Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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consisting of a number of compounds. Consumption

of shellfish containing the toxins may lead to illness

and/or death in humans. Details of the toxin groups,

their origin, and associated toxic syndrome are sum-

marized in Table 8.

0024 The source of toxins in bivalve molluscs is micro-

scopic planktonic algae, termed phytoplankton,

which are concentrated in bivalves by their filter feed-

ing mechanism and which form part of the animal’s

natural diet. Other types of shellfish (e.g., crabs,

lobsters), finfish, marine mammals, and seabirds

may also be contaminated with the toxins, generally

through the food chain. With the exception of shell-

fish, the other animals have not been associated with

human food poisoning, and may themselves become

ill or die due to the toxins. The toxins may also cause

physiological changes in some shellfish. However,

generally the changes are not obvious and contamin-

ated shellfish cannot be differentiated from their non-

toxic counterparts by their physical appearance.

tbl0008 Table 8 Details of shellfish toxins, their origin, and toxic syndromes

Tox in gr o up

Paralytic shellfish toxins Amnesic shellfish toxins Diarrhetic shellfish toxins Neurotoxic shellfish

toxins

Principal toxin Saxitoxin (carbamate toxin); Domoic acid Okadaic acid (OA) Brevetoxin

(molecular weight) (299.3) (311.14) (804.5) PbTx 1 (867.07)

Associated toxins Carbamate toxin (neo-STX);

gonyatoxins (GTX 1–4);

N-sulfocarbamoyl toxins

(B1, B2, C1–C4);

decarbamoyl toxins

(dc-GTX 1–4, dc-Neo,

dc-STX, do-STX, doGTX2,

doGTX3)

C5’ diastereomer;

Isodomoic acid,

mainly D, E, and F in

shellfish

OA compounds:

dinophysistoxins (DTXs),

OA; OA-diol esters;

others: YTXs; PTXs;

AZTs

PbTx 1–10

Chemical type Water-soluble

tertrahydropurine

Water-soluble

tricarboxylic acid

Polyethers Polyethers

Origin in relation to shellfish Alexandrium spp.

Gymnodinium catenatum

Pyrodinium bahamense

Pseudo-nitzchia spp. OA compounds:

Prorocentrum spp. and

Dinophysis spp. PTXs,

Dinophysis fortii YTXs,

Protoceratium reticulatum

AZTs, unknown

Gymnodinium breve

Type of phytoplankton Dinoflagellates Diatoms Dinoflagellates; AZPs

unknown

Dinoflagellates

Food-poisoning syndrome Paralytic shellfish

poisoning

Amnesic shellfish

poisoning (ASP)

Diarrhetic shellfish

poisoning (DSP)

Neurotoxic shellfish

poisoning (NSP)

Mode of action Block influx of sodium ions

in neuronal and muscular

voltage gated sodium

channels

Glutamate agonist,

binds to kainate-type

glutamate receptors

OA/DTXs inhibit serine/

threonine protein

phosphatases; YTX

hepatic and cardiac

damage; PTXs

hepatotoxic; AZTs

necrosis of pancreas

cells, liver, lymphocytes,

and myocardium

Influx of sodium ions

into voltage gated

sodium channels

Examples of symptoms Tingling of lips and tongue,

numbness, mild

headache, incoherent

speech, nausea,

vomiting, respiratory

difficulties, paralysis,

death

Nausea, vomiting,

diarrhea, headache,

memory loss,

seizures, coma,

death

Diarrhea, nausea,

vomiting, abdominal

pain and chills (OA,

DTXs and AZTs); PTX

and YTX not defined

Abdominal pain,

nausea, diarrhea,

chills, reversal

hot/cold,

headache,

muscle/joint pain

Time to onset 30 min–4 h 24–48 h 30 min–12 h (OA þ DTXs) 3–6 h

Life-threatening Yes Yes No No

Concentration reported to

cause symptoms

144–12 400 mg 100 g

1

60–295 mg OA 25–36 mg 100 g

1

; AZP

not defined

1600 mg 100 g

1

Regulatory limit 80 mg 100 g

1

20 mgg

1

Not detected by mouse

bioassay

20 MU 100 g

1

STX, saxitoxin; YTXs, yessotoxins; PTX, pectenotoxins; AZT, azaspiracid toxins; PbTx, brevetoxin; MU, mouse units.

SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans 5237

0025 Approximately 40 of the estimated 5000 phyto-

plankton species produce toxins. These occasionally

occur in large numbers causing discoloration of the

water column that has led to descriptions such as

‘red’ and ‘brown tides.’ However, large numbers of

algal cells need not be present for toxins to accumu-

late in shellfish. The toxic algae and hence shellfish

containing toxins occur worldwide, and it has been

suggested that they are increasing in distribution,

frequency, and intensity. Reasons postulated include

climate change, increased utilization of coastal

waters, and transport of toxic species via ships’ bal-

last water. The toxins are generally described as

secondary metabolites and their functional signifi-

cance to the algal cell is unknown. Inter- and intras-

pecies differences in the quantity of toxin produced

and the toxin profile exist, as do nontoxic variants

of toxic species. The pathways for toxin biosynthesis

have not been fully elucidated for most of the

toxin groups, although some steps have been charac-

terized. The genes involved are currently not

identified.

0026 The occurrence of specific algal species in some

areas, in some seasons, is fairly predictable (e.g.,

Alexandrium species, associated with paralytic shell-

fish toxins (PSTs), tends to occur in the summer

months in parts of the UK). Although, some field

data are available on environmental factors that trig-

ger the emergence of these toxic phytoplankton and

production of their toxins, it is currently not possible

to predict accurately the number of toxic cells and the

length of time for which these cells will remain in the

water column, nor their cellular toxin concentration.

All of these factors influence the total toxin burden in

exposed shellfish. Other factors which may influence

shellfish toxicity are rate of ingestion, degradation

to nontoxic compounds, biotransformation from

one toxin in a group to a less toxic compound in the

same group, and excretion.

Toxin Groups

0027Paralytic shellfish toxins PSTs are a group of

approximately 20 closely related tetrahydropurine

compounds of varying potency (Table 8). The dino-

flagellates involved in production of these toxins are

listed in Table 8. Saxitoxin (STX) was the first of

these compounds to be identified and is also con-

sidered the most potent. Some steps in the biosyn-

thesis of STX have been determined. These involve a

high-energy methyl group donor (S-adenosylmethio-

nine), a two-carbon unit (acetate), and an amino acid

precursor (arginine). However, the sequence of reac-

tions and exact number of biosynthetic steps have not

been fully defined. Additionally, controversy sur-

rounds suggestions that the dinoflagellate’s bacterial

flora are involved in the production/biotransforma-

tion of PSTs.

0028Shellfish contaminated with PSTs can contain dif-

ferent combinations of the compounds that can differ

from that of the dinoflagellate ingested. This is more

pronounced in some shellfish species (e.g., Spisula

solidissima) than others (e.g., M. edulis). PSTs are

also produced by some species of fresh-water cyano-

bacteria, e.g., Aphanizomenon flos-aquae, although

to date there are no reports of human food poisoning

from fresh-water shellfish due to these toxins. The

mode of action of PSTs is listed on Table 8, along

with details of the toxic syndrome, i.e., paralytic

shellfish poisoning (PSP) that they cause. PSP has

been known for centuries and the concentration of

PSTs reported to cause illness varies (Table 8). Out of

all the shellfish toxins, some consider PSTs to be the

most dangerous. This is due to the high concentra-

tions of PSTs that may occur in shellfish (Table 9) and

the fact that death can result within an hour.

0029Amnesic shellfish toxins Details of the amnesic

shellfish toxins (ASTs) are summarized in Table 8.

In this group, domoic acid (DA) is considered the

tbl0009 Table 9 Example of peak toxin concentration and depuration rates for some shellfish species contaminated with paralytic shellfish

toxins

Species Peak toxicity (mgSTXequiv.100g

1

) Time toregulatorylimit (weeks) Detoxification rate (% per day)

Crassostrea gigas 209–710 0.6–2.0 NA

Ostrea edulis 1000 6.4 4.0

Mytilus edulis 100–19259 0.6–15.6 4.8–15.4

Mytilus californianus 240–5300 2.8–9.0 5.1–8.9

Pecten maximus 2700 6.4 7.4

Placopecten magellanicus 809–6179

>52 0.2–0.6

Patinopecten yessoensis 6000–340000

>12–30 1.2–11.7

Regulatory limit, 80 m g saxitoxin (STX) equiv. 100 g

1

; NA, not available.

Includes data from different organs.

Data from digestive glands.

Modified from Bricelj MV and Shumway SE (1998) Paralytic shellfish toxins in bivalve molluscs: occurrence, transfer kinetics and biotransformation.

Reviews in Fisheries Science 6: 315–383.

