Further Trials to Extend

the Storage Life of Cod

and Mackerel using
Sodium Hypochlorite or
Ozone

Seafish Report No. SR501

March 1997
 The Sea Fish Industry Authority

Seafish Technology

Further Trials to Extend the Storage Life

of Cod and Mackerel
using Sodium Hypochlorite or Ozone

Seafish Report No. SR501 Author: R. Watson

March 1997
 The Sea Fish Industry Authority

Seafish Technology

Further Trials to Extend the Storage Life

of Cod and Mackerel
using Sodium Hypochlorite or Ozone

Seafish Report No. SR501 Author: R. Watson

March 1997

Summary

After capture and icing, the initial spoilage of fish is predominantly caused by natural
enzymatic processes. A few days after capture, bacterial spoilage becomes the most important
mechanism in the deterioration of fish. Seafish has identified the use of techniques such as
ozonation and chlorination which may enable improvement in fish quality by reducing the
rate of bacterial spoilage.

This report details a series of laboratory trials which follow on from initial work reported in
SR 498. Trials were carried out to determine the effect of sodium hypochlorite and ozone on
the quality of cod and mackerel in refrigerated seawater (RSW). In addition, trials were
carried out to determine the effect of ozone on boxed and iced fish, held in a gas flushed hold
or chill store. The rate at which ozone naturally decayed to a safe level was also determined,
along with the effectiveness of ozone as a surface disinfectant. A trial was also carried out
to determine the level of ozone in the hold of a fishing vessel fitted with an ozone generator,
during a fishing trip.

Results showed that both sodium hypochlorite and ozone gave only a small (approximately
1 Tony point) improvement in the quality of mackerel after extended storage (8 days). RSW
treatment had no effect on the quality of cod. Initial storage in chlorinated RSW gave no long
term benefits in quality when fish were later boxed and iced.

Storing boxed and iced fish in a high level of ozone (2ppm) in an ozonated chill store had no
effect on the sensory or microbiological quality of the fish. A level of 2ppm of ozone gas in
a sealed chill store took 8-9 hours to naturally decay to Omg/1. The level of ozone in the hold
of a fishing vessel reached over twice the current Health and Safety Executive (HSE)
occupational exposure standard. Caution must be employed to prevent crew from being
exposed to dangerous levels of toxic ozone gas.

Sodium hypochlorite (150mg/l) was significantly more effective than ozone gas (2mg/l) for
surface disinfection.

(SR501)
 Table of Contents

Page
Summary

1. Introduction 1

2. Trials Sequence 2

3. Equipment 3
3.1 Pilot Scale RSW Tanks 3
3.2 Chill Store 5
3.3 Ozone Generation and Monitoring Equipment 5

4. Common Experimental Methods 7

4.1 Chlorination of Water 7
4.2 Sensory Assessment 7
4.3 Microbiological Analysis 7
4.4 Fish Supply 7

5. Trial I - Determination of the Effect of Chlorine Dosing on the

Storage of Mackerel held in a Pilot Scale RSW Tank 8
5.1 Purpose 8
5.2 Method 8
5.3 Results 9
5.4 Discussion 9

6. Trial II - Determination of the Effect of Ozone Dosing on the

Storage of Cod and Mackerel held in a Pilot Scale RSW Tank .12
6.1 Purpose 12
6.2 Method 12
6.3 Results 12
6.4 Discussion 13

7. Trial III - Determination of the Effect of Ozone Gas on the Storage

of Boxed and Iced Cod and Mackerel held in a Refrigerated
Store 16
7.1 Purpose 16
7.2 Method 16
7.3 Results 16
7.4 Discussion 19

8. Trial IV - Determination of the Level of Ozone Gas in the Hold of a

Commercial Fishing Vessel Fitted with an Ozone Generator 20
8.1 Purpose 20
8.2 Method 20
8.3 Results 20
8.4 Discussion 22

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 9. Trial IV - Determination of the Effectiveness of Ozone Gas and
Sodium Hypochlorite for Surface Disinfection 23
9.1 Purpose 23
9.2 Method 23
9.3 Results 24
9.4 Discussion 26

10. Overall Discussion and Conclusions 27

11. References 30

Appendices
Appendix I - Torry Sensory Assessment Scoring Sheets

Figures
Figure 1 Pilot scale RSW tank
Figure 2 Stainless steel pilot scale RSW tank and water bath
Figure 3 The ozonation of RSW tanks
Figure 4 Ozone generation equipment
Figure 5 Rate of ozone decay at 0° C
Figure 6 The level of ozone recorded over a 7 day period
Figure 7 Changes in ozone concentration over a 6 hour period
Figure 8 Stainless steel plates
Figure 9 The effect of washing using 2mg/l ozone and 150mg/l of sodium hypochlorite

Tables
Table 1 The microbiological and sensory quality of mackerel stored in chlorinated RSW
Table 2 The microbiological and sensory quality of mackerel transferred from RSW after
2 days into untreated and chlorinated ice
Table 3 The microbiological and sensory quality of cod stored in ozone treated RSW
Table 4 The microbiological and sensory quality of mackerel stored in ozone treated RSW
Table 5 The microbiological and sensory quality of cod exposed to 2 mg/1 of ozone
Table 6 The microbiological and sensory quality of mackerel exposed to 2 mg/1 of ozone
Table 7 Disinfection of a stainless steel surface using 2 mg/1 ozone and 150 mg/1 sodium
hypochlorite

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1. Introduction

Despite diminishing fish stocks and the higher prices which can be obtained for top quality
fish, it is apparent that some fish of poor quality is still being landed into UK ports.

