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Journal of Food Protection, Vol. 85, No. 1, 2022, Pages 13–21

https://doi.org/10.4315/JFP-21-257
Copyright Ó, International Association for Food Protection

Research Note

Microbiological Quality and Heavy Metal Concentrations in Slipper

Oyster (Crassostrea iredalei) Cultured in Major Growing Areas in
Capiz Province, Western Visayas, Philippines: Compliance with
International Shellfish Safety and Sanitation Standards
ROLANDO PAKINGKING, JR. https://orcid.org/0000-0002-0914-4874,1* MA. LILIBETH HUALDE,1 ERNESTINA PERALTA,2
JOSEPH FAISAN,1 AND ROSELYN USERO3

1Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC AQD), Tigbauan 5021, Iloilo, Philippines; 2Institute of Fish

Processing Technology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao 5023, Philippines; and 3Negros Prawn
Producers Marketing Cooperative, Inc., Bacolod City 6100, Negros Occidental, Philippines
MS 21-257: Received 1 July 2021/Accepted 23 August 2021/Published Online 26 August 2021

ABSTRACT
The increasing demand for slipper oyster (Crassostrea iredalei) has propelled farmers to expand oyster cultivation areas in
the Philippines, chiefly for local consumption and feasibly for export overseas. As filter feeders, oysters can accumulate
pathogens from the surrounding waters, and these pathogens can cause foodborne diseases in consumers. Therefore, oyster
farming areas must be monitored for microbiological quality and heavy metal concentrations. In the present study, the
microbiological quality of oysters and their growing waters in the major oyster farming areas of the Cogon and Palina Rivers
and Cabugao Bay (in Roxas City and the Municipality of Ivisan, respectively, Capiz Province, Western Visayas, Philippines)
were examined monthly during the wet (May to October) and dry (November to April) seasons over 12 months. Regardless of
the sampling period, high levels of fecal coliforms in the water and Escherichia coli in oysters were found, clearly illustrating
that these oyster growing areas would meet only the class B standard under the European Union classification system and would
be considered “prohibited” for growing oysters under the U.S. classification system. Although Salmonella was occasionally
detected in oysters, Vibrio cholerae was not detected and Vibrio parahaemolyticus was within acceptable limits. The heavy
metal concentrations in oyster meat were also determined during the wet (July) and dry (March) seasons. Zinc and copper were
the most abundant metals detected, and concentrations of lead, cadmium, mercury, and chromium were below the regulatory
limits set by the European Union and the U.S. Food and Drug Administration. These oyster culture areas should be rehabilitated
immediately to improve the microbiological quality of the oysters. Oysters harvested from these sites must be depurated or
relayed to ensure quality and safety.

HIGHLIGHTS

High levels of human and animal fecal contamination occur in rivers and bays.

High fecal coliform and E. coli levels were found in oysters and water and regardless of season.

Salmonella was frequently detected in oysters during the dry season.

Oysters require postharvest treatment before being sold for human consumption.

Key words: Copper; Escherichia coli; Fecal coliforms; Oyster; Salmonella; Zinc

Oysters are a significant commodity in many coastal processed oyster products must enforce strict compliance
communities throughout Asia because of their affordability with food safety regulations. Because oysters are popularly
for low-income people (31). In the Philippines, oyster consumed raw or partially cooked, they can pose a health
production has dramatically declined over the past years due risk to consumers. The consumption of contaminated
to rising production costs and slack demand because of the oysters has been associated with outbreaks of acute
poor sanitary quality of oysters from farms located in gastroenteritis in both Eastern and Western Hemispheres
shallow coastal areas contaminated with domestic wastes (16).
(17, 23). Raw oysters are considered as luxury food in Oysters are filter feeders and can concentrate patho-
developed countries, and countries exporting live and genic microorganisms and toxins from the water and
sediment where they grow (22). Fecal coliform (FC)
* Author for correspondence. Tel: (63) 917 322 8772; Fax: (63) 33 bacteria counts are utilized extensively to predict sewage
3307011; E-mail: [email protected]. contamination and the presence of pathogens. Escherichia
14 PAKINGKING ET AL. J. Food Prot., Vol. 85, No. 1

