JOURNAL OF FOOD SCIENCE

FOOD MICROBIOLOGY AND SAFETY

Chlorine Dioxide Treatment of Seafoods

to Reduce Bacterial Loads
J.M. Kim, T.-S. Huang, M.R. Marshall, and C.-I. Wei

ABSTRACT MATERIALS & METHODS

Various seafoods were treated with fresh chlorine dioxide (ClO2) solutions (20,
40, 100, and 200 ppm total available ClO2) in 3.5% brine for 5 min, and bacterial Chlorine-demand-free water
loads and sensory quality were evaluated after 0, 3, and 7 d storage on ice. The Chlorine-demand-free water (CDF wa-
ClO2 treated groups at each time period had lower bacterial counts than non- ter) was prepared following the method of
treated and brine-treated groups. The differences in bacterial counts were sig- Ghanbari et al. (1982), by passing distilled
nificant, especially for groups treated with 100 and 200 ppm ClO2. Treated ClO2 water through two successive Barnstead
solutions contained very low or no bacterial loads. Treated red grouper and salmon deionizing units (Barnstead Inc., Dubuque,
with 100 or 200 ppm ClO2 developed skin discoloration (lighter color) and a choco- Iowa, U.S.A.) and then a glass column con-
late color in the gills. taining Porapak ® Q (Supelco Inc., Belle-
Key Words: chlorine dioxide, bactericidal, seafood safety, red grouper, salmon fonte, Pa., U.S.A.). This water was used to
prepare all reagents.
INTRODUCTION peeled vegetables and fruits to enhance Preparation of stock ClO2 and
CHLORINE DIOXIDE (ClO2) HAS BEEN freshness and extend shelf life. The U.S. working solutions
recognized as a bactericidal, viricidal, and Food and Drug Administration (FDA) al- A ClO2 stock solution was prepared by
fungicidal agent. It is widely used in Europe lows for ClO2 as a disinfectant at 3 ppm re- mixing 85% phosphoric acid (Fisher Scien-
as an alternative to chlorine (hypochlorous sidual in poultry chilling water that is in tific, Certified ACS) for 5 min at room
acid [HOCl] and hypochlorite [OCl-]) for contact with whole poultry carcasses (Fed- temperature with Oxine® concentrate, a 2%
drinking water disinfection (Symons et al., eral Register, 1995). ClO2 at 5 ppm can be chlorite solution (Bio-Cide International
1978) and in many drinking water treatment used in a sanitizing rinse for uncut, un- Inc., Norman, Okla., U.S.A.) (1:20, v/v), in
plants in the United States. Due to health peeled fruits and vegetables. The National a brown flask sealed with a glass stopper.
concerns related to trihalomethanes (THMs) Food Processors Association (NFPA) has The reaction mixture was then diluted with
and other reaction products generated during petitioned the FDA to allow ClO 2-treated 9 volumes of ice-cold CDF water. This
treatment with aqueous chlorine, alternative water as a rinse for cut or peeled fruits and stock solution was used to prepare various
disinfectants are needed. ClO2 treatment vegetables (Food Chemical News, 1994). working solutions (20, 40, 100 and 200
produces very little or no THMs in treated However, ClO 2 is not approved for treat- ppm) in 3.5% ice-cold brine. Brine was
water and is, therefore, a potential substitute ment of seafood because of the lack of tox- prepared by dissolving 35 g NaCl in 1 L of
for aqueous chlorine. The bactericidal activ- icological and safety information. CDF water. The stock solution was used to
ity of ClO2 is not affected by alkaline condi- Per capita seafood consumption rose prepare working solutions in 3.5% brine,
tions or organic compounds (Dychdala, steadily in the United States from 4.67 kg based on the content of total available chlo-
1991) and is being considered for applica- in 1960 to 7.03 kg in 1990 (U.S. Depart- rine (TAC) and ClO2 (TACD expressed as
tion in the food industry. ment of Commerce, 1960-1990). Fish and mg/mL ClO2), as determined by iodometric
As a bactericide, ClO 2 has been tested shellfish are common vehicles of food-

