218
Journal of Food Protection, Vol. 51, No.3, Pages 218-251 (March 1988)
Copyright' Intema~onal Association of Milk, Food and Environmental Sanitarians
Microbial Purification of Shellfish: A Review of
Depuration and Relaying
GARY P. RICHARDS
U.S. Department of Commerce, National Oceanic and Atmospheric Administration. National Marine Fisheries Service, Southeast Fisheries Center,
Charleston Laboratory, P.O. Box 12607, Charleston, South Carolina 29412
(Received for publication June 22, 1987)
ABSTRACT
A review of the literature on shellfish depuration and
relaying revealed wide diversity in microbial uptake and elimi-
nation among shellfish species and for different microorgan-
isms. Information on relaying of five commercial shellfish spe-
cies and on controlled purification (depuration) of 11 species
indicates that such processes are effective in reducing the levels
of bioconcentrated bacteria and viruses from shellfish. The
degree of bacterial and viral bioconcentration varies with shell-
fish species; however, the primary sites of bioconcentration are
the hepatopancreas and digestive diverticula. Low levels of
enteric viruses and coliphage may be sequestered in shellfish
hemolymph and tissues, thus protecting them from elimination
through depurative processes. Vibrio spp. appear to proliferate
when closely associated with intestinal cells of shellfish. Shell-
fish relaying techniques offer effective microbial depletion
provided water quality is acceptable and shellfish remain
physiologically active. The current body of literature on con-
trolled purification demonstrates a broad spectrum of conditions
under which shellfish are depurated. Optimal times, tempera-
tures and salinities for effective depuration vary among shellfish
species. Proper design and operation of depuration plants is
crucial to insure process integrity. Recirculating and flow-
through purification systems are effective in reducing the levels
of pathogenic and indicator microorganisms from shellfish, but
the extent to which they reduce viruses from shellfish is uncer-
tain. Studies are needed to validate the effectiveness of depura-
tion processes in eliminating pathogenic viruses and to address
the adequacy of indicator bacteria as measures of enteric virus
contamination.
Pollution of coastal waters can readily lead to contami-
nation of molluscan shellfish. Uptake and elimination of
some ofthese contaminants have been studied for a variety of
compounds including heavy metals (32,34,37,64, 100, 126,
147), petroleum hydrocarbons (51, 89, 102, 155), radio-
nuclides (28, 35) and toxins (11,13). Uptake and elimination
of potential human pathogens and indicator organisms from
shellfish are the subject of this review.
Microbial depuration is a dynamic process whereby
shellfish are allowed to purge themselves of contaminants
either in a natural setting or in land-based facilities. In the
JOURNAL OF FOOD PROTECTION. VOL. 51. MARCH 1988
natural setting, shellfish harvested from contaminated areas
are transferred to clean shellfish growing waters. This cleans-
ing process is known as relaying and is subject to natural
environmental conditions. Controlled purification is the
land-based process where shellfish are cleansed in tanks
usually containing flow-through or recirculating seawater.
Flow-through (also called once-through) systems are usually
shore-based since they rely on a constant supply of flowing
seawater. Recirculating systems are often, but not necessar-
ily, shore-based. French depuration plants often use a batch
(or fill and drain) process with large volumes of disinfected
seawater. In batch systems, shellfish are depurated until the
dissolved oxygen levels are depleted. Throughout this review
controlled purification will be referred to as depuration;
whereas depletion in a natural setting will be referred to as
relaying.
This review is intended to serve as a general reference on
bacterial and viral uptake and, more specifically, on their
elimination through depuration and relaying. Individual
species are discussed separately to allow quick reference to
those species of particular interest.
HISTORICAL PERSPECTIVE
The potential for disease outbreaks attributable to
shellfish ingestion has been recognized for over a century (re-
viewed by Richards, 141). Early reports of shellfish-associ-
ated illnesses focused on cholera (87) and typhoid fever
(9,76.1 14). Efforts to reduce pathogenic bacteria in shellfish
led to a study in the late 1800s showing that sewage-
associated bacteria could be purged by placing oysters in so
called "disgorging tanks", tanks of clean seawater, for a short
time (76). More sophisticated tanks were developed in France
using filtered seawater (47). Wells (172) effectively sanitized
oyster depuration waters with· calcium hypochlorite in an
effort to eliminate the need for filtration. In 1914, Johnstone
(93) reported on a flow-through system for depuration of
mussels. This system produced a 98.5% reduction in sewage
bacteria in 96 h.
Johnstone (92,93) conducted some of the earliest large-
scale relaying experiments. He determined that after 72 h,
mussels reduced their level of sewage bacteria by 99.6% (93).
 DEPURA TION AND RELAYING SHELLFISH
An early study by Phelps (132) considered the effects of
relaying on oysters. Only the shellfish liquor was tested for
Escherichia coli. It was concluded that relaying of oysters
was effective in rapidly reducing E. coli counts in oyster
liquor (132). Another paper on relaying demonstrated its
effectiveness in reducing Salmonella typhi from contami-
nated oysters within 3 weeks (99). Large outdoor "pits" or
ponds were reported useful for the depletion of E. coli from
native oysters (Ostrea edulis) and Portuguese oysters (Gry-
phaea angulata) (29). Numerous drawbacks to the relay of
mussels and other shellfish were set forth by Dodgson in 1928
(39). Included among these were the unavailability of clean
,waters in which to relay. the lack of cooperation by fishermen
in abstaining from premature harvesting. and the possibility
of shellfish recontamination.
Information on mussel (39.40) and oyster (40) purifica-
tion indicated the early successes of shellfish depuration.
Dodgson (40) pointed out that one mussel depuration plant
had been in operation in Conway, England since 1916 and
that newer plants were processing effectively. Early depura-
tion plants in the United States. such as the one at Inwood,
Long Island. New York, used sodium hypochlorite and water
changes to enhance oyster depuration (39). Another depura-
tion plant made its debut in 1931 in Newburyport. Massachu-
setts and is still operating today. By the early I 960s. depura-
tion plants had been established in England. Japan. France,
Spain. Portugal, Ireland and the United States (84.176).
Shellfish species commonly depurated in Europe were
mussels (Mytilus edulis). European flat oysters (Ostrea
edulis) and the Portuguese oyster (Crassostrea angu!afa)
(176). Other species depurated in small quantities in France,
Spain and Portugal included the palourde or carpet-shell
(Venerupis pullastra). praire (Venus verrucosa). clovisse
(Venerupis geographicus. Venerupis aurea) and the avignon
(Scombricularia plana) (176). Clams (Mya arena ria and
Mercenaria mercenaria) remain the primary species depu-
rated in the United States principally due to their higher
economic value over other shellfish species.
DEPURATION SYSTEMS
Water disinfection processes
Row-through depuration systems require seawater
treatment only if the available water is of questionable or
unacceptable sanitary quality. Recirculated water must be
disinfected to prevent bacterial build-up and recontamination
of the shellfish. Although seawater alone has bactericidal and
virucidal properties (2.23.111.121.135). it is ineffective in
handling large contaminant loads.
Water disinfection can be accomplished using hy-
pochlorites or chlorine. iodophores, ozone and ultraviolet
light. Chlorination is the oldest of the disinfection
procedures (24.172.173). Although effective in reducing
microbial levels. enteric viruses. such as polio, hepatitis A
and Norwalk viruses. appear less susceptible than bacteria to
chlorine inactivation (62.15.98,128). Chlorine, even at low
levels. can affect shellfish pumping (39.54). For this reason.
chlorinated water is usually dechlorinated using sodium thio-
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
219
sulfate, activated charcoal and/or vigorous aeration before
being added to shellfish depuration tanks (21). Kelly (95)
found that chlorinated seawater which was dechlorinated
with an excess of sodium thiosulfate decreased the activity of
two oyster species when compared with untreated seawater.
Iodophors have been used in some European countries
and in Italy in particular (21,22,49). Recirculating depura-
tion system using 0.1 to 0.4 mg iodophor/L of tank water
produced rapid bacterial reductions withOut any apparent
effect on shellfish feeding activity or edibility characteristics
(49).
Water disinfection can also be accomplished using ozone
(12.48.88,171). Ozonation is also effective in reducing
marine toxins from seawater (1 1.13). The use of ozone has
been limited primarily to depuration plants in France and
Australia. Some of the reasons for its limited use worldwide
is that it is toxic to shellfish; treated water must be vigorously
aerated before addition to depuration tanks and residual
levels must be measured.
Trace amounts of residual ozone. chlorine. and iodo-
phors entering the shellfish during regular shellfish pumping
activities could contribute to microbial degradation in situ.
However. shellfish tolerance to these compounds and their
byproducts is so low that a slight excess may inhibit normal
physiological processes.
Another disinfection process involves ultraviolet (UV)
light-irradiated seawater. This is a system capable of destroy-
ing microorganisms only when the organisms are in direct
contact with the light. Ultraviolet irradiation is effective in .
reducing bacteria and viruses (26.50.79). Because of this
effectiveness, it has been applied to the disinfection of
shellfish depuration waters (60.61.78,96.143.170,175). It
also does not leave a residual as in the other disinfection
methods and. therefore. cannot directly inhibit the physio-
logical processes of shellfish.
Depuration in the United States is conducted exclusively
with UV light disinfection. Depuration using UV light is
usually effective provided the water is of low to moderate
turbidity. the flow rate is adequate. and the UV lamps are
operating efficiently. The UV system must be cleaned regu-
larly to permit UV light penetration. If light penetration of
processing waters is impaired by high turbidity (96),
dinoflagellate blooms (125) or the white milky gametes
produced during shellfish spawning (16,52), depuration will
be ineffective. Even with adequate UV penetration, the
number of UV -resistant microorganisms can increase during
depuration (153) and may contribute to microbial increases
in process water.
Any of the above means of disinfection can produce
satisfactory depuration results, however each technique re-
quires safeguards to ensure process integrity. Proper equip-
ment and regular monitoring should permit the safe and
effective use of any of the systems.
An emerging technology known as UV -generated ozone
is under evaluation as an alternative water disinfection
process (10). Conventional UV lamps operate at a wave-
length around 254 nm; however. special systems are now
available which permit UV photooxidation in the ISO-nm
220 RICHARDS
range. Ionized gas (plasma) produced at 180 nm is commonly
called activated oxygen. It reportedly has some benefits to
aquaculture type systems over conventional ozone. Unlike
ozone, activated oxygen is nontoxic to fish and completely
removes nitrites fromthe water by converting them to nitrates
(personal communication with Water Management Inc.,
Englewood, CO). Activated oxygen reportedly oxidizes
dissolved organic matter in seawater, thus reducing biologi-
cal and chemical oxygen demand and turbidity, while in-
creasing dissolved oxygen levels in the water. An interna-
tional symposium entitled "Ozone and UV in Water Treat-
ment" was presented at Aquatech 86 (sponsored by the
International Ozone Association, Paris). The symposium
focused on UV-generated ozone technology. Application of
the principle to depuration plant processes is currently under
evaluation in the United States and abroad.
Water purification in Europe varies by country. Plants
in England use UV light. Ozone and chlorine are usually used
in France. Spain, which has mandatory depuration, uses all
three disinfection methods.
Plant design
In 1964, a series of status reports from individual states
provided guidance on depuration plant design and operation
(14,59,60). Construction of most depuration plants since the
late 1960s has followed the general guidance provided by
Furfari (52). These guidelines were based on information
available through the scientific literature and the general
experiences reported by depuration processors in the United
States, Canada and Europe (52). More recent and updated
guidance on depuration plant design is available
(21,22,53,125 ).
In general, plants should be constructed of corrosion-
resistant materials in a manner that will facilitate cleaning.
System designs should include plans for water treatment, i.e.,
temperature, salinity, turbidity and dissolved oxygen adjust-
ments, and filtration and disinfection requirements (52). If
clean seawater is not available for shore-based plants, water
may be obtained from seawater wells (115) or trucked in.
Plant operating procedures, including the regulation of flow
rates, washing tanks, shellfish culling and washing, and waste
handling, are necessary considerations to ensure adequate
depuration efficiency (21,22,52,53,156.166).
Water quality criteria
Minimum and maximum water quality guidelines must
be developed for each species of shellfish to be depurated.
Some of these criteria may be gleaned from the literature and
are mentioned later in this paper. Some criteria suggested for
depuration plants in the United States are as follows: mini-
mum temperatures of 1000C for Eastern oysters (Crassostrea
virginica) and hard clams (Mercenaria mercenaria) and 2°C
for soft clams (Mya arenaria) and maximum temperatures of
25°C for Eastern oysters and 20"C for hard and soft clams
(52,53,164); dissolved oxygen, 5.0 mg/L (min.) to satura-
tion (max.) and turbidity 0 to 20 Jackson turbidity units
(ITU) (52.53); salinity within 20% of harvest area salinities
and a pH 00.0-8,4 (52.53.164). The pH may be particularly
JOURNAL OF FOOD PROTECTION, VOL. 51, MARCH 1988
important in recirculating systems where excretion of feces
and other metabolic products from shellfish can cause a
reduction in water pH. A minimum flow rate of 1 gal/mini
bu (3.79 L/min/bu) is recommended (52). These criteria
should be updated to include optimal conditions for virus
depuration.
There is a growing concern that dissolved oxygen, as
measured by mg/L, does not adequately reflect the prevail-
ing conditions within a depuration system (personal commu-
nication, 1986 Interstate Shellfish Sanitation Conference
Depuration Committee). Temperature and salinity affect the
amount of oxygen that water can hold, so a more appropriate
measure of oxygen may be percent saturation. Standards
based on percent saturation are in effect in Europe (4,56).
Supersaturation of water with gases occurs when water
temperatures drop below the saturation point, causing visible
bubbles on the sides of the tank and impaired functioning of
the shellfish. Supersaturation of depuration tank water can
lead to shellfish mortality (16). Removal of oxygen from
depuration tank' water is a function of shellfish loading and
flow rates, temperature, salinity and shellfish activity.
Tank loading criteria
Depuration tanks should be designed and loaded with
shellfish in a manner which will provide uniform hydraulic
flow throughout the tank and throughout the baskets of
shellfish with minimum disturbance of shellfish by vibration
of turbulence (53). According to the U.S. Public Health
Service (52,166), tank loading should not exceed 1 bushel of
soft-shell clams (Mya arenaria) per 5 ft3 (0.14 m3) of tank
water, or 1 bushel of hard-shell clams (Mercenaria mer-
cenaria) or Eastern oysters (Crassostrea virginica) per 8 ft3
(0.233 m3) of tank water. Other countries frequently set
loading rates based on weight per unit area (e.g. kg/m2)
(Personal communication with Walter Canzonier).
To pump, bivalves open their shells by passive release of
the adductor muscle and a slight opening force exerted by the
elastic hinge ligament. Shell opening can be restricted by the
weight of shellfish stacked several layers high. Therefore, the
depth of shellfish within the baskets should be restricted to
shallow layers. Recommendations by Canzonier (22) are 15-
18 cm deep for mussels (Mytilus edulis) and only 3 or 4
shellfish high for oysters (no species mentioned). The soft-
shell clam and mussel may be stacked higher than some other
species because of their relatively light shell weight, while
heavy-shelled clams, such as Venus gallina, should be main-
tained only in shallow layers (21). Recommended guidelines
in the United States suggest that soft-shell clams (Mya
arenaria) be no more than 8 in. (20.3 cm) deep and that hard-
shell clams (mercenaria mercenaria) and Eastern oysters
(Crassostrea l'irginica) be no more than 3 in. (7.6 cm) deep
(recommendations of the 1986 ISSC Depuration Commit-
tee).
Good water circulatory patterns throughout the tank and
throughout the individual baskets are crucial for efficient
depuration (42,70), whereas dead spots within tanks or
baskets contribute to depuration failure (85).
 DEPURA nON AND RELAYING SHELLFISH
REGULATIONS
In the United States, regulations concerning depuration
are promulgated by individual states under the general
guidance of the National Shellfish Sanitation Program
Manual of Operations (164.165) and subsequent Food and
Drug; Administration interpretations. Under these guidelines
the quality of the source water from which shellfish are
obtained for depuration is considered critical to ensure
depuration effectiveness. Shellfish may be harvested from
areas classified as no worse than restricted «700 total
coliforms or <88 fecal coliforms per 100 ml of seawater)
(167). Other countries do not have guidelines restricting the
level of fecal contamination in the shellfish growing waters.
Shell stock entering depuration tanks in Europe and other
countries may be considerably more contaminated than shell-
"'Jd{ destined for depuration in the United States.
State regulations governing water quality parameters for
shellfish purification plants are currently in effect in Califor-
nia, Florida, Louisiana, Massachusetts, Maine, New Jersey.
New York, South Carolina and Texas. (Personal communi-
cation with Santo Furfari and John Hurst). Upon initial
startup, depuration plant operations and products are moni-
tored regularly during a process verification phase. If the
process is found effective in reducing levels of indicator
bacteria, sampling is reduced. State shellfish control agencies
regulate and monitor all aspects of shellfish depuration
including sample collection, transport. processing and distri-
bution. Products must meet end-product bacteriological
criteria which are enforced by the states. For soft-shell
clams. the general guideline approved in 1971 recommended
a median value of 50 fecal coliforms/l00 g of meats with no
more than 10% ofthe samples exceeding 130 fecal coliforms/
100 g (162). New end product standards for depurated
shellfish have been proposed (164). The proposed standards
for soft clams state that a single batch of depurated shellfish
with> 170 fecal coliforms/l 00 g or with an arithmetic mean
of duplicate samples > 125 fecal coliforms/100 g will be
rejected. For hard clams and Eastern oysters. a single batch
with > 100 fecal coliformslI 00 g or an arithmetic mean of
duplicate samples >75 fecal coliforms/l00 g will be cause for
rejection of the entire batch.
A minimum sampling scheduled and end product stan-
dards for evaluating depuration plant performance were
proposed in 1987 (164). The frequency of sampling would
depend on the variability of pollution in the shellfish harvest
areas. Depuration plant operations would be continually
evaluated to determine the efficacy of the process. Plant
compliance would be assessed based on the fecal coliform
results from 10 consecutively processed batches of shellfish.
The recommended geometric means and upper 10% criteria
for fecal coliform levels/100 g are: soft clams, geometric
mean of 50 and upper 10% of 130; hard clams and Eastern
oysters, geometric mean of 20 and upper 10% of 70. Plants
found not in compliance would be subject to closure until the
integrity of the process could be restored.
Numerous regulations affecting depuration plants and
processes are in effect in other countries; however. it is
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
221
beyond the purview of this review to comment on these ever
changing regulations.
ECONOMICS OF DEPURATION
Only limited information on the economic aspects of
depuration was found (52,83.156), and with the passage of
time since their publication. the dollar values require adjust-
ment to reflect inflation, added labor, raw material, and
processing costs. It is common knowledge among depura-
tion plant operators that shellfish subjected to depuration
