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    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Orgnization
    Geneva, 1984

         The International Programme on Chemical Safety (IPCS) is a
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        ISBN 92 4 154097 4 

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    1.1. Properties and analytical methods
        1.1.1. Chemical properties
        1.1.2. Methods of analysis for PSP in foods
     Biological methods
     Chemical methods
    1.2. Sources and occurrence
        1.2.1. Algal formation of toxins
     Oceanographic conditions associated
                      with blooms (red tide)
        1.2.2. Occurrence in seafood
     Accumulation in molluscs
     Accumulation in crustacea
     Transmission through zooplankton
                      to fish
     Accumulation in fish
    1.3. Exposure
    1.4. Metabolism
    1.5. Effects in animals
        1.5.1. Field observations
     Sea birds
        1.5.2. Experimental studies
     Acute toxicity
     Mode of action
    1.6. Effects on man
        1.6.1. Clinical studies
        1.6.2. Epidemiological studies


    2.1. Properties and analytical methods
        2.1.1. Chemical properties
        2.1.2. Methods of analysis for foodstuffs
     Biological methods
     Chemical methods
    2.2. Sources, occurrence, and exposure
        2.2.l Algae
        2.2.2. Occurrence in fish
        2.2.3. Environmental factors influencing the growth
              of causative dinoflagellates
        2.2.4. Human exposure
    2.3. Metabolism

    2.4. Effects on animals
        2.4.1. Experimental studies
        2.4.2. Mode of action
    2.5. Effects on man
        2.5.1. Clinical studies
        2.5.2. Epidemiological studies


    3.1. Properties and analytical methods
        3.1.1. Chemical properties
        3.1.2. Methods of analysis for tetrodotoxin in foods
    3.2. Occurrence and human exposure
    3.3. Mode of action
    3.4. Effects on animals
    3.5. Effects on man


    4.1. Properties and analytical methods
    4.2. Sources and occurrence
    4.3. Effects on animals
        4.3.1. Field observations
        4.3.2. Experimental animal studies
    4.4. Effects on man


    5.1. Sources and occurrence
    5.2. Chemical properties
    5.3. Analytical method
    5.4. Effects on animals - experimental studies
    5.5. Effects on man


    6.1. Dermatitis-inducing marine cyanophyte toxins
        6.1.1. Sources and properties
        6.1.2. Effects on animals
        6.1.3. Effects on man
    6.2. Freshwater cyanophyte toxins
        6.2.1. Sources, properties, analytical methods, and exposure
        6.2.2. Effects on animals
        6.2.3. Episodes of adverse effects reported in
              association with human exposure to toxic cyanophytes





Dr M.A. Arellano-Parra, Centro General de Intoxicaciones,
   Caracas, Venezuela

Dr R. Bagnis, Institut de Recherches Medicales Louis Malarde,
   Tahiti, Polynesie Française  (Chairman)

Professor A. Carpi de Resmini, Laboratory of Pathophysiology,
   Institute of Health, Rome, Italy

Dr J.M. Hughes, Center for Infectious Diseases, Centers for
   Disease Control, Atlanta, Georgia, USA

Dr C.Y. Kao, State University of New York, Downstate Medical
    Center, Brooklyn, New York, USA

Professor H.D. Tandon, All-India Institute of Medical
   Sciences, New Delhi, India

Dr K. Topsy, Chief Government Analyst of Mauritius,
    Vuillemain, Beau Bassin, Port Louis, Mauritius  (Vice Chairman)

Dr T. Yasumoto, Faculty of Agriculture, Tohoku University,
   Sendai City, Japan

Academician J. Zachar, Centre of Physiological Sciences,
   Slovak Academy of Sciences, Bratislava, Vlarska,


Dr R. Helmer, Scientist, Environmental Hazards and Food
   Protection, Division of Environmental Health, World Health
   Organization, Geneva, Switzerlanda

Dr A. Koulikovskii, Veterinary Public Health, Division of
   Communicable Diseases, World Health Organization, Geneva,

Associate Professor P. Krogh, Department of Microbiology,
   Royal Dental College, Copenhagen, Denmark  (Temporary
    Adviser) (Rapporteur)

Dr J. Parizek, Scientist, International Programme on Chemical
   Safety, Division of Environmental Health, World Health
   Organization, Geneva, Switzerland  (Secretary)

a  Technical Secretary for WHO of the Joint Group of Experts
   on the Scientific Aspects of Marine Pollution (GESAMP).


    While every effort has been made to present information in the 
criteria documents as accurately as possible without unduly 
delaying their publication, mistakes might have occurred and are 
likely to occur in the future.  In the interest of all users of the 
environmental health criteria documents, readers are kindly 
requested to communicate any errors found to the Manager of the 
International Programme on Chemical Safety, World Health 
Organization, Geneva, Switzerland, in order that they may be 
included in corrigenda, which will appear in subsequent volumes. 

    In addition, experts in any particular field dealt with in the 
criteria documents are kindly requested to make available to the 
WHO Secretariat any important published information that may have 
inadvertently been omitted and which may change the evaluation of 
health risks from exposure to the environmental agent under 
examination, so that the information may be considered in the event 
of updating and re-evaluation of the conclusions contained in the 
criteria documents. 

                           *  *  *

     A detailed data profile and a legal file can be obtained from 
the International Register of Potentially Toxic Chemicals, Palais
des Nations, 1211 Geneva 10, Switzerland (Telephone no. 988400 -


    Following the recommendations of the United Nations Conference 
on the Human Environment held in Stockholm in 1972, and in response 
to a number of World Health Assembly Resolutions (WHA23.60, 
WHA24.47, WHA25.58, WHA26.68), and the recommendation of the 
Governing Council of the United Nations Environment Programme, 
(UNEP/GC/10, 3 July 1973), a programme on the integrated assessment 
of the health effects of environmental pollution was initiated in 
1973.  The programme, known as the WHO Environmental Health 
Criteria Programme, has been implemented with the support of the 
Environment Fund of the United Nations Environment Programme.  In 
1980, the Environmental Health Criteria Programme was incorporated 
into the International Programme on Chemical Safety (IPCS).  The 
result of the Environmental Health Criteria Programme is a series 
of criteria documents. 

    The draft of the Environmental Health Criteria document on 
Aquatic (Marine and Freshwater) Biotoxins, prepared by Professor 
P. Krogh of Copenhagen, Denmark, was sent to focal points in member 
states and individual experts for comments. 

    A WHO Task Group on Environmental Health Criteria for Aquatic 
(Marine and Freshwater) Biotoxins met in Geneva from 12-17 
December, 1983.  Dr. J. Parizek opened the meeting on behalf of the 
Director-General.  The Task Group reviewed and revised the draft 
criteria document and made an evaluation of the health risks of 
exposure to aquatic (marine and freshwater) biotoxins. 

    The efforts of all who helped in the preparation and 
finalization of the document are gratefully acknowledged. 

                              * * *

    Partial financial support for the publication of this criteria 
document was kindly provided by the United States Department of 
Health and Human Services, through a contract from the National 
Institute of Environmental Health Sciences, Research Triangle Park, 
North Carolina, USA - a WHO Collaborating Centre for Environmental 
Health Effects. 


    In view of the complex character of the problem of aquatic 
biotoxins, the evaluation of the risks of adverse health effects 
made by the Task Group is presented together with the summary of 
information contained in the document. 


    This document deals with outbreaks of certain human diseases 
associated with human exposure to compounds produced by algae.  
Predators feeding on the algae become contaminated by these 
compounds which, in this way, enter the human food chain.  Diseases 
such as paralytic shellfish poisoning (PSP), ciguatera, and the 
more recently identified syndromes, neurotoxic shellfish poisoning 
(NSP) and diarrhoeic shellfish poisoning (DSP), are discussed in 
the document as well as the evidence of their association with 
dinoflagellate toxins present in human food.  Tetrodotoxin 
intoxication (pufferfish poisoning) is discussed because the 
compound, which is produced by certain fish in various areas of the 
world, has a similar action to that of saxitoxin, one of the main 
components causing PSP.  Direct dermal contact with toxins from a 
marine cyanophyte causes a particular type of acute dermatitis, 
observed in certain areas of the world.  No human disease has been 
identified as being a result of exposure to toxins from freshwater 
cyanophytes.  However, this topic has been included in the review 
for completeness. 

    Significant effects in non-human targets, associated with 
blooms of PSP-producing and NSP-producing dinoflagellates, and 
evidence of the involvement of PSP and NSP toxins in these 
outbreaks are also reviewed. 

Paralytic Shellfish Poisons

    The PSP toxins constitute a well characterized group of 
tetrahydropurines.  Saxitoxin was the first PSP component 
identified.  Subsequently, 12 other components, closely related in 
structure to saxitoxin, have been discovered in dinoflagellates 
and/or in shellfish. 

    The method used, so far, for the detection and quantitation of 
PSP components in environmental media such as food, and aimed at 
the assessment of exposure, is a bioassay using a rather unspecific 
endpoint (time to death of mice).  This method is not suitable for 
the measurement of the toxins in human tissues and fluids.  However, 
chemical methods such as those based on spectrophotometry and high 
pressure liquid chromatography are being introduced for this 

    The PSP components are produced by a well-defined group of 
dinoflagellates (mainly  Gonyaulax species), occurring in both 
tropical and temporate seas.  Molluscs, feeding on the algae, 
accumulate the toxins.  There is a report showing that the 

shellfish species accumulating PSP are resistant to the adverse 
effects of these compounds.  The highest concentrations of PSP in 
molluscs are found during algal blooms, a phenomenon that may be 
triggered by meteorological events.  However, shellfish also feed 
on non-motile algal cells (resting cysts) containing PSP, which are 
not bloom-related.  Another algal source of PSP in coral reefs is a 
red seaweed ( Janus sp.), which certain crabs feed on.  Finally, two 
reports have proved that even a freshwater cyanophyte,  Aphanizomenon 
 flos-aquae, can produce PSP components.  However, at present, there 
is no evidence that this would be of any significance with regard 
to human exposure, either directly or through entry of the compounds 
from freshwater cyanophytes into the food chain. 

    In marine ecosystems, transfer of PSP components from 
phytoplankton through zooplankton to fish has been observed, and 
fish kills and mass death of seabirds have been reported in 
association with blooms of PSP-producing dinoflagellates.  The PSP 
toxins were found in the zooplankton and in the gut of dead or 
diseased fish in these outbreaks, but only occasionally, in the 
muscle tissue of the same fish.  The available studies indicate 
that LD50 values for fish and birds are in the same range as those 
obtained in laboratory studies on mammals. 

    The only significant pathway of exposure to PSP, known at 
present for human beings, is the eating of predators contaminated 
by feeding on marine algal producers of the toxins.  The most 
significant food commodity is bivalve molluscs (mussels, clams, and 
oysters).  In shellfish, the highest concentrations of PSP have 
been found in the digestive organs (stomach and diverticula), but 
PSP has also been detected in other soft tissues. 

    Contamination with PSP is being monitored in shellfish-growing 
areas in several countries, and areas where the PSP level in the 
edible portion of the shellfish exceeds a certain value will be 
closed and will not be reopened before the level of shellfish 
contamination decreases below the action level. 

    Human exposure has also occurred through eating crabs 
contaminated by PSP in the coral reef ecosystems. 

    In spite of the above-mentioned evidence of PSP presence in 
dead or dying fish in association with blooms, no report is 
available, so far, that links consumption of finned fish by human 
beings with exposure to PSP. 

    There is no evidence of dermal exposure to PSP or exposure 
through drinking-water. 

    No data are available on the absorption, distribution, 
metabolism, and excretion of PSP toxins in animals, with the 
exception of limited information on PSP distribution in fish
and shellfish.

    Most of the studies on the toxicity of PSP have been performed 
as acute, single-dose experiments, using an extract from Alaskan 
butter clams containing saxitoxin.  In a number of animal species, 
the LD50 values by oral administration range from 100 to 800 µg 
saxitoxin/kg body weight.  When administered parenterally in mice, 
the LD50 is 3-10 µg saxitoxin/kg body weight, compared with 263 
µg/kg body weight by oral administration.  After parenteral or oral 
administration, the animals die within a few minutes in dyspnoea. 

    The systemic action of saxitoxin can be explained by a wide-
spread blockade of impulse generation in periferal nerves and 
skeletal muscles.  Saxitoxin affects the excitable membrane of 
single nerves and muscle fibres by blocking selectively the sodium 
channel through which the downhill movement of sodium ions accounts 
for the initiation of the electrical impulse.  Most probably 
saxitoxin (and the other PSP components) occupies a receptor on the 
outside surface of the membrane very close to the external orifice 
of the sodium channel. 

    Among the PSP components, saxitoxin, neosaxitoxin, gonyautoxin 
I, gonyautoxin III, and decarbamoyl saxitoxin exhibit lethal 
effects in the same range, whereas the remaining components 
(gonyautoxin II, IV, V, VI, VIII, VIII-epimer, sulfocarbamoyl 
gonyautoxin I & IV) are much less toxic. 

    Human intoxication associated with the consumption of shellfish 
containing PSP has been observed in many parts of the world.  About 
2500 cases of paralytic shellfish poisoning have been reported in 
the available literature.  There were 24 deaths among 905 PSP 
intoxications published in 1969-83.  The signs and symptoms in man 
may range from slight tingling and numbness about the lips to 
complete paralysis and death from respiratory failure.  Signs and 
symptoms appear rapidly within minutes to hours, and respiratory 
paralysis leading to death can occur within 2 - 12 h of consumption 
of the PSP-containing food.  The absence of hypotension was noted 
by the Task Group as being important for differential diagnosis.  
The results of animal studies indicate the existence of a dose-
response relationship.  However, the Task Group recognized serious 
difficulties in establishing the dose associated with the 
appearance of signs and symptoms and death.  These difficulties are 
based on such factors as the reliability of the bioassay used, so 
far, for estimation of the dose and uneven distribution of the 
toxins in the food consumed.  The Task Group noted the efficacy of 
monitoring the PSP content of shellfish at the production site as a 
preventive measure, and the need to search for other patients when 
observing a case of PSP intoxication. 

    The Task Group recognized also important effects of PSP 
exposure in non-human targets, represented by fish kill and mass 
death in seabirds. 

Ciguatera Toxins

    Consumption of a variety of tropical and subtropical fish has 
been associated with a human disease (ciguatera) charaterized by 
neurological, cardiovascular, and gastrointestinal symptoms.  Most 
of the toxicological research has been done using extracts from 
fish associated with outbreaks of ciguatera.  Recent research 
suggests that a group of toxins produced by the dinoflagellates on 
which the fish feed, could be transmitted through the fish to human 
beings as the causative agents.  This group produced by several 
species of dinoflagellates from coral reefs includes ciguatoxin, 
maitotoxin, and scaritoxin, and as shown very recently, okadaic 
acid.  These compounds have been chemically characterized but the 
chemical structure is only fully known for okadaic acid.  The 
extremely limited amounts available of these compounds is slowing 
down chemical and toxicological research as well as studies on the 
entry of these compounds into the food chain.  No chemical methods 
of analysis for these compounds in food or organisms are available 
at present.  Determinations for ciguatoxicity have so far been 
carried out by bioassay using mouse, cat, or mongoose.  More 
recently, a radioimmunoassay and a bioassay using mosquitoes have 
been developed.  The use and further development of these methods 
is limited by the scarcity of reference material. 

    Ciguatoxin and maitotoxin have been isolated from the 
biodetritus layer of coral reefs, from the dinoflagellate 
 Gambierdiscus toxicus collected from seawater, and from axenic 
cultures of  G. toxicus.  This dinoflagellate is attached to 
macroalgae in coral reefs.  In general, ciguatoxic fish species are 
limited to fish that feed on dinoflagellates and the detritus of 
coral reefs, particularly surgeon-fish, parrot-fish, and the larger 
reef carnivores that prey on these herbivores.  Ciguatoxin and 
maitotoxin have been identified in these fish from chemical and 
toxicological characteristics. 

    Purified extracts from ciguatoxic fish administered orally 
and parenterally to mice and orally to cats and mongooses produced 
acute effects within 48 h, characterized by diarrhoea, itching, 
inactivity, and death, after convulsive spasms.  In mice given the 
most purified ciguatoxin available at present, peritoneally, the 
LD50 value was 0.45 µg/kg body weight. 