Food Microbiology and Safety

to reduce bacterial populations in poultry
chiller water (Tsai et al., 1995), on pork
carcasses (Svoboda and Schwerdt, 1977),
fecal contaminated beef carcass (Cutter and
Dorsa, 1995), and in cucumber hydrocool-
ing water (Reina et al., 1995). ClO 2 treat-
ments extended the shelf life of chicken
broilers (Lillard, 1980) and reduced the in-
cidence of Salmonella spp. on poultry car-
casses (Thiessen et al., 1984). ClO 2 was
more effective than aqueous chlorine in
killing Listeria monocytogenes inoculated
on fish cubes (Lin et al., 1996). Conse-
quently, ClO 2 could serve as a processing
aid for seafood, poultry, red meat, and
Authors Kim, Huang, and Marshall are with the Food
and Environmental Toxicology Laboratory, Food
Science and Human Nutrition Dept., Univ. of Florida,
Gainesville, FL 32611-0720. Author Wei, formerly
with the Univ. of Florida, is now with the Nutrition
and Food Science Dept., 328 Spidle Hall, Auburn
Univ., Auburn, AL 36849-5605. Address inquiries to
Dr. C.-I. Wei (E-mail: cwei@humsci.auburn.edu).
© 1999 Institute of Food Technologists
borne diseases. They can be contaminated
with pathogenic and spoilage bacteria dur-
ing harvest, production, and distribution
due to improper handling and storage. Sea-
food is involved in an estimated 11% of
foodborne outbreaks in the United States,
and bacterial pathogens are involved in
about 25% of the disease outbreaks linked
to seafood (Bean and Griffin, 1990).
Puente et al. (1992) suggested that ClO2
had potential as an antimicrobial agent
against Vibrio parahaemolyticus in seawa-
ter. Kim et al. (1997, 1998) showed that
ClO2 treatment of fish fillets did not great-
ly affect their fatty acid composition nor
the contents of protein, fat, vitamins, and
minerals. Our objective was to determine
the effectiveness of using ClO2 in control-
ling bacterial loads and quality of various
seafood products over a 7-d storage. Such
information is important for government
regulatory agencies in their evaluation of
ClO2 in seafood processing.
Volume 64, No. 6, 1999—JOURNAL OF FOOD SCIENCE
and N, N-diethyl-p-phenylene-diamine
(DPD) ferrous titration methods (APHA,
1989). Both the stock and working ClO 2
solutions were freshly prepared on each
day of experiment.
Determination of the bactericidal
effectiveness of ClO2 solution on
various seafoods
Atlantic salmon (Salmo salar) aquacul-
tured in Chile and red grouper (Epi-
nephelus morio) harvested from the Gulf of
Mexico were purchased from a local sea-
food store (Gainesville, Fla., U.S.A.) with
no record of catch or storage history. Salm-
on were individually wrapped in plastic
and packaged in layers between crushed ice
in a polystyrene box (inside diameters: L
80 × W 20 × D 16.5 cm with 2-cm wall
thickness for the box and lid). The sealed
boxes were delivered by air transportation
to Miami, Fla., and then by truck to
Gainesville. Time from harvest to handling
in the laboratory was < 48 h. Red grouper
1089
 ClO2 Treatment of Seafoods to Reduce Bacterial Loads . . .
were shipped in ice by truck from Cedar
Key, Fla., to Gainesville. Time from har-
vest to handling in the laboratory was less
than 24 h. Calico scallops (Aequipecten
gibbus) were packed by Seasweet Scallop
Company (Cape Canaveral, Fla.), and
brown shrimp (Penaeus aztecus, headless
with shell, 74 tails/kg) were from Jubilee
Foods Inc. (Bayou La Batre, Ala.). The
shrimp sample reportedly received no pre-
vious sulfite or phosphate treatment.
Fish fillets, scallops and shrimp. Fish
fillets were prepared from whole red grou-
per and salmon, skinned, and cut into piec-
es (50g). After 54 pieces of red grouper and
salmon fillets were weighed, they were
dipped in an equal volume (1:1, w/v) of
ice-cold sterile brine for 1 min (pre-wash).
Following draining of excess liquid, 9 piec-
es were randomly chosen and treated with
4 volumes (1:4, w/v) of ice-cold sterile