coli is the principal FC, constituting ca. 95 to 98% of the barangay of Cabugao (Cabugao Bay, four sampling stations) in the
total FC population, which also includes Enterobacter and Municipality of Ivisan (Fig. 1). Selection of these sites was chiefly
Klebsiella strains. Autochthonous estuarine bacteria in the based on relative significance of oyster aquaculture as determined
family Vibrionaceae have been also implicated as causal by regional production volume from 2008 to 2011 (13, 14).
agents of food-related illnesses in humans (18). Vibrio
cholerae and Vibrio parahaemolyticus have been frequently Collection of oysters and water samples. Oysters are
farmed in these areas with the stake method. Oyster samples (n ¼
isolated from oysters. Because both clinical and environ-
25 to 30; shell height, 7 to 10 cm) were collected by hand from the
mental strains of Vibrio may be present in harvested oysters,
bamboo stakes at each sampling station monthly during the rainy
one cannot tell which is present unless someone becomes ill (May to October 2014) and dry (November 2014 to April 2015)
(16, 18). seasons. After harvest, oysters were cleaned and rinsed with
Although the safest approach to producing oysters is to distilled water, packed in plastic bags, stored in cooler boxes with
grow them in and/or harvest them from areas free from ice (5 to 108C), and transported within 3 h to the Fish Health
external sources of pollution, truly unpolluted oyster Laboratory (Southeast Asian Fisheries Development Center,
growing areas are scarce. Sourcing oysters from areas with Aquaculture Department, Tigbauan, Iloilo, Philippines). Near-
relatively low levels of pollution will ensure that the risk of surface (ca. 15 cm depth) water samples (500 mL) were collected
acquiring gastrointestinal diseases from fecal contaminants in sterile bottles and held at 5 to 108C until analyzed.
will be as low as can be attained without thorough cooking.
However, significant concentrations of metals such as Sample preparation for microbial analyses. The oysters
cadmium (Cd), lead (Pb), chromium (Cr), and mercury were removed from their shells with a sterile knife. The meat and
(Hg) in oyster flesh could threaten human health. These intravalvular fluid were aseptically collected in a sterile Erlen-
heavy metals are considered nonessential because of their meyer flask and homogenized in a sterile blender (Waring
Laboratory Blender 7010S, Thomas Scientific, Swedesboro, NJ)
high toxicity; they interfere with enzymatic reactions by
for 2 min.
changing the conformation of the enzymes, and they do not
participate in metabolic processes (5). Enumeration of FCs. A 10-mL water sample was added to a
The Bureau of Fisheries and Aquatic Resources of the tube with 90 mL of 0.1% peptone water, and then 10 mL of the
Philippines has recognized the need to develop a highly diluted sample was transferred into a five-tube set containing
competitive, sustainable shellfish industry capable of double-strength lactose broth (LB; Merck, Darmstadt, Germany).
meeting the growing consumer demand for shellfish Both 1- and 0.1-mL aliquots were transferred into a five-tube set
products that are high quality, safe, competitively priced, containing single-strength LB and incubated at 358C for 24 to 48
and produced in an environmentally responsible manner h. All single- and double-strength LB cultures included inverted
with maximum profitability in all production aspects of the Durham tubes. Tubes with gas production were inoculated into E.
industry. Thus, to satisfy the growing demand for oyster coli (EC; Merck) broth and incubated at 44.58C for 24 to 48 h. The
products in the Philippines and support national ambitions FC levels were determined using the most-probable-number
to develop export markets, Philippine authorities must (MPN) table and reported as MPN per 100 mL (1).
establish sanitary controls that comply with international
best practices, thereby ensuring that these oyster products Enumeration of E. coli. E. coli was enumerated with the
conventional five-tube MPN method (12). A 50-g sample of oyster
are safe and of high quality. In this study, the microbiolog-
meat was homogenized in 100 mL of 0.1% peptone. Dilution tubes
ical quality (FC counts in oyster culture water, levels of E.
(101 to 103) were prepared, and 2 mL of each dilution was
coli and V. parahaemolyticus, and presence of V. cholerae inoculated into each tube of lauryl tryptose broth (HiMedia,
and Salmonella) of oyster meat, concentrations of heavy Mumbai, India). Each tube containing an inverted Durham tube
metals (zinc [Zn], copper [Cu], Cd, Cr, Pb, and Hg) in was incubated at 358C for 24 h. Cultures from all tubes with
oyster flesh, and the physicochemical state of the culture turbidity and gas production were inoculated into EC broth and
waters were comprehensively examined. The results were incubated in a water bath at 44.58C for 24 h. A loopful of culture
used to determine how to classify the major oyster growing from EC broth tubes with turbidity and gas production was
areas in the province of Capiz, the top oyster-producing inoculated into tryptone broth (HiMedia), incubated at 358C for 24
province in Western Visayas, Philippines (15), in accor- h, and subsequently tested for indole production. The MPN table
dance with the classification standards prescribed by was used to determine the E. coli level (MPN/100 g) in the
relevant regulations in the European Union (EU) (11) and sample.
the United States (34). Information obtained in this study
will be useful for formulating guidelines for the Philippine Detection of Salmonella. A 25-g oyster sample was
National Shellfish Sanitation Program and to encourage homogenized in 225 mL of preenrichment broth and incubated
at 358C for 24 h. A 1-mL aliquot of this preenrichment culture was
production of safe high-quality oysters for local consump-
then transferred to tetrathionate broth (Merck) and incubated at
tion and export.
358C for 24 h. Selective enrichment cultures were streaked onto
xylose lysine deoxycholate agar (Merck) and incubated at 358C for
MATERIALS AND METHODS
24 h. Typical Salmonella colonies were screened for biochemical
Sampling sites. The major oyster farming areas examined in characteristics by culture on triple sugar iron agar and with the
this study are located in the province of Capiz, Western Visayas, indole, methyl red, Vogues-Proskauer, citrate test (2).
Philippines. These sites are in the barangays (villages) of Cogon
(site A: Cogon River, three sampling stations) and Cagay (site B: Enumeration of V. parahaemolyticus. A 50-g oyster sample
Palina River, five sampling stations) in Roxas City and the was homogenized in 450 mL of 0.1% peptone water supplemented
J. Food Prot., Vol. 85, No. 1 MICROBIOLOGICAL QUALITY AND HEAVY METALS IN SLIPPER OYSTER 15

