoxification rates became increasingly independent of the variables cited as the experiment progressed. One- and 2-compartment detoxification models, both with 2 variants (with fixed and variable detoxification rates depending on the environmental variables and body weight), were used to describe the detoxification kinetics observed. Neither of the 2 variants of the 1-compartment models correctly described detoxification. The 2-compartment models, on the other hand, particularly the environmentally controlled variant, fit the observed detoxification kinetics very well. There was only a slight difference between these last 2 models, which would suggest that the actual effect of the environmental variables considered in the detoxification process is unimportant.Versión del editor2,483

Identification of 19-epi-okadaic Acid, a New Diarrhetic Shellfish Poisoning Toxin, by Liquid Chromatography with Mass Spectrometry Detection

Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC–MS/MS).Versión del editor3,471

Differences in the toxin profiles of Alexandrium ostenfeldii (Dinophyceae) strains isolated from different geographical origins: evidence of paralytic toxin, spirolide and gymnodimine

Among toxin-producing dinoflagellates of the genus Alexandrium, Alexandrium ostenfeldii is the only species able to produce paralytic shellfish poisoning (PSP) toxins, spirolides (SPXs) and gymnodimines (GYMs). In this study we characterized and compared three A. ostenfeldii strains isolated from the Baltic, Mediterranean, and southern Chile Seas with respect to their toxin profiles, morphology, and phylogeny. Toxin analyses by HPLCeFD and LCeHRMS revealed differences in the toxin profiles of the three strains. The PSP toxin profiles of the southern Chile and Baltic strains were largely the same and included gonyautoxin (GTX)-3, GTX-2, and saxitoxin (STX), although the total PSP toxin content of the Chilean strain (105.83 ± 72.15 pg cell 1) was much higher than that of the Baltic strain (4.04 ± 1.93 pg cell 1). However, the Baltic strain was the only strain that expressed detectable amounts of analogues of GYM-A and GYM-B/-C (48.27 ± 26.12 pg GYM-A equivalents cell 1). The only toxin expressed by the Mediterranean strain was 13-desmethyl SPX-C (13dMeC; 2.85 ± 4.76 pg cell 1). Phylogenetic analysis based on the LSU rRNA showed that the studied strains belonged to distinct molecular clades. The toxin profiles determined in this study provide further evidence of the taxonomic complexity of this species.Preprin

Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture

A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.Versión del editor3,829

Evaluation of passive samplers as a monitoring tool for early warning of Dinophysis toxins in shellfish

From June 2006 to January 2007 passive samplers (solid phase adsorbing toxin tracking, SPATT) were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content (liquid chromatography–mass spectrometry, LC-MS) in picked cells of Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week−1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks

Changes in toxin content, biomass and pigments of the dinoflagellate Alexandrium minutum during nitrogen refeeding and growth into nitrogen or phosphorus stress

Two strains oi the paralytic shellfish toxin (PST) producing dinoflagellate Alexandrium minutum Halim (highly toxic ALl V and weakly toxic AL2V) were grown in batch culture with either nitrate or phosphate as the limiting nutrient. In comparison with cells of the strain AL1V, cells of AL2V grew at a similar C-specific Tale, had a higher C/N ratio, and lower ratios of chl a/chl C2and chl a/peridinin. Neither chlorophylls flor carotenoids could be used to estimate C-biomass, N-biomass or toxin content for this organismo The toxin profile for both strains was dominated (up to 95 %) by the gonyautoxin GTX4, with smaller proportions of GTX1, GTX2 and GTX3. The Tale of toxin synthesis for both strains was greatest 1 to 2 d after the N-refeeding of N-deprived cells, with the net Tale of toxin syn- .thesis exceeding that of C-biomass and cell division by a factor of up to 4. Toxin synthesis was not enhanced by short-term P-stress. N-stress alone led to a decrease in toxin cell-I, but P-stress followed by N-stress did not result in such a decline, implicating phosphorus in the regulation of toxin metabolism. Although arginine is a majar precursor for PST synthesis, taurine, glycine, glutamine, and cell N showed similar relations to that observed for arginine with respect to toxin contento Furthermore, the mole ratio of arginine/toxin could vary by a factor of up to 5 between AL1V and AL2V at peak values of toxin cell-1, and by more than 5 within a strain when growing under different conditions. These observations suggest that the relationship between free arginine content and toxin content is complex. No explanation for the higher toxin content of AL1V is apparent, except that AL1V has a higher N-content per cell and this may be conducive to a higher Tale of synthesis of the N-rich toxins.Publicado

Estudio de crecimiento y producción de toxinas en tres especies de Alexandrium: A. catenella, A. peruvianum y A. ostenfeldii.