brine (control) or freshly prepared ClO2 so-
lutions (20, 40, 100, or 200 ppm TACD) in
brine for 5 min with continuous stirring.
After an adequate amount of 1 N Na 2S2O3
solution was added to quench residual
chlorine in each ClO 2 solution, each fish
piece was removed, placed in separate
Whirl-Pak® bags (Nasco Inc., Fort Atkin-
son, Wis., U.S.A.), labeled, and stored with
crushed ice for 7 d at 5 °C.
Similarly, about 1800 g of scallops or
brown shrimp were dipped in an equal vol-
ume (1:1, w/v) of ice-cold sterile brine for
1 min (pre-wash). After draining the excess
liquid, portions (about 100 g) were re-
moved and randomly treated with 4 vol-
umes (1:4, w./v) of ice-cold sterile brine or
ClO2 solutions (20, 40, 100, or 200 ppm
TACD). Three 100 g portions were used
for each test group. Following the addition
of an adequate amount of 1 N Na2S2O3 so-
lution to quench residual chlorine and the
draining of excess liquid, 3 portions (about
30 g each) were removed from each group
Food Microbiology and Safety
and placed in separate sterile Whirl-Pak ®
bags. The bags were labeled and stored
with crushed ice for 7 d at 5 °C. The pH of
the test solutions was monitored before and
after dipping the fish fillets, scallops, or
brown shrimp. The experiments were re-
peated.
On each day of testing (day 0, 3, and 7),
3 pieces of red grouper or salmon, or 3
bags of scallops or brown shrimp were re-
moved from each group for quality evalua-
tion and bacterial enumeration. For the fish
fillets, bacterial enumeration was per-
formed by adding 9 volumes (1:9, w/v) of
sterile peptone water (0.1%) to each fillet
in a Whirl-Pak ® bag. The bag was placed
between the hands and rubbed back and
forth for 2 min before the peptone water
was removed and serially diluted with ster-
ile Butterfield’s buffer. For scallops and
shrimp, bacterial enumeration was per-
formed by homogenizing each sample at
high speed for 1.5 min with 9 volumes of
1090 JOURNAL OF FOOD SCIENCE—Volume 64, No. 6, 1999
0.1% peptone in sterile blender jars. The
homogenates were serially diluted with
sterile Butterfield’s buffer, and each diluent
was surface plated on quadruplicate plate
count agar (PCA) plates, containing 1.5%
NaCl. Pour plate method was also used for
some samples. Bacterial colonies on plates
were counted after incubation at 25 °C for
72 h.
Whole fish. Whole fish (18 each, gut-
ted) of salmon (about 3.6 kg each) or red
grouper (about 2.8 kg each) were washed
under running water for 2 min. The fish
were treated in a clean container with 4
volumes (1:4, w/v) of ice-cold sterile brine
or ClO2 solutions in brine (20, 40, 100, or
200 ppm TACD). After draining excess liq-
uid, each fish was labeled and placed on a
stainless steel table for sensory evaluation.
Three fish were used for each test group.
Following initial sensory evaluation, the
fish samples were stored for 7 d in a sea-
food display case (3 °C) with crushed ice.
Bacterial numbers on the whole fish
were determined by removing 2 skinned ar-
eas (3 × 3 cm2 each) from each fish, using
a sterile surgical blade and a pair of for-
ceps. For salmon, 2 additional muscle areas
(3 × 3 cm2 each) were removed from the
inside of the belly flaps of the correspond-
ing fish. The skin and muscle samples from
the same fish were each put in sterile
Whirl-Pak bags, weighed, and then added
with 9 volumes (1:9, w/v) of sterile 0.1%
peptone water. After the sample bags were
manually rubbed as described for 2 min,
the peptone water was removed and serial-
ly diluted with sterile Butterfield’s buffer
for bacterial enumeration using surface
plating or pour plate methods.
On day 3 and 7 of storage, skinned (and
salmon muscle) samples were removed
again from each fish for determination of
any time-related changes in bacterial num-
bers. Since each test fish was labeled indi-
vidually, the changes in bacterial numbers
for each fish over the 7-d period could be
