High performance liquid chromatography analysis of diarrhetic toxins inDinophysis spp. from the French coast

Diarrhetic shellfish poisoning has been a recurrent problem along the Brittany coast (France) since 1983. Okadaic acid (O.A.), the main toxin detected in mussels, is generally associated with the presence ofDinophysis cells in sea water. We report here the results of okadaic acid analyses by high performance liquid chromatography, on planktonic samples collected during the summer of 1991.D. sacculus, the majorDinophysis species, throughout this period, showed low okadaic acid content in raw extract, whereas toxic levels were 3- to 12-fold higher in sorted samples than in raw ones. A maximum O.A. concentration of 29.6 pg cell^-1 was found inD. sacculus/D. cf. acuminata extract. Similarly, higher O.A. levels were noted in raw samples when these two species were associated. Generally speaking, variations in raw samples were similar to those in sorted samples. Nevertheless, whenD. rotundata reached a concentration equal to that ofD. sacculus in sorted samples, the O.A. level was lower.     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Evidence of phagotrophy in Dinophysis fortii (Dinophysiales, Dinophyceae), a dinoflagellate that causes diarrhetic shellfish poisoning (DSP)

Food vacuoles were found in one species of pho‐totrophic Dinophysis, Dinophysis fortii Pavillard, collected in Okkirai Bay. Under transmission electron microscopy, almost 70% of observed food vacuoles were characterized by membranous profiles and contained large numbers of mitochondria. The mitochondria in the food vacuole had different morphologies from those in the D. fortii cytoplasm. This indicates that these vacuoles are not autolytic accumulation bodies, but ‘true’ food vacuoles. Identification of the origin of the contents failed, but the existence of large amounts of foreign mitochondria implies that the contents in the vacuoles were derived from eukaryotic prey. Other than the observation of the food vacuoles, bacterial cells were observed in the flagellar canal. Because the flagellar canal and connecting pusule sacs had been reported to relate to macromolecule uptake, the prey organisms of D. fortii were assumed to be both eukaryotic and prokaryotic organisms.     

Occurrence ofDinophysis spp. and toxic shellfish in the Northern Adriatic

The dynamics of the toxicity of the musselMytilus galloprovincialis was compared between two different shellfish farms, 5 km apart, but using the same cultivation technique. The main differences concerned the freshwater influx and the open aspect to the Gulf of Trieste. It is suggested that a deep closed bay and abundant fresh water inflow are the two main conditions for the low toxicity levels in mussels and for shorter periods of danger. A detailed study of the phytoplankton samples revealed the presence of eight species ofDinophysis in the area of both shellfish farms. During the period of the DSP outbreak in Slovenia (autumn and winter 1989).D. fortii andD. acuminata were the most frequentDinophysis species. There was a high positive correlation between the onset of mussel toxicity and the appearance ofDinophysis spp.     

Prorocentrum lima (Dinophyceae) in northeastern USA coastal waters: II: Toxin load in the epibiota and in shellfish

The seasonal variation in diarrhetic shellfish poisoning (DSP)-type toxins was followed in the epibiotic community and in shellfish between 41° and 44°N in coastal waters of the northwest Atlantic during a 2-year period. Low levels of okadaic-acid equivalents were detected at all stations in the <90 μm fraction of the collected epibiota as measured by the protein phosphatase inhibition assay, but only 3.5% of the samples had values greater than 100 ng (g dry weight of epibiota)⁻¹. No seasonal pattern could be detected due to differences in intensity, duration and timing of toxin content in the epibiota between the 2 years and between stations. Nevertheless, the concentration of DSP-type toxins in the epibiota correlated weakly but significantly with the abundance of Prorocentrum lima, when data from all stations were considered. A very limited toxin uptake by shellfish was measured at only one station in October and November 2001 and in June and July 2002 at times of maximum cell concentration of P. lima in the epibiota. Toxin levels in shellfish remained well below regulatory limits that would have required quarantine or bans on harvesting. Results from our 2-year survey suggest that, at this time, the threat of DSP events appears minimal. However, the presence of a known toxin producer and its demonstrated ingestion by shellfish would argue for further studies to better understand conditions leading to DSP outbreaks generated by an epiphytic dinoflagellate.     

