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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code FC1169

2. Project title

Defra aquaculture disinfectant listing scheme

3. Contractororganisation(s)

CefasWeymouth Laboratory,Barrack Rd, The NotheWeymouth, DorsetDT4 8UB

54. Total Defra project costs £ £61,890.00(agreed fixed price)

5. Project: start date................ 01 April 2005

end date................. 30 September 2006

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

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IntroductionUse of safe, effective disinfectants is very important for the implementation of effective biosecurity in intensive aquaculture, as for other farming operations. Important uses include treatment of equipment (e.g. tanks, nets or transportation containers) and effluent, limiting disease spread both within and between sites. Disinfectants are also required for decontamination of premises where there have been outbreaks of notifiable diseases (e.g. infectious salmon anaemia (ISA), spring viraemia of carp (SVC) or viral haemorrhagic septicaemia (VHS)).Under provisions of the Animal Health Act 1981, Defra maintain a list of disinfectants that are approved for use in the control of notifiable diseases of terrestrial animals and birds. This system of approval does not extend to the pathogens of fish and shellfish.Defra is proposing to develop a new scheme that will give disinfectant manufacturers the opportunity to prove that their products are effective against relevant aquaculture pathogens, in addition to terrestrial animal pathogens. Products that satisfy Defra criteria will be placed on a list, which will be freely accessible to farmers and aquaculture health professionals, aiding them in the selection of effective products.The scheme will have UK-wide relevance and is being developed in consultation with Defra funded this Cefas led R& D project (FC 1169) to develop applicable aquaculture disinfectant testing standards and to assist Defra with implementation of the scheme.

Objectives 1 and 3 Technical subcommittee workshops.A range of experts was gathered to recommend appropriate disinfectant testing standards at two workshops. The standards were developed in consultation with relevant experts from across the UK, with representatives from FRS Marine Laboratory, Aberdeen (an agency of the Scottish Executive Environment and Rural Affairs Department, SEERAD), and the Department of Agriculture and Rural Development, Northern Ireland (DARD) attending. The agencies responsible for the testing of disinfectants within the terrestrial scheme have also been consulted. A representative from The Norwegian Veterinary Institute also attended to provide advice (the Norwegians already have an established aquaculture disinfectant approvals scheme).

Objective 2 Disinfectant Testing StandardsUnder FC1169, a laboratory testing exercise was undertaken to develop aquaculture disinfectant testing standards as recommended by the technical subcommittee at the workshops. Two BS EN standards recommended for demonstrating a products efficacy against bacterial pathogens (a modified BS EN 1656) and viral agents (a modification of BS EN14675, substituting infectious pancreatic necrosis virus IPNV for bovine enterovirus) were tested. Contact time and temperature are both critical parameters for disinfectant testing. The current intention is to recommend that all volunteered products be tested in the presence of high organic loading for a contact time of thirty minutes at 4 °C. These conditions are the same as those specified in the Defra terrestrial disease scheme. As the activity of disinfectants decreases with a reduction in temperature, and the presence of organic material interferes with many disinfectants, they theoretically represent challenging conditions for the disinfectant. There may also be scope for manufacturers to submit data on product activity at shorter contact times.

Bacterial testing StandardEN1656 was modified to test Aeromonas salmonicida subsp. salmonicida, Yersinia ruckeri, Carnobacterium piscicola and Lactococcus garvieae, bacterial pathogens of aquaculture relevance, rather then the veterinary pathogens recommended in the standard.In summary, a standardised bacterial suspension (1.5 - 5 x108 CFU ml-1) is exposed to product prepared in hard water in presence of organic loading for a specified contact time and temperature. For a product to pass the test, it must demonstrate a greater than 5 log reduction in the bacterial count (equivalent to inactivating > 99.999% of the bacteria in the original test sample). Testing confirmed that the substituted organisms could be stored and readily resuscitated for use in testing , were not adversely affected by the assay conditions (except exposure to the dilution of disinfectant under test)

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and suspensions of the test organisms could be readily prepared for use in testing. All four organisms were also shown to be non sensitive to recovery using the standard recommended pour plate assay (that was easier to perform then the alternative spread plate assay). The modified standard was tested against four products with different chemistries: an iodophore containing product, a Chloramine T containing product, a combination peracetic acid hydrogen peroxide based disinfectant and a peroxygen system. It was shown that the preferred neutralisation dilution method was appropriate for testing all four products with suitable neutralisers identified. Repeat testing showed that, although there was some test-to-test variation in results around the higher end of the range of effective dilutions, dilutions of all four products could be reliably tested for bactericidal activity using the modified assay.

Viral testing standardA modification of the draft European standard BS EN 14675 (Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field) was validated with. Infectious Pancreatic Necrosis Virus (IPNV), Sp serotype, replacing Bovine Enterovirus Type 1, as a suitable model virus. IPNV is already successfully used in the aforementioned Norwegian scheme. It is highly relevant to UK aquaculture, provides a very resistant model and, from a practical aspect, reaches high titres when grown in cell culture. In summary, a standardised viral suspension (>1.0 x107 viral particles ml-1) is exposed to product prepared in hard water in presence of organic loading for a specified contact time and temperature. For a product to pass the test, it must demonstrate a greater than 4 log reduction in the bacterial count (equivalent to inactivating > 99.99% of the virus particles in the original test sample). The modified standard was tested against the same four products as used for the bacterial standard validation. With the exception of one product, where there were cell cytotoxicity problems, repeat testing showed that dilutions of three of the products tested could be reliably tested for virucidal activity using the modified assay.

Objective 4 Guidelines for manufacturers As part of the process to develop the testing standards, explanatory documents for forwarding to companies wishing to have their products listed have been drafted. These guidelines detail what data is required by Defra to support a product listing and also details the supporting Quality Assurance criteria (QA checklists that can be filled out by laboratories doing the testing).

Scheme Implementation (Objectives 6-8)Cefas has assisted Defra with scheme implementation, including visiting London to hold a meeting to discuss scheme implementation and providing advice on the form the scheme should take, including recommending appropriate experts to review test results submitted to Defra by companies.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

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IntroductionDisinfectants are vital tools in the implementation of effective farm biosecurity. They are used for the removal of potentially pathogenic microorganisms from surfaces, effluent and rearing facilities including equipment, tanks and clothing as well as the disinfection of gametes, principally ova. Disinfectants are also required for decontamination of premises where there have been outbreaks of notifiable diseases (e.g. infectious salmon anaemia (ISA), spring viraemia of carp (SVC) or viral haemorrhagic septicaemia (VHS)). Although their use may be well established and very effective in most cases, the rules governing their application and, often, what products should be selected for particular purposes are not so well understood. This lack of knowledge as to what may be used safely and effectively was brought into sharp focus by the ISA outbreak in Scotland in 1998 (Scottish Executive 1998). In this example, the absence of identified alternatives leads to the emergency use of hypochlorite at high concentrations (100-1000 mg L-1). This drastic action required a temporary derogation from the Scottish Environment Protection Agency (SEPA) which maintains a presumption against the discharge of effluents containing active residual chlorine, a presumption that has since been reinstated.

Regulations Governing Disinfectant UseHealth and Safety Disinfectants used to treat equipment are classed as biocides. In the past, their use in fish farming has generally been covered as for the other chemicals farmers routinely use. For instance, if the chemicals were hazardous to the operators or the environment standard Health and Safety at work procedures would need to be followed, e.g. the provisions of COSHH should be adhered to, including, where appropriate, production of Risk Assessments for their specific uses. Items such as ordering, storing, handling, using and disposing of disinfectants are all, to some extent, covered under these regulations. Discharge consent from the appropriate regulatory authority would also generally be required before such chemicals could be discharged.

Discharge consentsWith large volumes of disinfectant effluent being discharged each year into watersheds and the marine environment, discharge of disinfectant products is tightly controlled. In Scotland the discharge of disinfectants to the environment is controlled under the Food and Environment Protection Act 1985 Part II Deposits in the Sea, as amended by the Environment Protection Act 1990 (FEPA) and the Control of Pollution Act 1974 as amended by the Water Act 1989 and the Environment Act 1995 (COPA). A COPA discharge consent is issued by SEPA (Marine Laboratory Publication 1999). A similar process exists in England and Wales, with consents issued by the Environment Agency. Consents must be obtained for activities such as net and cage disinfection at both cage sites and shore bases and for effluent disposal at processing plants.

Biocidal Products Directive (98/8/EC)As well as these existing controls, authorisation of biocides is now also under the control of the Biocidal Products Directive (BPD; 98/08/EC; implemented into UK law through SI 2001 No. 880 and S.R. 2001 No. 422). In the future all biocides will be authorised for the particular use claimed (Marketing Claim) following a review of a dossier of data in support of that claim. In the UK such authorisations will be administered by the Health and Safety Executive’s Biocides & Pesticides Unit. However, as there are a great many chemicals already used as disinfectants and other biocides, it will take some time before they are reviewed against the updated criteria specified under BPD. There are four priority lists for review. The active ingredients in veterinary disinfectants (Product Type 3) will be reviewed by HSE under BPD, as part of the third priority list, starting in mid-2007. Industry had to ‘Notify’ those substances they intended to support for review, including the Product Types they intended to support them in, to the European Commission. If an active substance has been Notified for review in product type 3, products can be placed on the market (subject to any existing national rules) until the review is complete. Once the review is complete, products containing that substance will have to be authorised under the Directive or be removed from the market. A system of ‘mutual recognition’ can take place under the Directive, so

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that once a product is authorised in one Member State, that authorisation can be carried across to the other Member States. If industry did not want to support a substance through the review programme they had to option to ‘Identify’ it to the Commission, and products containing substances that have only been Identified – or those containing substances that have been Notified, but not for the relevant product type – could remain on the market until 1st September 2006.

Existing disinfectant listing and approval schemesAnimal Health Act 1981Under the provisions of the Animal Health Act 1981, Defra maintains a list of disinfectants that are approved for the control of diseases of terrestrial animals. For the purposes of this approval scheme, disinfectants are divided into 5 groups: (1) for use against tuberculosis, (2) for use against foot-and-mouth disease, (3) for use against swine vesicular disease, (4) for use against diseases of poultry and (5) for use under General Orders made under the Animal Health Act. Manufacturers may apply for their products to be approved for one or more of these groups. At the present time this system of approvals does not extend to pathogens of fish or shellfish.

