use of aqua des in aquaculture

PO Box 7, Warrington, Cheshire. England WA4 6HB Tel. +44 ( 0 ) 1925.643552 Fax. +44 ( 0 ) 1925 655856 E-Mail [email protected]

Doc No: Use of Aqua Des in Aquaculture Ref: JHM Date: 31/08/05 of 37

CHEMICALS SECTOR – PEROXYGENS DISINFECTION TECHNOLOGY TEAM

SOLVAY

SOLVAY on the web - http://www.solvay.com M-029-05

The use of Aqua Des in Aquaculture



CONTENTS An Introduction to Fish Farm Biosecurity Summary of Reports Appendix 1. Aqua Des testing against Infectious Pancreatic Necrosis virus- IPN virus 2. Disinfection Procedure for Aqua Des on Fish Production Facilities 3. Aqua Des Stability in Sea Water 4. Aqua Des Efficacy Data In The EN 1656 & EN 1657 Suspension Tests and EN 14349 Surface Test. 5. Aqua Des Efficacy Data in The Kelsey Sykes Test. 6. Environmental Impact of Aqua Des 7. Disinfection procedure against Infectious Salmon Anaemia – ISA virus (A Hard copy of this can be obtained from Jane Massey)



Introduction Intensive aquaculture is a rapidly growing business. The nature of fish production provides an ideal environment to which disease causing organisms can flourish and cause serious damage to productivity. Antimicrobial treatment is needed for infection / disease control in a number of areas in the fish industry where peracetic acid can be considered. High stocking in tanks pens or ponds means that the fish are vulnerable to infections and pests. Disinfection opportunities exist in the following areas: farm equipment and surface disinfection, well boats, algae control and fish processing facilities. This report has been prepared to summarise the data available on peracetic acid and to explain why there are market development opportunities for the product in the fish farming industry. Proven Efficacy Aqua Des has been tested and proved to be effective against a wide range of viral, bacterial and fungal disease causing organisms that effect fish production. (see appendix 4&5 Aqua Des Efficacy Data in the EN 1656 & EN 1657 Suspension Tests and EN 14349 Surface Test). Environmental Impact As well as being effective, it is important that the disinfectant causes no harm to the environment in which it is being used. Aqua Des is often referred to as environmentally friendly. (See appendix 6 the environmental impact of Aqua Des)

Standards are increasing for environmental discharges, residues of chemical treatments in fish, and disease / infection control as well as fish appearance. These apply across the industry from the fish farms to the final processing, packaging and transport. These standards are being driven and implemented by registration authorities, environmental agencies and by supermarkets. There is a trend towards adoption of HACCP (hazard analysis critical control point throughout aquaculture production to processing which will promote good hygiene practices and use of disinfectants. Based on existing information, peracetic acid can meet many of the requirements for chemical disinfection. In particular, environmental concerns relating to fish farm waste but also disinfectant and antibiotic treatments are critical. Substances which are toxic, persistent or liable to bioaccumulate have the potential to cause long term damage and are controlled by restrictions or bans on their use. For example, Environmental Quality Standards (defining annual mean concentration in coastal waters) are being set to impose restrictions in Scotland. In addition standards are set as an MRL (maximum residue



limit) for chemical residues in treated fish. All these measures favour chemicals such as peracetic acid which act then decompose rapidly to minimise residues in the environment and in fish. Disinfection Opportunities in the Fish Industry There are over 1000 fish and shellfish farming businesses in the UK operating on 1500 sites directly employing more than 3000 people. The main finfish species farmed in the UK are salmon (139,000 tonnes mainly in Scotland) and rainbow trout (16,000 tonnes) (2001 figures). The current estimate for EU fish farming is 520,000 tons of product that are produced throughout the entire European Union. Major producer states in the EU (2000)

United Kingdom -30% 161,000 Tonnes Greece - 12% 67,000 Tonnes Italy - 12% 65,000 Tonnes Spain - 9% 48,000 Tonnes Denmark - 7% 40,000 Tonnes Germany - 7% 36,000 Tonnes Major fish species reared in EU are Trout, Salmon, Seabream, Seabass, Turbot and Flatfish.

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Major Producer States Major Producer States in EUin EU



The fish farming industry is also very important in other regions of the world such as Chile, Asia, USA and Canada Data published by the United Nations indicate that total human consumption of fish is now over 100 million tonnes.

(Data source – The federation of European Aquaculture producers) Antimicrobial treatment is needed for infection / disease control in a number of areas in the fish industry where peracetic acid can be considered.

Farm equipment and surface disinfection Solvay has similar applications for peracetic acid in the animal health sector and similar products compete in fish farming, e.g. hypochlorite and iodophors. The high performance of peracetic acid and its activity against key problematic viruses, is a major advantage over Hypochlorite which will always be a cheaper option. Environmental pressures will favour peracetic acid and also situations where very rapid disinfection is required such as well boat disinfection where volumes are comparatively large. Data suggests that well boats played a major role in the spread of Infectious Salmon Anaemia in both Scotland and Norway.



Fish processing facilities There are a number of potential applications: equipment and surface disinfection, process water treatment including transport water as well as refrigerated storage and transport facilities. Peracetic acid can control human pathogens which may occur in fish such as Listeria species as well as fish spoilage micro-organisms.

Algae control and pollution control Algae are a current problem for fish farms, e.g. Red Tides caused by algal blooms have had severe effects on the culture of molluscs in Spain and Portugal. The algae can create a high BOD if the bloom dies, thus significantly reducing oxygen available to the fish. Oxygen depletion can also occur with the live bloom during nocturnal respiration. Under certain conditions, the algae produce toxins which can kill fish stock. Also the sheer density and sticky nature of the bloom can coat the gills and physically suffocate the fish. There are opportunities for peracetic acid for algal control and oxygenation. Egg and young fish treatment The current treatments for fungal control, malachite green and formalin are banned in many countries or their use is restricted with pressure to change to safer alternatives. Preliminary data show’s that peracetic acid can be effective but there is the issue that this application would have to be licensed as an approved medicine. Volumes for this application would be comparatively small.



