Toxin and toxic organisms
causing food poisoning
Dao Viet HaHead of Biochemistry Department Institute of Oceanography01 Cau Da – Nha Trang - Vietnam
WESTPAC workshop 2010.Dec.13
Marine products especially fish & shellfish: Important food resources in the Western Pacific &
the ASEAN countries
Aquacultural activities in Cam Ranh Bay, Khanh Hoa (Photo by Fukuyo, 2005)
Aquaculture becomes more important
Two types of toxic organism in aquatic environment
1.Toxic by taking toxic organisms such as microalgae by feeding activity (well-known)
example: FishBivalve…
2. Toxic by its own toxin production (unknown) (either symbiotic bacteria or encode in gene)
example: PufferBlue-ringed octopusGoby fishJelly fishConusCrab…
Two types of toxic organism in aquatic environment
1. Toxic by taking toxic organisms such as microalgae by feeding activity
example: FishBivalve…
2. Toxic by its own toxin production (either symbiotic bacteria or encode in gene)
example: PufferBlue-ringed octopusGoby fishJelly fishConusCrab…
Toxins in HAB organisms:
human poisoning
Characters of the accumulation type1.Toxic by taking toxic organisms such as microalgae by
feeding activity1) Toxin accumulation occurs during blooming of causative
microalgae 2) Toxins are excreted constantly, even during accumulation.3) Speed of accumulation and excretion is different
depending on species. 4) All filter feeding organisms have potential to become toxic
(cases of poisoning by a sea squirt were reported)5) Depending on microalgae fed, toxins in organisms differ;
Naturally poisoning caused by the organisms vary.PSP (paralytic shellfish poisoning)DSP (diarrehtic shellfish poisoning)ASP (amnesic shellfish poisoning)CFP (ciguatera fish poisoning) etc.
Characters of the accumulation type1.Toxic by taking toxic organisms such as microalgae by
feeding activity1) Toxin accumulation occurs during blooming of causative
microalgae 2) Toxins are excreted constantly, even during accumulation.3) Speed of accumulation and excretion is different
depending on species. 4) All filter feeding organisms have potential to become toxic
(cases of poisoning by a sea squirt were reported)5) Depending on microalgae fed, toxins in organisms differ;
Naturally poisoning caused by the organisms vary.PSP (paralytic shellfish poisoning)DSP (diarrehtic shellfish poisoning)ASP (amnesic shellfish poisoning)CFP (ciguatera fish poisoning) etc.
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Toxin
Origin of toxins are unicellular microalgae
Paralytic shellfish poison (PSP) Diarrhetic shellfish poison (DSP) Amnesic shellfish poison (ASP)Neurotoxic shellfish poison (NSP) Azaspiracid (AZA)
Toxin accumulation
PSP Toxin accumulates in relation to the abundance of causative plankton.Toxins are released, even during toxic plankton presence.
Max PSP toxin
North America
South China
Each shellfish species has different toxin accumulation character.
Scallop
Mussel Toxin excretion pattern of scallop varies from that of mussel:
Scallop keeps toxins much longer than mussel.
Certain amount of toxins remain in scallop very long time.
Toxin excretion
Toxin excretion
Toxin excretion: oyster, mussel: fast; clam, scallop: slow
Characters of the accumulation type1.Toxic by taking toxic organisms such as microalgae by
feeding activity1) Toxin accumulation occurs during blooming of causative
microalgae 2) Toxins are excreted constantly, even during accumulation.3) Speed of accumulation and excretion is different
depending on species. 4) All filter feeding organisms have potential to become toxic
(cases of poisoning by a sea squirt were reported)5) Depending on microalgae fed, toxins in organisms differ;
Naturally poisoning caused by the organisms vary.PSP (paralytic shellfish poisoning)DSP (diarrehtic shellfish poisoning)ASP (amnesic shellfish poisoning)CFP (ciguatera fish poisoning) etc.
toxi
city
(MU
/g)
May June July
Scallop
Oyster
Mussel
tunicate
Toxin accumulation speed varies species by species.