Norwegian Disinfectant Approvals SchemeThe Norwegians have regulations controlling the cleaning and disinfection of aquaculture sites (Norwegian Fisheries Regulations No. 194 and 195). The statutory basis for their aquaculture disinfectant approval scheme is No. 194. Their approval process is based upon a draft of BPD (98/8/EC) and they have published guidelines that are distributed to manufacturers wishing to have an aquaculture disinfectant approved. The guidelines state what claim-specific toxicity, ecotoxicity and efficacy data manufacturers should produce in support of an application to have a product approved as a ‘Technical Disinfectant’.

Published and other available information on the effectiveness of aquaculture disinfectantsThere are peer-reviewed and trade publications listing the effectiveness of a range of disinfectants against agents such as VHSV (7-9); infectious haematopoietic necrosis virus (IHNV, 10); infectious pancreatic necrosis virus (IPNV, 11, 9, 12, 13, 10); spring viremia of carp (SVCV, 9, 12, 14, 15); Renibacterium salmoninarum (12, 15, 16)However, the methods used in these different studies are not standard, making realistic comparison of the effectiveness of the different products, or active ingredients, investigated problematical. Also, as was the case with ISA, there was limited information about effective methods for control of some notifiable diseases and there is often no information as to the effectiveness of other, more modern biocides that may have better safety profiles than the active chlorine products often recommended.As a result of this, SEERAD recently funded work investigating the use of disinfectants against notifiable diseases of fish (17). The results of this ROAME have produced peer-reviewed publications (including 18) and updated our technical knowledge considerably in this area. The FRS Marine laboratory will shortly be publishing an updated disinfectant guide based on this work (D. Smail; personal communication) and the scientists involved in its drafting were invited to form part of the technical subcommittees convened under this project (Objectives 1 and 3). Cefas has also recently commissioned a series of student projects investigating the efficacy of disinfectants against a range of aquaculture pathogens under different conditions (19-21).

Establishment of an aquaculture disinfectant listing scheme.Representatives from the UK Health and Safety Executive (HSE), Defra Fisheries II, Defra Science Directorate, Defra AMED, Cefas, Fisheries Research Service (FRS) Aberdeen and Department of Agriculture and rural development Northern Ireland (DARD) all met in London (15/9/04) to discuss this and other issues relating to the use of disinfectants in aquaculture Appendix 1.It was clarified at this meeting that, when the Biocides and Pesticides Directive (BPD) (98/8/EC; implemented into UK law through SI No. 880/2001 and SR 2001/422) takes effect, HSE will oversee

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registration of all products marketed as aquaculture disinfectants (as with all other biocides), after assessing an application dossier in relation to that marketing claim. They will then issue authorisations for that proposed use. It is recognised that this HSE approval system may not be able to operate for a few years for already used products. For instance, active ingredients in veterinary disinfectants (Product Type 3) will not be reviewed by HSE under BPD, as part of the third priority list, until after 2007. The importance of such a scheme was reinforced by the recent outbreak of VHS in Yorkshire in 2006, where Defra were in the position of being unable to recommend disinfectants definitively proven to be efficacious at inactivating the causative virus. In Scotland and Northern Ireland, SEERAD and DARD are left in the same position; as was the case in 1998 when Scotland had an outbreak of ISA. It was recognised that acceptable standards have not yet been specified under BPD for efficacy testing of aquaculture disinfectants.As an outcome of this meeting Project FC1169 was conceived, with a proposal to resolve these problems partially at a UK level by initially introducing a listing system, similar to the Defra orders, whereby a company can submit evidence that a product is effective at inactivating a range of notifiable disease agents. It was envisaged that a letter would then be sent to the company confirming that their product is efficacious at a particular working concentration and it can then be listed as such. The project entailed setting appropriate testing standards and target organisms, assisting Defra in establishing the scheme and marketing the scheme to interested parties. This project used all the information generated under these initiatives (that included, in the case of (17), a full review of published literature on the effectiveness of disinfectants against aquaculture pathogens) to design practical and robust guidelines for manufacturers to enable the listing of their products. We also reviewed other disinfectant aquaculture approval schemes to see whether their standards may also be applicable for the UK context. In this respect, special attention was paid to the Norwegian approval scheme. A major UK user of biocides are marine farming operations; particularly cage rearing, transport and processing of Atlantic salmon. It has been claimed that full strength seawater affects the activity of iodophores and other disinfectants (29); although reference to this in primary literature is hard to locate. The antibacterial agents oxolinic acid, flumequine, sarafloxacin and oxytetracycline are all antagonised by seawater cations (30). An agreed seawater standard may need to be defined and assays modified to use it tested but, before this a series of tests were performed to assess the potential inhibitory effect of seawater on iodophore activity, using natural 35‰ sea water.Set-up of the scheme was restricted to developing standards for testing efficacy of disinfectants against viruses and bacteria, although the importance of other disease agents, as well as the development of egg sanitation procedures, is also recognised. It is envisaged that appropriate standards for testing against mycotic agents (such as Saprolegnia parasitica) and parasites (e.g. Gyrodactylus salaris) could also be developed in the future, and the scheme amended to include information as to the efficacy of products against them.

Objective 01.Objective as set out in the contract: ‘To convene a first workshop and produce draft standards on the basis of reviewed information and workshop discussions’

Extent to which objective 01 has been met. This objective was fully met over the course of the project with the workshop held shortly after the start of the project.

Methods used and the results obtained The first workshop was held in Weymouth where a range of different issues to do with establishing the scheme, as well as the specifics relating to the objective (see Appendix 2, minutes of meeting) were discussed. The meeting was well attended with representatives from UK government fish health laboratories (FRS Aberdeen, Cefas and DARDNI) as well as scientists responsible for the terrestrial

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disinfectant scheme (Veterinary Laboratory Agency (VLA) and the Institute of Animal Health (IAH) Pirbright) and the Norwegian Veterinary Institute (that oversees testing of products under their Regulation 194).It was decided that recommended standards should, as far as possible, follow available European (EN) disinfectant testing standards, modified for use with appropriate aquaculture pathogens. It was recognised that available time and resources prevented the creation of a long list of standards for testing disinfectants against a range of notifiable aquaculture pathogens. For this reason it was decided to develop single virus and bacterial testing standards, using model organisms that were suitable for use in the test systems to be recommended and were very resistant to the action of biocides. Demonstration of efficacy of a biocide against the model organisms will then provide a level of assurance of likely broad-spectrum activity. For testing virucidal efficacy, a modification of EN 14675, (Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field), substituting IPNV for bovine enterovirus as the model virus was recommended. For bacterial testing standards, a modification of EN 1656 (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary field), with a range of more relevant aquaculture pathogens substituted for the terrestrial pathogens specified in that standard (e.g. Aeromonas salmonicida subsp. salmonicida, Yersinia ruckeri, Carnobacterium piscicola and Lactococcus garvieae) recommended for testing. The attendees recommended that, before a scheme could be implemented, it was important to ensure that the recommended testing standards be tested in the laboratory first to provide Defra with confidence that data derived from tests using these standards were reliable and the tests were practical for testing laboratories to use. The laboratory validation tested a variety of different conditions, including the likely reproducibility of recommended testing standards (Objective 2).

Objective 2.‘To test and optimise the draft standards in the laboratory.’

Extent to which objective 02 has been met. This objective was fully met over the course of the project

Methods used and the results obtainedModifications of two available EN standards were tested: BS EN 1656, (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field) and BS EN 14675 (Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field - test method and requirements (phase 2/step 1)

Objective 2.1 Testing modified EN1656 IntroductionA modification of EN1656 was tested, as recommended by the Technical Subcommittee at the first workshop. EN1656 is a suspension based disinfection method. Testing is performed under high organic loading 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.The tests were performed as recommended in the standard with the following amendments (as recommended by the technical subcommittee at the first workshop): The test organisms recommended in the standard (Section 5.2.1. and all references thereafter) were

substituted by: Aeromonas salmonicida subsp. salmonicida (ATCC 14174), Yersinia ruckeri (ATCC 29473), Carnobacterium piscicola (ATCC 35586) and Lactococcus garvieae (NCIMB 702927).

The recommended temperature at which test plates are incubated prior to enumeration of viable colonies were changed from 36 or 37 °C to 22 °C 1 °C (Section 5.3.2.3 and all references thereafter).

Choice of experimental conditions (Section 5.5.1) mandatory conditions:

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1. For all dilutions, the temperature to be tested was 4 °C 1 °C (not 10 °C)2. For all dilutions, the contact time to be tested was 30 min 10 sec 3. For all dilutions, the Interfering substance to be tested was as described for dirty conditions (10g/l

yeast extract plus 10g/l bovine serum albumin solution) Products testedThe standard was tested using four different commercially supplied disinfectants: a Chloramine T containing disinfectant, an iodophore containing product (FAM 30), a peroxygen system (Virkon Aquatic ) and a hydrogen peroxide/peracetic acid system ( Proxitane® 5:23). All products were tested at least three times as recommended by the standard (with the modifications described above). For all products, the neutralisation dilution method was employed. Other elements of the validation concerned confirming that the substituted test organisms could be preserved and resuscitated using standard recommended protocols (or minor modifications).

Objective 2.1.1. Evaluate whether EN 1656 recommended strain preservation methods and for enumeration of surviving microorganisms are applicable for recommended substituted bacterial test strains.Materials and methodsStandard recommended bacterial storage methods were tested as well as the Standard recommended pour plate detection method (for bacterial counts, validations and tests). For this 1 ml of the test solution is added to approx 15 ml melted Tryptone Soy Agar (45 °C; TSA) and incubated under appropriate conditions until viable colonies can be discerned. Test solutions can also be enumerated by inoculating 1 ml of each dilution tested, in duplicate, onto TSA spread plates (= approx 10 plates each test, rather than 2).As there was a concern that the organisms to be tested using the modified standard may be sensitive to effects of being heated to 45 °C , we tested by plating, in triplicate, 100 microlitres of a bacterial suspension, using both methods, for all four organisms and comparing their recovery.ResultsStrains were stored using both the EN12353 recommended methods (onto glass beads and vials), as well as onto ProtectTM and successfully resuscitated more then 6 months after storage. There was no statistically significant difference in recovery for each method (P= 0.838; ANOVA). This suggests that the more straightforward pour plate method is an acceptable detection method. The pour plate method was used for all subsequent testing. We also plated cultures following 1000 & 800 ppm Chloramine T treatment using both methods and showed no difference in recovery (suggesting that pre-exposure to a disinfectant did not affect the sensitivity of the organisms to subsequent, transitory, heat stress).

