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Aquaculture and the environment

Partim: Harmful algal blooms

Prof. Dr. ir. Peter Bossier

Academic year 2012-2013

• Monographs on Oceanographic Methodology: Manual on
Harmful Marine Microalgae (Hallegraef, Anderson,
Cembella) 2003, Unesco Publishing, ISBN 92 3 103871
m: identification of HA
• identification of HA
Course content
• Harmful algal blooms: a global overview
• Characterising algal blooms
– Species involved
– Toxins involved
• Toxin detection
– Mouse assay
– Chemical methods
– Immunochemical methods
• Overview toxic levels/concentration algae that can cause
toxin accumulation
• Occurrence and monitoring of algal blooms
• Dynamics of algal blooms
• Ecology of algal blooms
• Sampling strategy

• Phytoplankton blooms, micro-algal blooms, toxic algae,

red tides, or harmful algae, are all terms for naturally
occurring phenomena.
• About 300 hundred species of micro algae are reported
at times to form mass occurrence, so called blooms.

• Nearly one fourth of these species are known to produce


• The scientific community refers to these events with a

generic term, ‘Harmful Algal Bloom’ (HAB), recognising
that, because a wide range of organisms is involved and
some species have toxic effects at low cell densities, not
all HABs are ‘algal’ and not all occur as ‘blooms’.
HAB: overview 1
• Species which produce basically harmless
water discolorations; however, under
exceptional conditions in sheltered bays,
blooms can grow so dense that they cause
indiscriminate kills of fish and
invertebrates due to oxygen depletion.

• Examples:
• Dinoflagellates: Gonyaulax polygramma
Overview 1
• Examples:
– Noctiluca scintillans,
Overview 1
cyanobacterium : Trichodesmium erythraeum
HAB organisms: overview 2
• Species which produce potent toxins that can find their
way through the food chain to humans, causing a variety
of gastrointestinal and neurological illnesses, such as:
• - Paralytic Shellfish Poisoning (PSP)
– dinoflagellates Alexandrium acatenella, A. catenella, A.
cohorticula, A. fundyense, A. fraterculus, A. minutum, A.
tamarense, Gymnodinium catenatum, Pyrodinium bahamense
var. compressum
– Alexandrium Gymnodinium catenatum
Overview 2
• - Diarrhetic Shellfish Poisoning (DSP)
– dinoflagellates Dinophysis acuta, D. acuminata, D.
fortii, D. norvegica, D. mitra, D. rotundata,
Prorocentrum lima )
• Dinophysis Prorocentrum lima
Overview 2
• - Neurotoxic Shellfish Poisoning (NSP)

– dinoflagellate Gymnodinium breve (New Zealand)

– Gymnodinium breve= Karenia breve
Overview 2
• - Ciguatera Fish Poisoning (CFP)

– dinoflagellate Gambierdiscus toxicus, Ostreopsis

spp., Prorocentrum spp.
• Gambierdiscus toxicus Ostreopsis
Overview 2
• - Amnesic Shellfish Poisoning (ASP)

– diatoms Pseudo-nitzschia multiseries , P.

pseudodelicatissima, P. australis
Overview 2
• - Cyanobacterial Toxin Poisoning
– Cyanobacteria such as Anabaena circinalis ,
Microcystis aeruginosa, Nodularia spumigena

– Anatoxin Anabaena
Overview 2: Microcystis
single cell colonial cyanobacteria

Toxin: microcystin
Overview 2: Nodularia

Toxin: nodularin
HAB overview 3
• Species, which are non-toxic to humans, but
harmful to fish and invertebrates (especially in
intensive aquaculture systems) by damaging or
clogging their gills.

• Examples:
– Diatom: Chaetoceros convolutus,
– Dinoflagellate: Gymnodinium mikimotoi,
– Prymnesiophytes: Chrysochromulina polylepis ,
Prymnesium parvum, P. patelliferum ,
– Raphidophytes: Heterosigma carterae , Chattonella
Overview 3
• Chaetoceros convolutus
Overview 3
Gymnodinium mikimotoi,
Proliferations of microalgae in marine or brackish waters can cause 1)massive
fish kills, 2)contaminate seafood with toxins, and 3)alter ecosystems in
ways that humans perceive as harmful.

A broad classification of HABs distinguishes two groups of organisms:

1. the toxin producers, which can contaminate seafood or kill fish, and
2. the high-biomass producers, which can cause anoxia and
indiscriminate kills of marine life after reaching dense concentrations.
Some HABs have characteristics of both.

Although HABs occurred long before human activities began to transform

coastal ecosystems, a survey of affected regions and of economic losses
and human poisonings throughout the world demonstrates very well that
there has been a dramatic increase in the impacts of HABs over the
last few decades and that the HAB problem is now widespread, and
serious. It must be remembered, however, that the harmful effects of HABs
extend well beyond direct economic losses and impacts on human health.
When HABs contaminate or destroy coastal resources, the livelihoods of
local residents are threatened and the sustenance of human populations is
• While harmful algal blooms, in a strict sense, are completely
natural phenomena which have occurred throughout
recorded history, in the past two decades the public health
and economic impacts of such events appear to have
increased in frequency, intensity and geographic distribution.

– One example, the increased global distribution of paralytic shellfish

• Until 1970, toxic dinoflagellate blooms of Alexandrium (Gonyaulux)
tamarense and Alexandrium (Gonyaulux) cutenella were only known
from temperate waters of Europe, North America and Japan (Dale and
Yentsch, 1978).
• By 1990, this phenomenon was well documented from throughout the
Southern Hemisphere, in South Africa, Australia, New Zealand, India,
Thailand, Brunei, Sabah, the Philippines and Papua New Guinea.

– Other species of the dinoflagellate genus Alexandrium, such as A.

cohorticulu and A. minutum, as well as the unrelated dinoflagellates
Gymnodinium catenatum and Pyrodinium bahamense var.
compressum have now also been implicated.
PSP- cases
in 1970 and
DSP- cases
in 1990 and
World distribution of reported presence of okadaates and/or
pectenotoxins in shellfish associated with the occurrence
Dinophysis spp.

Harmful Algae Volume 14 (2012) 87 - 106

ASP- cases
in 1990 and
Global distribution of ciguatera (CFP)
Occurrence of
PSP toxins
along the
coast in the
period 1991-
Japan 2002 report on “Harmful Algae Events”. DSP closures were
reported from areas JP-01, JP-02 (maximum) and JP-03. There were no
DSP outbreaks on the western coast despite the occurrence of
Dinophysis populations. Note the spatial segregation between the hot
spot region (JP-02) for DSP outbreaks (low biomass HABs) and those
(JP-03, JP-04, JP-05) with predominance of red tides (high biomass

Harmful Algae Volume 14 2012 87 - 106

Occurrence of
DSP toxins
along the
coast in the
period 1991-
Occurrence of ASP
toxins along the
european coast in
the period 1991-
Occurrence of azaspiracid
toxins along the european
coast in the period 1991-2000
Current situation

• Unfortunately, there are very few long term records of algal blooms at any
single locality. Probably the best data set refers to the concentration of PSP
toxins (µg saxitoxin equivalent / 100 g shellfish meat) in Bay of Fundy
(Canada) clams, which has been monitored by mouse bioassay since 1944
(White, 1987). Shellfish containing more than 80 µg PSP / 100 g shellfish
meat are considered unfit for human consumption.

• The Fig. shows evidence for a cyclic pattern of toxicity at this site with
increased frequency of toxic blooms in the late 1940s early 1960s, in the
late 1970s and early 1980s and possibly beginning again in the mid 1990s

• The issue of a global increase in harmful algal blooms

has been a recurrent topic of discussion at all major
conferences dealing with harmful algal blooms
(Anderson, 1989; Hallegraeff, 1993; Smayda, 1990).

• Four explanations for this apparent increase of algal

blooms have been proposed:
– increased scientific awareness of toxic species;
– increased utilisation of coastal waters for aquaculture;
– stimulation of plankton blooms by cultural eutrophication and/or
unusual climatological conditions; and
– transport of dinoflagellate resting cysts either in ships’ ballast
water or associated with translocation of shellfish stocks from
one area to another.
Case study: spreading of Prorocentrum
• Prorocentrum minimum was
documented for the first time in
Scandinavian waters during a
mass occurrence in the Oslofjord
in 1979.

• In the Kattegat it was first

observed in 1981.

• Since then P.m. has successively

increased its distribution into the
Baltic Sea. By 1984 it was found
north of the Island of Gotland. Cell When old plankton samples from
numbers in the open sea have the Norwegian coast were
been low, usually below 300 cells reanalysed, it became evident that
per liter.
P.m. occurred in this area before
1979, but was misidentified as P.
• In Danish fjords, Kiel Bight and
the mouth of the rive Warnow ( balticum. In the Oslofjord, which has
Germany), large blooms have been monitored extensively since
been recorded. Since then 1930, P.b. is recorded, but in
repeated massive blooms in the comparatively low concentrations.
Oslofjord with max concentrations
of several hunderd million cell per Real blooms were not reported untill
liter have occurred. 1979.
Case study: change of red tide in Galicia
• Only four red tides had been reported in Galicia
before 1976, and three of them were produced by
the non-toxic species Gonyaulax polyedra (1916,
1917, 1955).
• During the 50’s, phytoplankton of the Ria de Vigo
was intensively studied for several years and only
reported two red tides, one involving Ceratium
furca and the other G. polyedra, accompanied by
G. diacantha and G. spinifera and the ciliated
Mesodinium rubrum.
G. polyedra

•Phytoplankton was not studied on a regular basis again until 1977,

following an outbreak of PSP in 1976. Since then many red tides have been
reported, on the order of several per year. Protogonyaulax tamarensis (=
Alexandrium tamarense) has now become a regular species.
• Case history: geographic spreading
Southern New England (USA) has its recorded major red
tide outbreak in sept 1972, after hurrican “Carrie” which
disrupted the normal late summer water stratification. A
nutrient rich, well mixed environment resulted which is
highly suitable for dinoflagellate reproduction (up to 23x
106/l). Analysis confirmed the presence of PSP caused
by Protogonyaulax (Alexandrium) tamarensis var.
Excavata, which has since then been spreading south.

• Case history: spread by ships’ ballasting

The large chain-forming dinoflagellate
Gymnodinium catenatum, was never observed
during plankton monitoring in Tasmanian waters
in het period 1945-1980. It appeared first in
1980, and recurrent bloom have been noticed
ever since. Previously the species was only
known in Gulf of California, Argentina and Spain
• Cargo vessel ballast water was first suggested
as a vector in the dispersal of non-indigenous
marine plankton some 90 years ago. However, in
the 1980s the problem of ballast water transport
of plankton species gained considerable interest
when evidence was brought forward that non-
indigenous toxic dinoflagellate species had
been introduced into Australian waters into
sensitive aquaculture areas, with disastrous
consequences for commercial shellfish farm
operations (Hallegraeff and Belch, 1992).

• While the planktonic stages of diatoms and dinoflagellates show

only limited survival during the voyage in dark ballast tanks (Rigby
and Hallegraeff, 1994), their resistant resting spores are well
suited to survive these conditions.
– One single ballast tank thus was estimated to contain more than 300
million toxic dinoflagellate cysts which could be germinated into
confirmed toxic cultures.

• While the planktonic stages of diatoms and dinoflagellates show

only limited survival during the voyage in dark ballast tanks (Rigby
and Hallegraeff, 1994), their resistant resting spores are well
suited to survive these conditions.
Pellicle cyst


Harmful Algae Volume 14 2012 10 - 35

Schematic representation of the life cycle of heterothallic Alexandrium

species. Species have a haplontic life cycle, i.e. the motile vegetative
cells (1) are haploid. Under specific conditions, usually related to stress,
some vegetative cells can transform into a non-motile pellicle cyst (2) that
can rapidly switch back to the motile stage when conditions improve. The
sexual phase starts with the formation of gametes (3), which conjugate
(4) and form a diploid planozygote (5). Depending on environmental
conditions, the planozygote can transform into a resting cyst
(hypnozygote (6) or, for some species, can undergo meiosis and produce
a vegetative cell (1). Cysts can spend variable periods of time in the
sediments and, upon germination, release a motile cell termed a
planomeiocyte (7) which divides to produce vegetative cells (1).
• Many marine phytoplankton species produce dormant cysts or
resting spores during their life histories.

