Differential accumulation of paralytic shellfish toxins from Alexandrium minutum in the pearl oyster, Pinctada imbricata

To investigate the potential for differential accumulation of paralytic shellfish toxins (PSTs) in various tissues of the akoya pearl oyster, Pinctada imbricata, two feeding trials were carried out using the PST-producing dinoflagellate, Alexandrium minutum. When fed with A. minutum at concentrations between 100 and 1300 cells ml⁻¹, the maximum clearance by P. imbricata was shown to occur at a density of 300 cells ml⁻¹. When fed twice daily at this rate for up 12 days, P. imbricata accumulated analogues of gonyautoxins (GTXs): GTXs 1,4 and 2,3. The levels of GTXs in the viscera increased progressively on days 4, 8 and 12 to peak at 17.9 ± 4.47 μg STX-equivalent 100 g⁻¹ biomass. Following 12 days of depuration, in the absence of A. minutum, GTX levels fell by approximately 65% to 6.0 ± 2.20 μg STX-equivalent 100 g⁻¹ biomass. No GTX was found in the oysters at the start of the trial or in untreated controls. The accumulation of GTX was found to be tissue specific. No GTX was detected in the muscle tissue of P. imbricata during the feeding trial.     

Removal of Paralytic Shellfish Toxins by Probiotic Lactic Acid Bacteria

Paralytic shellfish toxins (PSTs) are non-protein neurotoxins produced by saltwater dinoflagellates and freshwater cyanobacteria. The ability of Lactobacillus rhamnosus strains GG and LC-705 (in viable and non-viable forms) to remove PSTs (saxitoxin (STX), neosaxitoxin (neoSTX), gonyautoxins 2 and 3 (GTX2/3), C-toxins 1 and 2 (C1/2)) from neutral and acidic solution (pH 7.3 and 2) was examined using HPLC. Binding decreased in the order of STX ~ neoSTX > C2 > GTX3 > GTX2 > C1. Removal of STX and neoSTX (77%–97.2%) was significantly greater than removal of GTX3 and C2 (33.3%–49.7%). There were no significant differences in toxin removal capacity between viable and non-viable forms of lactobacilli, which suggested that binding rather than metabolism is the mechanism of the removal of toxins. In general, binding was not affected by the presence of other organic molecules in solution. Importantly, this is the first study to demonstrate the ability of specific probiotic lactic bacteria to remove PSTs, particularly the most toxic PST-STX, from solution. Further, these results warrant thorough screening and assessment of safe and beneficial microbes for their usefulness in the seafood and water industries and their effectiveness in vivo.     

First detection of cyanobacterial PSP (paralytic shellfish poisoning) toxins in Spanish freshwaters

Presence of the paralytic shellfish toxins (PSTs) saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and gonyautoxin-5 was analyzed by mass spectrometry in 41 Spanish freshwaters and 13 strains of potential PST-producing planktonic cyanobacteria. Toxins were detected in five waterbodies, but were absent from the isolated strains. PST containing samples belonged to the same geographical region (South Western Spain) and were most likely related to the presence of the genus Aphanizomenon.     

Development of quantitative NMR method with internal standard for the standard solutions of paralytic shellfish toxins and characterisation of gonyautoxin-5 and gonyautoxin-6

The chemical analysis of paralytic shellfish toxins (PSTs) requires standard solutions with accurate concentration. The mouse toxicity in each toxin is also essential knowledge for the introduction of chemical analysis as an alternative method to mouse bioassay (MBA) in routine monitoring of shellfish. In this study, we developed the quantitative analysis of PSTs by nuclear magnetic resonance (NMR), using tert-butanol as an internal standard. Only proton signals with longitudinal relaxation time (T₁) of less than 2.5 s, including the internal standard, were used for quantitation of toxins. Our method showed good precision (<3%) and accuracy (slope: 1.0038, R²: 1.0000). The limit of quantitation (LOQ) at 5% relative standard deviation (RSD) was calculated to be 0.16 mM, which corresponded to 67 μg/mL as Saxitoxin (STX) diacetate form, while the limit of detection (LOD) was 0.04 mM. Gonyautoxin-5 (GTX5) and gonyautoxin-6 (GTX6) isolated from mussels were quantified by our method, and the toxicities of GTX5 and GTX6 were obtained by the MBA in which mice were standardized by STX provided from FDA. The specific toxicities of GTX5 and GTX6 newly calculated by the MBA were 120 MU/μmol (29 μg STX equiv./μmol) and 105 MU/μmol (25 μg STX equiv./μmol), respectively. These results are useful to convert the amount of GTX5 and GTX6 into the mouse toxicity, especially in the areas where the dinoflagellate Gymnodinium catenatum predominantly produces both toxins.     

