Biotoxins, aquatic (marine and freshwater) (EHC 37, 1984)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 37

AQUATIC (MARINE AND FRESHWATER) BIOTOXINS

This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
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

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CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR AQUATIC (MARINE AND FRESHWATER)
BIOTOXINS

SUMMARY WITH EVALUATION OF THE HEALTH RISKS OF EXPOSURE
TO AQUATIC BIOTOXINS AND RECOMMENDATIONS FOR FURTHER ACTIVITIES

INTRODUCTION: AQUATIC BIOTOXINS AND HUMAN HEALTH

1. PARALYTIC SHELLFISH POISONS

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

2. CIGUATERA TOXINS

2.1. Properties and analytical methods
2.1.1. Chemical properties
2.1.2. Methods of analysis for foodstuffs
2.1.2.1 Biological methods
2.1.2.2 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. TETRODOTOXIN (PUFFERFISH POISON)

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. NEUROTOXIC SHELLFISH POISONS

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. DIARRHOEIC SHELLFISH POISON

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. CYANOPHYTE TOXINS

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

7. EVALUATION OF HEALTH RISKS OF EXPOSURE TO AQUATIC BIOTOXINS

REFERENCES

WHO TASK GROUP ON AQUATIC (MARINE AND FRESHWATER) BIOTOXINS

Members

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,
Czechoslovakia

Secretariat

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

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

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).

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* * *

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 -
985850).

ENVIRONMENTAL HEALTH CRITERIA FOR AQUATIC (MARINE AND FRESHWATER)
BIOTOXINS

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.

SUMMARY WITH EVALUATION OF THE HEALTH RISKS OF EXPOSURE
TO AQUATIC BIOTOXINS AND RECOMMENDATIONS FOR FURTHER ACTIVITIES

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.

Introduction

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
purpose.

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.

Tetrodotoxin

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
saxitoxin.

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
animals.

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
failure.

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
episodes.

Recommendations

(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.

INTRODUCTION: AQUATIC BIOTOXINS AND HUMAN HEALTH

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
attention.

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
drinking-water.

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.

1. PARALYTIC SHELLFISH POISONS

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, pK_a 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
conditions.

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
||
O

2) neosaxitoxin -OH -H -C-NH₂
||
O

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

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

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

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

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
saxitoxin

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.

1.1.2.1. 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,
1980).

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.

1.1.2.2. 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
illumination.

Table 1. Relative toxicity of dinoflagellates of the genus
Gonyaulax^a
-------------------------------------------------------------------
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 1.2.2.1, 1.2.2.2).

1.2.1.1. 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,
1979).

1.2.2. Occurrence in seafood

1.2.2.1. 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 conditions^a
-------------------------------------------------------------------
Shellfish species Days after PSP concentration
feeding (µg/kg)^b
-------------------------------------------------------------------
Mya arenaria 0 ND^c
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.

1.2.2.2. 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).

1.2.2.3. 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 blooms^a
-------------------------------------------------
Number Mean Mean Mean mg/kg content^b
of fish length weight
sampled (cm) (g) gut muscle
-------------------------------------------------
17 21.6 110 1100 ND^c
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.

1.2.2.4. 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 1.5.1.2). 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

1.5.1.1. 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 1.2.2.2. 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).

1.5.1.2. 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, 1968-1976^a
---------------------------
Year PSP level^b
(µg/kg)
---------------------------
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

1.5.2.1. Acute toxicity

As mentioned in section 1.1.2.1, 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
parenterally.

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 administration^a
---------------------------------------------------------
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 ages^a
----------------------------------------------------
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 animals^a
-----------------------------------
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 1.1.2.1) (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 mice^a
-----------------------------------------
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.

1.5.2.2. 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):

Mild

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

Moderate

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;

Severe

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,
1983b,c).

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
(1978)

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
(1981)

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 NS^b local NS NS Anon (1972)
(Maine, New
Hampshire,
Massachusetts)

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
mentioned
above)
---------------------------------------------------------------------------------------------------------
^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.
2. CIGUATERA TOXINS

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 2.1.2.2, has not been applied under
practical conditions (Laigret et al., 1981; Parc et al., 1981).

2.1.2.1. 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.

2.1.2.2. 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
2.1.2.1. 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)
Spanish
mackerel

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

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
snapper
carrang
emperor
barracuda
(mainly)

Jamaica 250 0 grouper Local Bagnis (1978)
barracuda

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

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

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.
(1983)

US Virgin Islands 33 0 carrang Local Morris et al.
snapper (1982a,b)
------------------------------------------------------------------------------------------
3. TETRODOTOXIN (PUFFERFISH POISON)

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
extracts.

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 1.5.2.2.

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
animals^a
-------------------------------------------------------------------
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.

4. NEUROTOXIC SHELLFISH POISONS

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
1.1.2.1), 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
birds.

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 1.4.2.1). 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 B^a
-------------------------------------------------------------------------
Test LC₅₀ or EC₅₀^b,c Endpoint
organism LD₅₀^b (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.

5. DIARRHOEIC SHELLFISH POISON

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
completeness.

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,
1979).

6. CYANOPHYTE TOXINS

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,
1961).

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).

7. EVALUATION OF HEALTH RISKS OF EXPOSURE TO AQUATIC BIOTOXINS

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

REFERENCES

ABBOTT, B.C., SIGER, A., & SPIEGELSTEIN, M. (1975) Toxins
from the blooms of Gymnodinium breve. In: LoCicero, V.R., ed.
Proceedings of the First International Conference on Toxic
Dinoflagellate Blooms, Wakefield, Massachusetts, Massachusetts
Science and Technology Foundation, pp. 355-365.

ACRES, J. & GRAY, J. (1978) Paralytic shellfish poisoning.
Can. Med. Assoc. J., 119: 1195-1197.

ADACHI, R. & FUKUYO, Y. (1979) The thecal structure of a
marine toxic dinoflagellate Gambierdiscus toxicus gen. et sp.
nov. collected in a ciguatera-endemic area. Bull. Soc. Jpn.
Sci. Fish., 45: 67-71.

ADAMS, W.N. & MIESCIER, J.J. (1980) Commentary on AOAC
method for paralytic shellfish poisoning. J. Assoc. Off.
Anal. Chem., 63: 1336-1343.

ADAMS, J., SEATON, D.D., BUCHANAN, J.B., & LONGBOTTOM, M.R.
(1968) Biological observations associated with the toxic
phytoplankton bloom off the east coast. Nature (Lond.), 220:
24-25.

ALAM, M., OSHIMA, Y., & SHIMIZU, Y. (l982) About
gonyautoxins-I, II, III, and IV. Tetrahedron Lett., 23:
321-322.

ALCALA, A.C. (1983) Recent cases of crab, cone shell, and
fish intoxication on Southern Negros Islands, Phillipines.
Toxicon (Suppl. 3): 1.

ANDERSON, D.M. & CORBETT, M.D. (1979) Workshop: The role of
chelators and trace metals in toxic blooms. In: Taylor, D.L.
& Seliger, H.H., ed. Toxic dinoflagellate blooms, New York,
Elsevier, pp. 463-467.

ANDERSON, D.M. & MOREL, P.M.M. (1978) Copper sensitivity of
Gonyaulax tamarensis. Limnol. Oceanogr., 23: 283-295.

ANON (1957) Conference on shellfish toxicology, Washington
DC, US Department of Health, Eduation and Welfare, pp. 1-126.

ANON (1965) National shellfish sanitation program, manual of
operations, part 1: sanitation of shellfish growing areas,
Washington, DC, US Department of Health, Education and
Welfare, pp. 9, 19-20.

ANON (1972) Paralytic shellfish poisoning associated with
red tide - New England. Morb. Mortal. wkly Rep., 21: 325-326.

ANON (1973) The red tide - a public health emergency. New
Engl. J. Med., 288(21): 1126-1127.

ANON (1979) Paralytic shellfish poisoning and the mussel
quarantine order. Calif. Morb., 17(May 4).

