Download - Shellfish Toxicity
SHELLFISH TOXICITY: HUMAN
HEALTH IMPLICATIONS OF
MARINE ALGAL TOXINS
Ms. Bébhine Carey
PROTEOBIO, Mass Spectrometry Centre, Cork
Institute of Technology and The Environmental
Research Institute, University College Cork.
OUTLINE
Shellfish industry
Harmful algal blooms (HAB‟s)
Marine toxin syndromes
Diarrhetic Shellfish Poisoning (DSP)
Azaspiracid Shellfish Poisoning (AZP)
Amnesic Shellfish Poisoning (ASP)
Paralytic Shellfish Poisoning (PSP)
Neurotoxic Shellfish Poisoning (NSP)
Global increase in harmful algal blooms
SHELLFISH INDUSTRY
Rich source
protein, essential minerals, and
vitamins A and D
Mussels, clams and scallops are
cultured at sea
Bouchot culture method
Raft culture method
Bottom culture method
Rack and bag culture
HARMFUL ALGAL BLOOMS (HAB‟S)
Algae – usually autotrophic
organisms, unicellular or multi-
cellular
Largest – seaweed
Only organism to readily make
PUFAs
Plankton – mass of micro-
organisms , drift or float
Phytoplankton – diatoms,
dinoflagellates
How a toxic algal bloom occurs:
HARMFUL ALGAL BLOOMS (HABS)
Algae populations can increase rapidly
to form dense concentrations of cells
that may be visible.
“Red Tides”
Blooms are not always visible
Discolouration varies with species of
phytoplankton, size and concentration,
and depth
Phytoplankton generally proliferate
during the Summer months when the
water is calm and warmer.
HARMFUL ALGAL BLOOMS (HABS) Algal blooms can be non-toxic
providing energy to fuel food webs.
Small percentage produce
powerful toxins
Fish kills
Death of mammals and birds
Illness in humans
HAB also include blooms of non-
toxic species
Masses of algae die and decompose
Depletion of oxygen content in water
Oxygen levels become so low,
animals die or leave
MARINE TOXIN SYNDROMES
Bivalve molluscs filter large volumes of water
Mussels, clams, scallops
Phytoplankton
Toxic or non-toxic
Accumulate in digestive gland
Vectors
Five major toxic syndromes:
Diarrhetic Shellfish Poisoning (DSP)
Azaspiracid Shellfish Poisoning (AZP)
Amnesic Shellfish Poisoning (ASP)
Paralytic Shellfish Poisoning (PSP)
Neurotoxic Shellfish Poisoning (NSP)
Phytoplankton & Bacteria
Bivalves
Crustaceans
Fish
HumanMarine Mammals
Birds
MARINE TOXIN SYNDROMES
First recorded fatal case:
1793
Captain George Vancouver
Taboo to eat shellfish when the seawater became
phosphorescent due to dinoflagellate blooms
Paralytic shellfish poisoning
Lack of clinical testing methods
Gross underestimation of the incidence of human
poisonings from algal toxins
Many symptoms the same as bacterial or viral
infections
Little known about chronic exposure to these
toxins
DIARRHETIC SHELLFISH POISONING (DSP)
History:
First documented incidence of Diarrhetic Shellfish
poisoning, occurred in 1976 in North-eastern Japan.