Phytoplankton composition of the Kandalaksha Gulf, Russian White Sea: Dinophysis and lipophilic toxins in the blue mussel (Mytilus edulis)

Dinophysis acuminata and D. norvegica were observed in plankton net samples during the summer of 2002 from the Kandalaksha Gulf in the White Sea (North European Russia). Prorocentrum lima was found as an epiphyte on subtidal macroalgae in August, but not observed in plankton net samples. Protein phosphatase 2A (PP2A) inhibition measured 127.8 ng OA-equivalent/g of mussel (Mytilus edulis) hepatopancreas from samples collected a few days after when Dinophysis was recorded at a density of 1550 cells L⁻¹. Liquid chromatography–mass spectrometry confirmed presence of several classes of lipophilic shellfish toxins associated with Dinophysis spp. in the mussels including okadaic acid, dinophysistoxin-1, pectenotoxins and yessotoxins. No azaspiracid was detected. This represents the first identification of phycotoxicity in the White Sea.     

Seasonal distribution of species of the toxic dinoflagellate genus Dinophysis in Maizuru Bay (Japan), with comments on their autofluorescence and attachment of picophytoplankton

The seasonal distribution of the dinoflagellate genus, Dinophysis, in Maizuru Bay, Japan, was investigated from May 1997 to December 1999. Seven species of Dinophysis were detected, including the toxic species of Dinophysis acuminata and D. fortii. The most dominant species wasD. acuminata, detected year-around and more abundantly during periods when water temperatures were between 15 and 18 °C. No relationship was found between cell abundance of Dinophysis spp. and concentrations of dissolved inorganic nutrients. Phycoerythrin containing nano- and picophytoplankton (cryptophytes and cyanobacteria), suspected to be prey of mixotrophic Dinophysis, were enumerated simultaneously. A clear relationship was not found among the cell abundances of Dinophysis spp. and nano- and picophytoplankton. Autofluorescence of Dinophysis spp. (mainly D. acuminata and D. fortii) under blue-light excitation was usually of a yellow-orange color. Occasionally, Dinophysis spp. had red autofluorescencing and yellow-orange autofluorescencing particles. The proportion of cells possessing red autofluorescence tended to be higher in the warm season. Numerous coccoid cells of picophytoplankton (ca. 1–2 μm in diameter) were found attached to the cell surface of D. acuminata, D. fortii, etc. and food vacuole-like structures also observed. These observations suggest there is a close relationship between mixotrophic Dinophysis spp. and certain picophytoplankton. Based on our observations, the possibility that the picophytoplankton found to be attached onto Dinophysis cell surfaces are a food source for Dinophysis, and a source of DSP toxins, is discussed.     

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.     

Drastic hypothermia after intraperitoneal injection of okadaic acid,a diarrhetic shellfish poisoning toxin,in mice

The mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins had been used as the official method in Japan and also used in the world. In this study, hypothermia, one of the symptoms observed in mice after inoculation with DSP toxins, were characterized. Lethal and sublethal doses of okadaic acid (OA), a representative component of DSP toxins, were inoculated intraperitoneally into mice. Body-temperature changes over time were measured by an electronic thermometer or monitored by an infrared camera. Drastic hypothermia (<30°C in some mice) was observed in a few hours after administration of a lethal dose of OA. Dose-dependency was clearly seen between doses of OA inoculated and body-temperature decrease. Drastic hypothermia was also detected by using an infrared camera. These results suggest that hypothermia could be used as an index for the humane endpoint in experimental animal toxicological studies.     

Recent advances in analytical procedures for the detection of diarrhetic phycotoxins: a review

Okadaic acid and dinophysistoxins are produced by some marine unicellular algae from the plankton and also benthic microalgae and may accumulate in shellfish. These phycotoxins are involved in a gastrointestinal syndrome called diarrhetic shellfish poisoning (DSP), which occurs in humans after consumption of bivalve molluscs. Thousands cases of human poisonings in Europe were caused by consumption of toxic shellfish during the past decade. The rapid detection and the reliable determination of the main phycotoxins implicated in DSP are a major concern for governmental institutions in charge of the sanitary control of seafood safety. Analytical procedures for the detection and determination of DSP toxins can be classified as: bioassays, biochemical methods including immunoassays, or physicochemical methods. Although a large number of methods have been developed, none have been officially validated. A complete panel of tools for DSP toxin analysis should include screening, investigation, and confirmation methods. This paper presents a compilation of recent developments and optimisations of these methods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of okadaic acid,a major diarrheic shellfish toxin,in natural populations ofDinophysis spp. from the eastern coast of North America