Filter feeding organisms, such as scallop, mussel and sea skirt become toxic during toxic plankton blooming.
PSP plankton bloom
Characters of the accumulation type1.Toxic by taking toxic organisms such as microalgae by
feeding activity1) Toxin accumulation occurs during blooming of causative
microalgae 2) Toxins are excreted constantly, even during accumulation.3) Speed of accumulation and excretion is different
depending on species. 4) All filter feeding organisms have potential to become toxic
(cases of poisoning by a tunicate were reported)5) Depending on microalgae fed, toxins in organisms differ;
Naturally poisoning caused by the organisms vary.PSP (paralytic shellfish poisoning)DSP (diarrehtic shellfish poisoning)ASP (amnesic shellfish poisoning)CFP (ciguatera fish poisoning) etc.
Toxins caused human poisoning:1. Fish vector: Ciguatoxin,
2. Shellfish vector:
Paralytic shellfish poison (PSP) (saxitoxin and its derivatives) Diarrhetic shellfish poison (DSP) (dinophysistoxin, pectenotoxin, yessotoxin) Amnesic shellfish poison (ASP) (domoic acid) Neurotoxic shellfish poison (NSP) (brevetoxin) Azaspiracid shellfish poison (AZP)
Toxins caused human poisoning:1. Fish vector: Ciguatoxin,
2. Shellfish vector:
Paralytic shellfish poison (PSP) (saxitoxin and its derivatives) Diarrhetic shellfish poison (DSP) (dinophysistoxin, pectenotoxin, yessotoxin) Amnesic shellfish poison (ASP) (domoic acid) Neurotoxic shellfish poison (NSP) (brevetoxin) Azaspiracid shellfish poison (AZP)
Gambierdiscus toxicus
Human poisoning: Ciguatera Fish Poisoning
is most frequently reported seafood-toxin illness in the world.
illness results from eating reef fish containing ciguatoxins derived from benthic dinoflagellates of the genus Gambierdiscus growing in association with macroalgae in coral reefs in tropical and subtropical climates.
toxins are transferred through the food web by herbivorous and carnivorous fish.
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HOH3C H
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OHCH3H3C
HHHHH
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15
30
40
50
Characters of toxins responsible for ciguatera
Ciguatoxin main toxin for ciguatera fat soluble
Maitotoxin water soluble toxin
CTX activates the voltage-gated sodium channels in cell membranes which increases sodium ion permeability and depolarizes the nerve cell causing an array of neurological symptoms
under-reporting may be considerable; between10 and 50 thousand people per year are estimated to suffer from CFP
in some patients chronic symptoms may last for months
Human poisoning: Ciguatera Fish Poisoning
Ciguatera: 0.05 µg (mild symptom)
Acute: gastrointestinal, neurological and cardiovascular signs.
Symptoms (within a few hours of contaminated fish ingestion) in wide variation: numbness, headache, eresthism, dizziness, cyanosis, prickling of lips, tongue and throat, nausea, vomiting, metallic taste, dryness of mouth, abdominal cramps and diarrhea. Sever case: reversal of temperature sensation, general
motor incoordination, paralysis and convulsion.
Fatality rate is low but recovery is slow and it takes several months.
More than 30,000 cases per year, most commonly reported disease in tropical and sub-tropical countries.
Ciguatera
Ciguatera causative dinoflagellate
Gambierdiscus toxicus
Benthic dinoflagellates living on seaweeds
Ciguatera is known endemic in Australia and tropical islands, but quite few in Western Pacific Asia. Recently Philippines has toxic fish.
Mechanism of ciguatera
From: BFAR Philippinestoxin producer herbivore
omnivore
carnivore patient
Countermeasures to ciguatera
Prohibition of marketing of possibly toxic fish species is only practical countermeasure to prevent poisoning.
Measurement of toxin amount is possible by HPLC, but standard toxins are difficult to obtain.
ELISA kits have been developed, but effectiveness is limited. Kits are very expensive.
