Saxitoxin Analogues Research Articles

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C9H16N6O2. This structure differed from that of decarbamoyl STX (dcSTX; C9H16N6O3) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.

Paralytic toxin profile of the marine dinoflagellate Gymnodinium catenatum Graham from the Mexican Pacific as revealed by LC-MS/MS

The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (> 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations.

Identification and separation of saxitoxins using hydrophilic interaction liquid chromatography coupled to traveling wave ion mobility-mass spectrometry.

The aim of this work was to develop a reliable and efficient analytical method to characterise and differentiate saxitoxin analogues (STX), including sulphated (gonyautoxins, GTX) and non-sulphated analogues. For this purpose, hydrophilic interaction liquid chromatography (HILIC) was used to separate sulphated analogues. We also resorted to ion mobility spectrometry to differentiate the STX analogues because this technique adds a new dimension of separation based on ion gas phase conformation. Positive and negative ionisation modes were used for gonyautoxins while positive ionisation mode was used for non-sulphated analogues. Subsequently, the coupling of these three complementary techniques, HILIC-IM-MS, permitted the separation and identification of STX analogues; isomer differentiation was achieved in HILIC dimension while non-sulphated analogues were separated in the IM-MS dimension. Additional structural characteristics concerning the conformation of STXs could be obtained using IM-MS measurements. Thus, the collision cross sections (CCS) of STXs are reported for the first time in the positive ionisation mode. These experimental CCSs correlated well with the calculated CCS values using the trajectory method.

SxtA and sxtG gene expression and toxin production in the Mediterranean Alexandrium minutum (Dinophyceae).

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

Indirect quantitation of saxitoxin by HPLC with post-column oxidation and fluorometric detection.

The indirect identification and quantification of saxitoxin (STX) using other STX analogues by high-performance liquid chromatography with post-column oxidation and fluorescent detection (HPLC-FD) was investigated. Decarbamoylsaxitoxin (dcSTX) among the many STX analogues was selected as an external standard to identify and quantify STX. The retention time of STX in shellfish extracts by HPLC-FD was reproducibly estimated by using the retention time of dcSTX and the separation factor (α) between STX and dcSTX. Almost all of the columns tested to setup the method were useful to identify STX. Because a molar fluorescent coefficient of dcSTX was slightly different from that of STX, a factor used to correct the fluorescent coefficient in STX/dcSTX was determined to be 1.30. The indirect quantification of STX in scallop extracts by using the correction factor agreed to 80 - 100% precision with direct quantification using STX as an external standard.

Effect of temperature on growth and paralytic toxin profiles in isolates of Gymnodinium catenatum (Dinophyceae) from the Pacific coast of Mexico

The effects of temperature on growth, cell toxicity, toxin content, and profile of paralytic shellfish toxins was determined in eight isolates of Gymnodinium catenatum from several localities along the Pacific Coast of Mexico. The isolates were cultivated in modified f/2 media with Se (10−8 M), and a reduced concentration of Cu (10−8 M), under a 12 h:12 h day–night cycle with an irradiance of 150 μE m−2 s−1. Isolates were progressively adapted for three generations to each of the temperatures (16, 19, 22, 24, 27, 30, and 33 °C). The cultures were grown in 125 mL Erlenmeyer flasks with 60 mL of media and harvested by filtration in late exponential growth. Toxins were analyzed by HPLC with a post-column oxidation and fluorescent detection (FLD). G. catenatum isolates tolerate temperatures between 16 and 33 °C, with maximum growth rates of 0.32 and 0.39 div day−1 at 21 °C and 24 °C, respectively; maximum cell densities of 4700 and 5500 cells mL−1 were obtained at 27 and 21 °C, respectively. No effect of toxicity per cell with temperature was observed, varying between 10.10 and 28.19 pgSXTeq cell−1. Ten saxitoxin analogues were detected in all isolates, observing changes in the toxin profile with temperature. C1/2 toxins decreased from 80% mol at 16 °C to 20% mol at 33 °C, B1/2 toxins increased from 19% mol at 16 °C to 42% mol at 33 °C, and decarbamoyl toxins were more abundant at 21 °C. These results show that G. catenatum isolates from different regions of the Pacific coast of Mexico have a similar response to temperature and that this parameter can modify growth rate, cell density, and toxin profile of the species, particularly the decarbamoyl and sulfocarbamoyl toxins.

Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography–tandem mass spectrometry method

Hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5min with total analysis time of 9min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10ng/ml (n=3). Retention times for STX and GTX 1 were 6.47±0.03min and 4.44±0.01min (n=45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R2=0.9714 and R2=0.9768) in concentration ranges of 5–50ng/ml and 25–200ng/ml, respectively. For saxitoxin, calculated LOD was 3ng/ml and LLOQ 11ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC–MS/MS method are discussed.

Detection and confirmation of saxitoxin analogues in freshwater benthic Lyngbya wollei algae collected in the St. Lawrence River (Canada) by liquid chromatography–tandem mass spectrometry

The presence of cyanotoxins in benthic Lyngbya wollei algae samples collected in a fluvial lake along the St. Lawrence River, Canada, was investigated using a multi-toxins method. Hydrophilic interaction liquid chromatography (HILIC) and reverse phased liquid chromatography (RPLC) were coupled to triple quadrupole mass spectrometry (LC-QqQMS) for quantification and to quadrupole-time of flight mass spectrometry (LC-QqTOFMS) for screening and confirmation. The presence of two saxitoxin analogues, LWTX-1 and LWTX-6, was confirmed in benthic Lyngbya wollei algae samples. Concentration of LWTX-1 was between 209±5 and 279±9 μg g(-1). No other targeted cyanotoxin (such as anatoxin-a, nodularin, microcystin-LR, microcystins-RR and saxitoxin) was found in the samples. The presence of LWTX-6 was observed by using a screening approach based on an in-house database of cyanotoxins, an algorithm of identification and high resolution mass spectrometry measurements on the precursor and product ions. This work demonstrates the need for more research on the fate of benthic cyanotoxins in aquatic ecosystems such the St. Lawrence River.

Synthesis of Skeletal Analogues of Saxitoxin Derivatives and Evaluation of Their Inhibitory Activity on Sodium Ion Channels NaV1.4 and NaV1.5

Skeletal analogues of saxitoxin (STX) that possess a fused-type tricyclic ring system, designated FD-STX, were synthesized as candidate sodium ion channel modulators. Three kinds of FD-STX derivatives 4a-c with different substitution at C13 were synthesized, and their inhibitory activity on sodium ion channels was examined by means of cell-based assay. (-)-FD-STX (4a) and (-)-FD-dcSTX (4b), which showed moderate inhibitory activity, were further evaluated by the use of the patch-clamp method in cells that expressed Na(V)1.4 (a tetrodotoxin-sensitive sodium channel subtype) and Na(V)1.5 (a tetrodotoxin-resistant sodium channel subtype). These compounds showed moderate inhibitory activity towards Na(V)1.4, and weaker inhibitory activity towards Na(V)1.5. Uniquely, however, the inhibition of Na(V)1.5 by (-)-FD-dcSTX (4b) was "irreversible".

Preparation of Calibration Standards of N1-H Paralytic Shellfish Toxin Analogues by Large-Scale Culture of Cyanobacterium Anabaena circinalis (TA04)

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ±0.1 μmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 μmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50–90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.