ANON (1980) Ciguatera fish poisoning - Maryland. Morb.
Mortal. wkly Rep., 29: 610-611.

ANON (1981a) Ciguatera fish poisoning. St. Croix, Virgin
Island and the United States. Morb. Mortal. wkly Rep., 30:
138-139.

ANON (1981b) Annual Summary l978, Centers for Disease
Control, Foodborne Disease Surveillance, Atlanta, Georgia,
Bureau of Epidemiology, Bacterial Diseases Division, Enteric
Disease Branch, 53 pp.

ANON (1981c) Annual Summary l979, Centers for Disease
Control, Foodborne Disease Surveillance, Atlanta, Georgia,
Center for Infectious Diseases, Bacterial Diseases Division,
Enteric Diseases Branch, 40 pp.

ANON (1981d) [Testing methods for diarrhoeic shellfish
toxins,] Tokyo, Japan, Environmental Health Bureau, Sanitation
of Milk and Meat Division, Ministry of Health and Welfare, pp.
1-11 (in Japanese).

ANON (1982) Paralytic shellfish poisoning, Quebec, l98l.
Can. Dis. wkly Rep., 8-l8: 89-90.

ANON (l983a) Paralytic shellfish poisoning in Prachuap Khiri
Khan (Outbreak investigations, no. 6). Wkly. epidemiol. Rec.,
58: 349.

ANON (1983b) Annual Summary l980, Centers for Disease
Control, Foodborne Disease Surveillance, Atlanta, Georgia,
Center for Infectious Diseases, Bacterial Diseases Division,
Enteric Diseases Branch, 32 pp.

ANON (1983c) Annual Summary l98l, Centers for Disease
Control, Foodborne Disease Outbreaks, Atlanta, Georgia,
Bacterial Disease Division, Enteric Diseases Branch, 41 pp.

ANON (in press) Ciguatera poisoning - First confirmed report
in Canada. Can. Dis. wkly. Rep.

ARMSTRONG, I.H., COULSON, J.C., HAWKEY, P., & HUDSON, M.M.
(1978) Further mass seabird deaths from paralytic shellfish
poisoning. Br. Birds, 71: 58-68.

ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS (1980) Paralytic
shellfish poison biological method. In: Official methods of
analysis, 13th ed., Washington DC, AOAC, pp. 298-299.

ASTRACHAN, W.B. & ARCHER, B.G. (1981) Simplified monitoring
of anatoxin-a by reverse phase high performance liquid
chromatography and the subacute effects of anatoxin-a in
rats. In: Carmichael, W.W., ed. The water environment: Algal
toxins and health, New York, London, Plenum Press, pp. 437-446.

ASTRACHAN, W.B., ARCHER, B.G., & HILBELINK, D.R. (1980)
Evaluation of the subacute toxicity and teratogenicity of
anatoxin-a. Toxicon, 18: 684-688.

ATWELL, R.B. & STUTCHBURY, G.B. (1978) Toadfish poisoning
(tetrodotoxin) in the cat. Aust. vet. J., 54: 308-310.

AVARIA, S.P. (l979) Red tides off the coast of Chile. In:
Taylor, D.L. & Seliger, H.H., ed. Toxic dinoflagellate blooms,
Amsterdam, Elsevier, pp. 161-164.

AYRES, P.A. & CULLUM, M. (1978) Paralytic shellfish
poisoning. An account of investigations into mussel toxicity
in England, 1968-77, London, Ministry of Agriculture,
Fisheries and Food, p. 23 (Fisheries Research Technical Report
No. 4).

BADEN, D.G., MENDE, T.J., LICHTER, W., & WELLHAM, L. (1981)
Crystallization and toxicology of T34: A major toxin from
Florida's red tide organism (Ptychodiscus brevis). Toxicon,
19: 455-462.

BADEN, D.G., MENDE, T.J., BIKHAZI, G., & LEUNG, I. (1982)
Broncho constriction caused by Florida red tide toxins.
Toxicon, 20: 929-932.

BAGNIS, R. (1967) Les empoisonnements par le poisson en
Polynésie Française. Etude clinique et épidémiologique.
Rev. Hyg. Méd. soc., l5(7): 619-646.

BAGNIS, R. (1968) Clinical aspects of ciguatera (fish
poisoning) in French Polynesia. Hawaï med. J., 28(1): 25-28.

BAGNIS, R. (1969) Naissance et développement d'une flambée
de ciguatera dans un atoll des Tuamotu. Rev. Corps Santé,
10(6): 783-795.

BAGNIS, R. (1970) Recherches sur l'origine, la nature et
l'action physiologique des toxines ciguatérique en certains
biotopes coralliens de la Polynésie Française, Université de
Bordeaux I, Thèse d'université, 209 pp.

BAGNIS, R. (1974) Evolution d'une flambée de ciguatera aux
Iles Marquises. Méd. Armées, 2(2): 115-122.

BAGNIS, R. (1977) Modalités évolutives et biogénèse de la
ciguatera en Polynésie Française, Université de Bordeaux,
Thèse doctorat des Sciences, 128 pp.

BAGNIS, R. (1978) L'ichtyosarcotoxisme dans les Caraïbes.
Rapport de mission aux Antilles et à l'Ile de Paques (No.
941/IRM/K.10).

BAGNIS, R. (1980) Agressions naturelles sur les édifices
coralliens des Iles Marquises et ciguatera. Méd. océanienne,
12: 42-50.

BAGNIS, R. (1981a) L'ichtyosarcotoxisme de type ciguatera:
processus biologiques connus et perspectives au seuil des
années 80. Ann. Inst. Océanogr., Paris, 57(1): 5-24.

BAGNIS, R. (1981b) L'ichtyosarcotoxisme de type ciguatera:
phénomène complexe de biologie marine et humaine. Oceanol.
Acta, 4(3): 375-387.

BAGNIS, R. (1981c) Etude morphologique, biologique,
toxicologique et écologique de l'agent causal princeps de la
ciguatera, le péridinien "Gambierdiscus toxicus", Université
de Bordeaux II, Thèse de Doctorat d'Etat en Biologie Humaine,
180 pp.

BAGNIS, R. (1984a) Human features of ciguatera fish
poisoning. In: Proceedings of the Symposium on Seafood Toxins
in Tropical Regions, 26 September 1983, Kagoshima, Japan,
Kagoshima University Research Centre for the South Pacific,
16 pp.

BAGNIS, R. (1984b) Ciguatera outbreaks induced by
disturbances on coral reef ecosystems. In: Proceedings of the
Symposium on Seafood Toxins in Tropical Regions, 26 September
1983, Kagoshima, Japan, Kagoshima University Research Centre
for the South Pacific, 12 pp.

BAGNIS, R. & FEVAI, G. (197l) La ciguatera féline
expérimentale à Tahiti. Rev. Méd. vét., l22(6): 629-238.

BAGNIS, R. & LETOURNEUX, M. (1974) Une ciguatoxine commune à
divers poissons de récifs coralliens. Bull. Soc. Pathol. Exot.
Fil., 67(2): 209-217.

BAGNIS, R., BENNETT, J., NANAI, F., & THEVENIN, S. (1973)
Pollution marine et ciguatera dans l'atoll de Manihi. In:
Seminar of the South Pacific Commission on the Pollution of
the Lagoons, Guam, WP, l2 pp.

BAGNIS, R., CHANTEAU, S., & YASUMOTO, T. (1977) Découverte
d'un agent étiologique vraisemblable de la ciguatera.
C.R. Acad. Sci. (Paris), 28(l): l05-l08.

BAGNIS, R., KUBERSKI, T., & LAUGIER, S. (1979a) Clinical
observations on 3,009 cases of ciguatera (fish poisoning) in
the South Pacific. Am. J. trop. Med. Hyg., 28(6): 1067-1073.

BAGNIS, R., HURTEL, J.M., FUKUYO, Y., INOUE, A., & YASUMOTO,
T. (1979b) Quelques aspects morphologiques et biologiques du
dinoflagellé responsable probable de la ciguatera. C.R. Acad.
Sci. (Paris), 289(Série D): 639-642.