Summary of Reports Aqua Des testing against Infectious Pancreatic Necrosis virus- IPN virus At a temperature of 4º C Aqua Des showed a strong virucidal effect on IPN virus. A concentration of 500mg/l peracetic acid (1:100 dilution of Aqua Des) was found to be sufficient for a 4-log10 reduction in titre of IPN virus in less than 2 mins. At a concentration of 167 mg/l peracetic acid (1:300 dilution), a contact-time of 10 mins was required. Aqua Des testing against Infectious Salmon Anaemia – ISA virus Following the infectious salmon anaemia outbreak in Scotland in May 1998, there was a strong interest in evaluating alternatives to sodium hypochlorite for the disinfection of fish farming equipment. At a temperature of 4º C Aqua Des was found to be effective in the presence and absence of 1% serum at 1:80 and 1:250 final dilutions producing a greater than a 4-log10 reduction of the starting ISA virus infectivity. Aqua Des Stability in Sea Water Aqua Des was used to treat sea water at a range of concentrations that are typical for the fish farming industry to observe the run down. Concentrations of Aqua Des when diluted with sea water at <100mg/L gave a half life of 1-2 hours, concentrations >100mg/L gave a half life of 4-6 hours. Aqua Des Efficacy Data In The EN 1656 & EN 1657 Suspension Tests and EN 14349 Surface Test. Aqua Des has been tested against the wide range of bacterial and fungal disease causing organisms that effect fish production. When tested in the EN 1656 at a temperature of 4º C Aqua Des was found to be effective against A. salomomicida, C. piscicola, S. aureus and Y. ruckeri at a concentration of 5g/l. When tested in the EN 1657 at a temperature of 4º C Aqua Des was found to be effective against C. albicans at a concentration of 6g/l and 40g/l for A. niger. EN 14349 surface testing is a more stringent challenge than suspension testing, however, results were obtained which showed Aqua Des at a concentration of 5g/l to be effective against all bacteria except P. vulgaris. Even against this bacteria however, a degree of kill was demonstrated, even though the requirement of Log 4 was not achieved.



Aqua Des Efficacy Data in The Kelsey Sykes Test Kelsey Sykes testing is a disinfectant end point test. Inoculum is added to a solution of disinfectant in order to determine the end point of its action against a microorganism. Aqua Des 5 at 5g/l proved to be effective against S.aureus under the conditions tested.

Environmental Impact of Aqua Des Diluted Aqua Des solutions are degraded rapidly in the environment to carbon dioxide, oxygen and water. Therefore the product is not persistent, will not bioaccumulate and has no long term effects on aquatic or terrestrial ecosystems.



Appendix 1 Report on the testing of the effect of Aqua Des against the fish pathogen infectious pancreatic necrosis virus (IPN virus). Ann Kristin Øye and Espen Rimstad* Department of Pharmacology, Microbiology and Food Hygiene, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway

*Telephone + 47 2296 4766

*Fax + 47 2296 4818

*E-mail [email protected]

Introduction. Infectious pancreatic necrosis virus (IPN virus) is a small, naked dsRNA virus

belonging to the Birnaviridae family. Diseases caused by IPN virus occur globally in

aquaculture production. Although IPN virus is principally causing diseases in

salmonid fish, it has also been recognized as a pathogen in different marine fish

species as well as in bivalves.

Aqua Des contains 5 % w/w peracetic acid and approx. 20 % w/w hydrogen peroxide as well

as acetic acid and other minor ingredients. Both peracetic acid and hydrogen peroxide have

antimicrobial activity with peracetic acid being most efficacious at low concentrations and

cold conditions.

Materials 1. Virus that was tested: IPN virus, serotype Sp

2. Cell line used for cultivation of IPN virus: Chinook salmon embryo cells (CHSE-

214)

3. Growth medium/maintenance medium for the cell cultures: Eagle’s minimal

essential medium (EMEM) with 10 % fetal bovine serum in growth medium / 2 %

fetal bovine serum in maintenance medium; glutamine (4 mM), NaHCO3 (0,045

%), hepes (15 mM) and gentamicin (50 µg/ml).

mailto:[email protected]�



4. Washing of cells etc.: PBS de Boer (1,34 g Na2HPO4 · 2H2O, 0,39 g NaH2PO4 ·

2H2O, 8,5 g NaCl, Aq.dest ad 1000 ml, pH adjusted to 7,4, autoclaved at 121ºC,

15 min).

5. Dilution solution for Aqua Des: WHO-standard hard water (304 mg CaCl2, 139 mg

MgCl2 · 6H2O, Aq.dest ad 1000 ml, autoclaved at 121ºC, 15 minutes) with organic

soiling (1 % bovine serum albumine + 1 % yeast extract).

6. Neutralization solution: Sodiumthiosulfate (1 part 5 % Na2S2O3, 9 parts PBS de

Boer).

Methods

Preparation of cell cultures Two days prior to infection with the mixtures of IPN virus and Aqua Des dilutions,

CHSE-214 cells were seeded in 96-wells microtitre plates. The cells were incubated

at 20ºC. The cells were washed once with 100 µl PBS de Boer prior to infection.

Disinfecting procedure and infection of cells 1. One part virus solution was mixed well with 9 parts diluted Aqua Des at 4ºC in a

sterile tube. Dilutions from 1:100 to 1:500 were used for Aqua Des.

2. Samples of 100µl were removed after different incubation times in the range 0-15

min, and neutralized immediately by addition of 900 µl neutralization solution.

