Study of paralytic shellfish poisoning toxin profile in shellfish from the Mediterranean shore of Morocco

Since 1992, a monitoring program for bivalve molluscs contaminated by algal toxins was established at different stations along the Mediterranean Moroccan shores. The monitored stations were tested every 2 weeks. The presence of toxicity was determined using the mouse bioassay method. Toxin profile was carried out by HPLC/FD in selected contaminated tissues. According to the outcomes of this surveillance from 1994 to 1999, reliable information on toxicity of shellfish was obtained. They indicate that PSP is a recurrent toxicity in molluscs along the Mediterranean shore of Morocco. It has been noted a difference of PSP accumulation among individual shellfish. The cockle (Achanthocardia tuberculatum) presents toxicity throughout the year, while other specimens from the same area such as clam (Callista chione), warty venus (Venus gallina) and marine beans (Donax trunculus) accumulate it seasonally from January to April, after which they depurate the toxin. Moreover, the study of toxin profiles among individual shellfish was undertaken. It was found that shellfish presented a complex profile pointing to contamination by Gymnodinium catenatum.     

Comparative study on differential accumulation of PSP toxins between cockle (Acanthocardia tuberculatum) and sweet clam (Callista chione).

At the western Mediterranean coast of Morocco, the cockle (Acanthocardia tuberculatum) contained persistent high levels of paralytic shellfish toxins for several years, while other bivalve molluscs such as sweet clam (Callista chione) from the same vicinity were contaminated seasonally to a much lesser extent. In order to understand the causes of this prolonged contamination, a comparative study on PSP decontamination between sweet clam and cockle was conducted from November 2001 until June 2002. PSP toxicity was analysed by automated pre-column oxidation (Prechromatographic oxidation and LC-FD) in several organs of both species, namely digestive gland, foot, gill, mantle, muscle and siphon for sweet clams. The results showed that cockle sequester PSP toxins preferably in non-visceral organs (Foot, gill and mantle) contrary to sweet clam that sequester them in visceral tissues (digestive gland). The toxin profile of cockle organs indicated dominance of dcSTX, whereas sweet clam tissues contained especially C-toxins. Substantial differences in toxin profile between cockle and sweet clam, from the same area as well as from the composition of PSP toxin producer, Gymnodinium catenatum, confirm the bioconversion of PSP toxins in cockle.     

Protein markers of algal toxin contamination in shellfish

Filter-feeding bivalve molluscs are often contaminated by algal toxins. We have probed whether proteomic analysis of extracts from the digestive gland (DG) of mussels could be employed to identify biomarkers of contamination due to okadaic acid-group toxins. The protein extracts were obtained from 18 separate mussel samples and were analyzed by two-dimensional gel electrophoresis. When samples were divided into four different classes based on the content of OA-group toxins in the starting material, we found that two proteins varied as a function of OA contamination. By BLAST analysis, the two proteins were identified as a component of photosystem II and a subunit of NADH dehydrogenase. The analysis of peptide homologies showed that the peptide of photosystem II we detected in extracts from the DG of mussels contaminated by OA-group toxins is identical to its counterpart in Dinophysis algae, which are the producers of this group of toxins. We concluded that proteomic analysis can be used for the detection and identification of biomarkers of biotoxin contamination in shellfish, including both proteins expressed by the toxin producers and components that participate to the tissue response to the exogenous bioactive contaminant.     

Cytotoxic responses to unfractionated extracts from digestive glands of mussels.

The contamination of bivalve molluscs by lipophylic toxins is mainly detected by the use of unfractionated extracts from contaminated material in mouse bioassays. The development of alternate detection methods based on the use of cultured cells is hampered by difficulties related to the complexity of matrices including toxic compounds obtained from contaminated material. In this paper we have used unfractionated lipid extracts prepared from the digestive gland of mussels, which gave a negative response by the mouse bioassay, and have investigated their effects on functioning of MCF-7 cells. We show that altered growth was induced after addition of lipid extracts corresponding to less than 1mg of digestive gland per ml of culture medium. The cytotoxic effect was also confirmed by the analysis of the effect of the unfractionated extracts on four selected proteins, which were used as markers of general (actin), regulatory (mitogen activated protein kinase isoforms ERK1 and ERK2), as well as differentiative (alpha isoform of estrogen receptor) functions of the MCF-7 human breast cancer cell line.     