• Alternation between a dormant, benthic stage and a motile,

vegetative existence is a complex process that must be considered
in our efforts to understand and manage blooms of harmful algal
• Cyst germination provides an inoculum for many blooms, and cyst
formation can subsequently remove substantial numbers of cells in
later stages.

• Such cells have other important ecological roles with respect to

species dispersal, survival through adverse conditions, and genetic
recombination when sexuality is involved in their formation (Wall,
1971). Among the toxic or harmful marine phytoplankton species are
many that use this life history strategy.
• Most toxic or harmful species reproduce by asexual, binary division.
• Under certain conditions, however, sexuality is induced, involving a
series of developmental events that produce morphologically and
physiologically distinct cell types called gametes, zygotes, and
hypnozygotes (reviewed in Pfiester and Anderson, 1987).

• The term “cyst” is used to describe a non-motile cell which lacks

flagella and an ability to swim.

• Dinoflagellates form two different types of cysts –

– temporary cysts and
– resting cysts.
• The term “cyst” will refer to “resting cyst” or hypnozygote. The terms
“germination” and “excystment” will be used synonymously, as will
“cyst formation” and “encystment”.
Temporary cysts
• This non-motile cell is formed when motile, vegetative cells are
exposed to unfavorable conditions such as mechanical shock or a
sudden change of temperature or salinity. They are typically round
or oval-shaped protoplasts liberated by thecal rupture (ecdysis).
Initially, cell contents are the same as those of vegetative cells, but
through time, starch grains become apparent and pigments break
down and change their cellular distribution (Anderson, 1980).

• Temporary cysts are frequently observed in laboratory cultures,

especially in stationary growth phase. They are occasionally
observed in natural plankton samples, although it is always difficult
to ascertain whether the cysts were present naturally, or were
formed by the stresses of the sampling. When conditions become
favorable again, temporary cysts quickly re-establish a vegetative,
motile existence. The dormancy interval thus allows them to
withstand short-term environmental fluctuations. All planktonic
species can have a temporary cyst stage, and for most, this stage is
unrelated to the reproductive process.
Resting cyst
• This thick-walled, highly-resistant stage is occasionally
formed in cultures and routinely occurs in natural
plankton populations, often towards the end of a bloom
(Anderson et al., 1983; Lewis et al., 1979).

• Resting cyst formation begins with the sexual fusion of

gametes, which produce a swimming zygote
planozygote) that remains in the plankton for several
days before falling to the sediment as a non-motile cyst
(termed a hypnozygote). Under favorable conditions,
cysts can remain viable in sediments for 5-10 years,
sometimes even longer.
Dormancy vs. Quiescence
• It is important to use dormancy terminology with care. The literature
on seeds of higher plants defines
– “dormancy” as the suspension of growth by active endogenous
inhibition, and
– “quiescence” as the suspension of growth by unfavorable environmental
(i.e. exogenous) conditions. Thus dormant cysts cannot germinate, even
under optimal environmental conditions, while quiescent cysts are
competent to germinate, but are inhibited from doing so by some
environmental factor.
• Most cysts must proceed through a mandatory resting period
(lasting weeks to months, depending on species) before they are
capable of germination.
– This interval is generally considered a time for physiological
“maturation” (Pfiester and Anderson, 1987).
– The length of this mandatory interval varies considerably among
species (12 hrs to 6 months; Pfiester, 1977; Anderson, 1980), and for a
single species, can vary with the storage temperature as well.
– Thus cysts of A. tamarense stored at 4°C mature in 4-6 months,
whereas storage at warmer temperatures shortens the mandatory
interval to 3 months or less (Anderson, 1980).
Dormancy vs. Quiescence
• The duration of this process can have a significant effect on the
timing of recurrent blooms, as species with a long maturation
requirement may only seed one or two blooms per year, whereas
those that can germinate in less time may cycle repeatedly between
the plankton and the benthos and contribute to multiple blooms in a
single season.

• Recent study, however, suggests that some species such as

Gymnodinium catenatum and Pyrodinium bahamense may not
require this maturation period (Blackburn et al., 1989). Once a cyst
is mature and the dormancy interval is over, the resting state will
continue if external conditions are unfavorable for growth. Thus a
quiescent cyst cannot germinate until an applied external constraint
(such as cold temperature) is removed.
• Paralytic shellfish poisoning was unknown from the Australian
region until the 1980s when the first outbreaks appeared in the
ports of Hobart (Gymnodinium catenatum), Melbourne
(Alexandrium cutenella) and Adelaide (A. minutum).
• In Hobart, Tasmania, an examination of historic plankton samples,
cyst surveys in dated sediment depth cores (McMinn et al.,
unpublished) provided strong circumstantial evidence that the
toxic dinoflagellate G. catenatum was introduced after 1973.
• Furthermore, in Melbourne and Adelaide, genetic fingerprinting
using rRNA sequencing provided circumstantial evidence for the
genetic affinities between
– Australian and Japanese strains of A.cutenella and
– Australian and European strains of A. minutum (Scholin et al., 1993).
Some examples
• The impact of harmful microalgae is particularly evident when
marine food resources,e.g. aquacultures, are affected.

• Shellfish and in some cases finfish are often not visibly affected by
the algae, but accumulate the toxins in their organs. The toxins may
subsequently be transmitted to humans and through consumption of
contaminated seafood become a serious health threat.

• Although the chemical nature of the toxins is very different,

– they do not generally change or reduce significantly in amount upon
– neither do they generally influence the taste of the meat.

• Unfortunately, detection of contaminated seafood is not

straightforward, and neither fishermen nor consumers can usually
determine whether seafood products are safe for consumption. To
reduce the risk of serious seafood poisoning
– intensive monitoring of the species composition of the phytoplankton is
required in the harvesting areas
– in connection with bioassays and/or chemical analyses of the seafood
• In addition to posing serious health risks to consumers of seafood, some
microalgae may have devastating effects on fish and other marine life, both
in wild and aquacultures.

• Several species of micro algae belonging in different taxonomic groups can

produce toxins which damage fish gills by hemolytic effects.

• This has resulted in extensive fish kills with major economic losses. A
comprehensive economic analysis of the global impact of harmful algal
events on the aquaculture industry is not available,
– but the economic losses from single event in North America and
especially Japan have on several occasions amounted to more
than US$ 10 million. On one particular occasion the
raphidophyte flagellate Chattonella antiqua killed US$ 500
million worth of caged fish in Japan.
• In developing countries, seafood often constitutes an important or
even sole source of food and protein, especially in costal areas.

• With the increasing problems of overfishing, aquaculture may

become an increasingly important alternative for the supply of

• However, to minimize the risk of sea-food poisonings and the risk of

major economic losses due to fish kills, it is important to establish
– surveillance programmes and
– quality control of the seafood products
• which will often require expert assistance from countries which have
longstanding experience in this matter.
• Particularly in the tropics, people are often harrassed by diseases
and syndroms due to consumption of seafood contaminated by algal

• Some of these diseases may be fatal. There is currently no

international record of the number of incidents of human intoxication
caused by contaminated seafood.

• The numbers appearing in presentations at international meetings

are undoubtedly underestimates, as many cases and even fatalities
can be assumed to pass undiagnosed and hence unreported in the
official reports.
• Five human syndroms are presently
recognized to be caused by consumption
of contaminated seafood:
– paralytic shellfish poisoning - PSP
– diarrhetic shellfish poisoning – DSP
– neurotoxic shellfish poisoning – NSP
– ciguatera fish poisoning - CFP
– amnesic shellfish poisoning - ASP
Paralytic shellfish poisoning - PSP

• This is a life-threatening syndrome with

neurological effects.
• There is no known antidote to PSP. The known
global distribution has increased markedly over
the last few decades. Each year about 2000
cases of PSP are reported with 15 % mortality.
• Mild case: tingling sensation in fingertips,
• extreme case: muscular paralysis, death
Diarrhetic shellfish poisoning - DSP
• This is a wide spread type of shellfish poisoning which causes
gastrointestinal disturbances with diarrhea,
– vomiting, and
– abdominal cramps
– within 30 min to 12 h. (recovery after 3 days irrespective of medical

• If is not fatal, and the patients usually recover within a few days.

• There are thousands of reported incidents from developed countries,

e.g. 5000 in Spain in 1981 alone, but with the pathological picture of
DSP, many incidents may be regarded as an ordinary stomach
disorder, and therefore remain unreported.

• Chronic exposure to DSP is suspected to promote tumor formation

in the digestive system.
Neurotoxic shellfish poisoning-
• Until recently this syndrome has been restricted to the
Gulf of Mexico, but in 1993 it was reported also from
New Zealand.
• It is characterized by gastrointestinal and neurological
disturbances usually with recovery within a few days
(chills, headache, muscle weakness, joint pain).
• In severe cases:
– altered perception of cold and warm,
– breathing difficulties,
– double vision,
– trouble in talking and swallowing
• Toxic aerosols formed by wave action may cause
asthma-like symptoms.
Ciguatera fish poisoning - CFP
• This poisoning, transmitted by several tropical reef fish, is generally not
lethal, although fatalities have been documented.

• Ciguatera produces
– gastrointestinal,
– neurological and
– cardiovascular disturbances,
• and recovery often takes months or even years.

• It is widely distributed in the tropics; thus in the period 1960-1984, there

were a total of 24.000 cases of ciguatera in French Polynesia alone.

• Evidence is accumulating that disturbances of coral reefs by hurricanes,

tourist activity etc. increase the risk of ciguatera by providing more suitable
habitats for the benthic dinoflagellates (see causative organisms).
• There is at present no easy method to routinely measure the toxins
(ciguatoxin and maitotoxin) that cause ciguatera fish poisoning.
Amnesic shellfish poisoning -ASP

• This syndrome can be life-threatening.

• It is caused by domoic acid that accumulates in shellfish, but the

disease can apparently also be fish borne, so the risk to humans
may be more serious than previously believed.

• It is characterized by gastrointestinal and neurological disorders

including loss of memory, hallucination and confusion

• Human ASP intoxication is presently known primarily from Canada,

but the causative diatoms occur in many parts of the world, so
considerable care should be exercised during blooms of species of
the diatom Pseudo-nitzschia.

• Many of the syndromes and other harmful

effects pertain to occurrences of
• There is, however, an increasing number
of species recognized as toxic in other
algal classes, and harmful species are
now found in at least 5 groups of algae
Dinophycae (= Dinoflagellates)

• Species of Dinoflagellates are responsible

for, PSP, DSP, NSP, and ciguatera.
Dinophycae (= Dinoflagellates):
• PSP is caused by several species.

• Most cases are caused by

– Pyrodinium bahamense var. compressum and
– Alexandrium species .

• The latter genus comprises about 20 species

which are difficult to identify. They are
distinguished by small differences in the plate
structure of the plates covering the cell.
Pyrodinium bahamense var.
Dinophycae (= Dinoflagellates):PSP

Alexandrium minutum
• Cells of Alexandrium minutum, size between 17
et 29 µm.

• The genus Alexandrium counts about twenty

species; a lot of them occur in European waters

• Most of these species are non toxic except for

– A. ostenfeldii
– A. tamarense (not frequent in European waters) and
– Alexandrium minutum.
Fig. 1 Phylogenetic tree inferred by maximum likelihood analysis of
partial LSU rDNA (D1?D2 domains) of 21 nominal species of
Alexandrium . Analysis includes a subset of taxa included in the
maximum likelihood phylogenetic...
Donald M. Anderson , Tilman J. Alpermann , Allan D. Cembella , Yves Collos , Estelle Masseret , Marina Montresor

The globally distributed genus Alexandrium : Multifaceted roles in marine ecosystems and impacts on human health

Harmful Algae Volume 14 2012 10 - 35
lexandrium andersonii ( ), A. minutum ( ), A. tamutum ( ), A. peruvianum/A. ostenfeldii ( ), A. insuetum ( ), A. margalefii ( ), A. pseudogonyaulax ( ), A. taylori ( ), A. affine ( ),
A. catenella Group VI (♦), A. tamarense Group II (□), and Group III (▵).