3. The neutralized virus/disinfection solution was diluted 10-folds in serum free

medium and used to infect CHSE cells in 96-well plates, in 4 parallels, 50 µl/well.

The virus was allowed to adsorb for one hour before the addition of 100 µl

maintenance medium/well.

4. Infection was allowed to proceed at 15ºC until the cytopathic effect (CPE) was

evident (about 5 days).

Calculation of inactivation of virus The inactivation of virus was calculated as follows:

Inactivation = log(N/N0),

in which N is TCID50/50 µl in the sample and N0 is TCID50/50µl at time zero.

The time required for a 4-log10 titre reduction of IPN virus (log(N/N0) ≤ -4) was

calculated for different concentrations of Aqua Des.



Additional controls In addition to measurement of virucidal effect, every set up of the experiment also

included:

1. Cell culture control only medium added

2. Virus control 1 part virus + 9 parts medium

3. Neutralization effect neutralized disinfecting solution + virus

4. Cytotoxicity control neutralized disinfecting solution

Results The results of the different experiments are presented in Figure 1, and the time

required for a 4-log10 reduction in virus-titre for the different concentrations of Aqua

Des used are given in Table 1. Cytotoxic effects on the cell culture were commonly

observed for the 1:100 dilution of Aqua Des and several set-ups of experiments using

this dilution had to be discarded.

Each graph represents one set-up of experiments with four parallel-infected wells.



Figure 1a. The effect of a 1:100 dilution of Aqua Des on IPN virus.

Figure 1b. The effect of a 1:200 dilution of Aqua Des on IPN virus.

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Figure 1c. The effect of a 1:250 dilution of Aqua Des on IPN-virus.

Figure 1d. The effect of a 1:300 dilution of Aqua Des on IPN-virus.

Figure 1e. The effect of a 1:350 dilution of Aqua Des on IPN-virus.

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Figure 1f. The effect of a 1:400 dilution of Aqua Des on IPN-virus.

Figure 1g. The effect of a 1:500 dilution of Aqua Des on IPN-virus.

Table 1.

Time required for a 4-log10 titre reduction of IPN virus at different

concentrations of Aqua Des.

PAA concentration

mg/l

Dilution of Aqua Des Time

500 1:100 < 2 min

250 1:200 ~2 min

200 1:250 ~5 min

167 1:300 ~10 min

143 1:350 >15*

125 1:400 >15*

100 1:500 >15*

*Time required for a 4-log10 reduction in virus-titre not reached.

PAA = Peracetic acid

012345678

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TCID50/50 µl

Contact time (min)


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1: 50 0 d ilu tion of Aqua De s



Conclusion At a temperature of 4° C the disinfectant Aqua Des showed a strong virucidal effect

on IPN virus. A concentration of 500 mg peracetic acid (PAA) /litre (1:100 dilution of

Aqua Des) was found to be sufficient for a 4-log10 reduction in titre of IPN virus in

less than 2 min. At a concentration of 167 mg PPA/l (1:300 dilution), a contact-time

of about 10 min was required for a 4-log10 reduction in titre of IPN-virus. At lower

concentrations of PAA a 4-log10 reduction in IPN-virus titre was not reached during

15 min incubation time at 4° C.

Oslo, November 26, 2001

Espen Rimstad Ann Øye



Appendix 2 DISINFECTION PROCEDURE FOR AQUA DES ON FISH PRODUCTION FACILITIES This procedure can be used for cleaning and disinfection of fish farming and processing facilities included as part of the hygiene regime employed at the site. Such facilities include the following:

Well boats and fish farm vessels • deck area including railings, vessel sides, hatch covers, derricks, cranes etc • well system including water valves and pumps • hull and where possible below the water line • ballast tanks • hatchery equipment and tanks

Ancillary equipment • grading machinery and conveying equipment, feeder pipes, fish pumps and

piping, pallets • temporary buildings and shelters • cages and moorings, floats, raft platforms, barges • fish traps, seines, nets, containers, net washers etc

Processing room • building walls and floors, yards • tanks, equipment, conveyors Vehicles / Transport containers

Boot baths and wheel troughs Nets Safety and Material compatibility with Aqua Des should be considered for each item treated. Refer to Material Safety Data Sheet for Aqua Des and brochure: Aqua Des Peracetic Acid Solutions, Handling, Storage and Transport Information, for details.



Procedure for use of Aqua Des Treatment of surfaces 1. Remove live and dead fish. Where appropriate equipment should be dismantled prior to

cleaning. 2. Remove gross soiling by physical scraping or brushing followed by a cold wash. 3. Pressure cleaning with hot detergent to remove fats and oils. 4. A steam treatment can be applied at this stage. 5. A further cleaning step using Aqua Des at a dilution rate1 of 1:500 can be employed. 6. Routine disinfection is conducted with Aqua Des at a dilution rate1 of 1:200. For rough,

porous or dirty surfaces use a dilution rate of 1:125.

a) Application can be by high pressure spray at a rate of 0.25-0.3 litres per m2. Facilities and equipment should be allowed to dry naturally before re-use.

b) For smaller items of equipment and tanks etc, a soak treatment can be

employed. A minimum soak period of 10 minutes is recommended.

Where there is a risk of contamination or infection with specific diseases or microorganisms, then higher doses or longer soak periods can be recommended according to the problem identified – see Table 1 for recommendations.

No adjustment in dose applied is necessary with varying temperature.

Pay special attention to hard to clean places where contamination may persist.

1 Dilution with fresh water is desirable where available but seawater can also be used.

JH Massey 6/9/00 M-53-00



Boot baths and wheel troughs After cleaning which can use the above procedure, these should be filled with a solution of Aqua Des at a dilution rate of 1:200. Solutions should be replaced at least twice a week and preferably once a day.

