RESEARCH REPORT 2015 Bacteriological and Bio-toxin ...eastern-ifca.gov.uk/wp-content/uploads/2016/04/... · RESEARCH REPORT 2015 Bacteriological and Bio-toxin sampling 2015 L.E. RUTLAND

RESEARCH REPORT

2015

Bacteriological and Bio-toxin

sampling 2015

L.E. RUTLAND

2

Contents

1. Introduction Page3

2.0 Requirement under Regulation 854/2004 Page 4

2.1 Microbiological testing Page 4 -Sanitary Survey -Classification of production areas 2.2 Bio-toxin testing Page 7

-What is Shellfish poisoning and what are the toxic affects? 2.3 Amnesic shellfish poisoning (ASP) Page 9 -Causes -Clinical symptoms

2.4 Diarrhetic shellfish poisoning (DSP) Page 10 -Causes

-Clinical symptoms

2.5 Paralytic shellfish poisoning (PSP) Page 11 -Causes

-Clinical symptoms

3.0 Methods Page 12 -Collection and Processing of Live bivalve mollusc samples

-Method used to quantify Amnesic Shellfish Poison in Shellfish -Methods used to collect and analyse water samples for toxin-producing phytoplankton species -Collection of water samples -Processing of water samples

4.0 Results of the 2014/2015 monitoring programme Page 18 -E.coli flesh results -Bio-toxin results -Water sample test results -Flesh sample test results

5.0 Discussion Page 22

6.0 References Page 23

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1. Introduction

Shellfish form a major component of the landings

within our district. Within the Wash, Cockles and

Mussels are the main targets of fishermen with

cockles alone providing an average first sale

value of over one million pounds per year

(Jessop, 2014). Before shellfish can be harvested

and sold, bivalve production areas must be

classified to determine whether the shellfish

harvested from these areas are fit for human

consumption, this is a requirement under EC Regulation 854/2004.

It is well known that bivalves are responsible for a number of human diseases and this is largely due

to the nature of their feeding mechanism. Bivalves are filter feeders; they remove particles from

suspension by pumping water over their ctenidia. Particles are then selected and transported to the

mouth and latter digested (Ward, 1993). This feeding mechanism enables bivalve molluscs to

concentrate and retain bacterial and viral pathogens and contaminants found in there surrounding

waters; this can be common where shellfish beds are located close to point sources of pollution.

The Local authority is tasked with ensuring a sampling

regime is put in place so that periodic monitoring of the

shellfish production areas is achieved in order to check for

microbiological contamination, marine biotoxins and

chemical contamination.

Eastern IFCA collects both shellfish and water samples for bacteriological and biotoxin analysis on a

monthly basis on behalf of the local authority’s. The next section will outline the classification and

monitoring requirements for shellfish production areas.

Figure 1. Harvested Cockles from the Wash

Figure 2. Mytilus edulis

4

2.0 EU Requirement under Regulation 854/2004 to monitor shellfish

production areas

2.1 Microbiological testing

When an application is submitted for the establishment of a new shellfish production area the area

must first be classified. The classification process involves a number of steps which are outlined

below and detailed in the following section:

Sanitary survey

Classification of the production area

Establishment of a monitoring program

Bio-toxin monitoring

Sanitary Survey

A sanitary survey is carried out in order to evaluate the risk of microbiological contamination to

shellfish within the proposed production area and is carried out before a provisional classification

can be awarded. The survey considers the following information (Also summarised in figure 3).

location and extent of the bivalve mollusc fishery

Type of shellfishery (species, method of harvest, seasonality of harvest)

Location, type and volume of sewage discharges

Location of river inputs and other potentially contaminated water courses

Location of harbours and marinas

Hydrographic and hydrometric data

Existing microbiological data from water quality or shellfish monitoring undertaken in the

same area or adjacent areas.

Once the sanitary survey has been completed provisional classification of the harvesting area can be

made. The results of the sanitary survey provides the basis of the sampling plan and enables

Representative monitoring points (RMP) to be identified. RMP’s reflect the locations at which a

5

pollution event is most likely and thus routine monitoring of these sites should ensure detection of

such an event.

Figure 3. Requirements for the classification of shellfish harvesting areas

6

Classification of production areas The classification given to a production area determines the treatment that is required before the

shellfish may be marketed. For a provisional classification to be made a minimum of 10 samples

must be taken from the RMP over a 3 month period. Sampling is then continued on a monthly basis.