determined. The experiment with red grou-
per was repeated once (2 replicates), while
salmon was repeated twice (3 replicates).
Sensory evaluation of test seafood
samples
Seafood samples were evaluated on day
0, 3, and 7 at room temperature by a 10-
member panel, aged 24 to 48 yr (3 females,
7 males), from the Dept. of Food Science
and Human Nutrition at the Univ. of Flori-
da. Panelists met for 2 training sessions,
using fresh and stale samples and those that
had been treated with ClO2, in order to re-
view seafood attributes and establish uni-
form definition of terms and procedures for
sensory evaluation.
Seafood treated with brine, 20, 40, 100,
and 200 ClO2 were prepared. Fresh seafood
were used as a reference (control). A total
of 18 samples (triplicate for each group)
were used for each seafood. All samples
were coded with random 3 digit numbers.
Panelists were not informed as to treat-
ments. Panelists wore disposable gloves for
handling the samples and were instructed
to change gloves between samples. Panel-
ists recorded perceptions of each sensory
attribute on sensory evaluation sheets. The
degrees of abnormality (or defect) were
categorized into slight, moderate, and ex-
cessive with intensity ratings of 1 to 6 for
each category, where 1 = excellent, 3 to 4 =
fair, and 6 = very poor, respectively, for de-
scriptive terms. The terms were appearance
defects, discoloration, and formation of
odor for tested fish fillets, scallops, and
brown shrimp, and appearance defects, skin
discoloration, eye color, body damage, gill
and gut cavity, belly flaps, surface defects,
and formation of odor for tested whole red
grouper and salmon. The final gradings (A,
B, C) were made from the sensory evalua-
tion sheets, following those described by
the National Marine Fisheries Service
(NMFS) Fishery Products Inspection Man-
ual (NMFS, 1975). U.S. Grade A fish fillets
should (1) possess good flavor and odor
characteristic of the species and (2) comply
with the limits for defects for U.S. Grade A
quality as outlined in §263.104. U.S. Grade
B fish fillets should (1) possess reasonably
good flavor and odor characteristic of the
species and (2) comply with the limits for
defects for U.S. Garde B quality in accor-
dance with §263.104. U.S. Grade C fish fil-
lets should (1) possess minimal acceptable
flavor and odor characteristic of the species
with no objectionable off-flavors or off-
odors and (2) comply with the limits for
defects for U.S. Grade C quality in accor-
dance with §263.104.
Statistical analysis
Bacterial numbers in CFU/g sample
were transformed into log 10 for statistical
analysis. An analysis of variance (ANOVA)
was performed using the general linear
models procedure of the Statistical Analy-
sis System (SAS Institute Inc., 1995). Dun-
can’s multiple range test was used to obtain
pairwise comparisons among sample
means at a significance level of P = 0.05.
RESULTS & DISCUSSION
THE CONCENTRATION OF ClO2 IN BRINE
caused a dose-related decrease in pH (Table
1). Dipping of fish fillets, scallops, and
brown shrimp caused increases in pH in the
treated ClO 2 solutions. The treated 100
ppm and 200 ppm ClO2 solutions had less
increases in pH than those with 20 ppm
and 40 ppm ClO 2 solutions. Dipping of
scallops in ClO2 solutions caused more of
an increase in pH than dipping of shrimp,
fish fillets, or whole fish. Apparently, scal-
lops provided more proteins that interact
with ClO2 in the test solutions.
The bactericidal effectiveness of ClO2
Table 1 — Changes in pH of test solutions following treatment of various seafood samples (P < 0.05) bacterial numbers than the non-
After treatment treated control on day 7 of storage.
Before Grouper Salmon Whole Whole
The nontreated fillets of red grouper
treatment fillet fillet Scallops Shrimp grouper salmon (Table 3) and salmon (Table 4) and those
PWBa 6.10 ± 0.19 6.17 ± 0.03 6.23 ± 0.02 NDb ND ND ND