Presence of benzoate type toxins in Gymnodinium catenatum Graham isolated from the Mexican Pacific

Benzoate type toxins have been described as an important component of Gymnodinium catenatum cells. In this paper we study these toxins in a G. catenatum strain isolated from the Mexican coast. A partition of the toxins was done by solid-phase extraction on a COOH cartridge and detected by HPLC coupled to fluorescence after pre-column periodate oxidation. Two groups of the hydrophobic analogues of saxitoxin were identified: those containing a sulphate group in the benzoate moiety instead of a hydroxyl group like GC1/2 or GC3 and the hydroxy-benzoate analogues, with a sulphate group at the eleventh position of the STX core present or absent (GCs-GTX and GCs-STX analogues, respectively). These toxins are more abundant, in a relative basis, when comparing with a G. catenatum toxin content isolated from Portugal. This is the first report of the presence of these toxins in a Mexican strain.

Hydrolysis of hydroxybenzoate saxitoxin analogues originating from Gymnodinium catenatum

The paralytic shellfish poisoning (PSP) toxin producer Gymnodinium catenatum produces several hydrophobic analogues of saxitoxin (STX). These are poorly studied due to their recent discovery and lack of standards. It was previously observed these hydrophobic analogues could be partially hydrolysed, loosing its benzoate moiety during alkaline oxidation to obtain fluorescent products measurable by HPLC analysis. The hydrolysis reaction was further explored to study two practical aspects. One was the indirect measurement of these compounds through its hydrolysis products: the decarbamoyl analogues of STX. The second one was to simplify standard production of decarbamoyl analogues, which are commonly found in contaminated shellfish products. PSP analogues are unstable in alkaline media. The hydrolysis of benzoate analogues progressed rapidly with increasing base amount, but the decarbamoylgonyautoxin type hydrolysis products were short lived and converted into decarbamoylsaxitoxin type analogues. For a rapid estimation of the presence of these benzoate analogues in seafood, decarbamoylgonyautoxin type analogues can only be measured as decarbamoylsaxitoxin type equivalents. For production of standards, complete hydrolysis of hydroxybenzoate decarbamoylsaxitoxin analogues can render decarbamoylsaxitoxin and decarbamoylneosaxitoxin. Obtaining decarbamoylgonyautoxins was not suitable resorting to decarbamoylgonyautoxin type hydroxybenzoate analogues due to the prolonged reaction times required for complete hydrolysis and their instability at high pH. Hydrolysis of sulphated-benzoate analogues was best suited for obtaining decarbamoylgonyautoxins due to the very rapid hydrolysis time required.

New saxitoxin analogues in the marine environment: developments in toxin chemistry, detection and biotransformation during the 2000s

Scientific study of paralytic shellfish poisoning toxins (PSTs) started in the early XXth century. In the 1920s it was understood the link between the toxicity observed in mussels with certain microalgae species. The poison was eventually purified from the clam Saxidomus giganteus, taking its name from it: saxitoxin (STX). Along the 1970s and 1980s it was understood that other STX analogues existed, both in dinoflagellates and bivalves. These were grouped into three major occurring families: the carbamate, N-sulfocarbamoyl and decarbamoyl, depending on the variation of the side chain of the tetrahydropurine core. The deoxydecarbamoyl family was additionally recognised in the dinoflagellate Gymnodinium catenatum. Chemical research into these STX analogues was conducted worldwide during the 1990s mainly by HPLC with pre- or post-column oxidation and fluorescence detection. Implementation of fluorescence detection with spectral capabilities and mass spectrometry detection during the 2000s led to the recognition of new analogues. Metabolites originated by single or double hydroxylation at C11 position were found in mussels, and later suspected in other bivalves. Designated M1-M4, these present very low fluorescence, and can only be studied resorting to HILIC-MS. Three hydroxybenzoate analogues were characterised as an important toxin fraction of the dinoflagellate Gymnodinium catenatum, and named GC1-GC3. Later, many more analogues were suspected: the corresponding N1-hydroxyl variants of GC1-GC3 (GC4-GC6), di-hydroxybenzoate variants (GC1a-GC6a), and sulphate-benzoate variants (GC1b-GC6b). In bivalves, carbamoylase activity renders these analogues into decarbamoyl analogues. Other compounds with PST-like characteristics have been detected in bivalves from Angola, Argentina and Vietnam. Today, the range of naturally occurring STX derivatives, both in marine and freshwater environments, accounts to more than fifty structural variants. This poses a problem for carrying out food safety analysis based solely in chemical methods. Fortunately, most modifications to the side chain of the tetrahydropurine core result in diminished toxicity.