Nets 1. Remove live and dead fish, dry and transfer to net washer using normal containment

and hygiene measures. 2. Wash in net washer. 3. Transfer to disinfection vessel. Routine disinfection is conducted with Aqua Des at a

dilution rate1 of 1:200. For nets with a high level of fouling use a dilution rate of 1:125. Soak for a minimum of 30 minutes.

Where there is a risk of contamination or infection with specific diseases or microorganisms, then higher doses or longer soak periods can be recommended according to the problem identified – see Table 1 for recommendations.

No adjustment in dose applied is necessary with varying temperature.

Notes: • Care should be taken in disposal of waste disinfectant solutions and drainage. • This protocol can be modified to take account of conditions. Further advice is available from

Solvay Interox / Solvay Nordic as necessary. 1 Dilution with fresh water is desirable where available but seawater can also be used. JH Massey 6/9/00 M-53-00



Where there is a risk of contamination or infection with specific diseases or microorganisms, then higher doses or longer soak periods can be recommended according to the problem identified – see Table 1 for recommendations. Table 1 Disinfectant Treatments with Aqua Des

Microorganism Dilution of Aqua Des

Disinfection Time

Virus : General IPN Virus

1:100 1:100 1:300

30 minutes 2 minutes

10 minutes Bacteria: Escherichia coli Pseudomonas aeruginosa Salmonella typhimurium Staphylococcus aureus Streptococcus faecium (Enterococcus faecium) Spore-formers: Bacillus cereus Bacillus subtilis Clostridium sporogenes

1:200 1:200 1:200 1:200 1:200

1:20 1:50

1:200

10 minutes 10 minutes 10 minutes 10 minutes 10 minutes

1 hour 1 hour 1 hour

Fungi: Aspergillus spp. Penicillium spp. Candida albicans

1:50 1:50 1:100 1:200

15 minutes 15 minutes

2 hours 10 minutes

JH Massey 6/9/00 M-53-00



Appendix 3

SOLVAY

MEMO

CHEMICALS SECTOR – PEROXYGENS DISINFECTION TECHNOLOGY TEAM

SOLVAY on the web - http://www.solvay.com M-010-04

Date:

25th February 2004

To:

GE Williams

From:

JH Massey

Aqua Des Stability in Sea Water In Laboratory experiments, a sample of Sea Water (taken from the North Wales coast) has been treated with Aqua Des in order to observe the run down. Aqua Des was used to treat the sea water at a range of concentrations that are typical for the fish farming industry 5 mg/l, 50 mg/l, 100 mg/l, 250 mg/l and 300 mg/l PAA. Concentrations of Aqua Des when diluted with sea water at <100mg/L gave a half life of 1-2 hours, concentrations >100mg/L gave a half life of 4-6 hours This information is useful in assessing probable residual levels of Aqua Des for the fish farming industry. JH Massey

DISCLAIMER Whilst every care has been taken in the investigation of the topic considered in the report, it is neither

reasonable nor possible for the author to identify, define and address all the variations which recipients of this report may invoke in interpreting the information herein contained. All information contained herein is given in good faith but without warranty or guarantee. It is the responsibility of any recipient utilising any of the information contained herein to establish the appropriateness of the information with respect to his intended usage. If you have any queries about this report please contact the author.



Background A sample of sea water from the North Wales coast was collected for an experiment to observe the stability of diluted Aqua Des in sea water for its use in fish farming. Experimental Protocol The sea water was treated with the following concentrations of Aqua Des

5 mg/l (of PAA) 50 mg/l 100 mg/l 250 mg/l 300 mg/l

and examined over 48hrs at ambient temperature for residual peracetic acid using Reflectoquant and Merkoquant test strips. Results The results obtained are shown in Table 1and Graph 1. Conclusions/ Implications Concentrations of Aqua Des when diluted with sea water at <100mg/L gave a half life of between 1 and 2 hours, concentrations of >100mg/L gave a half life of between 4 and 6 hours. From other studies carried out the degradation of Aqua Des in seawater seems to be faster than the degradation in fresh water, which could be related to the high pH and salinity (ionic strength)1

1 Peracetic acid (Cas No. 79-21-0) and its equilibrium solutions, JACC No.40, European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), January 2001, ISSN –0733-6339-40, p30

. When water is added to Aqua Des the hydrolysis reaction below takes place: CH3CO3H + H2O -> H3CO2H + H2O2 Therefore measuring peracetic acid disappearance can give the hydrolysis rate and half-life for peracetic acid in seawater. Peracetic acid half-life calculated using method adapted from OECD guideline for testing of chemicals 111 ”hydrolysis as a function of pH”



This information is useful in assessing probable residual levels of Aqua Des for the fish farming industry.


Doc No: M-029-05 Ref: JHM Date: 20/02/09 of 37

Table 1 Stability of Aqua Des in Sea Water Concentration of PAA

Method of Analysis

Time 0 1hr 2hr 3hr 4hr 5hr 6hr 24hr 48hr

5ppm R 9.2 6.4 2.4 1.7 0.9 0.3 0 0 M ~10 5-10 0 0 0 0 0 0 50ppm R 48.6 53.1 39.7 20.7 15 11.6 9.5 0 0 M 50 50 ~40 20-30 20 10 5-10 0 0 100ppm R 124 100 86.2 77 71 21.6 16.2 0.6 0.4 M ~100 ~100 50-100 50-100 30-50 30 ~20 0 0 250ppm R 329 281 252 218 206 181 174 1.0 0.7 M 300-400 300-400 ~300 ~250 200-250 150-200 150-200 0 0 300ppm R 390 306 259 212 211 152 142 1.2 1.0 M 400-500 400-500 ~300 ~250 200-250 ~150 ~150 0 0 R = Reflectoquant M = Merkoquant Lab Book Reference – JHM A1/71-73 Half live calculation. Table Log10 (concentration) vs. time

