Accumulation Of Paralytic Shellfish Toxins Research Articles

Trophic transfer of paralytic shellfish toxins from clams ( Ruditapes philippinarum) to gastropods ( Nassarius festivus)

A local strain of the dinoflagellate Alexandrium tamarense (ATCI01), which predominantly produces C2 toxin, was fed to the clams ( Ruditapes philippinarum) under laboratory conditions. Concentrations of paralytic shellfish toxins (PSTs) in the dosed clams were determined by High Performance Liquid Chromatographic (HPLC) analyses, and the clams were homogenized and then fed to the gastropods ( Nassarius festivus). In the toxin accumulation phase, which lasted for 42 days, concentrations of PSTs increased in the snails gradually, reaching a maximum of 1.10 nmole g −1 at the end of the exposure period. The toxin content of the homogenized clams (food) was 13.18 nmole g −1, which was about 12-fold higher than the PST content in the snails. Between day 43 and day 82, the snails were fed with non-toxic clams, and this period represented the depuration phase. Accumulation and depuration rates of PSTs in the snails, N. festivus, were determined by fitting the experimental data to user-defined parameters program using a one-compartment model. Two different modeling approaches were used to derive the accumulation and depuration rates. The first approach is to derive both values from the data for the toxin uptake. The second approach is to derive depuration rate from the depuration data and then to derive uptake rate, allowing for toxin depuration, from the data for toxin uptake. The first approach yielded more consistent results for the toxin concentration at the end of the uptake period, when compared with the experimental data. The toxin uptake and depuration rates were 1.64 (pmole of toxin into snail per day) per (nmole g −1 of toxin in food) and 0.06 ± 0.02 day −1 (mean ± SE), respectively. The toxin profiles of snails were similar to the clams, but different from the algae. Besides C toxins (C1 and C2), dcGTX2 and dcGTX3 were also detected in both clams and snails. The β:α epimer ratio gradually decreased during trophic transfer and approached a ratio of 1:3 (26.4 mol%:73.6 mol% at day 42) in the snails, near the end of the accumulation period.

Accumulation of paralytic shellfish toxins (PST) from the cyanobacterium Aphanizomenon issatschenkoi by the cladoceran Daphnia magna

In order to access the effects of Paralytic Shellfish Toxins (PST) in freshwater environment, the accumulation of PST produced by the cyanobacteria Aphanizomenon issatschenkoi in juvenile Daphnia magna was investigated. D. magna was exposed to A. issatschenkoi cells (1.2×10 6 cells ml −1) for 6, 8, 12, 24 and 30 h and also to lyophilised material (1 mg ml −1) for 24 h. Survival and somatic growth of the juvenile D. magna was investigated, as was the activity of the biotransformation enzyme system glutathione-S-transferases (GSTs). Between 643±65.35 and 1170±51.72 pmol PST ml −1 were detected by HPLC-FLD in D. magna culture medium containing cells and 2745±64.61 pmol PST toxin ml −1, in the medium containing lyophilised material. PST were detected in D. magna tissues in cells exposure (between 6.51×10 −2±1.37×10 −2 and 3.78×10 −1±1.15×10 −2 pmol PST animal −1). In D. magna exposed to lyophilised material the mean (±SD) PST concentration was found to be 6.96×10 −3 (±3.84×10 −3) pmol PST animal −1. Following exposure to 1.2×10 6 cells ml −1 A. issatschenkoi fresh cells growth and survival of D. magna were reduced. D. magna exposed to the two A. issatschenkoi treatments (fresh cells and lyophilised material), showed a reduction in activity of the cytosolic glutathione-S-transferases (cGSTs). The results of this study indicate that D. magna can accumulate PST toxins and that the cyanobacterium A. issatschenkoi affects both the fitness and growth potential of juvenile D. magna.

