Partim: Harmful algal blooms

Prof. Dr. ir. Peter Bossier

Academic year 2012-2013

Aquaculture and the environment

Sources:• Monographs on Oceanographic Methodology: Manual on

Harmful Marine Microalgae (Hallegraef, Anderson, Cembella) 2003, Unesco Publishing, ISBN 92 3 103871 0

• http://ioc.unesco.org/hab/intro.htm• http://www.nmnh.si.edu/botany/projects/dinoflag/index.ht

m: identification of HA

• http://www.bi.ku.dk/ioc/default.asp: identification of HA• http://www.fao.org/docrep/007/y5486e/y5486e00.htm#Co

ntents• http://www.aesa.msc.es/crlmb/web/CRLMB.jsp• http://www.whoi.edu/science/B/redtide/rtphotos/

rtphotos.html

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

WHAT ARE HARMFUL ALGAE ?

• 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 toxins.

• 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’.

• 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

HAB: overview 1

Overview 1

• Examples:– Noctiluca scintillans,

Overview 1cyanobacterium : 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

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Overview 2• - Cyanobacterial Toxin Poisoning

– Cyanobacteria such as Anabaena circinalis , Microcystis aeruginosa, Nodularia spumigena

– Anatoxin Anabaena

Overview 2: Microcystissingle 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

antiqua.

Overview 3

• Chaetoceros convolutus

Overview 3Gymnodinium mikimotoi,

WHY ARE THEY HARMFUL ?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, and2. 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 beyond direct economic losses and impacts on human healthhealth. When HABs contaminate or destroy coastal resources, the livelihoods of local residents are threatened and the sustenance of human populations is compromised.

GLOBAL INCREASE OF ALGAL BLOOMS

• 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

poisoning. • 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.

GLOBAL INCREASE OF ALGAL BLOOMS:

PSP- cases in 1970 and

2000

GLOBAL INCREASE OF ALGAL BLOOMS:

DSP- cases in 1990 and

2000

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

GLOBAL INCREASE OF ALGAL BLOOMS:

ASP- cases in 1990 and

2000

Global distribution of ciguatera (CFP)

Occurrence of PSP toxins along the

european coast in the period 1991-2000

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 HABs).

Harmful Algae Volume 14 2012 87 - 106

Occurrence of DSP toxins along the european coast in the period 1991-2000

Occurrence of ASP toxins along the european coast in the period 1991-2000

Occurrence of azaspiracid toxins along the european coast in the period 1991-2000

Current situation

• http://www.ifremer.fr/depot/del/infotox/#infos

• http://algeinfo.imr.no/

GLOBAL INCREASE OF ALGAL BLOOMS• 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

GLOBAL INCREASE OF ALGAL BLOOMS

• 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 numbers in the open sea have been low, usually below 300 cells per liter.

• In Danish fjords, Kiel Bight and the mouth of the rive Warnow ( Germany), large blooms have been recorded. Since then repeated massive blooms in the Oslofjord with max concentrations of several hunderd million cell per liter have occurred.

When old plankton samples from the Norwegian coast were reanalysed, it became evident that P.m. occurred in this area before 1979, but was misidentified as P. balticum. In the Oslofjord, which has been monitored extensively since 1930, P.b. is recorded, but in comparatively low concentrations. Real blooms were not reported untill 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.

•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.

G. polyedra

• 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 water

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

TRANSPORT OF DINOFLAGELLATE CYSTS IN SHIPS’ BALLAST WATER OR ASSOCIATED WITH THE TRANSLOCATION OF SHELLFISH STOCKS.• 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).

TRANSPORT OF DINOFLAGELLATE CYSTS IN SHIPS’ BALLAST WATER OR ASSOCIATED WITH THE

TRANSLOCATION OF SHELLFISH STOCKS

• 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.

TRANSPORT OF DINOFLAGELLATE CYSTS IN SHIPS’ BALLAST WATER OR ASSOCIATED WITH THE

TRANSLOCATION OF SHELLFISH STOCKS

• 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.

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).

Harmful Algae Volume 14 2012 10 - 35

Pellicle cyst

planozygote

hypnozygote

Cysts• 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 species.

• 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.

Cysts • 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.

TRANSPORT OF DINOFLAGELLATE CYSTS IN SHIPS’ BALLAST WATER OR ASSOCIATED WITH THE

TRANSLOCATION OF SHELLFISH STOCKS

• 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

HARMFUL ALGAE AND MARINE FOOD RESOURCES

• 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

cooking; – 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

products.

HARMFUL ALGAE AND MARINE FOOD RESOURCES

• 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.

HARMFUL ALGAE AND MARINE FOOD RESOURCES

• 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 seafood.

• 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 adequate – surveillance programmes and – quality control of the seafood products

• which will often require expert assistance from countries which have longstanding experience in this matter.

TOXIC EFFECTS ON HUMANS

• Particularly in the tropics, people are often harrassed by diseases and syndroms due to consumption of seafood contaminated by algal toxins.

• 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.

TOXIC EFFECTS ON HUMANS

• 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, nausea;

• 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

treatment)

• 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- NSP

• 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.

WHICH ARE THE CAUSATIVE ORGANISMS ?

• Many of the syndromes and other harmful effects pertain to occurrences of dinoflagellates.

• 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

• 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. compressum

Dinophycae (= Dinoflagellates):PSP

Alexandrium

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

http://dx.doi.org/10.1016/j.hal.2011.10.012

 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

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 (▵).

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 solvents.

• It is stable in solution at neutral and acidic pH's, even at high temperatures, but alkaline exposure oxidizes and inactivates the toxin.