FIGURE 1. Map of Capiz Province showing the locations of the three major oyster culture sites and corresponding coordinates of the
sampling stations.

with 3% NaCl in a sterile blender. Decimal dilutions of up to 104 from the surface with a portable multiparameter meter (SmarT-
were prepared from this dilution, and 1 mL of each dilution was ROLL, In-Situ, Fort Collins, CO).
inoculated into one tube of a series of three tubes containing 0.1%
peptone water supplemented with 3% NaCl. All tubes were Detection of heavy metals in oysters. Oyster samples were
incubated at 358C for 48 h. Positive cultures were streaked on collected in July (wet season) and March (dry season) and
thiosulfate–citrate–bile salts–sucrose (TCBS; Merck) agar plates analyzed for total Cd, Cr, Pb, Cu, Zn, and Hg. At each collection
and incubated at 358C for 24 h. Typical colonies were submitted to time, four or five oysters (shell height, 7 to 10 cm) were collected
biochemical testing with API 20E (21). at 10 stations: three at Cogon River, three at Palina River, and four
at Cabugao Bay (30). The oysters were shucked, and the soft
tissues were collected in a glass beaker. Each composite sample
Detection of V. cholerae. A 50-g oyster sample was
was blended to produce a homogenized mixture. Triplicate
homogenized in 450 mL of 0.1% peptone water supplemented
aliquots (5 g) of the oyster macerate were used for Hg analysis
with 1% NaCl in a sterile blender and divided in two aliquots of
following the method of de Astudillo et al. (8). Concentrated nitric
250 mL each, one incubated at 358C and the other at 428C. After 6 acid (10 mL) was added to each macerate, and samples were left
and 18 h of incubation, aliquots of each portion were streaked in overnight to digest. Samples were allowed to reflux at 1308C for 3
duplicate on TCBS agar plates and incubated at 358C for 24 h. h, and then concentrated sulfuric acid (2.5 mL) and concentrated
Typical colonies were submitted to biochemical testing with API hydrochloric acid (1 mL) were added and allowed to reflux for
20E (21). another 3 h at 1308C. Potassium manganate (VII) solution (5% m/
V) was added dropwise to each sample. Hydroxylamine solution
Water physicochemical parameters. Water physicochemi- (50 μL, 10% m/V) was then added, and the digests were filtered
cal parameters of dissolved oxygen, temperature, pH, and salinity with a paper filter (no. 42, Whatman, Clifton, NJ) and made up to
were measured at each sampling station at ca. 1-m depth intervals volume with double-distilled water rinses of the residues. Total Hg
16 PAKINGKING ET AL. J. Food Prot., Vol. 85, No. 1

FIGURE 2. Monthly fecal coliform counts

(mean 6 SD, MPN/100 mL) in oyster
cultivation water obtained from sampling
stations in the Cogon and Palina Rivers
(Roxas City) and Cabugao Bay (Munici-
pality of Ivisan) in Capiz Province during
the wet (May to October) and dry (No-
vember to April) seasons.