often command a premium price in the marketplace. There
are several reasons for this. Depurated shellfish are less gritty
than nondepurated product because sand is purged, along
with microbial and chemical contaminants, from the shellfish
during the depuration process. This lack of grittiness makes
shellfish more palatable to some consumers. The flavor of
shellfish obtained from low salinity waters can be enhanced
by depurating them in higher salinity waters. Some buyers
of shellfish also feel there is less liability to them if they
obtain "cleansed" shellfish. On a similar note. some procure-
ment authorities who purchase shellfish for institutional con-
sumption now prefer the purchase of depurated products.
ILLNESSES FROM DEPURATED SHELLFISH
It is often assumed that depurated shellfish are bacteri-
ologically and virologically safe; however, this assumption is
false. The presence of a few pathogenic bacteria in shellfish
does not generally have public health significance, because
threshold levels necessary to cause illness far exceed the
levels present. Indigenous marine bacteria. such as Vibrio,
have been associated with outbreaks of gastroenteritis from
the consumption of depurated oysters (6). Vibrio does not
depurate well and may even multiply in depurating shellfish,
tank-water and plumbing systems (6,45,63).
Enteric viruses can be transmitted to humans by con-
taminated shellfish leading to outbreaks of gastroenteritis.
Norwalk virus illness and hepatitis A (46,123). Reviews on
outbreaks of shellfish-associated enteric virus illness have
been published by Gerba and Goyal (57) and Richards
(141,142). Enteric viruses are infectious even in very low
numbers (134,146), thus total virus depuration is essential to
ensure public safety. Depurated shellfish from England
were linked to widespread outbreaks of presumed viral illness
in the United States (163). The economic impact of a c1am-
associated outbreak of illness was depicted by Brown and
Folsom (15). Another study by Gill et al. (58) showed the
presence of small round viruses resembling Norwalk virus in
depurated oysters which met acceptable bacteriological cri-
teria. These oysters caused gastroenteritis in over 300 people
near London (127).
Grohmann et al. (65) conducted a unique epidemiologi-
cal study to determine the incidence of viral illness among
consumers of depurated and relayed oysters, Crassostrea
commercialis. The Commissioner of New South Wales and
the New South Wales State Fisheries instituted several
programs to reduce and better define the risks associated with
 RICHARDS
222
oyster consumption. Between 40 and 129 volunteers partici-
pated each week for 32 weeks by consuming ~6 depurated or
relayed oysters before marketing each lot. Fecal coliforms in
all batches were below the 230/1 00 g legal limit in Australia.
Oysters from Georges River and Brisbane waters were
tested. None of the volunteers became ill after eating Bris-
bane oysters, however 52 volunteers reported ill after eating
relayed or depurated oysters from the Georges River. Spe-
cific relay and depuration parameters were not provided. The
incubation period for illness ranged from <24 to 75 h with a
mean of 42 h. Symptoms were consistent with those of
Norwalk virus illness, primarily vomiting (67%), diarrhea
(56%) and nausea (56%). Recovery was usually complete in
36-48 h.
About 60% of the illnesses occurred during two weekly
periods which were preceded by heavy rains. Excluding
these two periods, the average sickness rate was 1%. No
bacterial pathogens were detected in the stools of ill volun-
teers; however, Norwalk virus was present in 8 of 25 (32%)
of the available stools. Antibody titers to Norwalk virus also
increased in 7 of 10 ill volunteers.
The epidemiological evidence clearly showed that Nor-
walk virus was present in depurated or relayed shellfish
which met bacteriological criteria acceptable in Australia. It
was suggested that Norwalk virus may persist in oysters for
longer periods than bacteria, requiring depuration for >48 h
(65). Studies on other viruses support the idea that viruses
depurate slowly from shellfish (20,120,148).
TECHNICAL ASPECTS OF SHELLFISH RELAYING
This section provides a review of the scientific literature
on relaying. Relatively little technical information is avail-
able on this subject, perhaps because relaying is not practiced
in many countries due to the unavailability of clean waters in
many areas as well as economic considerations. Available
literature covered the relay of five shellfish species, four of
which will be addressed separately. The fifth species, mussels
(Mytilus edulis), was discussed earlier under Historical Per-
spectives.
Soft-shell clams, Mya arenaria
Early bacteriological studies were conducted by Erdman
and Tennant (43) on depletion of coliforms from soft-shell
clams. They investigated the feasibility of relaying soft clams
from polluted areas to clean waters, but found that the
cleansing process was at the mercy of the weather. Under
favorable weather conditions initial coliform most probable
number (MPN) counts averaged 3,554/100 g for 29 lots of
clams. After 72-120 h, counts were reduced by 89.3% to 380
coliforms/lOO g. Coliform levels in seawater were also
measured during the relay period and were observed to
fluctuate erratically.
The clams quickly reflected increases in coliform levels
of seawater after heavy rainfall and run-off. In one instance,
l3 cm of rainfall caused coliform counts to increase in two
lots of clam meats from 7,933 and 2,467/100 g to 21,000 and
56,000/100 g, respectively, after 72-120 h, while mean
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
coliform counts in the seawater increased form 3.2/1 00 ml at
oh, to 173.1/100 ml after 24-48 hand 995.0/100 ml after 72-
120 h (43).
Eastern oysters, Crassostrea virginica
Becker (8) studied basket relaying of Mississippi oysters
and the effects of basket loading and relay times on the
reduction of coliform bacteria. Using stainless steel, pre-
sumably open mesh, baskets 14 in. (35.6 cm) on each side and
containing (a) a single layer of oysters (6.8 kg), (b) half a
basket (13.6 kg) of oysters, or (c) a full basket (27.2 kg) of
oysters, he determined that the single layers of oysters more
efficiently reduced their initial total and fecal coliform levels
than oysters obtained from the bottom layers of the one-half
full or full baskets. Counts were significantly higher
(P<O.O 1) in the full basket than in the one-half full or single
layer baskets. Becker speculated that the flow of water
through the baskets was physically impeded by the stacked
shellfish and that the excrement from the upper layers of
oysters could recontaminate lower-level shellfish during
periods when tidal flows were at a virtual standstill.
The length of relay was also critical for the single layer,
half- and full-baskets of oysters (8). About 95% of total and
fecal coli forms were eliminated in the single layer baskets in
48 h, however 96 h were required for similar reductions in the
half baskets, and full baskets showed no reductions over
initial counts after 96 h. In fact, coliform increases up to 2.6-
fold were reported in full baskets. Coliform increases may
have been the result of recontamination by oyster feces from
the upper layers or the outgrowth of coliform-like organisms
such as Klebsiella and Citrobacter species (67).
Water quality was also a variable factor during the study
(8). Bacterial water quality criteria of 70 total coliforms/l00
ml and 14 fecal coliforms/100 ml were exceeded on several
occasions.
An abstract published by Sobsey et al. (150) discussed
the depletion of bovine enterovirus type 1 from oysters
during basket relaying for up to 30 d. Over 99.99% of the
laboratory-acquired viruses were depleted in 30 d during the
spring and winter months. During summer and fall, only ca.
80% of the viruses were depleted in 30 d, but E. coli and
enterococcus elimination was >99.9%. Indicator bacteria
were not reliable for monitoring enteric virus elimination
from oysters (150).
Similarly, Kator and Rhodes (94) showed that oysters
and hard shell clams (Mercenaria mercenaria) relayed on-
bottom and in commercial size containers efficiently purged
fecal coliforms within 14 d, but that elimination of bovine
enterovirus type 1 was prolonged.
Supan and Cake published two abstracts (158,159) and
a paper (160) on an evaluation of containerized oyster
relaying in Mississippi Sound. The complete description of
these studies (160) discusses suspension relaying (shellfish
suspended in trays), on-bottom longline relaying (shellfish
placed on estuary floor in covered racks that are attached to
ropes for easy retrieval), and rack relaying (oysters in trays
within a specially designed rack which is placed on the
estuary floor). The latter was the most desirable method
 DEPURA TION AND RELAYING SHELLFISH
tested because it (a) provided the most consistent cleansing of
shellfish, (b) resisted burial even on less stable, soft bottoms
and (c) deterred theft because of its fully loaded weight
(3,(;--00 kg) and tray locking system (160). A fecal coliform
median value of 1,400/100 g was reduced to 45/100 g after
7 d (158,160). In another trial, 23,000 fecal coliforms/loo g
were reduced to 20/100 gin 10 d (158-160).
Reportedly, oyster mortalities>70% are not uncommon
with conventional (non-containerized) relaying techniques
due to physiological stress, shell damage, predators, and
smothering and clogging by sediments (160). Nearly 60% of
the oysters landed upside down when transplanted and these
Jysters are more susceptible to death by sediment intake and
Jill L:"'gging (66). Oysters mortalities in rack relay studies
averaged 1.6% compared to death rates of 19 to 30% for
control oysters relayed on bottom (160).
A recent study on relayed oysters was conducted by Cook
and Ellender (31 ). Shellfish were exposed to a feces suspen-
sion containing bacteria and viruses for 14 to 16 h in a 400-
L tank of aerated seawater. The oysters were relayed in
baskets. Microbial depletion occurred as follows: fecal coli-
forms, generally >99.9% reduction within 5 d; Salmonella
typhimurium, complete elimination of 103 to > I 0 5 organisms/
g in <II d; Salmonella montevideo, elimination of~ 104/g in
nearly 1 month; poliovirus type I (16-43,000 plaque forming
units (PFU)floo g of shellfish meat and liquor), >31 d in
some instances, but generally requiring 515 d for depletion
of low levels of virus (16-71 PFU/loo g).
Water temperature also affected the depletion rate of
relayed oysters (31). Oysters with high poliovirus counts
failed to effectively cleanse within 2 weeks at <20"C, but
elimination was effective in <I week at >25°C. Heavily
contaminated oysters retained viruses for >32 d at tempera-
tures <14°C. Salinities of relay waters ranging between 17.7
and 27.7 ppt appeared to have little effect on elimination of
fecal coli forms, S. typhimurium, S. montevideo, and polio-
virus I from relayed oysters (31).
Pacific oysters ,Crassostrea gigas
Oysters rapidly accumulated and eliminated total and
fecal coli forms at rates influenced by the levels of these
bacteria in the surrounding seawater (90). Quayle and
Bernard (138) conducted a study on the basket relay of
Pacific oysters. Baskets measured 62 x 62 x 30 cm high and
contained ca. 55 kg of oysters. Baskets loaded with 5, 10,20
and 40 kg of oysters demonstrated coliform and E. coli
reductions comparable to fully-loaded baskets. During the
first 24 h of relay, ca. 90% of the coliforms and E. coli were
eliminated. An additional 8% reduction was observed over
the next 24 h. E. coli levels as high as 186,000/100 depleted
to the E. coli level of the relay waters within 24 h (138).
Sydney rock oysters, Crassostrea commercialis
Son and Heet ( 152) described a study on the depletion
of bacterial pathogens from C. commercialis by relaying.
Relayed oysters were sampled at 0,2 and 6 d. Analyses were
conducted for E. coli, Salmonella, Bacillus cereus, Vibrio
parahaemolyticus. and Clostridium pelfringel1s. E. coli was
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
223
reduced from 590 and 600/100 g in duplicate tests to 80 and
180/100 g after relay for 2 d and to nondetectable levels
within 6 d. Salmonella was detected in only one oyster sample
before relaying and was not present after relaying. Mean B.
cereus counts were reduced from 335/g to 67 and 57/g after
relay for 2 and 6 d, respectively. V. parahaemolyticus counts
decreased from 18/g to 5 5/g in duplicate trials in 2 d and
remained 5 5/g for these same trials after 6 d. C. pelfringens
decreased from 18/g to 5 8/g in 2 d and to a nondetectable
level by day 6 (152).
More recently, Qadri et at. (139) relayed oysters which
were naturally contaminated with total and fecal coliforms
and artificially contaminated with E. coli. All oysters were
transported to a relatively clean estuary and relayed in wire
trays. Naturally contaminated oysters were relayed for I
week. Most oysters showed a substantial reduction in fecal
coliform MPNs. Total coliform levels fluctuated erratically
with nearly one-third of the samples (7 of 22) higher in total
coliforms after relay than before, possibly the result of
occasional pollution of the growing waters (139). Fifty
oysters, artificially contaminated for 10 h in a 50-L tank
containing IxlQ4 E. coli/ml of seawater, depleted 1.4 X 105
E. coli/100 g to I X 103/100 gin 1 week and to nondetectable
levels within 2 weeks.
SUMMARY OF SHELLFISH RELAYING STUDIES
Recontamination of relayed shellfish constitutes a poten-
tial drawback to effective relaying and influenced the results
of some studies (8,43,94,139). Heavy rains and the associ-
ated runoff may contaminate otherwise acceptable relay
waters rendering depletion ineffective.
Basket loading influenced the depletion rates oftotal and
fecal coliforms in one study on Eastern oysters (8), but not
in another study on Pacific oysters (138). This dichotomy
may be the result of differences between these shellfish
species relative to their ability to pump under heavy weight.
Temperatures and salinities may have differing effects
on various shellfish species. Temperature affected bacterial
depletion from Eastern oysters, but salinity reportedly did
not (31 ). Season appears to influence enteric virus depletion
(150).
Basket relaying may reduce shell breakage and shellfish
mortality over conventional relaying techniques (160). This
could be particularly useful for shellfish species with thin
shells or in areas where soft estuary bottoms could cause
smothering of the shellfish.
UPTAKE AND DEPURATION OF
MICROBIAL CONTAMINANTS
A detailed review of the scientific literature on the
uptake and depuration of microorganisms by shellfish is
presented in this section. Information on microbial uptake
parameters for specific depuration experiments are presented
in Tables 1, 3, 5, 7, 9, 12 and 14. Depuration parameters and
results are presented in Tables 2, 4, 6, 8, 10, 13 and 15. The
first set of tables contains information presented in the
 224 RICHARDS
literature concerning uptake parameters (type and level of
contamination, exposure period, and if uptake was conducted
in a static or flow-through system) for various shellfish
species. The latter set of tables delineates the types of
depuration systems used, water parameters, and contaminant
levels present in the specified shellfish before and after
depuration. Specific examples were selected at random, often
from extrapolations from graphs. For additional examples,
the reader should refer directly to the referenced literature. It
will become evident from examining these tables that condi-
tions vary greatly among the studies and that some informa-
tion, which would have facilitated comparison of data, is not
presented. Many of the depuration studies discussed in the
text are not referenced in the tables because of the unavaila-
bility of sufficient pertinent data.
Depuration rates of microbial contaminants may be
influenced by the manner in which shellfish accumulate the
contaminants, i.e., artificially in the laboratory or naturally,
over short or long periods, with high or low numbers of
contaminants present in the uptake water, etc. Therefore,
details on the uptake, tissue distribution and concentration of
microbial contaminants are provided. The major findings of
these studies are discussed under the specific shellfish head-
ings.
Many different analytical procedures were used in the
studies discussed in this section. Some of the older proce-
dures, particularly those for testing viruses in shellfish, may
have lacked the sensitivity and reproducibility of more recent
techniques. Therefore. the reader should exercise caution in
comparing and interpreting data from the various studies.
Hard-shell clams. Mercenaria mercenaria
The concentration of waterborne E. coli in hard clams
varied between 6.S and 8.S times the level of the surrounding
seawater when seawater concentrations were 10' to 105 E.
coli/I 00 ml (17). Bioaccumulation of E. coli was impaired in
higher turbidity waters (73.74). The uptake of E. coli and the
pathogen, S. typhimurium, occurred to a similar degree
(161). E. coli accumulation occurred primarily in the
digestive gland and, to a lesser extent, in the siphon tissues
(17). The greatest concentration of contaminating bacteria
(E. coli and S. typhimurium) appeared to be associated with
the feces and pseudofeces where they were tightly bound to
particulates (161). This binding appeared to be by physical
entrapment within fecal material rather than as a result of
ionic bonding (161).
Poliovirus type I also accumulated primarily in the
hepatopancreas (which includes the digestive diverticula)
(105,107) as well as in the siphon tissues (42). Virus
concentrations of 10 to lOO-fold have been achieved (149).
Liu et al. (105,107) showed that simple mechanical mincing
of the digestive diverticulum released SO-60% of the polio-
virus. This implied that the viruses were in a carriaoe state
rather than chemically bound to the tissues. These '"viruses
may be subject to physical entrapment rather than chemical
attachment, much like that observed for bacteria (161).
Viruses in the liquor were predominantly associated with the
hemolymph rather than the mantle cavity fluids (l05).
For bacterial viruses, Canzonier (20) dosed hard clams
with continuous low levels of E. coli phage S-13 (S-7 phage
particles/ml of seawater) for 16, 40, 160 and 184 h. Mean
counts for 20 clams ranged between 52 and 86/ml of digestive
gland for all time periods. The accumulation factor (ratio of
clam titer to seawater titer) was about IO-fold. Increasing
virus levels to 70 particles/ml of seawater resulted in a mean
accumulation factor of 2S-fold. In other trials, accumulation
factors were as high as I,IOO-fold (20).
Results of studies on uptake with subsequent depuration
of microorganisms from hard clams are shown in Tables I
and 2. The studies were conducted under a wide range of
depuration parameters. Those factors which affect the pump-
ing of clams appear to be the most significant to successful
depuration. Among these, temperature and salinity play an
important role.
The influence of temperature on depuration of hard
clams was evaluated for the following contaminants: E. coli
(19,63,73), poliovirus types I and 3 and Coxsackievirus type
B4 (108), V. pai"altaemolyticus and Vibrio harveyi (63) and
coliphage S-13 (20). In flow-through depuration systems, E.
coli, poliovirus I and 3, and Coxsackievirus B4 depurated to
nondetectable levels within 48 h at temperatures near 20°C
(19,73,107,108) (Table 2). At temperatures between 10 and
I SoC depuration was usually more protracted
(19,73,107,108) (Table 2). One study showed the net loss of
coliforms through elimination or natural inactivation at low
temperatures (129).
A study on the elimination of Vibrio and E. coli in a
recirculating depuration system concluded that E. coli was
rapidly eliminated at 8, 15 and 25OC, but that Vibrio was not
(63) (Table 2). The most rapid depuration of Vibrio was at
IS°C. At 8 and 2SoC, Vibrio reductions approximated I log
after 72 h.
V paraltaemolyticus levels oscillated throughout the
2SoC depuration cycles (63). Higher initial concentrations of
V. parahaemolyticus resulted in more substantial decreases in
counts within the first 6 h of depuration; however, after 6 h,
outgrowth of V. parahaemolyticus was greater in those clams
containing the lowest initial counts. It was speculated that
initial decreases in numbers of both V. parahaemolyticus and
V. hal1'eyi were made possible by a loose association between
the bacteria and the animal cells and the rapid shedding of
Vibrio via the feces (63). Subsequent persistence of \1ibrio
in hard clams may have been the result of a closer association
between the bacteria and the host's intestinal cells which
resulted in the apparent growth of Vibrio in clams.
Depuration of polio I was performed by Liu et al. (108)
at 7-8°C, 13-14°C and 19°C. Separate analyses of meats and
liquor indicated that very little depuration occurred at 7-8°C
even at 96 h. At 13-14°C, viruses were reduced from 50 and
100 PFU/ml of homogenate to nondetectable levels in the
liquor by 72 h. but were detectable in the meats even at 96 h.
Depuration at 19°C rendered viruses nondetectable in both
liquor and meats at 24 and 72 h, respectively (J08).
Hard clams containing similar levels of polio 3 were
depurated at 5-6°C. 14°C and 18-20°C (108) (Table 2). At 18-
2OVe, meats and liquor reached nondetectable virus levels
 DEPURA TION AND RELAYING SHELLFISH 225