Therefore, most countries do not have any countermeasure.
Toxins caused human poisoning:1. Fish vector: Ciguatoxin,
2. Shellfish vector:
Paralytic shellfish poison (PSP) (saxitoxin and its derivatives) Diarrhetic shellfish poison (DSP) (dinophysistoxin, pectenotoxin, yessotoxin) Amnesic shellfish poison (ASP) (domoic acid) Neurotoxic shellfish poison (NSP) (brevetoxin) Azaspiracid shellfish poison (AZP)
Mechanism of toxin accumulation
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Toxin
Excreted ToxinToxin producing microalgae
feeding microalgae
decomposing microalgae
excreting wastes
Gymnodinium catenatum
Alexandrium catenella
Pyrodinium bahamense
Human poisoning: Paralytic Shellfish Poisoning
PSP toxins produced from marine dinoflagellates of 3 genera, possibly the most geographically widespread marine biotoxins worldwide
in 1937, Sommer and colleagues established the association between toxic shellfish and blooms of the dinoflagellate Alexandrium catenella (then known as Gonyaulax catenella) along the California coast
toxins include the potent parent compound saxitoxin and approximately 20 variants of this basic structure which range considerably in toxicity
the regulatory limit for PSP toxins 80 µg STX equiv. 100 g-1 was established in the 1930s and is based on bioassays measuring toxic activity in mice
each year about 2000 cases of PSP are reported with 15 % mortality
levels of shellfish toxicity are dependent on the toxin profile and quota of the algae upon which they feed, the rate of uptake of algae by shellfish, capability of shellfish to transform, retain or depurate toxins
Human poisoning: Paralytic Shellfish Poisoning
Saxitoxin
0 500 1000 1500 2000 2500Specific toxicity (MU/mol)*
STXneoSTXdcSTX
GTX1GTX2GTX3GTX4
dcGTX2dcGTX3
GTX5(B1)C1C2C3C4
HPLC analyzer determines each toxin component as its concentration (mM)
248322951274
2468892
158418031617
1872
15160
23933143
Toxicity(MU/g) can be calculated = Concentration x Specific toxicity for each toxin component
Human poisoning: Paralytic Shellfish Poisoning
now realized that PSP toxins are more prevalent in food webs than previously thought
transfer of toxins to higher trophic levels has led to mortality events involving finfish, seabirds and marine mammals, e.g. whales having consumed toxic mackerel
shellfish typically serve as vectors of PSP toxins, but there are now many documented cases of shellfish mortalities
Paralytic shellfish poisoning (PSP)
Symptom is mainly paralysis of nerve system Tingling or burning sensation of lips, tongue and face starts
within ca. 30 min after ingestion. Paralysis of voluntary muscle extend to hands and legs. Patients keep conscious, but cannot move their body. Respiratory paralysis causes mortality within 12 hrs. High mortality (ca. 10%) Recover, if survival is over 12 hrs, within 48 hrs. No sequela.
No antidote. Facilitation of vomiting and urination is useful. Artificial respiration is the only a rescue.
Countermeasures to PSP
Prohibition of marketing of shellfish and other seafood possessing PSP toxins more than permitted level is the
only practical countermeasure to prevent poisoning.
Toxin amount is measured by mouse bioassay. Confirmation of toxins is by HPLC, but standard toxins are difficult to
obtain.
Most countries adopt 80 mg STX eq/100g edible body; Philippines takes 40 mg STX eq/100g edible body, because of monitoring difficulties.
ELISA kits have been developed, but effectiveness is still limited. Kits are very expensive.