Se estudió el crecimiento y el contenido en toxinas de 3 especies de dinoflagelados: Alexandrium catenella, A. peruvianum y A. ostenfeldii. A. catenella mostró tasas de crecimiento más elevadas que las otras dos especies. Las tres especies crecieron más rápidamente a 26ºC y menos a 15ºC aunque las tasas de crecimiento no mostraron grandes diferencias. Se observaron diferentes ventanas óptimas de crecimiento en relación a la salinidad en las tres especies. Las máximas tasas de crecimiento fueron a 32 y 37 de salinidad para A. catenella, a 25 de salinidad para A. peruvianum y a 10 de salinidad para A. ostenfeldii. Las tres especies produjeron toxinas PSP pero en muy diferentes cantidades, mostrando notables diferencias en el perfil de toxinas: (10 componentes A. catenella: C1, C2, GTX1-6, neoSTX y STX; 3 componentes A ostenfeldii y A. peruvianum: GTX2-3 y STX). La concentración de toxinas PSP en A. peruvianum fue muy baja. Se detectó también presencia de espirólidos (13-Desmetil-SPX C) en A. peruvianum y Gymnodimina-A en A. ostenfeldii

Palytoxins: Biological and chemical determination

Palytoxin (PLTX) is a marine polyether toxin with a very large and complex molecule that has both lipophilic and hydrophilic areas. It presents the longest continuous carbon atoms chain known to exist in a natural product second only to maitotoxin. This toxin was first isolated from Palythoa toxica and was subsequently reported in dinoflagellates of the genus Ostreopsis. Although PLTX has so far been associated with ciguateric fish poisoning (CFP), recent evidence suggests that PLTXs should be excluded from CFP toxins. NMR and LC–MS/MS techniques have enabled the isolation of 10–15 new analogues from dinoflagellates ever since their first discovery. Literature data on biological origin, poisonings and chemistry of certain naturally occurring PLTX analogues, commonly known as ostreocins, are detailed herein. This paper reviews all reported biological and chemical analysis methods to date for this group of compounds.Versión del editor2,924

Toxin profiles of natural populations and cultures of Alexandrium minutum Halim from Galician (Spain) coastal waters

The toxin profiles of three isolates and natural populations of the PSP agentAlexandrium minutum from several Galician rías (NW Spain) was obtained by HPLC. The toxin content of cultures of A. minutum is dominated by GTX4 (80–90%) and GTX4 (10–15%) with small amounts of GTX3 and GTX2 (less than 3% of each); similar results were obtained for natural populations of Alexandrium from three different Galician rías, where a mixture of A. lusitanicum Balech and A. minutum can occur. Important quantitative differences were found between the three isolates, one being highly and two weakly toxic. The results obtained from these isolates and natural populations of Alexandrium were very similar to those obtained from HPLC analyses of mussels intoxicated during a PSP outbreak in Ría de Ares (Rías Altas) in 1984, confirming that A. minutum (previously identified as Gonyaulax tamarensis Lebour and Alexandrium lusitanicum) was the PSP agent during the toxic outbreak in May 1984. Toxin profiles obtained from natural populations during different PSP outbreaks in different rías and from cultures are fairly consistent and suggest that at least from the toxin point of view, A. lusitanicum and A. minutum are identical, and that the toxin profile of A. minutum from Galicia can be used as a biochemical marker.Publicado

Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection

A rapid liquid-chromatography (LC) method is presented which uses fluorescence detection (FLD) for palytoxin analogues analysis in benthic dinoflagellates of the genus Ostreopsis. The amino-acidic reagent 6-aminoquinolyl-N-hydroxisuccinimidyl carbamate (AccQ) was used for fluorescence labelling followed by LC–FLD. The efficacy of the method is exemplified by comparison of the results of the quantification obtained by LC–FLD and the hemolytic assay performed for palytoxins for which a highly significant linear correlation was achieved (r2 = 0.9118). The derivatized palytoxin analogues were determined in the range of 0.75–25 ng. The proposed method was successfully applied to the determination and quantification of palytoxin analogues in 14 samples from different strains of Ostreopsis from different locations (Western Mediterranean Sea, Canary Islands, Madeira Islands and Southern coasts of Brazil). To confirm the chemical structure of the toxins, samples were also analyzed by liquid chromatography coupled with mass spectrometry (LC–MS) with a system that has a poorer sensitivity when compared with LC–FLD detection and the hemolytic assay. The successful use of this method with dinoflagellates is a good indicator of suitability for other types of marine samples.Versión del edito