Okadaic acid, one of the principal toxin components implicated in cases of diarrheic shellfish poisoning (DSP), was identified for the first time in natural phytoplankton assemblages from North American waters. During periods in late summer when significant quantities of okadaic acid were detected in net haul samples in the lower estuary and Gulf of St Lawrence in eastern Canada, the phytoplankton community consistently contained species of the dinoflagellate genusDinophysis Ehrenberg. The presence of okadaic acid was detected by screening dinoflagellate extracts with an enzyme-linked immunological assay (ELISA); positive results were confirmed by reverse-phase high-performance liquid chromatography (HPLC) separation, followed by fluorescence detection. Okadaic acid was only found in phytoplankton samples in which the photosynthetic dinophysoid speciesD. norvegica andD. acuminata were prominent; blooms of the related heterotrophic speciesD. rotundata exhibited no trace of okadaic acid, nor other suspected DSP components.     

Determination of paralytic shellfish poisoning (PSP) toxins in UK shellfish

A study was conducted to aid the interpretation of data generated by parallel testing of the qualitative Jellett Rapid Test (JRT) and the mouse bioassay (MBA) for detection of paralytic shellfish poisoning (PSP) toxins within the UK statutory shellfish biotoxin monitoring programme. A selection of stored sample extracts subjected to testing by MBA and/or JRT were further analysed by liquid chromatography with fluorescence detection (LC–FLD) to provide additional information on the concentrations of PSP toxins and toxin profiles.Results, from this study, demonstrate the potential of the JRT to effectively screen out PSP toxin negative shellfish samples and samples containing low concentrations of toxins from UK monitoring programmes. Additionally, data generated using LC–FLD highlights the potential of introducing alternative analytical techniques to completely replace the requirement for the MBA.     

Standardized extraction method for paralytic shellfish toxins in phytoplankton

The optimal conditions were established for extraction of paralytic shellfish toxins from a Danish clone of Alexandrium tamarense using extraction with acetic acid and HCl in the concentration range 0.01–1.0 N. Physical destruction of the cells was investigated microscopically to select the most efficient extraction procedure.The toxin content was quantitated by an automized isocratic reversed-phase high-performance liquid chromatography (HPLC) method. The best results as judged from the total amount of toxins and the toxin profile were obtained using 0.05–1.0 N acetic acid and 0.01–0.02 N HCl. Hydrochloric acid in the concentration range 0.03–1.0 N caused the amount of C1 and C2 toxins to decrease sharply and concomitant increase of gonyautoxins 2 and 3.The phytoplankton extracts with 0.1 to 0.5 N acetic acid or 0.01 N HCl were stable during 6 months at –20 °C, but the extracts with HCl 0.02 N underwent a change in toxin profile, although the total amount of toxins was constant.     

Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK

Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 10² to 10³ cells g⁻¹ fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.     

A procedure to estimate okadaic acid in whole dinoflagellate cells using immunological techniques

A single procedure to detect and estimate okadaic acid in isolated whole cells was developed based on immunofluorescence and microscope photometry. This procedure allows the study of variations in okadaic acid concentration per cell although it is no substitute for HPLC procedures. Cells from mid-log exponential and stationary phase from two different clonal cultures of the okadaic-acid-producing dinoflagellate Prorocentrum lima (PI 5V and PI 7V) were analyzed. The results showed that: (1) cells from saturated phase cultures contain more okadaic acid than those from exponentially-growing mid-log phase; (2) genetic differences exist in okadaic acid production between the clones used; (3) okadaic acid is synthesized continuously during the whole cell cycle.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

The effect of size and age on depuration rates of diarrhetic shellfish toxins (DST) in mussels (Mytilus edulis L.)