After capture and icing, the initial spoilage of fish is predominantly caused by natural
enzymatic processes. A few days after capture, the bacterial population on the skin and in the
gut cavity of the fish rapidly multiplies. From this point onwards bacterial spoilage becomes
the most important mechanism in the deterioration of the fish. Bacterial action on the low
molecular weight components of the fish muscle causes the familiar odours and flavours
associated with spoiled fish.

Previous Seafish work with disinfectants (Ref. 1) showed that the addition of a small amount
of sodium hypochlorite, slowed the rate of bacterial spoilage of mackerel held in a pilot scale
refrigerated seawater (RSW) tank. However, to be of practical use, this treatment would have
to be effective at a higher commercial fish to water ratio of up to 4:1. A similar treated RSW
trial using ozone had no effect. It was thought that repeating the trial with higher doses of
ozone may be effective.

A number of companies are now marketing ozone generators for use within the fish industry,
claiming that ozone gas pumped into a fish hold or chill store can improve the quality of
boxed and iced fish, and act as a powerful surface disinfectant. As ozone is a highly toxic and
corrosive gas, it is also important to determine how quickly it will decay naturally to a safe
level.

This report describes a series of laboratory trials to determine the effect of sodium
hypochlorite on the quality of mackerel in a treated RSW tank, with a commercial fish to
water ratio; also the effect of ozone on the quality of cod and mackerel stored in a treated
RSW system. Trials were also carried out to determine the effect of ozone gas on the quality
of boxed and iced fish in a chill store; the rate at which ozone naturally decays to a safe level
and the effectiveness of ozone gas for surface disinfection. In addition, a field trial was also
carried out aboard a commercial fishing vessel to monitor the level of ozone in the ozonated
hold during a fishing trip.

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 Further Trials to Extend the Storage Life of Cod and Mackerel

2. Trials Sequence

The following trials were carried out during the period August 1996 to January 1997. All
except Trial IV were carried out in the Seafish Laboratory, Hull.

Trial I - Determination of the Effect of Chlorine Dosing on the Storage of Mackerel

held in a Pilot Scale RSW Tank.

This trial was carried out to determine the effect of two chlorine dose levels on the sensory
and microbiological quality of mackerel held in treated RSW using a commercial 4:1 fish
to water ratio. In addition, to simulate commercial practice, fish were removed from treated
RSW storage after two days and stored in ice to determine if treated RSW had any long
term effect on quality. Chlorinated ice was also used to see if this added to any effect.

Trial II - Determination of the Effect of Ozone Dosing on the Storage of Cod and
Mackerel held in a Pilot Scale RSW Tank.

This trial was carried out to determine the effect of ozone on the microbiological and
sensory quality of cod and mackerel held in a pilot scale RSW system.

Trial III - Determination of the Effect of Ozone Gas on the Storage of Boxed and Iced
Cod and Mackerel.

This trial was carried out to determine the effect of ozone gas on the microbiological and
sensory quality of boxed and iced, and of exposed cod and mackerel held in an ozone gas
flushed, refrigerated chill store. The time taken for a high level of ozone gas to naturally
decay to a safe level was also determined.

Trial IV - Determination of the Level of Ozone Gas in the Hold of a Commercial

Fishing Vessel Fitted with an Ozone Generator.

This trial was carried out to measure the level of ozone in the hold of a commercial fishing
vessel fitted with an ozone gas flushing system. Levels were monitored over a 7 day period
of a fishing trip.

Trial V - A Comparison of the Performance of Ozone Gas and Sodium Hypochlorite

for Surface Disinfection.

This trial was carried out to compare the effectiveness of ozone gas and sodium
hypochlorite for the bacterial disinfection of a stainless steel surface contaminated with
spoiled fish slime.

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3. Equipment

3,1 Pilot Scale RSW Tanks

The design of the pilot scale RSW tanks used in Trials I and II is shown in Figure 1.

The tanks used in Trial I were constructed from a 1000 mm x 600 mm x 660 mm
Allibert No. 21250 Jumbox. The water in the tank was circulated using an Eheim 1060
pump with the flow rate regulated by an ABS ball valve. Pipework consisted of 25
mm reinforced flexible PVC hose.

In Trial II two 460 m x 730 mm x 480 mm deep, 316 stainless steel RSW tanks were
used. To maintain the RSW temperature between the ozonated tank and the control,
both tanks were sat side by side in a large recirculating water bath constructed from
an 1200 mm x 1000 mm x 765 mm Allibert No. 21626 pallet bin (Figure 2). The tanks
were placed inside a chill store (Figure 3). An Eheim 1060 pump was used to pump
the seawater from the ozone treated RSW tank through the ozone generator and the
refrigerated titanium heat exchanger (Section 3.3) then back into the treated tank.
Flow rate of the RSW was set to 0.51/min.

Tank

Valve

Pump

Figure 1 - Pilot scale RSW tank

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 Further Trials to Extend the Storage Life of Cod and Mackerel...

Water bath
RSWTanks

Valve

Pump

Figure 2 - Stainless steel pilot scale RSW tanks and water bath

Laboratory

i .—■

Figure 3 - The ozonation of RSW tanks

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3.2 Chill Store

All the trials were carried out inside an otherwise empty 4mx4mx3m walk in chill
store to provide chilling and temperature control.