Conc. of PAA time (hr) t/12

0 1 2 3 4 5 6 slope r2 kobs hours 5ppm 0.96 0.81 0.38 0.23 -0.05 -0.20 99% 0.46 2 50ppm 1.69 1.73 1.60 1.32 1.18 1.06 0.98 -0.14 95% 0.32 2 100ppm 2.09 2.00 1.94 1.89 1.85 1.33 1.21 -0.15 83% 0.33 2 250ppm 2.52 2.45 2.40 2.34 2.31 2.26 2.24 -0.05 98% 0.11 6 300ppm 2.59 2.49 2.41 2.33 2.32 2.18 2.15 -0.07 97% 0.17 4

T1/2 = 0.93/kobs Kobs = - slope x 2.303 when Log10 (Conc.) plotted against time


Doc No: M-029-05 Ref: JHM Date: 20/02/09 of 37

Graph 1

Stability of Aqua Des in Sea Water

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of 37 20 February 2009

Appendix 4 1 Introduction

Disinfectant testing may be divided into two categories: - Suspension testing offers a reliable means of evaluating the efficacy of disinfectant products under laboratory conditions. BS EN 1656, BS EN 1657 are established methods used to determine bactericidal and fungicidal activity respectively with reference to the veterinary industry. Suspension testing is often used as a means of studying a wide range of products to identify which one is the most effective. Surface testing presents a more stringent challenge to disinfectant products and is a closer representation of practical in-use conditions. It is typically used on a reduced number of products that have demonstrated superior performance under suspension testing. BS EN 14349 surface test is applicable to bacteria, again with reference to the veterinary industry.

2 Scope of Testing Aqua Des, supplied by Solvay was tested in accordance with a technical agreement drawn up between MGS Laboratories and Solvay. 3 Principle of Suspension Test Methods BS EN 1656 and BS EN 1657 are based upon the same principle. BS EN 1656 and BS EN 1657 testing is performed under dirty conditions using a sterile filtered 1.0% (in test concentration) solution of Bovine Serum Albumin (BSA) and 1.0% (in test concentration) Yeast Extract as the interfering substance. Product is mixed with the interfering substance and organism for the set contact time, and an aliquot then transferred to a neutraliser. This neutraliser is then enumerated to establish the number of viable organisms remaining, thus enabling the log kill to be established. 4 Principle of Surface Testing Surface testing is performed under dirty conditions represented by the use of sterile filtered 1.0% (in test concentration) solution of Bovine Serum Albumin (BSA) and 1.0% (in test concentration) Yeast Extract solution as the interfering substance. For veterinary applications, it is appropriate to test under these dirty conditions An inoculum suspension of strength appropriate for the organism under test and containing the appropriate level of BSA/Yeast Extract is prepared. A known small volume of the mixture is transferred to a clean, sterile sample of the surface under test and allowed to dry under suitable conditions. Once dry, the residue on the surface is treated with disinfectant solution of the appropriate concentration and allowed to stand for the required contact time. The surface is then transferred to a neutraliser solution and agitated to dislodge any remaining organisms from the surface. The solution is enumerated using standard microbiological techniques, and the result is compared to that obtained in a control experiment, in which water is


used in place of disinfectant solution. A log reduction of ≥4 for bacteria is required to pass the test.

5 Preparation

5.1 Preparation of test solutions Aqua Des was supplied by Solvay in concentrated form but diluted according to Solvay requirements (See results). All working solutions were prepared on a v/v basis from commercially available dehydrated Sea Water granules. Solutions for suspension testing are prepared at 125% of in test concentration to take account of dilution factors that are a feature of the BS EN 1650/1656 methods. Dilution factors do not apply to surface testing and solutions are made up at the required in test concentration. 5.2 Maintenance of organisms Testing was performed using organisms requested by Solvay in the Technical Agreement. All the organisms were received from NCTC & NCIMB (See results for organism details). They were checked for purity and inoculated into a broth. Following incubation, the broth was used to generate controlled organism slopes that were maintained as part of the MGS storage and maintenance of culture procedure.

5.3 Preparation of BSA/Yeast Extract solutions Interfering Substances were prepared to give the in test concentration required, and sterile filtered using a 0.22 micron filter.

5.4 Preparation of Aqua Des solution Stock solutions of Aqua Des were freshly prepared using Sea Water to requested concentrations as per Solvay requirements (See results). These solutions were used within 4 hours of preparation.


5.5 Preparation of Neutraliser solution The neutraliser solution used in the test programme was a formulation recommended by Solvay, prepared according to Solvay instructions, utilising Thiosulphate solution, and Catalase in an aqueous base.

5.6 Preparation of inoculum suspensions Bacteria for surface testing were cultured on Tryptone Soya Agar (TSA) for 24 hours before being suspended in Ringers & Peptone (R&P). The suspensions were used within four hours of preparation. C. albicans was cultured on Saboraud Dextrose Agar (SDA) for 24 hours before being suspended in R&P. The suspension was used within four hours of preparation. A. niger was grown on plates of SDA for a minimum of 5 days to encourage growth of spores. The plates were harvested into R&P, filtered through a sintered glass funnel and centrifuged at 2000 rpm for 20 minutes. The supernatant liquid was decanted and the spore pellet re-suspended in R&P. The suspension was examined microscopically to confirm absence of hyphae/germinating spores. 5.7 Preparation of test surfaces MGS Stainless steel coupons were used for the surface testing. The coupons were immersed in 5% Janitol solution for 15 minutes, rinsed well with sterile water, drained and transferred to 70% methanol solution. Approximately 2 hours before use, the surfaces were aseptically transferred to sterile dishes and dried at room temperature under laminar flow with occasional turning until completely dry. The clean surfaces were then stored at test temperature.