Distribution of Alexandrium species in the Mediterranean Sea, modified

from Penna et al. (2008). Open circles represent the sampled stations.
Colored circles, square, triangle, and diamond symbols represent the
species found by Penna et al. (2008) or by other authors, as defined and
based on nucleotide sequences and morphology Alexandrium
andersonii (), A. minutum (), A. tamutum (), A. peruvianum/A.
ostenfeldii (), A. insuetum (), A. margalefii (), A. pseudogonyaulax (), A.
taylori (), A. affine (), A. catenella Group VI (♦), A. tamarense Group II (□),
and Group III (▵).
Harmful Algae Volume 14 2012 10 - 35
Saxitoxin and gonyautoxin
Saxitoxins: characteristics and mode of action

• Saxitoxins are tricyclic compounds. They are molecules with two

guanidino groups with pKa's of 11.3 and 8.2, respectively. At
physiological pH then, the first-guanidino carries a positive charge,
whereas the second-guanidino group is partially deprotonated.
• Because of this polar nature, the saxitoxin molecule readily
dissolves in water and lower alcohols but is insoluble in organic

• It is stable in solution at neutral and acidic pH's, even at high

temperatures, but alkaline exposure oxidizes and inactivates the
• There are a number of STX variants generally divided into groups
based on their structure or organism of origin.
– The single sulphated STXs are known as gonyautoxins (GTX) and B-
– the doubly sulphated STXs are known as C-toxins.
– STX was the first known and has been the most studied toxic component
of paralytic shellfish poisoning (PSP).
Saxitoxin and its naturally
occurring relatives

Carbamoyl Saxitoxins

R1 R2 R3 R4 Rel.toxicity

Saxitoxin 35523-89-8 H H H OCONH2 1

Gonyautoxin 0.36
60508-89-6 H OSO3- H "

60537-65-7 H H OSO3- "

Neosaxitoxin 64296-20-4 OH H H "

Gonyautoxin I 60748-39-2 OH OSO3- H " 0.99
64296-26-0 OH H OSO3- " 0.73
Toxity of PSP molecules
Decarbamoyl Saxitoxins

Registry Rel. toxicity

Name R1 R2 R3 R4

Decarbamoyl 0.51
58911-04-9 H H H OH

86996-87-4 H OSO3- H " 0.65
gonyautoxin II

Decarbamoyl 0.75
gonyautoxin 87038-53-7 H H OSO3- "

Decarbamoyl No data
68683-58-9 OH H H "
Toxity of PSP molecules
Sulfamate Saxitoxins

Derivative Registry Rel. toxicity

R1 R2 R3 R4
Designation Number

B1 64296-25-9 H H H OCONHOSO3- 0;06

C1 80173-30-4 H OSO3- H " < 0.01

C2 80226-62-6 H H OSO3- " 0.1

B2 82810-44-4 OH H H " No data

Gonyautoxin 0.01
89614-45-9 OH OSO3- H "

Gonyautoxin 0.06
89674-98-6 OH H OSO3- "
Saxitoxins: characteristics and mode of
• This toxin blocks neuronal transmission by binding to the voltage-
gated Na+ channels in nerve cells, thus causing their neurotoxic
• STXs are highly toxic, killing guinea pigs at only 5 µg/kg when
injected i.m.

• The lethal doses for mice are very similar with varying adminstration
routes: i.p. (LD50 = 10 µg/kg), i.v. (LD50 = 3.4 µg/kg) or p.o. (LD50
= 263 µg/kg).

• The oral LD50 for humans is 5.7 µg/kg, therefore

– approximately 0.5 mg of saxitoxin is lethal if ingested and
– the lethal dose by injection is about ten times lower.
– The human inhalation toxicity of aerosolized saxitoxin is estimated to be
5 mg/min/m3.

• Saxitoxin can enter the body via open wounds and a lethal dose of
0.05 mg/person by this route has been suggested. Saxitoxin is 1,000
times more toxic than the potent nerve gas sarin
Saxitoxins: characteristics and mode of action (f)
• Saxitoxin is a potent neurotoxin that specifically and selectively binds the
sodium channels in neural cells. Thus, it physically occludes the opening of
the Na+ channel and prevents any sodium cations from going in or out of
the cell. Since neuronal transmittance of impulses and messages depends
on the depolarization of the inside of the cell, the action potentials are
stopped, impairing a variety of bodily functions, including breathing.

• Human nerves are especially sensitive to the toxins and in the early stages
of PSP, victims experience
– tingling and numbness of the mouth, tongue, face and extremities. Nausea and
vomiting may accompany the above symptoms.
– In severe cases, the patient will exhibit advanced neurological dysfunction such
as ataxia, weakness, dizziness, numbing of the lips, mouth and tongue, fatigue,
difficulty breathing, and sense of dissociation followed by complete paralysis.
The diaphragm may stop working and death can occur after cardio-respiratory
failure. Symptoms occur between 10 minutes and four hours after ingestion,
depending on the dose.

Identification of saxitoxin as a cause of intoxication by virtue of clinical
symptoms is not simple, because faulty identification of this toxin as nerve
gas poisoning may be fatal and administration of atropine would increase
fatalities. The laboratory detection and identification of compound is difficult
and is possible only in a well-provided analytical laboratory.
Dinophycae (= Dinoflagellates):DSP

• DSP is caused by several species of Dinophysis and

some species of Prorocentrum.

• Dinophysis is a large genus with some 200 described

species many of which pose considerable taxonomic

• Species are distinguished only by morphological features

such as size, shape, presence/absence of chloroplasts

• Thus the circumscription of many species is vague and a

comprehensive taxonomic revision of this genus is
Dinophycae (= Dinoflagellates):DSP

Diarrhetic shellfish toxins originate in

dinoflagellates, specifically

Prorocentrum lima,
Prorocentrum elegans,
Prorocentrum offmannianum
Prorocentrum concavum,
Dinophysis acuminata,
Dinophysis acuta,
Dinophysis fortii, (pectenotoxin)
Dinophysis hastata,
Dinophysis mitra,
Dinophysis rotundata,
Dinophysis norvegica and some strains
of Dinophysis tripos.
Protoceratium reticulatum (Yessotoxin) Dinophysis
DSP molecules
is a complex
lipophilic polyether
readily soluble in
many organic
solvents, degrading
in acid or base.

Okadaic group
• Diarrhetic shellfish poisoning (DSP) was first reported from the Tohoku
district in Japan.

• Since then, reports of DSP have emerged from every continent except
Africa and Australia.
• DSP has never resulted in a human fatality.

• As well as diarrhoea, other gastrointestinal symptoms include vomiting,

nausea and abdominal cramps, possibly becoming so severe that the
patient is incapacitated.

• Treatment usually is to simply make the patient as comfortable as possible

for the duration of the intoxication. Symptomatic treatment for severe
diarrhoea such as fluid replacement should be employed. Common anti-
diarrheals would provide little relief to DSP victims because much of the
diarrhetic effect is caused by epithelial destruction.

• Apart from this acute effect, chronic exposure may promote cancer as it
enhances skin tumours on mice when applied after a known carcinogen.
Mode of action of okadaic acid

•Okadaic acid potently inhibits serine/threonine phosphatases, enzymes

which dephosphorylate serine and threonine residues of other enzymes
receptors, switching them off or on as the case may be.


Dinophycae (= Dinoflagellates):NSP

• NSP is caused by Karenia (Gymnodinium) breve

• In addition to the human syndromes caused by

dinoflagellates, extensive fish kills have been
caused by several so-called unarmoured
species belonging in the genera Amphidinium,
Cochlodinium, and Gymnodinium.

• Identification of these is generally difficult and

requires examination of live cells.
Characterising algal blooms:
species and toxins involved, NSP

Gymnodinium breve
NSP molecules: brevetoxin
Brevetoxin (f)
• Brevetoxins (BVX) are neurotoxins produced by algae called Karenia brevis
(also called Ptychodiscus brevis and formerly Gymnodinium breve) from
which the toxin name is derived. The algae proliferate during red tide

• Brevetoxins and related toxins are believed to have been responsible for
massive fish kills from red tides in several regions. A long history of toxic
microalgal blooms exists in the Gulf of Mexico, blooms that have caused
massive fish kills and respiratory irritation in humans.

• It was later realized that the toxin in these blooms could also be passed to
humans via shellfish to cause a syndrome named neurotoxic shellfish
poisoning (NSP). Until cases were reported in New Zealand and Australia in
the early 1990s, reports of NSP were limited to the Americas.

• Victims of NSP can be misdiagnosed as suffering the fish-poisoning

syndrome caused by ciguatera. Typical symptoms are tingling in the face,
throat and digits, dizziness, fever, chills, muscle pains, abdominal cramping,
nausea and vomiting, diarrhea, headache, reduced heart rate and pupil
dilation. There have been no reported fatalities from NSP, although the toxin
kills test mammals when administered by various routes, including orally.
Brevetoxin (f)
• The brevetoxins are lipophilic 10- and 11-ring polyethers with molecular
weights around 900 Da [37]. There are two classes of brevetoxins,
– the first contains eight 6-membered rings and two heptameric and an 8-
membered ring (A type I brevetoxin,).
– The second class of brevetoxins has only 10 rings, with variation in the size of
the rings ranging from five bonds to nine bonds (A type II brevetoxin,)

These toxins depolarize and open voltage gated sodium (Na+) ion channels in
cell walls, leading to uncontrolled Na+ influx into the cell . Brevetoxins bind
to the ion channels of nerve and muscle tissue that selectively allows
sodium to pass into the cell. These sodium channels open during an action
potential in response to the change in the electrical potential across the cell
membrane. Brevetoxins change the voltage at which this opening occurs
nearer to the voltage threshold that triggers this process essentially making
the sodium channel, and consequently, the affected nervous and muscular
cells hyperexcitable.

Brevetoxins are unusually stable materials in the dry state. They are stable as
well as in different solvents (acetone, acetonitrile, alcohol, ethyl acetate or
DMSO), including water, where half-lives for active material range from 4-6
months. Solutions with a pH lower than 2 or higher than10 degrade the
• Ciguatera is caused by Gambierdiscus
toxicus, and
• perhaps also by some species of
Ostreopsis and Prorocentrum. Most of
these species can readily be identified by
trained taxonomists.
Gambierdiscus toxicus
Characterising algal blooms:
species and toxins involved, CFP

CFP (f)
• The term ciguatera originated in the Caribbean area to designate
intoxication induced by the ingestion of the marine snail Turbo pica (called
cigua), first described by a Cuban ichthyologist.

• Today, the term is widely used to denote a particular type of fish poisoning
that results from ingestion of primarily reef fishes encountered around
islands in the Caribbean and the Pacific.
• Current information points to one of the many polyether toxins, such as
ciguatoxin and related compounds, which are structurally similar to okadaic
• Ciguatoxin is produced by the Dinoflagellate Gambierdiscus toxicus and has
been isolated from the flesh and viscera of ciguatoxic fish.

• Ciguatoxin is not a single compound, but a class of compounds. At

present, 24 related ciguatoxins are known and these were found in different
fishes from the Pacific. They are low molecular weight, lipid polyethers.
They stimulate the enhancement of sodium ions through cell membranes
(nerve or muscle cells). In this way, the toxins affect the cells and create the
clinical symptoms seen in man.

CFP (f)
Ciguatoxin is regarded as a neurotoxin, but the clinical symptoms of ciguatera
poisoning can be classified into four broad groups: neurologic, cardiovascular,
gastrointestinal and general symptoms. Symptoms usually begin within10 minutes to
12 hours, but can occur up to 36 hours after eating a poisonous fish. The disease
commonly begins with nausea, vomiting and diarrhea, generalized weakness, a
decreased sensation to pain or touch, unusual or painful sensations produced by
ordinary stimuli, a burning or tingling of the hands and legs or around the mouth,
muscle pain and temperature reversal sensation (hot things feel cold and cold things
feel hot). Other less common symptoms include: chills, itching, dizziness, sweating,
headache and taste disturbances (a metallic taste or fuzzy sensation).
The nausea, vomiting, and other gastrointestinal symptoms last for
approximately 1 to 2 days. Weakness may last for 1 to 7 days. Neurologic symptoms
such as tingling or temperature reversal generally persist for up to a week, but it is
not unusual for these symptoms to periodically re-occur for a month or more. The
poisoned victim may note an increased or decreased heart rate. Medical personnel
may also note low blood pressure, dilated pupils, and irregular heart rhythm. These
symptoms resolve in two to three days [42].