3.3 Ozone Generation and Monitoring Equipment

Ozone (O3) is a highly toxic and reactive gas consisting of an unstable arrangement
of three oxygen atoms.Ozone will naturally decay fairly quickly, so it is generated as
and when required. Commercial ozone generators produce ozone from oxygen by
either UV light or high voltage electrical discharge. Ozone is a very effective
disinfectant. A low concentration of ozone will kill bacteria by directly oxiding and
destroying the bacterial cell wall.

Ozone is also toxic to humans. At very low levels, exposure can result in permanent
damage to the respiratory system. The current Health and Safety Executive (HSE)
occupational exposure standard (OES) is set at 0.2 parts per million (ppm) averaged
over a 15 minute reference period. To comply with COSHH regulations exposure
must not exceed this level (Ref. 2). It is prudent to reduce exposure to well below this
level to allow for fluctuations, and as a matter of good practice, all employers are
encouraged to keep exposure to well below the OES.

The ozone generation and water treatment equipment used in Trials II to IV is shown
schematically in Figure 4.

Oil-Gas
Destructor

Submersible
WATER IN Valve
Pump

Ozone gas Contact

monitor Column

Ozone
AIR IN Generator/ JREATED WATER
OUT
Control Box

Ozone/Air
Figure 4- Ozone generation equipment

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The equipment consisted of an RK2™ 2.5g/hr ozone generator and a 316 stainless
steel 1500 mm x 500 mm diameter seawater contact column, both supplied by Dryden
Aquaculture. The ozone generator used twin internal air dryers and an Octopus™
programmable controller/data logger. The controller had two probe inputs used to
measure pH and oxidation-reduction potential (ORP) (redox) of the treated water.
Water flow into the contact column was regulated using an Eheim 1060 submersible
pump and an ABS ball valve. Ozone not entering solution was destroyed by an
activated carbon filter in the off-gas destructor.

In trials III and IV ozone gas was taken directly from the generator and pumped into
the chill store. A Sabre Toxigard™ ozone gas monitor/logger was used inside the chill
store via an interface, to switch the ozone generator on or off to maintain the desired
concentration of ozone gas in the chill. A second Toxigard™ monitor was used in the
surrounding area to warn personnel of ozone leaks.

All pipework carrying ozonated air consisted of PTFE tubing. Pipework for the water
consisted of 25 mm reinforced flexible PVC hose.

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 Further Trials to Extend the Storage Life of Cod and Mackerel...

4. Common Experimental Methods

4.1 Chlorination of Water

The amount of free and total chlorine in a nominal (5% w/w available chlorine) sodium
hypochlorite solution was determined using a calibrated Hach DR2000
Spectrophotometer and DPD reagents. The water to be treated was then manually dosed
with the sodium hypochlorite solution to give the specified chlorine dose.

4.2 Sensory Sampling and Assessment

Raw and cooked sensory assessment was carried out on four fish from each treatment
using the Torry freshness scoring scheme (Appendix I). After filleting, the fillets were
rinsed in fresh water before being steamed for 20 minutes prior to assessment by a
minimum of three sensory assessment experts.

4.3 Microbiological Analysis

In trials I to HI two 150g samples offish flesh (made up of pieces of flesh taken from four
fish) were taken from each treatment. In trials I and n, two 150ml samples of RSW were
also taken from each treatment.

Fish and water samples were analysed by the Namas accredited PHLS Department of
Hull Royal Infirmary. Samples were analysed for Total Viable Count (TVC) at 37° C and
Pseudomonas sp.organisms were enumerated by using either a spiral plate count or
membrane filtration according to the standard methods detailed in HMSO document
No. 71.

4.4 Fish Supply

The cod used in the trials were small to medium sized gutted fish obtained from a single
haul of a local Bridlington or Scarborough day boat. The mackerel used in the trials were
whole fish from the English Channel fishery, landed into Portsmouth and transported to
Hull, boxed and iced in refrigerated transport.

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5. Trial I - Determination of the Effect of Chlorine Dosing

on the Storage of Mackerel held in a Pilot Scale RSW
Tank

5.1 Purpose
Previous Seafish laboratory trials (ref. 1) showed that regular 40 mg/1 (40 mg of chlorine
per kg of fish) doses of sodium hypochlorite could significantly slow the rate of
microbiological spoilage of mackerel, held in a pilot scale RSW system with a 1:1 fish
to water ratio.

This trial was carried out to repeat this earlier work using a higher 4:1 fish to water ratio
which may be encountered on some commercial RSW vessels.

Pilot scale RSW tanks were used to determine the effect of two levels of chlorine dosing:
an equivalent chlorine dose to the previous trial, 160 mg/1 (40 mg chlorine per kg offish)
and a lower chlorine dose of 40 mg/1 (10 mg per kg of fish).

In addition, to simulate commercial practice fish were removed from the treated and
untreated RSW after two days and kept in either normal ice or hypochlorite treated ice.

Sensory and microbiological assessment were used to determine the quality of the fish
in each treatment over an eight day period.

5.2 Method
Three pilot scale RSW tanks were each filled with 30 litres of 34 ppt artificial seawater
(ASW). The water in each was treated with sodium hypochlorite solution to give a
chlorine dose of 0 mg/1,40 mg/1 and 160 mg/1. Each tank was filled with 120 kg of fresh
mackerel. The water recirculation rate was set to 0.5 1/min (one tank exchange per hour)
before each tank was placed into the chill store, which was set to maintain the RSW
temperature at -1 ° C.