164 people affected
Diarrhoea, nausea, vomiting and abdominal pain
Caused by ingestion of mussels and scallops
The dinoflagellate, Dinophysis fortii was identified as the
source of the toxin
Toxin responsible was named Dinophysistoxin (DTX)
Legend in the region warned that mussels become toxic
during the month of the paulownia flowers (June)
DIARRHETIC SHELLFISH POISONING (DSP)
Categorised into three structural
groups:
Okadaic acid (OA) and
dinophysistoxins (DTX)
Pectenotoxin (PTX)
Yessotoxin (YTX)
Other derivatives of okadaic acid:
DTX 2 – an isomer of okadaic acid,
isolated from Irish mussels
DTX 3 – isolated from the digestive
gland of the scallop Patinopecten
yessoensis
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OH
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HO
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OH
O
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A
B C D
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CH3
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Toxins removed from DSP classification:
Yessotoxins – sulphated polyether compounds, were isolated from
scallops along with DTX1 and DTX3
Pectenotoxins – polyether macrolides, isolated from toxic scallops
Do not cause diarrhoea
Produce a very toxic response when injected i.p into mice
No case of human poisoning has been reported
DIARRHETIC SHELLFISH POISONING (DSP)
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Pectenotoxin 2
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Yessotoxin
Symptoms:
Diarrhoea, nausea, vomiting and abdominal cramps
Onset of symptoms can occur within 30 mins up to a few
hours
Complete recovery within 3 days
Chronic effects – potent tumour promoters
Toxicity studies – OA is two times more toxic than DTX2
Often confused as bacterial enterotoxin poisoning
Okadaic acid and dinophysistoxins are heat stable,
lipophilic compounds which accumulate in the
digestive glands of filter feeding bivalves
DIARRHETIC SHELLFISH POISONING (DSP)
Produced by the dinoflagellates Dinophysis spp. and
Prorocentrum spp.
Toxin profile varies within a single species
Europe – OA and DTX2
Japan – OA and DTX1
Regulatory limit for these toxins in Europe is 0.16 µg/g
DIARRHETIC SHELLFISH POISONING (DSP)
Prorocentrum Lima D. fortii D. acuminata D. acuta
Most recently discovered marine toxin
Several analogues have been identified
First confirmed incidence of Azaspiracid Shellfish poisoning,
occurred in 1995 in Netherlands.
Caused by consumption of mussels (M. edulis)
Cultivated in Killary Harbour, in the west of Ireland
At least eight individuals were affected
Abdominal pain, nausea, vomiting, diarrhoea – similar to DSP
Azaspiracid 1 (AZA1) isolated from these mussels – its structure
identified
AZASPIRACID SHELLFISH POISONING (AZP)
AZASPIRACID SHELLFISH POISONING (AZP) There are greater than 20 known analogues of azaspiracids
Symptoms:
Diarrhoea, nausea, vomiting and abdominal cramps
Recovery within 3 days
Toxicological studies
Induce widespread organ damage in mice
More dangerous than other classes of shellfish toxins
Target organs – liver, spleen, the small intestine
Carcinogenic
Chronic exposure – tumour formation in the lungs and malignant
lymphomas
Interstitial pneumonia
Shortened small intestinal villi
Protoperidinium crassipes was initially thought to be the
causative agent
Cannot produce their own food by photosynthesis
Predators of dinoflagellates
Do not proliferate into large blooms
Vector of AZA toxins
Azadium spinosum
Dinoflagellate
Identified as the producer of AZA toxins
AZASPIRACID SHELLFISH POISONING (AZP)
AZA 2 recently found in a sponge
(Echinoclathria sp.) – Japan
First report of AZA in Asia
Global outbreaks:
UK, Norway, France, Italy, Spain and Denmark
North Africa
Human intoxications due to AZP
shellfish that had been passed as „safe for human
consumption‟ using DSP mouse bioassays.
DSP mouse bioassay protocols result in the
extraction of only 5-40% of total azaspiracids present
in mussels.
AZASPIRACID SHELLFISH POISONING (AZP)
AMNESIC SHELLFISH POISONING (ASP)
History:
1987 in Canada
107 cases of shellfish poisoning
Vector responsible were mussels (M. edulis)
Cultivated on Prince Edward Island
Victims suffered gastrointestinal disturbances as well as unusual
neurological symptoms – memory impairment
3 people died within 18 days of admission
Neurological symptoms – headaches, confusion, disorientation,
seizures and coma
Permanent short term memory loss – Amnesic shellfish poisoning
Domoic acid was identified as the toxin responsible
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CH3
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COOH
CH3
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N
COOH
Epidemiological studies
Age-dependant response
Less than 40 years old – gastrointestinal problems
Greater than 50 years old – suffer from memory
loss
Onset of symptoms:
Gastrointestinal – within 24hrs
Neurological - within 48hrs
Symptoms can last for a few days - possibility of
permanent memory loss
Diatom of the Pseudonitzschia spp.