• Examination of the clinical symptoms in patients with pufferfish, shellfish (red

tide due to dinoflagellates) and polyether type toxin (ciguatoxin, okadaic acid,
brevetoxin and other polyether) poisonings shows that the symptoms overlap and the
causative toxins cannot be discriminated. In other words, there is no unique feature
that separates the clinical effect. The temperature reversal was supposedly unique
for ciguatoxin. Ciguatera fish poisoning is probably more important than any other
form of seafood poisoning. Its epidemiology is complex and it is impossible to predict
outbreaks. The ciguatoxins are not destroyed by cooking and, if consumed in
sufficient dose, can cause symptoms persisting for weeks, months or years [43].
Bacillariophycae (= Diatoms)
• ASP is caused by
– Pseudo-nitzschia australis,
– P. multiseries, and perhaps
– P. pseudodelicatissima.

• The two latter species are widely distributed in

both the Northern and Southern Hemispheres,
while the former occurs mostly in the Southern
• Species identification is difficult often requiring
electron microscopy, and misidentifications
probably occur in the literature.
Characterising algal blooms:
species and toxins involved, ASP

Pseudo-nitzschia australis

Pseudo-nitzschia multiseries
• Domoic acid (DA), was originally isolated from the
macroscopic red alga Chondria armata, locally known in
Japan as domoi.

• It was later identified as the cause of an unusual shellfish

poisoning syndrome, amnesic shellfish poisoning, that
first occurred on Prince Edward Island in Canada. The
source of the toxin was the diatom Pseudo-nitzschia
(previously Nitzschia) pungens forma multiseries.

• Other DA producing diatoms include Pseudo-nitzschia

seriata, P.multiseries, P. australis, P.
pseudodelicatissima, P.delicatissima, and P. turgidula
Domoic acid
• Domoic acid (MW=311; C15H21NO6) is a
tricarboxylic acid similar to the glutamate
receptor agonist kainic acid, being cyclised
analogues of L-glutamate.

• It is only mildly toxic to mammals, with an LD50 to

mice (intra-peritoneally) of 3.6 mg / kg.

• It is polar making it soluble in water and insoluble

in organic solvents. The three carboxylic acids
have pKa's of 2.1, 3.7 and 5.0 with the amino
group in the pentameric ring having a pKa of 9.8.
Domoic acid: ASP
Domoic acid
• Domoic acid is a potent amino acid neuroexcitant that acts
preferentially upon a sub-class of ionotropic glutamate receptors
found in nervous tissue, particularly the brain.

• These glutamate receptors are gated ion channels, which respond

to L-glutamate by opening and allowing the passage of cations.

• This action triggers many intracellular cascades either directly by

their introduction of cations or indirectly by the second messengers
produced by these cascades such as cyclic AMP, or reactive oxygen

• Induction of neuronal imbalance can cause the brain to malfunction

and may lead to lesions in the brain that can cause permanent

Cyanophycae (= Blue-green algae)

• In the marine environment, only few

species of blue-green algae cause
problems, e.g. species of
Trichodesmium and Nodularia, and
these have not been associated with
human syndroms so far.
• A wide variety of blue-green algae
cause serious problems in fresh and
brackish water environments and may
be a serious health problem where
surface water is used for drinking water
Harmful effect associated with marine microalgal blooms

• Marine fauna mortalities due to

– Physical damage (gills)
– Oxygen depletion
– Chemical; direct action or indirect action

• Human intoxications
– PSP known since 1793
– DSP known since 1978
– NSP known since 1970s
– ASP known since 1987
– CFP known since 16th century
Basic type of phytoplankton toxins
• Water soluble toxins
– Saxitoxin family (causing PSP, at least 18 forms)
– Some amino acids (domoic acid, mycosporine-like
amino acids
• Fat soluble toxins
– Okadaic acid and its derivatives
– pectenotoxins, DSP
– Polyethers: brevetoxins (NSP), ciquatoxin, ciguatera
– Yessotoxine
– Fatty acid and glycolipids
– Peptides (hepatotoxins from cyanobacteria)
HAB and impact on shellfish
• The potential of direct impact of toxin is of great concern
• Some toxins however have only minor effects on shellfish physiology
• Other (including uncharacterised) seem to have a bigger impact
• Karenia mikimotoi:
• Has been associated with direct killing of pearl oysters
• In Mytilus galloprovencialis:
• Reduced filtration rate when exposed to 5x 105 cell per ml
• Death at 5 x 106 per ml
• No mode of action has been establised
Domoic acid and fish behavior
• Research and field observations have provided evidence that fish are more
tolerant to domoic acid under ecologically relevant exposure conditions than their
piscivorous predators.
• This is an important distinction as more attention has been drawn to domoic acid
producing algal blooms and the potential for domoic acid to cause fish kills.

• Currently available data indicate that domoic acid producing algal blooms do not
cause fish kills or neuroexcitotoxic behaviors in fish.
• Neuroexcitatory behavioral effects have been documented in fish in laboratory studies
when fish were intraceolomically (IC) injected with domoic acid. In fact, with IC injection
as the mode of exposure all fish, bird, and mammal species tested to date show a similar
neurologic sensitivity to domoic acid in terms of behavioral excitotoxicity as quantified
by a 50% effective concentration (EC50) metric.

• However, IC injection is not an ecologically relevant mode of exposure. Dietary

consumption during toxic blooms is the route of exposure for fish. Results from
oral exposure experiments and observations from multiple highly toxic bloom
events have provided strong evidence that fish are not behaviorally affected by
domoic acid during natural bloom conditions, even though fish regularly contain
high levels of the toxin and act as vectors to seabirds and marine mammals.
Collectively, the data presented in this review suggest that fish are not significantly
impacted by domoic acid during typical toxigenic Pseudo-nitzschia blooms.
HARMFUL ALGAE Volume: 13 Pages: 126-130 Published: JAN 2012
Cyanotoxins: Bioaccumulation and Effects on
Aquatic Animals
• Cyanobacteria are photosynthetic prokaryotes producing cyanotoxins.
• These toxins can be classified into three main types according to their mechanism
of action in vertebrates:
– hepatotoxins,
– dermatotoxins and
– neurotoxins.
– Many studies on the effects of cyanobacteria and their toxins over a wide
range of aquatic organisms, including invertebrates and vertebrates, have
– acute effects (e.g., reduction in survivorship, feeding inhibition, paralysis),
– chronic effects (e.g., reduction in growth and fecundity),
– biochemical alterations (e.g., activity of phosphatases, GST, AChE, proteases), and
– behavioral alterations.
– Research has also focused on the potential for bioaccumulation and
transferring of these toxins through the food chain. Although the herbivorous
zooplankton is hypothesized as the main target of cyanotoxins, there is not
unquestionable evidence of the deleterious effects of cyanobacteria and their
toxins on these organisms.
• Source: MARINE DRUGS Volume: 9 Issue: 12 Pages: 2729-2772
Intoxication and detoxification in juveniles of Mytilus chilensis
exposed to paralytic shellfish toxins
AQUATIC LIVING RESOURCES Volume: 24 Issue: 1 Pages: 93-98

• Juveniles of the mussel Mytilus chilensis were exposed to a diet containing

paralytic shellfish poisoning (PSP) toxins produced by the dinoflagellate
Alexandrium catenella (strain ACC02).
• The feeding behaviour and the dynamics of intoxication and detoxification
were evaluated over an intoxication period of nine days, followed by a
detoxification period of eight days.
– A significant reduction in the feeding activity was measured during the
first days of exposure to the PSP toxins (days 0 and 2), followed by a
period of recovery observed on days 5 and 9, when the clearance rate
of the contaminated mussels significantly increased.
– During the detoxification period, the contaminated bivalves showed a
total recovery of clearance rate, and no significant differences were
observed between contaminated and control groups.
– The lower clearance rates observed over the first days of exposure
would produce a decrease in the energy intake and could affect the
rate of growth of juveniles. Despite this initial effect, the rapid
intoxication capacity of M. chilensis corroborates that this species is a
good indicator for the early detection of harmful algal blooms.
Effects of Alexandrium minutum exposure on nutrition-related
processes and reproductive output in oysters Crassostrea gigas

• This study assessed the effects of an artificial bloom of the toxin-

producing dinoflagellate, Alexandrium minutum, upon nutrition related
processes and reproductive output of the Pacific oyster, Crassostrea gigas.
• Oysters were exposed to A. minutum, Paralytic Shellfish Toxins (PST)
producer and compared to a control batch of oysters fed Isochrysis
galbana clone Tahitian (T.Iso).
• Spermatozoa in oysters exposed to A. minutum were morphologically and
functionally modified compared to spermatozoa of control oysters.
• Indeed, spermatozoa were less motile and had lower ATP content in
oysters exposed to A. minutum. Meanwhile, spermatozoa produced by
control oysters showed higher percentage of mortality and relative DNA
content than those produced by A. minutum exposed oysters.
• The results of this study suggests that an exposure of oysters to A.
minutum, can have consequences on spermatozoa fertility and
reproduction success.
• HARMFUL ALGAE Volume: 9 Issue: 5 Pages: 427-439(

• Identification of the causative species

• When harmful algae occur, an immediate and essential

action is reliable identification of the causative species
involved for a first assessment of the potential risks.

• Many species are difficult to identify and advanced

taxonomic training is necessary.
– Application of modern taxonomic concepts requires in some
cases electron microscopy for critical identification, and this
equipment is not generally available in many countries

– There is often also a scarcity of scientific literature and/or limited

access to libraries which is essential for both monitoring and
scientific work.
Morphology and identification
• Dinoflagellates mainly reproduce asexually via binary fission, but
some species reproduce sexually and form resting cysts.

• Their nutrition varies from

– autotrophy (photosynthesis) to
– heterotrophy (absorption of organic matter) to
– mixotrophy (autotrophic cells engulf prey organisms). These features
are species-specific.

• Dinoflagellate species are adapted to a variety of habitats:

– from pelagic to benthic,
– from temperate to tropical seas, and
– from estuaries to freshwater.
• Many species are cosmopolitan and can survive in variety of
habitats: in the plankton, or attached to sediments, sand, corals, or
macroalgal surfaces.
• Some species produce resting cysts that can survive in sediments
for an extended period of time, and then germinate to initiate blooms
Morphology and identification
• Dinoflagellates exhibit a wide divergence in morphology
and size that are essential features used to identify
species, as well as

• surface ornamentation (pores, areolae, spines, ridges,

– Armored or thecate species, those that possess a multi-layered
cell wall, can be distinguished from
– unarmored or athecate species, those that lack a cell wall.

• Surface morphology of thecate cells, often critical to

proper identification, can be discerned after cell fixation.

• However, identification of athecate species is mainly

based on live cells since many morphological features
may be destroyed by fixation (Steidinger & Tangen
Morphology and identification
• Another distinction used in dinoflagellate identification is
morphological cell type (Fig. A, B):

– 1. desmokont type where two dissimilar flagella are inserted

apically (e.g. Prorocentrum); and
– 2. dinokont type where two dissimilar flagella are inserted
ventrally (e.g. Alexandrium).

• Terminology to describe orientation is also used: the

forward end when the cell moves is called the apical
pole; the opposite end is the antapical pole
features of
Dinoflagellate morphology
• Although dinoflagellates can display considerable morphological variation, most share a
common anatomical pattern during at least one stage of their life cycle. Most dinoflagellates
have two flagella inserted into their cell wall via the flagellar pore(s) at approximately the
same location. In most dinoflagellates one of the flagella wraps around the cell and is known as
the transverse flagellum, while the other, longitudinal flagellum, extends tangentially to the
cell, perpendicular to the plane of the transverse flagellum.

•The beating of the longitudinal flagellum

and the transverse flagellum imparts a
forward and spiralling swimming motion,
and defines anterior (the direction of
swimming) and posterior (opposite to
anterior and the direction the longitudinal
flagellum is directed).
•The flagellar pore and point of flagellar
insertion defines ventral with the
opposite side dorsal. Left and right sides
of the cell are then defined as in most
• A depression often occurs on the
ventral surface at the point of flagellar
insertion, and is known as the sulcus.
The transverse flagellum often occurs in
a furrow known as the cingulum which
encircles the cell except where
interrupted by the sulcus on the ventral
Dinoflagellate morphology
• The cell wall of
dinoflagellates is
subdivided into multiple
polygonal amphiesmal
vesicles of varying
numbers (from half a
dozen to hundreds).
• In some dinoflagellates,
these vesicles are filled
with relatively thick
cellulose plates with
bounding sutures. When
this occurs, the cell wall
is referred to as a theca.
Identification tools
• Understanding HAB: detection and enumeration of specific species is

• Microscopic ID is standard method. But: need for expertise.