5.8 Preparation of inoculated test surfaces Immediately before use, 5ml of Interfering substance and 5ml of inoculum suspension were mixed aseptically to produce an inoculum test suspension with a BSA and Yeast Extract concentration of 1.0%. 0.05ml of test suspension was pipetted onto the centre of previously prepared individual test surfaces and maintained at 37oC in a current of air until completely dry. Drying time was approximately 30 minutes.


6 Testing 6.1 Suspension Testing All testing was conducted at 4oC +/-2oC and all timings controlled to +/-10 seconds. Concentrations of Aqua Des were used as requested by Solvay (see results). A contact time of 30 minutes was used for all organisms as listed in the results Testing was carried out according to the requirements of BSEN 1656 & BSEN 1657. 6.2 Surface Testing All testing was conducted at 4oC +/-2oC. Concentrations of Aqua Des were used as requested by Solvay (see results). A contact time of 30 minutes was used for all organisms as listed in the results Testing was carried out according to the requirements of BSEN 14939. All timings were controlled to +/-10 seconds with the exception of the crucial sonication step, which was controlled to +/-1 second.


7.Results 7.1 Suspension Results –BSEN 1656

TEST RESULTS (LOG REDUCTION) VALIDATION

(Pass criteria >50%) Organism Concentration Pass criteria 1% BSA + 1% yeast extract

A. salmonicida NCIMB 833

3.0g/l

Log 5

>6.4 PASS >100% (PASS)

4.0g/l >6.4 PASS >100% (PASS)

5.0g/l >6.4 PASS >100% (PASS)

C. piscicola NCIMB 2264

3.0 g/l

Log 5

>6.8 PASS 94% (PASS)

4.0 g/l >6.8 PASS 94% (PASS)

5.0 g/l >6.8 PASS 94% (PASS)

S.aureus NCTC 10788

1.5g/l

Log 5

<3.8 FAIL 95% (PASS)

3.0g/l <3.8 FAIL 95% (PASS)

5.0g/l >6.3 PASS 95% (PASS)

6.0g/l >6.3 PASS 95% (PASS)

8.0g/l >6.3 PASS 95% (PASS)

10.0g/l >6.3 PASS 95% (PASS)

Y. ruckeri NCIMB 12986

3.0g/l

Log 5

>7.0 PASS 80% (PASS)

6.0g/l 5.5 PASS 77% (PASS)

8.0g/l 6.0 PASS 76% (PASS)

10.0g/l >6.0 PASS 89% (PASS)


7.2 Suspension Testing –BSEN 1657

Organism Concentration

TEST RESULTS (LOG REDUCTION) VALIDATION

(Pass criteria >50%) Pass criteria 1% BSA + 1% yeast extract

A. niger NCPF 2275

20g/l

Log 4

<1.6 FAIL 80% (PASS)

30g/l 2.2 FAIL 80% (PASS)

40g/l >4.4 PASS >100% (PASS)

70g/l >4.4 PASS 100% (PASS)

100g/l >4.4 PASS 83% (PASS)

C. albicans NCPF 3179

4.0g/l

Log 4

<3.3 FAIL >100% (PASS)

6.0g/l >6.0 PASS 90% (PASS)

10.0g/l >6.0 PASS 99% (PASS)

14.0g/l >6.0 PASS >100% (PASS)

7.3 Surface Testing Results – BSEN 14349

Organism Concentration Pass criteria Result (Log Reduction) Validation

Stainless steel A B

E.hirae NCTC 12367

2.0 g/l

Log 4

<1.9 FAIL 78.1% 32

5.0 g/l 6.4 PASS 78.1% 1

7.0 g/l >6.4 PASS 78.1% NIL

P.aeruginosa NCTC 10332

2.0 g/l

Log 4

<0.1 FAIL 1.4% 30

5.0 g/l 4.1 PASS 1.4% 1

7.0 g/l >4.6 PASS 1.4% NIL

P.vulgaris NCTC 4175

2.0 g/l

Log 4

<1.4 FAIL 14.2% 20

5.0 g/l 2.3 FAIL 14.2% 1

7.0 g/l 5.6 PASS 14.2% NIL

S.aureus NCTC 10788

2.0 g/l

Log 4

<1.9 FAIL >100 % 142 *

5.0 g/l >6.4 PASS >100 % NIL

7.0 g/l >6.4 PASS >100 % NIL

*Validation failure because of product failure –see section 8.3 8. Validation 8.1 Suspension Validation There are three levels of Suspension testing Validation, namely Experimental Conditions Validation (ECV), Neutraliser Toxicity Validation (NTV), and Dilution Neutralisation Validation (DNV).


8.1.1 Experimental Conditions Validation (ECV) ECV is the basic level, to ensure the organism is not adversely affected by the interfering substance. Pass Criteria: ≥50% 8.1.2 Neutraliser Toxicity Validation (NTV) NTV checks that the neutraliser is not toxic to the organism. Pass Criteria: ≥50% 8.1.3 Dilution Neutralisation Validation (DNV) The most important, and the only validation quoted in the results, is DNV. This is a combined check of NTV and ECV. In addition, it tests for effective neutralisation of the dinsfectant. If the product has not been neutralised, the log kill would be artificially raised, thereby improving product performance. By performing the DNV, this risk is eliminated because neutralisation failure means DNV failure, thus invalidating the test results. Only the DNV result is quoted as NTV and ECV must pass in order for DNV to pass. Pass Criteria: ≥50% 8.2 Surface testing Validation 8.2.1 Recovery of Organism from Prepared Surfaces (A) The preparation and storage of inoculated surfaces is critical to the success of surface testing. BS EN 13697 literature makes reference to the following validation requirement :- Log (cfu in 0.05ml inoculum suspension) - Log (cfu recovered from control surface) ≥2 i.e. the number of organisms recovered from a test surface that has been inoculated, dried, exposed to water, neutraliser and sonication should be greater than 1% of the population placed on the surface during inoculation. 8.2.2 Residual Organisms on the Test Surface (B) Removal of residual organisms from the test surfaces is another critical stage in surface testing. Following sonication, surfaces exposed to active concentrations of product are aseptically removed from the neutraliser solution, rinsed with sterile water and transferred to a pre-poured agar plate. 0.1ml of sterile water is added and the surface