In Japan, domoic acid previously known as a
natural product
Anthelminthic and insecticidal properties
Originally discovered in a seaweed
AMNESIC SHELLFISH POISONING (ASP)
Marine animals
1998 - 70 sea lions washed up onto beaches in California
Suffering from neurological problems
47 animals died
DA identified in faecal samples and in nearby anchovies
Birds
1991 – DA poisoning report in Monterey Bay, California
Pelicans and cormorants behaving strangely
Vomiting, unusual head movements, scratching , many deaths
Vector – northern anchovy
AMNESIC SHELLFISH POISONING (ASP)
Alfred Hitchcock‟s “The Birds”.
Similar event in 1961 in Santa Cruz,
prompted production
Flocks of shearwaters – acting erratically,
flying into houses and cars, pecking
people, breaking windows, vomiting
Reported in the local newspaper
Clippings were included in Alfred Hitchcock's
studio proposal
Based on a book by Daphne du Maurier
Similar events have occurred since along
the same coastline
AMNESIC SHELLFISH POISONING (ASP)
PSP toxins are collectively called saxitoxins (STXs)
At least 21 analogues of these cyclic guanidines
Saxitoxin the most common
Dinoflagellates responsible:
Alexandrium, Gymnodinium, Pyrodinium species
PARALYTIC SHELLFISH POISONING (PSP)
HN
NHN
HNH
NH
O
NH
O
H2N
OH
OHAlexandrium tamarense Structure of STX
PARALYTIC SHELLFISH POISONING (PSP)
Symptoms:
Mild – tingling sensation or numbness of the lips, face and neck.
Prickly sensation in fingertips and toes
Severe – headache, nausea, vomiting, diarrhoea, muscular
paralysis and respiratory difficulty
High risk of death in the absence of artificial respiration
Onset of symptoms occurs rapidly
Lethal dose is 1-4 mg STX or equivalents
Saxitoxins clears from the blood within 24hrs
No organ damage or long term effects
Schedule 1 list of the Chemical Weapons Convention
History:
1927 – Northern California
102 people poisoned from eating mussels
6 deaths
PSP outbreaks have occurred on both the eastern and
western coastlines of North America
Alaska – badly affected
PSP events for more than 130 years
1987 – Cape Cod Bay
14 humpback whales
Mackerel
PARALYTIC SHELLFISH POISONING (PSP)
Have been detected in European waters, human
intoxications are rare
1970‟s – PSP intoxications from mussels cultivated in Spain,
Portugal and U.K
80-120 individuals
Repeated outbreaks in Chile and Argentina
21 PSP deaths reported in Chile since 1991
Rare identification of toxins in body fluids of victims
Philippines
2000 cases of PSP 1983-1998
115 deaths
PARALYTIC SHELLFISH POISONING (PSP)
NEUROTOXIC SHELLFISH POISONING (NSP) Marine dinoflagellate – Karenia brevis
Produce neurotoxins called brevetoxins
Is a naked dinoflagellate – no protective layer
Affect finfish, aquatic mammals and birds
Death of large manatees and bottlenose dolphins
Brevetoxin-producing HABs have caused problems in the Gulf of
Mexico for decades, records beginning in 1947
NSP is not geographically widespread
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HOO
Brevetoxin structure Karenia brevis
Symptoms:
Act in three ways depending on route of
exposure – onset 30mins to 3hrs
Symptoms persist for a few days
Oral - gastroenteritis, chills, sweats,
reduction in core temperature, hypotension,
arrhythmias, numbness, peripheral tingling
and in severe cases broncho-constriction,
paralysis, seizures and coma.
Inhalation – sea spray contains brevetoxins
due to delicate nature of Karenia brevis.