TABLE 1. Microbial uptake parameters for hard clam (Mercenaria mercenaria) depuratioll studies.
Exposure
Level! Static/
Contaminant Nat/Arta Period 100 ml flow-thru Reference
E. coli A 6-48 h 102-10" Ff Heffeman and Cabelli (73)
E. coli A 6-48 h 102_104 Ff Cabelli and Heffeman (19)
Polio 1 A 48 h 103 _10 7.5 S Liu et a1. (107)
Polio 3 A 48 h Ff Liu et al. (107.108)
Coxsackie B4 A 48 h Ff Liu et al. (108)
Polio 1 A 48 h Ff Liu et al. (108)
E. coli A 2h Ix 105 S Greenburg et al. (63)
V. parahaemolyticus A 2h Ix 105 S Greenburg et a1. (63)
V. harveyi A 2h IxlO 5 S Greenburg et al (63)
E. coli A Ff Canzonier (20)
Coliphage S-13 A 16-184 h 5-7xlQ2 Ff Canzonier (20)
S. typhimurium A 15min 5xlO6 S Timoney and Abston (161)
E. coli A 15 min 5xlO6 S Timoney and Abston (161)
• Abbreviation: A. artificially contaminated shellfish. Dash (-) indicates information was not provided.

within 24 h. AT 14°C. viruses became nondetectable in the clams in 48 h (104).

meats and liquor by 48 h. Clams depurated at 5-6°C required
72 and 96 h to reduce counts to nondetectable levels in the
liquor and meats. respectively. Limited work with similar
amounts of Coxsackievirus B4 showed that virus was cleared
from clam meats within 48 h at 20"C and by 72 h at 15°C ( 108)
as best determined using the analytical procedures of that era.
Depuration studies by Canzonier (20) conducted at 16°C
in a recirculating system showed that coliphage S-13 elimi-
nation did not correlate with E. coli elimination in hard clams
(Table 2). E. coli (ca. lOS CFU/ml) were reduced rapidly at
16°C to to CFU/ml and <I CFU/ml at 24 and 48 h.
respectively. Phage reductions proceeded slowly and in 6 d
were reduced by only I log at 16°C. At 24°C. phage was
reduced to a negligible level in 72 h. The pattern of phage
reduction was nearly identical in seawater and clams at both
temperatures. Canzonior (20) indicated that temperature is
the principal factor in coliphage S-13 and Staphylococcus
aureus phage P-80 inactivation in seawater; depuration has
no effect on low level phage reductions in hard clams. The
lack of correlation between viral and bacterial elimination is
noteworthy.
Salinity played an important role in the effectiveness of
depuration (Table 2). Salinities between 22 and 31 ppt were
optimal for E. coli depuration from hard clams (73). Clams
maintained in 31 ppt seawater effectively depurated at 20 ppt
salinity. but only after acclimating the clams to this lower
salinity for 4 weeks.
The effects of salinity on polio I depuration by hard
clams were evaluated by Liu et al. (108). At temperatures
fixed at 18-20"C and salinities of 23-28 ppt and 31 ppt. hard
clams depurated to nondetectable virus levels after 48 and 24
h: respectively. Clams maintained at salinities of 17-21 ppt
did not completely depurate even at 96 h (108). Under
favorable depuration conditions, polio, Coxsackie and echo
viruses efficiently depurated ( 104). About 99% of echovirus
" . tYpes I, 11 and 14 depurated from artificially contaminated
Shellfish conditioning. that is. allowing shellfish to
acclimate to the temperature and salinity of the process water.
appears necessary to ensure maximum depuration. Salinities
50-60% below the levels from which the shellfish were
harvested completely stopped the depuration process (108).
How rates also affected hard clam depuration (Table 2).
A flow rate of 13 ml/min/hard clam provided optimal
depuration of E. coli (73). This was roughly equivalent to 1
gal/min/bu (3.79 L/min/bu). How rates below 13 ml/min/
clam significantly reduced E. coli depuration efficiency (73).
At 3 ml/min/clam. clams stopped pumping; however. mor-
talities reportedly did not increase (18).
Turbidities from very low to 25 ITU did not affect
bacterial depuration efficiency in one study (19). but high
turbidities did enhance the depuration process in others
(73.74).
Depuration of S. typhimurium and E. coli from hard
clams was compared. Greater reductions were observed for
E. coli than S. typhimurium after 24 h (161) (Table 2).
Variations in pumping rates of individual clams may also
affect microbial depuration. It was estimated that ca. 1% of
depurated clams do not appreciably reduce their levels of
coliforms in 48 h (74). Hydraulic conditions within the tanks
and the physiological condition of the clams influenced
poliovirus 1 depuration (149).
Some of the published literature did not provide infor-
mation on sizes of the depuration tanks used. The approxi-
mate seawater capacities of those tanks for which information
was available were: 54 L (107.108,149). 114 L (63).227 L
(161) and 1.700 L (108.149). Size information is provided
to indicate whether commercial or laboratory-scale depura-
tion systems were employed. Water volume is important
since water in smaller tanks is more susceptible to tempera-
ture fluctuations unless thermostatically controlled. Water
volume and shellfish loading rates will also affect the pH and
dissolved oxygen levels in the system.

JOURNAL OF FOOD PROTECTION. VOL. 51. MARCH 1988

 N
N
0"1

TABLE 2. System design. water parameters. and microbiological results for the depuration of hard clams (Mercenaria mercenaria).
Water Parameters Dep.
,
Water b Contam. level/lOO gd
Temp. Sal. Flow rate time"
Contaminant System" treatment" (ac) (ppt) (ml/min/ciam) (h) Before After" Reference
E. coli Ff UV 21r 30-31 10-13 48 3xl1J4 NO Heffernan and Cabelli (73)
Cabelli and Heffernan (19)
E. coli Ff UV 15 30-31 10-13 72 3xl<r NO Heffernan and Cabelli (73)
Cabelli and Heffernan (19)
E. ('oli Ff UV 10 30-31 10-13 72 3xlO4 NO Heffernan and Cabelli (73)
Cabelli and Heffernan (19)
Polio Ff UV 18-21 ca.2LVh 24 5xIOJ/g NO Liu et aJ. (107) ~
Polio Ff UV 20-21.2 31.2-31.5 ca. 2LVh 24 IxlO2/g NO Liu et al. (107) (")
:t
Polio Ff UV 13-15 31-32 ca.2LVh 96 2xJ03/g NO Liu et al. (107.108) >
;.:1
Polio 3 Ff UV 18-20 31-32 ca.2LVh 24 Ix 102/g NO Liu et al. (l08) 'Vl='
Polio 3 Ff UV 14 31-32 ca.2LVh 48 IxI02/g NO Liu et al. (108)
Polio 3 Ff UV 5-6 31-32 ca.2LVh 96 IxI0 2/g NO Liu et a!. (108)
Coxsackie B4 Ff UV 20 30-31 ca.2LVh 48 4xlOZlg NO Liu et al. (108)
Coxsackie B4 Ff UV 15 30-31 ca.2LVh 72 4xlO2/g NO Liu et al. (108)
E. coli R UV+FILT 25 ca.O.5LVh 36 IxJ03/g <IO/g Greenberg et al. (63)
E. coli R UV+FlLT 15 ca.0.5LVh 24 IXlcP/g <IO/g Greenberg et al. (63)
E. coli R UV+FlLT 8 ca.0.5LVh 24 )xIOJ/g 5/g Greenberg et al. (63)
V. parahaemolyticus R UV+FlLT 25 ca.0.5LVh 72 2xlOJ/g 90/g Greenberg et al. (63)
V_ parahaemolyticus R UV+FlLT 15 ca.O.5LVh 72 2xlOJ/g ll/g Greenberg et al. (63)
V. paTOhaemolyticus R UV+FILT 8 ca.0.5LVh 72 2xlOJ/g 50/g Greenberg et al. (63)
 V. harveyi R UV+FILT 25 ca. O.5.6,1h 72 lxW3/g 100/g Greenberg et al. (63)
V. han'eyi R UV+FILT 15 ca. O.S.6,Ih 72 IxlOJ/g WIg Greenberg et al. (63)
V. harvey; R UV+FILT 8 ca. O.S.6,Ih 60 Ix103/g
~ 100/g Greenberg et al. (63)
~ E. l'Oli FI' UV 16 20-26 24 lx107 Ix103 Canzonier (20)
~ Coliphage S-13 FI' UV 16 20-26 144 33/g 9/g Canzonier (20)
t'o 0
c." Coliphage S-13 FI' UV 24 20-26 72 II/g ND Canzonier (20) t'l1
'tI
E. coli FI' UV 20 15 10 48 4.9x103 8.8xl ()2 Heffernan and Cabelli (73) c::
;g E. coli FI' UV 20 20 10 72 2x104 3x103 Heffernan and Cabelli (73)
;Q
)-
g E. coli Fr UV 20 20r \0 48 4.9x 1 03 ND Heffernan and Cabelli (73) g
;g E. coli FI'
z
UV 20 25 \0 24 4.9xlQ3 ND Heffernan and Cabelli (73) )-
c E. coli FI' UV 20 30 10 48 4.9x1OJ z
t;i ND Heffernan and CabelJi (73) 0
E. coli . Ixl()4
9 E. coli
Fr
Fr
UV 20 30-31 3
7
72
72 Ix 104
2xl()2 Heffernan and Cabelli (73) ;Q
t'l1
r-
~
UV 20 30-31 SO Heffernan and CabelJi (73) )-
E. coli FI' UV 20 30-31 13 24 Ix104 17 Heffernan and Cabelli (73) -<
<: Z
0
r-
E. ('oli Fr UV 20 30-31 27 48 Ixl()4 17 Heffernan and Cabelli (73) Cl
v,
.... S. typhimurillm R UV 20 22 ca. 1O.6,Ih 24 Ixl07 2xlOJ Timoney and Abston (161) ::c
E. coli R UV 20 22 ca. 1O.6,Ih 24 2xI07 2xl()4 Timoney and Abston (161) ~
s:: r-
::!l
)-
;Q
"Abbreviations: Fr, flow through; R, recirculating; S,. static; UV, ultraviolet light-irradiated seawater; FILT, filtered seawater; Cl Chlorine-treated seawater; CHAR, charcoal-filtered CIl
n seawater; ND, none detected. Dash (-) indicates information was not provided. ::c
::c
bListed as mUmin/shellfish, number of tank-water changes (~) per h, I/min/bu, or lib/kg.
1.0
oc <Usually the minimum amount of time required for consistent depuration to the indicated level.
oc
dCount/l00 g unless otherwise specified.
'Numbers in bold type indicate specific parameters under evaluation.
fJ)epuration after 4 wk acclimation of clams to 20 ppt salinity seawater.