    The only known pathway of human exposure to ciguatera toxins 
is through food, and with the exception of a marine snail, mainly 
through coral fish or fish predating on coral fish, such as 
surgeon- and parrot-fish, snappers, groupers, carrangs, barracuda, 
Spanish mackerel, and emperor.  The probability of exposure seems 
to be higher when eating large carnivorous fish, particularly the 
liver and other viscera.  Because of the focal entry of the toxins 
into the food chain, fish of the same species and the same size can 
be toxic in one place and not in another. 

    No data are available on the absorption, distribution, 
retention, and metabolism of the toxins involved in ciguatera. 

    The clinical picture of human disease (ciguatera) is quite 
variable.  Typically, symptoms occur within 1 - 6 h of ingestion of 
toxic fish.  Initial symptoms usually include nausea, malaise, and 
numbness and tingling of the lips, tongue, and throat.  Patients 
may later develop some or all of the following signs and symptoms: 
vomiting, abdominal cramps, diarrhoea, paraesthesia of the 
extremities, itching, myalgia, and arthralgia.  In more severe 
cases, ataxia, weakness, blurred vision, insomnia, sinus 
bradycardia, dysrhythmias, and hypotension may develop.  A symptom 
that is particularly suggestive of the diagnosis is the reverse 
perception of cold and hot.  The duration of illness is variable.  
Most of the patients recover within three days, but malaise, 
paraesthesia, pruritus, and ataxia may persist for weeks or even 
years in severe cases.  Patients repeatedly poisoned by ciguatoxic 
fish may develop a resurgence of ciguatera symptoms, even after 
eating fish containing little or no detectable toxin. 

    In the most severe cases, death results from circulatory 
collapse or respiratory failure.  Several thousand cases of 
ciguatera have been reported from tropical areas within the last 
two decades with case-fatality rates ranging from 0.1 to 4.5%. 

    Intoxications have also been reported outside the endemic areas 
and outside the tropical circumglobal belt due to the consumption 
of fish brought or imported from the endemic areas. 

    Limited data were available for evaluation of the dose-response 
relationship.  It is generally observed that one meal of toxic fish 
is sufficient to induce the disease in a human being, and, in one 
report, it has been suggested, on the basis of direct mouse assay 
determinations of ciguatoxicity in fish involved in intoxication, 
that oral intake of as little as 0.1 µg ciguatoxin can cause 
illness in an adult. 

    Of special interest are the studies showing an association 
between ciguatera outbreaks and naturally - occurring or man-made 
disturbances of coral reef ecosystems. 


    Intoxication by tetrodotoxin is different from the previously 
reported intoxication in several important aspects:  (a) the toxin 
is probably not an algal product, but appears to be produced by 
certain fish species and a few other animals; (b) human exposure 
is generally limited to consumption of certain fish species, the 
identification of which is feasible; with the passage of time, 
populations in the endemic areas have developed measures to prevent 
intoxication; (c) on the basis of the number of cases of 
intoxication, accidental tetrodotoxin poisoning does not appear to 
be an important public health problem; however, when intoxication 
does occur, the case fatality rate is high. 

    Tetrodotoxin is an aminoperhydroquinazoline compound.  Though 
the chemical structure of tetrodotoxin is entirely different from 
that of saxitoxin, the effects it induces in animals are very 

similar; the mouse assay developed for PSP has also been used for 
the detection of tetrodotoxin in the assessment of exposure.  
Recently, fluorescence spectrometric procedures for tetrodotoxin 
determination have been developed. 

    Tetrodotoxin has been found in fish of the family 
 Tetraodontidae (Pufferfish); the ovaries, liver, and intestines 
contain the highest amount with small amounts in the skin; the 
toxin has only occasionally been detected in the muscles of these 
fish.  The most toxic pufferfish are caught along the coasts of 
Japan and China.  Tetrodotoxin has also been found in the Japanese 
ivory shell and in the trumpet shell associated with fatal human 
cases.  In addition, the toxin has been identified in the skin of 
certain frogs and as the poisonous principal in the venom of the 
blue-ringed octopuses. 

    The signs of intoxication induced by tetrodotoxin in 
experimental animals are comparable with those caused by the PSP 
compounds.  However, for the same degree of neuromuscular 
paralysis, a systemic, lasting arterial hypotension is produced by 
tetrodotoxin, which is also a highly potent hypothermic agent.  The 
mode of action of tetrodotoxin is very similar to that of 

    In human beings, the onset of signs and symptoms of 
tetrodotoxin intoxication usually occurs from 10 to 45 min after 
ingestion, but may be delayed by 3 h or more.  Paraesthesia appears 
in the face and extremities and may be followed by sensations of 
lightness, floating, or numbness.  Nausea, vomiting, diarrhoea, 
and epigastric pain may also be present.  Later, respiratory 
symptoms become prominent with dyspnoea, shallow, rapid 
respiration, and the use of auxilliary muscles.  Cyanosis and 
hypotension follow, and convulsions and cardiac arrhythmia may 
occur.  In most instances, the victims retain consciousness until 
shortly before death, which usually takes place within the first 
6 h. 

    Occasional accidental intoxications including fatal outcome 
have been found associated with the consumption of pufferfish 
containing 0.5 - 30 mg tetrodotoxin/kg wet tissue fish.  In Japan, 
about 60 cases with 20 deaths occurred annually in the period 
1974-79.  Intoxications can occur, occasionally, even in non-
endemic areas.  Ten cases with three deaths were reported recently 
in an European country following consumption of imported frozen 
mislabelled pufferfish containing tetrodotoxin. 

Neurotoxic Shellfish Poisons

    Two forms of human disease have been reported in association 
with the red tides of the dinoflagellate  Gymnodinium breve around 
the coast of Florida.  In one form associated with consumption of 
shellfish contaminated with  G.  breve cells and/or toxins,
paraesthesia, alternating hot and cold sensations, nausea, 
vomiting, diarrhoea, and ataxia occur within 3 h; no paralysis has 
been observed.  The other form is an upper respiratory syndrome 
that has been reported in association with aerosols of  G. breve 

cells or toxins.  The rapidly reversable syndrome is characterized 
by conjunctival irritation, copious rhinorrhea, and nonproductive 
cough.  Four toxic components were isolated recently from cultured 
cells of  G. breve, and three of them have been determined 
structurally to be polyethers.  However, none of the toxic 
components has been chemically identified in food, air, or affected 
organisms.  Monitoring of shellfish as a food has been conducted 
using a mouse bioassay.  Fish kills and the mass death of seabirds 
have been observed in association with blooms of  G. breve in this 
area of Florida. 

Diarrhoeic Shellfish Poisons 

    Very recently, several toxic components have been isolated from 
shellfish associated with outbreaks of a syndrome in human beings, 
that is characterized by diarrhoea, nausea, vomiting.  Abdominal 
pain was reported in about half of the patients and chill in a 
limited number of cases.  Time from consumption of shellfish to the 
onset of illness ranged from 30 min to 12 h.  Five of the toxic 
components have been structurally elucidated as okadaic acid and 
derivatives, and polyether lactones. 

    Several species of dinoflagellates have been identified as 
organisms that produce okadaic acid and are also associated with 
disease outbreaks.  Human outbreaks involving more than 1300 cases 
have been reported from Japan with smaller outbreaks in Europe and 
South America. 

Dermatitis-Inducing Marine Cyanophyte Toxins

    Outbreaks of acute dermatitis in human beings after swimming in 
the sea during blooms of the filamentous marine cyanophyte  Lyngbya 
 majuscula have been reported repeatedly from two areas (Hawaii and 
Okinawa).  Two components that have been isolated from the algae 
and chemically identified as debromoaplysiatoxin and lyngbyatoxin A 
have been shown to induce inflammation when applied to the skin of 

    A gradation of the skin effect with the dose of 
debromoaplysiatoxin was observed in animal studies. 
Debromoaplysiatoxin was also shown to induce local skin effects on 
human volunteers at a concentration as low as 0.5 mg/litre.  
Histological studies confirmed the similarity between this skin 
effect and those associated with exposure to  L. majuscula. 

Freshwater Cyanophyte Toxins

    Blooms of certain freshwater cyanophytes  (Microcystis 
 aeruginosa, Anabaena flos-aquae, Aphanizomenon flos-aquae) in ponds 
and lakes have occasionally been observed to be associated with 
sudden death in farm animals after drinking the water, lesions 
consisting of either haemorrhages and liver damage, or respiratory 

    A few toxic components from these algae have been chemically 
characterized, but no report is available on the occurrence of the 
components in water.  Several studies have reported adverse human 
health effects associated with the blooms of the same cyanophytes 
in recreational and municipal water supplies.  At present, there is 
no evidence of the causal involvement of algal toxins in these 


   (a)  The limited availability of pure aquatic biotoxins, 
        with the exception of saxitoxin and tetrodotoxin, 
        inhibits progress in experimental toxicology, 
        analytical chemistry, phycology, clinical chemistry, 
        and ecotoxicology.  As a consequence, very limited 
        quantitative information is available on the
        exposure of human beings and non-human targets to
        algal toxins, and this severely affects monitoring
        and the establishment of preventive measures.

        Internationally coordinated efforts are needed to
        provide pure algal toxins in quantities to meet
        these needs;

   (b)  Methods of analysis for algal toxins in foods,
        in human and animal tissues and fluids, and in
        environmental media, should be subjected to
        international collaborative studies in order to
        assess the precision and accuracy of the methods;

   (c)  The surveillance and reporting of human and
        animal (domestic and wild) cases of algal
        toxin-related disease should be improved on a
        world-wide basis; and

   (d)  As most of the algal toxin-related diseases are
        associated with blooms, more information on the
        occurrence of blooms of toxic algae and the
        conditions producing blooms should be obtained on a
        world-wide basis.  On the basis of this information,
        attempts should be made to predict the occurrence of
        algal blooms and to provide early warning systems in
        affected areas.


    It has been known since ancient times that certain fish and 
shellfish are poisonous and can cause death when eaten.  The first 
Chinese pharmacopoeia, dated 2800 BC, records injunctions against 
eating pufferfish (Kao, l966).  European settlers in Northern 
America observed that various taboos and legends of the coastal 
Indians were associated with eating shellfish.  On the east coast, 
the Indians would not eat mussels, even when starving, and on the 
west coast, Indians maintained nightly lookouts for bioluminescence 
in the sea and would not eat shellfish when the sea was "glowing" 
(Dale & Yentsch, l978). 

    The chemical nature and biological basis for these food-borne 
intoxications have been elucidated over the last fifty years, 
beginning with the pioneering work of Meyer & Sommer on the 
etiology of paralytic shellfish poisoning (PSP) in California 
(Meyer et al., l928).  It is now evident that certain microscopic 
algae, present in phytoplankton, produce very potent toxins 
(phycotoxins, or algal toxins), which are chemical compounds mainly 
of low relative molecular mass.  Concentrations of phycotoxins in 
the sea or in fresh water are highest during an algal bloom or red 
tide, a phenomenon characterized by a sudden, rapid multiplication 
of algal cells caused by environmental factors not yet fully 
understood.  The phycotoxins are taken up by predators feeding on 
plankton, either directly as in the case of bivalve molluscs, or 
through several trophic levels as in fish.  These food items are 
then consumed by man. 

    Algal blooms, including those of toxic algae, have become a 
more frequent phenomenon throughout the world in the last decade 
or two.  The reason is not clear.  In some areas, it is believed 
that climatic and hydrographic factors are important.  Man-made 
pollution of the sea and freshwater and other human activities 
could change the aquatic environment in ways that provoke 
proliferation of toxin-producing algae.  However, it should be 
stressed that the occurrence of algal blooms is usually due to 
natural rather than man-made causes, though anthropogenic inputs 
are significant in some instances.  Furthermore, surveillance, 
detection, and reporting systems have improved in recent years, 
resulting in the more efficient accumulation of information 
concerning algal blooms on a world-wide basis.  As fish and 
shellfish constitute an important part of the world's food supply, 
and the main source of protein for certain communities, the 
apparently increasing contamination of food by aquatic biotoxins 
constitutes a specific chemical hazard deserving appropriate 

    Though also influenced by algal blooms, toxins from Cyanophytes 
(blue-green algae) constitute a different problem.  In this case, 
vectors are not known to be involved, and the toxins or microscopic 
toxic cells are brought into direct contact with human skin during 
swimming in the sea, or, in the case of freshwater, the toxins or 
toxic cells may possibly be transferred to the human organism 

through drinking-water.  Thus, the growth of blue-green algae in 
freshwater reservoirs may add to the difficulties of providing pure 

    The chemistry of some of the toxins is still only partly known.  
However, during the last decade, much progress has been made, e.g., 
the composition of the PSP complex has been elucidated, and some 
individual components chemically characterized; the structures of 
some of the neurotoxic and diarrhoeic shellfish poisons have been 
established.  In the absence of sufficient chemical knowledge in 
the past, most measurements have been made by a bioassay using 
mice, a procedure that is nonspecific in nature, but is still the 
only method in practical use for the quality control of seafood. 
This method is not sensitive enough for the analysis of clinical 
samples and specific and sensitive methods for detection should be 
developed on the basis of new knowledge. 

    Associations between aquatic biotoxins and human intoxication 
are based not on specific identification of the causal agent in the 
human body, but on the appearance of certain acute symptoms 
following the consumption of some food commodities containing the 
toxic principles.  Although the clinical features are variable, the 
neurological and gastrointestinal systems are commonly involved.  
Indeed, in some cases, the symptomatology of poisoning due to 
different biotoxins of this group could be similar and specific 
analytical methods to aid diagnosis would be desirable. 

    This document deals with algal toxins and tetrodotoxin.  It 
does not deal with other well-known disease entities involving 
waterborne agents that infect man directly or contaminate fish and 
shellfish, producing toxins during food preparation and storage.  
Thus, scombrotoxin is not dealt with, and shellfish allergens that 
cause allergic disorders in man, when the shellfish is consumed, 
have also not been included.  Other diseases caused by toxins not 
yet well defined and of uncertain origin (e.g., clupeotoxism, 
hallucinatory fish poisoning) are not discussed.  The hygienic 
aspects of fish and shellfish in general have been dealt with in 
three WHO publications (WHO, 1974, 1979, 1983) and in Wood (1976).  
The term "aquatic biotoxins" is used, following the example of the 
working group on aquatic biotoxins of the IUPAC Commission on Food 
Chemistry, dealing with methods of analysis for marine biotoxins 
(dinoflagellate toxins) and freshwater biotoxins (cyanophyte 
toxins) (Krogh, 1983). 

    For the purposes of this document, algae are uni- or 
multicellular organisms able to photosynthesize by means of 
chloroplasts, cell organelles containing chlorophylls, carotenes 
and xanthines.  Being eukaryotic cells, the algae are members of 
the Protista, one of the 5 kingdoms (Marqulis & Schwartz, 1982).  
The unicellular marine algae dealt with in this document all belong 
to the dinoflagellates.  Within several of the dinoflagellate 
genera there are species in which the cells do not contain 
chloroplasts, and thus are not true algae.  In this context 
however, all dinoflagellates are considered to be algae, and 
Dodge's monograph (1982) is used as a reference for dinoflagellate 

taxonomy.  In contrast to the eukaryotic dinoflagellates, the 
blue-green algae (Cyanophyte) consist of the more primitive (in 
morphological terms) prokaryotic type of cell, and in the above 
system are placed in Monera, and often named Cyanobacteria.  They 
are unicellular, colonial, or filamentous organisms, and occur in 
fresh water and seawater.  In this document, Komarek (1958) is used 
as reference for cyanophyte taxonomy. 


    Acute intoxication after consumption of shellfish is a syndrome 
that has been known for several centuries.  The etiology was first 
elucidated in this century, starting with investigations in 
California in the 1920s, following several episodes of fatal 
intoxications related to the consumption of mussels (Meyer et al., 
1928; Sommer & Meyer, 1937).  The term PSP, which is now widely 
used in describing this phenomenon, means paralytic shellfish 
poisons or paralytic shellfish poisoning.  A document dealing with 
paralytic shellfish poisoning has been published recently (Halstead 
& Schantz, 1984). 