Once monthly monitoring has been continued for 1 year full classification may be awarded to the

production area. There are three classes within this classification system, Class A, B and C (Figure 4).

Figure 4. Classifications of shellfish production areas

For a Class A classification Live bivalve molluscs collected within the production area must contain less than 230 E.coli per 100g of flesh and intra-valvular liquid.

In production areas with a Class A classification, live bivalve molluscs may be collected for direct human consumption and require no further processing.

For a Class B classification Live bivalve molluscs collected within the production area must not exceed the limits of a five-tube, three dilution Most Probable Number (MPN) test of 4 600 E.coli

per 100 g of flesh and intra-valvular liquid. In production areas with a Class B classification, live bivalve molluscs may be placed on the

market only after treatment in a purification centre or after relaying.

For a Class C classification Live bivalve molluscs collected within the production area must not exceed the limits of a five-tube, three dilution MPN test of 46 000 E.coli per 100 g of flesh and

intra-valvular liquid so as to meet the health standards required. In production areas with a Class C classification, live bivalve molluscs may be placed on the

market only after relaying over a long period so as to meet the health standards required

7

Adapted from (Nordsieck, 2011)

2.2 Bio-toxin testing

As well as analysing flesh samples for E.coli, flesh and water samples are also taken to look for toxin-

producing plankton and bio-toxins. The major toxin groups tested for during bio-toxin sampling are

Paralytic shellfish poisoning (PSP), Amnesic shellfish poisoning (ASP) and Lipophilic toxin responsible

for Diarrhetic shellfish poisoning (DSP). Table 2 highlights the permitted levels for each toxin tested.

What is Shellfish poisoning and what are the toxic affects?

Bivalves are suspension feeders that filter food from the

water column. Food enters the mantel cavity where

particles are captured by ciliated feeding structures.

Filtration is regulated to select particles based on size,

shape, nutritive value and chemical composition. (Arapov,

2010). The bivalve diet consists of phytoplankton, which

forms the primary food source, bacteria, detritus and

zooplankton and thus bivalves are important in the nutrient flux between benthic and pelagic

communities and are an important part of the marine food web.

Bivalve molluscs accumulate naturally occurring bio-toxins produced by marine algae and act as a

host for a wide range of parasites, these parasites have little or no effect within the bivalve host and

are transmitted to their definitive host (Humans and other vertebrates) by the consumption of raw

or undercooked shellfish where they can have serious effects on human health (Ben-Horin, 2015).

Toxin Annreviation Permitted levels of biotoxins

Paralytic shellfish poison PSP 800 micrograms/kilogram

Amnesic shellfish poison ASP 20 milligrams of domoic acid/kilogram

Diarrhetic shellfish poison DSP Must not be presant

Okadaic acid/Dinophysis toxins/Pectenotoxin OA/DTXs/PTXs 160 micrograms of Okadaic acid equivalents/kilagram

Yessotoxin YTXs 3.75 milligram of yessotoxin equivalent/killogram

Azaspiracid AZAs 160 micrograms of a zaspiracid equivalents/kilogram

Table 2. Permitted level of biotoxins

8

The major health conditions caused by Ingestion of

contaminated shellfish are Paralytic shellfish poisoning (PSP),

Amnesic shellfish poisoning (ASP) and Diarrhetic shellfish

poisoning (DSP). These are all caused as a result of ingestion

of shellfish contaminated with bio toxins produced by the

phytoplankton species which they consume.

These three major health conditions, there causes and clinical symptoms are discussed in the next

section.

9

2.3 Amnesic shellfish poisoning (ASP)

Cause

Amnesic shellfish poisoning (ASP) is caused as a result of consuming shellfish contaminated with

Domoic acid. Domoic acid is a neurotoxin which acts as a glutamate agonist and causes the up

regulation of glutamate production. In the body the over production of Glutamate results in the

excessive stimulation of neurons by action potentials which can result in the damage or apoptosis of

cells ultimately leading to permanent short term memory loss, and in some cases, death.