Human poisoning:Diarrhetic Shellfish Poisoningfirst documented in 1976 [scallops in Japan]
very common form of poisoning[5000 cases in Spain in 1981]traditional ‘DSP complex’
okadaic [OA] and dinophysistoxins [DTXs] acid polyethers that inhibit protein phosphatase, and are the only toxins of the DSP complex with diarrheogenic effects in mammals
pectenotoxins [PTXs] are polyether-lactones which are hepatotoxic to mice by intraperitoneal injection
yessotoxins [YTXs] are disulphated polyethers that are cardiotoxic to mice, but are not associated with human poisonings
diarrhetic lipophilic toxins have been confirmed in at least 12 mixotrophic or heterotrophic species of Dinophysis
occasionally DSP toxins in shellfish have been associated with concentrations of Dinophysis as low as a few hundred cells l-1
Dinophysis species
Human poiosning: Diarrhetic Shellfish Poisonin
Diarrhetic shellfish poisoning (DSP)
Symptom always includes diarrhea. Vomiting and stomachache associates in more than 60% cases. Fever is rare. Symptom starts within 15 min to 4 hrs after ingestion. No mortality. Recover within 3 days. No sequela (?) cancer? Main toxin, okadaic acid, is cancer promoting agent.
Only bivalves are vector. No antidote.
HOOC OOOHH3C
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2
7
Okadaic acid (OA)
Okadaic acid
one of DSP causative toxin no color, no smell, no taste, transparent heat stable, acid stable fat soluble
DSP occurrences and countermeasures
Occurrences in 1976 some hundreds cases in Japan by mussel in 1981-1983 several EU countries (Spain, France, Norway
etc.) had >8,000 cases by mussel Chile and New Zealand reported DSP recently in SEA very few report: Vietnam has several cases in Meretrix lyrata in Can Gio district from 1999 - 2002 causative food are only bivalves (mussel, scallop, clam, etc.)
Countermeasure prohibition of marketing of shellfish possessing DSP toxins
more than permitted level DSP toxin quantification by mouse assay in most countries. no monitoring in most SEA (only for foods for export)
Karenia brevis
Human poisoning: Neurotoxic Shellfish Poisoningcaused by brevetoxins produced by Karenia brevis [well known along the Gulf of Mexico, specifically the coast of Florida where it was first reported in 1844]
brevetoxins are lipid soluble cyclic polyethers which activate voltage-sensitive sodium channels causing sodium influx and nerve membrane depolarization [symptoms both neurological and gastrointestinal]
cell lysis allows toxins to be aerosolized by wind, wave and surf conditions causing respiratory and skin reactions in humans
Neurotoxic shellfish poisoning (NSP)
Symptom is neurological disorder such as tingling or burning sensation in mouth, which develops in the face, throat, and over the entire body, muscle paralysis and unusual temperature sensation within 1-2 hrs after ingestion. Patients had the sensation of being drunken. Diarrhea Patients keep conscious, but cannot move their body. Respiratory paralysis causes mortality within 12 hrs. No mortality Recover in a few days without any after effect. No antidote. At a red tide of K. brevis, another type of symptoms occur.
Neurotoxic shellfishpoisoning (NSP)
Characters of sick at red tide Responsible toxin is also brevetoxin Symptom appeared in people breathing wind containing volatile agents from dead red tide K. brevis cells is irritation of eyes and throats, accompanied by lacrimation and coughing. Mass mortality of fish and other marine organisms often occur at red tide bloom of K. brevis. Responsible agent is hemolytic compound, not brevetoxin.these are well described inhttp://www.floridamarine.org/features/default.asp?id=1018
Brevetoxin
Karenia brevis
Brevetoxin enhances inflow of Na+ inside nerve and muscle membrane.
Toxicity is strong. LD50 of brevetoxin B and
PbTX3 to mouse is 190 and 170 mg/kg, respectively.
Toxicity in shellfish is monitoredby mouse bioassay in USA,New Zealand, and Italy.
Amnesic shellfish poisoning (ASP)
Characters after ingestion of contaminated seafood
Poisoning cases was reported only in Canada with 108 patients including 3 mortality by softening of the brain. Symptom is vomiting, diarrhea, stomachache, headache, and diminution of appetite. In sever case, lost memory (amnesia), confusion, and lost of sense of balance and paralysis occur. In heavy case, patients lose conscious and die. Recover is slow. Amnesia is obvious. Mortality cases of birds and marine mammals were reported in USA, Argentine, and Mexico.