• Another constraint arises from difficulties in identifying and

distinguishing among morphologically similar species or strains. This
is a problem not only for those with limited taxonomic training, but also
for skilled taxonomists, since considerable time and effort are required
to identify a taxon if the distinguishing characteristics are difficult to
discern under the light microscope.
– For example, certain species of Alexandrium, such as A. tamarense, A.
fundyense and A. ostenfeldii are difficult to distinguish reliably under
conventional microscopy, without detailed critical taxonomic observations
of individual cells. Such fine levels of discrimination are often not feasible in
monitoring programs or studies that generate large numbers of samples for
cell enumeration, a situation encountered frequently in studies of HABs.
Identification tools
• A common problem in monitoring
programs focused on phytoplankton
species occurs when the “species of
interest” is only a minor component of the
planktonic assemblage.
• Many potentially useful measurements are
not feasible because of co-occurrence of
numerous organisms of other taxa, as well
as detritus.
Identification tools
• There is an additional complexity in the fact that cellular-
or strain-specific toxicity does not always track
identification based upon morphospecific criteria.

– For example, there are both toxic and non-toxic variants of the
dinoflagellate Alexandrium tamarense.

– Among certain strains of the diatom Pseudo-nitzshia multiseries

with the capacity to produce domoic acid, the induction of toxin
production by physiological stress may be required (Bates,
– Furthermore, although the toxin profile (relative composition of
various analogues) is typically rather constant and presumably
genetically fixed within a strain, the cell quota of toxin may vary
dramatically in natural populations as a result of multiple
extrinsic and intrinsic factors (Wright and Cembella, 1998).
Identification tools
• As a result of these problems and constraints, the
scientific community has been working towards the
development of species- or strain-specific “probes” that
can be used to label only the cells of interest so they can
then be detected visually, electronically, or chemically.

• Progress has been rapid and probes of several different

types are now available for many of the harmful algae,
along with techniques for their application in the rapid
and accurate identification, enumeration, and isolation of
individual species.
ID tools: lectins
• Lectins are non-enzymatic secretory proteins
(glycoproteins), from terrestrial origin, that bind non-
covalently to specific sugar residues at cell surfaces.
They are sugar specific.

• They can be fluorescently labelled, so that after binding

to a cell surface they can be detected by epifluorescence
microscopy (e.g. excitation 490 nm, emission 520nm)

• Cell are brought on a filter, allowed to incubate with the

lectin (15 min) and washed afterwards

• A variety of labelled lectins is commercially available.

Lectins: case study
• Lectins have been used to discriminate in Spain
– between Gymnodinium catenatum (toxic PSP producer) and
Gymnodinium impudicum (non toxic). (Costas and Rodas 1994)

• Using a series of lectins to differentiate between

– toxic and non-toxic Pseudo-nitzschia species. (Rhodes 1998)

• Differentiation
– between the toxic A. tamarense/A. catenella and the non toxic A.
fraterculus (Cho et al., 1999)

• Remark:
– Binding can be growth phase specific, strain specific (e.g. diatoms),
– in other species it is constant (some dinoflagellates and cyanobacteria)
Lectin-aided detection

In this case Gymnodinium was stained with FITC –labelled lectin from
Phaseolus limensis (bean) (excitation 490 nm, emission 520 nm
ID tools: antibodies
• The approach involves the use of antibodies that bind
specifically to proteins in the cell walls of the algal
species of interest.

• Antibodies are produced by inoculating cells of target

species into animals, which then produce antibodies in
response to the presence of the intact foreign organism
or compounds derived from it. The target molecule
against which the antibody is directed (termed an
antigen) is typically, but not necessarily, a cell-wall
protein. Fortunately, it is not necessary to purify specific
proteins in order to produce antibodies.
• To date no commercial antibodies are available
Species specific antibodies
• Applications of antibody probe technology to field populations are
limited to date. Experience with a
– PAb for the brown-tide organism Aureococcus anophagefferens,
– a MAb for the fish-killing alga Karenia mikimotoi and
– another for Alexandrium spp.,
suggest that immunofluorescence has a major role to play in HAB
monitoring and research programs.

• The antibody for the brown-tide organism has been used for
– cell enumeration and grazing studies, and
– to map the geographic distribution of this species over a large region
(Anderson et al., 1993). This antibody is now used for routine monitoring
of this harmful species.
– A MAb to the same organism is now available, as well, and it is being
used for whole cell assays and in a semi-automated ELISA plate format
• Finally, a MAb has been applied in field studies of Alexandrium
tamarense in the Gulf of Maine, USA (Townsend et al., 2001).

– This antibody is used in a whole cell format in which samples are

labelled and then the species of interest is counted by epifluorescence

– The antibody approach has greatly accelerated the counting of the

many field samples that are collected on multiple cruises during the
bloom seasons, since the samples can be scanned at low magnification.

– During these studies, a problem has arisen due to unexpected labelling

of A. ostenfeldii with the antibody. At certain times, this species co-
occurs with A. fundyense/tamarense in the Gulf of Maine, so cell counts
of the latter can be inaccurate if antibody labelling is used as the sole
identification criterion. As a result, enumeration of both species now
requires specification of both cell size and morphological criteria (food
Antibodies (f)
• A novel application of antibody technology was reported by Aguilera
et al. (1996), who used magnetic beads coupled to a MAb to A.
tamarense to separate the cells of this species from mixed plankton
samples after fixation.

• A more recent development of this method achieved

immunomagnetic separation of living cells, allowing several
different types of physiological analyses to be conducted on a target
species (e.g., rates of primary production and enzymatic activity, cell
quota measurements of chlorophyll, protein, etc.).

• In a direct application of this approach, A. tamarense/ fundyense

cells have been immunomagnetically isolated from Gulf of Maine
field samples and assayed for urease activity.
– This technology thus has great potential for species-specific
physiological measurements on HAB species for which cell surface
antibodies are available
Antibodies: summary
• In summary, antibody probes have been developed for a number of
key HAB species,
– though more emphasis has been placed on oligonucleotide probe
development (see below) in recent years.

• For some species (e.g., A. anophagefferens), antibody probes are

the method of choice for cell identification and enumeration, but for
others, cross-reactivity problems have limited applications.

• Finally, the use of magnetic beads with cell surface antibodies offers
the possibility of cell separation and thus species-specific
physiological measurements.
– There is thus sufficient benefit and potential for antibodies that
development efforts should be continued, in parallel with development
of oligonucleotide probe technologies.
NA probes
• In recent years, use of nucleic acid probe technology to detect
microorganisms has expanded considerably.

• This technology is used extensively in the detection of pathogenic

bacteria and other microbes, and is now being applied to HAB

• The procedure involves the detection of target nucleic acid

sequences by binding (hybridizing) those sequences to a short
strand of DNA containing a homologous complementary sequence.

• Extraordinary sensitivity and specificity are possible with well-

designed probes. Many DNA or RNA sequences can be targeted in
the organism of interest, including fragments of genes, spacer
regions between genes, repeated (non-transcribed) sequences, and
transcribed genes.
NA probes
• The first step in probe development is the identification of a unique
series of RNA or DNA bases that are only found in that organism.

• Typically,
– target genes have sequence domains that are highly conserved among
all organisms, and that are thus not useful in discrimination,
– as well as other domains that are variable to different degrees. It is the
latter that are the target areas for probe development.

• If resolution is sought at the genus, species, or even sub-species

levels, the most rapidly evolving, highly variable, domains are
targeted, such as those in
– the internal transcribed-spacer (ITS) regions of ribosomal RNA (rRNA).

• Short, contiguous segments (approximately 20 nucleotides) of

nucleic acid sequences are identified and serve as targets for
probes. “Oligonucleotide” probes are synthesized and used in a
variety of formats to detect the cells of interest.
Structure of rDNA

The nucleotidic polymorphism is not evenly distributed throughout the

ribosomal genes and the three regions evolve at different rates. ITS and
IGS are variable regions which mutate more frequently than the three
conserved coding subunit regions (18S, 5.8S, 28S). This generally
makes the former more informative for analyses of closely related
genomes, whereas the coding regions of the small and the large
ribosomal subunit are considered to be more useful for understanding
more distant relationships at the species/order level.
ITS: internal transcribed spacers; IGS; intergenic spacer; SSU: small
subunit; LSU: large subunit
NA probes
• Oligonucleotide (DNA) probes for identifying HAB
species applied in the whole cell format are typically
directed against
– sequences of the small subunit (18S or SSU), large subunit (28S
or LSU) and
– the internal transcribed spacers (ITS1, ITS2) of the rRNA cistron
(reviewed in Scholin, 1998).

• Much of this work, especially as it relates to field

surveys, has focused on species of
– Alexandrium,
– Pseudo-nitzschia, and
– Pfiesteria and Pfiesteria-like organisms, but
– Heterosigma akashiwo, Chattonella and Fibrocapsa, have also
been sequenced and targeted for probe design.
NA probes
• One way to use these probes is through fluorescent in
situ hybridization using intact cells that are either
– immobilized on a microscope slide or
– suspended in solution.

• In this “whole cell” format, the probe enters the cell and
binds to target sequences, excess probe is washed out,
and the complex is detected by fluorescence or

• As with antibodies, oligonucleotide probes can be

labelled with a variety of fluorescent dyes such as
fluorescein (FITC).
NA probes:
• Table 5.3 examples
NA probes: example
• Fig 5.4
• The picture shows that autofluorescence of chlorofyl
interfers strongly when certain filter set are used. For
instance using the uniR negative probe “the chroma
technology” was still detecting some cells, actually the
same as with autofluorescence. In this example FITC
labelling in combination with the “Omega Optical”
conditions produces the best results.

• Price: 1 to 5 euro per sample, probes are the least

expensive part of the assay (10 eurocent)

• More and more varieties for detection are being

developed such as real time PCR-based onces.
Identification tools
Molecular based probes: conclusions
• Molecular base probes are now routinely used in
many labs.
• There is no single optimal methodology
• The probes have to be ‘tuned” to the local needs
(such as the local varieties of strains to be
• With the exception of lectins, none of the probes
are commercially available at this stage.
Toxin detection tools
• Mouse assay methods
• Chemical methods
• Immunochemical methods
Mouse assay
• Whole animal bioassays provide a measure of total toxicity based upon the
biological response of the animal to the toxins.

• The general principle of most mammalian bioassays depends upon

intraperitoneal (i.p.) injection of an aqueous or organic shellfish extract.

• Standardized strains of laboratory mice of defined age, sex and weight are
frequently used in phycotoxin bioassays.

• The choice of extraction solvent depends on the solubility properties of the

toxins tested. Following injection of the extract, subsequent observations are
made to identify the time- and dose-dependent appearance of typical
symptoms (morbidity and mortality) caused by the toxins.

• Toxicity values are generally interpolated from standard curves,

– LD50 determinations over a fixed time (e.g. 24 h), or
– are derived from standard toxicity tables relating dosage to death times.

• Survival time of mice is generally used for the measurement of global toxicity,
expressed in mouse units (MU) which are converted into toxin specific units
(e.g. saxitoxin equivalents [STXeq]) based upon the toxicity response
calibrated with reference to toxin standards.

Mouse assay: advantages
• The principal advantage of a well administered and properly
calibrated mammalian bioassay, compared to physico-chemical
analysis or many in vitro methods, is that the toxicity determination
is directly relevant to human toxicity effects.

• Mammalian bioassays also screen inadvertently for the presence of

unknown or poorly defined toxic components in the extract matrix,
which may ultimately be found to have human health significance.
– For example, the first indications of the toxicity associated with the ASP
syndrome, later related to domoic acid (Wright et al., 1989), were
revealed in the course of routine AOAC mouse bioassays for PSP
toxicity using acidic aqueous mussel extracts from eastern Canada.
Skilled bioassay technicians noted that the aberrant symptoms of ASP
were distinguishable from the classic symptomology of PSP intoxication
and consequent death.
Mouse assay: disadvantages
• Whole animal bioassays have numerous inherent and operational
deficiencies when applied to the accurate quantitation of

• High capital investment and maintenance costs are often associated

with the installation and operation of bioassay facilities involving live
animals. Mammalian bioassays are labour intensive to perform and
they cannot be readily automated.

