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IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 1 of 12 Module VII Harmful Algal Blooms (4 hours) Contents 1.1. Suggested reading 1.2. The harmful algae 1.3. The algal toxins 1.4. Biological effects of the toxins 1.5. Coupling between occurrence of toxic algae and toxicity of shellfish and finfish 1.6. References References in bold are literature which will be made available during the practical part of the course; selected articles are included in the literature folder of the distant learning programme. 1.1. Suggested reading Present document and the following papers: Hallegraeff, G.M. 2010. Ocean climate change, phytoplankton community responses, and harmful algal blooms: a formidable predictive challenge. – J. Phycol. 46: 220-235. 1.2. The harmful algae Marine phytoplankton blooms - or 'red tides' - are naturally occurring phenomena. About 300 species are reported at times to form blooms with cell concentrations of several million per liter. It appears that boththe frequency, intensity and geographical distribution of algal blooms have increased over the last few decades (Smayda 1990, Hallegraeff 2004). The possible influence of climate changes has been reviewed by Hallegraeff (2010), and the possible role of ballast water dispersal in relation to harmful algal blooms was discussed by Smayda (2007). Close to one fourth of the bloom forming species is known to produce toxins that may be detrimental or even fatal to other flora and fauna including human beings. Presently, six human syndromes are recognized caused by algal toxins accumulated in fin- or shellfish. Despite the very different chemical nature of the toxins, these do not generally change or reduce significantly in amount upon cooking, and neither do they generally influence the taste of the meat. Unfortunately, detection of contaminated seafood is not straightforward, IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 2 of 12 and neither fishermen nor consumers can usually determine whether seafood products can safely be consumed. In recognition of the potential health risks associated with the consumption of contaminated seafood, the government authorities in many countries have imposed restrictions on certain seafood products for human consumption. These restrictions include analyses of the seafood products for algal toxins, and by many countries it is also required that monitoring programmes for the detection of harmful algae are established (Anderson et al. 2001). In addition to posing serious health risks to consumers of seafood, some toxic algae may have devastating effects on fish, both in the wild and in aquaculture. Several species of phytoplankton belonging in different taxonomic groups can produce toxins, which may result in extensive fish kills with major economic losses (Shumway 1990, Corrales & Maclean 1995, Zingone & Enevoldsen 2000). A comprehensive economic analysis of the global impact of phytoplankton blooms on the aquaculture industry is not available, but the direct economic losses from single events in North America and especially Japan and South East Asia have on several occasions amounted to more than 10 million US dollars. 'Red tides' is a concept previously applied by the international scientific community to events of harmful algal blooms (HAB), which is now the preferred terminology. It should be noted, however, that harmful effects are not necessarily associated only with toxin production by the microalgae. Some species may cause fish kills or other harmful events through secondary effects of intensive blooms, e.g. oxygen depletion or physical damage to fish gills. Such species include for example the dinoflagellate, P. micans, several species of the diatom genus Chaetoceros and a number of others (Hallegraeff et al. 2004). On the other hand, harmful effects may be caused by even very low cell concentrations of toxic species, e.g. Alexandrium spp. or Dinophysis spp., which are by no means reminiscent of a bloom as generally conceived. A bloom in this context may be defined as cell concentrations sufficiently high to cause harmful effects. In 1997, the IOC Intergovernmental Panel on Harmful Algal Blooms decided to establish a Task Team on Algal Taxonomy with the aim of providing an agreed reference list of harmful algal species, including correct citation of the author(s), date of valid publication, and a list of synonyms http://www.marinespecies.org/hab/ Further reading on HAB may be found in the GEOHAB Science Plan (2001). 