BAGNIS, R., HURTEL, J.M., CHANTEAU, S., CHUNGUE, E., INOUE,
A., & YASUMOTO, T. (1979c) Le dinoflagellé Gambierdiscus
toxicus, Adachi et Fukuyo, agent causal probable de la
ciguatera. C.R. Acad. Sci. (Paris), 289(Série D): 67l-674.

BAGNIS, R., BRONSTEIN, J.A., JOUFFE, G., FORESTIER, R.,
MEUNIER, J.L., LEJEAN, J., BRULEFER, D., PARC, F., & TETARIA,
C. (1979d) Complication neurologique de la ciguatera. Bull.
Soc. Pathol. Exot. Fil., 70(1): 89-93.

BAGNIS, R., CHANTEAU, S., CHUNGUE, E., HURTEL, J.H., YASUMOTO,
T., & INOUE, A. (1980) Origins of ciguatera fish poisoning:
a new dinoflagellate, Gambierdiscus toxicus, Adachi et Fukuyo,
definitely involved as a causal agent. Toxicon, 18(2):
199-208.

BANNER, A.H. (1975) Ciguatera: A disease from coral reef
fish. In: Jones, O.W. & Endean, R., ed. Biology and geology
of coral reefs, New York, Academic Press, pp. 177-213.

BANNER, A.H., SASAKI, S., HELFRICH, P., ALENDER, C.B., &
SCHEUER, P.J. (1961) Bioassay of ciguatera toxin. Nature
(Lond.), 189: 229-230.

BATES, H.A. & RAPOPORT, H. (1975) A chemical assay for
saxitoxin, the paralytic shellfish poison. J. agric. food
Chem., 23: 237-230.

BATES, H.A., KOSTRIKEN, R., & RAPOPORT, H. (1978) A chemical
assay for saxitoxin. Improvements and modifications. J. agric.
food Chem., 26: 252-254.

BAYLET, R., BECCARIA, C., NIAUSSAT, P.M., BOYER, F., &
GUILLAUD, M. (1978) Ichtyosarcotoxisme par ciguatoxine en
France. Pathol. Biol., 26: 95-97.

BERGMANN, J.S. (1982) Gambierdiscus toxicus and ciguatera in
South eastern Florida. In: Proceedings, V International IUPAC
Symposium on Mycotoxins and Phycotoxins, Vienna, l982, pp.
208-211.

BERGMANN, J.S. & ALAM, M. (1981) On the toxicity of the
ciguatera producing dinoflagellate, Gambierdiscus toxicus
Adachi and Fukuyo isolated from the Florida Keys. J. environ.
Sci. Health, A16: 493-500.

BHAT, R.V. (1981) A report on an outbreak of mussel
poisoning in coastal Tamil Nadu, India, Hyderabad, National
Institute of Nutrition, Indian Council of Medical Research,
pp. 1-9.

BLANKENSHIP, J.E. (1976) Tetrodotoxin: From poison to
powerful tool. Perspect. Biol. Med., 19: 509-526.

BORDNER, J., THEISSEN, W.E., BATES, H.A., & RAPOPORT, H.
(1975) The structure of a crystalline derivative of
saxitoxin. The structure of saxitoxin. J. Am. Chem. Soc., 97:
6008-6012.

BORISON, H.L., CULP, W.J., GONSALES, S.F., & MCCARTHY, L.E.
(1980a) Central respiratory and circulatory depression caused
by intravascular saxitoxin. Br. J. Pharmacol., 68: 301-309.

BORISON, H.L., ELLIS, S., & MACCARTHY, L.E. (1980b) Central
respiratory and circulatory effects of Gymnodinium breve toxin
in anaesthetized cats. Br. J. Pharmacol., 70: 249-256.

BOYER, G.L., SCHANTZ, E.J., & SCHNOES, H.K. (1978)
Characterization of 11-hydroxysaxitoxin sulphate, a major
toxin in scallops exposed to blooms of the poisonous
dinoflagellate Gonyaulax tamarensis. J. Chem. Soc. Chem.
Comm., pp. 889-890.

BUCKLEY, L.J., OSHIMA, Y., & SHIMIZU, Y. (1978) Construction
of a paralytic shellfish toxin analyzer and its application.
Anal. Biochem., 85: 157-164.

CALDWELL, D.E. & CALDWELL, S.J. (1978) A Zoogloea sp.
associated with blooms of Anabaena flos-aquae. Can. J.
Miocrobiol., 24: 922-931.

CARDELLINA, J.H., MARNER, F.J., & MOORE, R.E. (1979) Seaweed
dermatitis: Structure of lyngbyatoxin A. Science, 204: 193-195.

CARMICHAEL, W.W. (1981) Freshwater blue-green algae
(Cyanobacteria) toxins. A review. In: Carmichael, W.W., ed.
The water environment: Algal toxin and health, New York,
London, Plenum Press, pp. 1-14.

CARMICHAEL, W.W. & GORHAM, P.R. (1977) Factors influencing
the toxicity and animal susceptibility of Anabaena flos-aquae
(Cynaophyta) blooms. J. Phycol., 13: 97-101.

CARMICHAEL, W.W., BIGGS, D.F., & GORHAM, P.R. (1975)
Toxicology and pharmacological action of Anabaena flos-aquae
toxin. Science, 187: 542-544.

CARMICHAEL, W.W., GORHAM, P.R., & BIGGS, D.F. (1977) Two
laboratory case studies on the oral toxicity to calves of the
freshwater cyanophyte (blue-green alga) Anabaena flos-aquae
NRC-44-1. Can. vet. J., 18: 71-75.

CHANTEAU, S. (1978) Rôle d'un dinoflagellé benthique dans la
biogénèse de la ciguatera, Université de Clermont-Ferrand II,
Thèse de Spécialité, 63 pp.

CHANTEAU, S., BAGNIS, R., & YASUMOTO, T. (1976) Purification
de la ciguatoxine de la loche Epinephelus microdon
(Bleecker). Biochimie., 58: 1149-1151.

CHOU, H.N. & SHIMIZU, Y. (1982) A new polyether toxin from
Gymnodinium breve Davis. Tetrahedron Lett., 52: 5521-5524.

CHUNGUE, E. & BAGNIS, R. (1976) Distribution anatomique de
la toxicité du poisson perroquet de l'espèce Scarus gibbus.
Rev. int. Océanogr. méd., 35-36: 99-103.

CHUNGUE, E., BAGNIS, R., FUSETANI, N., & HASHIMOTO, Y.
(1976) Isolation of two toxins from a parrotfish Scarus
gibbus. Toxicon, 15(1): 89-93.

CHUNGUE, E., BAGNIS, R., & YASUMOTO, T. (1977) Le complexe
toxique des poissons perroquets. Biochimie., 59(8-9): 739-741.

CHUNGUE, E., BAGNIS, R., & PARC, F. (1984) The use of
mosquitoes (Aedes aegypti) to detect ciguatoxin in surgeon
fishes (Ctenochaetus striates). Toxicon, 22(1): 161-164.

COULSON, J.D., POTTS, G.R., DEANS, I.R., & FRASER, S.M.
(1968) Mortality of shags and other sea birds caused by
paralytic shellfish poison. Nature (Lond.), 220: 23-24.

DALE, B. (1977) Cysts of the toxic red-tide dinoflagellate,
Gonyaulax exavata (Braarud) Balech from Oslofjorden, Norway.
Sarsia, 63: 29-34.

DALE, B. & YENTSCH, C.M. (1978) Red tide and paralytic
shellfish poisoning. Oceanus, 21: 41-49.

DALE, B., YENTSCH, C.M., & HURST, J.W. (1978) Toxicity in
resting cysts of the red-type dinoflagellate Gonyaulax
excavata from deeper water coastal sediments. Science, 201:
1223-1225.

DEVLIN, J.P., EDWARDS, O.E., GORHAM, P.R., HUNTER, N.R, PIKE,
R.K., & STAVRIC, B. (1977) Anatoxin-a, a toxic alkaloid from
Anabaena flos-aquae NRC-44h. Can. J. Chem., 55: 1367-1371.