5238 SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans

main compound of concern. Little detailed toxico-

logical information exists on the other ASTs, al-

though it has been suggested that the isomers are

less toxic than DA, and the diastereomer is of equiva-

lent toxicity. DA has been detected in a range of

marine macroalgae and in some species of the diatom

genus Pseudo-nitzschia. Biosynthesis of DA is thought

to involve two different precursor units. One is

formed from the direct incorporation of acetate into

oxaloacetate during the Krebs cycle, forming an acti-

vated derivative, e.g., 3-hydroxyglutamic acid. The

other unit is thought to be an isoprenoid unit,

geranyl-pyrophosphate.

0030 Accumulation of DA-producing Pseudo-nitzschia

multiseries in M. edulis led to the first recorded inci-

dence of amnesic shellfish poisoning (ASP; Table 8)in

shellfish consumers in Canada in 1987. Gastroenter-

itis was the milder form of the illness and tended to

occur in those under 40 years of age. Severe neuro-

logical symptoms, such as short-term memory loss,

were experienced by older people, reportedly lasting

in some cases for a number of years. Those who died

suffered from other preexisting illnesses, e.g., dia-

betes. Although DA has subsequently been detected

in shellfish in countries other than Canada, no further

confirmed outbreaks of ASP have been reported.

0031 Diarrhetic shellfish toxins Diarrhetic shellfish toxins

(DSTs) were originally defined as okadaic acid (OA)

and related toxins called dinophysis toxins (DTXs;

Table 8) They were first suspected of causing shell-

fish-related illness (Table 8) in the Netherlands in

1961, and confirmed as being responsible for food

poisoning in Japan in the 1970s. The principal toxin

of this group, OA, has also been detected in the

sponges Halichondria okadai and H. melanodocia.

The method of detecting DSTs is a mouse bioassay

using a solvent extract. The nonspecific nature of this

technique has led to the inclusion of other compounds

in the DST group. These include fused polyether

compounds named yessotoxins (YTXs) and a group

of macrocyclic polyether lactones called pecteno-

toxins (PTXs). Some effects of these compounds in

rodents are summarized in Table 8. However, little

detailed toxicological and epidemiological informa-

tion is available and international debate is ongoing

on their inclusion in the DST group, their threat to

human health, and values, if any, for regulatory

limits. The biosynthesis of OA and the related YTXs

and PTXs is thought to occur by the successive add-

itions of acetate units to a growing polyketide chain,

mediated by the enzyme polyketide synthase (PKS).

Modification of PKS, involving for example oxidative

modification, methylation, and cyclization, results in

different structural types of the enzyme.

0032A further group of toxins, named azaspiracid

toxins (AZTs), is also currently included within the

DSTs. These are lipophilic, highly oxygenated poly-

ethers containing an unusual azaspiro-ring structure.

Although dinoflagellates are suspected as the source

of the toxin, undisputed evidence does not yet exist.

AZTs were first reported as causing food poisoning in

the 1990s with symptoms similar to those for OA

(Table 8). Effects of AZTs on rodents are summarized

in Table 8. There is strong support to separate this

group from the other DSTs and to name the illness

azaspiracid shellfish poisoning. Discussions are cur-

rently ongoing on what regulatory limits should be

set, given the limited extent of epidemiological data

available.

0033Neurotoxic shellfish toxins Neurotoxic shellfish

toxins (NSTs; Table 8) cause shellfish-related food

poisoning. However, they are more noted for finfish

and seabird kills than human illness. Produced by the

dinoflagellate Gymnodinium breve, they are infam-

ously known as the cause of the Florida red tides that

have been recorded for centuries. G. breve is typically

found in Florida waters, but similar forms have

been detected in waters elsewhere, particularly New

Zealand.

Monitoring and Methodology for Shellfish Toxins

0034A large number of countries worldwide monitor their

waters for toxic algae, the shellfish for toxins, or a

combination of both. Internationally accepted stand-

ards are recognized for PSTs and ASTs (Table 8).

Regulations for DSTs are less clear, with some coun-

tries using detection by mouse bioassay as criteria for

implementing harvesting closures and others suggest-

ing a limit of 8–16 mg 100 g

1

(Table 8). Within the

EU, limits are incorporated into directives with which

member states and countries exporting to the EU

must comply.

0035Some suggest that these regulatory limits can be

deemed effective as judged by the lack of shellfish

toxin-related food poisoning in countries with com-

prehensive monitoring programs. However, detailed

toxicological and epidemiological data supporting

these limits are lacking for many of the toxins,

hindered in some cases by lack of toxin standards

and appropriate methodology for toxin detection.

Additionally, questions relating to sampling proced-

ures for collection of an appropriate representative

sample from shellfish populations remain, e.g.,

sample size, frequency, definition of sampling area,

and number of samples per area.