Objective 2.1.2 Determine whether the experimental conditions affect the recovery of the test organisms. Materials and methodsAlthough the test organisms recommended in EN1656 are able to survive the recommended test conditions (exposure to high organic loading and incubation at 4 C for 30 min), it needed to be shown first that the alternative aquatic pathogens recommended for testing were also resistant to these conditions. EN1656 test validation procedures (A.4.1.2) were followed to confirm that there was no effect of the experimental conditions. For each organism, 1 ml of a bacterial suspension, Nv (6 x 102- 3 x 103 cfu/ml) was exposed to 1ml BSA + yeast and 8 ml hard water for 4°C 30 min and 1ml of the mixture then plated out and counted to determine A. To pass, A had to be 0.05 x Nv.Results.In repeated testing, there was no evidence that exposure of any of the test organisms to low temperature for 30 min (4 C) in the presence of high organic loading affected their subsequent recovery on solid media (A was always 0.05 x Nv).

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Objective 2.1.3 Determine whether dilution neutralisation is an appropriate method for inactivating bactericidal activity.EN1656 suggests two methods for inactivating the test substance at the end of the test to allow the surviving bacteria to be recovered and enumerated. The first recommended method is neutralisation dilution where, after the suspension of bacteria has been exposed to the test substance for the required period of time, the activity of the disinfectant is then inactivated by adding a sample of the test mixture to a neutraliser. This is the preferred method although, if an effective neutraliser cannot be identified, there are also recommended membrane filtration procedures (EN 1656 5.5.2.3). For membrane filtration, after the test, a sample of the test mixture is immediately vacuum filtered through a membrane with a large volume of rinsing fluid also added to remove the test substance from the bacteria. The rinsed bacteria are retained on the filter, which can then be transferred to solid media and incubated prior to enumeration of resultant colonies from those bacteria that survived the test. For this testing process, the suitability of the neutralisation dilution was assessed using the validation process recommended in EN1656 (A 4.1).ResultsA lecithin based neutraliser (Tween 80, 3% lecithin, 3 g L-1 sodium thiosulphate, 1 g L-1 histidine, 30 g L-1 saponin, 5 g L-1), 5 g L-1 Sodium thiosulphate and 5 g L-1 Sodium thiosulphate + catalase base were assessed as potential neutralisers. Effective neutralisers were identified for all four products tested which allowed testing to be done with the Standard recommended neutralisation method. All three neutralisers were shown not to be toxic towards any of the four test organisms. The lecithin based neutraliser was also shown to be effectively suppressing the activity of the Chloramine T –containing product, FAM 30, PVPI and Virkon Aquatic and was used for all subsequent testing of these products. 5 g L-1 Sodium thiosulphate + catalase base was shown to be an effective neutraliser of Proxitane® 5:23 and was used for all testing involving this product.

Objective 2.1.4 Determine likely reproducibility of modified test standard by repeating test 3 times with up to four different products. Materials and methodsAfter assessing that each product to be tested could be neutralised, the four test products were assayed using the modified EN 1656. For all four test organisms, the bacterial suspensions were prepared by inoculating them onto TSA slopes and culturing for up to 72h at 22 C. The bacterial suspension was then prepared by taking loopfulls of fresh culture and adding them to 10ml diluent (TSC) in a 100ml flask that contained 5g of glass beads. The suspension was then adjusted spectrophotometrically to between 1.5 x 108 to 5 x 108 cfu/ml using TSC. Target optical densities, derived from tests in our laboratory, for each organism are presented in Table 1, although each laboratory will need to test this independently as there is likely to be substantial variation from laboratory to laboratory (depending on type of spectrophotometer used etc.).

Table 1. Suggested optical densities to achieve bacterial suspensions of between 1.5 x 108 to 5 x 108 cfu/ml for the test organisms.

Organism Recommended optical density (OD650) Aeromonas salmonicida 0.1Yersinia ruckeri 0.1Carnobacterium piscicola 0.1Lactococcus garvieae 0.18

For testing, product test solutions were prepared in standardised hard water at 1.25 x the required test concentrations. For solid products, at least 1g of the product was dissolved in the standardised hard water recommended in the standard in volumetric flasks, with subsequent dilutions prepared using volumetric flasks. For liquid products, dilutions of the product were prepared on a v/v basis in the standardised hard water, again using volumetric flasks. As recommended in the standard, prepared dilutions were tested within an hour of preparation (test initiated). For the test procedure, 1.0 ml of the interfering substance for high level soiling conditions (10g/l yeast extract and 10 g/l bovine albumin) and 1 ml of a bacterial test suspension was added to a sterile glass test container and placed in 4 C water bath for 2 min, at the end of this time 8.0 ml of one of the product

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test dilutions was also added and the mixture briefly vortexed. The mixture was then incubated in the 4 C water bath for 30 min. All test reagents were equilibrated to 4 C before testing. Just before the end of the 30 min, the mixture was briefly vortexed and at 30 min, 1.0 ml of the test mixture was transferred to a tube containing 1.0 ml standardised hard water and 8.0 ml of a neutraliser shown to be appropriate for the particular product under test. The tube was then transferred to a water bath at 20C for 5 min. Immediately after the neutralisation period, a sample of 1.0 ml of the neutralised mixture was taken in duplicate and each sample transferred to a separate sterile petri dish. 12-15ml molten TSA added. The plates were incubated at 22 C for 48h and resultant colony forming units determined.

Product 1. Virkon AquaticAs can be seen in Table 2, The results of this testing exercise indicate that concentrations of Virkon Aquatic higher then 0.5% killed all four test organisms in the presence of high organic loading, although the relative reduction varied from test to test at this level (0.5%). A concentration of 1% passed on all occasions for all organisms except for Lactococcus garvieae, which failed on one test where the original bacterial test suspension was low. However, there had still clearly been a high level of inactivation in this test with a greater then 4 log reduction recorded (e.g. there were no viable colonies recovered from a ml of the test suspension).Both Gram positive organisms tested (Carnobacterium pisicola and L. garvieae) appeared to be more resistant to Virkon Aquatic then either of the Gram negative organisms tested. On all tests, there were no viable colonies cultured from the suspensions of Gram negative organisms after exposure to 0.5% Virkon Aquatic, while countable C. piscicola and L. garvieae colonies could still be recovered after exposure to this concentration in 2 /5 tests. A. salmonicida was apparently the most sensitive, with a nearly complete kill (greater then log 4 reduction) recorded in one 0.2 % test.

Table 2 Efficacy of a range of concentrations of Virkon Aquatic versus test organisms using the modified EN1656 (contact time of 30 min at 4C in the presence of interfering substance. Results are presented from three separate experiments. Where Ra was greater then 1.0 x 105 the product passed the test at the indicated test concentration (bold). On occasion, the original bacterial test concentration was too low to allow a 1.0 x 105 reduction to be demonstrated. Where this was observed but no viable colonies were recovered after the test (e.g. there had been effective killing) data are marked *

Test Organism Product conc. (% w/v)

Reduction in viability (Ra) at test concentration indicatedTest 1 Test 2 Test 3 Test 4 Test 5

A. salmonicidaNCIMB 833

0.01 <5.29 x 103

0.05 <5.29 x 103 <9.0 x 103

0.1 <5.29 x 103 <9.0 x 103 <1.32 x 104

0.2 <9.0 x 103 <1.75 x 104 >7.45 x 104*

0.5 >1.05 x 105 >1.8 x 105 >2.63 x 105 >3.5 x 105 >7.45 x 104*

1.0 >3.5 x 105 >7.45 x 104*

Y. ruckeriNCIMB 12986

0.01 <7.67 x 103

0.05 <7.67 x 103 <1.83 x 104

0.1 <7.67 x 103 <1.83 x 104 <1.63 x 104

0.2 <1.83 x 104 <1.98 x 104 <5.08 x 103

0.5 >1.53 x 105 >3.67 x 105 >3.27 x 105 >3.97 x 105 >1.05 x 105

1.0 >3.97 x 105 >1.05 x 105

C. piscicolaNCIMB 2264

0.01 <7.63 x 103

0.05 <7.63 x 103 <1.77 x 104

0.1 <7.63 x 103 <1.77 x 104 <3.01 x 104

0.2 <1.77 x 104 <2.13 x 104 <4.17 x 103

0.5 >1.63 x 105 >3.53 x 105 >6.03 x 105 <2.13 x 104 3.05 x 104

1.0 >4.27 x 105 >8.33 x 104*

L. garvieaeNCIMB 702927

0.01 <4.46 x 103

0.05 <4.46 x 103 <9.83 x 103

0.1 <4.46 x 103 <9.83 x 103 <9.23 x 103

0.2 <9.83 x 103 <9.67 x 103 <1.47 x 103

0.5 >8.93 x 105 >1.97 x 105 >1.85 x 105 <9.67 x 103 1.0 x 104

1.0 2.57 x 104 >2.94 x 104

a) R = reduction in viability (R = N x 10-1 /Na) where :N = number of cfu/ml of the bacterial test suspension exposed to the product at the concentration indicatedNa = number of cfu/ml in the bacterial test suspension after test procedure. Where 15 or less viable colonies were countable on each of the test plates, R was calculated on the basis that Na is less then 150, where there were more then 300 colonies, R was calculated on the basis that Na was more then 3000.

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Product 2. Proxitane® 5:23Concentrations of Proxitane® 5:23 higher then 0.2% killed suspensions of all four bacteria in the presence of BSA and yeast (with the exception of one test for L. garvieae), while concentrations of 0.1% and lower were less effective (Table 3). The Gram negative organisms tested appeared to be more sensitive to the effects of Proxitane® 5:23 then the Gram positive bacteria. In three separate tests, a 0.1% solution achieved a greater then 5 log kill against A. salmonicida and a greater than 4 log reduction was also shown in two out of the three tests against Y. ruckeri. By contrast, both C. piscicola and L. garvieae were shown to be resistant to 0.1% Proxitane® 5:23 on two out of three tests and surviving L. garvieae were recovered following exposure to 0.2% Proxitane® 5:23 in two out of three tests. All tests with 0.2% Proxitane® 5:23 showed that this concentration was bactericidal against all four organisms (except for one L. garvieae test).

Table 3 Efficacy of a range of concentrations of Proxitane® 5:23 versus test organisms using the modified EN1656 (contact time of 30 min at 4°C in the presence of interfering substance. Results are presented from three separate experiments. Where Ra was greater then 1.0 x 105 the product passed the test at the indicated test concentration (bold). On occasion, the original bacterial test concentration was too low to allow a 1.0 x 105 reduction to be demonstrated. Where this was observed but no viable colonies were recovered after the test (e.g. there had been effective killing) data are marked *.