Prior to storage, raw and cooked sensory assessment was carried out on the fish and
samples were taken for microbiological analysis.

After two days, 8 fish were removed from the 0 mg/1 dosed RSW tank and boxed in tap
water ice. Fish were also removed from the 160 mg/1 dosed RSW tank and eight fish were
boxed in tap water ice and eight fish were boxed in ice made from tap water dosed with
40 mg/1 of chlorine.

The treated RSW tanks were redosed with sodium hyphclorite after 2, 5 and 9 days
(which was as close to 48 hour intervals as possible).

Sensory and microbiological assessment of the RSW and boxed and iced fish was
repeated after 5 day and 10 days. (Fish were removed for assessment before the next
sodium hypochlorite dosing was carried out).

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 Further Trials to Extend the Storage Life of Cod and Mackerel...

5.3 Results
The results of the trial are shown in Tables 1 and 2 overleaf.

No difference in the raw sensory quality of fish held in the different RSW treatments was
observed during the trial. However, the chlorine dose of 160mg/l every 48 hours
somewhat reduced the rate of microbiological spoilage, resulting in treated fish having
a slightly better cooked odour and flavour than the control after 8 days. The lower
chlorine dose of 40 mg/1 had no such effect. Hypochlorite treatment at either level only
slightly reduced the bacterial loading of the mackerel flesh and the RSW.

No significant differences in the sensory or microbiological quality of mackerel held in

160 mgl/ treated RSW, then transferred to normal or hypochlorite treated ice and the
control was observed.

5.4 Discussion
In the earlier trial with a 1:1 fish to water ratio, a dramatic 3 Torry point difference
between the cooked flavour of fish kept in treated RSW and the untreated control was
observed after 8 days. This result has not been repeated in this latest trial at a commercial
4:1 fish to ice ratio.

Fish in treated RSW spoiled at a similar rate in both trials. However in this latest trial the
fish in the untreated RSW control tank spoiled at a much slower rate than that in the
earlier trial resulting only in a 0.75 Torry point difference between fish in treated and
untreated RSW after 8 days. The fish in this latest trial did not develop the distinctive
sour odours and flavour associated with the fish in the 1:1 RSW trial control tank.

It is suspected that some degree of anaerobic spoilage occurred in the 1:1 RSW control
tank. It is now thought that results of the 1:1 trial exaggerated the potential benefits of
using sodium hypochlorite dosing in an RSW system and a less than one Torry point
improvement in quality over an 8 day period is more realistic.

(SR50J) .9-
Key: ND - Not Detected
N/A - Not Applicable
TVC - Total Viable Count
Table 2 - The microbiological and sensory quality of mackerel transferred from RSW after 2 days into untreated
and chlorinated ice

5.1 x104 4.1 x103 7.00 6.50 6.75

Untreated RSW + ice

2.0x103 6.50 6.50 7.00

160 mg/l chlorinated 2.0 x104
RSW + ice

1.1 x104 2.0 x103 6.75 6.50 7.25

160 mg/l chlorinated
RSW + 40 mg/l treated
ice

3,8 x105 3.5 x103 5.00 5.00 5.00

Untreated RSW + ice

4.6 x 10s 3.5 x103 5.25 5.25 5.50

160 mg/l chlorinated
RSW + ice
8
1.8 x105 2.0 x103 5.25 5.00 4.50
160 mg/l chlorinated
RSW + 40 mg/l treated
ice
Key: TVC - Total Viable Count
 FISH Further Trials to Extend the Storage Life of Cod and Mackerel...

6. Trial II Determination of the Effect of Ozone Dosing

on the Storage of Cod and Mackerel Held in a Pilot
Scale RSW Tank

6.1 Purpose
An initial Seafish trial (Ref. 1) using ozone to treat both cod and mackerel in a pilot scale
RSW tank (12 mg/1 dose every 48 hours) showed that ozone had no significant effect on
either the sensory or microbiological quality of the fish. It was thought the high
concentration of organic material (blood, slime, scales, etc.) in the RSW neutralised the
ozone before it had chance to react with the fish.

This trial repeated the work using a much higher dose of ozone. Cod was treated using
an ozone dose of 75mg/l every hour whilst mackerel was treated with a low dose of 150
mg/1 every 48 hours to reduce the risk of accelerating oxidative rancidity in the oily fish.

6.2 Method
The two stainless steel RSW tanks and ozone generation equipment was set up as
described in Section 3.1.

Each RSW tank was filled with 30 litres of ASW. The water in the treated tank was
ozonated to give an electrode potential (ORP) of 850 mV (approximately 1.8 mg/1
ozone). Each tank was then filled with 30 kg. of fresh cod.

Prior to storage, raw and cooked sensory assessment was carried out on the fish, and
samples of fish and RSW water from each tank vere sent for microbiological analysis.
The ozone generator was left running continuously for the duration of the trial to give an
ozone dose of 75 mg/l/hr.

The trial was repeated using fresh mackerel. Mackerel in the treated RSW received an
ozone dose of 150 mg/1 after 0, 2,4 and 7 days.

For cod, the sensory and microbiological analysis was repeated after 4 and 11 days.
Mackerel assessment was repeated after 4 and 8 days.

6.3 Results
The results of the trial are shown in Tables 3 and 4 overleaf.

Addition of the cod caused the ORP to fall to 136mV. After 3 days ozonation, the ORP
remained constant at 340 mV and ozone gas gassed off from the surface of the treated
RSW tank and built up in the chill store.