scratched with a sterile pipette tip to dislodge any residual organisms. The surface is overlaid with agar and incubated. Pass citeria <100 cfu. 8.3 Validation Results One surface testing Residual Organism validation failure (B) was noted. Such failures have been encountered in previous testing and have always been associated with poor product performance. It is likely that the high population of residual organisms resulting from poor product performance overwhelms the mechanical removal process leading to validation failure. Under these circumstances and bearing in mind that the purpose of mechanical action validation is to ensure that product performance is not over-stated, it is our opinion that the test results may be accepted. It should be noted that where product performance was good validation results were all satisfactory. 9. Discussion 9.1 Suspension Testing As expected, Aqua Des at a concentration of 5g/l proved to be highly effective against the bacteria and yeast under the conditions tested. Action against A.niger spores required a concentration of 40g/l under the conditions tested. 9.2 Surface Testing Surface testing is a more stringent challenge than suspension testing, however, results were obtained which showed Aqua Des at a concentration of 5g/l to be effective against all bacteria except P. vulgaris. Even against this bacteria, however, a degree of kill was demonstrated, even though the requirement of Log 4 was not achieved.


Appendix 5

1 Introduction

Kelsey Sykes testing is a disinfectant end point test. Inoculum is added to a solution of disinfectant in order to determine the end point of its action against a microorganism.

2 Scope of Testing

Aqua Des, supplied by Solvay was tested in accordance with the Kelsey Sykes method provided by Solvay (See results). All working solutions were prepared using standard hardness water. These solutions were used within 4 hours of preparation. The inoculum was added to a 3ml solution of disinfectant over time, and transferred into multiple recovery broths in order to determine probability of kill. Testing was performed under dirty conditions, using organisms requested by Solvay in the Technical Agreement TA063. All the organisms were received from NCTC & NCIMB. They were checked for purity and inoculated into broths. Following incubation, the broths were used to generate controlled organism slopes that were maintained as part of the MGS storage and maintenance of culture procedure. The organisms were prepared by following the method supplied by Solvay. The organism chosen for final testing was determined in accordance with the same method.

3 Results

Organism Test Batch No Concentration Pass/Fail

S. aureus NCTC 10788

1

2.5g/l Fail

5.0g/l Pass*

7.5g/l Pass

2

2.5g/l Fail

5.0g/l Pass

7.5g/l Pass

3

2.5g/l Fail

5.0g/l Pass

7.5g/l Pass

*NB. This result was a borderline pass. In the light of the other 2 results, a satisfactory pass at 5.0g/l has been obtained. 4 Discussion

Aqua Des at the recommended concentration of 5.0g/l proved to be effective against S. aureus under the conditions tested. These results should be viewed in conjunction with the MGS report (TA 063) for BSEN 1656, 1657 and 14349 which provide quantitative rather than qualitative results.


Appendix 6 M-035-05

ENVIRONMENTAL IMPACT OF AQUA DES

Introduction Aqua Des is an active bactericide, fungicide and sporicide, which contains peracetic acid, hydrogen peroxide, acetic acid and water. Peracetic acid is always found in solution and in equilibrium with acetic acid and hydrogen peroxide: CH3COOOH + H2O <-----> CH3COOH + H2O2 The efficacy and also the toxicity of AQUA DES is mainly explained by the presence of peracetic acid in the product. Peracetic acid is able to oxidise different organic components of the tissue of all organisms. Based on this mode of action peracetic acid is potentially toxic for many organisms. Toxicity to aquatic organisms To investigate any potential effects of AQUA DES on the environment, SOLVAY conducted an extensive number of aquatic laboratory toxicity tests according to internationally accepted guidelines. Studies were performed with freshwater and saltwater organisms. A common way to express the toxicity of a compound is to determine the lethal concentration (LC50) of the compound. The LC50 is defined as the concentration which results in 50 % mortality of the organisms exposed during laboratory tests. This means that a very toxic compound has a low LC50 value. The results of the laboratory toxicity tests with PROXITANE® products are presented in the table on the next page. All LC50 values were based on concentrations of peracetic acid. Based on the peracetic acid content of the PROXITANE® products, the corresponding LC50 for the products can be calculated.


Species Product LC50 (mg/l) Reference

D. magna (water flea) PROXITANE® (15 %) 0.50 Douglas (1986a) C. crangon (shrimp) Oxymaster (12 %) 15 Tinsley et al. (1987a)

P. triqueter (embryos of marine tubeworm)

Oxymaster (12 %) 0.19 Dixon (1988)

C. gigas (embryos of oyster) Oxymaster (12 %) 0.28 Butler (1987) M. edulis (embryos of mussel) Oxymaster (12 %) 0.27 Fairhurst (1987)

O. mykiss (rainbow trout) PROXITANE® (15 %) 2.0 Douglas (1986b) P. platessa (plaice, marine fish) Oxymaster (12 %) 11 Tinsley et al. (1987b)