Causes irritation to the eyes and nasal
passages, respiratory problems
Skin – eye and nasal irritation
NEUROTOXIC SHELLFISH POISONING (NSP)
1987, the Gulf stream carried a Karenia
brevis bloom from Florida to North Carolina
48 people with neurological symptoms
Vector – oysters
1 person was hospitalised with severe
neurological symptoms
1993, New Zealand
186 individuals affected with gastrointestinal
symptoms and respiratory problems
Vectors – green mussels, cockles and oysters
New brevetoxin analogues were detected
NEUROTOXIC SHELLFISH POISONING (NSP)
MOUSE BIOASSAY VS. ANALYTICAL TECHNIQUES
Mouse Bioassay:
Inject replicate mice with extract of sample
Observe symptoms exhibited in mice
Measure time it takes for the mice to die – overall toxicity
Primitive, non-selective
Results dependant on mouse strain, gender, age and weight –
results cannot be reproduced by other labs
Analytical techniques
Can be validated
Robust
Does not harm animals
Sensitive and selective method
Regulatory limits:
AZP and DSP – 0.16 µg/g
ASP – 20 µg DA/g
NSP – 0.8 µg brevetoxin/g
PSP – 0.8 µg/g
MOUSE BIOASSAY VS. ANALYTICAL TECHNIQUES
0 1 2 3 4 5 6 7 8 9 10 11 12Time (min)
0
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tive A
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DTX2PTX2
GLOBAL INCREASE IN HAB‟S Positive effect:
critical food for filter-feeding bivalve shellfish
Negative effect:
Major human health implications
Environmental impact
Severe economic losses to aquaculture, fisheries, and tourism.
Factors affecting HABs growth are not well understood
Explanations for possible increase:
Increased scientific awareness
Increased utilisation of coastal waters for aquaculture
Eutrophication
Climate change
Increased global marine traffic
Italy, 1997
Canada,
DSP PSP ASP NSP AZP
DSPPSP
DSPPSP
DSPPSP
DSPPSP
DSP
PSPDSP
DSP
DSP
DSPPSP
PSP
PSP
PSP
PSP
AZAASP
AZAASP
AZA
AZA
AZA
ASP
ASPASP
ASP
ASP
ASP
ASP
NSP
NSP
INCREASED SCIENTIFIC AWARENESS Reports of HAB‟s, associated human illnesses, shellfish
closures – increased media attention
More and more research is being carried out in the field
Algae A. minutum only know to exist in Egypt until
1988
Now it has been reported in Australia, Ireland, France,
Spain, Turkey, Portugal, Italy, east coast of North America,
Thailand, New Zealand, Taiwan and Japan
DSP first documented in 1976 in Japan – Dinophysis
fortii
In Europe - D. acuminata, D. acuta, D. norvegica etc.
From 1976-1982, 1,300 DSP cases reported in Japan
In 1981, 5,000 DSP cases reported in Spain
Research is driven with advances in analytical technology
Mass spectrometry
Single MS
Triple quadrupole MS
Ion Trap MS
Time Of Flight MS
Orbitrap technology
Lower detection limits
Much higher selectivity of compounds
High mass accuracy
Higher resolution than leading TOF
INCREASED SCIENTIFIC AWARENESS
INCREASED COASTAL AQUACULTURE
Overfishing of coastal waters – aquaculture
is the solution
Increase in shellfish farming is resulting in
more reports of paralytic, diarrhetic,
neurotoxic and amnesic shellfish poisoning
As a direct result of increased aquaculture
there is also an increase in biotoxin
monitoring
Finfish culture – algae species which
damage gill tissue
Increased aquaculture activities can lead to
localised nutrient enrichment
EUTROPHICATION
Hong Kong harbour
Eight fold increase in HAB‟s from
1976-1986
Corresponded with increasing
human population and increase in
nutrient loading
1972, Seto Inland Sea,
Chattonella antiqua bloom
Fish farm
Killed 14 million cultured yellow tail
fish
Untreated sewage and industrial
waste from pulp and paper factories
Introduced effluent controls to
reduce chemical oxygen demand
(COD)
After a 4 year period:
Frequency of blooms have decreased
Changing land use – deforestation can also cause shifts in
phytoplankton species composition
Increasing concentrations of humic substances in land run off
Humic substances – end product of decayed matter
Acid rain:
Increase mobility of humic substances and trace metals in soil
River water draining from agricultural land
Rich in N and P
Stimulates diatom growth
River water draining from forest areas
Rich in humic and fulvic acids
Stimulate dinoflagellate blooms
EUTROPHICATION
Nitrogen:phosphorus ratio – Tolo
harbour, Hong Kong, 1980‟s
Molar ratio of N:P decreased nearly
50%
Human population and sewage
loading increased
Dinoflagellate blooms increased
Altered N:P ratios implicated in
shifts in diatom-dominant to
dinoflagellate dominant blooms
U.S , European and Asian coastlines
EUTROPHICATION
CLIMATE CHANGE
Greenhouse effect and Warming of Oceans
Dinoflagellate pyrodinium bahamense found
in coastal waters of the Atlantic and Indo-
West Pacific
Survey of its resting cyst fossils - indicate
much wider distribution in the past
In the Australasian region, dinoflagellate
does not extend further than Papua New
Guinea
100,000 years ago it was found as far south as
Sydney harbour
Concern that this toxic algae species will
return to Australian waters
Extreme climate events
NSP considered endemic to the Gulf of
Mexico and the east coast of Florida
1987 major Florida outbreak
Dispersed by the gulf stream northward into
North Carolina waters
1993, 180 shellfish poisonings reported in
New Zealand similar to Karenia brevis
Hidden plankton flora
Developed into a bloom
Triggered by unusual climatological
conditions
El Nino event – imbalance atmospheric
pressure and sea temperature results in a
shoaling thermocline
CLIMATE CHANGE
Extreme climate events
hurricanes
expand the existing distribution of cyst-producing toxic
dinoflagellates
Alexandrium tamarense in New England after a 1972
hurricane
Reef Disturbances
Ciguatera finfish poisoning well-known in coral reef
areas in the Caribbean, Australia, and French Polynesia
Rare disease two centuries ago
Now it has reached epidemic proportions especially in
French Polynesia
Reef disturbance by hurricanes, military, tourist
developments and coral bleaching (linked to global
warming) as well as future increasing coral damage due
to ocean acidification
Increasing the risk of ciguatera.
CLIMATE CHANGE
INCREASED GLOBAL MARINE TRAFFIC
Vector in dispersal of non-
indigenous marine plankton
Release of ballast waters
Algal cysts in ballast waters
Planktonic stages of dinoflagellates -
limited survival during the voyage
Resistant resting spores
One single ballast tank – 300 million
toxic dinoflagellate cysts
PSP was unknown in Australia up until the
1980‟s
First outbreak was in the ports of Hobart,
Melbourne and Adelaide
Hobart – examination of historic plankton samples,
cyst surveys in dated sediment depth cores
Toxic dinoflagellate G. Catenatum introduced after
1973
Genetic fingerprinting using rRNA sequencing
Genetic affinities between Australian and Japanese strains
of A. Catenella and Australian and European strains of A.
minutum
Severe economic damage to fisheries
INCREASED GLOBAL MARINE TRAFFIC
The International Maritime Organisation (IMO)
has introduced guidelines for ballast water
handling
Reballasting at sea, ballasting in deep water ,
disposal of ballast tank sediment away from sensitive
aquaculture or marine park areas
Ideally avoid ballasting during toxic dinoflagellate
blooms in ports
Use heat, electrical shock or chemical treatment
INCREASED GLOBAL MARINE TRAFFIC
Recent evidence
first reports of palytoxin and tetrodotoxin in European waters
Azaspiracids found in Japanese waters
Tetrodotoxin
Also called pufferfish poisoning
Paralytic toxin
Usually found in tropical and sub-tropical waters
September 2007 - Trumpet shellfish (Charonia lampas sauliae) from
the southern coast of Portugal
Symptoms include:
perioral numbness, acral numbness, nausea, vomiting, dizziness or
vertigo, weakness, ataxia, dyspnea, diaphoresis, and death from
respiratory failure
INCREASED GLOBAL MARINE TRAFFIC
Another possible vector:
Translocation of shellfish stocks from one
area to another
Faeces and digestive tracts of bivalves can
be loaded with:
Viable dinoflagellates
Resistant resting cysts
Japanese seaweeds:
Sargassum muticum (England, Netherlands,
Norway)
Undaria pinnatifida and Laminaria
japonica (Mediterranean)
Introduced to European waters through
Japanese oyster spat
INCREASED GLOBAL MARINE TRAFFIC
CONCLUSION
Impact on human health – increased in recent decades
Increase of HAB‟s
Increased marine traffic
Climate change
Eutrophication
Aquaculture
Increased scientific awareness
Improvements in analytical techniques have limited toxin
exposure
Clinical testing required
James et al. Shellfish toxicity: human health implications of
marine algal toxins, epidemiology and infections, 2010