IV
IV
-..J
 228 RICHARDS
Durgin et al. (42) showed that hard-shell clams were
capable of reducing feces-associated viruses in laboratory-
scale and commercial depuration systems. Polioviruses were
reduced from 300 PFU/shellfish to nondetectable levels
within 48 h in the laboratory-scale system. Depuration was
effective in eliminating naturally accumulated viruses within
48 h; however, much variability was noted among shellfish
processed in different batches (42).
Soft-shell dams, Mya arellaria
Cabelli and Heffernan (18) determined that accumula-
tion and elimination of E. coli in the soft clam followed a
pattern similar to that of hard clams. Accumulation of E. coli
achieved a steady state in which uptake and depletion were
equal. This occurred at a level ca. 3x that of the hard clam
which was an accumulation factor of 20-fold (18).
The bioaccumulation of poliovirus in individual clams
was highly variable according to Metcalf et al. (119,120).
With 0.09 to 34.0 PFU of polio type l/ml of seawater, virus
was concentrated up to 34-fold within the soft clam tissues.
Virus concentration occurred primarily in the hepatopan-
creas and siphon with very low levels found in the mantle,
adductor muscle, gills, pericardium and hemolymph
( 119,120).
Soft clams effectively depurated E. coli and coliforms at
8 to 16°C, however 12-13°C was optimal (3,18) (Table 4).
Coliform depuration was inhibited at 2°C (18). Arcisz and
Kelly (3) found that coliform levels did not steadily decline
in the meats or water during depuration at 2.5-4.5°C. At 13°C,
Salmonella schottmuelleri rapidly declined from 3,300/100
g to non detectable levels in 48 h (3) (Table 4). Information
on viral reductions as a function of depuration temperatures
could not be found in the scientific literature.
Optimal salinities and flow rates for effective bacterial
depuration were investigated by Cabelli and Heffernan (18)
(Table 4). At salinities between 15 and 30 ppt, 20 ppt was the
lower limit for good depuration of elevated temperature
coliforms. Optimal depuration occurred between 25 and 30
ppt (18).
TABLE 3. Microbial uptake parameters[or soft clam (Mya arenaria) depuration studies.
Exposure"
L.eveV
Contaminant Nat/Art Period 100 ml
E. coli A 6-10h Ixl0
E.T. coliforms b N NA NA
S. schoffnlllelleri A 6-IOh IxIO.1
Polio 1,2,3 A 20 h
Polio 1,2,3 A 20 h 20
Total coliforms N NA NA
Fecal coliforms N NA NA
E.T. colifornls N NA NA
:AbbreViations: A, artificially contaminated shellfish: N, naturally
informatIOn was not provided.
"Elevated temperature coli forms.
Flow rates of 3 to 24 milmin/soft clam or 1.2 to 9.6 gaIl
min/bu (4.5 to 36.3 L/min/bu) did not markedly affect
depuration rates, although at 3 milmin/clam shellfish mortal-
ity increased, an effect likely due to oxygen. depletion (18).
An earlier study by Goggins and coworkers (60,61) sug-
gested reduced depuration efficiencies for E. coli and coli-
forms in soft clams at flow rates < 1.1 L/min/bu and showed
the effects of reduced flow rates on dissolved oxygen levels
in process water. At 1.6 to 3.0 L/min/bu and at 15-200c, 35%
of the oxygen was depleted in their depuration system.
Lower flow rates (1.1 to 1.5 L/min/bu) caused the depletion
of 70% of the oxygen at the same temperatures (60,61).
Laboratory studies by Metcalf et al. (120) on the
depuration of poliovirus by soft-shell clams indicated the
persistence of low-level viral contamination after extended
periods of depuration (Table 4). Pools of 10 clams, contain-
ing 226 to 303 PFU of polio 1, depurated 80 to 88% of these
viruses within 48 h. The depuration rate was much slower
after 48 h and elimination of all viruses was generally not
achieved even llf 144h. With lowerlevels of virus (11.1-33.6
PFU/IO clams) depuration was incomplete in 2 of 6 trials
with 1.3 and 1.6 PFU present in clam pools after 72 h. It was
stated that unsuccessful depuration was probably the result of
just one or two clams not adequately pumping during the
depuration cycle (119,120). Metcalf et al. (120) mentioned
that shellfish size influences the volume of water pumped
through the clams' gills; that larger shellfish pump more
water than smaller ones. This may affect the rate of contami-
nant bioaccumulation and depuration.
Soft clams demonstrated more variability in depurating
feces-associated poliovirus than hard clams or oysters ac-
cording to Durgin et al. (42). In a laboratory-scale system, 30
PFU of poliovirus/clam were reduced to nondetectable levels
within 48 h. Depuration of poliovirus in a commercial plant
was reportedly effective within 48 h, however initial viral
counts were not stated (42).
Comparisons of coliforms, fecal coli forms and elevated
temperature coli forms were made by Cabelli and Heffernan
(18) on naturally contaminated clams (Table 4). Depuration
Static/
flow-thru Reference
4
S Arciszand Kelly (3)
NA CabelIi and Heffernan (18)
S Arcise and Kelly (3)
S Metcalf et al. (120)
S Metcalf et al. (119)
NA Cabelli and Heffernan (18)
NA Cabelli and Heffernan (18)
NA Pie) et al. (133) _
contaminated shellfish; NA, not applicable. Dash (_) indicates
 TABLE 4. System design, water parameters, and microbiological results for the depuration of soft clams (Mya arenaria).
Water earameters Dep
Water Temp. Sal. Flow Rate b timeC Contam. level/loo t
Contaminant System" treatment" ("C) (ppl) (ml/min/clam) (h) Before After" Reference
E. coli Ff none 2.5-4-5" 17 120 7xlO3 5xlO 2 Arcisz and Kelly (3)
E. m/i Ff none 13 17 72 1.7XI04 ND Arcisz and Kelly (3)
E. coli Ff none 15 I7 168 7.9x103 ND Arcisz and Kelly (3)
Cs E. coli Ff none 18 17 120 2.2Sx104 1.4xlOJ Arcisz and Kelly (3)
~ E.T. colifonns f Ff UV 2 30 I3 48 3xlOJ 2xlQ2 Cabelli and Heffernan (18)
~ E.T. coliforms Ff UV 8 30 13 48 3xI03 50 Cabelli and Heffernan (18) 0
1'"" tTl
c E.T. coliforms Ff UV 12 30 I3 48 3xIOJ ND Cabelli and Heffernan (18) "tl
c:::
."
~
E.T. coliforms Ff UV 16 30 13 48 3x103 50 Cabelli and Heffernan (18) ~
S. schottmuelleri ND ::!
g Ff none 2.5-4.0 I7 72 40 Arcisz. and Kelly (3) 0
z
S. schottmuelleri Ff none 3.5-4.5 17 96 3xI02 ND Arcisz and Kelly (3)
~ S. schottmuelleri Ff none 13 17 48 3.3xIOJ ND Arcisz. and Kelly (3) >
z
e> 0
~
r)
S. schottmuelleri Ff none 15 17 168 4.6xW ND Arcisz and Kelly (3) ;:0
:j S. schottmuelleri Ff none 18 17 48 3.3x103 ND Arcisz and Kelly (3) tTl
r
C
B.T. colifonns Ff UV 16-20 5 13 48 SxI03 4x103 Cabelli and Heffernan (18) >
:<: -<
< B.T. coliforms Ff UV 16-20 10 13 48 SxI03 5xlOl Cabelli and Heffernan (18) Z
0 a
r E.T. coliforms Ff UV 16-20 20 13 48 SXI03 2xI02 Cabelli and Heffernan (18) til
.... E.T. coli forms Ff UV 16-20 30 I3 48 Sx103 ND Cabelli and Heffernan (18)
:I:
tTl
r
Polio 1.2.3 R UV+FILT 1.5 I/min/bu 144 226-303g ~5g Metcalf et al. (120) r
3: :!l
>
;:0 Polio 1.2.3 R UV+FILT 1.5 l/min/bu 72 ~21.8g ~3g Metcalf et al. (119) VJ
:I:
Ii NDg
::z:: Polio 1.2.3 R UV+FILT 1.5 I/min/bu 24 33.63 Metcalf et a1. (119)
'0
QO
Total coliforms Ff UV 16-20 30 13 48 9xlO3 90 CabeUi and Heffernan (18)
Qo
Fecal coli forms Ff UV 16-20 30 13 24 IxlO J ND Cabelli and Heffernan (18)
E.T. colifonns Ff UV 16-20 30 13 24 2xI0 3 ND Cabe\li and Heffernan (18)
E.T. coliforms R UV 17-19 24-32 ca. I M1 48 1616 g 58 g Piel et al. (133)
E.T. colifonns R UV 17-19 24-32 ca. I M1 48 S93 g 421 Piel et al. (133)
B.T. colifonns R UV 17-19 24-32 ca. 1 M1 48 ~360& .::;16& Piel et al. (133)
>-f!See footnotes to Table 2.
fElevated temperature coliforms
'Summary data expressed as means.

I-.J
N
\0
 KII...t1AKU;)
230
for 24 h showed a reduction in each contaminant of ca. 1.5
log. Little if any further reductions occurred during the next
24 h of depuration (18).
A study was conducted at a commercial depuration plant
in Newburyport, Massachusetts (133). This plant has been
operating since about 1931 (84.133) purifying over 1.5
million bushels of soft shell clams between 1931 and 1974
(133). Using an elevated temperature plate count assay (72).
soft clams taken from restricted growing waters (70-700
coliforms/loo ml seawater) were evaluated at 0,24 and 48 h
(133). Depuration parameters are given in Table 4.
Zero and 24-h elevated temperature coliform counts
were equally reliable indicators of end product bacteriologi-
cal results (133). The study demonstrated with >99% confi-
dence that O-h shellfish with counts < 1,600 and/or 24-h
clams with counts <1,000 would satisfy the National Shell-
fish Sanitation Program criteria (162) (median $50 fecal
coliforms/l00 g of meat with $10% greater than 130/100 g)
after 48 h of depuration. Based on these results, regulatory
officials agreed to allow soft-shell clams with O-h counts of
<1000 or with 24-h counts <500 to be released without testing
at the completion of the 48-h depuration period (133).
The sizes of the depuration tanks were not reported for
some of the above studies. The approximate sea water capaci-
ties for those listed were: 60 L (119.120).200 L (3), and
12,000 L (133).
Hen clam. Venus gallina
A depuration study in Italy involving commercial and
laboratory-scale facilities was undertaken by Marcucci et al.
(112) to evaluate the reduction of several bacterial indicators
from naturally and artificially contaminated hen clams.
Clams were obtained from a variety of sources including
approved shellfish growing waters, heavily polluted canals.
boats arriving at the docks, and from flow-through tanks
spiked with raw sewage or clostridial spores.
After evaluating initial levels of clostridia. enterococci.
total and fecal coliforms, it was concluded that clostridial
levels were sufficiently high to permit its use as an indicator
of depuration process efficiency (112). Clostridial spores
tend to be more resistant to natural inactivation factors in the
marine environment than enterococci and coliforms. Even
though not all clostridia are of fecal origin or of public health
concern, the depuration of ciostridia would indicate the
dynamics of bacterial elimination and may more closely
reflect hazards associated with potential viral contamination
(112).
Casali et al. (25) conducted a series of experiments on the
hen clam to ascertain the depuration rates of clostridia.
Streptococcus faecalis. fecal coliforms and evaluated tem-
perature coliforms. Depuration was conducted in a commer-
cial plant in Italy in which polluted incoming seawater was
first disinfected with chlorine. then passed through sand and
charcoal filters and was subsequently used in flow-through
tanks. Loading densities were set at 16 kg/m2 , flow rate at 9
L/kg clam/h. Salinities ranged between 26 and 33 ppt, pH
levels were 7.1-8.8, chlorine residual ranged from 0.0 to 1.0
ppm at the inlet and 0.0 to 0.5 ppm at the outlet. Mortality
averaged 0.75% in clams without broken shells and generally
higher (0 to 30%) in clams with broken shells. Water tem-
peratures varied with season and ranged between 4 and 25 a C.
Within 24 h, S./aecalis reductions ranged from 47-87%
(mean 58.3%) and clostridial reductions ranged from 31-
94% (mean 81.5 % ), except when the water treatment system
malfunctioned. Fecal coliform and elevated temperature
coliform levels were too low in pre- and post-depurated
shellfish to draw any conclusions (25).
Enterococcus uptake and elimination experiments were
also conducted for hen clams in a laboratory system (25).
Clams artificially contaminated with S. faecalis (Table 5),
depurated 99% of the bacteria within 48 h (Table 6).
The study concluded that hen clams could be commer-
cially depurated of bacterial contaminants provided (a) it was
economically feasible (Venus clams command a low price),
(b) clams were carefully culled to remove dead or physically
damaged shellstock before depuration, (c) clams were dis-
tributed in relatively shallow layers in depuration tanks, (d)
depuration plants had adequate facilities to maintain proper
water disinfection and flow rates, and (e) better indicator
organisms were selected for use in monitoring process
efficiency. Fecal coliforms failed to be an effective indicator
of depuration efficiency in these tests (25).
Manila clam, Tapes japollica (Tapes philippinarum)
Vasconcelos and Ericksen (169) evaluated the natural
uptake of total and fecal coliforms and 20"C plate count
organisms by Manila clams in estuarine waters. Bacterial
increases above ambient water levels were 130 to 270-fold,
33 to 118-fold. and 16 to 30-fold, respectively.
Early studies, by Presnell et al. (137) involved the
accumulation and elimination of E. coli from Manila clams
(Tables 5 and 6). Accumulation averaged 25-fold over the
water levels in the fall and IS-fold in the winter. Variations
in seasonal uptake appeared to be associated with spawning
more than t~mperature or salinity changes. Depuration was
inconsistent for both seasons. Initial E. coli reductions were
rapid (1-2 logs in the first 24 h). Generally, E. coli decreased
from 1Q4-1OS/100 g to negligible levels within 96 h (137).
Hoff and Becker (81) evaluated the bioaccumulation and
depuration of polio 1 in Manila clams (Tables 5 and 6).
Poliovirus was continuously pumped into the tank to provide
200-300 polio/ml of seawater. Uptake was for 24 hand
depuration was for .$96 h. Bioaccumulation of crude (cell
culture-associated) poliovirus was 180x the level of virus in
the water. Filtered viruses (0.45-llm membrane) were con-
centrated only 3.6-fold. Clams containing I ()3 PFU of filtered
virus/g did not depurate to nondetectable levels (<5/g) until
96 h. Counts at 48 and 72 h were ca. 10 PFU/g. Crude
poliovirus at an initial concentration of ca. 104•3 PFU/g
depurated only to 102 PFU/g after 96 h.
Vasconcelos (168) compared the effects of flow rates on
Manila clam depuration. Comparable reductions in total and
fecal coliforms were achieved with flow rates ranging from
1 to 30 mlfmin/clam. Reductions of ca. 95% were obtained
after 48 h. Depletion of dissolved oxygen levels in process
water was observed in tanks receiving I and 10 ml/min/Clllm .
 DEPURATION AND RELAYING SHELLASH
TABLE 5. Microbial uptake parameters for (our shellfish species"·
Exposure
Species/
Contaminant Nat/Art Period
Hen clam
(Venus gallina)
S.faecalis A 9h
. Manila clam
(Tapesjaponica)
E. coli A 72h
Polio I A 24 h
Native littleneck clam
(Protothaca staminea)
E. coli A 72h
Olympia oyster
(Ostrea lurida)
E. coli A 24 h
Polio I A 24 h
Polio I A 48 h
'Abbreviation: A, artificially contaminated shellfish. Dash (-) indicates information was not provided.
These clams demonstrated siphon hyperextension and slow
retraction of siphons when touched. Clams receiving 20 and
30 ml/min/clam appeared normal and siphons retracted
rapidly.
Native littleneck clams, Protothaca staminea
The bioaccumulation and depuration of E. coli from
native littleneck clam was studied by Presnell et al. (137) and
Beck et al. (7). E. coli concentrations averaged 12 to 13-fold
over the water levels during the fall and 8 to 9.5-fold during
winter experiments (7.137). A comparison of E. coli uptake
in native littleneck clams and Manila clams (Tapes japonica)
showed considerably lower rates of uptake but more consis-
tent depuration by native littleneck clams (137). E. coli
levels 2: 5 X 103/100 g effectively depurated within 48 h
during summer and fall months (temperatures, 12.2-21.O"C;
salinities, 26.4-29.4 ppt), but required 96 h to depurate to 50
E. coli/100 g during the winter (4.4-8.9°C, 20.0-28.8 ppt)
(7). This confirmed the earlier work by Presnell et al. (137)
where artificially contaminated clams (Table 5) depurated
from initial E. coli counts as high as 5 x \04/100 g to nonde-
tectable levels within 48 h during the fall, but required >96
h to depurate during most of the winter samplings (Table 6).
Live clams, Villorita sp.
An innovative study in India by Balachandran and
SUrendran (5) involved the depuration of Villor-ita clams in
both chlorinated and non-chlorinated natural lake waters
. (salinity= 10.3 ppO, potable waters, and \0.3 ppt sodium
chloride solutions. A wide range of bacterial and some
proximate analyses were performed on the pre- and post-
. d~~urated clams. Depuration was conducted for 18 h without
diSInfection of the water except for the initial chlorination to
5 ppm in Some of the waters. It was implied that depuration
JOURNAL OF FOOD PROTECTION, VOL. 51. MARCH 1988
231
Levell Static/
100 ml flow-thru Reference
3-5x102 Ff Casali et al. (25)
Ff Presnell et al. (137)
2-3xIO'I Ff Hoff and Becker (81 )
Ff Presnell et al. (137-)
lxIOJ Ff Hoff and Becker (81 )
2-3xlO4 Ff Hoff and Becker (81 )
1.9x106 S DiGirolamo et al. (38)
was conducted in a static system.
Total plate counts, total coliforms and fecal streptococci
were reduced about one log from initial counts of 5.8 x 106/
g, 3.8 X 1Q4/g and 5.1 x 1Q4/g, respectively, regardless of the
type of water used or the presence of chlorine in the waters.
E. coli were reduced from 220/g to ~ 12/g for all water
treatments. In addition to good bacterial reduction in the
clams, depuration also removed sand and gritty matter (5).
Olympia oysters, Ostrea lurida
Beck et al. (7) determined that E. coli uptake in Olympia
oysters fluctuated with season. Ratios of oyster to water
MPNs were 8.1, 9.1 and 19.6 during winter, spring and fall,
respectively, in laboratory-contaminated oysters.
Temperatures 2:1 5°C and salinities> 25 ppt were associated
with higher levels of uptake. In a flow-through system. these
oysters depurated 5 X \OJ E. coli/lOO g to <50/100 g within
48 h during the spring and fall seasons (temperatures. 8.3-
19.O"C; salinities. 23.3-29.1 ppt), but did not depurate to
<100 E. coli/IOO g within 72 h during winter months (tem-
peratures, 7.2-9.O"C; salinities. 14.6-29.4 ppt).
Hoff and Becker (81 J simultaneously dosed Olympia
oysters with polio I (200-300 PFU/ml of seawater) and E.
coli (MPN of 1 x 103/100 ml of seawater (Table 5). Polio
concentrations in the oysters increased from 10 to 54-fold for
crude virus, 1 to 2.2-fold for centrifuged (essentially noncell-
associated virus). and 0.4 to 1.4-fold for filtered virus. E.
coli concentrations ranged from 10 to 43-fold. Oysters
subjected to crude preparations of poliovirus initially accu-
mulated virus to much higher levels (ca. WI/g) than oysters
subjected to filtered poliovirus (ca. \02.5/g). Depuration of
filtered virus to nondetectable levels required ~96 h. but
crude virus were detectable throughout the 96 h depuration
period (81). Crude virus more likely simulate those found
 N
W
N

