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.     

Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland.

The physiological response of the PSP toxin producing dinoflagellate Alexandrium minutum isolated from the Irish coast was assessed after modulating the initial concentrations of nitrate and phosphate in batch cultures. The cell growth in cultures of strain CK.A02 was primarily controlled by nitrate availability. In all experiments, only gonyautoxins 2 and 3 (GTX2 and 3) were synthesized along the different growth phases, with GTX3 dominating ( approximately 80%) at all stages, making the GTX2-3 toxin profile a possible population marker of A. minutum in Cork Harbour. The cellular toxin quotas remained low and relatively stable at around 2pgcell(-1), except when high N:P ratios were initially used for culture inoculations; in these conditions PSP toxins accumulated up to 14pgcell(-1). Due to the composition of the toxin profile, the toxicity of strain CK.A02 was generally relatively low (from 1.1 to 1.7pg STX eqcell(-1)) in comparison with strains from other geographic areas except when phosphate limiting culture conditions were applied (maximum of 12.5pg STX eqcell(-1)). Results showed that sufficient soluble protein quotas were necessary to observe the intra-cellular accumulation of PSP toxins in phosphate limiting conditions, highlighting also the requirement of adequate nitrogen supplies. The possible existence of localized toxicity hot spots in the field, linked to the accumulation of PSP toxins within A. minutum cells as a metabolic response to adverse environmental conditions, could potentially increase risks for shellfish farming operations.     

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.     

The effect of temperature on growth and production of paralytic shellfish poisoning toxins by the cyanobacterium Cylindrospermopsis raciborskii C10.

Cylindrospermopsis raciborskii is a cyanobacterium which produces either cylindrospermopsine or paralytic shellfish poisoning (PSP) toxins. We studied the effect of temperature on growth and production of PSP toxins by C. raciborskii C10, isolated from a freshwater reservoir in Brazil. We analyzed the extracellular and intracellular content of PSP toxins at two different temperatures: 19 and 25 degrees C. C. raciborskii C10 produces STX, GTX2, and GTX3 at both temperatures. dcSTX was also detected at 25 degrees C in the intracellular extracts obtained at the end of the stationary phase. The growth achieved at 25 degrees C and estimated by optical density at 700 nm was three times greater than at 19 degrees C. However, no significant differences were observed in the content of PSP toxins in either the cells or the extracellular media. The kinetics of accumulation of PSP toxins within the cells rather than in the media suggests an active PSP toxins-export process that is not related to cell lysis. The extracellular accumulation of PSP toxins at 19 degrees C suggested a biotransformation of STX to the epimers GTX2 and GTX3. The stability of the PSP toxins produced by C. raciborskii C10 was high enough for them to remain active in the media after 30 days (at 25 degrees C) or after 50 days (at 19 degrees C).     

Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.     

Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity,Toxin Profile,and Co-Occurrence with Cyclic Imines

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC–MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg⁻¹ for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg⁻¹ for the LC–MS/MS method, with a good correlation between both methods (r² = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.     

Analysis of paralytic shellfish poisoning toxin congeners by a sodium channel receptor binding assay.

This study was carried out to characterize the detection and quantitation of several paralytic shellfish poisoning (PSP) toxin congeners using a receptor binding assay (RBA). This involved competitive binding of the toxin congeners against tritium-labeled STX for receptor sites on rat brain sodium channels. Competitive binding curves were described by a four-parameter logistic equation. Half-saturation values (EC(50)) ranged from 4.38 nM for STX to 142 nM for GTX5. Receptor binding affinity was in the order STX>GTX1/4>neoSTX>GTX2/3>dcSTX>GTX5, and this was similar to the order of mouse toxicity of these congeners. Predicted toxin concentrations from observed STXeq values and EC(50) ratios relative to STX were within 20% or better of the actual concentrations used in the assay. In contrast predicted toxin concentrations using mouse toxicity ratios relative to STX did not provide a good match to actual concentrations, except for GTX1/4. This study has shown that the rat brain sodium channel RBA will provide a reliable integration of total toxicity of various PSP toxin congeners present in a sample.     