0036Most countries use a mouse bioassay for the detec-

tion of PSTs, DSTs, and NSTs in shellfish-monitoring

programs. Ethical considerations are the driving force

SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans 5239

behind the development of alternative methods, such

as immunoassays, cell assays, and analytical instru-

mental techniques, e.g., liquid chromatography–mass

spectrometry. In some cases progress is hampered by

the complexity of the toxins and the lack of toxin

standards, and to date, with one exception, no alter-

native method has been fully accepted for use in

routine monitoring. The exception is ASTs where a

high-performance liquid chromatographic technique

was developed at the time of the Canadian ASP out-

break and is now used routinely in many countries.

Depuration and Processing of Shellfish

Contaminated with Toxins

0037 Once shellfish are contaminated with toxins, options

for detoxifying or depurating the animals are limited.

They mainly involve commercial canning of some

shellfish species e.g., Mya arenaria and Mytilus edulis

contaminated with PSTs. However, the effectiveness

of these processes is dependent upon initial low levels

of toxin and caution is recommended in their use.

0038 Currently, commercial depuration systems are inef-

fective at cleansing shellfish of toxins, which leaves the

option of allowing shellfish to depurate naturally in

the environment. This can be problematic as rates of

detoxification vary considerably between species,

with some shellfish remaining toxic for a year or

more (Table 9). Depuration in some shellfish species

seems to be biphasic. It has been suggested that the

initial detoxification phase represents gut evacuation

of unassimilated toxin, whereas the second phase rep-

resents the release of toxins assimilated and incorpor-

ated in the tissue. The situation is made more complex

by changes in toxin profile observed during depur-

ation, particularly for PSTs. A role for biotransforma-

tion enzymes in this process has been suggested and

recent work has demonstrated the induction of glu-

tathione S-transferase in M. edulis exposed to PSTs.

Nevertheless, mechanisms involved in the depuration

of shellfish toxins are poorly understood and hinder

the development of commercial depuration processes.

Infectious Microbes

0039 A number of bacteria and human viruses responsible

for food poisoning are transmitted via the fecal–oral

route (Table 1). Infected individuals can shed large

numbers of these microbes in their feces. The link

between shellfish-transmitted disease and sewage pol-

lution in the marine environment has been known

since the late nineteenth century, with early docu-

mented evidence associated with the bacterial infec-

tion typhoid fever (Table 10). Of the different

shellfish species, bivalve molluscs are the main source

of microbial contamination that poses a health risk to

human consumers. This is exacerbated by traditional

consumption of some species raw or only lightly

cooked. As with shellfish toxins, there are no obvious

visible indications of contamination.

0040Ineffectively processed or untreated sewage, faulty

septic tanks, and feces from farm animals and seagulls

are a principal source of bacterial food-poisoning

agents in the marine environment. However, human

sewage is currently thought to be the only source of

human enteric viruses in marine waters. Sewage treat-

ment processes are only partially effective at remov-

ing viruses, and therefore coastal discharges are a

major source of these infectious agents in some

areas. Additionally, contamination of the marine en-

vironment by both bacteria and viruses is exacerbated

after heavy rainfall due to increased land run-off and

storm water discharges. However, not all infectious

agents concentrated in shellfish are from fecal

sources. Naturally occurring bacteria from the genus

Vibrio are also responsible for shellfish-transmitted

infection, particularly from raw oysters.

0041In recent years there have been fewer incidences of

infection due to shellfish consumption and this is

generally attributed to the success of the controls

now implemented in many countries for reducing

bacterial contamination of shellfish. When shellfish-

related food poisoning does occur, evidence from the

UK and USA suggests that the causative agent is not

identified in the majority of cases. However, enteric

viruses are often suspected because the clinical symp-

toms are frequently consistent with the epidemi-

ological criteria for viral gastroenteritis.

Bacterial Contaminants

0042Table 10 lists bacterial contaminants reported to

cause shellfish-related food poisoning. These can be

split into two main groups – bacteria where the prin-

cipal source of contamination is from animal or

human feces, and those which occur naturally in the

marine environment.

0043Salmonella spp. are commonly found in intestinal

tracts of animals and human carriers. Over 2000

different serotypes have been reported, most of

which are pathogenic to humans to varying degrees.