Test OrganismProxitane® 5:23 concentration (%

v/v)

Reduction in viability (Ra) at test concentration indicated

Test 1 Test 2 Test 3


0.05% <1.47 x 104 1.9211 x 104 ND0.1% >2.93 x 105 1.11 x 106 >1.3 x 105

0.2% >2.9 x 105 >2.0 x 106 >1.3 x 105

0.33% >2 x 106 ND ND0.5% >2.0 x 106 ND ND


0.05% <3.15 x 104 <1.35 x 104

0.1% <3.15 x 104 5.46 x 104 2.09 x 104

0.2% >6.3 x 105 >4.7 x 105 >2.7 x 105

0.3% >4.7 x 105 ND ND0.5% >4.7 x 105 ND ND


0.05% <2.56 x 104 <9.5 x 103

0.1% <2.52 x 104 2.51 x 105 <9.5 x 103

0.2% >5.03 x 105 >2.3 x 105 1.62 x 105

0.3% >2.3 x 105 ND ND0.5% >2.3 x 105 ND ND


0.05% <1.3 x 104 6.7 x 103

0.1% <1.3 x 104 5.4 x 105 <6.7 x 103

0.2% >5.6 x 105 1.26 x 105 9.37 x 103

0.3% >5.6 x 105 ND ND0.5% >5.6 x 105 ND ND


Product 3 Chloramine TThe Chloramine T product tested (Table 4) killed all four test organisms at dilutions lower then 0.1%. At 0.1% the product killed greater then 5 log bacteria in the majority of tests for all the organisms, although there were four marginal test failures at this dilution (R values that were less then 1 x 105 but still greater then 1 x 104). This indicates that, in the presence of high organic loading, the product was still able to kill > 99.99% of the bacteria it was in contact with after 30 minutes exposure at 4 C, even though the test was technically a failure.

Table 4 Efficacy of a range of dilutions of a Chloramine T containing product versus test organisms using the modified EN1656 (contact time of 30 min at 4C in the presence of interfering substance). Results are presented from three separate experiments. Where Ra was greater then 1.0 x 105, the

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product passed the test at the indicated test concentration (in bold). On occasion, the original bacterial test concentration was too low to allow a 1.0 x 105 reduction to be demonstrated. Where this was observed but no viable colonies were recovered after the test (e.g. there had been effective killing), data are marked by *.

Test Organism Product dilution(% v/v)

Reduction in viability (Ra) at test concentration indicated

Test 1 Test 2 Test 3


0.001 <1.89 x 104

0.01 <1.89 x 104

0.05 <1.89 x 104

0.08 >2.43 x 105

0.1 >2.0 x 104 2.97 x 104 >2.43 x 105

0.5 >2.0 x 104

0.7 >2.0 x 104

1.0 >2.0 x 104


0.001 <1.47 x 104

0.01 <1.47 x 104

0.05 <1.47 x 104

0.08 >3.1 x 106

0.1 >2.1 x 105 >2.93 x 105 >3.1 x 106

0.5 >2.1 x 105

0.7 >2.1 x 105

1.0 >2.1 x 105


0.001 <2.48 x 104

0.01 <2.48 x 104

0.05 <2.48 x 104

0.08 <1.62 x 104

0.1 >2.13 x 105 2.67 x 105 4.52 x 104

0.5 >2.13 x 105

0.7 >2.13 x 105

1.0 >2.13 x 105


0.001 <1.77 x 104

0.01 <1.77 x 104

0.05 <1.77 x 104

0.08 >1.17 x 105

0.1 >1.5 x 105 4.02 x 104 >1.17 x 105

0.5 >1.5 x 105

0.7 >1.5 x 105

1.0 >1.5 x 105


Product 4 FAM 30.FAM 30 at concentrations of 0.2% or lower failed to inactivate the four test organisms (Table 5). Although the test was not repeated at this concentration, 0.28-0.8 % was noted to kill both A. salmonicida and Y. ruckeri but the two Gram positive organisms tested (C. piscicola and L. garvieae) appeared to be more resistant with resistance to 0.28% noted for both bacteria and evidence of surviving organisms. All tests at 1% or higher resulted in an apparent complete kill of all four test organisms. 0.4 -0.8% tests showed complete clearing of the Gram negative bacteria tested, but some survival of C. piscicola and L. garvieae (although there was variation from test to test).

Table 5 Efficacy of a range of dilutions of FAM 30 versus test organisms using the modified EN1656 (contact time of 30 min at 4C in the presence of interfering substance). Results are presented from four separate experiments.

Test Organism Product dilution Reduction in viability (R) at test concentration indicated

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(% v/v) Test 1 Test 2 Test 3 Test 4


0.16 <9.63 x 103 <1.63 x 104 <2.95 x 104

0.2 <5.76 x 103

0.28 >4.1 x 105

0.4 >4.1 x 105

0.8 >1.93 x 105 >3.27 x 105

1 >1.15 x 105

1.6 >1.93 x 105 >3.27 x 105

2 >1.15 x 105

3.2 >1.93 x 105 >3.27 x 105


0.16 <1.40 x 104 <3.42 x 104

0.2 <3.78 x 104

0.28 >6.83 x 105

0.4 >6.83 x 105

0.8 >2.8 x 105 >6.83 x 105

1 >7.56 x 105

1.6 >2.8 x 105

2 >7.56 x 105

3.2 >2.8 x 105


0.16 <1.75 x 104

0.2 <1.09 x 104

0.28 <2.89 x 104

0.4 4.33 x105

0.8 8.27 x 104 >5.77 x 105

1 >1.63 x 105

1.6 >3.5 x 105 >5.77 x 105

2 >1.63 x 105

3.2 >3.5 x 105


0.160.2 <4.47 x 103

0.28 <1.22 x 104

0.4 1.92 x 105

0.8 >1.24 x 105 >2.43 x 105

1 >8.93 x 104

1.6 >1.24 x 105 >2.43 x 105

2 >8.93 x 104

3.2 >1.24 x 105

Objective 2.2 Testing modified EN14675 IntroductionA modification of EN14675 was tested, as recommended by the Technical Subcommittee at the first workshop. As for the modified EN 1656, the modified EN14675 is a suspension based disinfection method with testing performed under high organic loading 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.

In brief, for the tests, 1ml of interfering substance was mixed with 1ml of a suspension of > 107.5

TCID50 IPNV and equilibrated to the relevant test temperature. 8 ml of the product was then added to the mixture which was then left for the set contact time, thus enabling the relative reduction (log inactivation) to be established. The tests were performed as recommended in the standard with the following amendments (as recommended by the technical subcommittee at the first workshop): The test agent recommended in the standard (Section 4/5.2.1 and all references thereafter) should be substituted for: Infectious pancreatic necrosis virus (IPNV), serogroup A2, Spjarup (SP) isolate (ATCC). The assay required modifying for use with this different virus, details are as described in Appendix 8. Choice of experimental conditions (Section 5.5.1.1) mandatory conditions:

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1.For all dilutions, the temperature to be tested was 4 °C 1 °C (not 10 °C)2. For all dilutions, the contact time to be tested was 30 min 10 sec *3.For all dilutions, the Interfering substance to be tested was as described for dirty conditions (10g/l yeast extract plus 10g/l bovine serum albumin solution).

Products testedThe standard was tested using the same four different commercially supplied disinfectants: the Chloramine T containing disinfectant, an iodophore containing product (FAM 30), a peroxygen system (Virkon Aquatic) and a hydrogen peroxide/peracetic acid system ( Proxitane® 5:23). All products were tested at least three times as recommended by the standard (with the modifications described above). For all products, the neutralisation dilution method was employed. Other elements of the validation concerned confirming that the substituted test organisms could be preserved and resuscitated using standard recommended protocols (or minor modifications).

A modification of EN14675 was tested, as recommended by the Technical Subcommittee at the first workshop. As for the modified EN 1656, the modified EN14675 is a suspension based disinfection method with testing performed under high organic loading 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.

In brief, for the tests, 1ml of interfering substance was mixed with 1ml of a suspension of > 107.5 TCID50 IPNV and equilibrated to the relevant test temperature. 8 ml of the product was then added to the mixture which was then left for the set contact time, thus enabling the relative log reduction to be established. The tests were performed as recommended in the standard with the following amendments (as recommended by the technical subcommittee at the first workshop): The test agent recommended in the standard (Section 4/5.2.1 and all references thereafter) should be

substituted for: infectious pancreatic necrosis virus (IPNV), serogroup A2, Spjarup (Sp) isolate (ATCC). The assay required modifying for use with this different virus, details are as described in Appendix 8.

Products testedThe standard was tested using the same four different commercially supplied disinfectants: the Chloramine T containing disinfectant Chloramine T, an iodophore containing product (FAM 30), a peroxygen system (Virkon Aquatic) and a hydrogen peroxide/peracetic acid system ( Proxitane® 5:23). All products were tested at least three times as recommended by the standard (with the modifications described above). For all products, the neutralisation dilution method was employed. Other elements of the validation concerned confirming that the substituted test organisms could be preserved and resuscitated using standard recommended protocols (or minor modifications).

Objective 2.2.1. Evaluate whether EN 14675 can be readily modified to substitute IPNV for bovine enterovirus The standard for virus testing is based around using bovine enterovirus as the test virus, and appropriate cell lines for conducting the virus assays. From a literature survey and discussions with colleagues at workshops it was decided to use IPNV serotype Sp as the test organism. The rationale was based upon the fact that from the literature (and in-house experience) the virus is one of the more resistant viruses to inactivation by chemicals, it is readily available to testing laboratories from culture collections, it is present in many countries worldwide and should not present a problem should importation into a country be necessary, it is easy to cultivate and titrate, and it has relevance to aquaculture. The testing methods will follow EN14675 closely, but with modifications required by the use of that virus, such as use of 15°C for incubation of titrations rather than 37°C as required for bovine enterovirus.

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Objective 2.1.4 Determine likely reproducibility of modified test standard by repeating test 3 times with up to four different products. Materials and methodsThe assay was modified as described. Specific modifications entailed the use of Chinook salmon embryo (CHSE-214) cells to propagate and titrate the virus at 15°C. Preliminary tests were conducted as follows:1) to determine whether the initial dilution of the product for titration was sufficient to stop the action of the product and eliminate any cytotoxicity caused by it.2) in cases where the product was toxic on initial dilution, experiments were conducted with chemicals that might neutralise the product and eliminate the toxicity. The experiments also determined that the product/neutraliser mix was not toxic, and that the neutraliser itself did not inactivate the virus. The neutraliser used in the studies with bacteria was found to be cytotoxic, but sodium thiosulphate was effective at neutralising some products, and was not cytotoxic, and had no effect on the virus titre.3) to determine concentrations that would produce a clear pass, a clear fail, and a result on the pass/fail borderline with each product.