1.1.  Properties and Analytical Methods

1.1.1.  Chemical properties

    The chemistry of paralytic shellfish poisons (PSP) has been 
reviewed by Shimizu (1978) and Schantz (1980).  PSP are a group of 
toxins produced by dinoflagellates of the genus  Gonyaulax.  The 
first agent to be chemically characterized was saxitoxin, which, 
though it was initially discovered in shellfish in California, has 
since been found in greatest concentrations in the Alaskan 
butterclam,  Saxidomus giganteus, from which the name is derived.  
Saxitoxin has been shown to be a derivative of tetrahydropurine 
(Fig. 1) (Bordner et al., 1975; Schantz et al., 1975).  It is a 
white, very hygroscopic solid, soluble in water, slightly soluble 
in methanol and ethanol, but practically insoluble in most non-
polar organic solvents.  It is a very basic substance, with two 
titratable groups, pKa 8.2 and 11.5, and a relative molecular mass 
of 299 (Schantz et al., 1961).  Subsequently, several other toxins 
of the PSP group have been characterized chemically, including 1-
hydroxy saxitoxin (neosaxitoxin) (Shimizu et al., 1978); 11-hydroxy 
saxitoxin sulfate and the 11 beta-epimer of this compound (Boyer et 
al., 1978); 11-hydroxy neosaxitoxin sulfate and the 11 beta-epimer 
of this compound (Wichmann et al., 1981; Genenah & Shimizu, 1981) 
(Fig. 1).  The last four compounds, named gonyautoxins II, III, I, 
and IV, respectively, by Shimizu et al. (1976) and Alam et al. 
(1982), are slightly basic, but otherwise have properties similar 
to those of saxitoxin.  In general, PSP toxins are heat stable at 
acidic pH, but very unstable and easily oxidized under alkaline 

    Recently, a novel group of PSP compounds with a sulfocarbomoyl 
group has been isolated from both dinoflagellates and shellfish 
(Kobayashi & Shimizu, 1981; Hall, 1982; Harada et al., 1982a).  
These toxins have a low toxicity until hydrolysed to more potent 
forms (nos. 7 - 12, Fig. 1).  In addition, decarbamoylsaxitoxin, 
which previously had been made only in the laboratory, has been 
found in nature (Harada et al., 1983; Sullivan et al., 1983a).  
Thus, 13 PSP compounds are now known. 


                             R1       R2          R3
                             --       --          --

     1)  saxitoxin           -H       -H          -C-NH2

     2)  neosaxitoxin        -OH      -H          -C-NH2

     3)  gonyautoxin-I       -OH      -alphaOSO-3 -C-NH2

     4)  gonyautoxin-II      -H       -alphaOSO-3 -C-NH2

     5)  gonyautoxin-III     -H       -betaOSO-3  -C-NH2

     6)  gonyautoxin-IV      -H       -betaOSO-3  -C-NH2

     7)  gonyautoxin-V       -H       -H          -C-N-SO-3
                                                  || |
                                                   O H

     8)  gonyautoxin-VI      -OH      -H          -C-N-SO-3
                                                  || |
                                                   O H

     9)  gonyautoxin-VIII    -H       -betaOSO-3  -C-N-SO-3
                                                  || |
                                                   O H

    10)  gonyautoxin-VIII    -H       -alphaOSO-3 -C-N-SO-3
          epimer                                  || |
                                                   O H

    11)  sulfocarbamoyl      -OH      -betaOSO-3  -C-N-SO-3
          gonyautoxin-I                           || |
                                                   O H

    12)  sulfocarbamoyl      -OH      -alphaOSO-3 -C-N-SO-3
          gonyautoxin-IV                          || |
                                                   O H

    13)  decarbamoyl-        -H       -H          -H

1.1.2.  Methods of analysis for PSP in foods

    This subject has been reviewed by Krogh (1979).  The most 
commonly used procedure for PSP determination is a bioassay using 
mice, but this assay is not completely satisfactory, because of 
lack of sensitivity and pronounced variations.  However, several 
other alternative chemical procedures are being developed, some of 
which may be applicable to PSP monitoring programmes.  Despite the 
shortcomings of the mouse assay, the method is the only one 
suitable for regulatory purposes, where these limitations are of 
less significance.  Biological methods

    (a)  Mouse bioassay

    During the investigations that established the association 
between toxic shellfish and the toxin-producing dinoflagellates 
 (Gonyaulax catenella), Sommer & Meyer (1937) developed a bioassay 
for the PSP toxin.  It consisted of the intraperitoneal injection 
of mice with an acidified extract of shellfish tissues, and the 
determination of the rapidity of death following the injection.  By 
standardizing the conditions for the bioassay (mouse weight, pH of 
extract, and salt concentration), and introducing a purified 
saxitoxin standard (Schantz et al., 1958), a fairly reliable 
routine procedure was established.  When the assay was tested 
collaboratively (McFarren, 1959), a standard error of about 20% was 
observed.  The procedure has the status of an AOAC official final 
action method (Association of Official Analytical Chemists, 1980), 
and is, so far, the only method for assaying PSP that is in routine 
use by regulatory agencies all over the world (Adams & Miescier, 

    Because different strains of mice differ in their 
susceptibility to the PSP toxins, the sensitivity of the mouse 
colony used in the assay must be determined by calculating a 
correction factor (CF value) after intraperitoneal injection of the 
saxitoxin standard.  The acidified extracts of shellfish are 
screen-tested in a few mice, in order to determine the dilution of 
the extract that will kill mice of 19 - 21 g body weight within 5 - 
7 min, the conditions under which the assay is most sensitive.  In 
the main test, the time to death is converted into mouse units 
(MU), from which the concentration of toxin can be calculated using 
the CF value, assuming that the PSP toxins are saxitoxin or its 
derivatives.  Saxitoxin levels as low as about 400 µg/kg can be 
detected by the procedure, and the sensitivity is reduced with 
increased salt (NaCl) concentrations in the extract.  Near the 
detection limit, the toxin concentration may be underestimated by 
as much as 60% (Schantz et al., 1958). 

    Although Sommer & Meyer (1937) suggested that characteristic 
PSP symptoms, such as dyspnoea, could be used in the mouse test, 
these symptoms are subject to individual variations, and other 
factors, such as the rate of absorption, the site of injection, 
etc. (Kao, 1966).  The principle of the mouse bioassay is a 

measurement of the time to the last gasping breath, which is a 
clearer end-point.  The result of the mouse bioassay is non-
specific:  other agents can also cause death within 5 - 10 min 
following intraperitoneal administration.  Thus, the mouse bioassay 
cannot distinguish PSP from tetrodotoxin (Johnson & Mulberry, 1966, 
section 3), but confusion between PSP and other toxins is unlikely 
in cases where the origin of the sample is known. 

    (b)  Immunological assay

    Johnson & Mulberry (1966) developed an assay in which purified 
PSP (saxitoxin) was conjugated with proteins by formaldehyde 
condensation, and the antitoxin to the conjugate was produced in 
rabbits.  The antisera was used in haemagglutination and bentonite 
flocculation tests, with PSP extracted from spiked samples of 
butterclams, causing variable inhibition in the tests.  The 
haemagglutination-inhibition test was slightly more sensitive than 
the mouse assay, whereas the detection limit of the bentonite 
flocculation-inhibition test was comparable to that of the mouse 
assay.  Puffer fish poison (tetrodotoxin), which is detectable by 
the mouse assay, did not react in the tests.  The method suffers 
from some saturation phenomenon and is not useful for a 
quantitative determination of PSP in shellfish, because increasing 
amounts of PSP in extracts caused almost identical reactions.  Chemical methods

    Several spectrophotometric procedures involving various colour 
reactions of the PSP toxins have been developed.  The earliest 
procedure (McFarren et al., 1958, 1959) was based on the Jaffe 
reaction, which involved a colorimetric reaction with the guanidine 
moeity.  This procedure was inadequate, with a limit of detection 
of 1000 - 1500 µg/kg, and suffered from interference by other 
naturally occurring guanidine compounds. 
     A fluorimetric method has been developed for the determination 
of saxitoxin (Bates & Rapoport, 1975; Gershey et al., 1977; Bates 
et al., 1978) comprising acid extraction, clean-up on a weakly 
acidic resin column, and the alkaline oxidation of the eluate with 
hydrogen peroxide.  The fluorescent purine derivative of saxitoxin 
thus obtained was measured spectrophotometrically.  Levels as low 
as 4.0 µg saxitoxin/kg were measured in saxitoxin-contaminated 
shellfish.  Subsequent attempts to use this method in combination 
with chromatographic separation of various PSP toxins failed, 
because the N-1 hydroxy compounds, such as neosaxitoxin, and 
gonyautoxins I and IV, did not yield fluorescent products (Bates et 
al., 1978; Buckley et al., 1978).  In some contaminated shellfish, 
the latter compounds may comprise the major portion of the toxic 
material.  In a study comparing the mouse bioassay with a modified 
Bates-Rapoport procedure, the results of the latter were 11 -22% 
higher than those of the mouse bioassay (Shoptaugh et al., 1981). 

    Recently, a high pressure liquid chromatographic (HPLC) 
procedure has been reported, using separation of the toxins on a 
bonded phase cyano column and detection by fluorescence following 
alkaline oxidation with periodate (Sullivan & Iwaoka, 1983).  Six 
PSP components (saxitoxin, neosaxitoxin,, gonyautoxin I-IV) were 
identified and quantificted, with a good correlation with the 
mouse-bioassay method (Sullivan et al., 1983b). 

1.2.  Sources and Occurrence

1.2.1.  Algal formation of toxins

    The PSP toxins occur in, and are produced by, certain 
unicellular marine algae known as dinoflagellates, members of 
the phylum  Dinophyta.  Most of the PSP-producing dinoflagellates 
are found in the genus  Gonyaulax, including:   G. tamarensis, 
 G. catenella, G. acatenella, G. monilata, and  G. polyedra (Prakash,
1967; Schmidt & Loeblich, 1979).   G. excavata is considered to be a 
variety of  G. tamarensis (Taylor, 1975).  Other thecate toxin 
producers occur in the genus  Pyrodinium, such as  P. bahamense 
(Wall, 1975; Harada et al., 1982a) and  P. phoneus, though the 
latter organism was probably  G. tamarensis (Taylor, 1975).  Toxic 
 P. bahamense has been raised to varietal status, as  P. bahamense 
var.  compressa, compared to the non-toxic  P. bahamense var. 
 bahamense (Steidinger et al., 1980). 


    Dinoflagellates are among the major components of the marine 
phytoplankton.  They are single-celled organisms, 40 - 50 µm in 
diameter, and propelled by two flagellae; some are bioluminescent.  
In addition to the motile form, some dinoflagellates, such as 
 G. excavata, produce resting cysts (zygotes), as a result of sexual 
reproduction (Dale, 1977).  Lacking flagella, these cysts sink and 

accumulate at the sediment-water interface, where they overwinter.  
Under laboratory conditions, the transformation of motile cells 
into another type of cyst (temporary cysts) has been observed, 
resulting from environmental stress, such as low temperature (Fig. 
2).  Motile cells reproduce asexually by binary fission. 

    The toxin-producing species of the genus  Gonyaulax vary in
toxic potential, as indicated in Table 1.

    The toxic potential varies not only between species, but also 
between strains within species (Schmidt & Loeblich, 1979).  Toxic 
and non-toxic strains of  G. tamarensis have been encountered, even 
in the same locality (Yentsch et al., 1978).  From the biochemical 
point of view, these observations might indicate that PSP toxins 
are secondary metabolites similar to toxins produced by microscopic 
fungi (mycotoxins), but not much is known about the pathways by 
which PSP toxins are produced.  Thus, in a study of saxitoxin 
production by  G. catenella in axenic culture using a number of 14C-
labelled compounds likely to be precursors, such as guanidine and 
propionate, no clue to the biosynthesis was obtained, and it was 
concluded that, apparently, a highly specific pathway was in 
operation, not involving pathways for active C-1 and C-2 compounds 
(Proctor et al., 1975).  The highest yield of saxitoxin was 
obtained at a temperature of 12 - 13 °C during continuous 

Table 1.  Relative toxicity of dinoflagellates of the genus 
Species               Minimum number of cells required to produce
                      1 mouse unit of PSP (about 0.18 µg PSP)
 G. polyedra           1.7 x 105
 G. catenella          7 x 104
 G. catenella          5 x 104
 G. catenella          1.0 x 104
 G. acatenella         6 x 103
 G. tamarensis         4.5 x 103
a  Adapted from: Prakash (1967).

    PSP toxins are found not only in the motile cells of  Gonyaulax 
species, but also in the resting cysts, where levels 10 - 1000 
times higher than those in motile cells have been found (Dale et 
al., 1978).  In contrast, identical levels of PSP in cysts and 
motile cells of  G. tamarensis were measured by two other research 
groups (Oshima et al., 1982; White & Lewis, 1982). 

    Saxitoxin and neosaxitoxin have also been isolated from strains 
of a cyanophyte organism,  Aphanizomenon flos-aquae (Ikawa et al., 
1982) (section 6.2).  In addition, PSP components (gonyautoxin I, 
II, III) have been detected in a macroalga,  Jania sp., belonging to 
the red algae (Rodophyta) (Kotaki et al., 1983).  These red algae 
are eaten by crabs and snails, and PSP has been detected in these 
molluscs (sections,  Oceanographic conditions associated with blooms (red tide)

    The topic has been reviewed by Yentsch & Incze (1980). 
Contamination of shellfish with PSP toxins has traditionally been 
associated with the appearance of algal blooms, the so-called red 
tide.  Dinoflagellates are able to reproduce asexually at high 
rates, under the influence of environmental conditions, which have 
not yet been fully elucidated.  When a population of 
dinoflagellates develops quickly forming dense concentrations of 
from 104 cell/litre to 106 cell/litre, it is termed a bloom.  At 
levels of 106 cell/litre, the water can become discoloured 
depending on the participating algal species, hence the name "red 
tide", but it is important to realize that not all algal blooms are 
red coloured. 

    It is, however, also important to note that not all red tides 
are associated with toxic blooms and contamination of shellfish; 
they can also result from concentrations of non-toxic 
dinoflagellates or ciliates (McAlice, 1968).  Conversely, shellfish 
can still accumulate PSP when  Gonyaulax concentrations in the sea 
are below those found in algal blooms. 

    As red tides are essentially a coastal phenomenon, it has been 
suggested that land drainage might play a role in their initiation 
(Prakash, 1975).  Concentrations of chelators and trace metals may 
be involved.  Thus, Anderson & Morel (1978) reported that 
 G. tamarensis was more sensitive to Cu(II) ions than other members 
of the phytoplankton, and that it grew well, when the 
concentrations of Cu(II) ions were exceedingly low.  It has also 
been suggested that, by binding the copper ion, chelators could 
decrease its toxicity, whereas binding zinc and iron could increase 
the availability of these nutrients for growth (Anderson & Corbett, 
1979).  Field studies involving measurements of copper and iron 
during blooms support this hypothesis (Dale & Yentsch, 1978). 

    While blooms may result directly from the rapid growth of algal 
populations, physical (hydrographic) factors may transport existing 
populations to specific areas, where biological behaviour, such as 
positive phototaxis, can result in the formation of dense 
concentrations (Mulligan, 1975; Margalef et al., 1979; Seliger et 
al., 1979).  These phenomena may be triggered by meteorological 
events, such as rainfall and wind (Hartwell, 1975; Yentsch & 
Glover, 1977).  Recent data suggest that frontal zones, or 
discontinuities between water masses, are the factors most likely 
to influence the development of red tides.  These frontal zones may 
result from tide - or wind-generated convergences, or 
discontinuities. They are frequently marked by pronounced 
differences in the vertical stability of the two water masses 
(Pingree et al., 1975; Tyler & Seeliger, 1978; Yentsch & Mague, 

1.2.2.  Occurrence in seafood  Accumulation in molluscs

    (a)  Bivalves

    The topic has been reviewed by Yentsch & Incze (1980).  The PSP 
components are transferred to shellfish (mussels, clams, scallops) 
during filter-feeding, a characteristic feature of bivalves.  
During this process, food organisms in the seawater, such as 
 Gonyaulax cells, are transported from the gills in the mantle 
cavity to the oesophagus and stomach.  Digestion takes place in the 
stomach and its associated diverticula, often erroneously termed 
"liver" (Russel-Hunter, 1972).  The highest concentrations of PSP 
have been found in these digestive organs, apparently bound to 
melanin, but PSP is also found in other soft tissues of the 
bivalves.  The rate of PSP accumulation varies among shellfish 
species, as indicated in Table 2. 