1.3. The algal toxins Many species of microalgae are now known or suspected to produce toxins. In marine and brackish water environments most toxic species belong in the Dinophyceae, but also the Diatomophyceae, Haptophyceae, Raphidophyceae, and Cyanophyceae comprise toxic species. The algal toxins may cause damage to other flora and fauna directly or they may accumulate through the food web in e.g. shellfish or finfish thereby causing harm to their predators including humans (Backer et al. 2004, Hallegraeff 2004, Landsberg 2002). IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 3 of 12 Consumption of contaminated seafood may cause various gastrointestinal or neurological diseases in humans. The human syndromes and their most important symptoms are summarised in Table 1. There are currently no antidotes against these syndromes, but prolonged artificial respiration may save persons from even severe PSP, and treatment with mannitol may relieve the symptoms in case of ciguatera (Hallegraeff 2004). Shellfish and coral reef fish are causing most problems, but also other marine animals, e.g. birds, crabs, turtles, and sharks may accumulate algal toxins (Shumway 1990, Landsberg 2002, Shumway et al. 2003). IT IS IMPORTANT TO NOTICE THAT THE ALGAL TOXINS DO NOT GIVE BAD SMELL OR TASTE TO THE SEAFOOD, AND THEREFORECANNOT IMMEDIATELY BE DETECTED BY THE FISHERMEN OR THE CONSUMER, BUT ONLY THROUGH BIOASSAYS OR CHEMICAL ANALYSES. IT IS ALSO VERY IMPORTANT TO EMPHASIZE THAT THE ALGAL TOXINS ARE NOT DESTROYED BY COOKING AND THUS MAY BE PRESENT IN CANNED, FROZEN OR OTHERWISE PROCESSED SEAFOOD PRODUCTS. The highest toxin production in the algal cells may occur at different stages of growth in the different species. Thus in the species of Alexandrium studied so far, the toxin production is highest during the exponential growth phase (Cembella 1998 and references therein), while in species of Pseudo-nitzschia, the toxin production takes place during the stationary growth phase (Bates 1998 and references therein). A marked exception is Prorocentrum cordatum, which seems to be toxic only in the late stationary and senescent growth phase (Grzebyk et al. 1997). Table 1. Symptoms of algal toxin poisoning (modified from Hallegraeff 2004). Human syndrome Amnesic shellfish poisoning, ASP Azaspirazid poisoning, AZP Ciguateric fish poisoning, CFP Diarrheic shellfish poisoning, DSP Neurotoxic shellfish poisoning, NSP Paralytic shellfish poisoning, PSP Symptoms Mild Severe After 3-5 hours: nausea, vomiting, diarDecreasing reaction to deep pain, dizzirhoea, abdominal cramps ness, hallucinations, confusion, short-term memory loss, seizures Nausea, vomiting, severe diarrhoea, stomach cramps After 12- 24 hours: gastro-intestinal Neurological symptoms such as numbsymptoms such as diarrhoea, abdominal ness and tingling of hands and feet, cold pain, nausea, vomiting. objects feel hot to touch; difficulty in balance, low heart rate and blood pressure, rashes. In extreme cases death through respiratory failure. After 30 min.- a few hours (seldom more Chronic exposure may promote tumour than 12 hours): diarrhoea, nausea, formation in the digestive system. vomiting, abdominal pain. After 3-6 hours: chill, headache, diarrhoea, Paraesthesia, altered perception of hot and muscle weakness, muscle and joint pain, cold; difficulty in talking and swallowing nausea and vomiting Within 30 minutes: tingling sensation or Muscular paralysis, pronounced respiratory numbness around lips gradually spreading difficulty, choking sensation, death through to face and neck, prickly sensation in respiratory paralysis may occur within 2-24 fingertips and toes, headache, dizziness, hrs. nausea, vomiting, diarrhoea. Considerable clonal differences exist in many species ranging from highly toxic to low or even non-toxic clones. These differences are in some cases seen within the same IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 4 of 12 geographical area, while in other cases they relate to the geographical distribution. Such clonal differences are known from e.g. species of Alexandrium (Cembella 1998), Gymnodinium catanatum (Oshima et al. 1993), and species of Pseudo-nitzschia (Bates et al. 1998). 