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.     

The uptake, distribution and elimination of paralytic shellfish toxins in mussels and fish exposed to toxic dinoflagellates

We exposed green-lipped mussels Perna viridis and black sea breams Acanthopagrus schlegeli to toxic dinoflagellates Alexandrium fundyense to evaluate the accumulation, distribution, transformation, and elimination of paralytic shellfish toxins (PSTs) in a controlled environmental condition. The mussels were fed A. fundyense for 7 days followed by 3 weeks of depuration, and the fish were fed toxic clams (pre-exposed to the dinoflagellates) for 5 days followed by 2 weeks of depuration. The toxin content and the compartmental distribution of PSTs were monitored throughout the experiments by high-performance liquid chromatography with post-column fluorescence derivatization (HPLC-FLD). This is the first report to assess the biokinetics of PSTs in marine fish under dietary exposure. The hepatopancreas in the mussels and the viscera in the fish accumulated most of the PSTs. Differential elimination of each toxin was observed in the mussels. The C2 toxins were eliminated rapidly in all organs; except in hepatopancreas, the more potent toxins such as GTX4, were eliminated slower during the depuration period. The relative proportions of various PSTs in the mussels changed over time, suggesting toxin-specific uptake and elimination rates, or biotransformation preferences between toxins. In the fish, the ratio of C1/C2 was 3.0 times (p<0.01) higher when compared to the clam tissues, indicating that conversion from C2 to C1 might have occurred when the toxin was transferred from the clams to the fish. In summary, species differences in uptake, distribution and elimination of PSTs were observed between mussels and fish, and this may influence trophic transfer of algal toxins in marine organisms.     

PSP toxins from Aphanizomenon flos-aquae (cyanobacteria) collected in the Crestuma-Lever reservoir (Douro river, northern Portugal).

The presence of paralytic shellfish poisoning (PSP) toxins in cultures of Aphanizomenon flos-aquae, isolated from the Crestuma-Lever reservoir, was found by reversed phase high performance liquid chromatography employing two isocratic elution systems for the separation of PSP toxins. With the first isocratic elution protocol, the presence of apolar toxins as saxitoxin, decarbamoyl saxitoxin and neosaxitoxin not detected. On the other hand, GTX4, GTX1 and GTX3 as well as Cs toxins were present either in the Aphanizomenon flos-aquae cells collected directly from the bloom or in the other toxic isolates priorly cultivated in laboratory conditions.     

Interactions of paralytic shellfish toxins with xenobiotic-metabolizing and antioxidant enzymes in rodents.

Paralytic shellfish toxins (PSTs) are neurotoxins known to block voltage-gated sodium channels in intoxicated animals and humans. Their metabolism in mammalian systems and their effects on other receptors are not as well understood. In this study, we investigated the in vitro metabolism of two classes of PSTs, gonyautoxin 2/3 (GTX2/3) and C1/2 toxins (C1/2), using rat and mouse liver enzyme preparations. We also analyzed the effects of these toxins on several antioxidant and xenobiotic-metabolizing enzymes in mice. These toxins were selected for their prevalence in the coastal waters of Southern China. When the toxins were incubated with liver preparations containing Phase I and Phase II xenobiotic metabolizing enzymes and appropriate co-factors, no transformation of the toxins was detectable. When mice were given sub-lethal doses of GTX2/3, a loss of activity was observed in hepatic ethoxyresorufin-O-deethylase, penthoxyresorufin-O-deethylase, glutathione peroxidase and superoxide dismutase, but not in glutathione S-transferase, catalase and glutathione reductase. Exposure to the same mouse units of C1/2 caused only a slight reduction in the activity of penthoxyresorufin-O-deethylase and glutathione peroxidase. Our results indicated that these toxins may not be metabolized readily in mammals and that they may cause adverse effects other than sodium channel blocking.     

Detection of harmful cyanobacteria and their toxins by both PCR amplification and LC-MS during a bloom event.

We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, S?o Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in S?o Paulo.     