DILLENBERG, H.O. & DEHNEL, M.K. (1960) Toxic waterbloom in
Saskatchewan, 1959. Can. Med. Assoc. J., 83: 1151-1154.

DODGE, J. D. (1982) Marine dinoflagellates of the British
Isles, London, Her Majesty's Stationery Office, pp. 1-303.

ENGLEBERG, N. C., MORRIS, J. G., LEWIS, J., MCMILLAN, J. P.,
POLLARD, R. A., & BLAKE, P. A. (1983) Ciguatera Fish
Poisoning: A major common-source outbreak in the US Virgin
Islands. Ann. intern. Med., 98: 336-337.

FALCONER, I.R., BERESFORD, A.M., & RUNNEGAR, M.T.C. (1983)
Evidence of liver damage by toxin from a bloom of the
blue-green algae, Microcystis aeruginoza. Med. J. Aust., 1:
511-514.

FORRESTER, D.J., GASKIN, J.M., WHITE, F.H., THOMPSON, N.P.,
QUICK, J.A., HENDERSON, G.E., & WOODARD, J.E. (1977) An
epizootic of waterfowl associated with a red tide episode in
Florida. J. wildl. Dis., 13: 160-167.

FOXALL, T.L., SHOPTAUGH, N.H., IKAWA, M., & SASNER, J.
(1979) Secondary intoxication with PSP in Cancer irrorates.
In: Taylor, D.L. & Seliger, H.H., ed. Toxic dinoflagellate
blooms, New York, Elsevier, pp. 413-418.

GALLAGHER, J.P. & SHINNICK-GALLAGHER, P. (1980) Effect of
Gymnodinium breve toxin in the rat phrenic nerve diaphragm
preparation. Br. J. Pharmacol., 69: 367-372.

GENENAH, A.A. & SHIMIZU, Y. (1981) Specific toxicity of
paralytic shellfish poisons. J. agric. food Chem., 29:
1289-1291.

GERSHEY, R.M., NEVE, R.A., MUSGRAVE, D.L., & REICHARDT, P.B.
(1977) A colorimetric method for determination of saxitoxin.
J. Fish. Res. Board Can., 34: 559-563.

GILFILLAN, E.S. & HANSON, S.A. (1975) Effects of paralytic
shellfish poisoning toxin on the behaviour and physiology of
marine invertebrates. In: LoCicero, V.R., ed. Proceedings of
the First International Conference on Toxic Dinoflagellate
Blooms, Wakefield, Massachusetts, Science and Technology
Foundation, pp. 367-375.

GOLIK, J., JAMES, J. C., NAKANISHI, K., & LIN, Y. Y. (1982)
The structure of brevetoxin C. Tetrahedron Lett., 23:
2535-2538.

GORHAM, P.R. & CARMICHAEL, W.W. (1980) Phycotoxins from
blue-green algae. Pure appl. Chem., 52: 165-174.

GORYUNOVA, S.B. & DEMINA, N.S. (1974) [Algae-producers of
toxic substances,] Moscow, Nauka, pp. 1-256 (in Russian).

GRAUER, F.H. & ARNOLD, H.L. (1961) Seaweed dermatitis.
Arch. Dermatol., 84: 720-732.

GRINDLEY, J.R. & SAPEIKA, N. (1969) The cause of mussel
poisoning in South Africa. S. Afr. med. J., 43: 275-279.

GRUNFELD, Y. & SPIEGELSTEIN, M.Y. (1974) Effects of
Gymnodinium breve toxin on the smooth muscle preparation of
guinea-pig ileum. Br. J. Pharmacol., 51: 67-72.

GULBRANDSEN, R.-K.S. & AALVIK, B., (198l) Mytilotoxicosis.
Tidsskr. Norv. Laegeforen., 101: 452-454 (in Norwegian).

GUZMAN, L. & CAMPODONICO, I. (1975) Public Inst. Patagonia
Ser. Mon., 9: 1-6.

HALL, S. (1982) Toxins and toxicity of Protogonyaulax from
the North-east Pacific, Fairbanks, Alaska, US, University of
Alaska, PhD Thesis, pp. 1-196.

HALSTEAD, B.W. (1978) Poisonous and venomous marine animals
of the world, Princeton, Darwin Press.

HALSTEAD, B.W. & SCHANTZ, E.J. (1984) Paralytic shellfish
poisoning, Geneva, World Health Organization, pp. 60 (WHO
Offset Publication No. 79).

HAMMER, U.T. (1968) Toxic blue-green algae in Saskatchewan.
Can. vet. J., 9: 221-229.

HANNO, H.A. (1981) Ciguatera fish poisoning in the Virgin
Islands. J. Am. Med. Assoc., 245(5): 464.

HARADA, T., OSHIMA, Y., KAMIYA, H., & YASUMOTO, T. (1982a)
Confirmation of paralytic shellfish toxins in the
dinoflagellate Pyrodinium bahamense var. compressa and
bivalves in Palau. Bull. Jpn. Soc. Sci. Fish., 48: 821-825.

HARADA, T., OSHIMA, Y., & YASUMOTO, T. (1982b) Structures of
two paralytic shellfish toxins, gonyautoxins V and VI,
isolated from a tropical dinoflagellate, Pyrodinium bahamense
var. compressa. Agric. Biol. Chem., 46: 1861-1864.

HARADA, T., OSHIMA, Y., & YASUMOTO, T. (1983) Natural
occurrence of decarbamoylsaxitoxin in tropical dinoflagellate
and bivalves. Agric. Biol. Chem., 47: 191-193.

HARTWELL, A.D. (1975) Hydrographic factors affecting the
distribution and movement of toxic dinoflagellates in the
Western Gulf of Maine. In: LoCicero, V.R., ed. Proceedings of
the First International Conference on Toxic Dinoflagellate
Blooms, Wakefield, Massachusetts, Massachusetts Science and
Technology Foundation, pp. 47-68.

HASHIMOTO, Y. (1979) Marine toxins and other bioactive
marine metabolites, Tokyo, Japan Scientific Societies Press.

HELFRICH, Ph., PIYAKARNCHANA, T., & MILES, P.S. (1968) The
ecology of ciguateric reef fishes in the Line Islands. Occas.
Pap., Bernice P. Bishop Mus., 23(14): 305-370.

HESSEL, D.W., HALSTEAD, B.W., & PECKHAM, N.H. (1960) Marine
biotoxins. I. Ciguatera poison: some biological and chemical
aspects. NY Acad. Sci. Ann., 90: 788-797.

HILLE, B. (1975) The receptor for tetrodotoxin and
saxitoxin. Biophys. J., 15: 615-619.

HOKAMA, Y., BANNER, A.H., & BOYLAN, D.B. (1977) A
radio-immunoassay for the detection of ciguatoxin. Toxicon,
15:317-325.

HUGHES, J.M. (1979) Epidemiology of shellfish poisoning in
the United States, 1971-1977. In: Taylor, D.L. & Seliger,
H.H., ed. Toxic dinoflagellate blooms, New York, Elsevier, pp.
23-28.

HUGHES, J.M. & MERSON, M.H. (1976) Fish and shellfish
poisoning. New Engl. J. Med., 295: 1117-1120.

HUGHES, J.M., HORWITZ, M.A., MERSON, M.H., BAKER, W.H., &
GANGAROSA, E.J. (1977) Foodborne disease outbreaks of
chemical etiology in the United States, 1970-1974. Am. J.
Epidemiol., 105: 233-244.

IKAWA, M., WEGENER, K., FOXALL, T.L., & SASNER, J.Y. (1982)
Comparison of the toxins of the blue-green alga Aphanizomenon
flos-aquae with the Gonyaulax toxins. Toxicon, 20: 747-752.

JACKIM, E. & GENTILE, J. (1968) Toxins of a blue-green alga:
Similarity to saxitoxin. Science, 162: 915-916.

JOHNSON, H.M. & MULBERRY, G. (1966) Paralytic shellfish
poison: Serological assay by passive haemagglutination and
bentonite flocculations. Nature (Lond.), 211: 747-748.