Metabolites of saxitoxin analogues in bivalves contaminated by Gymnodinium catenatum

Bivalve metabolites of saxitoxin analogues, not present in microalgae, were recently described as an important toxin fraction in mussels contaminated by Alexandrium tamarense. These possess very low fluorescence, and require mass spectrometry detection. HILIC–MS was implemented to look for these metabolites in bivalves contaminated during Gymnodinium catenatum blooms at the Portuguese coast. The presence of M1 was tentatively identified in several bivalves, ranging from estuarine ( Mytilus galloprovinciallis, Cerastoderma edule and Ruditapes decussatus) to oceanic habitat ( Donax trunculus and Ensis spp.). It was hypothesized that M1 could contribute to an important fraction of the profile of STX analogues. M1 was more abundant in estuarine bivalves that retain longer PSP toxins, in the following order: mussels > cockles > clams. These data highlight that the study by fluorimetry alone of the carbamoyl, N-sulfocarbamoyl, and decarbamoyl families is manifestly insufficient to fully understand toxin dynamics in bivalves feeding on G. catenatum without a proper study of hydroxybenzoate and hydroxylated M-toxins.

Synthesis of bifunctional saxitoxin analogues by biotinylation

Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety.

Screening the toxicity and toxin content of blooms of the cyanobacterium Trichodesmium erythraeum (Ehrenberg) in northeast Brasil

Blooms of the cyanobacterium Trichodesmium occur in massive colored patches over large areas of tropical and subtropical oceans. Recently, the interest in such events has increased given their role in major nitrogen and carbon dioxide oceanic fluxes. Trichodesmium occurs all along the Brazilian coast and patches frequently migrate towards the coast. In this paper we screen the toxicity and toxin content of Trichodesmium blooms off the coast of Bahia state. Four samples, collected from February to April 2007, were analyzed. Organisms were identified and assessed for toxicity by means of several methods. Analogues of microcystins, cylindrospermopsins and saxitoxins were analyzed using HPLC. Microcystins were also assayed through ELISA. Results showed dominance of T. erythraeum, which makes up as much as 99% of cell counts. Other organisms found in smaller quantities include the dinoflagellates Prorocentrum minimum and P. rhathymum. Extracts from all samples delayed or interrupted sea urchin larval development, but presented no acute toxicity during a mouse bioassay. Saxitoxin congeners and microcystins were present at low concentrations in all samples, occurrences that had not previously been reported in the literature. Despite our finding of saxitoxin analogues and microcystins in Trichodesmium blooms, these toxins do not represent a potential harm to human health by primary contact. We conclude, based on our results and those reported in the recent literature, which differ from results published in 1963, that although toxins are present, there is no evidence that T. erythraeum blooms represent a threat to humans.

Atypical profiles of paralytic shellfish poisoning toxins in shellfish from Luanda and Mussulo bays, Angola