TABLE 6. System design. water parameters. and microbiological results for the depuration offour shellfish species.
Water parameters Dep.
Species! Water Temp. Sal. Row rate b timeC Contam. level/loo gd
Contaminant System" treatment" ("C) (ppt) (mt/min/clam) (h) Before After" Reference
Hen clam
(Venus gallina)
S. faecalis Ff CI 22-23 20 L/hJkg 48 1.05xl()6 IxlO4 Casali et al. (25)
S. faecalis Ff CI 22-23 20 L/hIkg 72 1.05xlQli 6xlO2 Casali et at. (25)
Manila clam
(Tapesjaponica) ~
(')
E. coli Ff UV 160 72 Ixl04 ~lxl02 Presnell et al. (137) ::c
Polio I Fr UV 15 L\Ih 96 3xt04/g 6xW/g Hoff and Becker (8/) >
Native littleneck clam ~
en
(Protothaca staminea)
E. coli UV Fall 160 48 5xJ04 ND Presnell et at. (137)
E. coli UV Winter 160 96 2x10 3 ~lxIOJ Presnell et at. (137)
.
:I
Olympia oyster
(Ostrea lurida)
)
t: E. coli Ff UV 15 Mt 48 4xJ04 ca.2xlO1 Hoff and Becker (81)
0 Polio I Fr UV 15Mt 48 lxl04/g 5/g Hoff and Becker (81)
'"'" Polio 1 S ALT 0 120 I.1XI04/g lxlOJ/g DiGirolamo et at. (38)
Polio I S FILT 0.35 Mt 72 3.6xlQ3/g 2.IXI02/g DiGirolamo et al. (38)
a-dSee footnotes to Table 2.
 DEPURATION AND RELAYING SHELLFISH 233

under natural conditions.
DiGirolamo et a!. (38) also studied polio I uptake and
depuration in Olympia oysters. The oysters accumulated 8.6
X ID-' PFU/g after 12 h exposure to seawater containing 1.9
X I ()4 PFU/ml of polio (Table 5). Extending the exposure to
48 h had no appreciable effect on the numbers of viruses
accumulated. Virus accumulation occurred primarily in the
digestive area (ca. 71 % or 1.4 x 1()4 PFU/g) at 12, 24 and 48
h of uptake. Only 20% was recovered from the mantle, gills
and palps. Two percent was found in the mantle cavity fluids
at 12 h and this increased to 6.2% (1.3 x 1(3) at 48 h. Some
general diffusion of virus from the digestive tract into the
body tissues was given as a possible explanation for this rise.
Poliovirus depuration in a stationary (static) system was
gradual with 16% (1.6 X I Q3 PFU/g) of the viruses remaining
in the oyster after 120 h (38). Virus reductions in a flow-
through system showed 24% of the total viruses accumulated
by the oyster still present after 24 h of depuration (38). By 72
h, less than I % of the total accumulated viruses remained in
the oysters.
Eastern oysters. Crassostrea virginica
A number of depuration studies have been undertaken to
determine the ability of Eastem oysters to both bioaccumu-
late and eliminate various microorganisms (42. 91.
104.116.122). Mitchell et a!. (122) observed up to 40-fold
concentrations of E. coli in oysters. Janssen (91) considered
the uptake of S. typhimurium and Franciscella tularensis by
oysters. Water containing 2 X lOs bacterialml yielded an
average of2.8 x 1()4 and 7.7 X 102 organisms/oyster, respec-
tively, after 48 h.
Metcalf and Stiles (117) evaluated the uptake and tissue
distribution of polio 1 and Coxsackie B3 viruses in oysters.
Uptake was performed in a static system. Viruses accumu-
lated in the digestive gland and, to a lesser extent, in the
stomach, mouth, esophagus and gills. These findings are
supported by more recent work where oysters, exposed to
poliovirus in a static system, concentrated more viruses in the
digestive tract than in the gills and mantle tissue, and these in
tum contained more than the mussel tissue (116).
Mitchell et al. (122) frrst showed that Eastem oysters can
concentrate polio up to 60x the level in the surrounding water
using a flow-through, virus uptake system (Table 7). This
flow-through system was shown superiorto the static systems
used in earlier uptake studies (71.117). more likely typifying
natural conditions.
Liu ( 104) reported on viral uptake studies using oysters
simultaneously contaminated with polio 1 and echo 1 viruses.
The viruses were accumulated at different rates. Virus uptake
appeared more efficient in oysters than in hard-shell clams,
which, was probably due to higher pumping rates in oysters
(104).
Oysters accumulated 25.6x more feces-associated virus
than stock virus, virtually all of which appeared in the
hepatopancreas and alimentary tract (42). Particle- or feces-
associated poliovirus were concentrated more efficiently by
oysters than laboratory-grown or purified virus preparations
(42.116).
The influence of temperature on oyster depuration was
evaluated in several studies (70,116.136.151) (Table 8).
Presnell and coworkers (136) concluded that oysters accli-
mated to warm waters (26.7-29.6°C) and then depurated in
warm (27.5°C) or cool (ca. 19.5°C) waters depurated at
different rates. Initial counts of 160,000 total and 790 fecal
coli forms were reduced >97.7% after 4 h of depuration at
27.5°C. When depurated in cooler waters, 4,900 total and
fecal coliforms required 24 h for reductions of 90 and 97%,
respectively.
Oysters were also acclimated to cool water (16.3-18 .8°C)
and then depurated in cool and warm (24.4-28.7°C) water
( 136). Even with salinities varying between 17 and 27 ppt,
total and fecal coliforms consistently depurated at a faster rate

TABLE 7. Microbial uptake parameters for Eastern oyster (Crassostrea virginica) depuration studies.
Exposure'
LeveV Staticl
Contaminant Nat/Art Period looml f1ow-thru Reference
Total colifonns N NA NA NA Presnell et at. (136)
Fecal colifonns N NA NA NA Presnell et at. (136)
Polio A 24 h 2.2-5.9xlQ4 FT Hamblet et at. (68)
Fecal colifonns N NA NA NA Haven et at. (70)
Total colifonns N NA NA NA Haven et at. (70)
S. typhimurium A 48 I X I OS Janssen (91)
S. typhimllrillm A 24 h 2x 107 Janssen (91)
Sh. flexlleri A 24 h 2x I 07 . Janssen (91)
F. tularensis A 24 h 2x 107 Janssen (91)
E. coli A 24 h Ixl()3 FT Mitchell et at. (122)
POlio I A 24 h 3xlO4 FT Micthell et at. (1f2)
Polio 1 A 24 h 1X I OS FT Mitchell et at. (122)
V. vulnificlIs A 24 h Ix 106 S Kelly and Dinuzzo (97)
.~lIlllificlts A 24 h bIOS S Kelly and Dinuzzo (97)
,"~Abbreviations: A, artificially contaminated shellfish; N, naturally contaminated shellfish; NA, not applicable. Dash (-) indicates
.r 1nfonnation was not provided,

JOURNAL OF FOOD PROTECTION. VOL 51, MARCH 1988

 234 RICHARDS
at the warm temperatures. Over 98% of the total and fecal
coliforms depurated in warm water within 24 h compared to
86.0 and 77.4%, respectively, in cold water.
In a study by Haven et al. (69.70) using a flow-through,
UV treatment system, total coliform depuration experiments
gave erratic results for temperatures ranging from 9.0 to
27.6°C, however fecal coliform MPN data showed more
regularity. At 14-29°C, fecal coliforms depurated from 104/
100 g to <50/100 gin 24 h.
The effects of temperatures on poliovirus depuration
were evaluated by Meinhold and Sobsey (116). Poliovirus
was not detected in oysters after depuration at 28°C for 3 d,
but was present in oysters depurated at 6°C for 5 d. The initial
concentration of viruses in these oysters was not given.
Another study (118) determined that enteroviruses did not
depurate from oysters which were dormant (at temperatures
from I-4°C). As temperatures increased, feeding also in-
creased with concomitant acceleration in depuration rates.
Recent preliminary studies by Sobsey et al. (151) com-
pared the reduction rates of laboratory-acquired polio type I
and hepatitis A viruses at 12, 17 and 23°C. Virus uptake tanks
containing 7-8 L of artificial seawater were inoculated with
polio and hepatitis A viruses to initial concentrations of 103
to 104 infectious particles per ml. Fifty oysters were placed
in the aerated water for 6-12 h at 23°C, following which, they
were depurated in tanks also containing 7-8 L of artificial
seawater. Poliovirus was rapidly reduced (>98%) within 3 d
at all temperatures. There was little or no active elimination
of hepatitis A virus at any temperature during the 5-d
depuration period.
Galtsoff (55) indicated that oyster filtration and shell
opening were reduced when oysters were placed in seawater
which deviated in salinity by 10% from the salinity at the
harvest site. Some reports on the effects of salinity on oyster
depuration conflict. Presnell et al. (136) showed that salini-
ties radically affected bacterial depuration rates, whereas
Haven et al. (70) concluded that salinities did not have a
differential effect on fecal coliform reductions. Comparison
of the salinity levels shows that the lower levels of salinity
evaluated by Presnell and coworkers (136) were not evalu-
ated in the study by Haven et al. (70).
Four salinity ranges were evaluated by Presnellet al.
(136): low (6.3-6.9 ppt), medium-low (9.3-11.8 ppt), me-
dium-high (16.6-18.0 ppt), and high (24.8-25.5 ppt) (Table
8). Increasing salinities resulted in progressive increases in
percent coliform reductions. Fecal coliforms depurated for
48 h gave the following reductions: low, 40.5%; medium-
low, 91.4%; medium-high, 96.7%; and high, 99.8%. Total
coliform reductions were also greater at the higher salinities.
Haven et al. (70) found that at salinities from 15.7 to 20.5 ppt
and temperatures from 14.7 to 25.8°C, initial fecal coliforms
ranging between 1,600 and 79,000/1 00 g generally depurated
to <50/100 g in 24-48 h.
Differenl;es in oyster depuration may be influenced by
the geographic area from which the oysters are obtained.
Oysters grow and reproduce within salinity ranges of <5 to
>32 ppt in the Chesapeake Bay area (69,70.125). so it is
possible that lower salinities are acceptable for oyster depu-
ration in the Chesapeake area. A minimum salinity for
depurating Gulf Coast oysters was set in a study by Huntley
and Hammerstrom (85) at 10 ppt although lower salinities are
frequently encountered in the Gulf. Depuration at this low
salinity was only marginally effective in a study by Presnell
et al. (136). Potential genetic differences between Crasso-
strea l'irginica obtained from the Chesapeake Bay and Gulf
Coast or shellfish ad apt ion to different geographic locations
could play an important role in the elimination of microor-
ganisms.
Only one study was identified on the depuration of
enteric viruses under different salinity regimes. Sobsey et al.
( 151) performed a laboratory uptake and depuration study
using polio type I and hepatitis A virus in 7-8 L tanks, as
discussed above. At 8, 18 and 28 ppt salinity and at 23°C,
<3% of the initial polioviruses remained after 2-3 d of
depuration. Hepatitis A virus was not extensively reduced in
oysters depurated at 8 or 18 ppt; 10-41 % of the hepatitis
remained after 5 d. Virus reductions were rapid and extensive
at 28 ppt and 23"C with ~5% of the viruses remaining within
2-3 d and only I % after 5 d.
Flow rates of 0.5, 1,3 and 5 L/oyster/h (8.3,16.7,50.0
and 83.3 ml/min/oyster) provided >98% reduction of total
and fecal coliforms, at ca. 28.5°C and ca. 20 ppt salinity, with
initial counts of 45 ,000 and 30,000/100 g, respectively (136)
(Table 8). This range of flow rates encompassed the recom-
mended flow rate of I gal/min/bu (3.8 L/min/bu or ca. I L/
oyster/h) as recommended by Furfari (52). Flow rates be-
tween I and 8 gal/min/bu (3.8-30.3 L/min/bu) provided
adequate oxygenation for depuration (70). No information
could be found on the influence of flow rates on the elimina-
tion of viruses from the Eastern oyster.
The total amount of water pumped by oysters during a
24-h period can range from 9 to 239 L (55). A 3-4 in. (7.6 to
10.2 cm) oyster may pump 34 L of seawater/h ( 109). Accord-
ing to Haven et al. (70) fecal coliform reductions did not
appear to correlate with pumping rate or waste product
excretion (biodeposits); oysters depurated equally well at
high and low pumping rates and with various levels of
excretion activity. Mean pumping rates for pools of 8 oysters
ranged from 1.4 to 10.5 L/h. At these pumping rates, total
colifonlls (984 and 3.055/1 00 g) and fecal coliforms (224 and
303/100 g) generally depurated to <50/100 g within 48 h.
The rate of biodeposition, excretion of feces and pseu-
dofeces, did not affect total or fecal col iform depuration (70).
Initial fecal coliform counts ~ 1,900/1 00 g depurated to ~50/
100 g within 24 h in oysters which produced high, low and
very low quantities of biodeposits as well as in those oysters
. producing no biodeposits. Similarly, total coliform levels as
high as 61,000/1 00 g usually depurated to ~230/1 00 g in 24
h regardless of the quantity of biodeposits produced. The
conclusions drawn in this study were that (a) pumping and
biodeposition rates are not good indices for measuring depu-
ration efficiencies and (b) oysters do not have to actively
pump or produce biodeposits to effectively depurate coli-
form organisms (70). "Dry controls" would be useful in the~e
types of studies to determine the fate of the contaminants In
closed, inactive shellfish during dry storage.
 TABLE 8. System design, water parameters, and microbiological results.for the depuration o.f Eastem oysters (Crassostrea virginica).
Water parameters
Dep.
Water Temp. Sal. Flow rate b timec Contam. level/I 00 gd
Contaminant System" treatment" ("C) (ppt) (ml/min/oyster) (h) Before After" Reference
Coliforms Fr UV 18.8-19.9" 20-22 66.7 48 4.9xW 3.3xl()2 Presnell et al. (136)
Coliforms Fr UV 26.9-27.7 20-22 66.7 48 1.6x I ()5 2.3xl()2 Presnell et al. (136)
Coliforms R UV 6.3-6.9 93 48 3.5x104 7.0xl03 Presnell et al. (136)
Coliforms R UV 9.3-U.8 93 48 1.6x 1()5 7.9x103 Presnell et al. (136)
Coliforms R UV 16.6-18.0 93 48 3.3x105 3.4x I ()2 Presnell et al. (136)
Coliforms R UV 24.8-25.5 93 48 3.3x104 45 Presnell et al. (136)
Fecal coliforms R UV 6.3-6.9 93 48 7.9x103 4.6x 10 3 Presnell et al. (136)
Fecal coliforms R UV 9.3-11.8 93 48 9.2x104 7 .9xl 03 Presnell et al. (136)
Ci Fecal coliforms R UV 16.6-18.0 93 48 3.3x103 1.1xl()2 Presnell et al. (136)
~ Fecal colifonns R UV 24.8-25.5 93 48 1.0x 104 20 Presnell et al. (/36)
~ Coli forms Fr UV 28.2-28.8 19.6-20.4 8.3 24 4.5xlQ4 45 Presnell et al. (J 36) o
~

C
..,.,
Coliforms Fr UV 28.2-28.8 19.6-20.4 16.7 24 4.5x104 1.3x 102 Presnell et al. (J 36) tg
Coliforms Fr UV 4.5x104 4.9x 1()2 c:
28.2-28.8 19.6-20.4 50.0 24 Presnell et al. (136) ::0
~ Coliforms Fr UV 28.2-28.8 19.6-20.4 83.3 24 4.5x104 78 Presnell et al. (136)
:>
...,
g Fecal colifonns Fr UV 28.2-28.8 19.6-20.4 8.3 24 3xlO4 NO Presnell et al. (136)
(5
;g z
Fecal coliforms Fr UV 28.2-28.8 19.6-20.4 16.7 24 3x104 45 Presnell et al. (J 36) :>
C z
t;1 Fecal colifonns Fr UV 28.2-28.8 19.6-20.4 50.0 24 3xlQ4 45 Presnell et al. (136) o
3xlO4
(j

~-<
::::! Fecal colifonns Fr UV 28.2-28.8 19.6-20.4 83.3 24 20 Presnell et al. (J 36)
3xlOJ
~ E. colig
E. colih
Fr UV+FlLT 20-21 25.6-26.0 50 28 9x 1()6 Presnell et al. (J 36)