Molecular Identification and Toxin Analysis of Alexandrium spp. in the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal Waters

The frequency of harmful algal blooms (HABs) has increased in China in recent years. Information about harmful dinoflagellates and paralytic shellfish toxins (PSTs) is still limited in China, especially in the Beibu Gulf, where PSTs in shellfish have exceeded food safety guidelines on multiple occasions. To explore the nature of the threat from PSTs in the region, eight Alexandrium strains were isolated from waters of the Beibu Gulf and examined using phylogenetic analyses of large subunit (LSU) rDNA, small subunit (SSU) rDNA, and internal transcribed spacer (ITS) sequences. Their toxin composition profiles were also determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All eight strains clustered in the phylogenetic tree with A. pseudogonyaulax, A. affine, and A. tamiyavanichii from other locations, forming three well-resolved groups. The intraspecific genetic distances of the three Alexandrium species were significantly smaller than interspecific genetic distances for Alexandrium species. Beibu Gulf isolates were therefore classified as A. pseudogonyaulax, A. affine, and A. tamiyavanichii. No PSTs were identified in A. pseudogonyaulax, but low levels of gonyautoxins (GTXs) 1 to 5, and saxitoxin (STX) were detected in A. tamiyavanichii (a total of 4.60 fmol/cell). The extremely low level of toxicity is inconsistent with PST detection above regulatory levels on multiple occasions within the Beibu Gulf, suggesting that higher toxicity strains may occur in those waters, but were unsampled. Other explanations including biotransformation of PSTs in shellfish and the presence of other PST-producing algae are also suggested. Understanding the toxicity and phylogeny of Alexandrium species provides foundational data for the protection of public health in the Beibu Gulf region and the mitigation of HAB events.     

Research on Marine Toxins in Taiwan

Some health issues of marine toxins in Taiwan are introduced here. From 1986 to 2002, the causative agents of seafood poisoning incidents have been reported to be tetrodotoxin (TTX, 30 outbreaks), paralytic shellfish poisons (PSP, 3 outbreaks), grass carp bile toxins (sporadically), ciguateric toxins (about 1 outbreak per year), excess dose of vitamin A (2 outbreaks), histamine (more than 5 outbreaks), pyropheophorbide a (1 outbreak), fish roe of Ascrossochelius paradoxus (1 outbreak), and allergens (sporadically). Among them, health issue of TTX in Taiwan is the most concerned problem. Therefore, TTX‐containing animals have been found to include puffer, goby, xanthid crab, gastropod, starfish, and flatworm. The dried dressed fish fillets produced from the non‐toxic puffers have been found to be adulterated by toxic puffers. The SDS‐PAGE and PCR methods for identifying species of puffers and their products have been developed. PSP was distributed in the purple clam, xanthid crab, and gastropod in Taiwan. The toxic alga Alexandrium minutum appeared in December–March. The toxin production of alga was affected by a variety of nutritional, environmental, and physiological factors. Most shellfish possessed high resistance to PSP, but the susceptibility of shellfish to the toxic alga was quite different depending on species. The food safety of animal bile juice is another important issue in Taiwan. The toxicity of bile juice was in the order of grass carp ? chicken ? snake. The major component of grass carp bile powder was 5α‐cyprinol sulfate (more than 90%) and those of other animals are bile salts. The toxic potencies of 5α‐cyprinol sulfate and 5α‐cyprinol induced acute kidney failure, but bile salts induced chronic liver dysfunction. Other marine toxins including diarrheic shellfish poisons (DSP), neurotoxic shellfish poisons (NSP), and amnesic shellfish poisons (ASP) are also very important from the health viewpoint in the world, though no poisonings due to those toxins have so far occurred in Taiwan.     

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.     

Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan.

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl-N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC-MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.     

Bacterial degradation of paralytic shellfish toxins.

Bacteria isolated from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture in vitro. Bacteria were isolated on marine agar and grown in marine broth supplemented with a mussel extract and an algal extract containing PSTs (saxitoxin, neosaxitoxin, gonyautoxins 2 and 3, decarbamoyl-gonyautoxins 2 and 3 and C1/C2 toxins). Toxin levels were measured before and after 5d of incubation, using high performance liquid chromatography (HPLC) and reduction of overall toxicity verified by mouse bioassays. Of the 73 bacterial cultures screened, seven isolates were designated "competent" PST degraders, individually reducing the overall toxicity of the PSTs by at least 90% within 3d. Most isolates degraded 100% of the saxitoxin and neosaxitoxin within 1-3d. In all cases, the overall kinetics of degradation of the toxicities was first order, as were the individual degradation kinetics of most of the individual toxins. This is the first report of nearly complete elimination of PSTs through bacterial action and may perhaps result in the development of a practical means to eliminate or reduce the risk of PSP intoxication associated with shellfish consumption.     

Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2.