KAINUMA, M. (198l) [Poisoning resulting from the ingestion
of poisonous fish and shellfish.] J. clin. Nutr., 59: 289-293
(in Japanese).

KAO, C.Y. (1966) Tetrodotoxin, saxitoxin and their
significance in the study of excitation phenomena. Pharmacol.
Rev., 18: 997-1049.

KAO, C.Y. (1972) Pharmacology of tetrodotoxin and
saxitoxin. Fed. Proc., 31: 1117-1123.

KAO, C.Y. (1983) New perspectives on the interaction of
terodotoxin and saxitoxin with excitable membranes. Toxicon,
Suppl. 3: 211-219.

KAO, C.Y. & NISHIYAMA, A. (1965) Actions of saxitoxin on
peripheral neuromuscular systems. J. Physiol. (London), 272:
50-66.

KAT, M. (1983a) Dinophysis acuminata blooms in the Dutch
coastal area related to diarrhetic mussel poisoning in the
Dutch Waddensea. Sarsia, 68: 81-84.

KAT, M. (1983b) Diarrhetic mussel poisoning in the
Netherlands related to the dinoflagellate Dinophysis
acuminata. Antonie van Leeuwenhoek, 49: 417-427.

KAWABATA, T. (1978) [Manual of methods for food sanitation,]
Tokyo, Environmental Health Bureau, Ministry of Health and
Welfare, Vol. 2, pp. 232-240 (in Japanese).

KIM, Y.S. & MARTIN, D.F. (1974) Interrelationship of Peace
River parameters as a basis of the iron index. A predictive
guide to the Florida red tide. Water Res., 8: 607-616.

KIMURA, L.H., HOKAMA, Y., ABAD, M.A., OYAMA, M., & MIYAHARA,
J.T. (1982) Comparison of three different assays for the
assessment of ciguatoxin in fish tissues: Radioimmunoassay,
mouse bioassay and in vitro guinea pig atrium assay. Toxicon,
20: 907-912.

KIRPENKO, Yu.A., SIRENKO, L.A., ORLOVSKIJ, V.M., & LUKINA,
L.F. (1977) [Blue-green algal toxins and the animal
organism,] Kiev, Naukova Dumka, pp. 1-251 (in Russian).

KIRPENKO, Yu.A., SIRENKO, L.A., & KIRPENKO, N.I. (1981) Some
aspects concerning remote after-effects of blue-green algae
toxins impact on warm-blooded animals. In: Carmichael, W.W.,
ed. The water environment: Algal toxins and health, New York,
London, Plenum Press, pp. 257-270.

KOBAYASHI, M. & SHIMIZU, Y. (198l) Gonyautoxin VII, a
cryptic precursor of paralytic shellfish poisons. J. Chem.
Soc. Chem. Comm., pp. 827-828.

KOMAREK, J. (1958) [A taxonomic revision of planktonic blue
algae of Czechoslovakia.] In: Komrek, J. & Ettl, H., ed.
[Algal studies,] Prague, The Czechoslovakian Academy of
Sciences (in Czech).

KONDRATEVA, N.V. & KOVALENKO, O.V. (1975) [A short
classification of toxic blue-green algae,] Kiev, Naukova
Dumka, pp. 1-64 (in Russian).

KOTAKI, Y., TAJIRI, M., OSHIMA, Y., & YASUMOTO, T. (1983)
Identification of a calcareous red alga as the primary source
of paralytic shellfish toxins in coral reef crabs and
gastropods. Bull. Jpn. Soc. Sci. Fish., 49: 283-286.

KROGH, P. (1979) Measurements of paralytic shellfish
poisons. A review of biological and chemical procedures.
Nord. vet. med., 31: 302-308.

KROGH, P. (1983) Algal toxins in seafood and drinking
water. Chem. Int., 5: 45-48.

KRUGER, G.H.L. & ELOFF, J.N. (1978) The effect of
temperature on specific growth rate and activation energy of
Microcystis and Synechococcus isolates relevant to the onset
of natural blooms. J. Limnol. Soc. S. Afr., 4: 9-20.

LAIGRET, J., PARC, F., CHANTEAU, S., CHUNGUE, E., & BAGNIS,
R. (1981) Ciguatera: absence actuelle d'immun-serum
specifique. Méd. Armées, 9(4): 337-339.

LAWRENCE, D.N., ENRIQUEZ, M.B., LUMISH, R.M., & MACEO, A.
(1980) Ciguatera fish poisoning in Miami. JAMA, 244(3):
254-258.

LIN, Y.Y., RISK, M., RAY, S.M., VAN ENGEN, D., CLARDY, J.,
GOLIK, J., JAMES, J.C., & NAKANISHI, K. (1981) Isolation and
structure of brevetoxin B from the "red tide" dinoflagellate
Ptychodiscus brevis (Gymnodinium breve). J. Am. Chem. Soc.,
103: 6773-6775.

LIPPY, E.C. & ERB, J. (1976) Gastrointestinal illness at
Sewickley, Pa. J. Am. Water Works Assoc., 68: 606-610.

MARGALEF, R., ESTRADA, M., & BLASCO, D. (1979) Functional
morphology of organisms involved in red tides, as adapted to
decaying turbulence. In: Taylor, D.L. & Seliger, H.H., ed.
Toxic dinoflagellate blooms, New York, Elsevier, pp. 89-94.

MARGULIS, L. & SCHWARTZ, K. V. (1982) Five kingdoms. An
illustrative guide to the phyla of life on earth, San
Francisco, W.H. Freeman and Company, pp. 1-20, 74-75.

MATSUI, T., HAMADA, S., & KONOSU, S. (1981) Difference in
accumulation of pufferfish toxin and crystalline tetrodotoxin
in the pufferfish, Fugu rubripes rubripes. Bull. Jpn. Soc.
Sci. Fish., 47: 535-537.

MATSUI, T., SATO, H., HAMADA, S., & SHIMIZU, Ch. (1982)
Comparison of toxicity of the cultured and wild pufferfish
Fugu niphobles. Bull. Jpn. Soc. Sci. Fish., 48: 253.

MCALICE, B.J. (1968) An occurrence of ciliate red water in
the Gulf of Maine. J. Fish. Res. Board Can., 25: 1749-1751.

MCCOLLUM, J.P.K., PEARSON, R.C.M., INGHAM, H.R., WOOD, P.C., &
DEWAR, H.A. (1968) An epidemic of mussel poisoning in
north-east England. Lancet, ii: 767-770.

MCFARREN, E.F. (1959) Report on collaborative studies of the
bioassay for paralytic shellfish poison. J. Assoc. Off. Anal.
Chem., 42: 263-271.

MCFARREN, E.F. (1960) Collaborative studies of the chemical
assay for paralytic shellfish poison. J. Assoc. Off. Anal.
Chem., 43: 544-547.

MCFARREN, E.F., SCHANTZ, E.J., CAMPBELL, J.E., & LEWIS, K.H.
(1958) Chemical determination of paralytic shellfish poison.
J. Assoc. Off. Anal. Chem., 41: 168-177.

MCFARREN, E.F., SCHANTZ, E.J., CAMPBELL, J.E., & LEWIS, K.H.
(1959) A modified Jaffe test for determination of paralytic
shellfish poison. J. Assoc. Off. Anal. Chem., 42: 399-404.

MCFARREN, E.F., SCHAFER, M.L., CAMPBELL, J.E., LEWIS, K.H.,
JENSEN, E.T., & SCHANTZ, E.J. (1960) Public health
significance of paralytic shellfish poison. Adv. food Sci.,
10: 135-179.

MCFARREN, E.F., TANABE, H., SILVA, F.J., WILSON, W.B.,
CAMPBELL, J.W., & LEWIS, K. H. (1965) The occurrence of a
ciguatera-like poison in oysters, clams and Gonyaulax breve
cultures. Toxicon, 3: 111-123.