Table 2.  Rate of PSP accumulation in two species of shellfish
fed  Gonyaulax excavata under laboratory conditionsa
Shellfish species      Days after               PSP concentration
                       feeding                  (µg/kg)b
 Mya arenaria           0                         NDc
                       7                         3110
                       14                        1350
 Mytilus edulis         0                         ND
                       7                         5370
                       14                        3110
a  Adapted from: White & Maranda (l978).
b  340 µg/kg was the limit of detection using the mouse bioassay.
c  ND = Not detected.

    Shellfish are generally not harmed by the presence of PSP 
toxins, though Gilfillan & Hansen (1975) noted some depression in 
the filtration rate of bivalves exposed to dense concentrations of 
 Gonyaulax cells.  Black mussels ( Choromytilus meridionalis) and 
white mussels  (Donax serra) in South Africa have often been found 
paralysed during red tides associated with  G. catenella (Popkiss et 
al., 1979). 

    PSP toxins may be transferred to shellfish not only through the 
motile cells, but also through the resting cysts, which may contain 
PSP (section 1.2.1).  Resting cysts have been identified in the 
digestive tract of molluscs (Ayres & Cullum, 1978). 

    (b)  Gastropods

    Amounts of PSP have been detected in the digestive glands of 
carnivorous gastropods, such as the rough whelk  (Buccinum 
 undulatum), under natural conditions as well as during studies in 
which PSP-containing digestive glands of scallops were fed to the 
gastropods (Caddy & Chandler, 1968). 

    Two species of turban shells ( Turbo argyrostoma, T. marmorata) 
and two species of top shells ( Toctus nilotica maxima, T. pyramis), 
inhabiting coral reefs, have been found to contain PSP toxins in 
the visceral regions (Kotaki et al., 1983).  Saxitoxin, 
neosaxitoxin, and a new toxin tentatively code-named TST, were 
predominant in the toxin profile.  The highest toxicity recorded 
was 4000 µg PSP/kg, although a marked regional and individual 
variation existed among specimens tested.  As the PSP-containing 
alga  Jania sp. was present in the stomach of the gastropods, this 
alga was presumed to be the source of the toxins.  Accumulation in crustacea

    PSP was not found in lobsters  (Homarus americanus) during
red tide episodes; however, when lobsters were fed PSP-containing 
clams under experimental conditions, PSP was found in the contents 
of the gut, but not in tissues (Yentsch & Balch, 1975).  PSP may 
accumulate in crabs (Schantz et al., 1975; Foxall et al., 1979).  
Xanthid crabs inhabiting coral reefs have been found to cause 
intoxication with a high fatality rate in Fiji, Japan, Palau, and 
the Philippines (Hashimoto, 1979; Alcala, 1983; Raj et al., 1983).  
The species most frequently implicated in poisoning is  Zosimus 
 aenus, which accumulates high levels of neosaxitoxin and saxitoxin.  
The source of toxin appears to be the PSP-containing alga  Jania sp.  
The marked regional and individual variation in toxicity was 
explained by the abundance of this alga in the habitat of the 
crabs.  Other species of coral-reef crabs, although too small to be 
regarded as food, also accumulate PSP toxins if  Jania sp. grows in 
their vicinity (Kotaki et al., 1983).  Transmission through zooplankton to fish

    Kill episodes have been observed in fish (herring, sand lance) 
spatially and temporally associated with blooms of toxic 
 G. excavata in the North Sea (Adams et al., 1968) and in the Bay of 
Fundy, Canada (White, 1977).  As these fish feed on zooplankton but 
not on dinoflagellates, it has been suggested that the zooplankton, 
feeding on the dinoflagellates, may act as vectors of PSP.   In the 
stomach of herrings from the Bay of Fundy kill, White (1977) was 
able to identify thecosomatous pteropods  (Limacina retroversa), and 
the stomach contents contained PSP.  In an experimental study, a 
similar amount of PSP (21 µg/fish) caused paralysis and death in 
herrings. Zooplankton collected during a bloom of toxic  G. excavata 
contained PSP, even 3 weeks after the bloom peak, when  Gonyaulax 
cells had disappeared, indicating accumulation in zooplankton 
(White, 1979).  This observation was subsequently confirmed in an 
experimental study using  Acartia clausii and  Balanys sp. (barnacle 

nauplii) as representatives of zooplankton grazing on PSP-producing 
 G. excavata (White, 1981a).  The PSP levels measured in the 
zooplankton were comparable with maximum levels commonly 
encountered in filter-feeding molluscs, e.g., 10 000 - 50 000 µg 
PSP/kg.  In a recent herring kill occurring during a bloom of toxic 
 G. excavata, another zooplankton organism,  Evadne nordmanni, was 
identified as the vector of PSP (White, 1980).  The PSP found in 
the dead or diseased herrings are listed in Table 3. 

Table 3.  Contents of PSP in Atlantic herrings 
from a kill during  G. excavata bloomsa
Number   Mean    Mean    Mean mg/kg contentb
of fish  length  weight    
sampled  (cm)    (g)     gut          muscle
17       21.6    110     1100         NDc
17       21.8    116     2450         ND
24       14.9    34      660          ND
14       16.4    39      50           330
11       15.6    35      2180         590
40       21.5    108     14 140       ND
a  Adapted from: White (1980).
b  300 µg/kg was the limit of detection using 
     the mouse bioassay.
c  ND = not detected.

    PSP was not detected in muscle tissue in most of the cases.  
According to the author, this was consistent with experimental 
data, which also showed that PSP was not found in muscle tissue of 
herrings killed by the oral administration of PSP.  Accumulation in fish

    PSP, as measured by the mouse bioassay, has been detected in 
sand-launce (970 µg/kg), involved in a mass death of sea birds 
(Nisbet, 1983) (section  Pufferfish, collected from areas 
with occasional PSP episodes, have been found to contain saxitoxin 
in the liver and roe, amounting to 0.2% of total toxicity, the main 
part being tetrodotoxin (Yasumoto, 1980). 

1.3.  Exposure

    All reported cases of PSP intoxication in free-living animals 
(section 1.5.1) and in human beings (section 1.6) have been 
associated with alimentary exposure to contaminated food.  The 
shellfish most often reported to contain PSP are clams and mussels 
and include members of the families  Mactridae (Spisula 
 solidissima), Myacidae (Mya arenaria), Mytilidae (Mytilus 
 californianus, Mytilus edulis, Modiolus modiolus), and  Veneridae 
( Protatheca staminea, Saxidomus giganteus, Saxidomus nuttalli) 
(Halstead, 1978).  Occasionally,  Spondylus butleri (Harada et al., 
1982), scallops, and oysters may be involved.  The contamination of 
these species can be focal and temporal.  In the Pacific area, some 

toxic crabs have been mentioned as being responsible for PSP-type 
outbreaks.  There have not been any reports on PSP cases associated 
with other routes of exposure, such as dermal exposure to seawater 
containing toxic algae, or respiratory exposure to droplets of such 
seawater.  Several countries have developed surveillance programmes 
for PSP contamination of shellfish (WHO, 1979).  In the USA, 
shellfish-growing areas are closed if the concentration of PSP in 
the edible portion of the shellfish equals or exceeds 800 µg 
PSP/kg, as measured by the mouse assay, until the concentration has 
decreased to below 800 µg PSP/kg (Anon., 1965).  The action level 
of 800 µg PSP/kg has been established on the basis of exposure data 
from earlier PSP outbreaks in Canada (Tennant et al., 1955; Anon., 
1957).  The 800 µg PSP/kg level is more than 10 times lower than 
the lowest level that has caused intoxication in these outbreaks. 

1.4.  Metabolism

    Data are not available on PSP absorption, distribution, 
metabolism, and excretion, probably because sensitive chemical 
methods for quantification have only recently been developed. 
However, there is one old study stating that the poison is quickly 
eliminated in the urine (Prinzmetal et al., 1932). 

1.5.  Effects in Animals

1.5.1.  Field observations  Fish

    Fish kills by PSP, produced during  G. excavata blooms, have 
been reported from Europe (North Sea) (Adams et al., 1968) and from 
the north-east coast of North America (White, 1977, 1980), as 
mentioned in section  The fish involved, sand eel 
( Ammodytes sp.) and herrings  (Clupea harengus harengus), do not 
feed on dinoflagellates, and zooplankton appears to have acted as a 
vector of PSP.  No reports describing pathological and 
microbiological findings from these fish kills seem to have been 
published, but, experimentally, oral administration of PSP to 
herrings was rapidly fatal, with oral LD50 values for herring,
pollock, flounder, salmon, and cod in the range of 400 - 755 µg 
PSP/kg (White, 1977, 1981b).  Sea birds

    Twice in the last decade, mass death of sea-birds associated 
with an algal bloom has occurred in the North Sea, off the north-
east coast of England.  In May 1968, a bloom of  G. excavata was 
associated with mass death, particularly of shags  (Phalacrocorax 
 aristotelis), but also of terns ( Sterna sp.) and cormorants 
 (Phalacrocorax carbo) (Coulson et al., 1968).  The pathological 
lesions observed in the dead birds included extensive inflammation 
of the alimentary tract and often haemorrhages at the base of the 
brain and elsewhere in the body, symptoms typical of PSP-induced 
death in birds.  As sea birds do not eat mussels, fish (e.g., the 
sand eel) appear to have been the vector for PSP.  In the second 

episode, in 1975, the loss of sea birds was recorded in detail by 
the monitoring of colour-ringed birds (Armstrong et al., 1978).  
Thus, a 62 - 64% mortality rate of shags was associated with the 
 G. excavata bloom in the spring of 1975, compared with an average 
annual mortality rate of 16%.  Increased mortality rates were also 
observed in the herring gull  (Larus argentatus), the cormorant, and 
the fulmar  (Fulmarus glacialis).  A monitoring programme for PSP in 
mussels was introduced in the United Kingdom (for the north-east 
coast) following the 1968,  G. excavata bloom (Ayres & Cullum, 
1978).  The maximum annual values for PSP in mussels over a 9-year 
period are listed in Table 4.  Thus, mass deaths in sea birds 
associated with PSP, never observed before in England, were 
encountered in 2 out of the 3 years with high maximal PSP levels in 
mussels, within the period 1968-76.  PSP were not actually detected 
in the sea birds and the sand lance, using the mouse assay, 
presumably because the levels were below the detection limit of the 
assay.  Consequently, Armstrong et al. (1978) recommended that a 
more sensitive chemical procedure for the determination of PSP 
should be introduced so that analyses for PSP could be performed 
directly on the birds.  Mass death in common terns  (Sterna  hirundo) 
was observed off the Massachusetts coast in June 1978 (Nisbet, 
1983).  PSP was detected in the sand-launce, the tern's principal 
food, and, at the same time, high levels of PSP were found in 
shellfish in this area. 

Table 4.  Maximum values of PSP in mussels ( M. edulis) from the
north-east coast of England, 1968-1976a
Year        PSP levelb 
1968        100 000                              
1969        12 250                           
1970        8000                             
1971        1000                             
1972        400                              
1973        400                              
1974        5500                             
1975        12 300                           
1976        1750                             
a  Adapted from:  Ayres & Cullum (1978).
b  Determined by the mouse assay.

1.5.2.  Experimental studies  Acute toxicity

    As mentioned in section, the principle of the mouse 
assay developed by Sommer & Meyer (1937) is measurement of time to 
death.  In the same paper, the authors suggested that signs 
charateristic of PSP intoxication, such as dyspnoea, could be 
observed after the intraperitoneal administration of toxin.  
Hypotensive effects have been observed to accompany the respiratory 
depression, implicating both central and peripheral actions (Watts 

et al., 1966).  In a study using a PSP preparation extracted from 
Alaskan butter clams, Wiberg & Stephenson (1960) determined the 
LD50 values for mice using three routes of administration 
(intravenous, intraperitoneal, and oral).  The determination was 
conducted on groups of 130 - 160 male mice per route, with 4 dose 
levels per route.  In addition, the intraperitoneal LD50 was 
determined in female mice, using groups of 70 animals and 2 dose 
levels.  The observation time was 4 h.  As indicated in Table 5, 
PSP is much less toxic when administered by the oral route than 

    Increasing the pH of the injection medium or the addition of 
sodium ions reduced intraperitoneal toxicity.  The sodium ion did 
not influence the oral or intravenous toxicity. 

Table 5.  LD50 following a single dose of PSP in the mouse
in relation to the route of administrationa
                          LD50 (µg PSP/kg body weight)             
Route of administration   (The 95% confidence limit                
                          in parenthesis)                          
                          male          female                     
intravenous               3.4                                      
                          (3.2 - 3.6)                              
intraperitoneal           10.0          8.0                        
                          (9.7 - 10.5)  (7.6 - 8.6)                
oral                      263                                      
                          (251 - 267)                              
a  Adapted from:  Wiberg & Stephenson (1960).

    A similar dependence of LD50 values on the route of 
administration was observed in rats of different ages (Table 6) 
(Watts et al., 1966).  A PSP extract from Alaskan butter clams was 
used in this study, with 2 routes of administration (oral and 
intraperitoneal).  Sixteen Osborne-Mendel rats (equal number of 
males and females in each group) were used for each dose level, and 
4 dose levels were used per age level and per route of 
administration.  The effect on respiration was studied in the rats 
administered PSP orally.  The newborns and weanlings responded with 
dyspnoea and a marked decrease in respiration rate throughout the 
study period, whereas the adult rats exhibited laboured breathing 
followed by a profound reduction in respiration rate within 5 min 
of treatment.  In addition, convulsions were observed in weanlings 
and adult rats, but not in newborns. 

Table 6.  LD50 following oral or intraperitoneal administration
of a single dose of PSP to rats of different agesa
                      LD50 (µg PSP/kg body weight)                
Age                   (The 95% confidence limits                  
                      are in parenthesis)                         
                      oral           intraperitoneal              
newborn (24 h)        64             5.5                          
(51 - 80)             (4.7 - 6.5)                                 
weanling (21 days)    270            8.3                          
(204 - 356)           (7.7 - 9.0)                                 
adult (60 - 70 days)  531            10.0                         
(490 - 576)           (8.5 - 11.8)                                
a  Adapted from:  Watts et al. (1966).

    Prior exposure to non-lethal doses of PSP seeems to lower the 
susceptibility of rats to lethal doses of PSP.  In a study using 
Sprague-Dawley rats (sex not indicated), the oral LD50 value for 
a purified PSP material was determined (McFarren et al., 1960).  
One group of rats was given a non-lethal dose of PSP (about one-
third of the LD50), 14 days before the test.  The LD50 for the 
pretreated rats was about 50% higher than that for untreated rats.  
No explanation was presented of the mechanism involved, and this 
observation has not been repeated by others. 

    Comparative data on LD50 values for various species of animals 
have been obtained following oral administration to animals of 
extracts of clams containing 1.6 - 4.0 mg PSP/kg, determined by the 
mouse assay (Table 7). 

Table 7.  Comparison of LD50 values following a single oral dose 
of PSP in various species of animalsa
Animal      LD50                                                  
            (µg PSP/kg body weight)                               
mouse       420                                                   
rat         212                                                   
monkey      400 - 800                                             
cat         280                                                   
rabbit      200                                                   
dog         200                                                   
guinea-pig  128                                                   
pigeon      100                                                   
a  Adapted from: McFarren et al. (1960).

    All the above-mentioned toxicological studies conducted until 
recently have been carried out using as PSP material the same 
extract from Alaskan butter clam prepared by Schantz et al. (1958).  
According to a more recent study (Genenah & Shimizu, 198l), it can 
be assumed, that on the basis of chemical analysis, the toxic 
component in this extract was saxitoxin. 