No significant differences were apparent in the raw appearance of ozonated and untreated
cod over the duration of the trial. Ozonated cod had a very faint or neutralised gill odour.
The cooked odour and flavour of cod kept in ozonated RSW was worse than the

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untreated control after 4 days. After 11 days, very faint burnt rubber/plastic odours and
flavours were detected. All samples had a strong salty flavour. Ozone treatment did not
significantly reduce the bacterial loading of the cod flesh. However, the ozone
dramatically reduced the bacterial loading of the treated RSW water.

For mackerel, the initial ozonation of clean seawater raised the ORP from 254 mV to
810 mV. Addition of the fish lowered the ORP to 0 mV for the duration of the trial.

No significant differences were apparent in the raw appearance of ozonated and untreated
mackerel observed over the duration of the trial Ozonated mackerel had a very faint or
neutralised gill odour. The cooked odour and flavour of ozonated mackerel was slightly
fresher than the control after 8 days. Ozone treatment did not significantly reduce the
bacterial loading of the fish but dramatically reduced the bacterial loading of the
seawater.

6.4 Discussion
Ozone dosing of RSW did not improve the sensory or microbiological quality of cod. It
is likely that using a higher dose would cause severe odour and flavour tainting.

For mackerel a regular 150 mg/1 dose of ozone gave a small improvement in the cooked
odour and flavour of the fish. As in the previous trial using sodium hypochlorite, the
ozone had little effect on the fish over the first few days of storage, when the spoilage is
predominantly enzymatic.

Although ozone dramatically reduced the level of spoilage bacteria in the RSW, it was
unable to greatly reduce the level of spoilage bacteria in the fish. It is thought that these
bacteria are protected by the skin and flesh, which rapidly neutralises the ozone.

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Key: ND • Not detected
TVC - Total Viable Count
N/A - Not Applicable
Key: ND - Not detected
TVC - Total Viable Count
N/A ■ Not applicable
I
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7. Trial III - Determination of the Effect of Ozone Gas on

the Storage of Boxed and Iced Cod and Mackerel held
in a Refrigerated Store

7.1 Purpose
It has been claimed that flushing a vessel hold or chill store with ozone gas can improve
the quality of boxed and iced fish, by slowing the rate of microbiological spoilage.

This trial was carried out to determine the effect on the sensory and microbiological
quality of cod and mackerel stored in a chill store containing a very high level of 2 ppm
of ozone. It was thought that the ice in a box of fish may prevent ozone gas from actually
reaching the fish, so in addition, fish were also laid outo on a tray directly exposed to the
gas. Sensory and microbiological assessment was carried out to determine the effect of
the ozone over an extended storage period.

Without a complex extraction/ozone destruction system, the level of ozone in a chill store
or hold would have to naturally decay to a safe level before personnel could enter. An
ozone logger was used to determine how long ozone gas would take to decay in an
undisturbed chill store at 0°C.

7.2 Method
A 50kg fish box was filled with 20 kg of cod and flake ice in a 3:1 ratio. The box was
placed in the refrigerated chill store (set to maintain 0°C), along with 10 fish laid out on
a plastic tray. The procedure was repeated for mackerel.

Ozone gas was pumped directly into the chill store to maintain a level of 2.0 ppm.
Duplicate boxes and trays of fish were placed in an environmental chamber operating at
precisely the same temperature as the chill store, as a control. Prior to storage, raw and
cooked sensory assessment was carried out on the fish and samples were sent for
microbiological analysis.

The sensory and microbiological assessment was repeated after 5 and 11 days. To
determine the rate of ozone decay, ozone was pumped into the empty chill store to
maintain a level of 2 ppm. The generator was then switched off and the level of ozone in
the chill was logged every 10 minutes.

7.3 Results
The results of the trial are shown in Tables 5 and 6 and Figure 5 overleaf.

During the trial no significant differences were observed in the raw and cooked sensory
quality of ozonated boxed and iced cod and the control.

After 5 days cod directly exposed to ozone had a very faint gill odour. After 11 days the
exposed side of the gills were blackened, with an atypical mushroom odour. The cooked

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odour of cod directly exposed to ozone was very faint after 5 days: after 11 days, 'byre
like' atypical off odours developed. No differences were observed in the cooked flavour
of cod directly exposed to ozone and the control. Ozone had no significant effect on the
microbiological quality of cod except for a slight reduction in the bacterial loading of the
flesh after 11 days directly exposed to ozone.

Ozone had no significant effect on the microbiological, raw or cooked sensory quality of
boxed and iced mackerel. Mackerel directly exposed to ozone had a faint gill odour after
5 days. After 11 days the exposed surface of the gills were blackened. The ozone had no
effect on the cooked odour or flavour or the bacterial loading of exposed mackerel.

All exposed cod and mackerel suffered from severe drying effects.

Ozone gas took 4.5 hours to decay naturally in the empty chill room from a concentration
of 2 ppm to the current HSE occupational exposure limit of 0.2 ppm. Ozone ga .ook 8-9
hours to fall from 2 ppm to 0 ppm.

Table 5 - The microbiological and sensory quality of cod exposed to 2 ppm of ozone

Key: TVC - Tc .; Viable Count

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 FISH Further Trials to Extend the Storage Life of Cod and Mackerel

Table 6 - The microbiological and sensory quality of mackerel exposed to 2 ppm of ozone

Key: ND - Not Detected

TVC -Total Viable Count

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Current COSHH Occupational exposure standard (OES)

Figure 5 - Rate of ozone decay at 0°C

7.4 Discussion
Ozone gas used at a relatively high concentration of 2 ppm had no significant effect on
the sensory or microbiological quality of either cod or mackerel boxed in ice.