MEDCOF, J.E., LEIM, A.H., NEEDLER, A.B., & NEEDLER, A.W.H.
(1947) Paralytic shellfish poisoning on the Canadian Atlantic
coast. Bull. Fish. Res. Board Can., 75: 1-32.

MEYER, K.F. (1953) Food poisoning. New Engl. J. Med., 248:
843-852.

MEYER, K.E., SOMMER, K.F., & SCHOENHOLZ, P. (1928) Mussel
poisoning. J. prev. Med., 2: 365-394.

MOORE, R.E. (1981) Toxins from marine blue-green algae. In:
Carmichael, W.W., ed. The water environment - Algal toxins and
health, New York, Plenum Press, pp. 15-23.

MORRIS, J.G., LEWIN, P., MARGRETT, N.T., SMITH, C.W., BLAKE,
P.A., & SCHNEIDER, R. (1982a) Clinical features of ciguatera
fish poisoning. A study of the disease in the US Virgin
islands. Arch. intern. Med., 142: 1090-1092.

MORRIS, J.G., LEWIN, P., SMITH, C.W., BLAKE, P.A., &
SCHNEIDER, R. (1982b) Ciguatera fish poisoning: Epidemiology
of the disease on St. Thomas, US Virgin islands. Am. J. trop.
Med. Hyg., 31: 574-578.

MULLIGAN, H.G. (1975) Oceanographic factors associated with
New England red tide blooms. In: LoCicero, V.R., ed.
Proceedings of the First International Conference on Toxic
Dinoflagellate Blooms, Wakefield, Massachusetts, Massachusetts
Science and Technology Foundation, pp. 23-40.

MURAKAMI, T., OSHIMA, Y., & YASUMOTO, T. (1982)
Identification of okadaic acid as a toxic component of a
marine dinoflagellate Prorocentrum lima. Bull. Jpn. Soc. Sci.
Fish., 48: 69-72.

MURATA, M., SHIMATANI, M., SUGITANI, H., ISHIMA, Y., &
YASUMOTO, T. (1982) Isolation and structural elucidation of
the causative toxin of the diarrheic shellfish poisoning.
Bull. Jpn. Soc. Sci. Fish., 48: 549-552.

MYNDERSE, J.S., MOORE, R.E., KASHIWAGI, M., & NORTON, T.R.
(1977) Antileukemia activity in the Oscillatoriaceae:
Isolation of debromoaplysia toxin from Lyngbya. Science, 196:
538-540.

NARAHASHI, T. (1972) Mechanism of action of tetrodotoxin and
saxitoxin on excitable membranes. Fed. Proc., 31: 1124-1132.

NARITA, H., NOGUCHI, T., MARUYAMA, J., UEDA, Y., HASHIMOTO,
K., WATANABE, Y., & HIDA, K. (198l) Occurrence of
tetrodotoxin in a trumpet shell, "Boshubora" Charonia
sanliae. Bull. Jpn. Soc. Sci. Fish., 47: 935-941.

NISBET, I.C.T. (1983) Paralytic shellfish poisoning: Effects
on breeding terms. Condor, 85: 338-345.

NOGUCHI, T., MARUYAMA, J., UEDA, Y., HASHIMOTO, K., & HARADA,
T. (1981) Occurrence of tetrodotoxin in the Japanese ivory
shell Babylonia japonica. Bull. Jpn. Soc. Sci. Fish., 47:
909-913.

NUKINA, M., TACHIBANA, K., KOYANAGI, L.M., & SCHEUER, P.J.
(1983) Structural studies on ciguatoxin. In: Symposium on
Seafood Toxins, Washington, DC, American Chemical Society
(Abstract no. 135).

NUNEZ, M.T., FISCHER, S., & JAIMOVICH, E. (1976) A
fluorimetric method to determine tetrodotoxin. Anal.
Biochem., 72: 320-325.

OHIZUMI, Y., SHIBATA, S., & TACHIBANA, K. (1981) Mode of the
excilatory and inhibitory actions of ciguatoxin in the guinea
pig vas deferens. J. Pharmacol. exp. Ther., 217: 475-480.

ONOUE, Y., NOGUCHI, T., NAGASHIMA, Y., & HASHIMOTO, K.
(1983) Separation of tetrodotoxin and paralytic shellfish
poisons by high-performance liquid chromatography with a
fluorometric detection using ophthalaldehyde. J. Chromatogr.,
257: 373-379.

OSHIMA, Y., SINGH, H.T., FUKUYO, Y., & YASUMOTO, T. (1982)
Identification and toxicity of the resting cysts of
Protogonyaulax found in Ofunato bay. Bull. Jpn. Soc. Sci.
Fish., 48: 1303-1305.

PARC, F., DUCOUSSO, R., CHANTEAU, S., CHUNGUE, E., LAFONT, E.,
& BAGNIS, R. (1981) Problèmes posés par la detection de la
ciguatoxine dans les tissus pisciaires. Méd. oceanienne, 14:
1-4.

PINGREE, R.D., PUGH, P.R., HOLLIGAN, P.M., & FOSTER, G.R.
(1975) Summer phytoplankton blooms and red tides along tidal
fronts in the approaches to the English Channel. Nature
(Lond.), 258: 672-677.

POCCHIARI, F. (1977) Trade of misbranded frozen fish:
medical and public health implications. Ann. Inst. Super.
Sanita, 13: 767-772.

POMPON, A., CHUNGUE, E., CHAZELET, I., & BAGNIS, R. (1984)
Ciguatera: une methode rapide, simple et fiable de détection
de la ciguatoxine. Bull. World Health Organ., 62(4): 639-645.

POPKISS, M.E.E., HORSTMAN, D.A., & HARPUR, D. (1979)
Paralytic shellfish poisoning. A report of 17 cases in Cape
Town. S. Afr. med. J., 55: 1017-1023.

PRAKASH, A. (1967) Growth and toxicity of a marine
dinoflagellate Gonyaulax tamarensis. J. Fish. Res. Board
Can., 24: 1589-1600.

PRAKASH, A. (1975) Land drainage as a factor in "red tide"
development. Environ. Lett., 9: 121-128.

PRAKASH, A., MEDCOF, J.C., & TENNANT, A.D. (1971) Paralytic
shellfish poisoning in Eastern Canada. Bull. Fish. Res. Board
Canada, 117: 1-88.

PRINZMETAL, M., SOMMER, H., & LEAKE, C.D. (1932) The
pharmacological action of "mussel poison". J. Pharmacol. exp.
Ther., 46: 63-73

PROCTOR, N.H., CHAN, S.L. & TREVOR, A.J. (1975) Production
of Saxitoxin by cultures of Gonyaulax catenella. Toxicon, 13:
1-9.

RAJ, U., HAQ, H., OSHIMA, Y., & YASUMOTO, T. (1983) The
occurrence of paralytic shellfish toxins in two species of
xanthid crab from Suva Barrier Reef, Fiji Islands. Toxicon,
21: 547-551.

RANDALL, J.E. (1958) A review of ciguatera tropical fish
poisoning with a tentative of explanation of its cause. Bull.
mar. Sci. Gulf Carib., 8(3): 236-267.

RAY, S.M. & ALDRICH, D.V. (1965) Gymnodinium breve:
Induction of shellfish poisoning in chicks. Science, 148:
1748-1749.

RAYNER, M.D. (1972) Mode of action of ciguatoxin. Fed. Proc.,
31: 1139-1145.

REYES-VASQUEZ, G., GERRAZ-REYES, E., & VASQUEZ, E. (1979)
Toxic dinoflagellate blooms in north-eastern Venezuela during
1977. In: Taylor, D.L. & Seliger, H.H., ed. Toxic
dinoflagellate blooms, New York, Elsevier, pp. 191-194.

ROY, R.N. (1977) Red tide and outbreak of paralytic
shellfish poisoning in Sabah. Med. J. Malay., 31: 247-251.

RUSSELL-HUNTER, W.D. (1972) A biology of lower
invertebrates, London, Collier-Macmillan, pp. 144-148.