• An additional drawback of mammalian bioassays is the high

variability among laboratories,

– due mainly to a number of variables that can affect the results, such as
specific animal characteristics (strain, sex, age, weight), general state of
health, diet, stress conditions, pH of the injected extracts, etc.

• Most of these parameters acquire special relevance when sample

toxicity levels are near the regulatory limit. For these reasons,
careful standardisation of the assay conditions is required to obtain
reproducible and reliable assessment of toxicity and to reduce
inconsistencies within and among laboratories.
Mouse assay: disadvantages
• Mammalian bioassays are susceptible to a host of artifacts and
inaccuracies which can bias the validity of the results.

• False reactions (positive or negative) can occur due to interference

– by substances coextracted during the sample preparation or
– to an inappropriate choice of extraction solvents or clean-up method.

• Many mammalian bioassays for phycotoxins have a poor dynamic

• dilution of the toxic analyte in the sample to achieve death times in
the working range of the assay also dilute other components in the
matrix and this can lead to non-linearity of the standard dose-
response curve.

• The assays tend to be more reliable for phycotoxins which yield a

low LD50 and short death times (i.e., high acute toxicity).
Mouse assay: disadvantages
• Compared to instrumental analytical methods, whole animal
bioassays are often
– much less sensitive (by up to five orders of magnitude) and
– Less precise (f20% is typical under optimal conditions).

• For example, for the AOAC mouse bioassay for PSP toxicity, the
acceptable regulatory limit for human consumption of shellfish
adopted in many countries (80 µg STXeq/100 g soft tissue) is only
twice the nominal toxicity detection limit (cu. 40 µgSTXeq/100 g). This
provides little security margin for technical errors. Moreover, no
definitive qualitative information is provided on the nature of the toxin
components. (AOAC:Association of Official Agricultural Chemists, US
funded organisation dedicated to analytical excellence, now changed
to analytical chemists)
Mouse assay: disadvantages
• This is particularly a problem for samples which contain multiple toxin
analogues which vary in specific toxicity, or where the co-occurrence of
different phycotoxins can lead to synergistic or antagonistic biological
responses, e.g. the simultaneous presence of a Na+ channel activator and a

• Nevertheless, despite the numerous technical and

ethical problems inherent to mammalian bioassays and
the poor information provided on specific toxin
composition, such assays are widely employed for
phycotoxin monitoring in seafood
• PSP is a neurotoxic syndrome resulting primarily from
the blockage of neuronal and muscular Na channels,
preventing propagation of action potentials.

• Symptoms: tingling of extremities, muscular

incoordination, respiratory distress, muscular paralysis

• Groups of compouds include STX and some more 24

different others:
– With their own specific toxicity (up to two order of magnitude)
– Their susceptibility to chemical conversion during sample
processing or storage
– Bioconversion in the shellfish
• The mouse bioassay for the determination of PSP
toxicity was first applied by Sommer and Meyer (1937) to
the assay of acidic extracts of mussels from California.

• In subsequent years, the general procedure has been

further standardized and validated in a series of inter-
collaborative studies (Association of Official Analytical
Chemists; AOAC, 1990).

• This reference method is the only procedure recognized

internationally for quantifying PSP toxicity and it is used
worldwide in PSP monitoring programs, albeit with some
variation in the acceptable regulatory limit for toxicity
• The PSP mouse bioassay for shellfish toxicity involves
– acidic aqueous extraction of the tissue (whole animal or selected organs)
followed by
– i.p. injection of 1 ml of the extract into each of three standardized mice.

• The mice are observed for classical PSP symptoms, such as

• jumping in the early stages, followed by
• death in 5 minutes by respiratory arrest.
• The time from initial injection to mouse death is recorded and the
toxicity is determined (in mouse units) from Sommer’s table.
– One mouse unit is refined as the amount of PSP toxin required to kill a 20
g mouse within 15 minutes. (100 MU: 1 min; 7.67 MU: 2 min; 3,7 MU: 3
– Internal calibration, relating STXeq to MU is regularly necessary.

• The bioassay is only quantitative when the mouse death occurs

between 5 and 7 minutes, with great variations to be expected above
or below these limits. Several dilutions may be needed to obtain an
extract concentration within this range. The precision of this assay is
often given as +20% (C.V.), but this must be regarded as optimal; if a
large dilution factor is required, this level of precision is substantially
for PSP
(1 MU is the amount injected
toxin which would kill a 20 g
mouse in 15 minutes and is
equivalent to 0.18 mg of
Sommer’s Table
Sommer’ Table
PSP mouse assay: pitfalls
• An important parameter influencing the PSP toxin bioassay results is
the pH during extraction.

• In the AOAC (1990) method, extraction in 0.1 M hydrochloric acid

followed by heating at 100°C, typically establishes pH ranging
between 2 and 4. The PSP assay procedure was initially designed to
quantify only STX, but at present about two dozen naturally-occurring
PSP analogues have been identified, which vary in toxicity, chemical
stability and relative abundance in shellfish and dinoflagellates.
PSP mouse assay: pitfalls
– Under the hot acidic conditions required in the AOAC protocol a
substantial proportion of the labile but low potency N-
sulfocarbamoyl toxins (Cl-C4, B 1, B2) are converted to their
respective high toxicity carbamate analogues (approximately 40-
60% conversion, depending upon the tissue matrix buffering
capacity). (e.g. conversion of C3 into GTX1)
– Epimerization also occurs with this hot acid treatment, resulting in
the conversion of p- to a-epimers, but this usually has a minor
effect on net toxicity.

• The PSP toxins are least stable at alkaline pH, yet heating under
strongly acidic conditions (e.g., pH 2) can also lead to chemical
transformation, with the degree of conversion depending upon the pH
(Nagashima et al., 1991). Between pH 3 to 4, all PSP components are
in a range of optimal stability. Low pH of the injected extract can also
lead to mouse bioassay artifacts caused by acidosis.
ASP mouse assay
• The AOAC mouse bioassay for PSP toxins can also detect domoic acid at
concentrations ca. 40 ppm and this procedure was used when ASP toxicity
was first identified in Canada in shellfish extracts from eastern Prince
Edward Island (Wright et al., 1989).

• The typical signs of the presence of domoic acid is a unique scratching

syndrome of the shoulders by the hind leg, followed by convulsions.
• The time of observation must be extended from 15 minutes to 4 hours.
Mouse deaths associated with mussels containing domoic acid were never
observed after 135 minutes (Quilliam et al., 1989; Todd, 1990).

• Although the AOAC extraction procedure can yield substantial recovery of

domoic acid, the tolerance level established in Canada and subsequently
adopted by certain other countries is 20 ppm, therefore the AOAC bioassay
procedure is too insensitive to be used with confidence for regulatory
purposes to quantify this toxin.

• For the routine detection of ASP toxins, the AOAC mouse bioassay has
been superseded by HPLC methods using diode-array/UV or fluorometric
detection (Quilliam et al., 1989; Pocklington et al., 1990) which have been
proven to be more sensitive and reliable tools
DSP mouse assay (reading)
• There is no general agreement concerning the appropriate testing
procedures to be applied for DSP toxins for regulatory purposes, and
standardization of methods represents a difficult administrative and
scientific challenge.
• One of the main problems arises from the different biological activity of the
three groups of lipophilic toxins currently included in the DSP toxin
complex (Yasumoto, 1990) –
– okadaic acid (OA) and
– analogues such as DTXl,
– DTX2, and
– acyl-derivatives (DTX3),
– polyether lactone pectenotoxins (PTX), and
– yessotoxins (YTX), disulfated polyether compounds.

• Only toxins belonging to the OA group produce diarrheic effects in

mammals (i.e., classic DSP symptoms), however PTX compounds are
reported to be hepatotoxic and YTX derivatives cause severe cardiac
damage in experimental animals. Although PTX and YTX are acutely toxic
to mice upon i.p. injection (Terao et al., 1990), the oral toxicity to humans
has not been established.
• Given that the human health risks of the two latter two groups of DSP toxins
are not well elucidated, they should remain included in DSP toxicity
monitoring programmes, at least until more toxicological data are available.
DSP mouse assay (reading)
• The official Japanese method for surveillance of DSP toxicity and also used
in some European countries, including Italy and the United Kingdom is
given below.
• In this method, a 20 g sample of homogenized hepatopancreas is extracted
thrice with 100 ml of acetone. The extracts are filtered, then the filtrate is
collected and the solvent removed by rotary evaporation. The residue is
made up to 20 ml with water and the suspension is extracted thrice with 50
ml of diethyl-ether.
• The combined organic layers are backwashed twice with small quantities of
water and evaporated to dryness.
• As in the original procedure described above, the residue is resuspended in
1% Tween 60 solution to a concentration of 5g hepatopancreas/ml Tween
60 prior to i.p. injection. The assay is very suitable for use with a wide
variety of shellfish tissues.
• With this procedure, possible PSP interferences are removed in the water
layer. High amounts of salts which are concentrated during the evaporation
step, and could cause artifactual mouse deaths, can also be removed
during the water washing step.
• A drawback of this extraction procedure is the poor solubility of YTX in
diethyl-ether; depending upon the pH and lipid content of the sample, this
toxic component might be lost in the water layer. A substantial improvement
is achieved if dichloromethane is used instead of diethyl-ether.
Dichloromethane has the advantage of solubilizing all the DSP toxins,
including YTX
NSP mouse assay (reading)
• The currently accepted method for the determination of NSP toxins
is the American Public Health Association (APHA, 1985) procedure
(originally Irwin, 1970), based on a diethyl- ether extraction of
shellfish tissue.The APHA protocol for NSP used extensively in the
United States, where the problem of NSP is most acute.

• After the detection of NSP in New Zealand in 1993, a management

strategy to monitor these toxins was developed by the MAF
Regulatory Authority. The sample preparation method used for the
detection of NSP and DSP toxins was based on acetone extraction
of these lipophilic components, followed by partitioning into
dichloromethane (Hannah et al., 1995).

– Sample extracts are prepared for mouse injection by suspension in a

sterile solution containing 1% Tween 60 to a final sample concentration
equivalent to 10 g/ml. The mouse bioassay is conducted by
administering 1 ml inoculations, and the bioassay results are calculated
in mouse units. Two or more deaths within 6 hours is suspect. NSP is
confirmed by APHA method and DSP by ELISA method.
Toxin detection tools
• Mouse assay methods
• Chemical methods
• Immunochemical methods
PSP detection by HPLC
• The post-column derivatization HPLC method for paralytic shellfish toxins
has a great advantage over other methods in its ability to quantitate each
toxin in a crude sample of small size.
• Simple clean-up procedures can avoid toxin transformations which easily
occurs during purification and concentration procedures.
• It is a powerful tool in research, especially for studies on toxin production
by dinoflagellates. The high sensitivity of the system enables to elucidate
the complete toxin profile of Alexandrium with samples as small as 100 cells
(Oshima et al., 1992).

• A clean-up procedure with a reverse-phase cartridge column minimizes the

errors involved in HPLC analysis.

• Use of carefully prepared standards is also essential for accurate evaluation

of toxin content. The major cause of discrepancy between the HPLC and
the bioassay methods is inaccuracy of the mouse bioassay.
• The mouse test is known to involve significant errors (McFarren, 1959; Park
et al., 1986), especially when testing samples of low toxicity, whereas the
replicated HPLC analyses showed less than 5% error.
• In conclusion, the mouse bioassay can be complemented by HPLC as a
regulatory measure for seafood safety.
for PSP:
PSP analysis by
HPLC: example
Toxin detection tools
• Mouse assay methods
• Chemical methods
• Immunochemical methods
Detecting toxins
• In theory, if not in practice, immunological methods for the detection of
phycotoxins have several advantages over chemical analytical methods for
the detection of particular toxins in phytoplankton in broad-scale screening

• The sensitivity of immunodiagnostic assays is typically orders of magnitude

greater than the corresponding mouse bioassay or chromatographic method
with fluorescence or mass-spectrometric detection.