Determination of paralytic shellfish toxins in Portuguese shellfish by automated pre-column oxidation.

Automated pre-column oxidation (the method of Lawrence) was implemented on a routine basis since the end of 1996 to study paralytic shellfish poisoning (PSP) toxins in Portuguese shellfish. Liquid chromatography confirmed the presence of PSP toxins when the known toxic algae were present: Gymnodinium catenatum and/or Alexandrium cf. lusitanicum. On the other side, it has eliminated PSP toxins as a possible recurrent contaminant in oysters from Sado estuary. These oysters were already known to contain high levels of some metals (mainly zinc, copper and cadmium) due to their location in a contaminated area and their particular physiology prone to accumulate metals. The presence of PSP toxins in Scrobicularia plana from Mondego estuary and Tellina crassa from the northern coast, during the absence of the above toxic microalgae in the water column, was confirmed. Unlike other shellfish, these two genera have the feeding habit of aspirating more sediment than organisms in suspension, and probably ingest from the sediment resting cysts of PSP producing microalgae. This is another route of contamination that may help to explain why after a bloom certain shellfish species maintain toxicity for long periods. The method revealed to have a fast implementation on a daily basis, short analysis time (around 20 min between samples), high sensitivity and robustness, and therefore, it is one of the best HPLC methods for screening a large number of shellfish samples for monitoring purposes.     

Differential dynamics of dinophysistoxins and pectenotoxins, part II: offshore bivalve species.

Different dinophysistoxin's profiles have been found repeatedly amongst some offshore bivalve molluscs. Species such as the clam, Spisula solida, esterify dinophysistoxins to a great extent, and contain always more okadaic acid (OA) than dinophysistoxin-2 (DTX2). In contrast, the clam Donax trunculus has a much higher percentage of non-esterified toxins, and often contains more DTX2 than OA. A detoxification experiment with D. trunculus and S. solida showed that the higher percentage of DTX2 in Donax was due to this toxin being eliminated more slowly than OA. The ester analogues of OA and DTX2 were eliminated faster than free OA and free DTX2 in D. trunculus. As D. trunculus esterifies OA to a greater extent than DTX2, the greater proportion of the free form of DTX2 explains why there is a gradual increase in total DTX2 over time. This slow elimination of free toxins contributes to wild D. trunculus specimens being, on average, six times more toxic than S. solida specimens on the Portuguese south coast. The commercial shellfish species more often monitored along the coast of Portugal between 2003 and 2004 (eight species in total) were examined for DTX2 content. The maximal percentage of total DTX2 in the total DSP toxins (OA + DTX2) was 40% in all species (clams, razors, cockles, oysters) except D. trunculus and Mytilus galloprovinciallis, in which it reached 70-90%. This phenomenon is seen only when contamination is due to the microalga Dinophysis acuta. The other important DSP-producer, Dinophysis acuminata, causes contamination only with OA. Current data suggest that D. acuta along the Portuguese coast always produces OA and DTX2 in a fixed ratio of 60:40. When this alga is dominant, contamination of bivalves occurs in a OA/DTX2 ratio of 60:40. In bivalves in which these toxins are esterified to a great extent, this profile is maintained due to the parallel elimination of OA and DTX2 esters at similar rates. However, in species with lower esterification, the relative proportion of OA and DTX2 rapidly decreases due to the selective retention of free DTX2. Elimination of pectenotoxin-2 seco acids (PTX2sa), the main pectenotoxin form found in both offshore species studied, followed an exponential decay. The half-lives of PTX2sas found in D. trunculus and S. solida were similar to that found in a previous study with the cockle, Cerastoderma edule, but longer than found in the blue mussel M. galloprovinciallis.     

Characterization of 9H-(1,3-dichlor-9,9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP)

Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157–9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.     

Paralytic shellfish poisoning toxin profile of mussels Perna perna from southern Atlantic coasts of Morocco

During the monitoring programme of harmful algal blooms established along the south Atlantic coast of Morocco, a bimonthly determination of harmful algae and phycotoxins analysis in Perna perna was carried out from May 2003 to December 2004. Results of mouse bioassay (in organs and whole flesh) showed a seasonal evolution of paralytic shellfish poisoning (PSP) toxin. The mussel's contamination was associated with the occurrence in water of Alexandrium minutum.The PSP toxin profile obtained with high-performance liquid chromatography (HPLC/FD) revealed the dominance of gonyautoxins GTX2 and GTX3 and a minority of GTX1, GTX4 and saxitoxin (STX). This profile explains that the toxicity was mainly associated with A. minutum.     