SANDERS, J.G. & RYTHER, J.H. (1979) Impact of chlorine on
the species composition of marine phytoplankton. In: Jolley,
R.L., Brungs, W.A., Cumming, R.B., & Jacobs, V.A., ed. Water
chlorination. Environmental impact and health effects,
Michigan, US, Ann Arbor Science, Vol. 3, pp. 631-639.

SCHANTZ, E.J. (1973) Seafood toxicants. In: Toxicants
occurring naturally in foods, Washington, DC, National Academy
of Sciences, pp. 425-447.

SCHANTZ, E.J. (1980) Phycotoxins from dinoflagellates. Pure
appl. Chem., 52: 183-188.

SCHANTZ, E.J., MCFARREN, E.F., SCHAFER, M.L., & LEWIS, K.H.
(1958) Purified shellfish poison for bioassay
standardization. J. Assoc. Off. Anal. Chem., 41: 160-168.

SCHANTZ, E.J., MOLD, J.D., HOWARD, W.L., BOWDEN, J.P.,
STRANGER, D.W., LYNCH, J.M., WINTERSTEINER, O.P., DUTCHER,
J.D., EALTERS, D.R., & RIEGEL, B. (1961) Paralytic shellfish
poison. VIII. Some chemical physical properties of purified
clam and mussel poisons. Can. J. Chem., 39: 2117-2123

SCHANTZ, E.J., GHAZAROSSIAN, V.E., SCHNOES, H.K., STRONG,
P.M., SPRINGER, J.P., PEZZANITE, J.O., & CLARDY, J. (1975)
Paralytic poisons from marine dinoflagellates. In: LoCicero,
V.R., ed. Proceedings of the First International Conference on
Toxic Dinoflagellate Blooms, Wakefield, Massachusetts,
Massachusetts Science and Technology Foundation, pp. 267-275.

SCHEUER, P.J. (1977) Marine toxins. Acc. chem. Res., 10:
33-39.

SCHEUER, P. J. (1982) Marine ecology - some chemical
aspects. Naturwissenschaften, 69: 528-533.

SCHMIDT, R.J. & LOEBLICH, A.R. (1979) Distribution of
paralytic shellfish poison among Pyrrhophyta. J. Mar. Biol.
Assoc., UK, 59: 479-487.

SELIGER, H.H., TYLER, M.A., & MCKINLEY, K.R. (1979)
Phytoplankton distributions and red tides resulting from
frontal circulation patterns. In: Taylor, D.L. & Seliger,
H.H., ed. Toxic dinoglagellate blooms, New York, Elsevier, pp.
239-248.

SHEUMACK, D.D., HOWDEN, M.E.H., SPENCE, I., & QUINN, R.J.
(1978) Maculotoxin: A neurotoxin from the venom glands of the
octopus Hapalochlaena maculosa identified as tetrodotoxin.
Science, 199: 188-189.

SHIMADA, K., OHTSURU, M., YAMAGUCHI, T., & NIGOTA, K. (1983)
Determination of tetrodotoxin by capillary isotachopheresis.
J. food Sci., 48: 665-680.

SHIMIZU, Y. (1978) Dinoglagellate toxins. In: Scheuer, P.J.,
ed. Marine natural products, New York, Academic Press, pp.
1-42.

SHIMIZU, Y., BUCKLEY, L.J., ALAM, M., OSHIMA, Y., FALLON,
W.E., KASAI, H., MAURA, I., GULLO, V.P., & NAKANISHI, K.
(1976) Structures of Gonyautoxin II and III from the east
coast toxic dinoflagellate Gonyaulax tamarensis. J. Am. Chem.
Soc., 98: 5414-5416.

SHIMIZU, Y., HSU, C., FALLON, W.E., OSHIMA, Y., MIURA, I., &
NAKANISHI, K. (1978) Structure of neosaxitoxin. J. Am.
Chem. Soc., 100: 6791-6793.

SHINNICK-GALLAGHER, P. (1980) Possible mechanisms of action
of Gymnodinium breve toxin at the mammalian neuromuscular
junction. Br. J. Pharmacol., 69: 373-378.

SHOPTAUGH, N.H., CARTER, P.W., FOXALL, T.L., SASNER, J.J., &
IKAWA, M. (1981) Use of fluorometry for the determination of
Gonyaulax tamarensis var. excavata toxins in New England
shellfish. J. agric. food Chem., 29: 198-200.

SIMON, B., MEBS, D., GEMMER, H., & STILLE, W. (1977)
[Symptoms of poison after the digestion of edible mussels.]
Dtsch med. Wschr., 102: 1114-1117 (in German).

SKULBERG, O.M. (1979) [Toxic effects of blue-green algae,]
Oslo, Norsk Institutt for Vannforskning, pp. 42 (Report No. 4)
(in Norwegian).

SOLOMON, A.E. & STOUGHTON, R.B. (1978) Dermatitis from
purified sea algae toxin (Debromoaplysia toxin). Arch.
Dermatol., 114: 1333-1335.

SOMMER, H. & MEYER, K.F. (1937) Paralytic shellfish poison.
Arch. Pathol., 24: 560-598.

SPIEGELSTEIN, M.Y., PASTER, Z., & ABBOTT, B.C. (1973)
Purification and biological activity of Gymnodinium breve
toxins. Toxicon, 11: 85-93.

STEIDINGER, K.A. (1975) Implications of dinoglagellate life
cycles on initiation of Gymnodinium breve red tides. Environ.
Lett., 9: 129-139.

STEIDINGER, K.A. (1979) Collection, enumeration and
identification of free-living marine dinoglagellates. In:
Taylor, D.L. & Seliger, H.H., ed. Toxic dinoflagellate blooms,
Amsterdam, Elsevier, pp. 435-442.

STEIDINGER, K.A., TESTER, L.S., & TAYLOR, F.J.R. (1980) A
redescription of Pyrodinium bahamense var. compressa (Böhm)
stat. nov. from Pacific red tides. Phycologia, 19: 329-337.

SUBCOMMITTEE ON LABORATORY METHODS FOR THE EXAMINATION OF
SHELLFISH (1970) Recommended procedures for the examination
of sea water and shellfish, 4th ed., Washington, DC, American
Public Health Association Inc., pp. 61-66.

SULLIVAN, J.J. & IWAOKA, W.T. (1983) High pressure liquid
chromatographic determination of toxins associated with
paralytic shellfish poisoning. J. Assoc. Off. Anal. Chem.,
66: 297-303.

SULLIVAN, J.J., IWAOKA, W.T., & LISBON, J. (1983a) Enzymatic
transformation of PSP toxins in the littleneck clam
(Protothaca staminea). Biochem. biophys. res. Commun., 114:
465-472.

SULLIVAN, J.J., SIMON, M.G., & IWAOKA, W.T. (1983b)
Comparison of HPLC and mouse bioassay methods for determining
PSP toxins in shellfish. J. food Sci., 48: 1312-1314.

TABETA, O. & KUMAGAI, H. (1980) J. Food Hyg. Soc. Japan, 21:
405-407.

TAYLOR, F.J.R. (1975) Taxonomic difficulties in the red tide
and paralytic shellfish poison studies: The "Tamarensis
complex" of Gonyaulax. Environ. Lett., 9: 103-119.

TENNANT, A.D., NAUBERT, J., & CORBEIL, H.E. (1955) An
out-break of paralytic shellfish poisoning. J. Can. Med.
Assoc., 72: 436-439.

TINDALL, D.R. (1983) Ciguatoxigenic organisms in the
Carribean. In: Symposium on Seafood Toxins, Washington, DC,
American Chemical Society (Abstract No. 137).

TORDA, T.A., SINCLAIR, E., & ULYATT, D.B. (1973) Puffer fish
(tetrodotoxin) poisoning, clinical record and suggested
management. Med. J. Aust., 1: 599-602.

TUSTIN, R.C., RENSBURG, S.F. VAN, & ELOFF, J.N. (1973)
Hepatic damage in the primate following ingestion of toxic
algae. In: Sounder, S.J. & Terblanche, J., ed. Liver, London,
Pitman Medical, pp. 383-385.