    The toxicity of the various PSP components has been compared, 
using freshly-isolated compounds and testing their toxicity by 
means of the AOAC mouse bioassay (section (Table 8). 

    The effects of saxitoxin on the nerves of bivalve molluscs have 
been studied (Twarog et al., 1972) and it appears that species 
known to accumulate PSP, such as  Mytilus edulis, M.  californianus, 
 Placopecten magellanicus, Saxidomus nuttalli, and  Mya arenaria are 
resistant to saxitoxin, whereas many other species are up to 100 
times more sensitive than the resistant species. 

Table 8.  Comparison of the lethal effects of the various PSP 
components, based on a single dose intraperitoneally administered 
to micea
PSP component                  Lethality              
                               (MU/µ mol)                              
saxitoxin                      2045                   
neosaxitoxin                   1617                   
gonyautoxin I                  1638                   
gonyautoxin II                 793                    
gonyautoxin III                2234                   
gonyautoxin IV                 673                    
gynyautoxin V                  136                    
gynyautoxin VI                 108                    
gynyautoxin VIII               277                    
gynyautoxin VIII epimer        20                     
sulfocarbamoyl gonyautoxin I   ND                     
sulfocarbamoyl gonyautoxin IV  ND                     
decarbamoyl saxitoxin          1378                   
a  Adapted from:  Genenah & Shimizu (1981), Wichmann et al. (1981), 
   Harada et al. (1982), and Harada et al. (1983).
ND = No data available.  Mode of action

    Both saxitoxin and tetrodotoxin (section 3) have been used 
extensively as experimental tools in neurobiology.  Of the various 
PSP components, only saxitoxin has been studied in detail as far as 
pharmacological effects are concerned, in part because the other 
components are usually not available in sufficient quantities for 
such studies.  The mechanism of the cellular and systemic actions 
of saxitoxin have been reviewed by Kao (1966, 1972, 1983), Evans 
(1975), and Narahashi (1972). 

    Nearly all the systemic actions of saxitoxin can be explained 
by a wide-spread blockade of impulse-generation in peripheral 
nerves and skeletal muscles.  Direct cardiac effects are usually 
minimal.  In mammals, these effects lead to paralysis, respiratory 
depression and circulatory failure.  In contrast to tetrodotoxin, 
saxitoxin typically induces less hypotension for the same degree of 
muscular paralysis, and the hypotension tends to be more transitory 
(Kao, 1972).  A depressant effect of saxitoxin on both the central 

vasomotor and respiratory centres was observed when the toxin was 
administered either directly into the cerebral ventricles or 
intravenously (Borison et al., 1980a).  However, under conditions 
of distribution equilibrium, such as those occurring in human 
poison victims who had ingested saxitoxin, the peripheral effects 
were the more important in accounting for the symptomatology 
(Borison et al., 1980a). 

    Extensive experiments on single nerves and muscle fibres have 
shown that saxitoxin, like tetrodotoxin, affects the excitable 
membrane by blocking selectively the sodium channel through which 
the downhill movement of sodium ions accounts for the initiation of 
the electrical impulse (Narahashi, 1972).  Recent studies with 
several other PSP compounds have shown that they act with a similar 
mechanism (Kao, 1983).  It had been suggested that saxitoxin blocks 
the sodium influx simply by plugging the sodium channel with one of 
the guanidinium moieties (Kao & Nishiyama, 1965; Hille, 1975). 
However, recent studies of some structure-activity relationships of 
saxitoxin and several of its analogues clearly demonstrate this 
postulate to be untenable.  Most probably, saxitoxin and its 
analogues occupy a receptor on the outside surface of the membrane 
very close to the external orifice of the sodium channel.  
Saxitoxin binds to the receptor site in part by electrostatic 
attraction between the cationic 7,8,9 guanidinium group and fixed 
anionic sites of the membrane, and by hydrogen-bonding involving 
the C-12 hydroxyl groups (Kao, 1983). 

1.6.  Effects on Man

1.6.1.  Clinical studies

    The signs and symptoms of PSP in man may range from a slight 
tingling and numbness about the lips to complete paralysis and 
death from respiratory failure (Meyer et al., 1928; Medcof et al., 
1947; McFarren et al., 1960).  Typically, the tingling sensation 
around the lips, gums, and tongue develops within 5 - 30 min of 
consumption.  In moderate and severe cases, this is regularly 
followed by a feeling of numbness in the finger tips and toes, and, 
within 4 - 6 h the same sensation may progress to the arms, legs, 
and neck, so that voluntary movements can be made only with great 
difficulty.  In fatal cases, death is usually caused by respiratory 
paralysis within 2 - 12 h of consumption of the PSP-containing 
food.  Typical symptoms, which may help in distinguishing the cases 
as mild, severe, or extreme, are (Prakash et al., 1971): 


    Tingling sensation or numbness around lips, gradually 
    spreading to face and neck; prickly sensation in
    fingertips and toes; headache, dizziness, nausea;


    Incoherent speech; progression of prickly sensation to
    arms and legs; stiffness and incoordination of limbs;
    general weakness and feeling of lightness; slight
    respiratory difficulty; rapid pulse;


    Muscular paralysis; pronounced respiratory difficulty;
    choking sensation; high probability of death in absence of
    ventilatory support.

    Sensitivity to PSP is so variable that estimates of the human 
dose resulting in death range from 500 µg to 1000 µg (Tennant et 
al., 1955) to 12 400 µg (Meyer, 1953). 

    There are no reports of late effects in survivors or of the 
effects of long-term, low-level exposure to PSP. 

1.6.2.  Epidemiological studies

    Cases of human intoxication, associated with the consumption of 
shellfish and supposed to be caused by PSP, have been known for a 
long time; according to Prakash et al. (1971) about 1600 cases have 
been reported, on a world-wide basis, up to 1970 most occurring in 
Europe, Japan, and North America.  In the last decade, however, a 
changing pattern of PSP distribution has emerged, with cases also 
being reported from developing countries (Table 9).  Whether this 
indicates a real increase in the number of annual PSP cases, or is 
the result of improved surveillance and reporting is not known. 
Data are available, from the USA, comparing outbreaks of PSP with 
those due to other chemical agents of food-borne disease.  In the 
period 1970-74, PSP constituted 4.3% of all reported outbreaks 
(Hughes et al., 1977).  When food-borne infectious agents 
(bacteria, viruses, and parasites) were considered in addition to 
chemical agents in 1978-81, PSP made up 1.1% of all outbreaks of 
food-borne diseases with known etiology (Anon, 1981a,b; Anon, 

Table 9.  More recent reports on the occurrence of PSP
Country         Number of  Number  Species of      Origin of  Concentration  Dinoflagellate  Reference
                people     of      shellfish       shellfish  of PSP         involved
                affected   deaths
Canada          2          0       Mussels         local      430 000 µg/kg                  Acres & Gray

Canada          5          1       Mussels, clams  local      21 000 µg/kg                   Anon (1982)

Germany,        19         0       Mussels         Vigo       12 000-40 000                  Simon et al.
Federal                             (Mytilus        (Spain)    µg/kg                          (1977)
Republic of                         edulis)                                       

India           98         1       Mussels         local                                     Bhat (198l)a

Malaysia        201        4       Clams           local                      P. bahamense    Roy (1977)a

Mexico          20         3       Mussels         local                                     Anon (1979)a

Norway          4          0       Mussels         local      400-4000 µg                    Gulbrandsen 
                                    (M. edulis)                ingested                       & Aalvik 

South Africa    6          2       Black mussels   local      84 000 µg/kg    G. catenella    Grindley & 
                                    (Chloromytilus                                            Sapeika
                                    meridionalis)                                             (1969)

South Africa    17         0       Black mussels   local      up to           G. catenella    Popkiss et 
                                                              72 830 µg/kg                   al. (1979)

Switzerland     23         0       Mussels         Vigo       20 000 µg/kg                   Zwahlen et 
                                    (M. edulis)     (Spain)                                   al. (1977)
Table 9.  (contd.)
Country         Number of  Number  Species of      Origin of  Concentration  Dinoflagellate  Reference
                people     of      shellfish       shellfish  of PSP         involved
                affected   deaths
Thailand        62         1       Mussels         local      9400 µg/kg                     Anon (1983)
                                   ( Mytilus sp.)

United Kingdom  78         0       Mussels         local      600-6000 µg     G. tamarensis   McCollum et 
                                   ( M. edulis)                ingested                         al. (1968)

USA             51         1       Mussels         local      3000-40 000                    Anon (1980)
(California)                       Oysters                    µg/kg                             

USA             33         0       NSb             local      NS             NS              Anon (1972)
(Maine, New

USA             26         0       Mussels, Clams  local      30 000-50 000   G. tamarensis   Anon (1973)
(Massachusetts)                    scallops                   µg/kg           

Venezuela       171        10      Mussels         local      790-33 000      G. tamarensis   Reyes-
                                                              µg/kg          Cochlodinium    Vasquez et 
                                                                             sp.             al. (1979)

Venezuela       9          1       Mussels         local                                     Anon          
                                    (Perma perma)                                             (1981c)a 
Table 9.  (contd.)
Country         Number of  Number  Species of      Origin of  Concentration  Dinoflagellate  Reference
                people     of      shellfish       shellfish  of PSP         involved
                affected   deaths
West Europe     120        0       Mussels         Vigo       12 000-40 000                  Zwahlen et 
(including                                         (Spain)    µg/kg                          al. (1977)
Federal Repub-
lic of Germany
and Switzerland
a  The diagnosis of the human cases has not been aetiologically confirmed, as no data were reported on 
   the presence of PSP components in the food associated with the disease.
b  NS = Not specified.

    A variety of fish inhabiting tropical and subtropical seas may 
become toxic, and, by ingestion, cause an intoxication in human 
beings named "ciguatera", which is characterized by neurological 
and gastrointestinal symptoms.  The term ciguatera is of Spanish 
origin, derived from cigua, which is a Carribean trivial name for 
a marine snail,  Turbo pica that, when eaten, is said to cause 
indigestion.  The principal toxin is ciguatoxin, but other toxic 
components have recently been identified.  The subject has been 
reviewed by Bagnis (1981a) and Withers (1982).  The disease 
ciguatera has been known since the 17th century and appears to be 
the most commonly occurring disease associated with seafood toxins 
(Hughes et al., 1977). 

2.1.  Properties and Analytical Methods

2.1.1.  Chemical properties

    The chemical properties of ciguatoxin have been reviewed by 
Scheuer (1982).  The chemical structure of ciguatoxin is still 
largely unknown.  The toxin has been extracted from the the moray 
eel liver and, after elaborate purification, has been obtained in a 
pure crystalline form as a white solid (Scheuer, 1982).  Ciguatoxin 
is a highly-oxygenated lipid, soluble in polar organic solvents but 
insoluble in water.  The relative molecular mass is estimated to be 
1111.7 ± 0.3, and possible molecular formulae are C53H77NO24 or 
C54H78O24, but other combinations cannot be excluded.a 

    Other toxic components isolated from ciguatoxic fish are 
maitotoxin (Yasumoto et al., 1971) and scaritoxin (Chungue et al., 
1976).  The chemical structure of the two components is unknown.  
However, scaritoxin resembles ciguatoxin in chemical and some 
chromatographic properties, but is distinguishable from it by DEAE-
cellulose column and by thin layer chromatography (Chungue et al., 
1977).  Maitotoxin is a highly oxygenated water-soluble compound 
with a large relative molecular mass and has no structural 
relationship with ciguatoxin and scaritoxin.b

a   Recent results of spectral and chemical studies of the 
    crystalline material has shown that ciguatoxin is a molecule
    with a structure resembling that of such polyethers as
    okadaic acid and brevetoxin C (Nukina et al., 1983).

b   In a recent study of dinoflagellates, isolated from      
    ciguatera areas in the Carribean using unialgal cultures,
    at least 5 different toxins were identified:  ciguatoxin, 
    maitoxin, okadaic acid, scaritoxin, and an unnamed toxin.
    All the compounds are thought to contribute to the       
    ciguatera syndrome in the Carribean (Tindall, 1983).     

2.1.2.  Methods of analysis for foodstuffs

    All analytical results for ciguatoxin and related components 
referred to in this document were obtained by biological methods.  
According to published information, the more recently developed 
radioimmunoassay, mentioned in, has not been applied under 
practical conditions (Laigret et al., 1981; Parc et al., 1981).  Biological methods

    This topic has been recently reviewed by Yasumoto et al. 
(1984).  For ciguatoxin, a mouse injection test, first mentioned 
by Banner et al. (196l), has since been modified by Kimura et al. 
(1982) and Yasumoto et al. (1984).  The method consists of injecting 
serially-diluted semipurified toxin extracts into mice and observing 
the mortality ratio for 24 h.  The results are obtained as mouse 
units, and one mouse unit is defined as the amount of toxin that 
kills a mouse (20 g body weight) in 24 h.  The method does not 
distinguish between ciguatoxin and scaritoxin. 

    A bioassay for ciguatoxin in fish has been developed on the 
basis of feeding cats or mongooses a ration containing 100 g of 
the fish to be tested per kg ration (Bagnis & Fevai, 1971; Banner, 
1975).  The cat is less satisfactory, because it may regurgitate 
part of the test meal.  Test animals were observed for 48 h, with 
the response rated from 0 (no response) to 5 (death within 48 h).  
Recently, a bioassay using mosquitoes  (Aedes aegypti) has been 
developed (Chungue et al., 1984; Pompon et al., 1984).  The 
procedure involves intrathoracic injection in mosquitoes of 
serially-dilated extract from fish, and the toxicity of the fish 
is expressed as mosquito LD50.  A good correlation between the 
mosquito bioassay and the mouse bioassay was observed.  All the 
tests described above appear to be non-specific and only 
semiquantitative at best.  Chemical methods

    A radioimmunoassay for ciguatoxin has been developed, using 
antibodies produced against a conjugate of human serum albumin and 
ciguatoxin isolated from toxic moray eel (Hokama et al., 1977).  
Results of the assay were correlated with those of the assays on 
mongoose, mouse, and guinea-pig atrium.  All the three assay 
procedures showed good correlation when ciguatoxin was present in 
fish tissues in high concentrations (Kimura et al., 1982). 

2.2.  Sources, Occurrence, and Exposure

2.2.1.  Algae

    A dinoflagellate,  Gambierdiscus toxicus, has been identified as 
the source of ciguatoxin and maitotoxin (Bagnis et al., 1977; 
Yasumoto et al., 1977a; Adachi & Fukuyo, 1979).  G. toxicus is an 
armoured dinoflagellate with two flagella, living around coral 
reefs, closely attached to macroalgae (Bagnis et al., 1979b), such 
as  Turbinaria ornata, Amphiroa sp., and  Jania sp.  Ciguatoxin and 

maitotoxin have been isolated from the biodetritus layer on coral 
reefs, from  G. toxicus collected from sea water, and from axenic 
cultures of  G. toxicus, using the mouse assay and some biochemical 
characteristics as identification procedures (Bagnis et al., 
1979b).  Fish, such as parrot-fish  (Scarus gibbus) and surgeon-
fish  (Ctenchaetus striatus), representatives of fish species likely 
to contain ciguatoxin and maitotoxin, feed on the layers of 
microorganisms and detritus colonizing coral beds, and thereby 
accummulate the toxins (Bagnis et al., 1980). 

    Subsequently, strains of  G. toxicus, able to produce ciguatoxin 
and maitotoxin, have repeatedly been isolated from macroalgae such 
as  Halimeda sp.,  Penicillus sp.,  Acetabularia sp. and  Gracilaria 
sp., and from coral reef off the coast of Florida, USA.  These 
findings elucidate the origin of toxicity of Florida Barracuda 
 (Sphyranea barracuda), a fish species often associated with cases 
of ciguatera in USA (Bergmann & Alam, 198l; Bergman, 1982).a 

    Surveys of coastal sea water in the Pacific (French Polynesia) 
have demonstrated that  G. toxicus is associated with  the  
occurrence of ciguatoxic fish (Chanteau, 1978; Bagnis, 1981c).  
A temporal fluctuation in the concentration of  G. toxicus cells 
in the sea water was observed, apparently associated with the death 
of corals caused by constructions in the lagoon, whereas no 
association was found with a number of trace elements in the sea 
water (Bagnis, 1977; Yasumoto et al., 1980a). 