Structural confirmation and occurrence of azaspiracids in Scandinavian brown crabs (Cancer pagurus).

In 2005 and 2006, azaspiracids were for the first time detected in brown crabs (Cancer pagurus) from the west coast of Sweden and the north and north-west coast of Norway. Azaspiracids are marine toxins that have been detected in blue mussels in Europe in recent years. On some occasions, they have been responsible for human intoxications with symptoms similar to those occurring by consumption of shellfish contaminated with okadaic acid group toxins. While the latter toxin group has been reported to accumulate in green crabs and brown crabs, azaspiracids have previously only been reported to occur in bivalve molluscs. LC-MS analysis of the hepatopancreas (HP) and roe of brown crabs revealed the presence of azaspiracid-1, -2 and -3, but only very low levels were detected in the white meat from the claws or the main shell. Mass spectral data were recorded using two different mass spectrometers, one with a triple-quadrupole mass analyzer and one with a linear ion-trap mass analyzer. The identities of the toxins were confirmed by comparing retention times and mass spectra of azaspiracid standards and the detected toxins. Levels detected ranged from 1.4 microg/kg tissue up to as much as 733 microg/kg tissue, although the majority of samples analyzed were below the suggested regulatory limit of 170 microg/kg HP. Higher levels were detected in HP compared with roe. Very little azaspiracids were detected in mussels from the same locations at the same time, and no proposed microalgal source of azaspiracids was reported in the water previous to or at the time of collection of the toxic crabs.     

Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish

Paralytic shellfish toxins in both cultured cells and natural phytoplankton blooms of the dinoflagellate Gymnodinium catenatum from inshore Tasmanian waters (Australia) were analyzed by high performance liquid chromatography, thin layer chromatography and electrophoresis techniques. The dinoflagellate toxins were dominated by low potency sulfocarbamoyl saxitoxin derivatives (98-99 mole% in total), including gonyautoxin VIII (C2) and its epimer (C1) and sulfocarbamoyl gonyautoxins I and IV (C3 and C4). Mussels and oysters contaminated by the dinoflagellate showed similar toxins, but contained larger proportions of C3 (40-57 mole%) and more potent carbamate toxins (7-23 mole% total).     

Dynamics of glutathione-S-transferases in Mytilus galloprovincialis exposed to toxic Microcystis aeruginosa cells, extracts and pure toxins

Molluscs and especially bivalves are able to accumulate dinoflagelates, diatoms and cyanobacteria toxins, and, being vectors for these toxins, transfer them along food chains. The data obtained from laboratory experiments showed that bivalve molluscs are resistant to cyanobacteria toxins. In this work, we wanted to test if Mytilus galloprovincialis organs react to microcystins and other cyanobacteria compounds by inducing or decreasing its GST activity. Acclimated mussels M. galloprovincialis were exposed to the toxic Microcystis aeruginosa M13 strain. Exposure of mussels to toxins was done in three ways: living Microcystis cells, crude Microcystis extracts and pure toxins. The measurement of soluble and microsomal GST activity in the different mussel organs was done by using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB). Analysis of the GST activity of the control mussels using CDNB as a substrate showed that cytosolic activity is much more significant than microsomal. Intact M. aeruginosa cells did not induce any significant response from the mussels, showing that these animals are quite resistant to the cyanobacteria if they are intact. On the other hand, cell extracts caused an important effect in the gut, in the gills and in the labial palps, although in different ways. There was an increase in GST activity in the gut and gills of mussels exposed to Microcystis extracts, showing a response of this detoxication pathway, but in the labial palps a severe reduction in GST activity occurred. Pure MC LR+YR induced an increase in GST activity in all organs but the labial palps. The results showed that other substances apart from microcystins may cause stress to mussels and affect detoxication enzymes such as GST.     