• 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-

toxins; – 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

Name Registry Number

R1 R2 R3 R4

Saxitoxin 35523-89-8 H H H OCONH2

Gonyautoxin II

60508-89-6 H OSO3- H "

Gonyautoxin III

60537-65-7 H H OSO3- "

Neosaxitoxin 64296-20-4 OH H H "

Gonyautoxin I 60748-39-2 OH OSO3- H "

Gonyautoxin IV

64296-26-0 OH H OSO3- "

N

Rel.toxicity

1

0.36

0.64

0.92

0.99

0.73

Toxity of PSP molecules

Decarbamoyl Saxitoxins

Name Registry Number

R1 R2 R3 R4

Decarbamoyl saxitoxin

58911-04-9 H H H OH

Decarbamoyl gonyautoxin II

86996-87-4 H OSO3- H "

Decarbamoyl gonyautoxin III

87038-53-7 H H OSO3- "

Decarbamoyl neosaxitoxin

68683-58-9 OH H H "

Rel. toxicity

0.51

0.65

0.75

No data

Sulfamate Saxitoxins

Derivative Designation

Registry Number

R1 R2 R3 R4

B1 64296-25-9 H H H OCONHOSO3-

C1 80173-30-4 H OSO3- H "

C2 80226-62-6 H H OSO3- "

B2 82810-44-4 OH H H "

Gonyautoxin C3

89614-45-9 OH OSO3- H "

Gonyautoxin C4

89674-98-6 OH H OSO3- "

Toxity of PSP molecules

Rel. toxicity

0;06

< 0.01

0.1

No data

0.01

0.06

Saxitoxins: characteristics and mode of action

• This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channels in nerve cells, thus causing their neurotoxic effects.

• 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 problems.

• Species are distinguished only by morphological features such as size, shape, presence/absence of chloroplasts etc.

• Thus the circumscription of many species is vague and a comprehensive taxonomic revision of this genus is needed. 

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

is a complex lipophilic polyether readily soluble in many organic solvents, degrading in acid or base.

Okadaic group

DSP molecules

DSP• 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.

DSP

Pectenotoxingroup

DSP

Yesso-toxingroup

Aza-spiracids

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

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 incidents.

• 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 toxins.

CFP

• 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

Ostreopsis

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 acid.

• 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 Hemisphere.

• 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

ASP• 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 species.

• Induction of neuronal imbalance can cause the brain to malfunction and may lead to lesions in the brain that can cause permanent injury.

•  

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 supplies.

• 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

Harmful effect associated with marine microalgal blooms

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 reported – 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(

WHAT TO DO ABOUT THEM ?

• 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, etc.). – 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 1996).

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

Basic morphologicaly

features of dinoflagellates

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 organisms.• 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 surface.

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

necessary

• 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, 1998).

– 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)

– HENCE NEED TO VALIDATE

Lectin-aided detection

In this case Gymnodinium was stained with FITC –labelled lectin from Phaseolus limensis (bean) (excitation 490 nm, emission 520 nm

FITC

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

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

Antibodies

• 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 microscopy.

– 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 vacuoles).

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 species.

• 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 radioactivity.

• As with antibodies, oligonucleotide probes can be labelled with a variety of fluorescent dyes such as fluorescein (FITC).

NA probes: examples• Table 5.3

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.

Na probe:

example

Identification toolsMolecular 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 detected)

• 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 phycotoxins.

• 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 range

• 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

blocker.

• 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• 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

PSP MOUSE BIOASSAY• 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

PSP MOUSE BIOASSAY• 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 min).

– 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 degraded.

Protocol for PSP

extraction(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

STXeq).

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.

HPLC conditions for PSP: example

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 programs.

• 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 preparation.

• 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 sites.

• 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 immuno-assays

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.

RidascreenAn 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 view.

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.

Exercise

• A sample contains 10 µg STX and 10 µg C1. Will the Ridascreen generate an overestimation or underestimation of the toxicity?

• 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, Canada).

• 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).

http://www.jellett.ca/global_occurrences.htm

Mist Alert system for PSP:

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 neuroreceptors).

• 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 matrices.

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 phosphorylase.

– 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

acute symptoms diarrhoea, paralysis

nausea, vomiting, diarhoea

tingling, hot and cold, dizziness

abdominal pain, nausea

vomiting, diarrhoea, confusion, memory loss

associated food shellfish shellfish shellfish fish shellfish

causative organism

Gymnodinium catenatum, Alexandrium, Pyrodinium bahamense var. Compressum

Dinophysis, Prorocentrum Karenia brevis Gambierdiscus

Pseudo-Nitzschia

Toxin molecular mechanism Na channel blockerPhosphorylase inhibitor Na channel activator Na/ Ca activator

Glutamate receptor agonist

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

Yessotoxin: 1mg/kgAzaspiricid: 160µg/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 (ASP):

• • 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 wastes.

– 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 level.

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 discharged.

• 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 diatoms.

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

conditions. – 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 biomass.

– 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

rias. • The inflow of warm waters coincides with very

weak upwelling ( which drives the nutricline below the photic layer) and favours the development of flagellates.

• 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 daysHarmful Algae Volume 14 2012 87 - 106

Dinophysis

• 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

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).

Harmful Algae Volume 14 2012 87 - 106

Case Study

The link between precipitation, river runoff, andblooms 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 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) 1992. Salinity and cell abundance data were only available as of mid-July 1992.

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) 1992.

• Salinity and cell abundance data were only available as of mid-July 1992

Case study

Case studyEnvironmental 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 stratified.

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 objective.

– 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 HAB monitoring scheme