in the oyster sample was determined with a cold vapor atomic seasons were compared with Student’s t test. The differences were
absorption spectrophotometer (AA-7000, Shimadzu, Kyoto, considered significant at P , 0.05.
Japan) with a hydride generator, and results were reported as
micrograms per gram of wet weight (8). For the analysis of Cd, Cr, RESULTS AND DISCUSSION
Pb, Cu, and Zn, the oyster samples were dried at 1058C for 72 h
No distinct pattern was established for increases in FCs
and thoroughly macerated with a mortar and pestle. Three
by season, wet (May to October) or dry (November to
replicates of each 1-g sample were then digested with 10 mL of
April) (Fig. 2). FC levels in the Cogon and Palina rivers
a solution of nitric acid and hydrochloric acid (4:1) initially for 1 h
were variable, with counts of 8 to 315 MPN/100 mL. The
at 408C and then 3 h at 1408C. The resulting solution was filtered
with a no. 42 Whatman paper filter. The metal concentrations were
mean (6standard deviation [SD]) annual FC levels for the
determined with the Melich method and analyzed with a graphite Cogon and Palina rivers were 43.3 6 11.5 and 76.9 6 2.7
furnace atomic absorption spectrophotometer (AA-7000, Shimad- MPN/100 mL, respectively. These values were significantly
zu), and results were reported as micrograms per gram of dry above the standard limit for an “approved” oyster growing
weight (3, 35). All chemicals used were of analytical grade. area (14 MPN/100 mL; 90% compliance, ,43 MPN/100
Quality assurance and quality control (recovery of spiked samples, mL) prescribed by the U.S. National Shellfish Sanitation
midstandards, method blank, and reagent blank) were conducted Program (NSSP) (34). Based on NSSP classification, these
during each analysis to guarantee accuracy and reliability of oyster farming areas generally were in the “restricted”
analytical results. Heavy metal concentrations below the limit of category (88 MPN/100 mL; 90% compliance, 260
detection were not subjected to statistical analysis. The concen- MPN/100 mL) (34). The EC counts in oyster meat obtained
trations of heavy metals in oysters per site during the wet and dry from the Cogon River were relatively high throughout the
J. Food Prot., Vol. 85, No. 1 MICROBIOLOGICAL QUALITY AND HEAVY METALS IN SLIPPER OYSTER 17

FIGURE 3. Monthly E. coli counts (mean

6 SD, MPN/100 g) in the meat and
intravalvular fluid of oysters obtained from
sampling stations in the Cogon and Palina
rivers (Roxas City) and Cabugao Bay
(Municipality of Ivisan) during the wet
(May to October) and dry (November to
April) seasons.

year (Fig. 3), whereas those in oyster meat from the Palina wastes from several fish ponds and residential houses
River were significantly higher in May, June, and October located upstream. We noted that the river was shallow and
(330 to 2,400 MPN/100 g). The mean annual EC counts the water was turbid, particularly during low tide, probably
were 476 6 17.5 and 1,137 6 172.7 MPN/100 g for oysters because the bamboo stakes and other aquaculture structures
from the Palina and Cogon rivers, respectively, significantly installed in the river impeded the natural movements of
above the limit for a class A (230 MPN/100 g; no results water downstream, and some houses along the river banks
exceeding 700 MPN/100 g) classification as recommended have no septic tanks. Like the Palina River sites, the Cogon
by the EU Shellfish Harvesting Area Classification Criteria River sampling site was densely populated and the river was
(SHACC) (11). used as a navigational lane for commercial boats that
The high FC and EC counts frequently encountered in transport their products to Roxas City. Because the oyster
the water (stations 1 through 5) and oyster meat (stations 3 growing areas were close to the mouth of the river, the high
through 5) obtained from the Palina River could be FC and EC counts could be attributed to domestic wastes
attributed to the constant flushing of effluents and domestic that were dumped directly into the mouth of the river.
18 PAKINGKING ET AL. J. Food Prot., Vol. 85, No. 1

TABLE 1. Detection of Salmonella in oyster meat collected from three sampling sites in Capiz Province during the wet and dry seasons
Salmonella detectiona

Wet season Dry season

Site Station May June July Aug. Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr.

Cogon River 1   þ    þ  þ þ þ þ
2   þ   þ     þ þ
3   þ   þ þ þ þ  þ þ
Palina River 3 þ         þ  
4 þ     þ þ þ þ  þ 
5      þ þ þ  þ þ 
Cabugao Bay 1 þ   þ  þ þ  þ þ þ þ
2 þ  þ   þ þ þ  þ þ þ
3 þ   þ  þ þ þ þ þ þ 
4 þ     þ þ   þ þ 
a
þ, presence; , absence.