The results showed that the lowest LC50 values were found for the smallest aquatic organisms (water fleas and embryos). Small organisms, e.g. water fleas, algae and embryos of mussels, are probably more sensitive to peracetic acid because they have a high surface to body weight ratio. The remaining species which were tested showed LC50 values which were higher than 1 mg/l. Acetic acid and hydrogen peroxide are less toxic than peracetic acid, which means that the toxicity of PAA products will be explained mainly by the presence of peracetic acid in the product. Therefore it can be assumed in general that a PAA product with a two times higher peracetic acid concentration is also two times more toxic. Fate in water In surface water peracetic acid will be hydrolysed. The degradation products formed by hydrolysis are acetic acid and hydrogen peroxide, both of which are easily (bio)degradable substances. In water the degradation rate of peracetic acid depends on temperature, concentration and the presence of organic matter in the water. Mainly the microorganism (e.g bacteria) will be responsible for the degradation of peracetic acid in surface water. However, a relatively high concentration of peracetic acid in surface water could kill the microorganisms and in this case chemical degradation could be more important. In general the half-life of peracetic acid will be less than 1 day assuming initial concentrations of about 1 mg/l. The biodegradation of peracetic acid has been studied using several experimental models (Gerike et al., 1990). At a peracetic acid concentration of 90 mg/l an inhibition of the degradation was found due to microbial toxicity at this concentration. However a rapid degradation of peracetic acid was found in a Closed Bottle Test, when solutions of 2-5 mg/l were inoculated with adapted bacteria. Soil After a spillage of AQUA DES to the soil, peracetic acid will degrade rapidly due the reactivity of the compound. Laboratory experiments showed half-lifes of several minutes when peracetic acid was applied at soil samples packed in columns (McAdam, 1997). The compound will in most cases not penetrate the soil to a significant extent. It should be realised that, similar to the effect on waste water biological treatment plants, the effect on soil flora and fauna depends on the volume and speed of product release. A strong correlation between iron content and decomposition rate is known to exist.


Bioaccumulation Bioaccumulation is defined as the net accumulation of a substance by an organism as a result of uptake from all environmental sources. Due to the rapid degradation of peracetic acid in the environment and due a rapid metabolism of the compound in the organism, peracetic will not bioaccumulate. The octanol/water partition coefficient is sometimes used to estimate the bioaccumulation potential of a compound. The calculated octanol/water partition coefficient for peracetic acid is very low, which confirms the low bioaccumulation potential of this compound (Thus, 1994). Conclusion It should be prevented that AQUA DES enters directly the aquatic environment because peracetic acid concentrations of 1 mg/l will already affect the aquatic ecosystem. However, diluted peracetic acid solutions are degraded rapidly in the environment to carbon dioxide, oxygen and water. Therefore the product is not persistent, will not bioaccumulate and has no long term effects on aquatic or terrestrial ecosystems. A.G. Berends C.G. de Rooij Toxicologist Head Chemical Safety and Toxicology Department If you need more information on the environmental characteristics of AQUA DES please contact A.G. Berends and/or C.G. de Rooij. SOLVAY S.A. DCT/ES - Toxicology and Chemical Safety Rue de Ransbeek 310 B-1120 Brussels Tel. + 32 2264 3398 Fax. + 32 2264 2990 E-mail: [email protected] The content of this document has been prepared by SOLVAY with all possible care and from the available scientific information. It is provided for information only. SOLVAY cannot accept any responsibility or liability and does not provide a warranty for any use or interpretation of the material contained in the publication.


References Butler R, 1987. Determination of the 48 hour median effect concentration (EC50) of Oxymaster to the pacific oyster (Crassostrea gigas) in terms of larval survival and development. WRc Environment (GB) CO 1643-M/EV 8687. Dixon D, 1988. Comparative study of the toxicity and genotoxicity of Oxymaster and hypochlorite. Report for Solvay Interox. Douglas MT, Pell IB, 1986a. The acute toxicity of PROXITANE 1507 to Daphnia magna. Huntingdon Research Centre Ltd., report n° LPT 44/851641. Douglas MT, Pell IB, 1986b. The acute toxicity of PROXITANE 1507 to rainbow trout. Huntingdon Research Centre Ltd., report n° LPT 43/851643. Fairhurst F, 1987. Determination of the 48 hour median effect concentration (EC50) of Oxymaster to the common mussel (Mytilus edulis), interms of larval survival and development. WRc Environment (GB) CO 1644-M/EV 8687. Gerike P, Gode P, 1990. The biodegradability and inhibitory threshold concentration of some desinfectants. Chemosphere, 21 (6): 799-812. Maunuksela, J., 1997. Telefax on experimental trials with peracetic acid and waste water treatment plant. Finnish Peroxides, Voikkaa, Finland. McAdam, J., 1997. Peracetic acid in the presence of soil. Internal memorandum of SOLVAY Interox, Widnes, United Kingdom. Poffé R, de Burggrave A, Houtmeyers J, Verachtert H, 1978. Disinfection of eflluents from municipal sewage treatment plant with peroxy acids. Zbl. Bakt. Hyg., I.Abt. Orig. B167: 337-346. Thus JLG, 1994. Calculation of the octanol/water partition coefficient of peracetic acid. Telefax of SOLVAY Duphar, Weesp, The Netherlands. Thus JLG, Groeneveld, AHC and JMTh van der Laan-Straathof, 1994. Possible biodegradation of peracetic acid (preliminary experiment). SOLVAY Duphar Int. Doc. No. 56834/16/97. SOLVAY Duphar, C.J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands. Tinsley D, Sims I, 1987a. The acute toxicity of Oxymaster to brown shrimp (Crangon crangon) under semi-static conditions. WRc Environment (GB) CO 1649-M/EV 8687. Tinsley D, Sims I, 1987b. The acute toxicity of Oxymaster to plaice (Pleuronectes platessa) under semi-static conditions. WRc Environment (GB) CO 1650-M/EV 8687 .

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