The most serious illness is typhoid fever resulting

from infection from Salmonella typhi (Table 10). A

number of cases of this acute systemic disease occurred

in the early 1900s, due to contaminated oysters from

grossly polluted waters. Nowadays, modern sanita-

tion practices, improved control systems, and a re-

duced number of human carriers mean this disease

has not been reported in relation to shellfish from

developed counties for a number of years. Gastroen-

teritis is a more common Salmonella-related illness,

although again as a consequence of modern practices

5240 SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans

is not often caused by shellfish (Table 10). Gastroen-

teritis has also been reported from consumption of

shellfish contaminated with species of Campylo-

bacter and enterotoxigenic strains of Escherichia

coli (ETEC: Table 10). (See Salmonella: Salmonel-

losis; Campylobacter: Properties and Occurrence;

Escherichia coli: Food Poisoning by Species other

than Escherichia coli.)

0044 Hemorrhagic strains of E. coli, particularly strain

0157, causes hemorrhagic colitis bloody diarrhea,

with 10% of cases developing serious complications,

such as hemolytic uremic syndrome. This may lead to

death, particularly in children and the elderly. Al-

though to date there are no reports of infection via

shellfish from this pathogen, they remain of concern

given their prevalence in the environment, association

with cattle, and their low infective dose. Research is

required to assess their survival in the marine environ-

ment, their potential to accumulate in shellfish

exposed to farm run-off, and the risk to the consumer

of shellfish. However, it must be borne in mind that

other bacterial pathogens, including Yersinia entero-

litica and Clostridium perfringens, may be isolated

from molluscan shellfish, although no cases of shell-

fish-borne illness due to these species have been

reported. Bacteria such as Shigella spp. and Staphylo-

coccus aureus have also been associated with shell-

fish-borne infection, but generally by contamination

from infected food handlers. (See Clostridium: Oc-

currence of Clostridium Perfringens; Shigella;

Staphylococcus: Properties and Occurrence.)

0045Vibrios, particularly species of Vibrio parahaemo-

lyticus and V. vulnificus, are also causative agents

of illness due to the consumption of raw shellfish.

However, unlike the bacteria described above, these

species are prevalent naturally in warm estuarine

waters throughout the world. Attempts to recover

these bacteria can be complicated by their reported

ability to form a viable but nonculturable state.

V. parahaemolyticus tends to cause gastroenteritis,

tbl0010 Table 10 Microbes reported to cause shellfish-related food poisoning and a summary of the symptoms

Type Name Description Type of illness Incubation

period

Symptoms Length of illness

Bacteria Salmonella typhi Gram-negative Typhoid fever 1–2 weeks Fever, chills, headache,

constipation, diarrhea

(20%), muscle pain

1–2 weeks; can

be fatal

Bacteria Salmonella spp. Gram-negative Gastroenteritis 12–36 h Diarrhea, nausea,

occasionally vomiting,

abdominal pain, chills,

moderate fever,

drowsiness

2–7 days; low

number of

fatalities

Bacteria Campylobacter spp. Gram-negative Gastroenteritis 2–5 days Bloody diarrhea, nausea,

malaise, fever, and

severe abdominal pain

3–5 days; low

number of

fatalities

Bacteria Escherichia coli Gram-negative Gastroenteritis 12–72 h Diarrhea, abdominal

cramps, nausea, and

chills

2–3 days

Bacteria Vibrio

parahaemolyticus

Gram-negative Gastroenteritis 4–48 h Profuse diarrhea,

abdominal pain, nausea,

sometimes fever, and

occasional vomiting

2–5 days

Bacteria Vibrio vulnificus Gram-negative Primary septicemia < 24 h Fever, abdominal pain,

vomiting, diarrhea,

sepsis

50% mortality

Bacteria Vibrio cholerae non-O1 Gram-negative Gastroenteritis Diarrhea, abdominal

cramps, occasionally

vomiting and fever

Virus Norwalk-like viruses Single-stranded

RNA; 30–35 nm

diameter

Gastroenteritis 1–4 days Nausea, vomiting,

abdominal pain,

occasional diarrhea

and low-grade fever

12–48 h

Virus Hepatitis A Single-stranded,

RNA c.27nm

diameter

Hepatitis 2–6 weeks Fever, headache, nausea,

vomiting, diarrhea,

abdominal pain,

jaundice

Up to several

months

Mortality rate highest in people with other predisposing illnesses, e.g., diabetes.

Data source: Eley AR (1992) Microbial Food Poisoning. Hackney CR and Pierson MD (eds) (1994) Environmental Indicators and Shellfish Safety. London:

Chapman and Hall.

SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans 5241

whereas V. vulnificus is a particularly invasive patho-

gen which may cause primarily septicemia within

24 h of exposure (Table 10). Raw oysters containing

large numbers of the latter bacterial species tend to be

the main source of infection, which is characterized

by a mortality rate of 50% in susceptible individuals.