treated with brine and ClO2 solution at 20
Brine 6.04 ± 0.15 6.07 ± 0.04 6.09 ± 0.06 6.14 ± 0.07 6.61 ± 0.25 6.19 ± 0.01 6.16 ± 0.04 or 40 ppm were usually considered very
ClO2 good quality (Grade A), showing no ap-
20 ppm 3.74 ± 0.12 5.06 ± 0.09 4.56 ± 0.04 5.71 ± 0.09 5.67 ± 0.07 4.01 ± 0.01 4.10 ± 0.08 pearance or discoloration defects. Fishy
40 ppm 3.34 ± 0.11 4.53 ± 0.08 4.00 ± 0.06 5.29 ± 0.18 4.53 ± 0.08 3.41 ± 0.09 3.56 ± 0.06
100 ppm 3.01 ± 0.08 3.87 ± 0.08 3.41 ± 0.11 4.55 ± 0.32 3.53 ± 0.12 3.11 ± 0.01 3.12 ± 0.04 odor was not noted in any of the test fillets
200 ppm 2.72 ± 0.07 3.15 ± 0.11 3.01 ± 0.03 3.77 ± 0.29 3.05 ± 0.19 2.77 ± 0.04 2.78 ± 0.08 on day 0. However, the nontreated red
aPWB = The pre-washing brine was the ice-cold sterile brine used for dipping the fish fillets at 1:1 (w/v) for 1 min. grouper fillets, and those treated with brine
bND = Not determined
or 20 ppm ClO2 started to rate lower in
muscle consistency and discoloration fol-
lowing cold storage for 3 d. These defects
was demonstrated in the treated ClO2 solu- bers were not significant (P > 0.05) among worsened after 7 d. Furthermore, an unde-
tions with seafood samples (Fig. 1). The the 6 groups. The number of microorgan- sirable fishy odor developed with some of
treated 100 ppm and 200 ppm ClO 2 solu- isms in each group increased following the test fillets. A rusty color occurred with
tions with scallops, brown shrimp, and fish storage of the fillets for 3 or 7 d. However, the treated red grouper fillets at the 100 or
fillets contained no viable bacteria, al- the 6 groups showed no difference in bacte- 200 ppm ClO 2 treatment, possibly due to
though low bacterial loads were detected in rial numbers following storage for 3 and 7 the reaction of fish organics with ClO 2.
the 20 ppm and 40 ppm ClO2 solutions. d. Except for the salmon fillets treated with Similar observations were noticed with the
None of the treated ClO 2 solutions used 200 ppm ClO 2, differences in bacterial nontreated salmon fillets. Salmon fillets
with whole fish contained any viable bacte- numbers among the other 5 groups on day treated with brine or ClO2 solutions at 20
ria. Thus, less organic compounds were re- 0 were not significant. The bactericidal ef- or 40 ppm were rated better in quality than
leased from the whole fish to react with fect of ClO 2 was still evident among the nontreated controls. Discoloration of salm-
ClO2, which in turn provided more active various treatment groups following cold on fillets also occurred after treatment with
ClO2 for effective bactericidal activity. The storage for 3 and 7 d. Salmon fillets treated ClO2 solutions at 100 or 200 ppm.
results of this study also indicated that the with 100 ppm or 200 ppm ClO2 had lower Note that quality varied with individual
20 ppm ClO2 solution was an effective dis-
infecting solution for rinsing the whole
fish.
Table 2 — Changes in bacterial numbers over time on treated seafood samples with differ-
Fish fillets ent concentrations of ClO2 solutions
Washing of red grouper and salmon fil-
lets with brine slightly reduced the initial
bacterial loads of the pre-washed fillets (no ClO2 treatment log10 CFU/g
treatment group). Compared to the non- Sample (ppm) Day 0 Day 3 Day 7
treated control and brine treated groups, Grouper fillet No treatment 4.82 ± 0.52C 6.34 ± 0.47B 7.95 ± 0.35A
treatment of red grouper fillets with ClO2 Brine 4.87 ± 0.43C 6.24 ± 0.46B 7.86 ± 0.37A
solutions on day 0 caused a dose-related 20 4.77 ± 0.61C 6.21 ± 0.35B 7.76 ± 0.66A
40 4.76 ± 0.44C 5.93 ± 0.56B 7.62 ± 0.37A
decrease in the natural microflora (Table 100 4.55 ± 0.52C 5.99 ± 0.57B 7.45 ± 0.37A
2). However, differences in bacterial num- 200 4.28 ± 0.36C 5.66 ± 0.85B 7.49 ± 0.51A
Salmon fillet No treatment 4.38 ± 0.57Ba 5.13 ± 1.00B 7.58 ± 0.45Aa
Brine 4.04 ± 0.51Ca 5.53 ± 1.17B 7.11 ± 0.45Aab
20 4.02 ± 0.56Ca 4.93 ± 0.56B 7.07 ± 0.41Aab