Domoic acid is produced by a number of species of marine diatoms of the genus Pseudo-nitzschia

and is thought to be a mechanism used to eliminate excess photosynthetic energy when it is no

longer required by cells (Mos, 2001). Shellfish become contaminated following direct filtration of the

plankton or by feeding directly on contaminated organisms (Jeffery, 2004). Human contamination

results from the consumption of affected shellfish.

Clinical symptoms Within 24 to 48 hours of consuming contaminated shellfish the following symptoms can be expected:

Serious cases may result in seizures, coma or death (Todd, 1993).

Pseudo-nitzschia (Baker, 2012) Chemical structure of Domoic acid (Wikipedia, 2009)

• Headaches • Dizziness • Confusion

• Memory loss

• Vomiting

• Diarrhoea

• Nausea

• Abdominal cramps • Disorientation

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2.4 Diarrhetic shellfish poisoning (DSP)

Cause

Diarrhetic shellfish poisoning (DSP) is caused as a result of Okadaic acid which is a naturally occurring

biotoxin produced by the dinoflagellate’s Dinophsis and Prorocentrum lima. Okadaic acid increases

the permeability of the intestinal epithelia which is thought to contribute to the diarrhoea caused by

shellfish poisoning (Tripuraneni, 1997).

In a similar way to Amnesic shellfish poisoning, shellfish can accumulate Okadaic acid resulting in

contamination. Subsequent consumption of the infected shellfish can result in Diarrhetic shellfish

poisoning.

Clinical symptoms

Shortly after the consumption of contaminated Shellfish the following symptoms may be observed:

(Alexander, 2008)

Diarrhoea

Nausea

Vomiting

Abdominal pain

Prorocentrum Lima

Ninophysis Spp Images from: (Hayashi,K et al, 2007)

Chemical structure of Okadaic acid Image from: (Wikipedia, 2006)

11

2.5 Paralytic shellfish poisoning (PSP)

Cause

The dinoflagellate responsible for PSP is Alexandrium Spp. They produce a complex of 12

neurotoxins including carbamate toxins, neosaxitoxin and gonyautoxins (Arapov et al, 2010). These

toxins are able to block the voltage-gated sodium channels which are involved in the propagation of

action potentials and so affect the central nervous system. During algal blooms feeding Shellfish

filter the dinoflagellates which upon digestion release PSP that become concentrated in the digestive

organs and become unsafe for human consumption.

Clinical symptoms

Tingling sensation

Numbness around lips

Prickly sensation in the fingertips and

toes

Headaches

Dizziness,

Nausea,

Vomiting and

Diarrhoea

Temporary blindness

Giddiness

Incoherent speech

Respiratory difficulties

Tightness around the throat

Muscular paralysis

Death through respiratory paralysis

Taken from (Van Egmond et al, 2004)

Alexandrium Spp Images from: (Hayashi, 2007)

Chemical structure of PSP toxin (Van Egmond et al, 2004)

12

3.0 Methods Collection and Processing of Live bivalve mollusc samples

As is evident from the descriptions above, shellfish poisoning is a serious human health hazard and

must be taken seriously. This is why we are required to carry out monthly monitoring of shellfish

production areas on behalf of the local authority.

The monthly monitoring is in place to check the microbiological content of live bivalve molluscs from

each production area and to monitor the levels of toxin-producing plankton in the waters of the

production areas. If values fall outside set thresholds then it is the responsibility of the competent

authority to close the production area so that live bivalve molluscs cannot be harvested in order to

prevent risk to human health.

The required number of shellfish are collected from the relevant RMP (See table 3 and figure ) and

then rinsed using freshwater of potable quality to remove most of the mud and sediment. These are

then allowed to drain and are placed into polythene bag which is labelled with the relevant sampling

information and securely tied. This is then placed into a cool box along with the sample sheet and a

temperature logger. Samples must reach the testing lab within 48h and must not have exceeded a

temperature of 10°C.

SITE Food Authority Bed Classification Sample species and quantity requred

Ouse Mouth Borough council of Kings Lynn and west Norfolk Pending Mussels 35

Ouse Mouth Borough council of Kings Lynn and west Norfolk B-LT Cockles 50

Nene Mouth Fenland District Council B-LT Cockles

Black Buoy Boston Borough Council B-LT Cockles

North Lays Boston Borough Council B-LT Cockles

Toft Boston Borough Council B-LT Mussels 35 and 1 ltr Water

Welland Wall Boston Borough Council B-LT Mussels 35

Stubborn Sands Borough council of Kings Lynn and west Norfolk B-LT 1 ltr Water

LT- long term classification

Table 3. Table showing the sampling locations, bed classifications and sampled species within the Wash

13

The methods used to process flesh samples are explained bellow.

Figure 5. Chart showing the location of the Representative monitoring points within the Wash and the species sampled

14

Toxin group tested Methods employed

ASP liquid chromatography (LC) with Ultra-violet (UV)

PSP liquid chromatography (LC) with fluorescence (FLD) detection

Lipophilic toxins liquid chromatography (LC) with tandem mass spectrometry (MS/MS)

Method used to quantify Amnesic Shellfish Poison in Shellfish

The specific methods used to test for each of the toxins can be found in the Food standard’s agency

protocols. The table below details each of the techniques.

An outline of the procedure used to analyse shellfish toxins is shown in figure and uses the example

of ASP. In order to determine whether shellfish contain toxic concentrations high enough to cause

Amnesic Shellfish Poison in humans the level of domoic acid in shellfish tissue is quantified. This is

achieved using an extraction method followed by anion exchange before analysis using HPLC.

Table 4. methods used for routine toxin analysis

15

Figure 6. Procedure for the analysis of ASP in shellfish

16

Methods used to collect and analyse water samples for toxin-producing

phytoplankton species

As phytoplankton form an important food source for shellfish it is important to ensure that the

water surrounding production areas does not contain toxic species in concentrations above a certain

trigger level. Currently the following species are recorded when observed in water samples:

Alexandrium species11

Dinophysis species

Pseudo-nitzschia species

Prorocentrum lima

Prorocentrum minimum

Lingulodinium polyedrum

Protoceratium reticulatum

Protoperidinium crassipes / curtipes

Collection of water samples

Water samples are collected on a monthly basis and are taken close to the location of the shellfish

sampling site. Ideally water samples are taken at high tide and are collected using a bucket which

must be rinsed three times prior to taking a sample. If using a bucket, surface water is taken

however if possible a Tube sampler may be used in order to obtain water samples from a depth

greater than 2 meters.

Following collection of the water sample a 500 ml subsample is taken and placed into a brown

Nalgene bottle which is also rinsed three times. 2.5ml of Lugol’s Iodine is then added to the sample

bottle and agitated to ensure even mixing before the bottle is sealed and sent to Cefas for analysis

where samples are stored at room temperature until they are processed.

17

Toxin Toxin producing algae (trigger Level)

ASP Pseudo-nitzschia spp (150,000 cells/L)

LTsDinophysiaceae (100 cells/L)

Prorocentrum lima (100 cells/L)

PSP Alexandrium spp (Presence)

Processing of water samples

Analysis of the water samples follows the standard operating procedures drawn up by the UK

national reference laboratory for marine bio-toxins. For each toxin measured a Trigger‘ level in terms

of cell concentrations is used which identifies weather further action is required. Table 5 below

shows these trigger levels and figure 7 outlines the process used to analyse the water samples.

Table 4. Trigger levels for toxin producing algae

Figure 7. Procedure for the analysis of water samples

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Sample location Sample type Number of samples collected

Black Buoy Flesh 11

Nene Mouth Flesh 9

North Lays Flesh 10

Ouse Mouth Flesh 8

River Ouse Flesh 2

Stubborn Sand Water 10

Toft Water 10

Toft Flesh 9

Welland Wall Flesh 12

4.0 Results of the 2014/2015 monitoring programme

This section will outline the results of the bacteriological and the bio-toxin monitoring carried out in

2015. The table below shows the sampling statistics for all sampling conducted between January and

December 2015 . In total 81 samples including both water samples and flesh samples were collected

during 2015 with 15 samples missed due to poor weather or issues with vessels. Three additional

water samples were requested as levels of phytoplankton in the samples were beyond trigger levels.

All other samples were within limits and did not require further action to be taken.

E.coli flesh results

The graphs below shows the E.coli flesh results for each sample location visited during 2015. A large

amount of variability was observed in the E.coli levels at each site and a large amount of variability

between sites.

All sites showed clear peaks in the levels of E.coli however these picks were not comparable

between sites in terms of both extent and timing.

The largest values for E.coli, in the range of 3300-4600 E.coli per 100g of flesh, were observed at

Ouse mouth, Nene mouth and Welland wall. These three sites are located closest to river mouths

and as such are closer to the pollution source. Toft, North lays and Black Buoy are located further

from the river mouths and have E.coli values in the range of 18-1000 E.coli per 100g of flesh. These

sites may experience a dilution effect as you move away from the pollution source which may

account for the lower values.

Table 5. breakdown of all results fom 2015

19

78 20 18

45 78

170

20

130

230

780

0

100

200

300

400

500

600

700

800

E.co

li p

er 1

00

g

North Lays

690

130

490 490

170

230 230

20

690

0

100

200

300

400

500

600

700

E.co

li p

er

10

0g

Toft

330

780

1300

1100

780 690

490

780

130

330

780

0

200

400

600

800

1000

1200

E.co

li p

er 1

00

g

Black Buoy

20

1700

450

490 330

490

2200

490

780

330

780

490

3300

0

500

1000

1500

2000

2500

3000

E.co

li p

er 1

00

g

Welland Wall

3300

1300

130 110 330

230

4600

780 0

500

1000

1500

2000

2500

3000

3500

4000

4500

E.co

li p

er

10

0g

Ouse Mouth

230

780

3300

780

110

780

2300

490 690

0

500

1000

1500

2000

2500

3000

E.co

li p

er 1

00

g

Nene Mouth

21

Bio-toxin results

Water sample test results

Table 6 shows the results of the bio-toxin analysis. As with the E.coli results no

thresholds were reached whereby further action was required. Of the 21 samples

analysed for bio-toxins, 9 samples contained phytoplankton species at a

detectable level. The species identified were Pseudo-nitzschia spp and Dinophysis

spp both of which are abundant and widespread species.

Sampling Point

Date Sample Collected

Alexandrium spp (cells

L-1)

Dinophysis spp (cells L-

1)

Prorocentrum lima (cells L-1)

Pseudo-nitzschia spp (cells L-

1)

Lingulodinium polyedrum (cells L-1)

Protoceratium reticulatum (cells L-1)

Prorocentrum Cordatum (cells L-1)

Stubborn Sand

20/01/2015 Not present Not present

Not present Not present

Not present

Not present

Not present

Stubborn Sand



Not present

Not present

Not present

Stubborn Sand

21/04/2015 UNABLE TO ANALYSE TOO MUCH SEDIMENT

Stubborn Sand


Not present 4400 Not present

Not present

Not present

Stubborn Sand



Not present

Not present

Not present

Stubborn Sand



Not present

Not present

Not present

Stubborn Sand



Not present

Not present

Stubborn Sand



Not present

Not present

Stubborn Sand

17/11/2015 Not present 200 Not present 1200 Not present

Not present

Not present

Stubborn Sand

15/12/2015 Not present 200 Not present Not present

Not present

Not present

Not present

Toft 19/01/2015 Not present Not present


Not present

Not present

Not present



Not present

Not present

Not present



Not present

Not present



Not present

Not present



Not present

Not present



Not present

Not present

Not present



Not present

Not present

Not present

Toft 14/09/2015 Not present 40 Not present 160 Not present

Not present

Not present

Toft 07/12/2015 Not present 40 Not present Not present

Not present

Not present

Not present



Not present

Not present

Not present

22

Flesh sample test results

The flesh sampling returned values that were at the reporting Limit or the Limit

of quantitation and therefore did not require further action.

5.0 Discussion

Throughout 2015 133 samples including both water and flesh samples were

collected and processed. Of these samples 3 produced results that required an

additional sample to be taken, however no further action was required.

The action levels for toxins in shellfish flesh were not exceeded and therefore no

closures were put in place throughout 2015.

Missed samples are an area of concern as failure to collect samples can ultimately

result in closure and therefor every effort should be made to ensure that all

samples are collected on a monthly basis to enable rapid detection of a pollution

event and to ensure that fisheries remain in open to harvesting.

23

6.0 References

Arapov, J., Ezgeta-Balić, D., Peharda, M., Ninčević Gladan, Ž. (2010) Bivalve feeding-How and what

they eat? Croatian Journal of Fisheries. 68 (3), 105 - 116.

Baker, A. L. (2012) Phycokey - an image based key to Algae (PS Protista), Cyanobacteria, and other

aquatic objects. University of New Hampshire Center for Freshwater Biology. /phycokey.htm 25 Jan

2016. . Available from: http://cfb.unh.edu/phycokey .

Ben-Horin, T., Bidegain, G., Huey, L., Narvaez, D. A. & Bushek, D. (2015) Parasite transmission

through suspension feeding. Journal of Invertebrate Pathology. 131155-176.

Bricelj, V. M. , Lee, J. H. , Cembella, A. and Anderson, D. M. (1990) Uptake kinetics of paralytic

shellfish toxins from the dinoflagellate Alexandrium fundyense in the mussel Mytilus edulis. Marine

Ecology-Progress Series. 63177-188.

Buck, J. (2012) Sewage Outlet at Penrhyn Bay. Available from: <div

xmlns:cc="http://creativecommons.org/ns#" xmlns:dct="http://purl.org/dc/terms/"

about="http://s0.geograph.org.uk/geophotos/03/05/71/3057127_f7eb2b5b.jpg"><span

property="dct:title">Sewage Outlet at Penrhyn Bay</span> (<a rel="cc:attributionURL"

property="cc:attributionName" href="http://www.geograph.org.uk/profile/12987">Jeff Buck</a>) /

<a rel="license" href="http://creativecommons.org/licenses/by-sa/2.0/">CC BY-SA 2.0</a></div> .

Cefas. (2015) Sanitary surveys. Available from: https://www.cefas.co.uk/cefas-data-hub/food-

safety/sanitary-surveys/ .

Hayashi, K., Jacox, J., Glanz, J., Alvarado, N., Kudela, R., Rosen, B. & Coale, S. (2007) phytoplankton

identification. Available from: http://oceandatacenter.ucsc.edu/PhytoGallery .

Jan Alexander, Guðjón Atli Auðunsson, Diane Benford, Andrew Cockburn, Jean-Pierre Cravedi,

Eugenia Dogliotti, Alessandro Di Domenico, María Luisa Fernández-Cruz, Johanna Fink-Gremmels,

Peter Fürst, Corrado Galli, Philippe Grandjean, Jadwiga Gzyl, Gerhard Heinemeyer, Niklas Johansson,

24

Antonio Mutti, Josef Schlatter, Rolaf van Leeuwen, Carlos Van Peteghem, Philippe Verger. (2008)

Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European

Commission on marine biotoxins in shellfish – okadaic acid and analogues. The EFSA Journal. 5891-

62.

Jeffery, B., Barlow, T., Moizer, K., Paul, S. & Boyle, C. (2004) Amnesic shellfish poison. Food and

Chemical Toxicology. 42 (4), 545-557.

Mos, L. (2001) Domoic acid: a fascinating marine toxin. Environmental Toxicology and Pharmacology.

9 (3), 79-85.

Nordsieck, R. (2011) The homepage on gastropods, bivalves and other molluscs. Available from:

http://www.molluscs.at/bivalvia/ .

Todd, E. C. (1993) Domoic acid and amnesic shellfish poisoning-a review. Journal of Food Protection.

56 (1), 69-83.

Tripuraneni, J., Koutsouris, A., Pestic, L., De Lanerolle, P. & Hecht, G. (1997) The toxin of diarrheic

shellfish poisoning, okadaic acid, increases intestinal epithelial paracellular permeability.

Gastroenterology. 112 (1), 100.

Van Egmond, H. P., Van Apeldoorn, M. E. & Speijers, G. J. A. (2004) Marine biotoxins. Rome, Food

and agriculture organization of the United Nations. FAO Food and nutrition, Paper 80.

Ward, J. E., MacDonald, B. A., Thompson, R. J. & Beninger, P. G. (1993) Mechanisms of suspension

feeding in bivalves: Resolution of current controversies by means of endoscopy. Limnology and

Oceanography. 38 (2), 265-272.

Wikipedia. (2009) Domoinic Acid Structural Formulae. Available from:

https://en.wikipedia.org/wiki/Domoic_acid#/media/File:Domoinic_Acid_Structural_Formulae.png .

Wikipedia. (2006) Structure of Okadaic acid. Available from:

https://en.wikipedia.org/wiki/Okadaic_acid#/media/File:Okadaic_acid.svg .

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