o<
Fr UV 20-21 25.6-26.0 50 28 9xl()6 3xlOJ Presnell et al. (J 36) zCl
E. coil; Ff UV 19-24 20-24 50 24 IxlO7 lxl()5 Presnell et al. (136)
r- en
Ut E. colii Fr UV 19-24 20-24 50 24 lxlO7 8x104 Presnell et al. (J 36) ::c
3:
Poliol Ff UV+FlLT 25.3-27.3 17.9-22.0 48 4x103/g NO/g Hamblet et al. (68) ~:!l
:>
::0
Polio' Ff UV+FlLT 25.3-27.3 17.9-22.0 48 4x10 3/g ND/g Hamblet et al. (68) en
n Coliforms Ff UV ~26 I I. 7 I/min/bu 24 ~2. Ixl0 3 50 Haven et al. (70) ::c
:c
E. coli Ff UV 19.4-24.6 8.5-23.0 33.3 48 lxlO4 NO Mitchell et al. (122)
~
00 S. typhimurium R UV+CHAR ambient 15 21d 3.5x10Jf 32f Janssen (91)
S. typhimurium R UV+CHAR ambient 15 28 d 2xlQ4f 1.2x I 04f Janssen (91)
S . .flexneri R UV+CHAR ambient 15 24d 4.9x103f NOf Janssen (91)
F. wlarellsis R UV+CHAR ambient 15 9d 7.7x103f 3xlOJf Janssen (91)
F. tillarensis R UV+CHAR ambient 15 18 d 7.7x103f NOf Janssen (91)
V. \'ulnificus S none 14 7d l4/g 3/g Kelly and Oinuzzo (97)
V. l'1l1nijiclls s none 14 16d 14/g NO Kelly and Dinuzzo (97)
V. l'UlllijiCIlS s none 14 7d 28/g 7/g Kelly and Oinuzzo (97)
V. vlll"ificlls s none 14 16d 28/g O.I/g Kelly an d Oinuzzo (97)
Polio I Ff UV 20.7-24.6 16.5-19.5 33.3 72 5xW/g NO Mitchell et al. (122)
Polio I Fr UV 19.4-23.2 8.5-23.0 33.3 72 5xlQ4/g 3/g Mitchell et al. (122)
·'"See footnotes to Table 2.
N
fCount/oyster. VJ
U\
g-'Turbidities of process water: 88.5-9.0 Jackson turbidity units (JTU), hI5.8-22.8 JTU, iI9.4-24.1 JTU, i63.9-72.4 JTU, k8-2l ppm, solids, '54-80 ppm solids.
 236 RICHARDS
Seemingly contradictory results were reported in this
same study when it was shown that feces and pseudofeces
collected from oysters depurating for 4 h contained ex-
tremely high levels of total and fecal coliforms (2.5 x I ()6 total
and 1.5 x lOS fecal coliforms/l 00 g of feces and 6.7 x 1()4 total
and 5.6 x 103 fecal coliforms/lOO g of pseudofeces) (70).
Subsequent collections at 24 and 48 h showed huge count re-
ductions as the total contaminant loads in the oysters dimin-
ished. It was not possible to compare counts in oyster meats
with counts from feces or pseudofeces since the number of
oysters from which 100 g of the biodeposits were obtained
was not given. But, there was evidence that coliforms did not
multiply in the biodeposits or, if they did, the death rate
exceeded the rate of microbial replication (70).
Sobsey et al. (151) evaluated the effects of an algal food
source on polio and hepatitis A virus depuration. Oysters fed
the marine alga, lsochlysis galbana, consumed an appre-
ciable amount of alga, but did not depurate more readily than
nonfed oysters. This supports the findings of Haven et al. (70)
who, as previously reported, found that the rate ofbiodeposi-
tion had no effect on oyster depuration. Indeed, the consump-
tion of alga should lead to an increase in the rate ofbiodeposi-
tion, but apparently without an increase in the rate of
depuration. These findings suggest that many of the bacterial
and viral contaminants may not be associated with the
alimentary tract.
Information on the effects of pumping rates on virus
depuration is not available. However, pumping rates may
have influenced one study by Akin et al. (1) which compared
the accumulation and depuration of polio 1 by individual
oysters. Virus accumulation for 4 h was highly variable
among individual oysters (ranging from 5,400 to 32,000
PFU/ml homogenate in one trial and 34 to 22,000 PFU/ml in
another). This variability implied that some oysters pumped
better than others. The variability was reduced as the period
of bioaccumulation increased. Virus depuration was also
highly variable. One experiment showed 190 to 4,200 PFU/
ml homogenate remaining after depuration for 4 h while
another had 97 to 10,200 PFU remaining after 4 h.
Flow and pumping rates can affect the availability of
dissolved oxygen in depuration process water. At 26°C,
oysters effectively depurated 2,100 fecal coliforms!100 g to
~50!1 00 g in 24 h at dissolved oxygen levels > 1.8 mg/L
(69,70). At <1.8 mg/L depuration progressed more slowly.
Depuration was effective in reducing 2,100 fecal coliforms/
100 g to <50/100 gin 72 h with dissolved oxygen levels as
low as 0.6 mg/L (69,70). Depuration was more effective at
high dissolved oxygen levels (7.5-9.5 mg/L) (70). Oxygen
uptake by Eastern oysters has been measured as 2.8 mg/h/4
in. (10.2 cm) oyster at 24-25°C (55). Haven et al. (70)
recommended that dissolved oxygen levels be maintained at
2 mg/L throughout the depuration cycle. Supersaturation of
process water by over-aeration and drops in temperature can
cause oyster mortality (125).
Turbidity had little or no effect on bacterial depuration
(68.70.77.136). provided it was not so' high that water
disinfection processes were impaired. Depuration at mean
turbidities of 8.8, 19.3, 22.6 and 69.7 JTU provided similar
E. coli reductions in a flow-through system (136) (Table 8).
Total and fecal colifonns depurated well with total solids
varying between I and 100 mg/L in the process water (70).
Hamblet et al. (68) and Hill et al. (77) determined the
effects of seawater turbidity on poliovirus uptake and elimi-
nation. In 24 h oysters accumulated 3x more virus in low
turbidity seawater (16-24 mg/L) than they did in high turbid-
ity seawater (54-77 mg/L) with accumulation factors of 18x
and 5x. respectively. Turbidities between 8 and 80 mg/L did
not influence virus elimination (68,77). Low-level polio-
virus contamination depurated to nondetectable levels within
48 h when oysters were exposed to high or low turbidity
seawater in a flow-through system (68) (Table 8).
An interesting study by Haven et al. (69,70) was con-
ducted to determine the effects of disease-associated physio-
logical stress on oyster depuration. Oysters were infected
naturally with various levels of Haplosporidium nelsoni,
formerly called Minchinia nelsoni, (causative agent of the
shellfish disease known as MSX) and both naturally and
artificially with Perkinsus marinum (causative agent of an-
other shellfish disease called dermo). Typically, H. nelson;
causes physiological stress in oysters reflected by lower
weight meats and occasional death. Perkinsus marinum
causes necrosis of epithelial and connective tissue in heavily
infected oysters. Both conditions were expected to suppress
bacterial depuration rates. Surprisingly, total and fecal coli-
form reductions occurred at relatively the same rates in
un infected controls, naturally- and artificially-infected ani-
mals, and in oysters infected simultaneously with both
pathogens. The severity of infection did not appear to alter
the depuration of total or fecal coliforms (69,70).
Oyster size (2-5 in., 5.1-12.7 cm) and food supply
(chlorophyll levels in water) had no demonstrable affect on
fecal coliform depuration from oysters (70,129). Oysters
obtained from four different river systems of the Chesapeake
Bay also did not show any differences in depuration rates
(70).
Perkins et al. (129) found that C. l'irginica could usually
be depurated within 48 h to an MPN of <18 fecal coliformsl
100 g with <10% of the samples exceeding 78 fecal coli- ,
forms/IOO g. This success was based on the depuration of
marginally contaminated oysters in an efficient system. Such
levels are comparable to the newly proposed National Shell-
fish Sanitation Program guidelines (164) for end product
criteria which indicate a geometric mean of 20 fecal coli-
forms/lOO g with not more than 10% exceeding 70/100 g.
Other studies on bacterial depuration (Table 8) showed
acceptable reductions in coliforms and E. coli provided
initial (0 h) counts were not excessive. S. typhimur;um,
Slzigellaj7exneri and F. tularcnsis required prolonged depu-
ration to effectively reduce their levels (91) (Table 8).
Vibrio l'Ulni/icus depurated slowly from oysters with
complete elimination after 16 d (97) (Table 8). These find-
ings suggest that V. l'ulni/icus may be present in oysters as a
consequence of uptake and accumulation during nonn al
filtration processes rather than by multiplication within the
oyster. Barrow and Miller (6), however. found that vibrios
persisted throughout commercial depuration of oysters and
 DEPURATION AND RELAYING SHELLFISH 237

speculated that replication of vibrios greatly exceeded their
rate of UV light-inactivation. Steslow et al. (157) indicated
that oysters artificia\1y infected with fecal coliforms, V.
VU 111 ificus , and non-Ol V. cholerae depurated more rapidly
than environmentally contaminated oysters.
Akin et al ( I ) reported that polio 1 counts were signifi-
cantly reduced after depuration for 24 h. Liu (104) indicated
that poliovirus (70-2, III PFU/g) depurated to negligible
levels in 72 h, but that echovirus (40-2,740 PFU/g) did not
depurate completely with 6-318 PFU/g remaining after 72 h.
Depuration experiments were, presumably, performed under
favorable conditions. Mitchell et al. (122) defined the para-
meters of their depuration study and showed 3-4 log reduc-
tions in polio I counts within 72 h (Table 8).
Durgin et a!. (42) performed depuration experiments in
tanks containing 60 L of recirculated seawater which was
UV -treated and charcoal filtered. Oysters were capable of
reducing feces-associated poliovirus in this laboratory-scale
system, from 300 PFU/oyster to nondetectable levels within
48 h. This study also evaluated commercial depuration
processes in a flow-through (1.6-5.0 gal/min/bu; 6.1-18.9 L/
min/bu), 1,324-L tank in which in flowing seawater was UV-
treated. Ten bushels of oysters could be depurated in each
batch. Commercial depuration practices were effective in
eliminating bioaccumulated polioviruses within 48 h in
oysters as well as in hard- and soft-shell clams.
Tank sizes varied among the depuration studies. The
approximate water capacities of the tanks were: 7-8 L (151),
10 L (97), 33 L (70), 50 L (91), 260 L (136), 738 L (122),
and 1324 L (42). The depuration of oysters in different size
and shape tanks containing from 367 to 1730 L of seawater
was also monitored by Haven et al. (69,70). Depuration of
fecal coliforms was comparable regardless of tank dimen-
sions or capacities.
Pacific oysters, Crassostreagigas
Kelly (95) showed that the bioaccumulation ofE. coli by
Pacific oysters was low, ranging from no increase to 9-fold
increases over ambient water MPNs. By comparison Kelly
obtained substantially higher uptakes in Olympia oysters
(Ostrea lurida). Beck et al. (7) also identified low bioaccu-
mulation of E. coli by Pacific oysters and higher concentra-
tions in Olympia oysters. During winter, spring and fall
experiments, mean E. coli concentrations were 2.6, 2.2 and
4.2-fold, respectively, for Pacific oysters. The highest oyster
to water ratio in all experiments was 6.1 compared to 37.5 for
Olympia oysters. Vasconcelos and Erickson (169) deter-
mined that the accumulation and elimination of total and
fecal coliforms occurred at a reduced level in Pacific oysters
compared to Manila clams. Total coliform concentrations
were ~37x the level in the water column and 20"C plate count
microorganisms were ~76x ambient water levels.
The reduction of E. coli, S. /aecalis, and C. pelfrillgens
in artificia\1y contaminated oysters depurated for 48 h in a
commercial UV -light treatment system was evaluated by
Madden et al. (110). At IO"C, the initial count of each
bacterium dropped 90% after 16.6,23.6, and 13.6 h, respec-
tively. This signifies the possible resistance of S.jaecalis to
the effects of UV light depuration.
Several studies on the uptake and subsequent depuration
of microorganisms are shown in Tables 9 and 10. One study,
by Hoff and Beck (80), compared E. coli and coliphage
accumulation in artificially contaminated oysters. They
found that neither contaminant was accumulated to any
appreciable level, suggesting that optimal conditions for
uptake were not achieved. Total contaminant levels were, in
all instances, below the levels found in the water. Phage
present in the oyster liquor (~490/ml) and meats (~35/g)
were depurated to nondetectable levels within 26 h (80)
(Table 10).
In another study on coliphage accumulation and depura-
tion in C. gigas, Hoff et a!. (82) allowed 2-3 d for phage
uptake in a 10-1 stationary system. Fresh seawater and phage
were added at 24-h intervals to give 3 X 108 PFU of coliphage/
100 ml of seawater. Phage accumulation was as high in
washed meats and liquor after IS min as it was throughout the
72-h uptake period. No virus concentration occurred. In fact,
shell liquor phage counts, regardless of accumulation time,
averaged 2.8 X 106/ml and meats were consistently lower,
averaging 6.8 x 103/g.
Phage depuration was conducted in a flowing seawater
system (Table 10) for up to 14 d (82). For each trial, 99% of
the phage were eliminated from the meats after depuration
for 24 h. Phage elimination from shellfish liquor also de-
clined rapidly, remaining about the same or only slightly
higher than those of meats. Examination of feces and pseu-
dofeces revealed that most phage were excreted within 24 h,
but some could be detected after 72 h or longer.

TABLE 9. Microbial uptake parameters/or Pacific oyster (Crassostrea gigas) depuration studies.
Exposure'
Level! Static!
COntaminant Nat/Art' Period 100 m 1 flow-thru Reference
COliphage C-2 A 72 h FI' Hoff and Beck (80)
ColiphageC-2 A 48-72h 3x108 S Hoffetal.(82)
Polio 1 A 48 h 1.Sx 106 S DiGirolamo et al. (38)
Cricket paralysis virus A 72 h Ixl()6 S Scotti et al. (148)
Fecal colifonns N 1 wk b NA NA Buisson et al. (16)
~io 1 A 24 h 2 -3x 104 FI' Hoff and Becker (81)
~Abbreviations: A, artificially contaminated shellfish; N, naturally contaminated shellfish; NA, not applicable. Dash (-) indicates
Information was not provided
bOysten; were relayecl from c')ean to contaminated growing areas for 1 wk.

JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988

 N
W
00

TAB LE 10. System design, water parameters, and microbiological results for the depuration of Pacific oysters (Crassostrea gigas).
Water parameters
Dep. Contam. leveVIOO gd
Water Temp. Sal. Flow rateb timec
Contaminant System8 treatment8 ("C) (ppt) (ml/min/oyster) (h) Before Afte~ Reference
Coliphage C-2 Ff none 160 18 35/g ND Hoff and Beck (80)
Coliphage C-2 Ff none 160 48 2x 1()6/g 80/g Hoff et al. (82)
Polio 1 S' none 0 120 2x104 /g 2X\03/g DiGirolamo et al. (38)
Cricket paralysis virus R UV+FILT 2.5L\1h 96 3xlQ4/g 1.5x I OJ!g Scotti et al. (148) C!
Fecal colifonns R UV+FILT 5.'Jf ±20%1 2.5L\1h 72 8xl03 5x)()2 Buisson et al. (16) (')
::c
Fecal colifonns R UV+FILT 10.2 ±20%8 2.5L\1h 72 4xlOJ Ixl()2 Buisson et al. (16) ~
Fecal colifonns R UV+FILT 17.0 ±20%8 2.5L\1h 72 4xlOJ 40 Buisson et al. (16) t:l
til

Fecal coli forms R UV+FILT 20.3 ±20%g 2.5L\1h 72 5xl Q4 60 Buisson et al. (16)
Fecal coli forms R UV+FILT 21.0 ±20%g 2.5L\1h 24 8x I ()2 20 Buisson et al. (16)
Fecal coliforms R UV+FILT 21.0 ±20%g 2.5L\1h 72 2xlOJ 30 Buisson et al. (16)
Polio I Ff UV 66.7 96 IxlQ4/g ND Hoff and Becker (81 )
a-dSee footnotes to Table 2.
'Water changed at 12 h intervals.
fN umbers in bold type indicate specific parameters under evaluation.
g±20% of harvest site salinity.
 DEPURA nON AND RELAYING SHELLFISH
Uptake and distribution of viruses by C. gigas have been
studied by several investigators (38,81.148). Hoff and
Becker (81) evaluated polio I uptake by continually adding
virus to give a concentration of 200-300 PFU/ml of seawater
in a flow-through system (Table 9). Crude poliovirus prepa-
rations were concentrated 38 to 39-fold while filtered virus
preparations were concentrated only 1.0 to 1.4-fold.
More recently, DiGirolamo et al. (38) conducted polio-
virus accumulation and depuration studies on Pacific and
Olympia oysters. Uptake was achieved in ~48 h in 3.5 L of
nonflowing but aerated seawater (I3°C, 28 ppt salinity)
which contained 1.9 X 10" PFU of polio/ml. Pacific oysters
accumulated 4.6 X \03 virus/g after 12 hand 1 x 1000/g after
48 h. Most of the virus was found in the digestive area and
feces after 12, 24 and 48 h of uptake. Mantle, gills, palp, and
mantle cavity fl uids were pooled. The pools contained ca. 1%
of the virus at 24 hand 16% at 48 h. The body contained the
least virus at 12 and 24 h (ca. 0.5%), but rose to 16% at 48
h. The depuration phase of these experiments on C. gigas
were performed in a closed system in which seawater was
replaced at 12-h intervals. Only one-log reductions were
observed in this essentially static system even after 120 h
(Table 10).
Accumulation and elimination of a small virus, resem-
bling polio and hepatitis A viruses, were studied by Scotti et
al. (148) using New Zealand-grown Pacific oysters. The
cricket paralysis virus, a picornavirus of insects, was used in
this study. Indirect uptake experiments were performed with
32P-labeled viruses, which were added to lO-L tanks of UV
sterilized seawater at ambient temperatures for 24 h. Ten
oysters were allowed to bioaccumulate the viruses and
seawater was sampled periodically to determine reductions in
radioactivity. Maximum virus uptake was achieved in 12 h.
About 50% of the viruses were accumulated in 24 h. A
substantial amount of virus apparently adsorbed to the oyster
shells or the walls of the containers (even though containers
were treated to reduce virus adsorption). Similar uptake ex-
periments with non-labeled virus showed rapid uptake in C.
gigas tissues. Virus bioaccumulation averaged I X 10" and 0.5
X 1000/g of oyster after 6 and 16 h, respectively. The weight
or size of the oysters did not influence virus uptake; large
oysters (26-44 g) accumulated about the same level of virus
as small oysters (9.3-23 g).
Scotti et al. (148) exposed oysters to cricket paralysis
virus for 3 d and then depurated them in a 40-L laboratory
system as shown in Tables 9 and 10. When individual oysters
were assayed after depuration for 10 d, they showed tremen-
dous virus variability ranging from essentially no reduction
(ca. 1000/g remaining) to 2-log reductions.
Another study was performed in a commercial depura-
tion plant (148). Oysters exposed to both E. coli and cricket
paralysis virus were pooled and assayed after depuration for
$,9 d. Depuration parameters were not given, but E. coli
levels were reduced over 3 logs in 3 d. No appreciable
r~ductions were observed in virus levels even after depura-
tion for 9 d. It was concluded that bacterial indicators should
not be used as indicators of viral contamination.
Seasonal effects on depuration rates of artificially con-
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
239
taminated oysters were evaluated by Beck et al. (7). E. coli
levels around 3 x 103/100 g depurated to nondetectable levels
in a flow-through system within 48 h during the spring (12.0-
19.O"C, 23.3-29.0 ppt). During the fall (8.3-17.2°C, 25.4-
29.1 ppt) and winter (7.2-9.O"C, 14.6-29.4 ppt), similar E.
coli levels depurated to ~501100 g in nand 96 h, respec-
tively.
Buisson et al. ( 16) performed depuration experiments in
a laboratory-scale, recirculating system. The parameters are
described in Table 10. Experiments were conducted over an
entire year in process water adjusted to approximate ambient
growing water temperatures. Reportedly, salinities were
within 20% of growing water salinities, but it is not known
if the minimum salt concentration (20.5 ppt) for the effective
purification of C. gigas (4) was maintained. Tank loadings of
I, 2 or 3 oysters/L did not give significant differences (P <
0.01) in depuration rates after 1,2, or 3 d. A minimum flow
of 2.5 cycles/h was adequate to ensure low fecal coliform
concentrations in the water «2/100 ml).
Table II gives times and temperatures required to
depurate specific levels of fecal coliforms from C. gigas to
~230/100 g. Buisson et al. (16) provide an interesting plot
graph which depicts times, temperatures and contaminant
loads at which oysters can always, sometimes, and never be
depurated. Interpolations from this graph give temperatures
and corresponding fecal coliform loads which could be
depurated in 24 h under the conditions specified by Buisson
et al. (16).
Oyster spawning contributed to unsuccessful depuration
because the water turned milky white and interfered with UV
light penetration (16). Supersaturation of process water with
gases also contributed to unsuccessful depuration and high
oyster mortalities (16).
Finally, oysters which had accumulated crude (ca. 10"1
g) and filtered (ca. 102.5Ig) poliovirus in a static system effec-
tively depurated in a flow through system to nondetectable
levels in 96 h for the higher level (Table 10) and in 24-48 h
for the lower level according to Hoff and Becker (81).
Conversely, oysters which bioaccumulated poliovirus in a
flow-through system and were depurated in a static system
showed gradual viral reductions with 3.3 X 103 PFU/g (21 %)
of the accumulated viruses remaining in the oysters after 120
h (38).
Laboratory depuration studies were conducted in tanks
ranging from 40 L (16.148) to only 3.5 L (38). The com-
mercial depuration tank used by Madden et al. (110) was
5,500 L. Other tank sizes were not given, including the size
of the commercial tank used by Scotti et al. (148).
Sydney rock oyster, Crassostrea commercialis
A review of oyster depuration and rationale for the
depuration of the Sydney rock oyster was presented by Fleet
(49). Illnesses attributable to the consumption of sewage-
polluted shellfish in Australia and the general success in
depurating other species of oysters provided the tenor for his
review.
Studies on the bioaccumulation and tissue distribution of
contaminants by the Sydney rock oyster were not available in
 240 RICHARDS

TABLE II. Times and temperatures required to depurate fecal coli/arms to <2301100 g in C. gigas"
Initial conc. fecal Time (days)
colifonns/lOO g 2 3 >3
<1,000 12.7 -21.O"C
1,000- 10,000 17.0-21.O"C lO.2°C
10,000-100,000 20.0-21.O"C 11.6-12.7°C
>100,000 10.0-IS.O"C
"Taken from Buisson et al. (16).