Food Microbiology and Safety

40 3.79 ± 0.42Cab 4.94 ± 0.84B 6.96 ± 0.55Aab
100 3.74 ± 0.65Cab 4.62 ± 0.44B 6.91 ± 0.77Ab
200 3.31 ± 0.44Cb 4.75 ± 0.60B 6.47 ± 0.39Ab
Scallops No treatment 4.99 ± 0.19a 4.98 ± 0.28a 5.40 ± 0.45a
Brine 4.80 ± 0.09Bab 4.68 ± 0.13Bb 5.59 ± 0.25Aa
20 4.80 ± 0.18ABab 4.54 ± 0.10Bb 4.86 ± 0.38Ab
40 4.74 ± 0.23ABb 4.56 ± 0.11Bb 4.95 ± 0.35Ab
100 4.43 ± 0.15Cc 4.15 ± 0.16Bc 4.65 ± 0.20Ab
200 4.44 ± 0.22Ac 3.92 ± 0.43Bc 4.74 ± 0.42Ab
Whole grouper (skin) No treatment 5.34 ± 0.61Ca 6.39 ± 0.52Ba 8.03 ± 0.53Aab
Brine 4.66 ± 0.59Cb 6.07 ± 0.47Bab 8.14 ± 0.57Aa
20 4.42 ± 0.37Cb 5.99 ± 0.35Bab 7.71 ± 0.53Aabc
40 4.65 ± 0.61Cb 5.77 ± 0.79Babc 7.89 ± 0.74Aab
100 4.06 ± 0.37Cb 5.51 ± 0.41Bbc 7.16 ± 0.32Ac
200 4.13 ± 0.40Cb 5.13 ± 0.45Bc 7.35 ± 0.45Abc
Whole salmon (skin) No treatment 3.32 ± 0.69C 4.76 ± 0.93B 6.44 ± 0.78A
Brine 3.31 ± 0.74C 4.82 ± 0.44B 6.46 ± 0.49A
20 3.18 ± 0.83C 4.76 ± 1.00B 6.26 ± 1.08A
40 3.36 ± 0.91C 4.82 ± 1.29B 6.13 ± 1.17A
100 2.93 ± 0.96C 4.63 ± 0.83B 6.02 ± 0.83A
200 2.75 ± 1.14C 4.58 ± 0.70B 6.05 ± 1.12A
Whole salmon (muscle) No treatment 3.96 ± 0.81Ca 5.29 ± 0.78Ba 7.21 ± 0.73Aa
Brine 3.69 ± 0.75Cab 5.33 ± 0.69Ba 7.15 ± 0.56Aa
20 2.94 ± 0.89Cbc 4.38 ± 0.83Bab 6.55 ± 0.89Aab
Fig. 1—Residual bacterial numbers in 40 2.71 ± 1.06Ccd 4.06 ± 1.34Bb 6.11 ± 0.95Abc
treated ClO2 and brine solutions following 100 1.96 ± 1.16Cd 3.34 ± 1.17Bb 5.50 ± 1.20Ac
dipping with various seafood samples. 200 1.89 ± 1.05Cd 3.44 ± 1.25Bb 5.73 ± 0.69Abc
PWB=Pre-Washing Brine. This was the ice- A-CWithin each row of the test seafood, means followed by the same letter are not significantly different from each other
cold sterile brine used for dipping the fish at P = 0.05.
fillets, shrimp, and scallops at 1:1 (w/v) for a-dWithin each column of the test seafood, means followed by the same letter are not significantly different from each
1 min. other at P = 0.05. Data are means ± standard deviations from two trials each having triplicate samples.

