shellfish toxicity

48
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.

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Page 1: 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.

Page 2: Shellfish Toxicity

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

Page 3: Shellfish Toxicity

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

Page 4: Shellfish Toxicity

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:

Page 5: Shellfish Toxicity

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.

Page 6: Shellfish Toxicity

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

Page 7: Shellfish Toxicity

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)

Page 8: Shellfish Toxicity

Phytoplankton & Bacteria

Bivalves

Crustaceans

Fish

HumanMarine Mammals

Birds

Page 9: Shellfish Toxicity

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

Page 10: Shellfish Toxicity

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)

Page 11: Shellfish Toxicity

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

HOO

O

OH

OH

O

O OO

HO

H

OH

O

O

A

B C D

E

FG31

35

CH3

H

Page 12: Shellfish Toxicity

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)

O

O

CH3

CH3O

OO

CH3

H3COH

OH

O

O O

O

OH

H3C

O

OCH3

CH3

Pectenotoxin 2

O

OO

O

O

O

O

O O

O

O

CH3

CH3

CH3

OH

CH3

H3C

NaO3SO

H3C

HO

NaO3SO

Yessotoxin

Page 13: Shellfish Toxicity

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)

Page 14: Shellfish Toxicity

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

Page 15: Shellfish Toxicity

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)

Page 16: Shellfish Toxicity

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

Page 17: Shellfish Toxicity

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)

Page 18: Shellfish Toxicity

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)

Page 19: Shellfish Toxicity

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

COOH

CH3

H

COOH

CH3

H

N

COOH

Page 20: Shellfish Toxicity

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)

Page 21: Shellfish Toxicity

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)

Page 22: Shellfish Toxicity

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)

Page 23: Shellfish Toxicity

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

Page 24: Shellfish Toxicity

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

Page 25: Shellfish Toxicity

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)

Page 26: Shellfish Toxicity

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)

Page 27: Shellfish Toxicity

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

O

O

O

O

O

O

O

O

O

O

O

HOO

Brevetoxin structure Karenia brevis

Page 28: Shellfish Toxicity

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)

Page 29: Shellfish Toxicity

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)

Page 30: Shellfish Toxicity

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

Page 31: Shellfish Toxicity

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

100

Rela

tive A

bu

nd

an

ce

OA

DTX2PTX2

Page 32: Shellfish Toxicity

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

Page 33: Shellfish Toxicity

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

Page 34: Shellfish Toxicity

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

Page 35: Shellfish Toxicity

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

Page 36: Shellfish Toxicity

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

Page 37: Shellfish Toxicity

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

Page 38: Shellfish Toxicity

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

Page 39: Shellfish Toxicity

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

Page 40: Shellfish Toxicity

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

Page 41: Shellfish Toxicity

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

Page 42: Shellfish Toxicity

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

Page 43: Shellfish Toxicity

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

Page 44: Shellfish Toxicity

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

Page 45: Shellfish Toxicity

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

Page 46: Shellfish Toxicity

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

Page 47: Shellfish Toxicity

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

Page 48: Shellfish Toxicity

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