Prolonged direct exposure to ozone damaged the sensitive gill tissue of both cod and
mackerel, causing atypical gill odours to develop.

Workers would not be able to enter the empty chill store ozonated with 2 ppm of ozone
for at least 5 hours if the gas were allowed to decay naturally to a safe level, but if there
were an extraction system or a mass of fish in the store this period would be reduced.

It was noted that some plastics hardened and became brittle and objects made from
natural rubber disintegrated within 24 hours of being exposed to ozone gas. Long term
ozonation of a factory, chill store or fish hold could cause a variety of structural,
mechanical or electrical failures unless suitable ozone resistant materials were employed
throughout.

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8. Trial IV - Determination of the Level of Ozone Gas in

the Hold of a Commercial Fishing Vessel Fitted with an
Ozone Generator

8.1 Purpose
Ozone generators have already been installed on a number of fishing vessels in the UK
fleet. The ozone gas is pumped into the fish hold with the aim of improving fish quality.

An ozone logger was used to record the level of ozone in the hold of a vessel over 7 days
of a fishing trip.

8.2 Method
The vessel investigated was a 28m, modern full shelterdecked trawler with an 8m x 5m
x 3 m fish hold. Access to the hold was via a lm x lm hatch. The nominal capacity of the
ozone generator installed was 0.6g/hr. The operational procedure consisted of switching
the ozone generator on with the hatch closed and the hold sealed, to allow the level of
ozone to build up. When access to the hold was required the generator was turned off
and the hatch was opened for 30 minutes to 1 hour to allow the ozone in the hold to
disperse.

A Toxiguard™ ozone logger was positioned in a central location attached to the ceiling
of the hold. The logger recorded the ozone concentration every 10 minutes over a 7 day
period.

8.3 Results
The level of ozone recorded over the 7 day period is shown in Figure 6. The initial ozone
peak is shown in detail in Figure 7. The rate at which ozone built up in the hold is shown
in Section 1 of Figure 7. The level of ozone with hatch closed and the ozone generator
switched off is shown in Section 2. Section 3 shows the level of ozone with the hatch
open and the ozone generator switched off.

The maximum level of ozone recorded in the hold was 0.5 ppm.

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 Further Trials to Extend the Storage Life of Cod and Mackerel...
0.6-r
Current HSE Occupational exposure standard 0.2 ppm
cvjcvicvicjcvicgcvicviojcvicvioioi cvi
Figure 6 - Level of ozone recorded over a 7 day period
Section 3
Cuircnl HSE Occupational cxposunj standard 02 ppm)
1:43 am 6:43 am 7:43 am
Figure 7 - Changes in ozone concentration over a 6 hour period
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 FISH Further Trials to Extend the Storage Life of Cod and Mackerel...

8.4 Discussion
The level of ozone in the upper area of the hold quickly reached nearly three times the
HSE occupational exposure standard of 0.2 ppm. As ozone is 1.6 times heavier than air
it is likely that the concentration of ozone at the bottom of the hold was much higher. The
results indicate that ozone would take many hours to naturally decay to a safe level.
Opening the hatch, quickly removed the ozone from the upper part of the hold. However
30 minutes to 1 hour may not be sufficient time to reduce ozone levels in the lower part
of the hold, due to the limited air exchange through the small hatch. Unfortunately the
design of the vessel is such that with the hatch open, the ozone in the hold can only
escape onto the working deck,an enclosed area with limited ventilation where crew carry
out fish gutting, washing and grading operations. This could result in personnel on the
working deck being exposed to a level of ozone higher than the current OES. A powerful
extraction and/or ozone destruction system should be used to remove the ozone from the
hold quickly and safely.

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 Further Trials to Extend the Storage Life of Cod and Mackerel...

9. Trial V - Determination of the Effectiveness of Ozone

Gas and Sodium Hypochlorite for Surface Disinfection

9.1 Purpose
It has been claimed that ozone gas can be used to disinfect fish processing or storage
areas.

This trial was carried out to compare the performance of ozone gas on its own and ozone
gas and sodium hypochlorite (bleach) in conjunction with washing, for disinfecting a
stainless steel surface contaminated with spoiled fish slime.

Sodium hypochlorite was used at a concentration of 150 mg/1, the level recommended by
Seafish for general disinfection purposes. Ozone gas was used at a high concentration of
2 ppm overnight (12 hrs). Swabbing was carried out to determine the microbiological
loading of the treated and untreated surface.

9.2 Method
Two 50 cm x 30 cm polished stainless steel plates were washed and copiously rinsed with
tap water. A 30 ml sample of slime was collected from 20 day old cod, stored at 0° C.
The slime was mixed with 20 ml of deionized water and thoroughly mixed. A 25 ml
aliquat of slime solution was evenly lawned onto the surface of each stainless steel plate.