Volume 64, No. 6, 1999—JOURNAL OF FOOD SCIENCE 1091

 ClO2 Treatment of Seafoods to Reduce Bacterial Loads . . .

fish prior to filleting. Quality variation of Table 3—Gradinga of grouper fillets following treatments with ClO2 solutions or brine and
then stored at 4 oC for 0, 3 or 7 d
fish samples also occurred between batches
used for different trials on different test Experiment #1 Experiment #2
dates. Such individual- and batch-varia- Replicate Replicate
tions of test samples would affect the over- Treatment Day I II III I II III
all evaluation of the bactericidal effective-
ness of ClO2 and its affect on fish quality, Control 0 A A A A B B
3 A A A B B B
especially since the number of test samples 7 C B C B B B
was small. Brine 0 A A A A A B
3 A B B B B B
7 C C C B B B
Calico scallops 20 ppm 0 A A A B A A
Scallops treated with ClO2 solutions at 3 A A B B A B
40 ppm, 100 ppm, and 200 ppm on day 0 7 B C C B B B
40 ppm 0 A A A B A B
had lower bacterial numbers than the non- 3 A A A B A B
treated control (Table 2). On day 3 of stor- 7 B B B B B B
age, the scallops treated with 100 ppm and 100 ppm 0 B A B B B B
3 B B B B B B
200 ppm ClO2 had lower bacterial numbers 7 C B C B B B
than the groups treated with brine or 20 200 ppm 0 B B B B B B
ppm or 40 ppm ClO 2 solution. The latter 3 C B C B B B
7 C B C B B B
treatments also had lower bacterial num-
aSee text for definitions of grades
bers than the untreated control. Compared
to day 0 samples, the groups treated with
100 ppm and 200 ppm ClO2 had lower bac-
Table 4—Gradinga of salmon fillets following treatments with ClO2 solutions or brine and
terial numbers following 3 d storage. The then stored at 4 °C for 0, 3 or 7 d
bacterial numbers continued to increase af-
Experiment #1 Experiment #2
ter 7 d. However, the ClO2 treated groups
still had significantly lower numbers than Replicate Replicate
the others. Treatment Day I II III I II III
A fishy odor rather than a seaweed odor Control 0 A A A A A A
occurred with the nontreated scallops, 3 B B B A A A
which partly caused their initial Grade B 7 C C B A A B
Brine 0 A A A A A A
rating (data not shown). However, treat- 3 A A A B A A
ment of scallops with brine or ClO 2 solu- 7 B B A B B B
tions at 20 ppm or 40 ppm appeared to re- 20 ppm 0 A A A A A A
move the odor and, thus, resulted in a bet- 3 B B B A A B
7 B B B A B B
ter rating for treated scallops. Scallops 40 ppm 0 A A A A A A
treated with 100 ppm or 200 ppm ClO2 so- 3 B A A A A A
lution had an unappealing rusty color, 7 B A B A A A
100 ppm 0 A A B B B B
which may have been due to the oxidation 3 B B B B B B
of scallop proteins by ClO2.The occurrence 7 B B B B B B
of the rusty color and fishy odor contribut- 200 ppm 0 B B B B B B
3 B B B B B B
ed to their lower rating, following cold 7 B C B B B B
storage for 7 d. The nontreated scallops de- aSee text for definitions of grades.
teriorated following storage for 3 or 7 d,
producing discoloration and odor. The scal-
Food Microbiology and Safety

lops treated with brine or ClO2 solution at Table 5—Changes in bacterial loads (aerobic plate counts) of shrimp following treatment
20 ppm or 40 ppm maintained better quali- with ClO2 at various concentrations and then storage at 4 °C and -20 °C for 3 and 7 d
ty than the nontreated control after 3 or 7 d log10 CFU/g
storage (data not shown). Odor formation
and discoloration also occurred with these ClO2 treatment Day 3 Day 7
groups but to a lesser extent than the non- (ppm) Day 0 4 °C -20 °C 4 °C -20 °C
treated control. No treatment 5.97 ± 0.19a 6.99 ±0.14a ND 8.26 ± 0.14a ND
Brine 5.56 ± 0.15b 6.90 ± 0.11a 5.74 ± 0.12a 8.19 ± 0.09ab 5.61 ± 0.18a
Brown shrimp 20 5.64 ± 0.12b 6.77 ± 0.26a 5.39 ± 0.32b 8.15 ± 0.13ab 5.23 ± 0.15b
40 5.19 ± 0.19c 6.76 ± 0.51a 5.42 ± 0.16b 8.03 ± 0.17b 4.98 ± 0.23bc
The number of natural microflora in 100 5.42 ± 0.25b 6.24 ± 0.30b 5.32 ± 0.12b 8.00 ± 0.18b 4.86 ± 0.20c
each group of brown shrimp increased, fol- 200 5.13 ± 0.13c 6.08 ± 0.12b 4.75 ± 0.23c 8.03 ± 0.17b 4.52 ± 0.21d
lowing storage at 4 °C for 3 or 7 d. Com- a-dWithin each column of the test seafood, means followed by the same letter are not significantly different from each