Regardless of the season, .80 to 90% of the water and stations were high during the rainy season, the levels
oyster meat samples obtained from both the Palina and significantly dropped to acceptable levels (230 MPN/100
Cogon rivers had FC and EC counts above the standard g) during the dry season (Fig. 3). Siltation is widespread in
limit for an “approved” oyster growing area as prescribed the culture area due to numerous bamboo stakes installed
by the NSSP and above the standard for a class A close to each other (ca. 0.15 to 0.3 m apart), which is still
designation as prescribed by the SHACC. Similar findings the popular method of growing oysters in Cabugao Bay.
were previously reported by Duncan et al. (9). However, Because some oysters attached to bamboo stakes are
these researchers collected samples only twice in some of exposed to sunlight during low tide, pathogenic bacteria
these sites, once during the wet season and once during the such as E. coli and Salmonella could proliferate in oysters
dry season; hence, their data on the FC and EC loads of the postharvest when flushing would not be feasible. Although
representative samples were applicable only at the time of the EC counts generally were 54.8 6 67.1 to 56.3 6 63.9
collection. In contrast, our data were generated over a 1- MPN/100 g during the dry months (Fig. 3), these data could
year period and provide some concrete evidence pertinent to not be used as basis to certify the area as class A because the
the microbiological designations of these sites in conjunc- SHACC states that E. coli levels in oyster meat must be
tion with the NSSP and SHACC. consistently 230 MPN/100 g regardless of the period or
The FC counts (2 to 79 MPN/100 mL) in the majority season. The data on E. coli loads in oysters harvested from
of samples obtained from Cabugao Bay were significantly the three major growing areas studied clearly indicate that
lower than those in samples from the Palina and Cogon these areas belong in the class B category of the SHACC
rivers. FC counts in the water samples periodically taken (4,600 MPN/100 g in 90% of samples; no results .46,000
from the sampling stations in Cabugao Bay were within the MPN/100 g) (11).
standard limit except in October and December (Fig. 2). The V. parahaemolyticus counts in oyster meat collected
annual mean water FC count for all the sampling stations monthly from all sampling stations in Cogon and Palina
studied was 10.1 6 3.4 MPN/100 mL, indicating that the rivers and Cabugao Bay were ,100 MPN/g, which is
levels of FCs in the cultivation water was within the within the maximum limit for V. parahaemolyticus in fresh
prescribed limit for an “approved” oyster growing area as and frozen bivalve mollusks (19). All oyster samples
defined by the NSSP (34). collected from the sampling stations in the Cogon and
The EC counts in oysters from Cabugao Bay were high Palina rivers and Cabugao Bay were negative for V.
in May and October (1,100 to 1,200 MPN/100 g) but cholerae. In contrast, Salmonella was occasionally detected
significantly lower from November to April (170 MPN/ in oyster samples collected from the sites studied, although
100 g). The annual mean EC count for all the sampling the majority of positive samples were collected during the
stations was 285 6 5.9 MPN/100 g for oysters collected dry months (Table 1). The presence of Salmonella during
from Cabugao Bay, still slightly above the criterion for the the dry months was persistent in the water containing high
class A category. Because of the heavy rainfall that occurred levels of FCs. However, oysters obtained from all sampling
days before our samples were collected in October, large stations in Cabugao Bay in May were positive for
quantities of domestic wastes and organic matter coming Salmonella even though FC counts in the water were low.
from residential houses and fish ponds along the shoreline Oyster samples from the Cogon River were all negative for
of Cabugao Bay contributed to the high FC counts in the Salmonella, although the FC counts in the water were
water samples tested. Before our samples were collected in significantly high. These observations corroborate the
December, waste water from nearby fish ponds containing findings of Brands et al. (6), who found no correlation
high amounts of organic matter had been released into the between the of FC levels and the presence of Salmonella in
bay. Although EC loads in oyster meat from the sampling the rearing water and meat of oysters, respectively.
J. Food Prot., Vol. 85, No. 1 MICROBIOLOGICAL QUALITY AND HEAVY METALS IN SLIPPER OYSTER 19

TABLE 2. Concentrations of heavy metals in oyster meat collected from the three sampling sites in Capiz Province during the wet and dry
seasons
Mean (6SD) concn (μg/g)a

Cogon River Palina River Cabugao Bay

Metal Wet Dry Wet Dry Wet Dry

Pb ,0.003 ,0.003 ,0.003 ,0.003 ,0.003 ,0.003

Hg ,0.002 ,0.002 ,0.002 ,0.002 ,0.002 ,0.002
Cr ,0.010 ,0.010 ,0.010 ,0.010 ,0.010 ,0.010
Cd 0.04 6 0.02 0.72 6 0.05* 0.04 6 0.01 0.67 6 0.07* 0.02 6 0.01 0.44 6 0.06*
Cu 87.11 6 9.57 84.41 6 8.47 89.23 6 7.97* 67.71 6 7.23 52.47 6 9.07 58.81 6 10.7
Zn 100.27 6 27.8 82.79 6 19.0 123.71 6 17.0 90.79 6 30.6 71.22 6 16.6 47.88 6 11.7
a
Limits of detection (μg/g dry weight): Pb, 0.003; Hg, 0.002; Cr, 0.01; Cd, 0.002; Cu, 0.01; Zn, 0.002. Regulatory limits (μg/g) set by the
EU (10): Pb, 1.5; Hg, 0.5; Cd, 1.0; by the Interstate Shellfish Sanitation Conference (33): Cr, 13; and by the FDA (32): Cu, 100; Zn, 150.
* P , 0.05.