No antidote
domoic acid
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Responsible toxins of ASP
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kainic acid
Main toxin, domoic acid, is water soluble amino acid. Domoic acid and kainic acid compete with glutamic acid
to react to a receptor of nerve, and connect to it more than 10 times stronger than glutamic acid.
Glutamic acid is stimulant transmitter in the central nervous system. By domoic acid, glutamic acid cannot work.
Domic acid breaks memory center of brain irrecoverably.
glutamic acid
ASP causative organisms
The genus Pseudo-nitzschia has more than 33 species. 22 of them are non-toxic or not confirmed its toxin productivity. Pseudo-nitzschia spp.
Pseudo-nitzschia produces domoic acid only late-log to stationary growth phases
13 diatoms are confirmed its toxin productivity 11 Pseudo-nitzschia species
P. australisP. callianthaP. delicatissimaP. fraudulentaP. galaxiaeP. multiseriesP. multistriataP. pseudodelicatissimaP. pungensP. seriataP. turgidula
1 Nitaschia species: N. navis-varingica 1 Amphora species: A. coffaeiformis
has also caused intoxications in wildlife [sea lions, whales, sea otters, sea birds]
has however caused significant closures of fisheries, e.g. recreational razor clam harvesting
since 1987 monitoring has successfully prevented other human incidents of ASP
Human poisoning: Amnesic Shellfish Poisoning
Countermeasures to ASP
Prohibition of marketing of seafood possessing ASP toxins more than permitted level (20mgDA/kg edible part) is the only practical countermeasure to prevent poisoning.
At the time of poisoning occurrence in Canada, patients took 60-110 mg had mild symptom, but those 115 - 290 mg had serious poisoning.
Toxin amount is measured by LC (Liquid Chromatography).
ELISA kits have been developed for monitoring purposes.
Most of countries, except EU, North America, and Oceania, do not have ASP monitoring system.
Human poisoning: Azaspiracid Shellfish Poisoning
Protoperidinium crassipes identified as a potential producer of AZA, however the heterotrophic nature of this dinoflagellate requires consideration of its potential role as a vector rather than progenitor of these toxins
Protoperidinium crassipes
AZAs are marine polyether toxins also co-extracted with other lipophilic toxins, providing a single response in conventional mouse bioassays for DSP
caused human illness in 1995 in the Netherlands following ingestion of shellfish from Killary Harbour, Ireland [symptoms typical of DSP but DSP toxins were below regulatory levels]
Azaspiracid Shellfish Poisoning (AZP)
Protoperidinium crassipes
AZP toxicity in mussels is monitored by mouse bioassay or LC/MSin Europe.
Found in Netherlands in 1995 by mussels harvested in Ireland. So far reported only from mussels in Ireland water. Main symptoms such as nausea, vomiting, diarrhea and stomach cramp were similar to DSP, but responsible toxinwas different, neither OA nor DTXs. Causative organisms are dinoflagellates such as Protoperidinium crassipes.