TWAROG, B.M., HIDAKA, T., & YAMAGUCHI, H. (1972) Resistance
to tetrodotoxin and saxitoxin in nerves of bivalve molluscs. A
possible correlation with paralytic shellfish poisoning.
Toxicon, 10: 273-278.

TYLER, M.A. & SELIGER, H.H. (1978) Annual subsurface
transport of a red tide dinoflagellate to its bloom area:
Water circulation patterns and organism distribution in the
Chesapeake Bay. Limnol. Oceanogr., 23: 227-246.

WALL, D. (1975) Taxonomy and cysts of red tide dinoflagellates.
In: LoCicero, V.R., ed. Proceedings of the First International
Conference on Toxic Dinoglagellate Blooms, Wakefield,
Massachusetts, Massachusetts Science and Technology Foundation,
pp. 249-255.

WATTS, J.S., REILLY, J., DACOSTA, F.M., & KROP, S. (1966)
Acute toxicity of paralytic shellfish poison in rats of
different ages. Toxicol. appl. Pharmacol., 8: 286-294.

WHITE, A.W. (1977) Dinoflagellate toxins as probable cause
of an atlantic herring (Clupea harengus harengus) kill, and
pteropods as apparent vector. J. Fish. Res. Board Can., 34:
2421-2424.

WHITE, A.W. (1979) Dinoflagellate toxins in phytoplankton
and zooplankton fractions during a bloom of Gonyaulax
excavata. In: Taylor, D.L. & Seliger, H.H., ed. Toxic
dinoflagellate blooms, New York, Elsevier, pp. 381-384.

WHITE, A.W. (1980) Recurrence of kills of atlantic herring
(Clupea harengus harengus) caused by dinoflagellate toxins
transferred through herbivarous zooplankton. Can. J. Fish.
aquat. Sci., 37: 2262-2265.

WHITE, A.W. (198la) Sensitivity of marine fishes to toxins
from the red-tide dinoflagellate Gonyaulax excavata and
implications for fish kills. Mar. Biol., 65: 255-260.

WHITE, A.W. (1981b) Marine zooplankton can accumulate and
retain dinoflagellate toxins and cause fishkills. Limnol.
Oceanogr., 26: 103-109.

WHITE, A.W. & LEWIS, C.M. (1982) Resting cysts of the toxic,
red tide dinoflagellate Goynaulax excavata in Bay of Fundy
sediments. Can. J. Fish. aqua. Sci., 39: 1185-1194.

WHITE, A.W. & MARANDA, L. (1978) Paralytic toxins in the
dinoflagellate Gonyaulax excavata and in shellfish. J. Fish.
Res. Board Can., 35: 397-402.

WHO (1974) Fish and shellfish hygiene, Geneva, World Health
Organization, pp. 1-62 (Technical Report Series No. 550).

WHO (1979) Report of the WHO expert consultation on
paralytic shellfish poisoning, Berlin (West), 5-8 December
1978, Geneva, World Health Organization, pp. 1-14 (VPH/79.14).

WHO (1983) Training course on fish poisoning, Suva, Fiji,
11-25 January 1983, Manila, Regional Office for the Western
Pacific, World Health Organization (Report (WP) PDV/ICP FSP/
002).

WIBERG, G.S. & STEPHENSON, N.R. (1960) Toxicologic studies
on paralytic shellfish poison. Toxicol. appl. Pharmacol., 2:
607-615.

WICHMANN, C.F., BOYER, G.L., DIVAN, C.L., SCHANTZ, E.J., &
SCHNOES, H.K. (1981) Neurotoxins of Gonyaulax excavata and
Bay of Fundy scallops. Tetrahedron Lett., 22: 1941-1944.

WITHERS, N.W. (1982) Ciguatera fish poisoning. Ann. Rev.
med., 33: 97-111.

WOOD, P.C. (1976) Guide to shellfish hygiene, Geneva, World
Health Organization, pp. 1-80 (WHO Offset Publication No. 31).

YASUMOTO, T. (1980) [Ciguatera.] Igaku no ayumi, 112: 886
(in Japanese).

YASUMOTO, T. & KANNO, K. (1976) Occurrence of toxins
resembling ciguatoxin, scaritoxin, and maitotoxin in a turban
shell. Bull. Jpn. Soc. Sci. Fish., 42: 1399-1404.

YASUMOTO, T. & SCHEUER, P.J. (1969) Marine toxins of the
Pacific-VIII ciguatoxin from moray eel livers. Toxicon, 7:
273-276.

YASUMOTO, T., HASHIMOTO, Y., BAGNIS, R., RANDALL, J.E., &
BANNER, A.H. (1971) Toxicity of the surgeonfishes. Bull.
Jpn. Soc. Sci. Fish., 37: 724-734.

YASUMOTO, T., NAKAJIMA, I., BAGNIS, R., & ADACHI, R. (1977a)
Finding of a dinoflagellate as a likely culprit of ciguatera.
Bull. Jpn. Soc. Sci. Fish., 43: 1021-1026.

YASUMOTO, T., NAKAJIMA, I., CHUNGUE, E., & BAGNIS, R.
(1977b) Toxins in the gut contents of parrotfish. Bull. Jpn.
Soc. Sci. Fish., 43: 69-74.

YASUMOTO, T., OSHIMA, Y., & YAMAGUCHI, M., (1978) Occurrence
of a new type of shellfish poisoning in the Tohoku district.
Bull. Jpn. Soc. Sci. Fish., 44: 1249-1255.

YASUMOTO, T., INOW, A., OCHI, T., FUJIMOTO, K., OSHIMA, Y.,
FUKUYO, Y., ADACHI, R., & BAGNIS, R. (1980a) Environmental
studies on a toxic dinoflagellate responsible for ciguatera.
Bull. Jpn. Soc. Sci. Fish., 46: 1397-1404.

YASUMOTO, T., OSHIMA, Y., SUGAWARA, W., FUKUYO, Y., OGURI, H.,
IGARASHI, T., & FUJITA, N. (1980b) Identification of Dynophysis
fortii as the causative organism of diarrheic shellfish poisoning.
Bull. Jpn. Soc. Sci. Fish., 46: 1405-1411.

YASUMOTO, T., NAKAMURA, M., OSHIMA, Y., & TAKAHATA, J.
(1982) Contruction of a continuous tetrodotoxin analyzer.
Bull. Jpn. Soc. Sci. Fish., 48: 1481-1483.

YASUMOTO, T., RAJ, U., & BAGNIS, R. (1984) Seafood
poisonings in tropical regions, Sendai, Japan, Tohoku
University, pp. 1-74.

YENTSCH, C.M. & BALCH, W. (1975) Lack of secondary
intoxification by red tide poison in the American lobster
Homarus americanus. Environ. Lett., 9: 249-254.

YENTSCH, C.M. & GLOVER, H. (1977) Progress towards an
environmental predictive index for toxic dinoflagellate
blooms. In: Wilt, D.S., ed. Proceedings of the Tenth National
Shellfish Sanitation Workshop, Washington, DC, US Department
of Health, Education and Welfare, pp. 142-151.

YENTSCH, C.M. & INCZE, L.S. (1980) Accumulation of algal
biotoxins in mussels. In: Luts, R. & Incze, L.S., ed. Mussel
culture in North America, New York, Elsevier, pp. 223-246.

YENTSCH, C.M. & MAGUE, F.C. (1979) Motile cells and cysts:
Two probable mechanisms of intoxication of shellfish. In:
Taylor, D.L. & Seliger, H.H., ed. Toxic dinoflagellate blooms,
New York, Elsevier, pp. 127-130.

YENTSCH, C.M., DALE, B., & HURST, J.W. (1978) Co-existence
of toxic and nontoxic dinoflagellates resembling Gonyaulax
tamarensis in New England costal waters (N.W. Atlantic).
J. Phycol., 14: 330-332.

ZWAHLEN, A., BLANC, M.-H., & ROBERT, M. (1977) Epidémie
d'intoxication par les moules. Schweiz. med. Wschr., 107:
226-230.

    See Also:
       Toxicological Abbreviations