Depuration or elimination of diarrhetic shellfish toxins (DST) was followed for 73 days in 1- and 2-year-old mussels. The age groups also differed in size, providing a broad approach to studying the effect of the differences in physiology accompanying the differences in size. Content of DST was analysed both on groups and individual mussels. Environmental variables were measured to evaluate their effect on depuration.We found no significant differences in elimination rate of DST between 1- and 2-year-old mussels under natural conditions. This suggests that size and age do not affect the elimination rate of the DST. The present study is the first study on the effect of age and size on the elimination rate of algal toxins in bivalves. The natural variations in food levels and temperature were not found to affect the elimination rate of DST.The digestive gland weights in the 1-year-old mussels increased four times while the DST content per individual decreased eight times. This demonstrated that dilution of toxins due to tissue growth could have an important contribution to declines in toxin concentrations. Changes in tissue mass are affected by environmental variables via growth or starvation, and when such changes lead to concentration or dilution of toxins this does not reflect the accumulation or removal of toxins from the tissues. We hence suggest that when evaluating the actual elimination capacity of the mussels, as in the present study, the total content of toxins per individual should be used, rather than toxin concentrations.The 1-year-old mussels had faster growth compared to the 2-year-old mussels in both total soft tissue and digestive glands. The mechanism of DST elimination is still unknown. If this process involves metabolism of the toxins, one could expect the rates of elimination to follow overall metabolic rates. However, the results from the present study suggest that large differences in growth rates, which also include difference in feeding and metabolic rates, do not affect the elimination rate of DST. Our results support the assumption that the depuration rates cannot be accelerated, even in artificial systems, as a cost-effective way to solve the problem with toxic mussels for the industry.     

Palatability and fatality of the dinoflagellate <Emphasis Type="Italic">Prorocentrum lima</Emphasis> to <Emphasis Type="Italic">Artemia salina</Emphasis>

Prorocentrum lima is a toxic alga that produces both intra-cellular and extra-cellular toxins, including okadaic acid (OA) and dinophysistoxins (DTXs). Nauplii of the brine shrimp Artemia salina were exposed to both the cell and cell-free culture medium of P. lima in order to test the hypotheses that the extra-cellular medium is toxic to brine shrimp and that the P. lima cell is palatable but fatal to it. Artemia cysts incubated in the cell-free medium hatched, but mortalities were recorded for nauplii that hatched in, and metanuaplii exposed to, test solutions (autoclaved filtered seawater + cell-free medium) that contained at least 50% of the cell-free medium. Animals exposed to cells of P. lima readily fed on the cells. Some, especially among the Day 1 nauplii, ingested only one cell before dying, while others ingested more than one cell, up to six cells in the case of Day 3 nauplii, before dying. Day 3 nauplii were readily and heavily impacted by the P. lima cells. Survival analysis was used to evaluate survivorship of Day 1 to Day 3 nauplii exposed to cells of P. lima. Estimates were made of tD50s for the different age groups. Comparisons of the tD50s showed that the tD50s for Day 1 and Day 2 nauplii did not vary significantly, but they each varied significantly from the tD50 for the Day 3 nauplii. The possible ecological implications of the findings are discussed.     

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on OA and DTX1

It has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads^® SP850, Sepabeads^® SP825L, Amberlite^® XAD4, Dowex^® Optipore^® L-493 and Diaion^® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72 h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media.     

Diversity of Rhizobia isolated from various Hedysarum species

Cultural and physiological properties, serology, plasmid profiles and infective traits were determined for 23 strains of rhizobia isolated from various Hedysarum species: H. coronarium (common name: sulla) (16), H. carnosum (1), H. alpinum (3), H. mackenzii (2) and H. pallens (1) from Portugal, Spain, Tunisia, Alaska and Israel. Strains isolated from H. alpinum, H. mackenzii and H. pallens have slow growth rates on yeast-extract mannitol medium and were unable to nodulate H. coronarium plants, whereas the latter were effectively nodulated by all sixteen fast growing strains from sulla. Regardless of the country of origin all H. coronarium strains fell into one serogroup and were not serologically related with strains of other Hedysarum species. The RAPD (random amplified polymorphic DNA) fingerprinting method which was carried out on five H. coronarium and three H. alpinum strains allowed distinction to be made among serologically related rhizobia. No particular plasmid profile pattern was observed in relation to the host or geographical origin of the strains.