• Immunoassays made be configured with picogram detection limits and this

approach is very amenable to automation (multi-channel manifolds and
micro-plate readers) with little concern for complicated sample clean up and

• In the last few decades, there have been several concerted attempts to
produce reliable immunodiagnostic test kits for various phycotoxins.
Detecting toxins
• Many of these efforts have been hampered by

– the lack of purified toxins for conjugation and

– difficulties in producing stable immunogens from relatively low
molecular weight toxins (e.g., saxitoxin [STX], domoic acid [DA]).

• Since toxin conjugates for immunization are typically

prepared from only a single, readily available derivative,
whereas toxigenic phytoplankton usually contain a suite
of chemically related derivatives, cross-reactivity is
important in the development of immunological methods.
Detecting toxins
• Antibody methods of toxin detection are “structural assays” that are
dependent upon the conformational interaction of the analyte (toxin)
with a molecular recognition factor, such as the epitopic binding
• Thus, cross-reactivity in immunoassays is limited to components
with compatible epitopic sites and may not reflect relative biological
activity or specific toxicity.
• Such assays yield only an integrated quantitative value representing
a group of toxins, whereas the components may vary widely in
specific toxicity.
• The lack of broad-spectrum cross-reactivity for toxic, naturally
occurring analogues has been a major drawback to the use of
quantitative immunoassays for screening phycotoxins in naturally
contaminated samples; however, this is being overcome by second
generation antibodies, where more care is taken in designing the
antibody ‘receptor site’.
Types of
Detecting toxins
• An ELISA method for the detection of STX
in shellfish is commercially available as a
test kit (RIDASCREENR, R-Biopharm
GmBH, Darmstadt, Germany). This assay
is frequently used for toxin assays in
shellfish but has not been evaluated with
respect to performance with phytoplankton
extracts or intact cells.
An established and reliable detection method for
Saxitoxin toxins has been the "mouse mean death
time bioassay“. The limitations of this procedure are
the high variability of the results and low sensitivity
(approx. 37 µg PSP/100 g of sample).

• This low sensitivity is particularly problematic given

the allowable limits of 40 and 80 µg PSP/100 g of
sample respectively set by the individual member
states of the EU.
• In addition, this method is to be critically
reconsidered from the animal protection point of
Ridascreen: Test principle
• The basis of the test is the antigen-antibody reaction.

• The microtiter wells are coated with antibodies directed against saxitoxin
loaded antibodies

• To PSP standards or sample solutions,

– PSP enzyme conjugate and anti-PSP antibodies are added.
– Free PSP and PSP enzyme conjugate compete for the PSP antibody binding
sites (competitive enzyme immunoassay).
– At the same time, the anti-PSP antibodies are also bound by the immobilized
capture antibodies.
– Any unbound enzyme conjugate is then removed in a washing step.

• Enzyme substrate (urea peroxide) and chromogen tetramethylbenzidine)

are added to the wells and incubated.
– Bound enzyme conjugate converts the colorless chromogen into a blue product.
The addition of the stop solution leads to a color change from blue to yellow.

• The measurement is made photometrically at 450 nm (optional reference

wavelength. The absorption is inversely proportional to the saxitoxin
concentration in the sample.
Sensitivity of the Ridascreen test
Ridascreen: Specificity
• The specificity of the RIDASCREEN® Saxitoxin test was determined
by analyzing the cross-reactivities to corresponding toxins.

• Cross-reactivity:
• Saxitoxin .........................................................100 %
• Decarbamoyl saxitoxin .............................. 10 - 30 %
• Gonyautoxins II, III, B1, C1 und C2.............. 10 - 30 %

• The specificity of the RIDASCREEN® Saxitoxin assay was

established by analyzing the cross reactivity to the corresponding
toxins. Because of the fact, that pure standard substances are not
available, a contamination of the corresponding toxins with saxitoxin
is possible. Therefore, the results obtained indicate approx. the
above mentioned or less cross reactivities.
• A sample contains 10 µg STX and 10 µg
C1. Will the Ridascreen generate an
overestimation or underestimation of the
• What is the difference in sensitivity
between the mouse assay and
Ridascreen of PSP?
Mist Alert system for PSP
• The recently developed MIST Alert™ (Jellett Biotek, Dartmouth,
Canada) has been shown to be highly effective for the detection of
PSP toxins in shellfish (Laycock et al., 2002) and plankton matrices
(Silva et al., 2001; 2002).

• This lateral flow immuno-chromatographic (LFI) assay is available

on a platform similar to that of a common home pregnancy test kit
and permits screening for PSP toxins in <20 minutes.

• The polyclonal antibodies have been well characterised for their

cross-reactivity and limit of detection for multiple PSP toxins
standards (CRMP, IMB, National Research Council, Halifax,
• All STX analogues commonly found in shellfish, including the N-
sulfo-carbamoyl derivatives, are detected, albeit at somewhat
reduced sensitivity for the N-1-OH toxins (Laycock et al., 2002).
Mist Alert system for

Possible assay outcomes

Mist Alert system for ASP
• Antibodies produced by AgResearch (Hamilton, New
Zealand) have been well-characterized by ELISA
(Garthwaite et al., 1998) and are now incorporated into
the ASP toxin assay known as the MIST Alert™ for
ASP (Jellett Biotek Ltd., Dartmouth, Canada), an
immunochromatographic assay based on the same
platform as previously described for PSP toxins.

• This test has also been recently applied with success to

the detection of ASP toxins in samples from toxigenic
cultures of Pseudo-nitzschia multiseries and natural
plankton assemblages containing toxic diatoms (A.
Cembella et al., unpubl. data). The nominal detection
limit is 2 to 10 ng on the test strip.
Functional probes (reading)

• In contrast to structural assays that depend upon a

molecular recognition factor that may or may not be
correlated with specific toxity,
• functional assays are based upon the biochemical action
of the toxin (e.g., binding to the ion channels of
• Quantitation therefore tends to correlate well with the
specific toxicity of the analyte, in spite of differences from
whole animal responses that exist due to variation in
mechanisms of toxin uptake and conversion in the body.
• For matrices that contain several toxic components with
a similar mode of biological activity, but which vary in
specific potency, such assays should yield an accurate
estimate of net toxicity.
Functional probes (reading)

• Among these functional assay methods are

– cell culture (cytotoxicity) assays,
– neuroreceptor assays, and
– enzymatic activity tests.

• Like the antibody methods, these techniques are less

controversial and more practical alternatives to
bioassays using live mammals.
• Such assays can be performed using simple extraction
procedures as they have greater specificity than whole
animal assays and do not require the removal of agents
such as heavy metals, which can contribute to false
positive responses.
Functional probes (reading)

• Very low detection limits (<10–12 M) may be attained with

functional assays for phycotoxins and the methods are reasonably
easy to automate for multiple parallel analyses.

• As for the antibody methods, most functional techniques for toxins

in phytoplankton are merely variants of the same assay as
developed for use with shellfish or human tissue matrices, and are
applied only to the bulk assay of extracted toxins.

• Non-specific binding is one of the problems associated with

functional assays, and its importance cannot be overemphasized.
• Non-specific binding (e.g., to a neuroreceptor) can be defined as
extraneous interaction with the ligand (i.e., toxin) resulting from the
presence of bindable non-target components in the sample matrix.
• This spurious binding of components (fatty acids, proteins, etc.) is
unrelated to the analytes of interest (toxins), but, fortunately, tends
to be somewhat less in phyoplankton extracts than in shellfish tissue
Functional probes (reading)

• Functional in vitro enzymatic assays for phycotoxin detection are

comparatively rare, but
– the specific inhibition of protein phosphatase Type 1 (PP1) and Type 2A
(PP2A) by certain DSP toxin analogues (OA and DTX1) has been
exploited in the development of a phosphatase radioassay using 32P

– This assay has been applied to assay naturally-contaminated mussel

tissue, extracts of cultured Prorocentrum lima, and net tow material from
natural phytoplankton assemblages. The same enzyme inhibition assay
is also useful for the detection of microcystins, a class of phycotoxins
produced by certain cyanobacteria, and other toxins capable of
inhibiting PP1.
• A useful version of this PPase assay, based on colorimetric detection, has
been applied for the assay of DSP toxins in shellfish and plankton (Tubaro et
al., 1996), and further refinements have been recently incorporated.
• A fluorescence-detection version of this assay has also been developed and
successfully used for the detection of DSP toxins (Vieytes et al., 1997).
– Although the fluorimetric assay offers better sensitivity, and may be
preferred in direct comparisons with the colourimetric version, it requires
a fluorescence plate reader. The enzyme-inhibition assay for DSP
toxins are applied to bulk extracts of plankton or other tissues and no
cell-specific probe variation is currently available.
Summary: effects, organism, legal limits

Disease and conditions PSP DSP NSP CFP ASP

Incubation time 5-30min < 24h 30'-24h <24h <24h

nausea, diarrhoea,
vomiting, tingling, hot and abdominal pain, confusion,
acute symptoms diarrhoea, paralysis diarhoea cold, dizziness nausea memory loss

associated food shellfish shellfish shellfish fish shellfish

Gymnodinium catenatum,
Alexandrium, Pyrodinium
bahamense var. Dinophysis, Pseudo-
causative organism Compressum Prorocentrum Karenia brevis Gambierdiscus Nitzschia

Phosphorylase receptor
Toxin molecular mechanism Na channel blocker inhibitor Na channel activator Na/ Ca activator agonist

below detection
Legal limit in EU 80µg/100g limit 160µg OA/kg 20µg/g

Yessotoxin: 1mg/kg
Latest EU regulations
• Regulation (EC) No 853/2004
• establishes that food business operators must ensure that live bivalve
molluscs placed on the market for human consumption must not contain
marine biotoxins in total quantities (measured in the whole body or any part
edible separately) that exceed the following limits:
• • 800 micrograms per kilogram for paralytic shellfish poison (PSP):
• • 20 milligrams of domoic acid per kilogram for amnesic shellfish poison
• • 160 micrograms of okadaic acid equivalents per kilogram for okadaic acid,
dinophysistoxins and pectenotoxins in combination:
• • 1 milligram of yessotoxin equivalents per kilogram for yessotoxins:
• • 160 micrograms of azaspiracid equivalents per kilogram for azaspiracids.
The ecology of algal blooms
• species appear to be stimulated by “cultural
eutrophication” from domestic, industrial and agricultural

– The Fig. below illustrates an 8-fold increase in the number of red

tides per year in Hong Kong Harbour in the period 1976 to 1986
(Lam and Ho, 1989). This increase (mainly Gymnodinium
nagusakiense, Gonyaulax polygramma, Noctiluca scintillans and
Prorocentrum minimum) shows a striking relationship with the 6-
fold increase in human population in Hong Kong and the
concurrent 2.5-fold increase in nutrient loading, mainly contributed
by untreated domestic and industrial waste.

The ecology of algal blooms
The ecology of algal blooms
– A similar experience was noted in the Seto Inland Sea, one of
the major fish farm areas in Japan (Okaichi, 1989).
– Between 1965 and 1976 the number of confirmed red tide
outbreaks (mainly Chattonella antiqua, since 1964; and
Gymnodinium nugusukiense, since 1965)
• progressively increased 7-fold,
• concurrent with a 2-fold increase in the COD (chemical oxygen
demand) loading, mainly from untreated sewage and industrial
waste from pulp and paper factories.
– During the most severe outbreak in 1972, a Chattonella red tide
killed 14 million cultured yellow-tail fish.
• Effluent controls were then initiated
– to reduce the chemical oxygen demand loading by about half,
– to introduce secondary sewage treatment, and
– to remove phosphate from house-hold detergents.
– Following a time-lag of 4 years, the frequency of red tide events in the
Seto Inland Sea then decreased by about 2-fold to a more stationary
The ecology of algal blooms
Time series of nutrient and Pseudo-nitzschia in the
Mississipi delta. A) nitrate nitrogen increase. B)
densities of Pseudo-nitzschia
The ecology of algal blooms
• A similar pattern of a long-term increase in nutrient loading of
coastal waters is evident for the North Sea in Europe (Smayda,
1990) .