Glutathione-S-transferase activity of Fucus spp. as a biomarker of environmental contamination

Coastal zones are important areas from both ecological and economical points of view. However, in the last decades, in several regions of the globe, they have been increasingly impacted by complex discharges of contaminants and by marine traffic accidents. The Portuguese Atlantic coast is particularly exposed to these contaminants due to the proximity of important navigation routes. Several rocky shore organisms have been tested and used as bioindicators of environmental contamination. However, to the best of our knowledge Fucus spp., which are key species in rocky shore communities, have not been used as bioindicators in monitoring studies based on biomarkers. The objective of this study was to investigate the potential of glutathione-S-transferase (GST) activity of several Fucus species (Fucus ceranoides, Fucus spiralis var. platycarpus, Fucus spiralis var. spiralis and Fucus vesiculosus var. vesiculosus) to discriminate sites with different contamination levels along the Portuguese Northwestern coast, between the Minho river estuary and the Aveiro's Lagoon, as an environmental biomarker. With the exception of F. spiralis var. spiralis, for which a confusing pattern of activity was found requiring further analysis, all the other species and varieties showed higher GST levels in more contaminated sites than in less contaminated ones, indicating that Fucus spp. are suitable for use as bioindicators and their GSTs as biomarkers of environmental contamination in coastal zones and estuaries.     

Physiological cost of tolerance to toxicants in the European flounder Platichthys flesus, along the French Atlantic Coast

Physiological and genetic responses of flounder Platichthys flesus populations were investigated along the French Atlantic Coast in one moderately contaminated estuary (Ster) and three contaminated estuaries (Seine, Loire and Gironde). The focus of this study was to explore the relationship between stress resistance and energetic trade-offs, in order to detect possible differential physiological capacities or performances between individuals carrying particular alleles or genotypes (allozyme data) characterised as “tolerant” or “sensitive”. A general reduction of the relative fecundity, the growth rate and the condition factor was highlighted in contaminated fish populations, suggesting that survival in such polluted systems implies energetic costs for fish thus reducing the energy available for particular functions. A lower observed heterozygosity was also detected in contaminated populations with respect to the Ster, suggesting a general decrease in genetic variability in response to chemical stress (with an exception for the Seine estuary). This study confirmed the previously detected relationship between PGM 85, AAT1 95 alleles and reduced DNA damage in contaminated fish [Marchand, J., Tanguy, A., Laroche, J., Quiniou, L., Moraga, D., 2003. Responses of European flounder Platichthys flesus populations to contamination in different estuaries along the Atlantic coast of France. Mar. Ecol. Prog. Ser. 260, 273–284] and furthermore suggested that, reduced fecundity and condition factor associated to the individuals carrying the previous alleles, were also reflecting the cost of resistance to stress in polluted populations. The cost of tolerance to stress as well as the high gene flow from neighbouring populations less exposed to contamination may explain the apparently moderate increase of the suspected “tolerant” alleles in contaminated flounder populations.     

Paralytic shellfish poisoning due to ingestion of Gymnodinium catenatum contaminated cockles--application of the AOAC HPLC official method

The potent paralytic shellfish toxins (PSTs) produced by Gymnodinium catenatum have appeared irregularly since the onset in 1986 of a monitoring program aimed at preventing contaminated bivalves from the Portuguese coast to reaching the consumer. In years where high contamination levels were attained, sporadic episodes of human poisonings were also recorded, as in 1994. The reappearance of high contamination led to the appearance of new cases during 2007. This study reports details of toxin ingestion, symptomatology and toxin presence in the fluids of one of these victims, an adult male who ingested several kilograms of cockles.In cockle samples collected the week before and during the week when the intoxication took place, the major PSTs detected by the HPLC method based on AOAC Official Method 2005.06 belonged to the sulfamate (81–68 molar percent) and decarbamoyl groups (19–32 molar percent), comprising GTX5, GTX6, C1,2, C3,4, dcNeo, and dcSTX. In the patient urine sample sulfamate and decarbamoyl derivatives were also found, comprising by GTX5 (28%), GTX6 (25%), dcSTX (24%) and dcNeo (22%), but no C toxins and no dcGTX2,3 were detected. Compared to the cockle samples, there was an increase in the proportion of dcSTX, dcNeo and GTX5 (molar percentage) in the urine sample, but not of GTX6. Overall, compounds which had the presence of an O-sulfate at C11 were absent in urine while being relatively abundant in the bivalve (36.5–47.0 molar percent). In blood plasma PSTs were not detected.     