ResultsThe modified procedures for preparation of IPNV for use in the assay and detection using were found to be appropriate.One potential problem was encountered in that the standard specifies that a titre/ml > 107.5 TCID50 IPNV is needed for use in the assay. The average IPNV Sp titre in all testing was only 1.06 x 107 TCID50/ml which is less then the standard specifies. However, on a pragmatic basis that titre was still sufficient to determine a >4log drop in infectious virus titre, so it is recommended that the standard be relaxed to specify ‘should ideally be above >1.0 x 107 TCID50/ml to enable a 4 log reduction even where a defree of cytotoxicity is present’.A larger number of experiments than reported here were carried out, which included scoping studies and tests to overcome the problems of precipitation and cytotoxicity than reported below. Only the final inactivation trials are reported here. The large number of those non-reported test also meant that the manpower and funds were not available to complete some of the replicate test required.

Product 1. Virkon AquaticThis product was effective at producing ≥4log drop in titre at concentration of 0.5% (Table 6). A concentration of 0.2% produced results on the pass/fail borderline. Product 2. Proxitane® 5:23It was difficult to determine the borderline pass/fail concentration for this product. (Table 7) Several attempts were made to determine that concentration, but when conducting replicate studies the concentration previously determined was not always effective at producing the borderline value. However, concentrations producing a clear pass and a clear fail were determined.Product 3. Chloramine TTests of Chloramine T were problematic in that, when tested under conditions of high soiling, precipitation occurred at concentrations >1.5%, but initial tests showed that that concentration was ineffective at inactivating the virus (Table 8). There was only a 1.5 log drop at a Chloramine T concentration of 2%. It considered that conducting tests in conditions where precipitation occurred was unsatisfactory, as the precipitation could have reduced the efficacy of the product. However, in a preliminary test, 2.4% Chloramine T caused a 3.17 log drop in virus titre (test fail), but 3.2% caused a 4.17 log drop in titre (borderline pass/fail). A test was conducted in which there was low soiling (and no precipitation during the test), and a concentration of Chloramine T that caused >4 log drop in titre was determined. Only one test was conducted.Product 4. FAM 30FAM 30 was found to be extremely cytotoxic, and no neutraliser was found that would neutralise the toxicity. However, from the tests conducted, some qualitative results were obtained. At a concentration of 220 ppm, toxicity of FAM 30 was only apparent at the 10-2 dilution of the titration, and so a titre was

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obtained, but that concentration did not reduce the titre of IPNV. At a concentration of 440 ppm, toxicity of FAM 30 was apparent up to the 10-4 dilution of the titration. At higher dilutions the cell monolayers were in good condition, and did not exhibit cytotoxicity or viral cytopathic effect. Therefore 440 ppm reduced the virus titre, but the exact log reduction could not be determined. A concentration of 880 ppm was cytotoxic up to the 10-6 dilution of the titration.Product 5. BuffodineFAM 30 is an iodophore, and since no meaningful results were obtained with that product, another iodophore, Buffodine was tested. That too was cytotoxic in initial test but the toxicity was eliminated on neutralisation. Low levels of toxicity were encountered at the higher product concentrations (1 and 2%), but this did not affect the reading of the results. The product did not inactivate the virus to any significant effect at the highest concentration tested (2%) and, due to the aforementioned toxicity, higher concentrations could not be tested (Table 9).

Table 6 Efficacy of a range of concentrations of Virkon Aquatic versus IPNV using the modified EN14675 (contact time of 30 min at 4C) in the presence of interfering substance. Results are presented from three separate experiments.

Test ProductConcentration (% v/v)

Titre (control) (TCID50/ml)

Log titre(control)

Titre (test) (TCID50/ml)

Log titre (test)

Log drop Result

1 2.58 x 107 7.411 0.1 5.56 x 107 7.74 0.0 Fail1 0.2 2.58 x 103 3.41 4.0 Pass1 0.5 5.56 x 101 1.74 5.67 Pass2 3.47 x 106 6.582 0.1 5.56 x 106 6.74 0.0 Fail2 0.2 8.15 x 102 2.91 3.67 Fail2 0.5 1.76 x 101 1.24 5.33 Pass3 1.2 x 106 6.083 0.1 3.78 x 106 6.58 0.0 Fail3 0.2 3.78 x 102 2.58 3.50 Fail3 0.5 1.76 x 101 1.24 4.83 Pass

Table 7 Efficacy of a range of concentrations of Proxitane® 5:23 versus IPNV using the modified EN14675 (contact time of 30 min at 4C) in the presence of interfering substance. Results are presented from three separate experiments.

Test ProductConcentration (% v/v)


Log titre(control)


Log titre (test)

Log drop Result

1 5.56 x 106 6.741 0.16 5.56 x 106 6.74 0.0 Fail1 0.276 No result1 1.6 1.76 x 102 2.24 4.5 Pass2 3.78 x 106 6.582 0.16 2.58 x 106 6.41 0.17 Fail2 0.276 2.58 x 102 2.41 4.17 Pass2 1.6 1.76 x 102 2.24 4.33 Pass3 5.56 x 106 6.743 0.16 3.78 x 106 6.58 0.17 Fail3 0.276 2.58 x 102 2.41 4.33 Pass3 1.6 1.76 x 102 2.24 4.5 Pass

Table 8 Efficacy of a range of concentrations of Chloramine T versus IPNV using the modified EN14675 (contact time of 30 min at 4C) in the absence of interfering substance. Results are presented from one experiment.

ProductConcentration (% v/v)


Log titre(control)


Log titre (test)

Log drop Result

5.56 x 106 6.740.008 2.58 x 106 6.41 0.33 Fail0.04 3.78 x 106 6.58 0.17 Fail0.08 3.78 x 106 6.58 0.17 Fail0.4 2.58 x 106 6.41 0.33 Fail0.8 1.76 x 106 6.24 0.50 Fail1.2 2.58 x 105 5.41 1.33 Fail1.6 2.58 x 103 3.41 3.33 Fail3.2 1.76 x 102 2.24 4.5 Pass

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Table 9 Efficacy of a range of concentrations of Buffodine versus IPNV using the modified EN14675 (contact time of 30 min at 4C) in the presence of interfering substance. Results are presented from one experiment.

ProductConcentration (% v/v)


Log titre(control)


Log titre (test)

Log drop Result

2.58 x 107 7.410.1 2.58 x 107 7.41 0.0 Fail

0.125 1.76 x 107 7.24 0.17 Fail0.16 2.58 x 107 7.41 0.0 Fail0.25 2.58 x 107 7.41 0.0 Fail0.5 3.78 x 107 7.58 0.0 Fail1.0 1.20 x 108 8.08 0.0 Fail2.0 2.58 x 107 7.41 0.0 Fail

DiscussionEN 14674 was successfully modified to allow the use of IPNV Sp as the test virus. However, problems with the testing procedure were highlighted during the test procedure. The greatest problem was the cytotoxicity of some of the products, although that could be overcome in some instances solely by dilution of the product upon titration, or by the use of a chemical neutraliser such as sodium thiosulphate. However, in the case of FAM 30, dilution and the use of chemical neutralisers were ineffective at reducing the toxicity of the product. This was compounded by the relative resistance of IPNV to the product, which meant that increasing concentrations of the product were needed which could not be neutralised. A similar situation occurred with Chloramine T which precipitated with the interfering substances. Again a problem was that in order to inactivate the virus, relatively high concentrations of the product had to be tested, and they increased the level of precipitation. However, in principle the method is suitable for testing disinfectants against IPNV.

Objective 2.3 Determine level of required test replication for modified EN1656 and EN 14675Variation from test to test was noted for all products tested. One would like to say something about the distribution of R (the reduction ratio) based on this data, but many factors combine to prevent our knowing an appropriate form or guessing its parameters. The form of the distribution may vary with the product, the target organism and the starting concentration. In practice, we are interested only in whether R was sufficiently large to pass the test at a given concentration, and can assume that near to the minimum effective concentration there will be a certain probability of passing (call it p) and hence a probability of failing (1-p). At higher (more effective) concentrations p will become almost 1, and at greater dilutions p will become very small. This was demonstrated, empirically, in the test procedures against the different products. Manufacturers should have little difficulty obtaining a test result at each extreme, but what procedure should they adopt in the middle to find the second positive result? One approach would be to carry out many further trials and hence build up a more complete empirical sampling distribution of R. This would be very expensive and provides more information than is necessary. Again, as a practical point, one would want p still to be near to 1 in the region of the lower reported concentration. This suggests that manufacturers should carry out one test and, if it fails, unless some failure of protocol has been identified, should increase the concentration until a clear pass is obtained with a single try. It may be objected that this still leaves p fairly undefined, but it for the manufacturers to decide the concentration at which they are “confident” that a similar independent test would again produce a positive outcome. It should also be appreciated that, as was demonstrated in the seawater testing experiments, the specified testing conditions were extremely rigorous (high organic loading and low temperature). Thus, even if it could be demonstrated that a concentration may ‘pass’ in one test but fail in the next, it is very probable that any concentration shown to inactivate more then 5 log of a bacterial suspension under these, harsh, conditions in a particular trial is likely to be able to kill bacteria under field conditions.Further, the likely recommendations are that, before a dilution of a product can be listed as efficacious against bacterial pathogens, it will be requirement that the dilution it shall need to be demonstrated to

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kill all four of the test organisms under formal Quality Assurance audit conditions (Objective 4). The practicalities of testing will mean that a laboratory testing a product will need to perform initial ‘range finding’ tests to determine concentrations that are likely to pass at the first attempt in an attempt to avoid potentially costly and time consuming retesting (e.g. p will be near to 1).

Objective 2.4 General conclusions and recommendations from laboratory testingIn general, the modified standards were both shown to be effective assays for measuring the bactericidal and virucidal activity of a range of disinfectants against relevant aquaculture pathogens. With the exception of the cytotoxicity problem encountered with FAM 30, there were no major technical problems encountered in the laboratory in terms of deploying the assay, however for laboratories wishing to use the modified assay some aspects need to be carefully addressed if they are to achieve reliable, reproducible results. Preparation of bacterial and viral suspensionsAlthough every effort was made to standardise the preparation conditions, variation in the concentration of bacteria and viruses in the prepared inocula from test to test were noted. On occasions, this variation was enough to render particular test results void. Unless the bacterial suspension used for the assays can be shown to have been between 1.5 – x 108 – 5 x 108 cfu/ml for the bacterial test suspension (N) and 6 x 102 – 3 x 103 cfu/ml for the bacterial suspension used in validation procedures (Nv), the relevant tests will need to be repeated. Bacterial suspensions were adjusted spectrophotometrically and, based on OD650 readings adjustment of the bacterial suspension to the following optical densities resulted in acceptable performance (> 7/8 tests valid).Preparation of reagents and checking equipment.Great care should be taken to ensure test reagents are prepared as specified in the standard. Also, temperatures of water baths in particular and incubators should be checked using independently calibrated thermometers.