TABLE 12. Microbial uptake parameters for Sydney rock oyster (Crassostrea commercialis) depuration studies.
Exposure"
LeveV Staticl
Contaminant Nat/Art Period 100 ml flow-thru Reference
APCb N NA NA NA Souness and Fleet (153)
Col iforms N NA NA NA Souness and Fleet (153)
E. coli N NA NA NA Souness and Fleet (153)
E. coli A 1.5 h S Rowse and Fleet (145)
S. charity A 1.5 h S Rowse and Fleet (145)
APC N NA NA NA Souness et al. (154)
E. coli N NA NA NA Sou ness et al. (154)
APC N NA NA NA Son and Fleet (152)
E. coli N NA NA NA Son and Fleet (152)
S. typhimurillm A 6h S Son and Fleet (152)
S. senftenberg A 6h S Son and Fleet (152)
B. cereus A 6h S Son and Fleet (152)
v. parahaemolyticlls A 6h S Son and Fleet (152)
c. perfringe1ls A 6h S Son and Fleet (152)
"Abbreviations: A, artificially contaminated shellfish; N, naturally contaminated shellfish; NA, not applicable. Dash (-) indicates
information was not provided.
bAPC, aerobic plate count.

the published literature on depuration. This is in part because
many of the depuration studies relied on the use of naturally
contaminated oysters (152-154) for which bioconcentration
data cannot be obtained. Two studies used artificially con-
taminated oysters in at least a portion of their work (145,152)
(Table 12), however neither of these studies specifically ad-
dressed the concentration or tissue distribution of microbes
within the oysters.
Rowse and Fleet (144) determined that oyster feces serve
as a reservoir of concentrated, viable bacteria which may
recontaminate waters in depuration tanks and may contribute
as a source of oyster recontamination. Agitation of tank water
can also resuspend fecal deposits and recontaminate oysters
during purification. Shallow tank depuration systems, such
as the one described by Souness et al. (154), could increase
the possibility of shellfish recontamination by feces-associ-
ated microorganisms (144), unless hydraulic parameters are
appropriate.
Souness and Fleet (153) evaluated the effects of water
temperature, flow rates, oyster density, and pumping rates on
the depuration of C. commercialis in laboratory (ca. 40 L)
and pilot-scale (350 L) depuration systems. A water tem-
perature of 25°C provided optimal pumping by oysters as de-
termined by dye uptake experiments. Pumping rates were
reduced at < 150C and ~30"C.
Two temperature ranges were evaluated in laboratory
depuration trials, 18·20"C and 27-29°C (153) (Table 13).
Interestingly, the rate of reduction in aerobic plate counts,
total coliforms, and E. coli decreased at the higher tempera-
tures. Similar findings were obtained by Rowse and Fleet
(145) in a 4,OOO-5,OOO-L commercial depuration system
(Table 13). E. coli and Salmo1lella charity depurated more
efficiently within the temperature range of 18-22°C, than at
either 13-17°C or 24-27°C (145). These findings suggest that
oyster pumping rates may not be the principal factor affecting
microbial depuration of the Sydney rock oyster. Rowse and
Fleet (145) also showed that physiological shock was not
apparent in winter-harvested oysters depurated in warmer
waters (18-22°C), or in summer-harvested oysters purified in
cooler waters (18-22°C).
Bacterial buildup was observed in the recycled process
water after depuration at elevated temperatures and with flow
rates of 1-2 cycles (past the UV tube) per h (153). At 25°C,
20-fold increases in aerobic plate counts occurred at 1 cycle/
h, whereas only 10-fold increases were observed with 2
cycles/h. Reducing the temperature to 20"C and increasing
the flow to 3 cycles/h provided reductions in aerobic plate
counts of the water as well as aerobic plate count, colifonn
and E. coli levels in the meats. Microbial levels of the water
also increased in pilot scale studies (400-L tanks) with flowS
<3 cycles/h and at ~ 25°C. Microbial buildup in the water was
not the result of inefficient UV treatment, but rather the
consequence of microbial multiplication in the system. Over
99% of waterborne bacteria were destroyed after exposure to
the UV light: however, the numbers ofUV-reslstan . t micro-
.
organisms reportedly increased during the 48-h depuration
 DEPURA nON AND RELAYING SHELLFISH
cycle (153).
Depuration trials using 2 or 4 oysters!L of tank water
showed no significant differences in depuration rates (153).
Other studies also reported that depuration was effective with
loadings of 4 oysters!L (152,154).
The effects of salinity on shellfish depuration were
studied by Rowse and Fleet ( 145). At 18~22°C, low salinities
(16-20 ppt) gave slow and inconsistent bacterial reductions
and high oyster mortalities (ca. 30% during 48 h of depura-
tion), whereas higher salinities (32-36 ppt and salt-supple-
mented seawater at 43-47 ppt) produced rapid and consistent
depuration of E. coli and S. charity with low mortalities
(Table 13). Rapid exposure to lower salinities (16-20 ppt)
was physiologically stressful to the oysters causing reduced
activity and increased mortalities. Conversely, the sudden ex-
posure to increased salinity seawater did not impair pumping
and purification processes and retarded spawning during the
summer months.
Two systems (ca. 4,000 and 5,000 L) for the commercial
depuration of Sydney rock oysters were evaluated by Souness
et al. (154) (Table 13). Aerobic plate counts generally
decreased lO-fold within 48 h, while E. coli was effectively
eliminated from initial levels as high as 1.1 X IOS/1 00 g.
Son and Fleet (152) described a study on the depuration
of the following pathogens: Salmonella spp., B. cereus, C.
perfringens, and V. parahaemolyticus (Table 13). Aerobic
plate counts and E. coli analyses were also conducted (Table
13). A laboratory-scale (20 L) depuration system effectively
reduced E. coli to nondetectable levels within 48 h (Table
13). Aerobic plate counts decreased one log, but seldom to
levels below IQ4/g possibly due to the persistence of an
indigenous gut flora in oysters (30.152,170). High levels of
S. typhimurium and S. senftenberg dramatically decreased
(about 4 logs) within 2 d. C.peljl-ingensdecreased from IOS/
g to 5/g in 48 h. B. cereus, a bacterium associated with
oyster-borne food poisoning (86), and V. parahaemolyticus
depurated to low levels within 48 and 72 h, respectively ( 152)
(Table 13).
Eyles and Davey (45) focused on the commercial depu-
ration of indicator and pathogenic bacteria from C.
commercialis. A well-maintained depuration plant which
used UV -treated and recirculated seawater was evaluated for
its ability to reduce naturally accumulated coliforms, E. coli,
V. parahaemolyticus, V. cholerae, Salmonella and total
aerobic microorganisms. Aerobic plate counts of depurated
oysters (4.8 x 102/g) were significantly lower (P < 0.01) than
counts from nondepurated oysters (1.2 X 103/g). Salmonella
warragul was isolated from only one sample of unpurified
~ysters. Depuration contributed to highly significant reduc-
tIons (P < 0.001) in coliform and E. coli levels, although
three batches of oysters contained E. coli in excess of the 2.3/
g standard of Australia's National Health and Medical Re-
search Council (124). Failure to effectively depurate these
batches was attributed, in part, to a malfunctioning UV-
treatment system and excessive initial E. coli levels (45).
There was no significant difference (P > 0.1) between
mean V. parahaemolyticus counts in unpurified and depu-
fated oysters (45). Maximum counts among individual
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
241
samples were 48/g before and after depuration. Non-Ol V.
cholerae was detected both before and after depuration. It
was speculated that V. parahaenwlyticus and V. cholerae may
persist throughout the depuration process (45). This refutes
earlier work by Son and Fleet ( 152) who showed rapid Vibrio
reductions in laboratory-inoculated oysters. Eyles and
Davey (45) used naturlllly contaminated shellfish. These
conflicting results may signify the importance which the
mode of contaminant uptake and the origin of the contami-
nant have on the depuration process.
Eyles and Davey (45) also evaluated 54 oyster samples
depurated in 25 different plants. E. coli was detected in 19%
of the depurated samples and exceeded the Australian stan-
dard in three cases (2.5, 3.5 and 30/g). V. parahaemolyticus
was detected in 39% ofthe depurated samples obtained from
13 of the 25 plants and from oysters harvested from five
different estuaries. Most (75%) of the oysters containing V.
parahaemolyticus were detected during the warmest months
of the year. V. cholera was isolated from only one depurated
sample.
It is commercial practice in Australia to transport and
store in-shell oysters at ambient temperature for 2-3 weeks
after harvest (152,154). Depuration followed by storage at
ambient temperatures for 3 weeks increased oyster survival
rates when compared to nondepurated and stored oysters
(154). Nonpurified oysters exhibited ca. 40% mortality,
whereas mortality was ca. 10% in depurated and stored
oysters (154). Culling of weak and damaged shellfish before
and after commercial depuration would reduce the mortality
rate of stored oysters, but the extent of oyster cuIling in this
study is unknown.
Mussels, Mytilus edulis
Mussel depuration has been extensively practiced in
European countries where mussels are a valued food source.
Their commercial value is great with ca. 100,000 metric tons
of mussels having been depurated in Spain alone (101).
Unfortunately, much of the research has not been published
or is not generally available for review. Consequently, there
are many gaps in the reporting of microbial uptake and
depuration by the mussel.
Dodgson (39) produced an extensive report on mussel
and, to a lesser extent, oyster physiology and purification.
Early experimental data and conclusions advanced by
Dodgson are still credible today and served as a basis for the
development of depuration plants throughout Europe and
elsewhere.
Dodgson (39) was one of the first scientists to discount
the effects of chlorine on mussel purification by taking
exception to earlier work by Johnstone (93) and others who
supported the use of chlorine. Dodgson concluded that
chlorine levels between 0.25 and 5.0 ppm altered normal
physiological activities of mussels. Chlorine was found
acceptable in sanitizing outer shell and inner tank surfaces.
Studies on mussel physiology (39) showed that mussels
pumped at an average rate of 45 L/24 h with maximal
pumping rates as high as 64 L/24 h. Mussels were found to
open and pump more efficiently at dusk or in the dark than
 TA.BLE 13. System design, water parameters, and microbiological results for the depuration of Sydney rock oysters (Crassostrea commercialis),
N
Water parameters N
Water Temp. Sal. Flow rateb
Del!.
timeC eontam. leveVIOO gd """'
Contllrni lIant System" treatment" ("C) (~pt) (cycles/h) (h) Before After" Reference
APC f R UV 18-20· 31 I 48 7xlO6 2X\06 Souness and Fleet (153)
APC R UV 27-29 31 48 7xl()6 5.2xl()6 Souness and Fleet (J 53)
CoJiforms R UV 18-20 31 24 I.lx \0" 1.6x I ()l Souness and Fleet (153)
Coliforms R UV 27-29 31 48 l.lxl<J4 2.4x103 Souness and Fleet (153)
E. coli R UV 18-20 31 48 1.5x I 03 90 Souness and Fleet (153)
E. coli R UV 27-29 31 48 1.5x103 2.3xl()2 Souness and Fleet (153)
E. coli R UV 13-17 32·36 3 48 9.3x I 03 3 Rowse and Fleet (145)
E. coli R UV 18-22 32-36 3 48 9.3x 1Q3 4 Rowse and Fleet (145)
E. coli R UV 24-27 32·36 3 48 9.3x I Q3 7 Rowse and Fleet (145)
S. charity R UV 13-17 32·36 3 48 2.3xl Q3 7 Rowse and Fleet (145)
S. charity R UV 18-22 32-36 3 48 9.3x I Q3 3 Rowse and Fleet (145)
S. cnarity R UV 24-27 32-36 3 48 2.3x10 3 93 Rowse and Fleet (145)
E. cofi R UV 18-22 16-20 3 48 3.3x104 1.5x I 0 3 Rowse and Fleet (145)
E. cofi R UV 18-22 43-471 3 48 4.8xl()l 9 Rowse and Fleet (145)
S. charity R UV 18-22 16-20 3 48 1.5xl()l 4.8xl()2 Rowse and Fleet (145)
S. charity R UV 18-22 43-471 3 48 1.5x I <J4 6.5 Rowse and Fleet (145)
APC' R UV 20 3 48 9.lx107 3.3xl()6 Souness et al. (154)
E. ('of;A R UV 20 3 48 1.1xl<J4 NO Souness et al. (154) 22
()
APe; R UV 21 3 48 2.5xlQ6 1.2x I Q6 Souness and Fleet (153) :r:
;I>
;:0
E. colii R UV 21 3 48 1.Ixl (}'I NO Souness and Fleet (153) I:'
en
APC R UV 18·22 3 48 2xlO7 2xl()6 Son and Fleet (152)
E. col; R UV 18-22 3 48 IxlO4 NO Son and Fleet (152)
S. tyrJhimuriuTn R UV 18-22 3 60 9xl<J4 9 Son and Fleet (152)
S. s~l1ttenberg R UV 18-22 3 72 2xl(}'l 36 Son and Fleet (152)
B. cereus R UV 18-22 3 48 Ix 106 7xl()2 Son and Fleet (152)
V. pamhaemolyticus R UV 18-22 3 72 9xlO7 8xl02 Son and Fleet (152)
C. p€ifringens R UV 18-22 3 48 IX\07 5x)()2 Son and Fleet (152)
APC R UV 25 31 1 48 IxlO9 9xl()6 Souness and Fleet (153)
APC R UV 25 31 2 48 Ix 109 IxlQ6 Sou ness and Fleet (153)
APC R UV 20 31 3 48 2xlO 7 2xlQ6 Souness and Fleet (153)
Coliforms R UV 25 31 1 48 1.1xl<J4 1.5x I 0 3 Souness and Fleet (153)
Coliforms R UV 25 31 2 48 1.1xl04 4.3xl()2 Sou ness and Fleet (153)
Coliforms R UV 20 31 3 48 4.6x 10 3 90 Souness and Fleet (153)
£. coli R UV 25 31 1 48 1.5xl03 2.lx102 Souness and Fleet (153)
E. coli R UV 25 31 2 48 1.5x 10 3 NO Souness and Fleet (153)
E. cofi R UV 20 31 3 24 1.5xl03 40 Souness and Fleet (153)
''''See footnotes to Table 2.
rAPC. aerobic plate count.
gSalt·supplemented seawater.
h6,OOO-L commercial depuration tank ..
il\.OOO-L commercial depuration tank.
 DEPURATION AND RELAYING SHELLASH
in the daylight. Even at cold temperatures (0-3.9"C) mussels
vigorously pumped at dusk, but not during the day. Natural
water temperatures at which mussels actively fed and passed
feces and pseudofeces ranged between 0-26°C. High mortali-
ties occurred as a result of high temperatures of the depura-
tion waters (l8.3°C) or of air temperatures (21.1 0C) during
storage. To circumvent this shortfall, depuration was avoided
during the summer months. Dodgson (39) also showed,
through extensive feeding experiments. that food could pass
through the mussel's alimentary tract in as little as 1.25 h.
Mussels remained relatively hardy under conditions of
oxygen deficiency and deprivation (39). A series of experi-
ments demonstrated that mussels could survive in apparently
good health in sealed jars of water for 4 weeks. The distinct
odor of H,S was apparent upon opening the jars on day 26.
but the m~ssels remained active. Because of their ability to
survive under anoxic conditions, mussels were touted as
facultative anaerobes. In a commercial, static depuration
system containing 150,000 mussels in 160,000 L of seawater,
dissolved oxygen, as determined by the Winkler method, fell
from 6.5 cclL to <2.0 cclL in 12 h and to nearly 0 cclL in 22
h at relatively constant temperatures (7.8-1 O.O"C). The pH of
the process water in this commercial system gradually
dropped from 8.1 at 0 h to 7.1 after 22 h (39).
Dodgson (39) traced the evolution of one of England's
earliest depuration facilities at Conway. The historical as-
pects in the development of this plant are not important here,
but some of the early findings concerning bacterial reduc-
tions in mussels over various time intervals are important.
The Conway plant was constructed in 1914 as a mussel
purification plant. During its early operations, chlorinated
(but not dechlorinated) seawater was used in a static system.
Actual levels of residual chlorine in the tank water were not
given but were expected to be quite low. During the first year
of operation (1916-1917), bacteriological results indicated
that optimal cleansing of mussels occurred within 48 h; tests
for coliform organisms showed reductions of about 2 orders
of magnitude within 48 h with little or no further reduction
by 72 h. Based on this and subsequent supportive data, 48-h
depuration intervals became universally accepted as the
benchmark for the depuration of all shellfish species.
Depuration during Dodgson's era was generally accom-
plished by means of static depuration systems. Dodgson (39)
reported on one rather unconventional flume system for
mussel depuration. Mussels were placed in a 4 ft X 6 in. X 6
in. (1.2 m X 15.2 em X 15.2 cm) inclined trough. Chlorinated,
but aged, seawater flowed down the trough and over the
mussels at a rate of ca. 45.5 L/min. This process permitted
shellfish pumping and effectively washed away shellfish
excretions. This system of purification was faster than static
systems as evidenced by substantial coliform reductions
within 0.5 h and virtually complete elimination within 24 h.
Rough handling of shellfish often causes shell damage.
The effects of rough handling were evaluated by Dodgson
(39). In one study, sacks of mussels were dropped from 4 or
more ft (1.2 m) in the air. A large number of mussels at the
,bottom of the sacks were damaged or killed. Individual
'mussels dropped from 5-6 ft above wooden grids into depu-
JOURNAL OF FOOD PROTECTION. VOL. 51, MARCH 1988
243
ration tanks functioned normally. Based on these findings,
fishemlen were allowed to pour mussels out of sacks from
a height of 3-4 ft.
Other early works by Dodgson (40), Clegg and Sher-
wood (27). Johnstone (93), Petrilli (130.131), Reynolds
(140) and Wood (174.177) provide some general informa-
tion on mussel depuration. Wood (174) compared contami-
nant uptake and depuration of mussels with European oysters
(Ostrea edufis) and found that during cold weather, mussels
continued to actively pump at 1.5-2.1 °C even though oysters
stopped pumping at these low temperatures. Reynolds (140)
effectively depurated mussels in 3-in. (7.6 em) layers within
48 h, but found that 6-in. (15.2 cm) layers would not always
depurate satisfactorily.
The uptake of polio 3 and Coxsackie A9 was studied by
Duff (41). Viruses were added to 6 L of aerated seawater at
18.5-22.0"C. Virus uptake by the mussel and inactivation in
the seawater was evaluated for up to 22 d. Within 6 d ~98%
of each virus was bioaccumulated. Virus inactivation ap-
peared to occur in the mussel, with Coxsackie A9 being much
more susceptible than polio type 3. Overall, poliovirus
uptake was 3-fold greater than Coxsackie uptake. Duff (41 )
speculated that possible differences in surface charges be-
tween the viruses may account for increased polio adsorption
to shellfish mucus.
Depuration of poliovirus 2 from artificially contami-
nated mussels was studied by Crovari (33) (Tables 14 and
15). Depuration was conducted in glass containers with up to
10 L of seawater. Fresh seawater was added to each container
dropwise from a barrel above the container. When the glass
container reached a water level equal to 10 L (ca. every 6 h)
a siphon automatically released and the container was emp-
tied. The analytical procedures for detecting poliovirus were
not as sensitive as today's and could only give a qualitative
indication of virus presence. Nevertheless, mussels depurated
for 0,6, 12, 18 and 24 h were consistently positive for viruses.
No polio was detected in shellfish depurated for 48 and 72 h.
The effects of algal suspensions on the pumping rates of
mussels were studied by Davids (36). This is mentioned here
because acceleration or inhibition of pumping rates due to
food supply could affect flow-through depuration systems.
Davids showed that mussels measuring 2.9 cm in length
pumped between 0.35 and 1.05 L/h at 18°C, and that pumping
rates accelerated to as high as lAO L/h after the transfer of the
shellfish from concentrated algal suspensions to clean seawa-
ter. Pumping rates were more rapid in dilute suspensions of
Isochrysis and Nitzchia (30,000-40,000 cells/ml) than in just
seawater. In contrast, Chlorella quickly reduced pumping
rates. Davids concluded that mussels do not like Chlorella as
a food source.
Ledo et al. (101) performed depuration studies on natu-
rally contaminated mussels in Spain (Table IS). The depura-
tion of potential bacteriological indicators was evaluated in
commercial plants which used chlorinated (2 plants) or
untreated (l plant) seawater. Process water ranged from 13
to 19°C with salinities from 31.7 to 34.3 ppt. Chlorine-treated
water contained 3 ppm chlorine which was dechlorinated by
aeration before use. The untreated seawater plant was of
 ... ,
244 RICHARDS
TABLE 14. Microbial uptake parameters/or mussel (Mytilus edulis) depuration studies.
&posure a
Contaminant Nat/Art Period
Polio 2 A 3h
APCb N NA
Total coliforms N NA
Fecal coliforms N NA
E. coli N NA
Fecal streptococci N NA
aAbbreviations: A, artificially contaminated shellfish; N, naturally contaminated shellfish; NA, not applicable. Dash (-) indicates
information was not provided.
bAPC, aerobic plate count.
flow-through design. It was not stated if plants using chlori-
nated water had recirculating or flow-through systems.
Depuration results after 48 h were similar for both
systems (101). Total aerobic counts decreased lO-fold but
seldom to levels <1()3. Reductions of other microorganisms
in both systems ranged as follows: total coliforms, 30.2-
38.6%; E. coli, 89.0-91.5%; and fecal streptococci, 74.0%
(chlorinated) and 87.0% (untreated). Untreated seawater was
most effective in reducing fecal coliforms (90.1 % vs.
63.4%). Other bacteria present in the pre-depurated mussels
in decreasing order of prevalence were: Enterabacter, E.
coli, Citrabacter, Yersinia, Salmonella, Klebsiella, Proteus,
and Serratia. The latter five bacteria were detectable in ca.
8% of the samples before depuration and were nondetectable
after depuration. The frequency of Enterabacter isolates
diminished after depuration for 48 h (from 60 to 50%).
Citrabacter was detected with nearly equal frequency both
before (48%) and after (50%) depuration.
Fecal streptococci were present at substantially higher
levels than E. coli and fecal coliforms in pre- and post-
depurated shellfish (101). After depuration, fecal strepto-
cocci counts were one log higher than E. coli. This suggests
• that current indicator organisms may not truly reflect the
safety of depurated products. Marcucci et al. ( 112) found less
variability in clostridia counts among mussels obtained from
various locations in Italy. They suggested that relatively
hardy clostridial spores might be a better indicator of depu-
ration efficiency particularly for depuration-resistant enteric
viruses. Another study showed greater persistence of clos-
tridia spores than E. coli and S.faecalis vegetative cells in the
marine environment (110).
SUMMARY OF SHELLFISH DEPURATION STUDIES
AND GENERAL CONCLUSIONS
Many factors influence the ability of shellfish to depu-
rate. Some factors are peculiar to a specific shellfish species,
whereas others relate to all species. Optimal depuration
temperatures and salinities vary with shellfish species. Like-
wise, other factors affecting the shellfish physiology or
pumping rates must be evaluated on a species-by-species
basis if depuration is to be effective. The geographic area
from which shellfish are harvested may also affect shellfish
depuration. Some shellfish apparently bioconcentrate micro-
JOURNAL OF FOOD PROTECTION, VOL. 51, MARCH 1988
Level! Static/
IOOml flow-thru Reference
ca. lx10 5 Crovari (33)
NA NA Ledo et al. (101)
NA NA Ledo et al. (101)
NA NA Ledo et al. (101)
NA NA Ledo et al. (101)
NA NA Ledo et al. (101)
organisms at different rates and to different levels than other
shellfish species (7,95,169). There are, however, prevailing
similarities in the uptake, tissue distribution, and depuration
of microbial contaminants among different shellfish species.
Some of the similarities are discussed below.
Basically, W~ know that shellfish bioconcentrate bacte-
ria and viruses particularly in flow-through, laboratory-
uptake systems. Similar abilities to concentrate contaminants
would be expected in the natural environment. E. coli was
concentrated 6.5 to 8.5-fold in hard-shell clams (17) and 40-
fold in Eastern oysters (122). Poliovirus concentrations ca.
50-fold and higher were common for several shellfish species
(81,119,120,122,149). Coliphage concentrations as high as
l,lOO-fold have been reported (20). Evidence suggests that
particle- or feces-associated viruses are more readily bioac-
cumulated than purified (essentially single) virus particles
(42,81,116). There is also some evidence to suggest that
naturally acquired microbial contaminants depurate more
slowly than those laboratory-induced (74,157). Microbial
concentration occurs primarily in the hepatopancreas and
digestive diverticula (17,38,42,105,107,119,120), and with
clams, also in the siphon (17,42,119,120).
We also know that shellfish can be at least partially
purified of bacterial and viral contaminants given the proper
set of conditions. The rate of cleansing is proportional to the
level of pollution (103,108,120), more highly contaminated
shell stock taking longer to depurate than lightly contami-
nated shellfish.
The rate of depuration decreases with time. Buisson et al.
(16) offered two possible explanations for this phenomenon.
The first pertains to the passage of contaminants through the
digestive system as a result of feeding. As food is depleted in
the recirculating depuration waters, oysters cease to excrete
contaminants. This theory was not supported by the studies
where the presence or absence of food in the depuration
waters showed no marked effect on depuration (16,70,129).
A more likely explanation advanced by Buisson et aI.
(16) is that bacteria are depurated at rates influenced by
microbial adsorption to or association with the digestive tract
or other tissues. Weakly attached bacteria are released and
depurated first, whereas more firmly attached or engul~ed
organisms are depleted more slowly (or decline due to dle-
off). This concept gains support by work of DiGiorlam~ et
al. (38) suggesting diffusion of poliovirus from the digestive
TABLE 15. S~stem desis.n. water e.arameters. and microbiolos.ical results tor the dee.uralion ot mussels (M~ti1us edulis).
Water Parameters
Dep.
Water Temp. Sal. time· Contam. l~veVIOO g t:)
sg
COl1taminant System' treatment' ("C) (ppt) flow rateb (h) Before After' Reference
~
Polio 2 Sd none 18-22 ()d 48 present ND Crovari (33) >
:j
APCC CI 13-19 31.7-34.3 48 2xl()6 9xl0' Ledo et al. (101) 0
Total colifonns 13-19 48 9xl()3 5xl03 Ledo et al. (101)
z
CI 31.7-34.3 >
FecaJ coJifonns CI 13-19 31.7-34.3 48 IxlO3 3x 1 ()2 Ledo et al. (101) ~
E. coli CI 13-19 31.7-34.3 48 6xl()2 60 Ledo et a!. (101)
Fecal streptococci CI 13-19 31.7-34.3 48 3xl0l txt 03 Ledo et at. (101) '"
p:!
>
APC Ff none 13-19 31.7-34.3 48 5xtO' 2xlO' Ledo et al. (101) -<