Salmonella can contaminate shellfish via human feces (e.g., Water parameters of the three study sites did not
sewage), particularly in the local population where the significantly vary over 12 months except for salinity. Water
bacteria are excreted by either clinically ill patients or temperature was 28 to 348C, pH was 8.0 to 8.8, and
carriers (30). In the Philippines, official reports of typhoid dissolved oxygen was 3.3 to 5.2 mg/L. The salinity of the
and paratyphoid fever due to consumption of bacterially oyster cultivation water generally was 28 to 34 ppt except in
contaminated or improperly cooked oysters are rare, October, in which heavy rainfall reduced the salinity in the
perhaps because the majority of the cases, particularly Cogon River and Cabugao Bay to ca. 22 ppt. In the Palina
those in isolated areas, are not properly diagnosed or River, salinity likewise dropped to ca. 4 ppt due to increased
reported. However, Salmonella still causes a large number freshwater inputs.
of shellfish-associated outbreaks in the Philippines. In summary, this study has provided the first compre-
The concentrations of Cd, Pb, Hg, and Cr in oysters hensive information clearly illustrating that the microbio-
collected from the three sampling areas were below the logical quality of oysters and the water quality at the oyster
limits of detection and therefore complied with the growing sites are poor and far below the limits recom-
regulatory limits (Table 2) set by the EU (10) and the mended in the NSSP and SHACC. In the years since this
U.S. Food and Drug Administration (33). These low metal study was conducted, periodic monitoring by the competent
concentrations may be attributed to the fact that Capiz authority (the Bureau of Fisheries and Aquatic Resources)
Province lacks industrial factories that often release in these oyster growing areas has revealed no apparent
effluents containing these toxic metals into the cultivation
changes, and these oyster growing areas would still be
water of the oyster growing areas. In contrast, Zn and Cu
categorized as class B according to EU standards and
concentrations in oysters were high during the wet season
“prohibited” according to U.S. standards (28). Thus, export
but still within allowable limits (32). The high Zn and Cu
of Philippine oysters to the EU or the United States
concentrations in oyster flesh are not surprising because
currently is not possible. Oysters harvested from these areas
these metals are regarded as essential nutrients (7). As an
must be treated by depuration or relaying or must be cooked
essential element for normal growth and development (24),
with an approved method as stipulated in the SHACC
Cu is present in aquatic invertebrates, and its bioaccumu-
lation can increase with animal size (26). Zn and Cu exist as guidelines (11) to render these oysters safe for human
ions (Zn2þ and Cu2þ) and act as natural cofactors in consumption. Because sanitary protocols stipulated in the
enzymatic reactions inside cells, and oysters acquire high Philippine National Standard on the Code of Practice for
concentrations of these metals from their diet (29). High Cu Live and Raw Bivalve Molluscs (27) have just been
and Zn concentrations in oysters are mostly related to circulated for adoption, government law enforcement
anthropogenic activities (20). Azlisham et al. (4) reported agencies and the private sector should work in concert to
high concentrations of Zn followed by Cu in the flesh of strictly implement these regulations and all laws pertinent to
oysters collected from Setiu Lagoon, Terenggau, Malaysia. the indiscriminate dumping of effluents and wastes from
These high concentrations could be associated with domestic and industrial sources to mitigate the pollution
effluents coming from Cu- and Zn-based agricultural problems besetting the oyster industry. Rehabilitation of
products such as feed additives, liming materials, inorganic these oyster areas, assessment of production techniques, and
fertilizers, and antibiotics that have been widely applied in introduction of appropriate stock management schemes are
nearby shrimp and fish rearing ponds (7, 25). Although the urgently needed and should be implemented by both the
concentrations of Cr, Cd, Hg, and Pb were below the private sector and government agencies, such as the newly
allowable limits, periodic monitoring of these toxic metals established National Shellfish Sanitation Program of the
in oysters should continue because these metals have been Bureau of Fisheries and Aquatic Resources, to sustain the
commonly implicated in human poisonings (5). long-term viability of the Philippine oyster industry.
20 PAKINGKING ET AL. J. Food Prot., Vol. 85, No. 1