Toxin variability in cultured and natural populations of Alexandrium tamarense from southern South America – Evidences of diversity and environmental regulation

Cell content and composition of paralytic shellfish toxins of 10 cultured strains and 6 natural populations of Alexandrium tamarense from the Argentine sea, were analyzed. These data were compiled with previously published data into a comprehensive view of the toxin composition of the complex A. tamarense/Alexandrium catenella from southern South America.The N-sulfocarbamoyl derivatives C1,2 were predominant in almost all the cultured strains. The second major derivatives were GTX1,4, although the GTX1,4/C1,2 ratio varied largely. Some strains contain relatively high amounts of GTX2,3 (up to 29%) and/or neoSTX (up to 24%). In all strains STX was a minor component (0–4.4%) whereas GTX5 was present only in Alexandrium catenella isolates. Similarity analysis based upon toxin profiles showed that cultured strains from Argentine, Brazil, Chile and Uruguay clustered together. However, whereas some strains from the same geographic area exhibited different toxin profiles, and consistently fell out in separate subgroups, strains from Chile are grouped in a unique subgroup. In contrast to cultured strains, C1,2 were minor components among field populations. The highly toxic GTX1,4 were predominant in all spring field populations (69.1–93.6%). Moreover, their toxin cell content (163.9–261.4 fmol cell⁻¹) and toxicity (68.2–93.0 pg STX equiv. cell⁻¹) were several times higher that showed by the cultured strains. Field populations are more closely related to one another than to the cultured strains. However a less toxic and morphologically distinctive autumn population, contained GTX2,3 as the quasi unique (88.5%) toxin derivative clustered separately. Variability in toxin content and composition of A. tamarense field populations were well correlated with in situ temperature and nitrate concentration. Whereas toxin cell content and GTX1,4 (mol %) increased following saturation functions, GTX2,3 (mol %) decrease exponentially with the increase of the in situ nitrate concentration.     

Salinity effect on growth and toxin production of four tropical Alexandrium species (Dinophyceae).

Four tropical PSP toxins-producing dinoflagellates, Alexandrium minutum, Alexandrium tamiyavanichii, Alexandrium tamarense and Alexandrium peruvianum from Malaysian waters were studied to investigate the influences of salinity on growth and toxin production. Experiments were conducted on constant temperature 25 degrees C, 140 microE mol m(-2) s(-1) and under 14:10 light:dark photo-cycle with salinity ranged from 2 to 30 psu. The PSP-toxin congeners, GTX 1-6, STX, dcSTX, NEO and C1-C2 were analysed by high performance liquid chromatography. Salinity tolerance of the four species in decreasing order is A. minutum>A. peruvianum>A. tamarense>A. tamiyavanichii. Specific growth rates and maximum densities varied among these species with A. minutum recorded as the highest, 0.5 day(-1) and 6 x 10(4) cells L(-1). Toxin content decreased with elevated salinities in A. minutum, the highest toxin content was about 12 fmole cell(-1) at 5 psu. In A. tamiyavanichii, toxin content peaked at optimal growth salinity (20 and 25 psu). Toxin content of A. tamarense, somehow peaked at sub-optimal growth salinity (15 and 30 psu). Results of this study implied that salinity fluctuation not only influenced the growth physiology but also toxin production of these species.     

Accumulation and depuration profiles of PSP toxins in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Alexandrium catenella.

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella (Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4 x 10(7)cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.     

Non-selective retention of PSP toxins by the mussel Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels, Mytilus galloprovincialis, were contaminated by paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense. Temporal variations in the toxin content and the profile of mussels during the feeding experiment were monitored by high-performance liquid chromatography (HPLC). The toxin profile of mussels was compared with that of A. tamarense to clarify the mechanism of uptake of toxins in mussels. The prominent toxins in mussels and A. tamarense were N-sulfocarbamoyl toxins (C1,2) and carbamate toxins, gonyautoxin-1,4 (GTX1,4). The toxin profiles of both mussels and A. tamarense were almost constant throughout the experimental period. There were no remarkable differences in the toxin proportion between mussel and A. tamarense. These results indicate that mussels do not selectively accumulate particular toxins.     