1.4. Biological effects of the toxins A detailed description of toxin chemistry and analytical methods is beyond the scope of this course. The chemical nature and the biological effects of algal toxins in the marine environment are summarised in Table 2. Extensive accounts of algal toxins, their effects, analytical methods etc. may be found in Falconer (1993), Botana (2000), Landsberg (2002), and Hallegraeff et al. (2004). Table 2. Summary of the most important toxins produced by marine microalgae and their biological effects with selected references. The human syndromes referred to in Table 1 are indicated in bold in the first column. Toxins and their effects on the environment Saxitoxins and saxitoxin derivatives, heterocyclic guanidines, about 20 naturally occurring derivative are known, causing PSP Chemical affinity Alcaloids Causative species Biological effects References Alexandrium spp., Gymnodinuim catenatum, Pyrodinium bahamense var. compressum Trichodesmium spp Neurotoxic, block the + Na channels of neuronal and muscular cells, preventing depolarisation and propagation of the action potential Shimizu (2000) Long & Carmichael (2004) Domoic acids (DA), 10 congeners, causing ASP Amino acids Pseudo-nitzschia spp, Nitzschia navisvaringica, Amphora caffeaeformis Both neurogical and gastrointestinal effects, strong glutamate agonist Azaspirazids, 3 congeners, causing AZP Polyether compounds Azadinium spinosum Neurotoxic symptoms with slow pro-gressive paralysis in mice, DSPlike symptoms in humans Brevetoxins, 2 groups of toxins with 10 naturally occurring analogues, causing fish kills and NSP Polyether compounds Karenia brevis, Chattonella spp. Ciguatoxins (CTX), more than 20 analogues are known, main cause of CFP Polyether compounds Gambierdiscus toxicus Neurotoxic, bind to the + Na -channel resulting in persistent activation of nerve cells; some toxin analogues with haemolytic effects Complex neurotoxic effects primarily due to + activation of the Na channel of cell membranes Bates (1998), Skov et al. (1999), Nijjar & Nijjar (2000), Van Dolah (2000), Quilliam (2004a) Tillmann et al. 2009, Satake et al. (1998), Ito et al. (2001), James et al. (2001), Quilliam (2004b) Baden & Adams (2000), Quilliam (2004b) Wright & Cembella (1998), Lewis et al. (2000), Lewis (2004) IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 5 of 12 Cooliatoxin Polyether compounds Coolia monotis Galacto-lipids, fatty acids Polyether compounds Gymnodimine Polyether compounds Amphidinium carterae, A. operculatum, Karenia mikimotoi, Chrysochromulina polylepis Karenia mikimotoi, Gymnodinium spp. ? Maitotoxins (MTX), 3 congeners, implicated in CFP Polyether compounds Gambierdiscus toxicus Okadaic acid (OA), dinophysis toxins (DTX-1 and DTX-2), at least 8 conge-ners but some are believed to be precursors or shellfish metabolites, casuing DSP Palytoxins Polyether compounds Dinophysis spp, Prorocentrum spp Polyether compounds Ostreopsis lenticularis, O. siamensis Pectinotoxins, 10 analogues are known Prorocentrolide, 2 analogues Polyether compounds Polyether compounds Dinophysis spp. Prymnesins Polyether compounds Prymnesium parvum Haemolytic, ichthyotoxic Spirolids, 4 naturally occurring analogues Polyether compounds Alexandrium ostenfeldii Yessotoxins, 6 congeners are known Polyether compounds Lingulodinium polyedrum, Protoceratium reticulatum Gonyaulax. spinifera Chattonella marina, C. antiqua, Fibrocapsa japonica, Olisthodiscus luteus, Heterosigma akashiwo, Cochlodinium polykrikoides ’Fast-acting toxins’, neurological symptoms including convulsions in mice, effects on humans unknown Non-diarrhetic, apparently interfering with lipid membranes, cardiotoxic Reactive oxygen compounds Prorocentrum lima, P. maculosum Neurological effects, blocking of unmylenated nerves, causing acute respiratory distress in mice Haemolytic, ichthyotoxic Holmes et al. (1995), James et