ACKNOWLEDGMENTS 16. Flick, G. J., and L. A. Granata. 2010. Biological safety of fresh and
processed shellfish. SRAC Publication 4910. Southern Regional
We express our heartfelt gratitude to Southeast Asian Fisheries Aquaculture Center, Mississippi State University, Delta Research and
Development Center/Aquaculture Department, Bureau of Fisheries and Extension Center, Stoneville.
Aquatic Resources—Region VI, Negros Prawn Producers Cooperative, the 17. Food and Agriculture Organization of the United Nations. 2012. The
local government units of the Municipality of Ivisan and Roxas City, and state of world fisheries and aquaculture—2012. Food and Agriculture
collaborators from Palina Greenbelt Ecopark, Barangay Cabugao, and Organization of the United Nations, Rome.
Barangay Cogon. This study was funded by the Department of Science and 18. Froelich, B. A., and R. T. Noble. 2016. Vibrio bacteria in raw oysters:
Technology, Philippine Council for Agriculture, Aquatic and Natural managing risks to human health. Philos. Trans. R. Soc. Lond. B 371.
Resources Research and Development (study code 6286-T-RD-DOST03). https://doi.org/10.1098/rstb.2015.0209
19. International Commission on Microbiological Specifications for
REFERENCES Foods. 1986. Their significance and methods of enumeration, p.
19–30. In F. S. Thatcher and D. S. Clark (ed.), Microorganisms in
1. American Public Health Association. 2012. Recommended proce-
foods, 2nd ed., vol. 1. University of Toronto Press, Toronto, Ontario,
dures for the examination of seawater and shellfish, 21st ed.
Canada.
American Public Health Association, Washington, DC.
20. Jara-Marini, M. E., J. N. Tapia-Alcaraz, J. A. Dumer-Gutiérrez, L.
2. Andrews, W. H., H. Wang, A. Jacobson, B. Ge, G. Zhang, and T.
García-Rico, J. García-Hernández, and F. Páez-Osuna. 2013.
Hammack. 2007. Salmonella, chap. 5. In Bacteriological analytical
Comparative bioaccumulation of trace metals using six filter feeder
manual. Available at: https://www.fda.gov/Food/FoodScience
organisms in a coastal lagoon ecosystem of the central-east Gulf of
Research/LaboratoryMethods/ucm070149.htm. Accessed 3 June
California. Environ. Monit. Assess. 185:1071–1085. https://doi.org/
2021.
10.1007/s10661-012-2615-z
3. Azevedo, J. A. M., A. B. Barros, P. R. B. de Miranda, J. G. da Costa,
21. Kaysner, C. A., and A. DePaola. 2004. Vibrio, chap. 9. In
and V. X. Nascimento. 2019. Biomonitoring of heavy metals (Fe, Zn,
Bacteriological analytical manual. Available at: https://www.fda.
Cu, Mn, Cd and Cr) in oysters: Crassostrea rhizophorae of mangrove
gov/food/foodscienceresearch/laboratorymethods/ucm070830.htm.
areas of Alagoas (Brazil). Braz. Arch. Biol. Technol. 62. https://doi.
Accessed 3 June 2021.
org/10.1590/1678-4324-2019180211
22. Lee, R., A. Lovatelli, and L. Ababouch. 2008. Bivalve depuration:
4. Azlisham, M., V. J. Vedamanikam, and N. A. M. Shazilli. 2009.
fundamental and practical aspects. FAO Fisheries Technical Paper
Concentrations of cadmium, manganese, copper, zinc, and lead in the
551, Food and Agriculture Organization of the United Nations,
tissues of the oyster (Crassostrea iredalei) obtained from Setiu
Rome.
Lagoon, Terengganu, Malaysia. Toxicol. Environ. Chem. 91:251–
23. Lovatelli, A. 1988. Status of oyster culture in selected Asian
258.
countries. Available at: http://www.fao.org/docrep/field/003/
5. Balali-Mood, M., K. Naseri, Z. Tahergorabi, M. R. Khazdair, and M. AB716E/AB716E11.htm. Accessed 3 June 2021.
Sadeghi. 2021. Toxic mechanisms of five heavy metals: mercury, 24. Ochoa, V., C. Barata, and M. Carmen-Riva. 2013. Heavy metal
lead, chromium, cadmium, and arsenic. Front. Pharmacol. content in oysters (Crassostrea gigas) cultured in the Ebro Delta in
12:643972. https://doi.org/10.3389/fphar.2021.643972 Catalonia, Spain. Environ. Monit. Assess. 185:6783–6792. https://
6. Brands, D. A., A. E. Inman, C. P. Gerba, C. J. Mare, S. J. Billington, doi.org/10.1007/s10661-013-3064z
L. A. Saif, J. F. Levine, and L. A. Joens. 2005. Prevalence of 25. Páez-Osuna, F., and C. C. Osuna-Martínez. 2015. Bioavailability of
Salmonella spp. in oysters in the United States. Appl. Environ. cadmium, copper, mercury, lead, and zinc in subtropical coastal
Microbiol. 71:893–897. lagoons from the southeast Gulf of California using mangrove