2.2.2.  Occurrence in fish

    The occurrence of ciguatoxin in fish has been reviewed by 
Banner (1975) and WHO (1983).  More than 400 species of bony 
fish have been reported in the literature to have caused ciguatera 
(Halstead, 1978).  In general, ciguatoxic species are limited to   
fish that feed on algae and the detritus of coral reefs,           
particularly the surgeon-fish  (Ctenochaetus striatus), parrot-fish 
 (Scarus gibbus), and the larger reef carnivores that prey on these 
herbivores (Bagnis, 1981b).  Thus, the larger carnivores such as    
moray eels, snappers, groupers, carrangs, Spanish mackerels,       
emperors, certain in-shore tunas, and barracuda are most toxic.    
Ciguatoxin has been detected in the contents of the gut, the liver,
and the flesh (muscle tissue) of surgeon-fish (Yasumoto et al.,    
197l), and parrot-fish, groupers, and snappers (Bagnis &           
Letourneux, 1974), by means of the mouse assay and chromatography 
(Chanteau et al., 1976; Yasumoto et al., 1977b).  The highest     
concentrations of ciguatoxin were found in the liver and other    
viscera (Helfrich et al., 1968; Chungue & Bagnis, 1976).  Not all 
the fish in a single population contained equal levels of toxin.  
Furthermore, even when the flesh did not contain detectable levels

a   Recently it has been reported that three dinoflagellate
    species  (Gambierdiscus toxicus, Prorocentrum concavum, and
     Prorocentrum rhathymum) are producers of toxins that
    contribute to the ciguatera syndrome in the Carribean
    (Tindall, 1983).

of ciguatoxin, the liver contained an appreciable amount, as 
demonstrated in the moray eel (Yasumoto & Scheuer, 1969).  
Ciguatoxin has also been found in the viscera of a turban shell 
( Turbo argyrostoma, a marine snail), a food item that has 
occasionally caused ciguatera-like intoxication in man (Yasumoto & 
Kanno, 1976). 

2.2.3.  Environmental factors influencing the growth of
causative dinoflagellates

    Randall, in a review in 1958, had already suggested that the 
occurrence of ciguatera might have an environmental background.  
Randall also refined an earlier hypothesis that the disease 
agent was transmitted from herbivorous to carnivorous fish, and he 
suggested, without any direct proof, however, that the causative 
organism was a benthic blue-green alga.  He further mentioned that 
disturbances of the coral reef caused creation of new surfaces to 
support vigorous growth of the hypothetically toxic cyanophyte 
organism.  Evidence supporting the hypothesis of an environmental 
influence on ciguatera has subsequently been provided in a series 
of investigations in French Polynesia by Bagnis and co-workers 
(Bagnis, 1969, 1974, 1977, 1980, 1981b; Bagnis et al., 1973, 1974, 
1980).  Thus, natural disturbances of the coral reefs, such as 
hurricanes and storms, or man-made disturbances, such as blasting 
of reefs, crashing of ship anchors, and building of piers or 
wharfs, provide conditions for growth of the macroalgae to which 
 G. toxicus cells are attached, resulting in increased 
dinoflagellate populations.  These disturbances, which cause 
increased numbers of ciguatoxic fish and increased toxin levels in 
the affected fish resulting in increased incidence rates of 
ciguatera, may have long-lasting effects, up to 10 - 15 years after 
the disturbance took place.  At this point, no information is 
available on the environmental influence on the other 
dinoflagellates ( P. concavum and  P. rhathymum) associated with 
ciguatoxic fish. 


2.2.4.  Human exposure

    The only known pathway of human exposure is through the 
consumption of contaminated fish, with the exception of a marine 
snail.  In the past, this exposure was limited to the circumglobal 
tropical and subtropical belt shown in Fig. 3.  However, recent 
evidence (section 2.5) has shown that interregional transport of 
fish can result in human exposure in other parts of the world. 

2.3.  Metabolism

    Data on absorption, retention, distribution, and metabolism of 
ciguatoxin in human beings or animals are not available because 
reliable chemical methods of analysis are lacking. 

2.4.  Effects on Animals

2.4.1.  Experimental studies

    Ciguatoxin, extracted from fish, purified to some extent by 
solvent extraction, and injected intravenously or intraperitoneally 
into mice, produced acute effects characterized by diarrhoea, 
retching, inactivity, and death after convulsive spasms (Bagnis, 
1970; Banner, 1975).  Similar effects were observed within 48 h, 
when cats or mangooses were fed a ration containing ciguatoxin.  
The effects on mice, cats, and mongooses were used as a basis for 
the biological determination of ciguatoxin, decribed in section  The pronounced toxicity of ciguatera toxins is 
noteworthy.  Thus, ciguatoxin has an LD50 (ip) in mice of 0.45 
µg/kg body weight (Scheuer, 1982), while the MLD (ip) in mice for 
maitotoxin is 0.15 µg/kg body weight (Yasumoto et al., 1984). 

2.4.2.  Mode of action

    Few relevant data on the mode of action are available because 
of the restricted availability of purified toxins of this group.  
The pharmacological action of ciguatoxin is related to its direct 
effects on excitable membranes rather than to its 
antichlolinesterase properties.  Ciguatoxin has a potent 
depolarising action due to a selective increase in sodium  
permeability in the nerve cells and striated muscle, which can be 
counteracted by calcium ions (Rayner, 1972).  The effect of 
ciguatoxin on smooth muscle can be explained by a potent releasing 
action of the toxin on endogenous norepinephrine from adrenergic 
nerve terminals and a potentiating effect on the postsynaptic 
membrane (Ohizumi et al., 1981). 

2.5.  Effects on man

2.5.1.  Clinical Studies

    The clinical picture is quite variable.  Typically, symptoms 
occur within 1 - 6 h of ingestion of toxic fish.  Initial symptoms 
usually include nausea, malaise, and numbness and tingling of the 
lips, tongue, and throat.  Patients may later develop some or all 

of the following signs and symptoms:  vomiting, abdominal cramps, 
diarrhoea, paraesthesia of the extremities, itching myalgia, and 
arthralgia.  In more severe cases, ataxia, weakness, blurred 
vision, insomnia, sinus bradycardia, dysrhythmias, and hypotension 
may develop (Bagnis 1968; Morris et al., 1982a).  A symptom that 
particularly suggests the diagnosis is alternating sensations of 
cold and hot (Bagnis, 1967).  The duration of illness is variable. 
Most of the patients recover within three days, but malaise, 
paraesthesia, pruritus, and ataxia may persist for weeks or 
even years in severe cases (Hughes & Merson, 1976; Bagnis et al., 
1979d).  Patients repeatedly poisoned by ciguatoxic fish may 
develop a resurgence of ciguatera symptoms even after eating fish 
containing little or no detectable toxin (Bagnis, 1984a). 

    On the basis of mouse-assay analyses of toxic fish recovered 
from patients meals, Yasumoto suggested that the oral intake of 
as little as 0.1 µg (10 MU) of ciguatoxin can cause illness in an 
adult (Yasumoto, 1980; Yasumoto et al., 1984). 

    In the most severe cases, death results from circulatory 
collapse or respiratory failure.  Halstead (1978) reported a case-
fatality rate of about 12%, but mentioned that limited inability 
statistics were available.  This publication includes an intensive 
review of case reports on ciguatera, going back to the beginning of 
the last century.  However, 3 deaths due to ciguatera occurred 
among 3009 cases (corresponding to a case-fatality rate of 0.1%) in 
French Polynesia (Bagnis et al., 1979a).  No deaths occurred among 
184 cases reported to the Centers for Disease Control, USA, in 
1970-74 (Hughes & Merson, 1976) or among 33 patients in the US 
Virgin Islands in 1980 (Morris et al., 1982a); 3 out of 67 patients 
died in outbreaks that were reported from Puerto Rico in 1981, 
corresponding to a case fatality rate of 4.5% (Anon, in press). 

2.5.2.  Epidemiological studies

    Cases of ciguatera are commonly encountered throughout the 
Caribbean area and much of the Pacific area, in the zones between 
latitudes 35° N and 35° S (Fig. 3).  The location and other 
characteristics of recently-reported outbreaks are summarized in 
Table 10. 

    The annual incidence of ciguatera intoxication on the Virgin 
Islands on the basis of emergency room admissions for 3 years was 
3.6 cases per 1000 (Morris et al., 1982b).  Results of a household 
survey suggested that the true annual incidence was actually 7.3 
per 1000.  These cases were diagnosed for the characteristic 
combination of gastrointestinal and neurological symptoms.  Hanno 
(1981) estimated that the true incidence on the Virgin Islands 
might be as high as 30 per 1000.  In the South Pacific, incidence 
rates vary from 1 case per 10 000 in Wallis, Futuna, Naury, Guam, 
Salomons, and Cook Islands to 4 - 5 cases per 1000 in Tuvalu and 
French Polynesia (Bagnis, 1984a).  In the USA, ciguatera accounted 
for 22% of all outbreaks of food-borne chemical diseases reported 
in the period 1970-74 (Hughes et al., 1977).  When food-borne 
infectious agents (bacteria, viruses, and parasites) were 

considered in addition to chemical agents in 1978-81, ciguatera 
made up 8.4% of all reported outbreaks of food-borne diseases of 
known etiology (Anon, 1981c; Anon 1983b,c).  Of the 67 ciguatera 
outbreaks reported during these 4 years, 34 (51%) occurred in 
Hawaii, 11 (16%) in Puerto Rico, 5 (7%) in the US Virgin Islands, 
and 5 (7%) in Florida (Anon., 1981b,c; Anon., 1983c,d). 

    Cases of ciguatera have also been encountered outside the 
circumglobal belt, where the organism  G. toxicus is present and 
where ciguatoxic fish are traditionally caught.  Thus an outbreak 
of ciguatera occurred in Maryland, USA, involving 12 persons 
showing symptoms, and two persons who were hospitalized because of 
hypotension.  The intoxication was due to a fish (grouper) that had 
been transported from Florida to the restaurant in Maryland, where 
the episode took place (Anon, 1980).  In France, ciguatera has 
been diagnosed in association with the consumption of ciguatoxic 
fish imported frozen from China, Province of Taiwan (Baylet et al., 
1978).  In Canada, an outbreak of ciguatera was observed that 
involved two persons and was associated with the consumption of 
barracuda brought by tourists from Jamaica (Anon, in press). 

Table 10.  Recent reports on the occurrence of outbreaks of ciguatera
                   Number of  Number   Species        Origin
Country            people     of       of fish        of fish          Reference
                   affected   deaths
Bahamas            14         0        barracuda      Local            Anon (1982)

Canada             2          0        barracuda      Jamaica          Anon (1983b)

Cuba               100        0        moray eel      Local            Bagnis (1978)

Fiji               925        1        snapper        Local            Yasumoto
                                       barracuda                       et al. (1984)

France             2          0        not specified  China (Province  Baylet et al.       
                                       (frozen fish)  of Taiwan)       (1978)

French Polynesia   3009       3        surgeon-fish   Local            Bagnis et al.       
New Caledonia                          parrot-fish                     (1979)
(South Pacific)                        grouper
Jamaica            250        0        grouper        Local            Bagnis (1978)

La Reunion         367        0        snapper        Salya de
(Indian Ocean)                                        Malha

USA (Florida)      129        0        grouper        Local            Lawrence et al.
                                       snapper                         (1980)

USA (Maryland)     12         0        grouper        Florida          Anon (1980)

USA (Shipboard)    24         0        barracuda      Gulf of Mexico   Barkin (1974)

US Virgin Islands  51         0        snapper        Local            Engleberg et al.
US Virgin Islands  33         0        carrang        Local            Morris et al.
                                       snapper                         (1982a,b)

    In contrast to other biotoxins included in this document, 
tetrodotoxin, according to present knowledge, is probably not 
produced by algae, but by certain fishes and a few other animals.  
On the basis of the number of human victims involved yearly, 
tetrodotoxin poisoning is not an important public-health problem.  
However, in contrast with most other algal intoxications discussed 
below, the illness in tetrodotoxin intoxication is severe and the 
mortality rate is high.  Furthermore, increase in world trade has 
led to cases of the shipping and sale of misbranded toxic fish to 
countries where tetrodotoxin poisoning had previously been unknown 
(Pocchiari, 1977).  Thus, the magnitude of tetrodotoxin poisoning 
as a public-health problem is influenced less by the number of 
human victims involved than by its potential threat to human life 
and health. 

    The history of tetrodotoxin poisoning has been reviewed in some 
detail by Kao (1966).  In recent years, considerable interest in 
tetrodotoxin has developed among natural-product chemists and 
neurobiologists.  For the former, there are challenging problems 
related to its isolation and purification as well as to its 
structure.  As regards the latter, tetrodotoxin remains the most 
important and most widely-used tool for selectively blocking the 
sodium channel. 

3.1.  Properties and Analytical Methods

3.1.1.  Chemical properties

    Schantz (1973) and Scheuer (1977) have reviewed the chemistry 
of tetrodotoxin.  The compound has been obtained from an extract 
of pufferfish viscera in the form of colourless crystal prisms that 
are slightly soluble in water.  It is an aminoperhydroquinazoline 
compound (Fig. 4), with a relative molecular mass of 319.  It has a 
guanidinium group with a pKa of 11.6, and a unique intramolecular 
hemilactal bond.  The toxin is unstable at pH levels above 8.5 and 
below 3. 

3.1.2.  Methods of analysis for tetrodotoxin in foods

    Though of entirely different chemical structure, tetrodotoxin 
induces toxic effects very similar to those of saxitoxin (section 
1.5), and the mouse bioassay developed for PSP has also been 
used for tetrodotoxin (Kao, 1966; Schantz, 1973).  In Japan, a 
modification of the mouse bioassay is now in operation as an 
official method (Kawabata, 1978), and a chemical assay has been 
developed (Nunez et al., 1976) on the basis of the production of 
fluorescent compounds of tetrodotoxin by alkali treatment.  In this 
method, there is a linear relation between the fluorescence-
intensity and the concentration of tetrodotoxin in the range of 
0.34 - 10 µg/ml.  A continuous analyser, using the same reaction, 
was constructed by Yasumoto et al (1982).  In this method, which is 
more sensitive and specific than the previous method, the toxin, 
after separation from contaminants on an ion-exchange column, is 
converted to fluorescent compounds by heating in a solution of 

2N sodium hydroxide.  Both the retention time of the toxin and the 
intensity of fluorescence are recorded automatically on a 
fluorimeter.  A linear relation exists between the intensity of 
fluorescence and the concentration of tetrodotoxin in the range of 
0.02 - 4 µg toxin/mg.  The results are reproducible within a 
variation of 3%. 


    Another continuous analyser was constructed by Onoue et al. 
(1983) in which 1-phthalaldehyde was used as the reagent to 
produce a fluorescent derivative from tetrodotoxin.  This method 
has the advantage of differential detection of tetrodotoxin from 
PSP toxins.  It suffers from the need for laborious pretreatment 
of the extract, because the reagent is more reactive with amino 
acids which are present in an overwhelming abundance in the 

3.2.  Occurrence and Human Exposure

    The occurrence of tetrodotoxin has been reviewed by Kao (1966) 
and Blankenship (1976).  It is mainly found in the ovaries, liver, 
and intestines of various species of pufferfish, lesser amounts 
being found in the skin; the body muscle is usually free of the 
toxin, with the exception of  Lagocephalus lunaris lunaris, which 
often contains fatal amounts of tetrodotoxin in the muscle tissue 
(Tabeta & Kumagai, 1980).  The most toxic pufferfish are members 
of the family  Tetraodontidae, but not all the species in this 
family contain the toxin.  The most toxic ones are caught along the 
coasts of China and Japan, and the meat of these species is 
considered a delicacy.  The amount of toxin in the roe is related 
to the reproductive cycle, and is greatest just before spawning 
(early summer).  Tetrodotoxin has also been found in the skin of a 
group of newts of the genus  Taricha, native to northern California 
and southern Oregon, in the USA.  It has been detected in the skin 
of Central American frogs of the genus  Atelopus.  In addition, 
tetrodotoxin has been identified as the poisonous principal in the 
venom of the blue-ringed octopuses,  Hapalochlaena maculosa and 
 H. lunata of southern Australia, involved in human fatalities 
through bites (Freeman & Turner, 1970; Sheumack et al., 1978).  
Tetrodotoxin has also been found in the Japanese ivory shell, 
 Babylonia japonica, and in the trumpet shell, associated with fatal 
human cases following consumption (Narita et al., 1981; Noguchi et 
al., 1981). 