al. (2001) ‘Fast-acting toxin’ which is toxic to mice, ichthyotoxic Wright & Cembella (1998), James et al. (2001) Legrand (1998), Estacion (2000), Van Dolah (2000), Rossini (2000), Van Dolah (2000) Neurotoxic affecting Ca2+ influx, the precise mechanism of action unknown Inhibitors of protein phophatase enzymes, OA presumably a tumor promotor Depolarising cell membranes affecting both cardiac, smooth, and sceletal muscles, and myelinated nerves, cytotoxic Non-diarrheic, hepatotoxic ’Fast-acting toxins’, Impairment of the respiratory and osmoregulatory functions of the gills Yasumoto et al. (1990) Wright & Cembella (1998), Tosteson (2000) Draisci et al. (2000) Wright & Cembella (1998), James et al. (2001) Igarishi et al. (1996), Wright & Cembella (1998) Cembella et al. (2000), Richard et al. (2001) Draisci et al. (2000), Rhodes et al. (2006) Landsberg (2002) IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 6 of 12 Kalkitoxin Nodularins Cylindrospermopsins Lipidic – thiazoline compound Cyclic pentapeptids Lyngbya majuscula neurotoxic Nodularia spumigena Alcaloids Umezachia natans hepatotoxic with inhibition of proteins (PP) Phosphatases 1 and 2A Hepatotoxic, with inhibition of glutathione and protein synthesis. Long & Carmichael (2004) Long & Carmichael (2004) Long & Carmichael (2004) Most algal toxins are neurotoxic, but also toxins with gastro-intestinal effects and cytotoxins, hepatotoxins, and dermatotoxins are known; the latter 3 types are produced by cyanobacteria and found in fresh and brackish water habitats. Neurotoxins. The PSP toxins comprise the largest and also the best characterised group of neurotoxins. They may be divided into 3 group: 1) the highly toxic carbamate toxin (saxitoxin, neosaxitoxin, gonyautoxins), 2) the decarbamoyl toxins of intermediate toxicity, and 3) the weakly toxicN-sulfocarbamoyl toxin. The PSP toxins block the Na+ channels of neuronal and muscular cells thereby preventing depolarisation and propagation of the action potential, which is essential for the transmission of nerve impulses and muscle contraction. The peripheral nervous system is particularly affected (Shimizu 2000). The ASP toxins are strong competitors to kainate and bind with high affinity to the glutamate receptors in the brain. Pertinent activation of the kainate glutamate receptors result in increased intracellular concentration of Ca2+, which in turn may result in cell rupture. Particularly the hippocampus, the site of learning and memory, part of the brain is affected by domoic acid (Nijjar & Nijjar 2000). The ciguatoxins (CTX) which are fat-soluble and the water-soluble maitotoxins (MTX) are the most potent algal toxins known. The CTXs bind to the sodium-channels making these permeable to Na+ thereby inducing depolarisation of cell membranes (the opposite effect of PSP toxins) (Lewis et al. 2000). The biological activity of the MTXs is Ca2+ dependent causing membrane depolarisation and influx of Ca2+into the cells, although the exact mechanism of action is unknown (Estacion 2000). The CTX produced by the microalgae Gambierdiscus toxicus may be transformed though the food web (herbivorous-carnivorous fish) into more oxidised forms, which are up to 10-fold more toxic than the CTX toxin produced by the alga (Lewis 2001); thus there may be a bio-magnification of the toxic effects of CTX toxins. The biological effects of some NSP toxin analogues are similar to those of CTX s. Toxins with gastro-intestinal effects. The DSP toxins inhibit protein phophatase enzymes, which regulate a large and diverse array of cellular processes. The diarrhoea associated with DSP is presumably caused by hyper-phosphorylation of proteins in the epithelia of the intestine resulting in disturbance of the water balance (Van Dolah 2000 and references therein). There is mounting evidence that okadaic acid is a tumour promotor (Rossini 2000 and references therein). IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 7 of 12 Pectenotoxins (PTXs) and Yessotoxins (YTXs) are usually grouped with the DSP toxins because these toxins often co-occur and are extracted together from shellfish. The molecular basis of the activity of these toxins is virtually unknown. None of them cause diarrhoea; PTXs