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean=1.4×10 9 cells l −1, S.D.=0.29×10 9 cells l −1) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PST l −1, S.D.=9.9 nmol PST l −1). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus. Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 μg PST 100 g −1 by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi. Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day −1) and those fed with A. falcatus (8.2% day −1) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean=1.4×10 9 cells l −1 , S.D.=0.29×10 9 cells l −1 ) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PST l −1 , S.D.=9.9 nmol PST l −1 ). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus . Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 μg PST 100 g −1 by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi . Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day −1 ) and those fed with A. falcatus (8.2% day −1 ) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.

Accumulation of paralytic shellfish toxins in the scallop Patinopecten yessoensis caused by the dinoflagellate Alexandrium catenella in Otsuchi Bay, Iwate Prefecture, northern Pacific coast of Japan

: The survey on scallop toxicity in association with causative dinoflagellates was conducted when the cultured scallops Patinopecten yessoensis were found to accumulate paralytic shellfish toxins in late November of 1997 at Otsuchi Bay, Iwate Prefecture, Japan. Alexandrium catenella was observed in high density at the beginning of the survey. The cell density of A. catenella decreased and completely disappeared in early December. The scallop toxicity also decreased with decrease of the cell density of A. catenella. The toxin profile of the scallop was similar to that of natural and cultured cells of A. catenella isolated from the Bay, showing that scallop toxicity is due to A. catenella. This is the first case in which paralytic shellfish toxins were confirmed in scallops during a bloom of A. catenella in Sanriku coast of Iwate Prefecture.

The purine degradation pathway: Possible role in paralytic shellfish toxin metabolism in the cyanobacterium Planktothrix sp. FP1

The paralytic shellfish toxins (PSTs) are potent neurotoxic alkaloids and their major biological effect is due to the blockage of voltage-gated sodium channels in excitable cells. They have been recognised as an important health risk for humans, animals, and ecosystems worldwide. The metabolic pathways that lead to the production and the degradation of these toxic metabolites are still unknown. In this study, we investigated the possible link between PST accumulation and the activation of the metabolism that leads to purine degradation in the filamentous freshwater cyanobacterium Planktothrix sp. FP1. The purine catabolic pathway is related to the nitrogen microcycle in water environments, in which cyanobacteria use traces of purines and ureides as a nitrogen source for growth. Thus, the activity of allantoicase, a key inducible enzyme of this metabolism, was used as tool for assaying the activation of the purine degradation pathway. The enzyme and the pathway were induced by allantoic acid, the direct substrate of allantoicase, as well as by adenine and, to a lower degree, by urea, one of the main products of purine catabolism. Crude cell extract of Escherichia coli was also employed and showed the best induction of allantoicase activity. In culture, Planktothrix sp. FP1 showed a differential accumulation of PST in consequence of the induction with different substrates. The cyanobacterial culture induced with allantoic acid accumulated 61.7% more toxins in comparison with the control. On the other hand, the cultures induced with adenine, urea, and the E. coli extract showed low PST accumulation, respectively, 1%, 38%, and 5% of the total toxins content detected in the noninduced culture. A degradation pathway for the PSTs can be hypothesised: as suggested for purine alkaloids in higher plants, saxitoxin (STX) and derivatives may also be converted into xanthine, urea, and further to CO 2 and NH 4 + or recycled in the primary metabolism through the purine degradation pathway.

Characterisation of bacterial communities associated with toxic and non-toxic dinoflagellates: Alexandrium spp. and Scrippsiella trochoidea

Several dinoflagellate species have been shown to produce potent neurotoxins known as paralytic shellfish toxins. Evidence is also accumulating that marine bacteria associated with dinoflagellates play a role in the accumulation of paralytic shellfish toxins. In this study, the diversity of bacteria in cultures of both toxic and non-toxic dinoflagellates, Alexandrium spp. and Scrippsiella trochoidea, were compared using colony morphology, restriction fragment length polymorphisms, denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA genes and, ultimately, sequence determination of the 16S rRNA genes. The results suggest that a number of different bacterial species are associated with dinoflagellates, some of which are common to each of the dinoflagellate cultures examined, whereas others appear to be unique to a particular dinoflagellate. The phylogenetic diversity of the bacteria observed was limited to two bacterial phyla, the Proteobacteria and the Cytophaga-Flavobacter-Bacteroides (CFB). Although phylum level diversity was limited, many distinct phylogenetic clades were recovered, including members of both the α- and γ-subclasses of the Proteobacteria. Additionally, several of the bacterial phylotypes isolated were not closely related to any published bacterial species but, rather, were identical to isolates characterised from Alexandrium cultures 4 years earlier. Finally, many of the bacteria isolated from the dinoflagellate cultures were related to microorganisms with known surface-associated life histories (e.g. the CFB phylum, Hyphomonas, Caulobacter and some members of the Roseobacter clade including Ruegeria algicola).