Objective 2.5 Evaluate effects of seawater and organic loading on iodophore activity.IntroductionConcern has been expressed that disinfectants, particularly iodophores, are inactivated by seawater (35‰). This has potential practical implications in that many aquaculture operations take place in seawater (e.g. marine salmon farming). If disinfectants are significantly affected by the action of seawater, then it would also likely be necessary to recommend modifications of testing standards to allow for these possible interfering effects when evaluating the efficacy of disinfectants to be deployed on marine sites.

Materials and MethodsA series of experiments were carried out testing the relative effect of addition of seawater or organic loading on the bactericidal activity of both FAM 30 (an iodophore that also contains a range of other active ingredients) and povidone iodine (PVPI; Sigma). The products were tested at a range of concentrations against both A. salmonicida and C. piscicola using a further minor modification of the modified EN1656 standard used for the main study (e.g. with or without seawater and/or organic loading). For all tests, as well as plating out 1ml of the test suspension in duplicate, 0.1ml was also plated out after exposure to the disinfectant. The experiments are summarised in table x, in all cases treatment order was randomised and separate solutions prepared for each replicate. The seawater used for all experiments was full strength (34 ppt) seawater drawn from Weymouth harbour and filter sterilised prior to use.As can be seen in Tables 10 and 11, although organic loading predictably had a major effect on the activity of both PVPI and FAM 30 bactericidal activity (5-10 fold difference), there was no evidence in any of the series of experiments that seawater had a noticeable effect on the efficacy of either PVPI or FAM 30 against either Aeromonas salmonicida or Carnobacterium piscicola.

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Table 10 Effect of seawater and organic loading on bactericidal activity of PVPI against A. salmonicida

Experiment TreatmentReduction in viability ® at test concentration indicated

100 250 500 1000 2500 5000

Experiment 1(n =1 per treatment)

Seawater + organic loading <4.03 x 102 <4.03 x 102 <4.03 x 102 >8.06 x 104

Seawater only <4.03 x 102 >8.06 x 104 >8.06 x 104 >8.06 x 104

Hard water + organic loading <4.03 x 102 <4.03 x 102 <4.03 x 102 >8.06 x 104

Hard water only <4.03 x 102 >8.06 x 104 >8.06 x 104 >8.06 x 104

Experiment 2(n = 3 per treatment)

Seawater + organic loading <3.53 x 102 <3.53 x 102 <3.53 x 102

Seawater only 1.67 x 103 >7.6 x 104 >7.6 x 104

Hard water + organic loading <3.53 x 102 <3.53 x 102 <3.53 x 102

Hard water only <3.53 x 102 >7.6 x 104 >7.6 x 104

Table 11 Effect of seawater on bactericidal activity of FAM 30 against A. salmonicida and C. piscicola with or without organic loading, or with or without seawater.

Experiment TreatmentReduction in viability ® at test concentration indicated

100 250 500 1000 1600 4000Experiment 1Activity of FAM 30 versus A. salmonicida(n =3 per treatment) in presence of interfering substance.

Seawater + organic load <1.22 x 103 >2.43 x 105

Hard water + organic load <1.22 x 103 >2.43 x 105

Experiment 2Activity of FAM 30 versus A. salmonicida(n =3 per treatment)

Seawater only <3.55 x 102 3.87 x 103 >7.14 x 104

Hard water only <3.55 x 102 8.73 x 103 >7.14 x 104

Experiment 3Activity of FAM 30 versus C. piscicola(n =3 per treatment)

Seawater only <5.13 x 103 >1.02 x 106 >1.02 x 106

Hard water only <5.13 x 103 >1.02 x 106 >1.02 x 106

Objective 3 To reconvene the experts at a second workshop and produce definitive standards and guidelines’

Extent to which objective 3 has been met. This objective was fully met with the workshop held in September 2005 at FRS Aberdeen. Definitive standards and guidelines have been drafted for Defra’s approval (See Appendices 6-8).

Methods used and the results obtainedThe data produced under Objective 2 were reviewed by the technical subcommittee (See Appendix 3). In general, as no major problems were encountered with either of the recommended modified assays, the technical subcommittee recommended that the modified EN standards (EN 1656 modified to include testing against A. salmonicida, Y. ruckeri, C. piscicola and L. garvieae and EN 14675 modified to use IPNV as test virus) be used to determine whether disinfectants for use in aquaculture are efficacious against relevant aquaculture bacterial or viral pathogens.The technical subcommittee discussed some of the problems encountered and recommended solutions. Firstly, it was recognised that strict adherence to the standard meant ensuring that the majority of test procedures should be done within 10 seconds (e.g. test exposures, neutralisations etc.). It was discussed

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by the subcommittee and agreed that, occasional failures to perform a procedure within 10 seconds, would not constitute a test failure, although testing laboratories should still try to adhere to this criterion.This also brought up the important issue of Quality Assurance. It was recognised that it was vital for any Defra endorsed listing scheme that Defra should have confidence that particular test results reported by a company should have been achieved by the relevant testing laboratory having performed the test as specified by the standard (testing conditions, preparation of reagents etc.). Instead of Defra specifying a laboratory to run the tests, it was suggested that acceptable quality control could be obtained by requiring testing laboratories to adhere to internationally recognised quality systems (e.g. ISO17025) and also for tests in support of the highest dilution to be listed to be audited.

Objective 4 ‘To assist Defra with setting up of scheme, to include organising a third workshop to market the scheme to industry and other stakeholders.’Extent to which objective 4 has been met. This objective has been partially met.Methods and ResultsRepresentatives from Defra have been continually updated as to the progress of the scheme throughout its development and have attended both workshops. Defra remain committed to implementing the scheme, including requesting a no cost extension to the project to allow Cefas to continue to provide them with assistance in implementation. A meeting was recently held in London to discuss how the scheme can be implemented and a number of actions arose, including identifying resource within Defra to implement the scheme (Appendix 4).

Objective 5 ‘To write articles for Trade journals’Extent to which objective 5 has been met. This objective has been met. One article has already been placed in Finfish news, a further article is planned when a timetable for implementation is finalised with Defra.

Objective 6 ‘To assist Defra in publication of guidelines and standards for dissemination to industry.’Extent to which objective 6 has been met. This objective has been met.

Objective 6.1 Publication of guidelinesGuidelines detailing how the recommended testing standards should be performed have been drafted (Appendices 6 and 8). Objective 6.2 Quality Assurance As recommended by the second technical subcommittee, appropriate quality assurance procedures have been designed to give Defra confidence that data produced by a testing laboratory has been produced a specified by the particular standard. The Cefas Quality Assurance Department performed an audit of both the modified EN1656 and modified EN 14675 and from this produced Quality Assurance checklists (see Appendix 7) that can be forwarded to companies wishing to test their products to assist them in the production of data acceptable to Defra.

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Objective 7 To assist Defra in production of website detailing scheme.Extent to which objective 6 has been met. This objective has been partially met.

Details of the methods used and the results obtainedAs part of a meeting to discuss implementation of the scheme Cefas, how information on dilutions of products shown to be efficacious against aquaculture pathogens can potentially be listed on the Defra website was discussed.

Objective 8. To assist Defra in determining appropriate charging regimes to entail recovery of costs associated with listing a product (administration costs and expert review of submitted applications).Extent to which objective 6 has been met. This objective has been partially met.

Methods and results As part of a meeting to discuss implementation of the scheme, it was suggested to Defra that members of the technical subcommittee (Objectives 1 and 3) could potentially provide expert reviews of submitted applications. A potential framework for the scheme was also suggested to Defra for their consideration , as well as potential

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Appendix 1 Disinfectants in Aquaculture MeetingMinutes of Meeting 15 September 2004

Venue: Room 806a, 1a Page Street

Present:

Pansy Barrett (PB) Defra (Fish II)Graham Bell (GB) HSEDavid Graham (DG) DARDNIDavid Fraser (DF) FRSTrevor Hastings (TH) FRSDavid Verner-Jeffreys (DVJ) CEFAS (Chair)Ivy Wellman (IW) Defra (AMED)Neil Auchterlonie (NA) Defra (FScU, Minutes)

Agenda Item 1. Introductions and Background to Proposal

1. DVJ welcomed everyone to the meeting, which was the first attempt to gather interested parties together to discuss the official recognition of disinfectants in aquaculture; standards of testing, and to identify an agency prepared to take responsibility for their authorisation.. DVJ commented that biosecurity is an important issue in intensive aquaculture and effective disinfectants are a necessity for good husbandry practice, as well as the disinfection of sites following outbreaks of notifiable diseases

Agenda Item 2. Objectives of the Meeting

2. DVJ described the main objective of this work as to ultimately develop a scheme, or schemes, for authorisation, or listing, of aquaculture disinfectants (needs to be agreed who does this) and testing standards. Money will be required to set up and run such a scheme. DF commented that FRS was in agreement that such a disinfectants authorisation scheme was needed and would be required under the revised Directive EC91/67.

Agenda Item 4. Overview of Defra Disinfection Order

3. IW outlined the statutory basis of testing under the Diseases of Animals (Approved Disinfectants) Order. Currently there is a system in place to approve veterinary disinfectants (under the 1981 Animal Health Act). To be listed, disinfectants have to demonstrate that they are effective in laboratory tests (conducted by VLA and IAH laboratories) against specific diseases (Foot and Mouth disease, Swine Vesicular disease, Diseases of Poultry and Tuberculosis) or seek approval for “general orders” use. The general orders tests effective action against specified bacteria (Salmonella cholerae suis) with disinfectants approved for Orders regulating certain activities such as movement and transportation of animals.

4. Disinfectant manufacturers submit an application form, MSDS and cheque to cover current fees to AMED and a sample of the disinfectant to the laboratory. The relevant testing is carried out by the VLA or IAH laboratory, who then communicates the results of the test back to AMED. AMED then inform the manufacturer whether the product has

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passed or failed the test. Disinfectants are only approved when they are listed on the Amendment Order, through the Statutory Instrument procedure.