– Since 1955 the phosphate loading of the River Rhine has increased 7.5-
fold, while nitrate levels have increased 3-fold. This has resulted in a
significant 6-fold decline in the Si:P ratio, because long-term reactive
silicate concentrations (a nutrient derived from natural land weathering)
have remained constant.
– More recently, improved wastewater treatment has been causing
decreases in the ammonia:nitrate ratio of River Rhine discharge
(Riegman et al., 1992). The nutrient composition of treated wastewater
is never the same as that of the coastal waters in which it is being

• There is considerable concern (Officer and Ryther, 1980; Ryther

and Dunstan, 1971; Smayda, 1990) that such altered nutrient ratios
in coastal waters may favour blooms of nuisance flagellate species
which replace the normal spring and autumn blooms of siliceous
The ecology of algal blooms
The ecology of algal blooms
Development of blooms
• Casual factors: presence and/or advection of species, inoculum,
cysts etc can encourage outbursts when conditions are favourable.

• Enhancement factors:
– cell multiplication can be encouraged by hydrological and climatic
– It has been recognized that blooms appear in warm and stable waters
where mixing is absent; and after heavy rain fall, not because of a drop
in salinity but because of stratification.
– This stability of the water column is important because it favours the
accumulation of algae at the optimal depth for growth.
– Wind-driven currents, tides, upwelling, downwelling, divergencies and
convergencies as well as other frontal boundary structure may be
prerequisited for blooms to occur.
– While nuisance blooms are virtually always coastal phenomena, it has
been suggested that favourable physical and hydrological conditions
must act synergistically with nutrient enrichment to yield maximum
– Allochtonous and terrigenous nutriet loadings are almost invariably
linked to nuisance bloom developments.
Development of blooms
• While dinoflagellates can develop in nutrient-poor
waters, diatoms require waters with high concentrations
of nutrients. This means that diatoms consume more
nutrients than dinoflagellates, and the high division rate
of diatoms allow them to outcompete dinoflagellates.
• However, some dinoflagellates are known to grow in
nutrient-rich waters, e.g. Prorocentrum
Blooms in relation to hydrography

• In the North west of Spain there are four bays (rias) linked with
rivers that are rich in nutrients. Hence together with local
hydrological features (upwelling) the rias themselves are loaded with
nutrients. Because of this they support immense mussel, oyster and
fish culture. Ria de Vigo is one of the four oceanic bays which
support a major raft-suspension blue mussel aquaculture industry in
Spain ( more than 200 000 ton)
Blooms in relation to hydrography
• In this area, there are intensive upwelling periods,
especially during summer. It is observed that blooms
occur between July and October (late summer/autumn)
when upwelling is weak or absent and when a mixture of
autotrophs and heterotrophs occur.
• When upwelling occurs, jets of cold North Atlantic waters
are injected into the rias. This lowers the water
temperature in the ria, while it is warmer offshore.
• Intense upwelling favours the development of diatoms.
This is because strong upwelling destroys stratification
which allows nutrients from the bottom to be loaded into
the photic zone.
Blooms in relation to hydrography
Blooms in relation to hydrography
• When the upwelling ceases in autumn, offshore
water flow into the rias.
• This increases the temperature of the water in the
• The inflow of warm waters coincides with very
weak upwelling ( which drives the nutricline below
the photic layer) and favours the development of
• With the nutricline pushed into the deeper waters,
out of the photic zone, It is only the actively
swimming dinoflagellates (like the chain forming
Gymnodinium catenatum) which are better
adapted to vertical migration, that can penetrate
the deep waters and take up nutrients and flourish
to the disadvantage of diatoms.
Blooms in relation to hydrography
Ria’s in Galicia

Warm water approaching the coastal areas in october because of

cessation of upwelling
Simplified conceptual model describing the growth strategy of mixotrophic Dinophysis spp. in
response to availability/absence of prey during its growing season. (1) Optimum growth when
prey is available and light intensity is high. (2) Mesodinium is no longer available
and Dinophysis division continues for several doublings; division rates will depend on light
intensity. (3) If suboptimal conditions remain, Dinophysis growth decreases, gametes are
formed and cells accumulate starch deposits as storage product. (4) Long periods of prey
limitation may prevent growth completely (μgross = 0) and even lead to cell death. Blue lines
indicate time forwards in the absence of prey. Red lines indicate that each stage is reversible
provided the population of Dinophysis matches a patch of Mesodinium (lag phase of 2–4 days
Harmful Algae Volume 14 2012 87 - 106
• Harmful algal events arising from contamination of shellfish with
phycotoxins occur in one of two ways.
• Either a population of harmful plankton is carried into a shellfish
production site with local currents, or else
• a resident toxic algal population exists in a bay which can persist
year on year due to a dormant overwintering stage in its life cycle.
• Most harmful events arising from infestation by Dinophysis are due
to transport of cells into bays used for shellfish production.
Steidinger (1975) recognised different phases in the development of
harmful algae populations: initiation, growth, and maintenance
(plateau). She also recognised transport of blooms as being highly
important, as it can cause the initiation (bloom import) or termination
(bloom export) of harmful events. Recognition of these aspects of
Dinophysis blooms wherever they arise is necessary to improve
prediction of the events and mitigation of their effects.
Dinophysis blooms
• By “initiation” we understand here the time when
cells of Dinophysis start being detected by
quantitative methods (>10–102 cells L−1). Much
of the available information points to an origin of
Dinophysis populations on the continental shelf.
None of this however shows how Dinophysis
survives through environmentally
disadvantageous circumstances such as the
winter months of temperate regions.
Dinophysis blooms
• A common feature observed in most seasonal
samplings of Dinophysis populations is that
numbers start to increase when there is water
column stability (Maestrini, 1998).
– Raine et al. (2010b) noted that blooms of D.
acuminata and D. acuta around southwestern Ireland
occur during the months of June–mid-September
when the water column is thermally stratified.
– In Mutsu Bay (northeast Japan), a temperature
threshold has been established (8 °C) above which
the annual occurrence of D. fortii starts (Ozaka, 1985
cited in Maestrini, 1998).
Daily Ekman transport and seasonal distribution of temperature and D.
acuta cell concentrations in Ría de Pontevedra (Galician Rías). The
dashed line separates two scenarios for D. acuta blooms: under stratified
conditions in the upwelling season, with cell maxima found in the
pycnocline (left) and at the end of the upwelling season in homogeneous
water columns and cell maxima near the surface (right).
Harmful Algae Volume 14 2012 87 - 106
Harmful Algae Volume 14 2012 87 - 106

Transport of Dinophysis along and into the coasts of north-western Europe. (A–C) The system of currents
along the north-western Atlantic coast of the Iberian peninsula illustrated by Escalera et al. (2010). Grey thick
arrows are the poleward counter current of the Eastern Boundary Current system. (A) In summer the
Portuguese Coastal Current (long black arrows) flows south, promoted by upwelling favourable northerly
winds. Cross shelf transport of plankton arises during upwelling relaxation (thin grey arrows). (B) During
autumn a narrow inshore flow of warm water is found (dotted lines) and transports harmful dinoflagellates
north towards the Galician Rías (NW Spain). This prevails during the whole autumn–winter season except
when (C) upwelling-favourable winds blow. (D) A baroclinic coastal flow occurs around the perimeter of the
Celtic Sea (Hill et al., 2008) in summer. This takes the form of strong, narrow jets which are found over bottom
fronts (see inset in D) and transport plankton along the coastline. The alongshore flows transport plankton
populations towards aquaculturally important bays, indicated in (C) and (D). Exchanges of water and plankton
between the coastal shelf and bays (E) are caused by downwelling, whereby populations including Dinophysis
are carried into the bays with warm surface water during upwelling relaxation (Iberia) or wind-forcing from the
predominantly south-westerly winds that occur in south-western Ireland. The dynamics are maintained by flow
reversal (lower diagram) caused either by resumption of upwelling favourable winds (Iberia) or by winds
blowing axially along the bays towards the open ocean (southwest Ireland).
Case Study

The link between precipitation, river runoff, and

blooms of the toxic dinoflagellate Alexandrium
tamarense in the St. Lawrence

• Andréa M. Weise, Maurice Levasseur, François J. Saucier,

Simon Senneville,
Esther Bonneau, Suzanne Roy, Gilbert Sauvé, Sonia Michaud,
and Juliette Fauchot
• Can. J. Fish. Aquat. Sci. 59: 464–473 (2002)
Case study: study area
Case Study

Interannual variability of (a) temperature (°C), (b) salinity (‰), and (c) Alexandrium tamarense cell
abundance (cells·L–1) at Sept-Îles between mid-May and the end of October, 1989–1998. Data
were only available as of mid-June 1990 and mid-July 1989 and 1992. The dotted line at 1000
cells·L–1 represents the concentration at which shellfish generally become toxic in the St.
Lawrence (80 μg STX eq·100 g meat–1).
Case study

Abundance ranking of Alexandrium tamarense vs. temperature (°C) and

salinity (‰) at Sept-Îles between mid-May and the end of October, 1990–1998.
No salinity data were available for 1989. The circle encompasses most of the
highest cell concentrations.
Case study
• Association between heavy
rainfall, river runoff, salinity, and
Alexandrium tamarense blooms
at Sept-Îles during 1992 and

• Daily Moisie River runoff (m3·s–

1; solid line) and rainfall (mm;
bars) during (a) 1996 and (d)
• salinity (‰) during (b) 1996 and
(e) 1992;

• A. tamarense cell
concentrations (cells·L–1) during
(c) 1996 and (f) 1992. Salinity
and cell abundance data were
only available as of mid-July
Case study
• Association between
heavy rainfall, river
runoff, salinity, and
Alexandrium tamarense
blooms at Sept-Îles
during 1992 and 1996.
• Daily Moisie River runoff
(m3·s–1; solid line) and
rainfall (mm; bars) during
(a) 1996 and (d) 1992;
• salinity (‰) during (b)
1996 and (e) 1992;

• A. tamarense cell
concentrations (cells·L–1)
during (c) 1996 and (f)
• Salinity and cell
abundance data were
only available as of mid-
July 1992
Case study
Case study
Environmental conditions
preceding the 1994
sustained bloom at Sept-
Îles. (a) Daily Moisie
River runoff (m3·s–1)
(solid line) and rainfall
(mm) (bars), (b) salinity
(‰), (c) wind speed (m·s–
1), and

(d) A. tamarense cell

abundance (cells·L–1).
Case study: conclusions
• The analysis of this 10-year data set indicates that the timing of A.
tamarense blooms at Sept-Îles is related to a combination of key
environmental variables,
– notably temperature,
– salinity,
– rainfall,
– Moisie River runoff, and
– wind,

• whereas the magnitude of blooms is controlled by the duration of

these favourable conditions promoting bloom development.

• The fact that significant interannual variability in both the timing and
magnitude of A. tamarense blooms is observed over the 10-year
period demonstrates that this proper combination of du Saint-
Laurent factors is not met every year.

• The results reveal that there was a “window” when A. tamarense

cells were present in the surface waters every year, i.e., the last two
weeks of June and the first week of July. This is a period when the
water column has warmed substantially and is more likely to be
Sampling strategy: IFREMER
• In order to meet its objectives, the Rephy network assures the surveillance
of two matrices, namely water and shellfish:
– Water samples for monitoring phytoplankton species, allowing for the detection
of toxic species, and
– If necessary shellfish samples for measuring toxines in consumable products

• With respect to consumer risk, the strategy is based on the detection of

toxic species in the water, which can incentivate the analysis for
phytotoxines in shellfish

• The REPHY network contains a series of sampling sites distributed over the
entire French coast.

• The sampling strategy and the nature of observations is linked to desired


– A better knowledge of the environment can be obtained by a minimal number of

sampling points that are revisited on a regular basis during the year.
– The protection of the customer: here the number of sampling points need to be
bigger, but they do not need to be sampled all year around.

• Also enumeration of the toxic species is sufficient for consumer protection,

while for a better knowledge of the environment it is necessary to
enumerate all species present in the samples
Sampling strategy: IFREMER
• Concentration of cells that trigger DSP analysis
Dinophysis spp. > 500 cells/l
– Remark: there seems to be a rather poor
correlation between average cell concentration
and DSP toxicity, mainly due to patchy
occurrence of Dinophysis

• Concentration of cells that trigger PSP analysis

• Alexandrium minutum >10 000 cells/l
• Alexandrium catenella / tamarense >5000 cells.l

• Concentration of cells that trigger ASP analysis

• Pseudo-nitzschia spp. > 100 000 cells/l
Relation between cell number per
liter and toxicity levels in molluscs
HAB monitoring programme:
Danish example
New Zealand