Characterisation of bacterial communities associated with toxic and non-toxic dinoflagellates: Alexandrium spp. and Scrippsiella trochoidea

Several dinoflagellate species have been shown to produce potent neurotoxins known as paralytic shellfish toxins. Evidence is also accumulating that marine bacteria associated with dinoflagellates play a role in the accumulation of paralytic shellfish toxins. In this study, the diversity of bacteria in cultures of both toxic and non-toxic dinoflagellates, Alexandrium spp. and Scrippsiella trochoidea, were compared using colony morphology, restriction fragment length polymorphisms, denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA genes and, ultimately, sequence determination of the 16S rRNA genes. The results suggest that a number of different bacterial species are associated with dinoflagellates, some of which are common to each of the dinoflagellate cultures examined, whereas others appear to be unique to a particular dinoflagellate. The phylogenetic diversity of the bacteria observed was limited to two bacterial phyla, the Proteobacteria and the Cytophaga-Flavobacter-Bacteroides (CFB). Although phylum level diversity was limited, many distinct phylogenetic clades were recovered, including members of both the α- and γ-subclasses of the Proteobacteria. Additionally, several of the bacterial phylotypes isolated were not closely related to any published bacterial species but, rather, were identical to isolates characterised from Alexandrium cultures 4 years earlier. Finally, many of the bacteria isolated from the dinoflagellate cultures were related to microorganisms with known surface-associated life histories (e.g. the CFB phylum, Hyphomonas, Caulobacter and some members of the Roseobacter clade including Ruegeria algicola).

Winter accumulation of paralytic shellfish toxins in digestive glands of mussels from Arcachon and Toulon (France) without detectable toxic plankton species revealed by interference in the mouse bioassay for lipophilic toxins

Since January 1993, neurological symptoms and rapid deaths (5 to 10 min) were typically observed in the mouse bioassay of acetone extracts of digestive glands from Arcachon and Toulon (France) during the winter season. It was assumed initially that a new lipophilic toxin was present because tests using the AOAC mouse bioassay for paralytic shellfish toxins on acid extracts of whole shellfish meat were negative, no known lipophilic toxins were detected and no toxic phytoplankton species were observed in the area during the poisoning events. In this study, however, preparative isolation of the toxic factor from toxic mussel digestive glands has revealed the presence of paralytic shellfish toxins, the principal ones being gonyautoxins-2 and -3 at Arcachon and gonyautoxins-1, -4, -2 and -3 at Toulon. The toxin concentrations recorded were below levels harmful to consumers and therefore represent a false positive in the mouse bioassay for lipophilic toxins based upon acetone extraction. The origin of the toxins remains to be determined. Copyright © 1999 John Wiley & Sons, Ltd.

Winter accumulation of paralytic shellfish toxins in digestive glands of mussels from Arcachon and Toulon (France) without detectable toxic plankton species revealed by interference in the mouse bioassay for lipophilic toxins.

Since January 1993, neurological symptoms and rapid deaths (5 to 10 min) were typically observed in the mouse bioassay of acetone extracts of digestive glands from Arcachon and Toulon (France) during the winter season. It was assumed initially that a new lipophilic toxin was present because tests using the AOAC mouse bioassay for paralytic shellfish toxins on acid extracts of whole shellfish meat were negative, no known lipophilic toxins were detected and no toxic phytoplankton species were observed in the area during the poisoning events. In this study, however, preparative isolation of the toxic factor from toxic mussel digestive glands has revealed the presence of paralytic shellfish toxins, the principal ones being gonyautoxins-2 and -3 at Arcachon and gonyautoxins-1, -4, -2 and -3 at Toulon. The toxin concentrations recorded were below levels harmful to consumers and therefore represent a false positive in the mouse bioassay for lipophilic toxins based upon acetone extraction. The origin of the toxins remains to be determined.