5. IW emphasised that this was a voluntary scheme, but there are disease-specific EU directives that require Member States to use only ‘approved’ disinfectant in a disease outbreak. There are perceived market benefits accruing from the international recognition of the disinfectant products meeting Defra’s approved standard, a situation which potentially benefits the disinfectant manufacturers which have chosen to seek approval of their products by this scheme.

6. IW said that the scheme would carry on administering the approval scheme until transfer to administration by HSE when present national practice is superseded by regulation under Biocidal Products Directive (BPD) 98/8/EC, but the final process has not been agreed yet.

7. A discussion followed during which GB pointed out that disinfectants are biocides and are subject to regulation under the Biocidal Products Directive (BPD) 98/8/EC. GB stated that he was not aware that there is a current requirement in EU legislation to have an “approved” disinfectant for good husbandry practice, and asked if this was a requirement for a notifiable disease outbreak. DF and TH replied that the wording under the Diseases of Fish Act and the EU legislation pertinent to fish disease doesn’t currently use the word “approved” however the draft revision of EC Directive 91/67 does. GB stated that he regards this as central to the issue – does the EU legislation require for the use of an “approved disinfectant” or just a “disinfectant”. If the requirement was for an ‘approved disinfectant’ then UK legislation was required to implement this requirement in the UK. If not, then the UK Regulations were a ‘UK national scheme’. On the basis of the discussions GB concluded that there was no EU legislation in this area at the present time and ‘approval’ was currently via a UK national scheme.

8. DF summarised the FRS experiences during outbreaks of the notifiable diseases Infectious Salmon Anaemia (ISA) and Viral Haemorrhagic Septicaemia (VHS) during which manufacturers would ask if their product could be used for disinfection of fish farm sites. Due to a lack of technical data, these questions could not be readily answered, DF commented that long term it would appear sensible to have a list of authorised or approved disinfectants analogous to that of terrestrial systems. DG asked if a 2-tier process would be workable whereby all disinfectants satisfy the regulations under the BPD, but there could exist a separate approval process for pathogens of interest to the UK. GB answered that all products would need to be authorised under BPD but that the standard of efficacy required to obtain that authorisation would be for the regulatory authority to determine. A higher standard might be required for pathogenic organisms

9. A discussion led by GB followed regarding the current status of disinfectants and the consequences following the publication of the second review regulation under BPD by the EC. GB described the review process for the active substances used in biocidal products – Industry were required to identify active substances used in biocidal products and notify those that they wished to support in the review programme. Then member states were given an opportunity to do the same and to take the responsibility as notifier for active substances that they considered ‘essential’ in their territory and had not been notified by industry. At the appropriate time HSE had asked all Departments if there were active substances that they wished to support in this way. Maureen Nowak (AMED, Defra) had been approached to do this – there is the possibility that the part of Defra responsible for fish may not have been consulted. TH emphasised the

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importance of this issue to those doing contingency plans for an outbreak of notifiable disease. IW noted that the AMED legal advisor would appreciate contact with the opposite number in Fisheries on this topic.

10.The BPD review programme is now starting to have an effect - some active substances used in aquaculture products have not been identified and products containing them should have been withdrawn from the market already. Others that have not been notified will have to be withdrawn from the market in September 2006. Products based on new active substances will have to go through an application and authorisation process before they can be placed on the market.

11.The BPD doesn’t specify efficacy tests, although it does give a substantial amount of guidance. TH asked if Defra efficacy tests could be transposed to the BPD. GB replied that Defra was part of the decision-making process and that their requirements would have to be taken into account. A lot of work would need to be done on efficacy standards and what might constitute “pass rates”, e.g. disinfectants required to have action against human, animal, fish pathogens are likely to be required to have efficacy at the upper end of the scale. PB asked if the UK knew anything about the efficacy standards expected by the other member states, and GB responded that this was unknown at the present time. The review programme was to be undertaken in four ‘priority lists’. The active substances used in aquaculture disinfectants were on the third list of these and is due to start in 2007. Following this review, products would require authorisation in each member state in which they were to be supplied. In the UK the assessment will concentrate on the UK situation. This would be the opportunity for Departments to input into the authorisation process – HSE is the regulatory authority on behalf of the Departments – if products need approval in this area, Defra would have a key input and could require that the efficacy of a product reached a required standard. GB added that HSE, as the Competent Authority would have a list of products authorised under the BPD, and an agency with responsibility in certain areas could abstract from that list.

12.Further discussion centred on the role of the HSE and the potential role of the relevant Departments. GB reiterated that HSE is the regulatory authority, but a Department with a relevant interest could theoretically set guidelines – HSE are the housekeepers, but would have to seek specialist knowledge from other Departments. GB mentioned that in fact a process of consultation with SEERAD and Defra had been initiated in the past in relation to terrestrial animals, although IW added that this process has stalled.

13.Discussion then focussed on the environmental aspects of disinfectants, and particularly environmental risk assessments and discharge consents. GB pointed out from the HSE perspective, the current procedure established by AMED and the proposal for fish diseases is actually based on efficacy rather than environmental impact – at the present time these are general chemicals which are subject to the legislation on general chemicals, including CHIP, COSHH and relevant environmental law. Once regulated under the terms of the BPD, an authorisation would only be granted if it could be shown that products could be used without significant risk. TH noted the difference with the law governing the regulation of veterinary medicines, where environmental impact is paramount concern.

14.Some time was spent discussing the various issues attached to the topic of a UK approval scheme. The salient points were:

The existing DEFRA/AMED scheme and the Norwegian model for aquaculture disinfectant approval are good starting points, however it is necessary to develop a scheme within the UK context.

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There is no perceived problem in setting up a scheme designed to list disinfectants considered acceptable for use in outbreaks of notifiable fish diseases, and the HSE would probably have to accept any Department’s approach in this manner, although the standards and criteria for testing are of key importance.

No active substances or products used in veterinary disinfectants have been approved under the BPD at the current time – active substances which have been notified by manufacturers appear in the 2nd review regulation. Veterinary disinfectants are classified as Product Type 3 and the active substances used in them are on the 3rd priority list for review.

This review of 3rd priority list active substances will commence in 2007. Notifiers of the active substances have to submit a dossier to support Annex 1 inclusion of active substances. Once included on Annex I products would then need to be authorised within member states. In the UK, ultimately authorisation would be granted on the advice of the relevant Departments and the Biocides Consultative Committee.

Defra “approval” under the AMED scheme was noted as having good marketing potential in other countries.

The possibility of tagging aquaculture disinfectants onto the AMED disinfectant database has already been asked of the Defra IT unit which constructed the system. Due to the extremely customised nature of the database, the technical issues involved would make this unlikely, although the relevant IT contacts are readily available to discuss options. Legally there may be difficulties in including fish in the scheme – the Animal Health Act specifically excludes fish.

In terms of aquaculture, there are only a small number of active substances that have been supported under the review programme that will be available from 2006 onwards.

“Approval” means two things – Is it safe? Is it efficacious? Within a UK ‘approval’ scheme established for fish disinfectants we would test efficacy, but we don’t know if the product is safe. Under BPD environmental safety would be assessed , but in the interim there remains the possibility that a product approved on efficacy grounds could subsequently be found to be environmentally unsafe.

Under BPD the inclusion of an active substance on Annex I would be for 10 years in the first instance and then subject to review. At the current time we don’t know what is involved. It may be that in the future different environmental standards will apply.

Agenda Item 6. Authorisation process/regulatory issues

15 DVJ commented that there is a reference to “approved disinfectants” in the draft document of the new Fish Health Regulations. GB reiterated that HSE is the regulatory body for biocides under the BPD, and although the HSE would seek advice on efficacy from other Departments this was unlikely to take place for a few years. DVJ then asked if it would be possible to get an aquaculture disinfectant system running in the interim. GB suggested instigating a ‘list of products or chemicals shown to be efficacious against a range of significant aquaculture pathogens’. This was to ensure that such a ‘listing’ could not be construed as being an ‘approval’ and that it only applied to efficacy. It was noted that such a system would have to be of sufficient standard that it wouldn’t be superseded by BPD at the end of the decade. One of the problems is that no one has yet assumed ownership for such a scheme.

16.DVJ discussed the need to set up testing standards, and asked how would decisions be made on such standards and could we easily identify the relevant people. IW volunteered that people who do the current testing for AMED would be happy to discuss the current system, and one of the IAH staff has already held discussions with David Smail of FRS on an aquaculture specific test. It was proposed to set up a Technical Sub-

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Committee to address the need to define a standard, and it was agreed that this would consist of David Graham (DARD), representatives from FRS and CEFAS (to include D Smail), a representative from Fish II (Defra) and a representative from VLA or IAH. GB suggested that HSE – David Dillon – should be copied into any of the communications from this sub-committee.

17. The identification of testing standards for efficacy was then discussed, and it was agreed that the sub-committee should investigate the situation in other countries within the EU, Norway, Canada, USA and Australia to see what tests already exist. DVJ noted that the OIE would be a good contact for this work, and would refer the matter to Barry Hill at Weymouth.

18. Discussion centred on generic disinfection compounds, such as sodium hydroxide and quicklime, that are occasionally used by the industry for disinfection. It is unclear how many generic compounds are currently listed, and if those that are listed will be available for use in a few year’s time. GB mentioned that for commodity chemicals such as these there is no practical method of restricting their sale for general use. Consequently, chemicals, such as sodium hydroxide, would still be available for users as disinfectants, as they are freely available for a wide range of other applications. GB explained that BPD is a marketing directive and if the manufacturer does not place a chemical on the market for a specific purpose, then a purchaser can purchase it and use it for any purpose. GB also commented that there is a derogation under the BPD that permits an “emergency’ use for 120 days for limited and controlled use of an unauthorised product. It could be envisaged that during an outbreak of an exotic notifiable fish disease, such use may be authorised. GB also mentioned the work the EU was doing that might enable an ‘essential use’ status to be achieved. To achieve this, if the review programme resulted in there being no active substances suitable for fish disease control, this is a potential route that may be taken for a single active substance. However, there is a need to demonstrate that there is no other viable alternative. It should be possible for industry or government to sponsor the application for essential use status of important generic chemicals.

Post meeting note: Stephen Irving (CEFAS) identified two categories here: Compounds which are off patent and sold as proprietary formulations by more than one

company. The precedent in the pesticide industry is that for these, they form a consortium to finance the work. As the scheme registers the product (not just active ingredient) their investment is therefore covered as, although any company can sell the active ingredient, only those products tested are registered.