V. cholerae is separated into two groups on the basis

of the O1 somatic antigen. Strains possessing the O1

antigen were previously believed to derive only from

human feces, but are now known to exist in low

numbers in some estuarine waters. These strains are

responsible for cholera, and shellfish have been impli-

cated as a vector for the disease. V. cholerae non-O1

are ubiquitous in some coastal waters and are fre-

quently identified as a cause of gastroenteritis from

raw shellfish (Table 10). (See Vibrios: Vibrio cho-

lerae; Vibrio parahaemolyticus; Vibrio vulnificus.)

Viruses

0046 Viral pathogens may also cause food poisoning due

to consumption of contaminated bivalve molluscs

(Table 10). Epidemiological evidence suggests that

the Norwalk-like viruses (NLV), also known as

small round structured viruses, are the most common

cause of infection. This group of viruses consists of a

genetically diverse virus strain, separated into gen-

ogroups I (including the prototype Norwalk virus)

and II. With an infectious dose thought to be as low

as 10–100 virions, an US FDA risk assessment esti-

mated that 100 000 cases of seafood-related NLV

gastroenteritis occur per year. The immunological

response to NLV infection is not well understood

and appears to be short-term, with individuals be-

coming susceptible to reinfection within 6 months to

1 year. Patients may also suffer from a mixed infec-

tion due to viruses of both genogroups.

0047 The most serious virus infection linked to shellfish

consumption is hepatitis A. This virus is extremely

stable and is currently thought to replicate in the liver.

Unlike the other enteric viruses, hepatitis A has a long

incubation period of about 2–6 weeks and causes a

serious debilitating disease (Table 10). It is a self-

limiting infection and, although it rarely causes

death, infected people may be incapacitated for sev-

eral months. Young children frequently experience

only mild illness, whereas full symptoms develop in

the majority of infected adults. Recovery is complete

and leads to long-term immunity from reinfection.

Occasionally, epidemics due to hepatitis A in shellfish

occur. In Shanghai, China, during 1988, approxi-

mately 300 000 cases of hepatitis A were traced to

clams from a sewage-polluted area. It is also interest-

ing to note that incidences of gastroenteritis, followed

by hepatitis A infection, have been reported from

contaminated shellfish.

0048Astroviruses have also been implicated in out-

breaks relating to oysters. However, detailed epidemi-

ological data are lacking and the importance of these

organisms in shellfish-related food poisoning is

not clear. Other viruses, such as rotaviruses, adeno-

viruses, and enteroviruses, have been detected in

shellfish, but to date they have not been linked with

infectious disease following seafood consumption.

Despite their name, enteroviruses (e.g., polioviruses)

do not commonly cause gastroenteric symptoms, e.g.,

diarrhea and vomiting, although they do replicate in

the intestinal tract and are passed via the fecal–oral

routes. These viruses are often found in shellfish but

have not been reported to cause infection.

Parasites

0049Helminth parasites have been implicated in human

infection, primarily from consumption of raw or

under cooked finfish and, more rarely, from crabs

and bivalves. Protozoan parasites of the genera Giar-

dia and Cryptosporidium cause human disease and

are generally associated with contaminated fresh

water. Both occur in detectable numbers in raw and

treated sewage. There is a dearth of information

regarding the survival of these organisms in sea

water and their potential to contaminate shellfish.

Monitoring, Methodology, and Use of Indicators for

Infectious Microbes

0050Bacteria Most countries have implemented sanitary

controls for the production of bivalve shellfish. In the

EU, these are applied via the European Directive 91/

492/EEC (Table 11) and in the USA by interstate

trading agreements set out in the FDA National Shell-

fish Sanitation Program Manual of Operations.

Third-country imports into the EU and the USA

have to comply with these standards. These regula-

tions include assessment of fecal pollution in shellfish

(determined by measuring bacterial indicators), pro-

cessing requirements for shellfish from contaminated

areas, conditions for processing and dispatch estab-

lishments, and marketing documentation.

0051The bacterial indicators generally involve meas-

urement of fecal coliforms or E. coli in shellfish

(EU) or shellfish-growing waters (USA). The concept

is that if indicators are present in sufficiently high

numbers, there is a high probability that specific

pathogens are also present. The appropriateness of

these indicators has been intensely debated over the

years. Nevertheless, fecal coliform/E. coli indicators

are used routinely worldwide and their implementa-

tion, along with specific processing methods, has

resulted in a decrease in shellfish-related bacterial

food poisoning.