Accumulation of paralytic shellfish toxins by surfclams, Spisula solidissima (Dillwyn, 1897) in the Gulf of Maine: seasonal changes, distribution between tissues, and notes on feeding habits.

Accumulation of paralytic shellfish poisoning (PSP) toxins by surfclams, Spisula solidissima, was studied over a period of two years at two inshore locations in southern Maine and at six stations on Georges Bank in the Gulf of Maine. Whole animals as well as individual tissues (siphon, mantle, digestive gland, foot, adductor muscle, gill) were analyzed for PSP-toxicity levels using the standard AOAC mouse bioassay. Analyses of gut contents were carried out on surfclams from both inshore and offshore locations to identify the type of particles ingested. Surfclams feed primarily on phytoplankton and detrital material characteristic of the overlying seawater and surface sediment. No evidence was found for any selection based on particle size or type. Elevated levels of PSP toxins were noted in surfclams from Georges Bank more than two years after initial toxification. Toxins were not evenly distributed among the various tissues of surfclams. Initially, maximum toxicity among surfclam tissues was found in digestive glands; however, subsequent analyses of samples collected later in the year indicated that toxicity in gill and mantle tissues had increased relative to initial values. No toxicity was detected in adductor muscles. Surfclams are characterized by a high variation in total toxin load among individual animals, with a tendency for decreasing variation as toxin levels increase. Archived data from the Main Department of Marine Resources revealed annual and seasonal patterns of toxin accumulation by surfclams, i.e., toxin accumulation is an annual event, with initial increases in toxicity usually occurring in early spring.

Accumulation of paralytic shellfish toxins by green mussel Perna viridis by feeding on cultured cells of Alexandrium cohorticula isolated from the Gulf of Thailand.

Twelve clones of Alexandrium cohorticula were newly isolated from the Gulf of Thailand and made cultured. All the clones were found to be toxic. However, the toxicity levels per cell were different from clone to clone even when cultured under the same conditions. The green mussel Perna viridis which were fed on the cultured cells of A. cohorticula accumulated about 50% of the toxin contained in the supplied cells, showing that A. cohorticula may cause bivalve toxicity, when it blooms in the environment.

Report of the oceanographic observations off Kyoga-misaki in Japan Sea from October to December, 1957 : Japan, Maizuru Marine Observatory, Oceanographical Section, 1961. Bull. Maizuru Mar. Obs., 7: 29–36

A model was developed in this paper in order to study and compare the paralytic shellfish toxin (PST) transfer and accumulation of two different potential PST vectors in the planktonic community, the heterotrophic dinoflagellate in its red form Noctiluca scintillans and the copepod Acartia clausi. Different factors that influence the toxin transfer such as toxin synthesis, grazing on toxic and non-toxic food and population size of PST producers and vectors were considered in the model. Moreover, a laboratory experiment was conducted in order to calculate the detoxification rates of Noctiluca fed on Alexandrium catenella. According to the model results, the two grazers showed a significant difference mainly in the timing of the PST accumulation. Noctiluca exhibited a rapid response to the grazing of Alexandrium with high initial toxin accumulation, followed by a reduction to zero concentration of toxins in a period of almost two days. In contrast, Acartia showed a considerable delay in comparison to Noctiluca to accumulate the same amount of toxin in the population. This delay is linked to the slower reproduction rates that characterize the copepod. The range of the initial values used for the sensitivity analysis of the model is representative of the coastal environment of a Galician ria (embayment located at the NW of Iberian Peninsula), where the two grazers and Alexandrium may co-exist. The model for Acartia showed less sensitivity to these key parameters probably due to the time delay in accumulation of significant amount of toxins. Both grazers showed a rapid (50 h) reduction of ingested toxin, suggesting inefficiency to transfer toxins through predation in the food web.