7. Campbell, N. A., J. B. Reece, and L. G. Mitchell. 1999. Biology, 5th oysters (Crassostrea corteziensis and Crassostrea palmula). Arch.
ed. Addison, New York. Environ. Contam. Toxicol. 68:305–316. https://doi.org/10.1007/
8. de Astudillo, L. R., I. C. Yen, and I. Bekele. 2005. Heavy metals in s00244-014-0118-3
sediments, mussels and oysters from Trinidad and Venezuela. Rev. 26. Pan, K., and W. X. Wang. 2009. Biodynamics to explain the
Biol. Trop. 53(Suppl. 1):41–53. difference of copper body concentrations in five marine bivalve
9. Duncan, P. F., M. N. Andalecio, E. Peralta, L. Laureta, A. R. Hidalgo, species. Environ. Sci. Technol. 43:2137–2143. https://doi.org/10.
and R. Napata. 2009. Evaluation of production technology, product 1021/es802888u
quality and market potential for the development of bivalve mollusc 27. Philippine National Standards, Bureau of Agriculture and Fisheries
aquaculture in the Philippines. Australian Centre for International Standards. 2011. Bureau of Agriculture and Fisheries product
Agricultural Research, Canberra. standards for live and raw molluscs. PNS/BAFPS 89:2011, ICS
10. European Commission. 2006. Commission Regulation (EC) No 67.120.30. Bureau of Agriculture and Fisheries Standards, Quezon,
1881/2006 of 19 December 2006 setting maximum levels for certain Manila, Philippines.
contaminants in foodstuffs. Off. J. Eur. Union L 364:5–24. 28. Regidor, S. (Bureau of Fisheries and Aquatic Resources, Philip-
11. European Commission. 2007. Commission Regulation (EC) No pines). 2021. Personal communication.
1441/2007 of 5 December 2007 amending regulation (EC) No 2073/ 29. Silva, C. A. R., P. S. Rainbow, B. D. Smith, and Z. L. Santos. 2001.
2005 on microbiological criteria for foodstuffs. Off. J. Eur. Union L Biomonitoring of trace metals contaminants in Potengi Estuary, Natal
322:12–29. (Brazil) using the oyster Crassostrea rhizophorae, a local food
12. Feng, P., S. D. Weagant, M. A. Grant, and W. Burkhardt. 2002. source. Water Res. 35:4072–4078.
Enumeration of Escherichia coli and the coliform bacteria, chap. 4. 30. Souza, D. S., A. P. Ramos, F. F. Nunes, V. Moresco, S. Taniguchi, D.
In Bacteriological analytical manual. Available at: https://www.fda. A. Leal, S. T. Sasaki, M. C. Bícego, R. C. Montone, M. Durigan, A.
gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064948. L. Teixeira, M. R. Pilotto, N. Delfino, R. M. Franco, C. M. Melo, A.
htm. Accessed 3 June 2021. C. Bainy, and C. R. Barardi. 2012. Evaluation of tropical water
13. Fisheries Statistics of the Philippines. 2010. Fisheries statistics of the sources and mollusks in southern Brazil using microbiological,
Philippines 2008–2010. Available at: https://psa.gov.ph/sites/default/ biochemical, and chemical parameters. Ecotoxicol. Environ. Saf.
files/fish_stat_2008-2010.pdf. Accessed 3 June 2021. 76:153–161.
14. Fisheries Statistics of the Philippines. 2011. Fisheries statistics of the 31. Szuster, B. W., K. Chalermwat, M. Flaherty, and P. Intacharoen.
Philippines 2009–2011. Available at: https://psa.gov.ph/sites/default/ 2008. Peri-urban oyster farming in the upper Gulf of Thailand.
files/fishriestat_2009-2011.pdf. Accessed 3 June 2021. Aquac. Econ. Manag. 12:268–288.
15. Fisheries Statistics of the Philippines. 2015. Fisheries statistics of the 32. U.S. Food and Drug Administration. 2001. Products hazards and
Philippines 2013–2015. Available at: https://psa.gov.ph/sites/default/ controls guidance, 3rd ed. U.S. Food and Drug Administration, Silver
files/FStatPhil13-15docx.pdf. Accessed 3 June 2021. Spring, MD.
J. Food Prot., Vol. 85, No. 1 MICROBIOLOGICAL QUALITY AND HEAVY METALS IN SLIPPER OYSTER 21

33. U.S. Food and Drug Administration. 2007. National Shellfish Food and Drug Administration, Center for Food Safety and Applied
Sanitation Program. Guide for the control of molluscan shellfish. Nutrition, College Park, MD.
Interstate Shellfish Sanitation Conference, Columbia, SC. 35. Yap, C. K., A. R. Azmizan, and M. S. Hanif. 2011. Biomonitoring of
trace metals (Fe, Cu, and Ni) in the mangrove area of peninsular
34. U.S. Food and Drug Administration, Center for Food Safety and Malaysia using different soft tissues of flat tree oyster Isognomon
Applied Nutrition. 2009. National Shellfish Sanitation Program. alatus. Water Air Soil Pollut. 218:19–36. https://doi.org/10.1007/
Guide for the control of molluscan shellfish: 2007 revision. U.S. s11270-010-0621-8