5242 SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans

0052 One of the main advantages of these bacterial indi-

cators is the relative ease by which they can be

detected and quantified utilizing traditional bacteria

culture techniques. For example, in the UK, a five-

tube, three-dilution, most probable number, two-

stage technique with minerals-modified glutamate

broth and chromogenic medium for detecting b-

glucuronidase activity, is used for the detection of

E. coli in shellfish.

0053 Viruses People in a number of countries have con-

tracted viral infections after consuming shellfish that

comply with bacterial indicator regulations. This sug-

gests bacterial standards are inappropriate controls

for a viral hazard in shellfish; yet virus standards for

bivalve molluscs do not currently exist. Develop-

ments of such regulatory limits are hampered largely

by lack of appropriate, reliable, robust, and easy-

to-use methods.

0054 The most common methods for the detection of

viruses is electron microscopy and cell culture, neither

of which has proved very successful in the detection

and quantification of enteric viruses, such as NLVs

and hepatitis A, in shellfish. Electron microscopy is

not sufficiently sensitive and many of these viruses are

considered nonculturable (NLV) or difficult to culture

(hepatitis A). In recent years, immunoassays have

become available, although the success of application

to shellfish has been limited. Molecular techniques,

such as polymerase chain reaction (PCR), combined

in some instances with sequencing of the amplified

product or hybridization of the product with probes,

seem to be the most promising approach to date.

Such methods have been developed for hepatitis A

and NLV detection in shellfish. Their use, however,

is not straightforward because shellfish extracts

contain substances inhibitory to PCR that may

cause false negatives. This has resulted in the use of

time-consuming, laborious extraction procedures

that are problematic to apply in a routine monitoring

scenario. Additionally, it is uncertain that PCR results

indicate the presence of viable infectious viruses.

However, some workers argue that this is not an

issue because viral RNA is unlikely to survive for

any length of time in shellfish without a complete

protein coat. Others suggest use of antibody-capture

techniques aimed at recovering the whole virion,

prior to PCR. This also has the potential advantage

of reducing complex extraction techniques. However,

for NLVs, application of immunocapture techniques

is hindered by the lack of suitable immunological

agents. Although PCR-based methods have advanced

research on viruses in shellfish, further work is re-

quired to simplify, improve, and standardize the

methodology. It has been suggested that their routine

use is a number of years in the future and that

consideration should be given to utilizing viral

indicators.

0055Bacteriophages are the most frequently suggested

viral indicators because of their physical and genomic

similarity to human enteric viruses, their abundance

in sewage effluents, and the relative ease in which

they are quantified. Methodology generally utilizes

plaque assays, although it should be noted that these

are not always robust. Use of general somatic bac-

teriophage of coliforms has also been proposed.

However, standardization and reproducibility of

methods utilizing this diverse group are problematic.

Male specific (Fþ) bacteriophages are another con-

tender as viral indicators and have been used by some

researchers to study shellfish depuration. Studies have

shown that Fþ elimination kinetics reflects that of

enteric viruses and that it is a more effective indicator

of viral presence in depurated shellfish than E. coli.

However, one criticism of Fþ bacteriophage is that

their distribution is not restricted to human effluents,

tbl0011 Table 11 Regulatory limits implemented on biological contaminants of shellfish using EU regulations as an example

Harvestinglimits for bacteria Harvesting limits for toxins End-product standard

Classification Microbialstandard (per100 g

shellfish)

Treatment required Toxin Limit

A < 230 Escherichia coli or

< 300 FC

None PSTs 80 mg 100 g

1

Negative for Salmonella in 25 g of

bivalve flesh

B < 4600 E. coli or < 6000 FC in

90% of samples

Depuration or relayed DSTs Not detected in

bioassay

< 230 E. coli (or < 300 FC) 100 g

1

C < 60 000 FC 100 g

1

Relayed for at least

2 months

ASTs 20 mgg

1

Below harvesting limits for toxins

Ns > 60 000 FC 100 g

1

Prohibited

Microbes and toxins only.

Mouse bioassay methodology not defined.

FC, fecal coliforms; PSTs, paralytic shellfish toxins; DSTs, diarrhetic shellfish toxins; ASTs, amnesic shellfish toxins; Ns, not stated but often called

class D. Data source: Council Directive 91/492/EEC and 97/61/EC.

SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans 5243

thereby causing doubt as to their suitability as human

virus indicators in rural areas primarily impacted

with animal fecal contamination. Currently, research-

ers are attempting to optimize systems to differentiate

between animal and human Fþ bacteriophage. As an

alternative, the bacteriophage of the obligate anaer-

obe Bacteroides fragilis has been proposed as an indi-

cator of human specific pollution.