pared to the nontreated control, shrimps
treated with ClO 2 solutions on day 0 had
lower numbers of natural microflora (Table
5). Shrimps treated with ClO2 at 100 or
200 ppm had lower bacterial numbers than
the other 4 groups following 3 d storage.
This dose-related bactericidal effect of
ClO2 also occurred with samples stored 7
d. Compared to nontreated shrimps and
those treated with 20 ppm or 40 ppm ClO2,
shrimps treated with 100 ppm or 200 ppm
other at P = 0.05. Data are means ± standard deviations from two trials each having triplicate samples. ND = Not
determined.
ClO2 had slight discoloration (melanosis) In separate trials, brown shrimp with a
and chlorine smell. The blackening wors- bacterial load of 5.87 log10 CFU/g were
ened with these 2 groups after 3 and 7 d treated with ClO 2 solution at 20, 40, 100,
storage. Minor melanosis and odor forma- or 200 ppm and, then, stored at -20 °C for
tion also occurred with nontreated shrimp 1 wk. Compared to the nontreated control,
and those treated with 20 and 40 ppm ClO2 ClO2-treated shrimp showed a dose-related
following cold storage for 3 or 7 d. decrease in bacterial numbers following

1092 JOURNAL OF FOOD SCIENCE—Volume 64, No. 6, 1999

storage for 3 or 7 d (Table 5). The extended
storage at -20 °C of the ClO 2-treated sam-
ples further reduced the bacterial loads.
Whole fish (red grouper and
salmon)
The bactericidal effectiveness of ClO2
was demonstrated in all treated ClO2 solu-
tions with whole red grouper and salmon
(Fig. 1). However, the dose-related de-
creases in bacterial numbers with the treat-
ed fish as determined from the skinned (for
both fish) or muscle areas (for salmon
only) only occurred on day 0 (Table 2). The
washing of the whole fish with brine only
slightly reduced initial bacterial loads when
compared to the no treatment control. The
storage of treated samples for 3 and 7 d
caused time-related increases in bacterial
numbers in all test groups. For most red
grouper treated with 100 ppm or 200 ppm
ClO2 and then stored at 4 °C for 3 or 7 d,
there were lower bacterial numbers on the
skin than the nontreated and brine-treated
groups (Table 2). Similar results also oc-
curred with treated salmon, especially
when the bacterial loads were determined
from the muscle areas (Table 2). Variation
in initial bacterial loads occurred with the
test fish used in a trial and in different
batches of fish used in separate trials.
Therefore, the bactericidal efficacy of ClO2
was not clearly demonstrated from analysis
of data from separate trials using small
numbers of whole fish.
The sensory quality of the ClO2 treated
red grouper and salmon was less favorable
following storage at 4 °C for 3 or 7 d (data
not shown). The nontreated red grouper
and salmon and those treated with brine
also demonstrated some degree of quality
deterioration after 7 d. Discoloration
(bleaching) of the skin occurred with the
treated red grouper and salmon at 100 ppm
and 200 ppm ClO 2, possibly due to the
combined effects of oxidation of ClO 2 and
low pH (2.72 to 3.01). Although the fishy
odor did not occur from these samples, a
light chocolate color developed in the gills
due to interactions between blood and
ClO2, which was considered as a major
quality defect of the treated fish. The fish
eyes also changed color following treat-
ment with ClO2 solutions. Such changes
were not noted with the nontreated or
brine-treated samples.
CONCLUSION
RESULTS DEMONSTRATE THAT ClO2 WAS
very effective in reducing microorganisms
in water used for washing and handling
seafood. Therefore, ClO2 solution could be
used as a chlorine alternative to wash sea-
food products for reducing bacterial loads,
enhancing/preserving freshness, extending
shelf life, and improving safety. ClO2 could
also be used in a depuration system to re-
duce seafood pathogens. Our results sug-
gest that ClO2 should be approved by regu-
latory agencies as an alternative treatment
for seafood products.
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