The disinfection treatment of the plates is shown in Figure 8 overleaf. Plate 1 was divided
into three sections, each section contained ten 5 cm x 4 cm rectangular areas. The first
section was left as the control. The second section was washed with a sterile scrubbing
brush and tap water for 30 seconds, followed by a 10 second rinse with tap water. The
third section was washed with a sterile scrubbing brush and a 150 mg/1 solution of
sodium hypochlorite followed by a 10 second rinse with tap water. Plate 1 was placed in
a chill store operating at 0° C. Plate 2 was divided into two sections, each section
contained ten 5 cm x 4 cm rectangular areas. The first section was left as a control. The
second section was scrubbed with tap water for 30 seconds and rinsed for 10 seconds
with tap water. Plate 2 was placed in a second chill store operating at the same
temperature with an ozone input from the ozone generator. A Sabre Toxigard™ ozone
monitor/logger was used to maintain the level of ozone in the chill at 2 ppm. Great care
was taken to avoid cross-contamination between the various sections of the plates during
washing.

After 12 hours the plates were removed. Four 20 cm3 areas were selected at random from
each section and swabbed using a sterile cotton wool swab, moistened with sterile
deionized water. The swabs were then sent for microbiological analysis.

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 FISH Further Trials to Extend the Storage Life of Cod and Mackerel

Plate 1-Chill Plate 2-Ozonated chill

; Wash15Cmg/i
Control Wash : •; sodium hypochlorite

Figure 8 - Stainless steel plates

9.3 Results
The results of the trial are shown in Table 7 and graphically in Figure 9 overleaf.

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Table 7 - Disinfection of a stainless steel surface using 2 ppm ozone and 150 mg/l sodium
hypochlorite

TVC - Total Viable Count

1.00E+07

1.00E+00

Q TVC ■ Pseudomonas spp

Figure 9 - The effect of washing using 2 ppm ozone and 150 mg/l sodium hypochlorite

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 FISH Further Trials to Extend the Storage Life of Cod and Mackerel...

9.4 Discussion
Washing with 150mg/l sodium hypochlorite was significantly more effective than
washing followed by ozonation.

The use of ozone gas without washing had little effect. It is thought that washing removed
the protective layer of slime/dirt which slightly improved the effectiveness of ozonation.

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 Further Trials to Extend the Storage Life of Cod and Mackerel

10. Overall Discussion and Conclusions

Keeping mackerel in RSW at a commercial 4:1 fish to water ratio and dosed with 160mg/l
of chlorine only gave a small 0.75 Tony point improvement in the cooked flavour of the fish
over an 8 day period. The dramatic 3 Torry point improvement in quality observed in an
earlier trial with mackerel in a 1:1 fish to water ratio was not repeated. In this trial, the control
fish spoiled at a much slower rate without developing the sour odour and flavours as in the
earlier trials. It is likely that some degree of anaerobic spoilage took place in the control tank
of the earlier trial, exaggerating the benefits of using sodium hypochlorite in an RSW system.

Keeping mackerel in sodium hypochlorite treated RSW for 2 days had no long term effect
on quality when fish was subsequently transferred into normal or chlorinated ice.

It is unlikely that chlorination of RSW would find a commercial use due to the relatively
small improvement to be gained in fish quality, and the fact that the benefit only becomes
apparent after longer storage periods (8 days) when the fish is of poor quality anyway. The
capital costs associated with chlorine generation and safety monitoring equipment is likely
to be high. To repeat the dosing level used in this trial, a large 1 kg/hr chlorine generator
would be required for a 1,000 tonne capacity RSW vessel.

Ozone did not improve the quality of cod kept in RSW. Using a relatively high level of ozone
did not improve the sensory or microbiological quality of the fish after 5 days and resulted
in faint but undesirable off flavours developing on extended storage. The lack of any positive
improvement in quality mirrors the findings of other workers investigating the effects of
ozone treatments on the quality of cod (Ref. 3,4,5). However, as with sodium hypochlorite,
using ozone to treat mackerel in RSW resulted in a slight improvement of the cooked odour
and flavour of the fish after 8 days. The reason why ozone has an effect on mackerel but not
on cod is not clear, but may be due to the intrinsic nature of the fish. The thick natural slime
layer or the thicker skin of cod may prevent the disinfectant reaching the spoilage bacteria.

As with sodium hypochlorite, the benefits of commercial ozone treated RSW system would
be limited due to the small improvement in quality which is only achieved after extended
storage. The capital cost of ozone generation equipment and the associated monitoring
equipment would be high. To repeat the dose level received by the mackerel in this trial a
1,000 tonne capacity RSW vessel would require a massive 75 kg/hr ozone generator or a
large 3.5 kg/hr generator continuously dosing the tanks. The effect of a small continuous dose
of ozone has not been investigated and may not have any effect.

Storing cod or mackerel in a highly ozonated chill store (2 ppm) did not improve the
microbiological or sensory quality of boxed and iced cod or mackerel. This is not surprising
as it is unlikely that ozone could significantlypenetrate the ice which surrounded the fish.
However, fish directly exposed to ozone gas also showed no improvement in quality, and
atypical blackening of gill tissue occurred after 8 days.

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 Further Trials to Extend the Storage Life of Cod and Mackerel

The incorrect use of ozone in this application has obvious health and safety implications.
Logging the level of ozone gas showed that without extraction and/or ozone destruction
equipment it would take approximately 4-5 hours before the level of ozone in a 2 ppm gas
flushed empty hold or chill store would naturally fall to the current occupational exposure
standard of 0.2ppm. This rate of ozone decay would be significantly slower in less humid
conditions or at a lower temperature or greater if the room were full of fish or had an
extraction/ozone destruction system. However, it is prudent to reduce exposure to well below
this level as a matter of good practice. An effective ozone monitoring system would be
required to protect workers and detect leaks in surrounding areas. Further information on the
occupational hazards of ozone is available from HSE (Ref.2). The long-term use of ozone gas
in a processing area, chill store or fish hold could also result in structural, mechanical or
electrical failures unless suitable ozone resistant materials are employed throughout.