    There has not been any report linking the presence of 
tetrodotoxin in these animals with algae or microbes, but it is 
noteworthy that pufferfish raised artificially in ponds do not 
contain tetrodotoxin (Matsui et al., 1981, 1982). 

    Human exposure is generally limited to consumption of certain 
fish species, the identification of which is feasible.  This is 
more difficult with frozen fish flesh.  In international trade with 
frozen fish from areas where tetrodotoxin-containing fish are 
caught, special care should be taken to avoid transport of 
contaminated fish flesh (section 3.4). 

3.3.  Mode of Action

    The mode of action of tetrodotoxin is very similar to that of 
saxitoxin and is dealt with in section 

3.4.  Effects on Animals

    The effects of tetrodotoxin, either in contaminated fish or in 
a purified form, have been tested experimentally on a large variety 
of animal species (Table 11).  In all animals, with few exceptions, 
the signs of intoxication are generally the same, and comparable 
with those caused by the PSP compounds.  These effects involve 
primarily the peripheral neuromuscular system, which is paralysed 
to different extents because of interference with the generation 
and conduction of electrical impulses (section 1.5).  There are 3 
manifestations of tetrodotoxin intoxication that appear to be 
somewhat different from those due to PSP compounds.  Tetrodotoxin 
is a highly potent emetic agent, so that vomiting is frequently 
observed in both cats and dogs and also in man.  For the same 
degree of neuromuscular paralysis, the systemic arterial 
hypotension produced by tetrodotoxin is significantly greater and 
lasts appreciably longer than that produced by the PSP toxins.  
Lastly, tetrodotoxin, acting through a central mechanism, is a 
highly potent hypothermic agent. 

    Pufferfish and taricha newts containing tetrodotoxin are 
resistant to the action of tetrodotoxin. 

    Two field cases of tetrodotoxin intoxication have been reported 
in which cats had been fed a diet of pufferfish containing an 
unknown level of tetrodotoxin (Atwell & Stutchbury, 1978).  The 
cases were characterized by paralysis, ataxia, and respiratory 
depression, and the symptoms could be reproduced in cats by feeding 
liver and flesh contaminated with tetrodotoxin. 

Table 11.  Comparative lethality of tetrodotoxin in various 
                                  Minimum lethal dose
                                  (µg tetrodotoxin/kg body weight)
Plaice  (Paralichthys olivaceus)   0.5                      
Dragonfly                         1.3                      
Carp                              2.0                      
Pigeon                            2.7                      
Rat                               2.7                      
Sparrow                           4.0                      
Guinea-pig                        4.5                      
Frog                              5                        
Hen                               6                        
Rabbit                            8                        
Mouse                             8                        
Dog                               9                        
Cat                               10                       
Turtle                            46                       
Eel                               80                       
Toad (Bufo)                       200                      
Snake (non-poisonous, species     450                      
 not given)
a  Adapted from:  Kao (1966).

3.5.  Effects on Man

    In man, the onset of symptoms of tetrodotoxin intoxication 
usually occurs from 10 to 45 min after ingestion, but may be 
delayed by 3 h or more.  Paraesthesia appears in the face and 
extremities and may be followed by sensations of lightness, 
floating, or numbness.  Nausea, vomiting, diarrhoea, and epigastric 
pain may also be present.  Later, respiratory symptoms become 
prominent with dyspnoea, shallow, rapid respiration, and the use of 
auxilliary muscles.  Cyanosis and hypotension follow, and 
convulsions and cardiac arrhythmia may occur.  In most instances, 
the victims retain consciousness until shortly before death, which 
usually takes place within the first 6 h (Torda et al., 1973).  In 
Japan, the average annual number of tetrodotoxin cases for the 
period 1974-79 was 60, with 20 deaths (Kainuma, 1981).  In the USA, 
two non-fatal outbreaks were reported in the period 1970-74 (Hughes 
et al., 1977).  In Italy, 10 cases of tetrodotoxin intoxication 
were observed, with 3 deaths, following the consumption of frozen 
pufferfish imported from China, Province of Taiwan, mislabelled as 
angler fish (Pocchiari, 1977).  Samples of the pufferfish contained 
from 0.5 to 30 mg tetrodotoxin per kg wet tissue. 


    A disease in human beings associated with red tides involving 
the dinoflagellate  Gymnodinium breve has been encountered around 
the coasts of Florida, USA, named neurotoxic shellfish poisoning 
(NSP).  According to symptoms and mode of exposure, two syndromes 
can be identified:  (a) NSP associated with the consumption of 
shellfish containing cells or metabolites of toxic  G. breve.  The 
symptoms are predominantly neurotoxic in nature and resemble PSP, 
except that paralysis does not occur; (b) NSP characterized by 
respiratory symptoms and associated with exposure to aerosols of 
 G. breve cells (Hughes & Merson, 1976). 

    There is however much less data available for this disease, 
compared with the other diseases caused by dinoflagellate toxins 
and tetrodotoxin.  Thus, the  G. breve toxins have never been 
chemically identified in food (and air) in episodes involving human 
beings, and only a limited number of toxicity studies on animals 
have been conducted so far. 

4.1.  Properties and Analytical Methods

    The chemical properties of toxins obtained in earlier 
investigations have been reviewed by Shimizu (1978).  More 
recently, 4 toxic components have been isolated from cultured 
cells of  G. breve and the stucture was determined for three of 
them, named brevetoxin B, brevetoxin C, and GB-3 (Lin et al., 1981; 
Chou & Shimizu, 1982; Golik et al, 1982).  These components share 
the same skeleton made up of a single carbon chain locked into a 
rigid ladder-like novel structure consisting of 11 continous 
transfused ether rings (Fig. 5).  The compounds are soluble in 
organic solvents but are unstable in chloroform; this has caused 
difficulties in isolating the toxins in earlier investigations.  
The toxins are not fluorescent and do not have properties that make 
detection and quantification easy.  Hence, no chemical method for 
analysis exists.  During the purification procedure, a fish 
bioassay has been employed (Lin et al., 1981).  A mouse bioassay 
has been developed comparable to the assay used for PSP (section, but involving more elaborate extraction and clean-up 
procedures and an observation time of 6 - 24 h (Subcommittee on 
laboratory methods for the examination of shellfish, 1970; 
Spiegelstein et al., 1973).  The disadvantage is that no standard 
preparation of  G. breve toxins is available for standardization of 
the mouse bioassay. 

4.2.  Sources and Occurrence

    The NSP toxic compounds have been isolated exclusively from 
 G. breve, a non-thecate (naked) dinoflagellate, encountered around 
the coasts of Florida, USA, particularly during red tides, which 
are initiated in offshore waters primarily in the late summer and 
autumn months (Steidinger, 1975).  The iron content of the water 
might be used as a predictive guide, as a maximum of iron has been 
observed immediately preceding red tides (Kim & Martin, 1974). 
Taxonomically, the organism has recently been transferred to 
 Ptychodiscus brevis (Steidinger, 1979). 


4.3.  Effects on Animals

4.3.1.  Field observations

    A disease has been observed in fish and birds that is thought 
to be caused by  G. breve toxins, because the episodes have occurred 
in close association with blooms of  G. breve cells, and because 
similar symptoms were observed after feeding  G. breve cells to 

    Every 3 - 4 years, blooms of  G. breve occur on the west coast 
of Florida, causing massive fish kills.  The fish species involved 
are mainly tomtate fish  (Haemulon aurolineatum) and striped mullet 
 (Mugil cephalus) (Forrester et al., 1977).  The fragile naked cells 
of  G. breve rupture on passage through the gill processes of the 
fish, releasing the toxins, which readily pass through the gill 
surfaces with lethal effect, if the  G. breve cell concentration is 
sufficiently high.  Fish that swim into a red-tide area will 
continue actively for a while, then will suddenly lose balance, 
gasping at the surface before becoming passive on the bottom 
followed by a terminal struggle.  Death occurs without pathologic 
lesions (Abbott et al., 1975). 

    Mass death of sea birds and mass fish kills have been observed, 
associated with red tides, off the west coast of Florida (Forrester 
et al., 1977).  The birds involved were double-crested cormorants 
 (Phalacrocorax auritus), redbreasted mergansers  (Mergus merganser), 
and lesser scaup  (Aythya  affinis).  The signs shown by the affected 
birds included weakness, reluctance to fly, clear nasal discharge, 

viscous oral discharge, oil gland dysfunction, diarrhoea, dyspnoea, 
tachypnoea, tachycardia, and hypotension. 

4.3.2.  Experimental animal studies

    In a study using white Pekin ducklings, force-fed with tissues 
of clams that had been filter-feeding on toxic  G. breve cells and 
with sea water containing  G. breve cells, the birds showed ataxia 
and spastic movements within 3 days, and died within 5 days 
(Forrester et al., 1977).  Similar signs, including death within 
6 - 22 h, were observed in male white Leghorn chicks fed tissues of 
oysters  (Crassostrea virginica) that had been filter-fed in the 
laboratory on toxic  G. breve cells (Ray & Aldrich, 1965). 

    Mice are susceptible to  G. breve toxin preparations 
administered intravenously, intraperitoneally, or sub-cutaneously, 
showing signs similar to those observed in mice administered PSP 
(section  A bioassay was also developed using the 
mosquito fish  (Bambusia affinis), which seems to be very 
susceptible to toxic  G. breve (Spiegelstein et al., 1973). 

    During  in vitro experiments, spasmogenic effects of  G. breve 
toxin preparations through the stimulation of the post-ganglionic 
cholinergic nerve fibre have been elucidated in muscle preparations 
of guinea-pig ileum (Grunfeld & Spiegelstein, 1974).  The results 
of further  in vitro experiments have shown that  G. breve toxin 
preparations depolarize the resting membrane potential by increasing 
sodium permeability in rat phrenic nerve diaphragm preparations 
(Gallagher & Shinnick-Gallagher, 1980; Shinnick-Gallagher, 1980).  
In cats, administered  G. breve toxin preparations intravenously 
(after vagotomy) and intracerebroventricularly, regular 
breathholding and hypertension with tachycardia was observed, 
leading ultimately to respiratory and circulatory failure (Borison 
et al., 1980b). 

    The acute effects of a crystalline preparation of brevetoxin B 
(named T34) have been observed during  in vivo and  in vitro studies, 
summarized in Table 12 (Baden et al., 1981). 

    Crystalline preparations of 2 toxic components from  G. breve, 
named T17 and T34, the latter being identical to brevetoxin B, 
were injected intratracheally into guinea-pigs.  T17, at doses 
ranging from 0.001 to 0.080 mg/kg body weight increased the 
resistance to pulmonary inflation at all doses.  The pulmonary 
response to T17 differed slightly from those to histamine and 
acetylcholine in its longer persistence at peak levels.  The rate 
of onset was, however, equally rapid in all cases.  T17, 
(0.02%) administered at 0.01 mg/kg body weight, caused 
bronchoconstriction approximately equivalent to that caused 
by 0.05 µg acetylcholine/kg.  The studies using T34 (0.20%) 
were not pursued because the concentration necessary to produce 
bronchoconstriction equivalent to that of 0.050 µg atropine/kg 
body weight was 0.05 mg/kg (Baden et al., 1982). 

Table 12.  Comparative toxicity of brevetoxin Ba
Test                LC50 or          EC50b,c        Endpoint
organism            LD50b            (mg/litre)     measured
                    (24 h)                      
Mosquito fish       0.011 mg/litre                  death
 (Gambusia affinis)  (0.005 - 0.023)                  

Mouse (ip)          0.20 mg/kg                      death
                    body weight                     
                    (0.15 - 0.27)

Tissue culture
 KB tumour                           0.26           cell growth (protein
                                     (0.23 - 0.29)  determination)

 B 388 lymphocytic                   0.32           cell growth (cell
       leukaemia                     (0.12 - 0.89)  number)

 L 1210 lymphoid                     0.42           cell growth (cell
        leukaemia                    (0.17 - 1.03)  number)

Sea urchin egg                       8.9            division of
                                     (6.5 - 12.2)   fertilized eggs
a  Adapted from: Baden et al. (1981).
b  The 95% confidence limits are in parenthesis.
c  EC50 = concentration in the median causing 50% inhibition.
4.4.  Effects on Man

    In human beings, consuming shellfish contaminated with  G. breve 
cells, paraesthesia, alternating sensations of hot and cold, 
nausea, vomiting, diarrhoea, and ataxia occur within 3 h (McFarren 
et al., 1965).  Paralysis has not been observed, and the disease 
(NSP) appears to be milder than PSP (Hughes & Merson, 1976).  In 
the USA, in the period 1970-74, 2 outbreaks (a total of 5 cases) of 
NSP were recorded, both associated with the consumption of clams; 
no deaths occurred.  The concentrations of NSP in the clams were in 
the range 30 - 118 MU/100 g (Hughes et al., 1977; Hughes, 1979).  
In USA, shellfish containing any detectable level of NSP per 100 g, 
as determined by the mouse assay, is considered potentially unsafe 
for human consumption (Subcommittee on laboratory methods for the 
examination of shellfish, 1970). 

    An upper respiratory syndrome of NSP has been reported, 
associated with aerosols of  G. breve cells and/or toxins, in 
coastal areas of Florida, USA (Hughes & Merson, 1976).  The rapidly 
reversible syndrome is characterized by conjunctival irritation, 
copius rhinorrhoea, and nonproductive cough. 


    An intoxication characterized by gastrointestinal disturbances, 
often occurring as outbreaks associated with the consumption of 
shellfish, and consequently named diarrhoeic shellfish poisoning 
(DSP), has been reported from several parts of the world, including 
the Far East, Europe, and South America.  The identification of the 
toxin-producing algal organisms, and the characterization of the 
chemical structure of some of the algal toxins present in the 
shellfish involved have been achieved very recently.  Information 
on such aspects as analytical procedures and toxicology is 
therefore limited at present.  However, as many hundreds of DSP 
cases have been reported, it is included in the document for 

5.1.  Sources and Occurrence

     Dinophysis fortii, an armoured marine dinoflagellate, has been 
identified as a producer of DSP in Japan (Yasumoto et al., 1980b), 
whereas  D. acuminata is suspected of being the toxin producer in 
recent outbreaks in the Netherlands, based on epidemiological 
evidence.  DSP has not been detected in cells of  D. acuminata 
because attempts to cultivate the organism isolated from Dutch 
waters have been unsuccessful (Kat, 1983a,b).  Cases in Chile were 
associated with the occurrence of  D. acuta, though detailed 
information is not available (Guzman & Compodonico, 1975).  
Occurrence of one of the DSP toxins, okadaic acid, has been 
confirmed in a benthic dinoflagellate,  Prorocentrum lima (Murakami 
et al., 1982), though involvement of this species in DSP has never 
been known. 

    Species of  Dinophysis are distributed widely but seldom form 
red tides.  It has been noted that in the presence of  D. fortii at 
a low cell density of 200 cells/litre, mussels and scallops become 
toxic enough to affect man.  The infestation period in Japan ranges 
from April to September (Yasumoto et al., 1978). 

5.2.  Chemical Properties

    The presence in shellfish of 9 toxic components has 
been recognized and the chemical structures of 5 components have 
been established (Murata et al., 1982; Yasumoto et al., 1984).  
These toxins are classified into two groups:  okadaic acid and its 
derivatives named dinophysistoxins, and the novel polyether 
lactones named pectenotoxins (Fig. 6).  The chemical structure for 
dinophysistoxin-2 is not yet known because of its limited 
availability, while pectenotoxin-3, -4 and -5 are closely related 
to pectenotoxin-1, in chemical structure. 