0056 An alternative to bacteriophage is the detection of

other human viruses that are either widely prevalent

in polluted waters and/or easier to detect than viral

pathogens. Human enteroviruses have been used in

this context, particularly for water. However, methods

of detection are currently complex. Human adeno-

viruses have also recently been suggested but further

work is required to determine their prevalence in

relation to NLVs and hepatatis A. Overall, more

work is needed to assess further prevalence of poten-

tial indicators compared to the viral pathogens, to

improve methods, and to develop standards to apply

before the most appropriate viral indicator approach

can be developed.

Depuration and Processing of Shellfish Containing

Infectious Microbes

0057 Shellfish may be harvested and marketed directly

without any further processing providing they meet

specific standards, including limits on the numbers of

bacterial indicators. However, if they exceed these

limits, depuration and/or relaying are permissible up

to certain concentrations of the indicators. To provide

an example of the type of regulations imposed, the

criteria set within the EU are summarized in Table 11.

0058 Depuration procedures for treatment of microbial

contaminants of shellfish were first developed during

the 1920s. These involve placing bivalves in clean sea

water in tanks away from the source of microbial

contaminants and allowing their natural excrement

process to remove contaminants within the mollusc.

The tanks contain static, flow-through, or recycled

sea water sterilized with ultraviolet irradiation, ozone,

or chlorination. Depuration is implemented for 1–7

days, although 2 days is more common. The effect-

iveness is assessed generally by a reduction in fecal

coliform or E. coli numbers to specific concentrations

(e.g., to class A in the EU). However, shellfish from

depuration plants meeting these limits may still con-

tain human enteric viruses at concentrations suffi-

cient to cause infection. Recent work suggests that

during depuration viruses are eliminated at much

lower rates than bacteria and sea-water temperature

is critical.

0059 Relaying requires that shellfish are removed from

their original contaminated harvesting site and placed

in an area with reduced microbial contaminants for at

least 2 months to allow natural depuration to occur.

Practical difficulties associated with this process

include problems in finding suitable waters and

obtaining ownership rights to these waters. One add-

itional issue that is rarely addressed is the possibility

that shellfish may be contaminated with the resting

stage of toxin-producing dinoflagellates. Moving

such shellfish to a ‘clean’ site risks seeding that site

with toxic dinoflagellates. Shellfish may also be sold

as a processed product which have been commercially

heat-treated. These modern, regulated methods (e.g.,

raising internal shellfish meat temperature to 90



for 1.5 min) are effective at reducing both the bacter-

ial and virus loading in contaminated shellfish.

Future Perspectives

0060The impact of contaminants on shellfish as a resource

can be severe in terms of both the viability of the

associated industries and the health of the human

consumers. The highly nutritious and economic value

of shellfish argues for the continuing improvement of

its husbandry and the monitoring of contamination

by pollutants, toxins, and microbes in order to pre-

vent spoilage, waste, and ill health. Vigilance and

awareness of existing and changing contamination

threats are imperative. The selection of statutory

tissue levels and contaminants to be monitored must

be responsive to changing and improving information

on toxicity, toxic mechanisms, production of new

chemicals, occurrence of ‘new’ toxins, and increasing

hazards from viruses. Automation, new analytical/

biological techniques and the production of appropri-

ate standards will increase the number and efficiency

by which contaminants can be routinely measured.

These may include biological measurements (i.e., bio-

markers) of chemical exposure, such as inhibition of

the shellfish cholinesterase (EC 3.1.1.8) activity as

an indicator of contamination by organophosphate

pesticides, and induction of 7-ethoxyresorufin O-

deethylase (EROD) activity in cell systems as an indi-

cator of PAHs. The development of new technology

based on immunorecognition and molecular bio-

logical techniques is also likely to become increas-

ingly important in the future. Efforts must be made

to prevent contamination of this valuable resource

from occurring and, in the case of toxins and pollu-

tants, models should be produced to assist regulatory

authorities to predict their accumulation and depur-

ation in shellfish. A greater understanding of the bio-

logical processes in shellfish, once contaminated, is

also required in order to develop efficient and safe

treatment processes. Additionally, the standardiza-

tion of statutory contaminant levels and analytical

procedures between national and international bodies

5244 SHELLFISH/Contamination and Spoilage of Molluscs and Crustaceans

will facilitate regulation and monitoring of the shell-

fish industry to the consumer’s benefit.

See also: Parasites: Occurrence and Detection;

Salmonella: Salmonellosis; Vibrios: Vibrio

parahaemolyticus; Vibrio vulnificus; Viruses