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography—multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53±5%), OA (26.5±2.3%) and total pectenotoxins (20.8±4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.     

Metabolic transformation of dinophysistoxin-3 into dinophysistoxin-1 causes human intoxication by consumption of O-acyl-derivatives dinophysistoxins contaminated shellfish

This paper describes for the first time a massive intoxication episode due to consumption of shellfish contaminated with 7-O-acyl-derivative dinophysistoxin-1, named Dinophysistoxin-3 (DTX-3). 7-O-acyl-derivative dinophysistoxin-1, a compound recently described in the literature, was found in shellfish samples collected in the Chilean Patagonia fjords. This compound does not inhibit Protein Phosphatases and also does not elicit the symptoms described for Diarrheic Shellfish Poisoning (DSP). The data showed here, give evidence of metabolic transformation of 7-O-acyl-derivative dinophysistoxin-1 (DTX-3) into Dinophysistoxin-1 (DTX-1, Methyl-Okadaic acid) in intoxicated patients. This metabolic transformation is responsible for the diarrheic symptoms and the intoxication syndrome showed by patients that consumed contaminated shellfish, which showed only the presence of 7-O-acyl-derivative dinophysistoxin-1. Patients fecal bacterial analysis for the presence of enteropathogens was negative and the mouse bioassay for DSP, performed as described for regulatory testing, was also negative. The HPLC-FLD and HPLC-MS analysis showed only the presence of DTX-3 as the only compound associated to DSP toxins in the contaminated shellfish samples. No other DSP toxins were found in the shellfish sample extracts. However, the patient fecal samples showed DTX-1 as the only DSP toxins detected in fecal. Moreover, the patient fecal samples did not show DTX-3. Since 7-O-acyl-derivative dinophysistoxin-1 (DTX-3) was the only compound associated to DSP toxins detected in the shellfish samples, an explanation for the diarrheic symptoms in the intoxicated patients would be the metabolic transformation of DTX-3 into DTX-1. This transformation should occur in the stomach of the poisoned patients after consuming 7-O-acyl-derivatives dinophysistoxin-1 (DTX-3) contaminated bivalves.     

Issues in fishery products safety in the united states

Most seafoods available to the U.S. public are wholesome and unlikely to cause illness in the consumer. Nevertheless, there are areas of risk. The major risk of acute disease is associated with the consumption of raw shellfish, particularly bivalve molluscs. For persons living in tropical areas, there is a risk of ciguatera; other natural toxins (paralytic shellfish poisoning, neurotoxic shellfish poisoning, etc.) have been associated with shellfish from endemic areas. Finally, there are less well-defined risks of acute and chronic disease related to environmental contamination of aquatic food animals. Dealing with such risks on a short-term basis requires improvements in the present system of regulatory control. In the long term, amelioration and eventual elimination of some hazards require strengthening and more effective application of control measures to prevent the disposal of human and industrial waste into offshore marine and fresh waters. © 1993 John Wiley & Sons, Inc.     

Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum

In a recent survey of paralytic shellfish poisoning (PSP) toxins in Gymnodinium catenatum Graham extracts, using LC with postcolumn oxidation and fluorescence detection, three novel saxitoxin analogues were revealed in isolates from several locations, including Australian waters. We have named them as G. catenatum toxins, GC1 (1), GC2 (2), and GC3 (3). The compounds were isolated from a culture of the Australian strain by LC-MS-guided fractionation employing a C18-silica column and hydrophilic interaction chromatography. The unusual structures of these novel compounds were characterized by low- and high-resolution MS, MS/MS, and NMR spectroscopy. GC3 (3) was found to be the 4-hydroxybenzoate ester derivative of decarbamoylsaxitoxin, while GC1 (1) and GC2 (2) are the epimeric 11-hydroxysulfate derivatives of GC3 (3).     

New Trends in the Occurrence of Yessotoxins in the Northwestern Adriatic Sea

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers’ revenues and the availability of local fish, causing a major economic loss in Italy’s main shellfish sector. Methods: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. Results: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. Conclusion: In this regard, it is essential to develop and activate, as soon as possible, an “early warning” system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations.