SHELLFISH TOXICITY: HUMAN

HEALTH IMPLICATIONS OF MARINE ALGAL TOXINS

Ms. Bbhine Carey PROTEOBIO, Mass Spectrometry Centre, Cork Institute of Technology and The Environmental Research Institute, University College Cork.

OUTLINE

Shellfish industry Harmful algal blooms (HABs) 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 (HABS)

Algae usually autotrophic

organisms, unicellular or multicellular

Largest seaweed Only organism to readily make PUFAs

Plankton mass of microorganisms , 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 nontoxic 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 Birds Fish

Marine Mammals

Human

Crustaceans

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:

O HO OH O

Okadaic acid (OA) and dinophysistoxins (DTX) Pectenotoxin (PTX) Yessotoxin (YTX)

B
O OH

C
O O

CH3

O
31

F
O H

G
35

D E

O OH

HO

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

DIARRHETIC SHELLFISH POISONING (DSP)

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
CH3 O O O O OH CH3 O CH3 CH3 O H 3C O O O CH3
NaO3SO H 3C O O O NaO3SO O O O CH3 CH3 O O CH3 H 3C HO O

H3C

OH OH O O

Yessotoxin

O CH3 OH

Pectenotoxin 2

DIARRHETIC SHELLFISH POISONING (DSP)

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 g/g

Prorocentrum Lima

D. fortii

D. acuminata

D. acuta

AZASPIRACID SHELLFISH POISONING (AZP)

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)

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

AZASPIRACID SHELLFISH POISONING (AZP)

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.

AMNESIC SHELLFISH POISONING (ASP)

CH3 COOH

History:

1987 in Canada 107 cases of shellfish poisoning

H CH3

COOH N H COOH

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

AMNESIC SHELLFISH POISONING (ASP)

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 Hitchcocks 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

PARALYTIC SHELLFISH POISONING (PSP)

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

H 2N O O HN HN
Alexandrium tamarense

NH NH NH OH OH

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

PARALYTIC SHELLFISH POISONING (PSP)

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

1970s 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

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

HO O O O O O O O O O

O O O

Brevetoxin structure

Karenia brevis

NEUROTOXIC SHELLFISH POISONING (NSP)

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

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

MOUSE BIOASSAY VS. ANALYTICAL TECHNIQUES

100 Relative Abundance

Analytical techniques

DTX2

PTX2

Can be validated Robust

Does not harm animals

Sensitive and selective method

OA
1 2 3 4 5 6 7 8 9 Time (min) 10 11 12

00

Regulatory limits:

AZP and DSP 0.16 g/g ASP 20 g DA/g

NSP 0.8 g brevetoxin/g

PSP 0.8 g/g

GLOBAL INCREASE IN HABS

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

AZA DSP PSP

Canada,
PSP ASP NSP ASP DSP ASP

AZA AZA ASP DSP PSP PSP

AZA ASP Italy, 1997

DSP

DSP PSP ASP

PSP ASP

AZA ASP

PSP

DSP

PSP DSP DSP

ASP PSP NSP DSP PSP

DSP

PSP

ASP

NSP

AZP

INCREASED SCIENTIFIC AWARENESS

Reports of HABs, 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

INCREASED SCIENTIFIC AWARENESS

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 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 HABs 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

EUTROPHICATION

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, 1980s

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

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

CLIMATE CHANGE

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.

INCREASED GLOBAL MARINE TRAFFIC

Vector in dispersal of nonindigenous 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

INCREASED GLOBAL MARINE TRAFFIC

PSP was unknown in Australia up until the

1980s

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

CONCLUSION

Impact on human health increased in recent decades Increase of HABs

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