It was found that the actual level of ozone in the hold of a vessel using ozone gas flushing can
exceed the HSE occupational exposure level. The level of ozone at the top of the hold
reached 0.5 ppm. As ozone is heavier than air, it is likely that the concentration at the base
of the hold was much higher. The current practice of venting the gas in the hold through the
working deck (an enclosed processing area with limited ventilation) is unacceptable. An
ozone extraction/destruction system should be installed to protect the crew and allow rapid
access into the hold. The current hold venting time of 30 minutes to 1 hour is thought to be
insufficient for a 120m3 well of ozonated air to evacuate through a small hatch which would
induce little air movement. The time taken for ozone to reach a safe level throughout the hold
must be determined. As this time could vary with humidity/airflow and temperature, it would
be prudent for crew entering the hold to carry a personal ozone monitor/alarm.

It is unlikely that ozone will find wide spread commercial use in a gas flushed storage rooms
for fish due to its ineffectiveness, although ozone is an excellent deodorizing agent. Reports
of such systems significantly improving the quality of boxed and iced fish may be subjective,
influenced by an odourless or unusually fresh smelling hold or chill store.

Ozonation alone, even at a relatively high concentration of 2 ppm did not effectively kill
bacteria on a stainless steel surface. Scrubbing and rinsing with tap water to remove the
majority of dirt and organic material slightly improved the effectiveness of ozone. However,
scrubbing with the recommended 150 mg/1 level of sodium hypochlorite followed by rinsing
was far more effective than washing and ozonation. Sodium hypochlorite achieved almost
full bacterial sterilisation, killing 99.9999% of all bacterial and all the Pseudomonas sp. (fish
spoilage bacteria) present.

It is unlikely that ozone gas flushing would be used commercially for general surface
disinfection, due to its relatively poor performance and the potential health risks and
corrosion problems, as discussed for the previous application. It is possible that ozonated
water could be used to achieve a similar degree of disinfection to chlorinated water.
However, technical and safety problems may be encountered such as ozone coming out of
solution and the long term effects of ozonated water in contact with unprotected skin are not
yet fully understood.

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 Further Trials to Extend the Storage Life of Cod and Mackerel

The future of chlorine and ozone for some applications within the food industry is uncertain.
Both chlorination and ozonation can produce harmful chemical by products as a result of
directly reacting with the fish or reacting with organic material in the water which is then
transferred to the fish. In recent years the US Food and Drug Administration (FDA) has
raised questions about the safety of ozone for washing seafood and the use of treated ice
(Refs. 6 and 7). It seems likely that legislation to restrict the use of disinfectants in contact
with food will prevail.

Considering the limited effect of disinfectants on the quality offish and the associated safety
and technical problems which can arise, it is likely to be preferable to land premium quality
fish by reducing microbial spoilage through shorter trip length and a high refrigeration
capacity. Alternatively, the product could be frozen at sea.

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 Further Trials to Extend the Storage Life of Cod and Mackerel

11. References

1. WATSON, R.B. 1996. " Initial Trials to Extend the Storage Life of Cod and
Mackerel using Sodium Hypochlorite or Ozone to Treat Ice and Refrigerated Water",
Seafish Report No. 498.

2. "OZONE: Health Hazards and Precautionary Measures", HSE Guidance Note EH38
(revised 1996).

3. RAVESI, E. 1987. "Ozone Treatments of Fresh Atlantic Cod, Gadus morhuay\

Marine Fisheries Review 49 (4), pp. 37-42.

4. VYNCKE, W. 1981. "Ozonated Washing Water. No Effect on Shelf Life of Cod

Fillets", paper presented at the 1 lth meeting of West European Fish Technologist
Association.

5. WIGNALL. 1988. "An Evaluation of Ozonated Ice as a Preservative for Fish" (Torry
Research Station Confidential Report TRS/4/19/36.

6. AHMED, F.E. 1991. "Seafood Safety", Institute of Medicine, National Academy

Press, ISBN 0-309-04387-5 p. 125

7. NELSON, W. 1982. "The use of Ozonated Ice to Extend the Shelf Life of Fresh
Alaskan Fish". Report submitted to the Alaskan Department of Commercial
Economic Development Office, Commercial Fish Development, Anchorage.

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 Further Trials to Extend the Storage Life of Cod and Mackerel

Appendix I

Torry Sensory Assessment Scoring Sheets

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H&FSSH Further Trials to Extend the Storage Life of Cod and Mackerel

(SR50J)
Apn 1 - i
 Freshness Score Sheet for Iced Cod - Raw Fish

s:
 FISH Further Trials to Extend the Storage Life of Cod and Mackerel..

(SR50J) App 1 - iii

 FISH Further Trials to Extend the Storage Life of Cod and Mackerel

Mackerel Scoring Scales

(Revised April 1986)

Raw Fish

(SR50J) App 1 - iv
 FISH Further Trials to Extend the Storage Life of Cod and Mackerel....

Mackerel Scoring Scales

Raw Fish

(SR501) App1 - v
 FISH Further Trials to Extend the Storage Life of Cod and Mackerel.

Mackerel Scoring Scales

Cooked Fish Odours

(SR50]) App 1 - vi
 FISH Further Trials to Extend the Storage Life of Cod and Mackerel....

Cooked Fish Flavours

(SR501) App 1 - vii