Cyanotoxin Screening in BACA Culture Collection: Identification of New Cylindrospermopsin Producing Cyanobacteria

Microcystins (MCs), Saxitoxins (STXs), and Cylindrospermopsins (CYNs) are some of the more well-known cyanotoxins. Taking into consideration the impacts of cyanotoxins, many studies have focused on the identification of unknown cyanotoxin(s)-producing strains. This study aimed to screen strains from the Azorean Bank of Algae and Cyanobacteria (BACA) for MCs, STX, and CYN production. A total of 157 strains were searched for mcy, sxt, and cyr producing genes by PCR, toxin identification by ESI-LC-MS/MS, and cyanotoxin-producing strains morphological identification and confirmation by 16S rRNA phylogenetic analysis. Cyanotoxin-producing genes were amplified in 13 strains and four were confirmed as toxin producers by ESI-LC-MS/MS. As expected Aphanizomenon gracile BACA0041 was confirmed as an STX producer, with amplification of genes sxtA, sxtG, sxtH, and sxtI, and Microcystis aeruginosa BACA0148 as an MC-LR producer, with amplification of genes mcyC, mcyD, mcyE, and mcyG. Two nostocalean strains, BACA0025 and BACA0031, were positive for both cyrB and cyrC genes and ESI-LC-MS/MS confirmed CYN production. Although these strains morphologically resemble Sphaerospermopsis, the 16S rRNA phylogenetic analysis reveals that they probably belong to a new genus.     

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.     

Comparison of paralytic shellfish poisoning toxin between carnivorous crabs (Telmessus acutidens and Charybdis japonica) and their prey mussel (Mytilus galloprovincialis) in an inshore food chain.

Paralytic shellfish poisoning toxin in two shore crab species, Telmessus acutidens and Charybdis japonica, were compared with the toxin in the prey mussel Mytilus galloprovincialis and causative dinoflagellates Alexandrium tamarense, all having been collected at Onahama, Fukushima Prefecture, in the northern part of Japan. When the toxicities were detected in mussels by mouse bioassays, 73.7% of the sampled T. acutidens were toxic in the hepatopancreas. T. acutidens has been found to become toxic for three years, therefore, it can be concluded that the crab commonly and repeatedly accumulate the toxins via the food chain at Onahama. C. japonica was also expected to be a possible vector species, because small quantities of the toxins were detected in eight specimens of the crab by HPLC analysis. By the comparison of the toxin profiles in the dinoflagellates, mussels and the crab T. acutidens, reductive conversions of GTX1 and GTX4 were observed when the toxins passed through the three species in the food chain. But increases of STX and neoSTX by further reductive process were not observed in the crab. The absence of the STX group toxins in the crab suggests that the crab eliminates the toxin before such reductive process occur.     

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.     

A combination fluorescence assay and Folin-Ciocalteau phenol reagent assay for the detection of paralytic shellfish poisons

Paralytic shellfish poison (PSP) profiles of crude shellfish extracts were determined by linear gradient elution from a Bio-Rad AG-50-X4 strong cation-exchange resin mini-column. STX, GTX2 and GTX3 were detected by fluorescence assay. NeoSTX and GTX1/GTX4 were detected with a Folin-Ciocalteau phenol reagent assay. The major toxicity associated with extracts of Mytilus edulis and Mya arenaria collected during a 1972 red tide off Hampton, New Hampshire, was due to the presence of GTX1/GTX4, with some activity associated with neoSTX, GTX2 and GTX3. STX was also present. Correlations to mouse toxicity are provided.     

Comparative efficiencies of different non-toxic microalgal diets in detoxification of PSP-contaminated oysters (Crassostrea gigas Thunberg)

Experimental PSP contamination of adult Pacific oysters (Crassostrea gigas) by the toxic dinoflagellate Alexandrium minutum Halim (120 cells.mL-1 continuously maintained in each flume) was carried out in a recirculated seawater system to obtain toxin levels above the safety threshold. In these conditions, 150 to 300 micrograms STX.eq.100 g-1 of shellfish tissues were produced at 16 degrees C within 8 to 15 days, corresponding to field values observed along French coasts. Diets based on non-toxic flagellates or diatoms were then used to detoxify the contaminated oysters. Despite large individual variations in toxin levels at the end of the contamination period, detoxification times were of the same order of magnitude (3 to 4 days), reaching a toxin level equal to or less than the safety threshold. These variations were most likely related to marked individual variability in valve and/or clearance activities. No significant differences in detoxification rates were found when oysters were fed Isochrysis galbana, Tetraselmis suecica, Thalassiosira weissflogii, or Skeletonema costatum. The different biochemical compositions of each algal species appeared to have no significant effect on detoxification rates. GTX2/GTX3 were the dominant compounds found in shellfish tissues during depuration, whereas C toxins were quite low (< 2 micrograms STX.eq.100 g-1) and STX or NeoSTX undetectable. These results do not suggest any bioconversion of paralytic toxins but indicate good correlation between the toxin composition of Alexandrium and oyster tissues.     

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.