Marine biotoxins from microalgae can accumulate in shellfish and lead to poisoning of human consumers as well as fish, marine mammals and sea birds. Toxicological assessment of the toxins and development of analytical methods require large amounts of high-purity toxins and their metabolites. Although these toxins can be obtained in limited amounts from contaminated shellfish or from microalgal cultures, difficulties arise when the toxin-producing microalga is difficult to culture or its identity is not known. To circumvent this problem, we have developed a large-scale method for solid-phase extraction (SPE) of lipophilic biotoxins from natural microalgal blooms in seawater. To enhance subsequent purification of toxins adsorbed on the column, we included a filtration step to release the toxins from the cells while removing insoluble compounds and cellular debris. The efficacy of the method was illustrated by extraction and purification of okadaic acid and dinophysistoxin-2 from a high-density Dinophysis acuta bloom in Spain and from a mixed bloom containing low densities of D. acuta in Norway. Isolation of the toxins adsorbed on the SPE column was simple and efficient, and results obtained so far indicate that the method is potentially applicable to a wide range of microalgal toxins such as azaspiracids, pectenotoxins, spirolides and microcystins. The method should also be useful for harvesting toxins from large-scale microalgal cultures, and for bioprospecting for and isolation of bioactive natural products from marine and freshwater environments.     

Uptake, distribution and depuration of paralytic shellfish toxins from Alexandrium minutum in Australian greenlip abalone, Haliotis laevigata

Farmed greenlip abalone Haliotis laevigata were fed commercial seaweed-based food pellets or feed pellets supplemented with 8 × 10⁵Alexandrium minutum dinoflagellate cells g⁻¹ (containing 12 ± 3.0 μg STX-equivalent 100 g⁻¹, which was mainly GTX-1,4) every second day for 50 days. Exposure of abalone to PST supplemented feed for 50 days did not affect behaviour or survival but saw accumulation of up to 1.6 μg STX-equivalent 100 g⁻¹ in the abalone foot tissue (muscle, mouth without oesophagus and epipodial fringe), which is ∼50 times lower than the maximum permissible limit (80 μg 100 g⁻¹ tissue) for PSTs in molluscan shellfish. The PST levels in the foot were reduced to 0.48 μg STX-equivalent 100 g⁻¹ after scrubbing and removal of the pigment surrounding the epithelium of the epipodial fringe (confirmed by both HPLC and LC-MS/MS). Thus, scrubbing the epipodial fringe, a common procedure during commercial abalone canning, reduced PST levels by ∼70%. Only trace levels of PSTs were detected in the viscera (stomach, gut, heart, gonad, gills and mantle) of the abalone. A toxin reduction of approximately 73% was observed in STX-contaminated abalone held in clean water and fed uncontaminated food over 50 days. The low level of PST uptake when abalone were exposed to high numbers of A. minutum cells over a prolonged period may indicate a low risk of PSP poisoning to humans from the consumption of H. laevigata that has been exposed to a bloom of potentially toxic A. minutum in Australia. Further research is required to establish if non-dietary accumulation can result in significant levels of PSTs in abalone.     

Variety of PSP toxins in four culture strains of Alexandrium minutum collected from southern Taiwan.

Paralytic shellfish poisoning (PSP) toxin profiles were compared among four culture strains of Alexandrium minutum. GTX-1, 2, 3 and 4 are the PSP toxins that occur in A. minutum, and other PSP toxins were not detected. When comparing the toxin profile of four A. minutum strains, GTX1 and 4 were the major toxins in Amtk1, Amtk2, and Amtk4, but in Amtk7, GTX3 and 2 were the major toxins. The results indicate that strains with various toxin profiles exist in southern Taiwan, and suggest that the comparison of the toxin profiles between strains at different localities is difficult. Additionally, the toxin profiles of A. minutum strains cultured in the same environment were different, suggesting that it was owing to the intrinsic nature of toxic algae.     

Combined Effects of Temperature and Toxic Algal Abundance on Paralytic Shellfish Toxic Accumulation,Tissue Distribution and Elimination Dynamics in Mussels Mytilus coruscus

This study assessed the impact of increasing seawater surface temperature (SST) and toxic algal abundance (TAA) on the accumulation, tissue distribution and elimination dynamics of paralytic shellfish toxins (PSTs) in mussels. Mytilus coruscus were fed with the PSTs-producing dinoflagellate A. catenella under four simulated environment conditions. The maximum PSTs concentration was determined to be 3548 µg STX eq.kg⁻¹, which was four times higher than the EU regulatory limit. The increasing SST caused a significant decline in PSTs levels in mussels with rapid elimination rates, whereas high TAA increased the PSTs concentration. As a result, the PSTs toxicity levels decreased under the combined condition. Additionally, toxin burdens were assessed within shellfish tissues, with the highest levels quantified in the hepatopancreas. It is noteworthy that the toxin burden shifted towards the mantle from gill, muscle and gonad at the 17th day. Moreover, variability of PSTs was measured, and was associated with changes in each environmental factor. Hence, this study primarily illustrates the combined effects of SST and TAA on PSTs toxicity, showing that increasing environmental temperature is of benefit to lower PSTs toxicity with rapid elimination rates.