*~
Total coJifonns Ff none 13-19 31.7-34.3 48 8xlO3 5xl03 ~ et al. (101)
Fecal coJifonns Ff none 13-19 31.7-34.3 48 4xJ()2 30 Ledo et al. (101) en
E. coli ' Ff none 13-19 31.7-34.3 48 3xl()2 30 Ledo et al. (101)
Fecal streptococci Ff none 13-19 31.7-34.3 48 IxlO3 2x 1 ()2 Ledo et al. (101) :!l
en
a-cSee footnotes to Table 2. :c:
flWater changed at 6 h intervals. Dropwise addition of water.
•APC. aerobic plate count.

N
t;
 246 RICHARDS
tract to the body. Virus levels in the body tissues increased
from 0.5% after uptake for 12 and 24 h, to 16% after 48 h.
The presence of low-level, virus contamination in shellfish
hemolymph has also been observed (38,82 ,J OS,
106,119,120). . appropriate indicator, but research to address its effectiveness
Canzonier (20) speculated that viral accumulation in
shellfish is by two related but distinct processes. The first is
where food and microbial contaminants enter the mantle
cavity, are collected and entrapped and subsequently enter the
digestive tract. In such an event most viruses pass through the
digestive system and are expelled in the feces. A second
process involves the exposure of shellfish to small numbers
of virus particles over prolonged periods. In such instances,
particles may be sequestered in the digestive gland and
associated tissues and, to a lesser extent, in the hemolymph.
Sequestered particles would not be subject to rapid release
and could persist for periods comparable to those observed
for virus inactivation in seawater as influenced by tempera-
ture.
Other evidence supports Canzonier's concept. For in-
stance, Liu et al. (107) found that poliovirus depurated more
rapidly from the digestive diverticula than from the hemo-
lymph (24 vs. 72 h). Work by Greenberg et al. (63) suggests
that the initial rapid reduction of Vibrio in depurating clams
may be the result of a loose association of the Vibrio with the
animal cells, but that a closer association with the cells results
in a persistence and subsequent outgrowth of Vibrio. Perhaps
for these same reasons, shellfish are unable to purge total
aerobic bacteria below certain levels (101,152-154). Pain
(127) reiterated the beliefs of some scientists that mobile
scavenger cells caned haemocytes pick up viruses from the
digestive system and migrate into other tissues of the shell-
fish. Physiological phenomenon responsible for sequesteri-
zation may vary with different viruses and bacteria accord-
ing to shellfish species (127). The slow release of attached or
sequestered pathogens is of public health concern, particu-
larly for viral pathogens which may be infectious at very low
levels.
RESEARCH NEEDS
In view of recommendations made by the 1986 Interstate
Shellfish Sanitation Conference Depuration Committee, a
Microbiology Workshop held at the Virginia Institute of
Marine Science (Gloucester Point, VA, May 28-30, 1986)
and this review of the depuration literature, the following
recommendations for additional research are provided.
Much research is needed to delineate the role of environ-
mental parameters on the depuration of microbial contami-
nants from shellfish. Infomlation is generally available on
parameters necessary for E. coli and poliovirus reductions
from most of the commercially valuable shellfish species.
Specific information on the depuration of other pathogens,
i.e., hepatitis A and Norwalk viruses, Vibrio spp., and most
of the enteric bacterial pathogens is needed.
Research is required to reassess the adequacy of indica-
tor organisms as predictors of shellfish sanitary quality and
safety, Fecal coliforms. E. coli and enterococci have obvi-
JOURNAL OF FOOD PROTECTION. VOL 51. MARCH 1988
ous deficiencies as indicators. The predictive relationship of
current bacterial indicators to the presence of major patho-
gens such as Vibrio spp. and enteric viruses needs further
evaluation. Clostridium spp. have been suggested as a more
as a potential indicator of pathogenic bacteria and viruses is
required.
More sensitive, reliable, and universally accepted assay
techniques must be developed for virus analyses. Currently,
standard methods do not exist for detecting low levels of
hepatitis A, Norwalk, and rotaviruses from naturally con-
taminated shellfish (141). Recent advances in the develop-
ment of gene probes for detection of polio, hepatitis A, and
other viruses in marine water and shellfish may provide the
key to the development of cost-effective virus screening
techniques (910,113). Numerous poliovirus extraction and
assay techniques are available for specific shellfish species
(57), however, the reliability of the methods for detecting
viruses in naturally contaminated shellfish remains un-
known.
Epidemiological studies to determine the actual health
risks associated with ingestion of depurated and relayed
shellfish would provide important information to assist in the
regulation of the industry. Current depuration and relaying
guidelines and standards are not based on hard epidemiologi-
cal evidence of safety, but rather on the perception of safety.
If consumers are perceived to remain healthy after eating
shellfish products, the regulations are perceived to be effec-
tive. Unfortunately, sporadic foodbome illnesses do occur;
but because of poor reporting, are not officially linked to a
specific food. Most illnesses go undetected by regulatory
officials and do not contribute to the data base needed for
reevaluation of regulations or guidelines. Epidemiological
investigations such as the one performed in New South Wales
(65) would provide a clue to the actual health risks associated
with the consumption of depurated and relayed shellfish.
Eyles (44) reviewed the literature on the accumulation
and elimination of viruses by oysters and mentioned several
problems associated with past experiments. He pointed out
that (o) most depuration experiments used shellfish contain-
ing unnaturally high initial virus levels which could mean an
unrealistically long depuration time, (b) many of the virus
detection procedures have been relatively insensitive and
viruses could be present in samples recorded as virus free, and
(c) purified virus preparations, frequently used in uptake and
depuration experiments, are not necessarily taken up by
shellfish in the same manner as solids-associated viral con-
taminants.
Depuration research should be standardized to enable
comparison of research data among investigators. Parameters
should be consistent between commercial- and laboratory-
scale systems taking into consideration the temperature,
salinity, turbidity, flow rates, tank sizes, loading factors,
contaminant loads and uptake period (for artificially con-
taminated shellfish), physiological status and size of the
shellfish, and the specifics of the depuration system (f1ow-
through vs. reCirculated, water disinfection process. etc.).
 DEPURA TJON AND RELA YTNG SHELLFISH
The above information. in addition to being standardized.
should be disclosed in research reports and publications to
permit comparison of data. Current literature contains many
gaps in the data. in part. due to under-reporting. This makes
comparison of research results very difficult if not impos-
sible.
Future depuration and relaying studies should include
internal viral and/or bacterial controls to facilitate inter- and
intra-laboratory comparisons. Bacteriological studies should
incorporate E. coli or fecal coliforms into any research
involving bacterial depuration so that the effectiveness of the
depuration system can be determined in terms of conven-
tional indicators and the results compared with previous
studies. Likewise virological studies should include E. coli.
as a measure of process effectiveness. and poliovirus type 1
as an internal. benchmark control to facilitate comparison
with other studies.
Depuration was not intended for grossly polluted shell-
fish or for shellfish harvested from grossly polluted waters.
Therefore. upper limit (0 h) bacteriological criteria for
shellfish are needed to ensure adequate reductions of bacte-
rial contamination within certain periods. Criteria should be
based on the upper levels of bacteria which can reasonably be
expected to depurate within a given time frame in a properly
functioning depuration plant. Viral levels should also be
evaluated and compared with bacterial criteria for possible
institution of upper level limits for viruses. Criteria should
take into account the effects of low temperatures on shellfish
pumping rates and the inadequacies of the fecal coliform
indicator at low temperatures.
ACKNOWLEDGMENTS
The author gratefully acknowledges the assistance of the following
individuals: Walter Canzonier and Santo Furfari for technical review of the
manuscript; John C. Hoff, John Bemiss and Jennifer Sample for technical
assistance; Sylvia McCorkel and Lois Winemiller for literature retrieval; and
Jan Carson and Jerry Fewox for typing the manuscript.
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