are strongly hepatotoxicwhile the most important effect of YTXs seems to be on the cardiac muscle. The chemical structure of YTXs is closely related to MTXs (Draisci et al. 2000). Fast-acting toxins. Gymnodimine, prorocentrolids, spirolids, and pinnatoxins are called fastacting toxins because they rapidly kill mice in bioassays. The origin of pinnatoxins is unknown, while the others are producedby microalgae. The possible effects of these toxins on humans are unknown. Ichthyotoxins. Several species of harmful algae are known cause fish kills. The toxic principles involved include various haemolytic and reactive oxygen compounds as well as several compounds not yet characterized. (see Landsberg 2002 and Hallegraeff et al. 2004, and reference therein). Haemolytic compounds are produced by different species of unarmoured dinoflagellates, e.g. Amphidinium carterae, A. operculatum, and Karenia mikimotoi, but also Alexandrium tamarense may produce haemolytic compounds, which may cause fish kills. Shellfish mortality has been associated with blooms of Heterocapsa circularisquama, and Oda et al. (2001) suggested that this was caused by a haemolytic toxin but other unknown compounds may alsobe involved. Other species that may cause fish kills through production of haemolytic toxins are the haptophytes Prymnesium spp. and Chrysochromulina leadbeateri and C. polylepis. Reactive oxygen compounds (e.g. superoxides and hydrogen peroxide) are produced by several species of raphidophytes, which have caused severe fish kills on several occasions. A number of unmamoured dinoflagellates, e.g. Karlodinium micrum, and some species related to Karenia mikimotoi may cause fish kills but the toxic compounds are unknown 1.5. Coupling between occurrence of toxic algae and toxicity of shellfish and finfish In most cases where algal toxins cause human illness, the vector is shellfish or in case of CFP finfish (Shumway 1990, Landsberg 2002). Planktonic microalgae are food for filter feeding shellfish, and if toxic species are ingested, the algal toxins accumulate in the tissue of the shellfish. The shellfish do not seem to be severely affected by the toxins, but in some cases, the filtration rate decreases. Rates of toxin accumulation and depuration depend on the species of shellfish. Mussels accumulate andeliminate the algal toxins fast (Peperzak et al. 1995), while scallops (e.g. Saxidomus giganteus, Spisula solidissima, Siliqua patula) may retain the algal toxins in their tissue for several years (Fernandez et al. 2004). One of the main difficulties in the monitoring and management of HAB is the apparent lack of a direct and simple relationship between the occurrence of potentially toxic algal species and toxicity in shellfish. There may be many reasons for this apparent discrepancy including insufficient knowledge of the autecology of both the algae and the shellfish species involved and also insufficient sampling programmes and data analysis. One parameter, which is rarely considered in monitoring programmes, is relative cell abundance. This may, however, be important because the filtration rate of the shellfish depends on the food available, viz. the IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 8 of 12 phytoplankton biomass. Thus, Dahl & Johannessen (2001) found good relationship between the concentration of DSP toxins in blue mussels (Mytilus edulis) and increasing ratios of Dinophysis acuta (cells l-1) to chlorophyll a. Ciguatoxins accumulate in coral reef fish, and at least 400 species of fish may cause ciguatera. Maitotoxins are even more potent than ciguatoxins, but being water-soluble they are not believed to play a significant role in CFP (Estacion 2000, Lewis 2001). IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 9 of 12 1.5. References Anderson, D.M., Andersen, P., Bricelj, V.M.,Cullen, J.J., Rensel, J.E. 2001. Monitoring and management strategies for harmful algal blooms in coastal waters. - APEC #201-MR01.1, Asia Pacific economic program, Singapore, and Intergovernmental Oceanographic Commission technical series no. 59, Paris, 268 pp. Backer, L.C., Fleming, L.E., Rowan, A.D. & Baden, D.G. 2004. Epidemiology, public health and human deceases