Azadinium spinosum
AZP remains a rare illness and only five intoxication events reported to date [may be a high percentage of under reporting]
EU regulatory limit of AZAs 160 µg kg-1
Tillmann et al. (2009) recently identified Azadinium spinosumas the source of AZA
Human Health: Azaspiracid Shellfish Poisoning
Protoceratium reticulatum
Lingulodinium polyedrum
Human Health: Yessotoxins
Gonyaulax spinifera
YTXs have been associated with DSP as they are simultaneously extracted with DSP toxins and give positive results when tested with the mouse bioassay
recent evidence indicates that they should be excluded from the DSP toxins group as they do not cause diarrhea and date no human intoxication has been reported
yessotoxins [marine polyether toxins] known to be produced by 3 dinoflagellate species
YTXs have been found to be potent cytotoxins prompting the EU to establish a maximum permitted level of 1 mg YTX equiv. kg-1
Palytoxin
is a large, complex polyhydroxylated compound; one of the most potent toxins so far known
Human poisoning: Palytoxin
first isolated from the soft coral [Palythoa toxica] and subsequently from many other organisms including the benthic dinoflagellate, Ostreopsis siamensis
has caused mortalities of molluscs and echinoderms
cases of human deaths have been related to the consumption of crabs, sea urchins and fishOstreopsis siamensis
Microcystis aeruginosa
toxic cyanobacteria more common in freshwater, e.g. Microcystis aeruginosaproduces potent hepatotoxins called microcystins
WHO set an advisory limit of 1µg microcystin-LR equiv. l-1drinking water
toxic Microcystis blooms threaten wildlife and farm animals, human health, and recreational activities
Human poisoning: Hepatotoxicity
Poisoning Problems in Western Pacific
PSP: Serious 1980s and 1990s, but few cases after 2000; now recurrence problem causative species increases; area increasesDSP: Toxic dinoflagellates are detected, but no
monitoring on toxicity in shellfishASP: Toxic diatoms are detected, but no
monitoring on toxicity in shellfishNSP, AZP: No studies Ciguatera: 1997- Philippines and Hong Kong;
several studies on benthic dinoflagellates, but few on toxicity of fishes
Two types of toxic organism in aquatic environment
1. Toxic by taking toxic organisms such as microalgae by feeding activity
example: FishBivalve…
2. Toxic by its own toxin production (either symbiotic bacteria or encode in gene)
example: Puffer,Blue-ringed octopus,Goby fish,Snail,Horseshoe crab, Xanthid rab…
Toxic animals
Some common marine puffer fish in SEA (Species name upper from left; Takifugu oblongus, Lagocephalus inermis, L. lunaris, middle from left; frL.
suezensis, Torquigener gloerfelti, T, brevipinnis, lower from left; Arothron hispidus, A. nigropunctatus, Canthigaster valentine)
Horseshoe crab Carcinoscopius rotundicauda
Goby fish Youngeichthys nebulosa
Blue-ringed octopusHapalochlaena lunulata
Marine toxic snails
Some toxic snails in Vietnam: From left, up to down:Charonia tritonis, Nassasius conoidalis, Oliva cericea, Polinices didyma, Nassasius catus*, Tutufa bufo, Tubo chysostomus, Tectus pyramis*:Causative species for human poisonings in VN
Chemical structure of Tetrodotoxin
Symptoms of poisoning•Main symptom of TTX poisoning is paralysis, whichis similar to PSP, which appears in 0.5 - 5 hoursafter eating. Death may occur within 1.5 - 8 hourswith lethal dose to human (50 kg) about 2 mg. Ifpatients survive for 8 hours, they will recoverwithout any residual longer effects.
Regulation•No regulation of TTX in toxic animals such aspuffers has been set in most countries. In Japan,10 MU is regarded as a limit of safe-consumptionlimit for puffer meat and skin (Kodama and Sato,2005).
・TTX and PSP occur in various but limited species in high concentration.
・A low level of TTX and PSP can be detected commonly in aquatic animals
・Causative organisms could be microorganisms.
Bacterial production of toxins
TTX: Yasumoto et al. (1986)Noguchi et al. (1986)
PSP: Kodama et al. (1988)
ProblemsToxin production is very lowBacteria do not always produce toxins
Kodama et.al. 2000
Characters of toxicitySpecies variationSeasonal variationGeographical variationIndividual variation (very wide in case of
puffer)Organ variationOccasionally (marine snails)
So, poisoning is unprediction, difficult to regulate/control
Numbers of poisoning cases, victims and death by eating puffer in Vietnam
Deaths by eating puffer is 42,9% of deaths by food poisoning Japanese cases are in ()
year case nos patients casualty1999 30 (20) 105 (34) 16 (2)
2000 27 (29) 129 (40) 29 (0)
2001 48 (31) 211 (52) 29 (3)
2002 49 (37) 210 (56) 29 (6)
2003 22 (38) 82 (50) 24 (3)
total 176 (155) 737 (232) 127 (14)
Importance:
Education to local peoples on scientific facts.