Currently Angola does not possess a monitoring programme for shellfish contamination with marine biotoxins. Among other potentially toxic microalgae, the presence of Gymnodinium catenatum and Pyrodinium bahamense has been reported at the Angola coast, two species associated worldwide with production of paralytic shellfish poisoning toxins (PSTs). A preliminary assessment of contamination with PSTs was carried out by HPLC with pre-column derivatization in samples of Semele proficua from Luanda Bay and samples of Senilia senilis from Mussulo Bay, collected between June 2007 and February 2008. An unusual profile was found, not matching any of the 10 oxidation products expected from the known hydrophilic PSTs normally reported in marine dinoflagellates: the N-sulfocarbamoyl, the decarbamoyl or the carbamoyl analogues of saxitoxin (STX). Four major compounds were noted, and designated A through D. These were not autofluorescent, additionally A and D presented much stronger response after peroxide oxidation than after periodate oxidation. Fluorescence emission and ultraviolet absorption maxima were similar to oxidation products of STX analogues. Separation carried out in two different C18 columns clearly showed the retention times did not match the oxidation products of standards. In the period of August through October 2007, samples of S. proficua from Luanda Bay presented a strong cross-reaction with a commercial antibody towards STX, in the range of 2600–5800 STX equiv./kg, while for the same period levels in the samples of S. senilis from Mussulo Bay ranged between <30 and 340 STX equiv./kg. This reaction was directly proportional to the presence of the four unknown fluorescent peaks. The presence of these compounds was very persistent throughout the studied period, in particular in Luanda Bay. This contamination might not be characteristic of a dinoflagellate blooming, but could be related to a persistent cyanobacterial contamination, due to the strong sewage pollution of the area.

Isolation and Structure Elucidation of New and Unusual Saxitoxin Analogues from Mussels

Chemical analyses of plankton and highly toxic mussel samples collected in eastern Canada during an intense bloom of the dinoflagellate Alexandrium tamarense established the presence of a complex mixture of paralytic shellfish poisoning (PSP) toxins. Application of a newly developed technique, hydrophilic interaction liquid chromatography-mass spectrometry, confirmed the identities of the known toxins and revealed the presence in the mussels of five saxitoxin analogues (M1-M5) that were not present in the plankton. Four of these compounds were isolated and their structures established by tandem mass spectrometry, 1D- and 2D-NMR spectroscopy, and chemical interconversion experiments. One of these was found to be 11beta-hydroxysaxitoxin (M2), while the other three were found to be new saxitoxin analogues, namely, 11beta-hydroxy-N-sulfocarbamoylsaxitoxin (M1), 11,11-dihydroxy-N-sulfocarbamoylsaxitoxin (M3), and 11,11-dihydroxysaxitoxin (M4). Compound M5 remains unidentified because of insufficient material for characterization.

Assessment of Specific Binding Proteins Suitable for the Detection of Paralytic Shellfish Poisons Using Optical Biosensor Technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0-1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.

Pigment profile and toxin composition during a red tide of Gymnodinium catenatum Graham and Myrionecta rubra (Lohman) Jankowski in coastal waters off Mar del Plata, Argentina

Microscope observations of samples, collected in autumn 2003 during a red tide in the coastal waters of Mar del Plata, Argentina, suggest that the phytoplankton community consisted mainly of the chain-forming dinoflagellate Gymnodinium catenatum (89 000 cells l−1). However, the unusually high concentration of chlorophyll a (171μg l−1) and the relative abundance of alloxanthin (73%) among the carotenoid pigments measured by high-performance liquid chromatography (HPLC) indicated that cryptophyceans predominated in these samples. Re-examination of Lugol's preserved plankton samples under high magnification revealed the presence of a low number of recognisable individuals of the ciliate Myrionecta rubra, together with a high abundance of cytoplasmic contents of their broken cells. Analysis of toxin composition of these samples by HPLC with fluorescence detection showed that, as in most of the G. catenatum strains reported from different geographical origins, the toxin profile was dominated (82%) by the less potent N-sulfocarbamoyl toxins. However, the toxin profile of the studied G. catenatum population exhibited certain atypical aspects: it contained the toxins C1,2, GTX4, GTX2,3 and dcGTX2,3, while lacking GTX5, GTX6, C3, C4 and the recently described hydroxybenzoate saxitoxin analogues GC1–3. The toxin profile of this population differed from those reported for Uruguayan strains, despite their geographic proximity. These results suggest that the observed G. catenatum bloom was a local event, which was not directly related to the transport of an established population from the estuarine region of the Río de la Plata. No harmful effects attributable to this red tide were observed on the Mar del Plata coast.