5.3.  Analytical Method

    A mouse bioassay, using intraperitoneal injection of toxin 
extracts and a 24-h observation period, is being used as a 
regulatory measure to monitor shellfish toxicity in Japan, and 
shellfish with a DSP toxin level exceeding 50 MU/kg are banned from 

harvesting or sale (Anon, 1981d, personal communication, Yasumoto, 
1983).  In a rat bioassay used in the Netherlands for monitoring 
purpose, the material to be tested is included in the diet and 
observations of diarrhoeal symptoms and reduced feed intake are 
recorded (Kat, 1983a,b). 


5.4.  Effects on Animals - Experimental Studies

    Mice injected with toxic extracts of DSP shellfish 
intraperitoneally show inactivation and general weakness, and die 
within 30 min - 48 h, depending on the dose given.  On the basis of 
intraperitoneal administration, chicks are less sensitive.  
Vomiting was observed by Yasumoto et al. (1980b) in cats fed toxic 
mussels and scallops.  When rats  (Rattus norvegicus) were fed DSP 
toxic shellfish as part of the diet, diarrhoea and reduced feed 
intake were observed (Kat, 1983b). 

5.5.  Effects on Man

    During the period 1976-82, more than 1300 people were diagnosed 
as DSP cases in Japan.  Frequency of signs and symptoms were: 
diarrhoea (92%), nausea (80%), vomiting (79%), abdominal pain 
(53%), and chill (10%).  The time from consumption of shellfish to 
the onset of illness ranged from 30 min to several hours, but 
seldom exceeded 12 h.  About 70% of patients developed symptoms 
within 4 h.  Suffering may last for 3 days but leaves few 
after-effects.  In the Netherlands, more than 30 cases were 

encountered in the DSP outbreak in 1981 (Kat, 1983b).  Cases of 
gastrointestinal disorders have been observed in Chile in 1970 and 
1971, apparently associated with blooms of  Dinophysis sp. (Avaria, 


6.1.  Dermatitis-Inducing Marine Cyanophyte Toxins

6.1.1.  Sources and properties

    The subject of cyanophyte toxins has been reviewed by Moore 
(1981).  Contact with the filamentous cyanophyte  Lyngbya majuscula, 
when swimming in the sea, can result in a type of dermatitis called 
"swimmers itch" or "seaweed dermatitis", as reported from Hawaii 
(Grauer & Arnold, 1961) and Japan (Okinawa) (Hashimoto, 1979).  
Two skin-toxic components have been isolated from  L. majuscula, 
i.e., debromoaplysiatoxin and lyngbyatoxin A.  In crystalline form, 
debromoaplysiatoxin consists of colourless needles and has a 
melting point of 105.5° - 107.0 °C and a relative molecular mass of 
592 (Mynderse et al., 1977) (Fig. 7).  Lyngbyatoxin A is a tan-
coloured gummy solid in crystalline  form, with a relative 
molecular mass of 437 (Cardellina et al., 1979) (Fig. 7). 
Debromoaplysiatoxin has also been isolated from two other species 
within the cyanophyte family Oscillatoriaceae, i.e.,  Oscillatoria 
 nigroviridis and  Schizothrix calcicola (Mynderse et al., 1977). 

6.1.2.  Effects on animals

    The toxicity of debromoaplysiatoxin has been studied on the 
skin of mice and rabbits (Solomon & Stoughton, 1978).  The toxin 
was dissolved in 100% ethanol to make 0.5%, 0.05%, 0.005%, 0.0005%, 
and 0.00005% solutions.  Groups of hairless, female mice (strain 
HRS/J) were used, with 3 animals per group; 10 µl of solution was 
applied to the back of each mouse.  Each solution (10 µl) was 
applied to separate areas of the shaved back of a New Zealand 
white rabbit.  In the mice, there were petechial haemorrhages 
within 1 h of application of 0.5% debromoaplysiatoxin; by 24 h, the 
area was pale and oedematous, and by 5 days there was a firm crust, 
which took 2 - 3 weeks to heal.  A dose-effect relationship was 
observed, and even the smallest dose (0.00005%) induced mild 
oedema. Histologically, the changes over 24 h of application of 
0.5% solution included almost complete destruction of the 
epidermis, oedema, coagulation of collagen, infiltration throughout 
the dermis and deep into the subdermal muscle of polymorphonuclear 
leukocytes and erythrocytes.  The dermal and subdermal blood 
vessels showed mural fibrinoid necrosis with polymorphonuclear 
infiltration and leukocytoclasis.  The histological changes seen in 
mice treated with smaller doses were similar but less notable.  In 
mice treated twice with the 0.005% solution with a 2-week interval, 
the macroscopic reaction was similar to that seen in mice receiving 
only one treatment.  In the skin of the rabbit, there was also a 
dose-effect relationship, and the lowest dose induced a reaction 
consisting of mild erythema.  Histologically, the changes were 
similar to those seen in the mice.  Thus, 10 µl of the 0.00005% 
solution (5 pg debromoaplysiatoxin) induced skin inflammation in 2 
animal species, and the authors concluded that no other agent is 
known that will induce inflammation when applied in such a small 
amount.  No details of the skin testing of lyngbyatoxin A appear to 
have been published. 


6.1.3.   Effects on man

    Cases of acute dermatitis after contact with  L. majuscula 
have been reported from Hawaii (Grauer & Arnold, 1961) and Japan 
(Hashimoto, 1979).  In Hawaii, more than 125 cases were received 
for treatment and hundreds of mild cases were suspected in the 
period July-August 1958.  The clinical picture is characterized by 
the gradual onset of itching and burning within a few min to a few 
h after swimming in the sea, where fragments of the alga are 
suspended.  Visible dermatitis and redness develops after 3 - 8 h, 
followed by blisters and deep desquamation.  The eruption affects 
the region of the body not covered by the swimming trunks.  
Histologically, the lesions were described as acute, vesicular 
dermatitis, characterized by superficial desquamation, oedema of 
the epidermis with vesicles within the epidermis.  Occasionally, 
the vesicles contained polymorphonuclear leukocytes and 
erythrocytes, and the deepest portion of the epidermis was 
infiltrated by polymorphonuclear leukocytes (Grauer & Arnold, 

    These lesions have been reproduced by applying solutions 
of debromoaplysiatoxin on the skin (Solomon & Stoughton, 1978).  
The compound was dissolved in 100% ethanol to obtain 0.5%, 0.05%, 
0.005%, 0.0005%, and 0.00005% solutions, which were applied on 
the skin of the two investigators.  The lowest concentration 

with which dermatitis developed in 6 - 12 h was the 0.05% 
solution.  Histological studies confirmed the similarity between 
the dermatitis induced experimentally and that associated with 
 L. majuscula, mentioned above. 

6.2.  Freshwater Cyanophyte Toxins

    Although reports on disease induced in farm animals by toxic 
cyanophytes in drinking-water are known from the last century, the 
elucidation of the chemical nature of these cyanophyte toxins has 
been progressing very slowly.  The first full documentation of the 
chemical structure of a freshwater cyanophyte toxin (anatoxin-a) 
was published in 1977 (Devlin et al., 1977), and a chemical method 
of analysis for this toxin was subsequently reported (Astrachan & 
Archer, 1981).  There are indications of intoxications induced in 
animals, as field cases in farm animals, and as experimentally-
induced disease in a variety of animal species, which can be 
related to certain species of cyanophytes living in freshwater.  
There are adverse effects in human beings, which may be related 
to drinking water containing high concentrations of cyanophytes. 
For these reasons, freshwater cyanophyte toxins have been included 
in this monograph.  It can be anticipated that more information on 
the chemistry and occurrence of the cyanophyte toxins will be made 
available in the near future, as much research is in progress this 
field (Carmichael, 1981). 

    Most of the available information on toxic freshwater 
cyanophytes is concerned with the following 3 species:   Microcystis 
 aeruginosa, Anabaena flos-aquae, and  Aphanizomenon flos-aquae.  
 M. aeruginosa is a coccoid blue-green alga, and the onset of 
 Microcystis blooms in lakes is correlated with temperature, with 
blooms occurring when the water temperature reaches 19 - 20 °C, an 
observation supported by experimental data (Krüger & Eloff, 1978).  
 Anabaena flos-aquae and  Aphanizomenon flos-aquae are filamentous 
blue-green algae. 

    The cells of blue-green algae are embedded in mucilage 
containing bacteria, mainly gram-negative, which may play a role as 
producers of vitamins and metal chelators.  Zoogloea bacteria have 
been found closely associated with  Anabaena flos-aquae preceeding 
the peak of a bloom (Caldwell & Caldwell, 1978), whereas members of 
the  Enterobacteriaceae have been shown to depress toxin production 
by  Anabaena flos-aquae (Carmichael & Gorham, 1977).  All 3 algal 
species occur ubiquitously (Kondrateva & Kovalenko, 1975) and toxic 
blooms of these algae have been reported from many countries. 

    The topic has recently been reviewed by Goryunova & Demina 
(1974), Kirpenko et al. (1977), Gorham & Carmichael (1980), and 
Carmichael (1981). 

6.2.1.  Sources, properties, analytical methods, and exposure

     Microcystis aeruginosa toxin

    Several toxic preparations have been isolated from 
 M. aeruginosa, which contain peptides, carbohydrates, and other 
compounds, and have relative molecular masses ranging from 1300 to 
19 400.  Two compounds of low relative molecular mass have recently 
been isolated, one a peptide with hepatotoxic properties, the other 
causing respiratory arrest in mice (Carmichael, 1981). 

     Anabaena flos-aquae toxin

    A toxin, anatoxin-a, which when administered to mice orally or 
intraperitoneally caused acute effects including signs of paralysis 
and death, was isolated from  Anabaena flos-aquae and chemically 
characterized as an alkaloid (Fig. 8), with a relative molecular 
mass of 165 (Devlin et al., 1977).  A method of analysis for 
anatoxin-a has been developed, involving high performance liquid 
chromatography, with 90% recovery in the concentration range 1 - 
500 mg/kg, and a limit of detection of 0.1 mg/kg (Astrachan & 
Archer, 1981).  Furthermore, three toxic preparations, namely 
anatoxin b, c, and d, have also been isolated (Carmichael & Gorham, 
1977).  Anatoxin-a acts as a potent nicotinic agonist paralysing 
peripheral muscles by a depolarizing neuromuscular blockade 
(Carmichael, 198l). 


     Aphanizomenon flos-aquae toxins

    Purification of a toxic factor from  Aphanizomenon flos-aquae, 
possessing electrophysiological properties similar to those of 
saxitoxin, resulted in several closely interrelated fractions, 
which were similar to saxitoxin on the basis of chromatography and 
infrared spectroscopy (Jackim & Gentile, 1968).  Recently, two 
toxic compounds, identical to saxitoxin and neosaxitoxin on the 
basis of paper electrophoretic and thin layer chromatographic 
properties, were isolated from strains of  A. flos-aquae (Ikawa et 
al., 1982). 

    No studies are available on the possibility of the passing of 
freshwater cyanophyte toxins into the human food chain or of their 
bioconcentration by predators similar to dinoflagellate toxins.  
The possibility of human exposure to cyanophyte toxins through 
recreational and municipal water supplies has been considered in 
association with cyanophyte blooms (Carmichael, 1981; section 

6.2.3), however, no reports are available on the chemical 
identificaton and quantification of cyanophyte toxins in 
recreational and municipal water supplies. 

6.2.2.  Effects on animals

     Microcystis aeruginosa cells and toxins

    Field cases of  Microcystis intoxication in farm animals, 
particularly in cattle, have been reported, as a result of drinking 
water from lakes containing blooms of  M. aeruginosa (Hammer, 1968; 
Skulberg, 1979).  The cases were acute, characterized by 
haemorrhages, photosensitization, and liver damage including 
necrosis of hepatocytes and moderate proliferation of bile duct 
epithelia.  Liver damage characterized by panlobular hepatocytic 
necrosis, superimposed haemorrhage, connective tissue 
proliferation, and pleomorphic hepatocytes developed in laboratory 
studies on vervet monkeys given lyophilised  M. aeruginosa cells 
orally for 6 - 7 months (Tustin et al., 1973).  Gonadotoxic and 
embryotoxic effects, as well as mutagenic effects in the bone 
marrow, were reported in rats orally administered an extract of 
 M. aeruginosa collected from a bloom (Kirpenko et al., 1981). 

     Anabaena flos-aquae cells and toxins

    Field cases of sudden death in cattle have been reported, 
associated with drinking water from lakes containing blooms of 
 Anabaena flos-aquae (Hammer, 1968; Carmichael et al., 1977).  Death 
occurred within a few hours of ingestion of a lethal bolus, and the 
signs observed were characteristic of respiratory failure.  Toxic 
 Anabaena flos-aquae cells administered orally to calves, rats, 
ducks, and goldfish caused death as a result of respiratory arrest 
(Carmichael et al., 1975).  Using a toxic extract from the cells, 
it was concluded that the main effect was the production of a 
sustained post-synaptic depolarizing neuromuscular blockade.  No 
adverse effects on food consumption, growth, blood cells, serum 
enzymes, hepatic mixed function oxidase and morphological changes, 
were observed, when anatoxin-a was administered to female Sprague-
Dawley rats (20 animals per group) in the drinking-water at 5.1 and 
0.5 mg/litre for 7 weeks, or when anatoxin-a was injected 
intraperitoneally into 18 female rats (0.016 mg/rat) for 21 days.  
Female golden hamsters (5 - 6 animals per injected group, 7 - 9 
animals per control group) were injected intraperitoneally with 
anatoxin-a (0.125 and 0.2 mg/kg body weight) 3 times per day on day 
8 - 11, or on day 8 - 14, or on day 12 - 14 of the gestation 
period.  Decreased fetal weights were observed in most groups 
compared with the controls, and hydrocephaly was observed in one 
litter from the group administered 0.125 mg anatoxin-a/kg body 
weight on day 12 - 14 (Astrachan et al., 1980). 

     Aphanizomenon flos-aquae cells and toxins

    The following lethal doses were observed after peritoneal 
injection of a toxic extract from  Aphanizomenon flos-aquae cells: 
in killifish  (Fundulus heteroclitus), 0.5 mg/kg body weight; in 

sheepshead minnows  (Cyprinodon variegatus), 0.5 mg/kg; and in mice, 
8 mg/kg (Gentile & Maloney, 1969). Copepods, ostracods, and 
cladocerans were unaffected by toxin concentrations of 2 g/litre in 
the water environment. 

6.2.3.  Episodes of adverse effects reported in association
with human exposure to toxic cyanophytes

    During blooms of cyanophytes  (Microcystis sp.,  Anabaena sp. and 
 Aphanizomenon flos-aquae) in Canadian lakes in June-July 1959, many 
cases of acute death in domestic animals (dogs, cattle, horses) 
were encountered associated with the drinking of lake water 
(Dillenberg & Dehnel, 1960).  In addition, 12 people became ill 
after swimming in the lakes, with headache, nausea, and 
gastrointestinal upsets.  In the vomitus and stools of one of the 
patients, cyanophyte cells  (Microcystis sp.,  Anabaena circinalis) 
were identified; no other microbial causative agents were found in 
this patient. 

    In August 1975, a water-borne outbreak of gastrointestinal 
disease occurred in Sewickley in Pennsylvania, USA, affecting 62% 
of the population (size 8000).  Bacterial agents could be excluded, 
and it was assumed that blue-green algae present in the uncovered 
drinking-water reservoir were the cause, although a viral etiology 
could not be completely excluded.  The water contained more than 
100 000 blue-green algae cells per ml, dominated by  Schizothrix 
 calcicola and  Lyngbya spp; no test for algal toxicity was conducted 
(Lippy & Erb, 1976).  Elevation of gamma-glutamyl transpeptidase and 
of alanine aminotransferase, indicating toxic liver injury, as 
measured in a community in Australia, was found to be associated 
with blooms of toxic  M. aeruginosa in the drinking-water reservoir.  
No such enzyme changes were measured in an adjacent control 
population (Falconer et al., 1983). 


    In view of the character of the problem, the health risk 
evaluation is presented together with the summary at the beginning 
of the document. 


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    See Also:
       Toxicological Abbreviations