associated with harmful marinealgae. – In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (eds), Manual on Harmful Marine Microalgae. – Monographs on oceanographic methodology 11, Unesco, Paris, pp. 723-750. Baden, D.G. & Adams, D.J. 2000. Brevetoxin: chemistry, mechanism of action, and methods of detection. – In: Botana, L.M. (ed.) 2000. Seafood and freshwater toxins, Marcel Dekker, NewYork, pp. 505-532. Bates, S.S. 1998. Ecophysiology and metabolism of ASP toxin production - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI series, Series G, Ecological Sciences, vol. 41, Springer-Verlag, Berlin, pp. 267-292. Bates, S.S., Garrison, D.L. & Horner, R.A. 1998. Bloom dynamics and physiology of domoic-acid-producing Pseudo-nitzshcia species. - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI series, Series G, Ecological sciences, vol. 41, Springer-Verlag, Berlin, pp. 405-426. Botana, L.M. (ed.) 2000. Seafood and freshwater toxins. - Marcel Dekker, New York, 798 pp. Carstensen, J., Henriksen, P. & Heiskanen, A-S. 2007. Summer algal blomms in shallow estuaries: definitions, mechanisms and link to eutrofication. – Limnol. Oceanogr. 52: 370-384. Cembella, A.D. 1998. Ecophysiology and metabolism of paralytic shellfish toxins in marine microalgae. - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI series, Series G, Ecological Sciences, vol. 41, Springer-Verlag,Berlin, pp. 381-426. Cembella, A.D., Lewis, N.I. & Quilliam, M.A. 2000. The marine dinoflagellate Alexandrium ostenfeldii (Dinophyceae) as the causative organism of spirolide shellfish toxins. – Phycologia 39: 67-74. Corrales, R. A. & Maclean, J. L. 1995. Impacts of harmful algae on sea farming in the AsiaPacific areas. - J. App. Phycol. 7: 151-162. Dahl, E. & Johannesen, T. 2001. Relationship between occurrence of Dinophysis species (Dinophyceae) and shellfish toxicity. - Phycologia 40: 223-227. Draisci, R., Lucentini, L., Mascioni, A. 2000. Pectinotoxins and yessotoxins: chemistry, toxicology, pharmacology, and analysis. - In: Botana, L.M. (ed.), Seafood and freshwater toxins, Marcel Dekker, New York, pp. 289-324. Estacion, M. 2000. Ciguatera toxins: mechanism of action and pharmacology of maitotoxin. In: Botana, L.M. 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Prymnesin-2: a potent ichthyotoxin and hemolytic glycoside isolated from the red tide alga Prymnesium parvum. - J. Am. Chem. Soc. 118: 479-480. Ito, E., Satake, M., Ofuji, K., McMahon, T., Silke, J., James, K. & Yasumoto, T. 2001. Small intestinal injuries in mice caused by a new toxin, azaspirazid, isolated from Irish mussles. - In: Hallegraeff, G.M., Blackburn, S.I., Boalch, C.J. & Lewis, R.J. (eds), Harmful Algal Blooms 2000, Intergovernmental Oceanographic Commission of Unesco, Paris, pp. 395-398. James, K.J., Furey, A., Satake, M. & Yasumoto, T. 2001. Azaspirazid poisoning (AZP): a new toxic shellfish syndrome in Europe. - In: Hallegraeff, G.M., Blackburn, S.I., Boalch, C.J. & Lewis, R.J., (eds), Harmful Algal Blooms, Intergovernmental Oceanographic Commission of Unesco, Paris, pp. 250-253. Landsberg, J.H. 2002. The effects of harmful algal blooms on aquatic organisms. - Rev. in Fish. Sci. 10: 113-390. Legrand, A.M. 1998. 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(ed.), Seafood and freshwater toxins, Marcel Dekker, New York, pp.419-447. IOC Science and Communication Centre on Harmful Algae University of Copenhagen Harmful algal blooms, page 11 of 12 Long, B.M. & Carmichael, W.W. 2004. Marine cyanobacterial toxins. - In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (eds), Manual on Harmful Marine Microalgae, Monographs on Oceanographic Methodology 11, Unesco, Paris, pp. 279-296. Nijjar, M.S. & Nijjar, S.S. 2000. Ecobiology, clinical symptoms, and mode of action of domoic acid, an amnesic shellfish toxin. - In: Botana, L.M. (ed.), Seafood and freshwater toxins, Marcel Dekker, New York, pp. 325-358. Oda, T., Sato, Y., Kim, D., Muramatsu, T., Matsuyama, Y. & Honjo, T. 2001. Hemolytic activity of Heterocapsa circularisquama (Dinophyceae) and its possible involvement in shellfish toxicity. – J. Phycol. 37: 50. Oshima, Y., Blackburn, S.I., Hallegraeff, G.M. 1993. 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