Commonly used chemicals (e.g. NaOH) that have many uses. For these, it is difficult (clearly). As a policy- do we want to register these types of materials? They are dangerous (H and S issues), cheap and if a list of products registered contains something like this, despite its lower efficacy, some will use it because it is cheap. It is also very difficult to guarantee use rates (lack of appropriate labelling) and thus the end point efficiency of the treatment. Going this route, government will have to finance the efficacy package BUT in doing so there is a potential liability on safety grounds and efficacy grounds.

19. It was noted that the establishment of the list of efficacious products for control of fish diseases should be similar in some respects to the Defra scheme: products containing active substances that have not been supported under BPD cannot be listed. Similarly any product subsequently shown to be unsafe can be removed. Although not everybody was happy with the term, to some extent a ‘Health warning’ should be attached to the listing, saying in effect that that the list is only concerned with efficacy, no guarantee as to any other aspects of proposed use (such as safety or future authorisation under BPD) are implied.

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20. Action Point: DVJ to examine the list and identify any potential aquaculture chemicals which may be lost in the next few years and clarify AMED advised sanitation procedures during control of notifiable terrestrial diseases.

21. Action Point: IW and PB to help to arrange meeting between the legal representatives of AMED and Fish II.

Agenda Item 7. Funding Issues22. DVJ asked about the possibility of funding the initial research to set up an approval scheme. NA replied that until it had been decided which policy group would take ownership for such a scheme, it was difficult to offer advice on this aspect. GB summarised three levels where he envisaged a requirement for money: 1. The cost of the research to establish the efficacy standard and the admin systems; 2. The cost of individually testing products to the standard; 3. The costs of the evaluation of the submissions from the manufacturers. Under the BPD, industry were responsible for the costs of 2 and the evaluation costs at 3 would be charged by way of an application fee. It is important that such work is not under-charged as it could be construed as state support to the industry. The policy would be to retrieve the full economic cost, currently approximately £300 per day. IW commented that Defra’s policy is to operate approval regimes on a cost recovery basis, but that AMED’s approval system does not, at the moment, as the current Fees Order was set in 1991. A consultation exercise is underway prior to updating fees to recover the cost of the tests.

Agenda Item 8. Implementation23. The implementation of an approval scheme was discussed. DVJ pointed out that this also depends on who takes on the scheme – potentially it could even be the industry itself. IW commented that AMED publish a list on the Defra website, and TH agreed that this would be a relatively easy method of publication.

24. The issue of confidentiality in relation to the publication of company and product information was discussed. The issue centres around what would be a voluntary scheme in the first instance – companies may not want to provide product information since they may feel that this gives their competitors an advantage. Under the BPD there is a system of data protection. Care would need to be taken to ensure that the protection of data under BPD was not compromised by its earlier submission under the proposed voluntary scheme. Under an approval scheme, companies could be asked for the relevant product information, and if they refuse to supply, it would need to be pointed out that they would need to supply these details in the future (and would need to have a good reason to withhold the information in the interim). IW commented that the current terrestrial scheme published on the website only lists the company address, telephone number, name of product and what the product is approved for.

25. Advertising. All agreed that this could be easily done by publicity at industry forums and production of articles for inclusion in the trade press.

Agenda Item 9. Other interested parties26. Discussion focused on other stakeholders which may need to have an input into an approval scheme. GB commented that there is a stakeholders’ group meeting held between the HSE and the biocides industry on 8th December in London. Some of the manufacturers attending may be involved with aquaculture disinfectants, and this would be a good opportunity to discuss the pertinent issues. Other interested parties identified were the EA, SEPA, OIE and the aquaculture industry. TH commented that Committee for Aquaculture Health (CAH) meetings may be the best forum to inform others of this proposed work.

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Action Points: DG to liaise with environmental regulatory body in N. Ireland. DG and TH to inform policy units of proposed work. GB and possibly DVJ to communicate proposed approval scheme to biocides industry representatives at the December meeting. DVJ to circulate copies of minutes from this meeting and other background information to other interested parties, when identified.

Agenda Item 10. AOB27. NA highlighted the use of chemicals by the aquaculture industry to disinfect eggs. It was agreed that this was also of importance and chemicals marketed with this claim would also probably also be captured by the BPD (98/8/EC), although clarification from VMD should probably be sought (in case they are considered as medicines).Post meeting note: DVJ recently visited VMD and the situation is not straightforward.

Article 1 of Directive 65/65/EEC defines a "medicinal product" as:‘Any substance or combination of substances presented for treating or preventing disease in human beings or ANIMALS’. Or, ‘any substance or combination of substances which may be administered to human beings or ANIMALS with a view to making a medicinal diagnosis or to restoring, correcting or modifying physiological functions in human beings or animals is likewise considered a medicinal product.’ On this basis, as eggs are not considered ‘animals’ under the terms of relevant UK legislation it could be argued that chemicals used to treat them might therefore not be considered medicines. However, VMD have treated certain egg treatments as medicines in the past (e.g. they have granted a Marketing Authorisation (MA) to a company for a product used to control saprolegniasis in salmonid eggs). They note that, although the egg may not technically be an ‘animal’ when the product is applied, the product may affect the animal’s future development (particularly if it is absorbed by the developing embryo). This may affect both the developing animal and the eventual consumer (in the case of food species). The continued surveying (statutory and non statutory) for the potential illegal use of the antifungal treatment Malachite Green by the residues section of VMD shows how concerned they are about the use of unauthorised egg treatments. However, if a company markets a product claiming to ‘clean’ an egg, rather than improve the health of the animal, or protect against infection, these would not be treated as medicines. Comparison was drawn to udder treatments. Where a company markets an udder wipe with a general ‘cleaning’ claim, even if that includes an iodophore component (< 3000 ppm), VMD have not treated them as medicines, they would instead be treated as disinfectants. Apparently VMD treats disinfection of poultry eggs in the same way (if the claim is ‘cleaning’ then they do not consider them medicines). However products marketed with claims that include ‘protect against mastitis’ would be considered medicines. If VMD decide a claim about a particular egg treatment is not medicinal but, instead, refers to a claim for a disinfectant, then the BPD would therefore be the relevant regulatory umbrella, as the marketing of all disinfectants, with certain explicit exceptions, is now controlled under the provisions of 98/8/EEC). It is suggested that any company wishing to market an egg disinfectant should contact VMD who will assess the particular marketing claim for their product and advise whether it is a medicinal claim, or not. If the claim is considered medicinal, the company would require a Marketing Authorisation from VMD before that product could then be sold or distributed in the UK.

28. DVJ concluded the meeting, and thanked all for their input.

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Appendix 2 First Technical Subcommittee Workshop 18-19 April 2005

Establishment of a UK Aquaculture Disinfectant listing schemeFirst Technical Subcommittee Workshop 18-19 April 2005

CEFAS Weymouth Laboratory, Room 107a

Present: N. Auchterlonie (NA) - DEFRA D. Graham (DG) - DARDA. Husby (AH) - Norwegian National Veterinary InstituteI. McLaren (IM)- VLAD. Smail (DS) - FRS AberdeenS. Williams (SW) - IAH Pirbright

P. Dixon (PD), M. Algoet (MA), K. Way (KW), D. Alderman (DA), A. Reese (AR) - CEFAS Weymouth

Chair: D. Verner-Jeffreys (DVJ) - CEFAS Weymouth

Minutes: N. Ridout (NR) -CEFAS Weymouth

SummaryThe workshop reviewed and recommended appropriate standards for use in testing aquaculture disinfectants.

1. It was decided that recommended standards should, as far as possible, follow available European (EN) disinfectant testing standards, modified for use with appropriate aquaculture pathogens.

2. It was recognised that available time and resources prevented the creation of a long list of standards for testing disinfectants against a range of notifiable aquaculture pathogens. To preserve the integrity of Defra approvals in general, it was recognised as vital that any standards recommended by Defra are thoroughly tested before being recommended. For this reason it was decided to develop single virus and bacterial testing standards, using model organisms that were suitable for use in the test system and were very resistant to the action of biocides. Demonstration of efficacy of a biocide against the model organisms will then provide a level of assurance of likely broad-spectrum activity.

3. Viruses: It was decided to test the feasibility of modifying prEN 14675 for use with Infectious Pancreatic Necrosis Virus (sp serotype). IPNVsp is a suitable model as it is already used in EN 14675-based approvals in Norway (so we have information already about its suitability for use in the recommended test system) and is a very resistant notifiable organism, highly relevant to UK aquaculture.

4. Bacteria: It was decided to test the feasibility of modifying EN 1656 (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary field). It was suggested that model organisms should be as per Norwegian guideline recommendations (Aeromonas salmonicida subsp. salmonicida (ATCC 14174), Yersinia ruckeri (ATCC 29473), Carnobacterium piscicola (ATCC 35586), except Lactococcus garvieae be substituted for Staphylococcus aureus (ATCC 6538)

5. Testing Conditions for both viruses and bacteria: ‘high’ soiling, 4°C and a contact time of 30 min were recommended as mandatory testing conditions. The option of also including other information about effectiveness at lower contact times and higher temperatures may also be possible (EN standards allow for this). To avoid confusion for both users and manufacturers wishing to compare products, it is recommended that mandatory conditions are imposed though.

6. Seawater. The possible interfering effects of seawater on biocide activity should be controlled for where biocides are to be used in seawater sites. It was decided to test the effects of various formulations of seawater for use as a diluent in testing standards.

7. Validation of testing standards. It was proposed to test up to four different aquaculture disinfectants, each representing a different mode of action chemistry, with the recommended standards. Tests will be repeated at least three times and, subject to available resources, different contact times, and use of different ‘seawater’ diluents will all be tested. It is hoped that R. salmoninarum will also be included, as well as the other bacterial isolates tested using the modification of EN 1656. Comparison can then be made with the resistance of R. salmoninarum to the biocides tested and the other organisms, providing information on the likely validity of substituting Carnobacterium piscicola in particular as a surrogate for R. salmoninarum.

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8. ‘Fungi’. The importance of developing appropriate standards against oomycete (aquatic ‘fungal’) pathogens, such as Saprolegnia parasitica, as well as the agents of EUS (A. invadens) and crayfish plague was recognised. Available standards will be reviewed and recommendations for research in support of developing suitable standards recommended.

9. Parasites. The difficulties of specifying suitable standards for testing disinfectants against notifiable parasitic agents (e.g. Bonamia) were noted. However the importance of having appropriate methods of disinfecting sites, stock or equipment potentially contaminated with Gyrodactylus salaris, as part of contingency plans to deal with any potential introduction of G. salaris into the UK, was also recognised. Contact with Norwegian experts in this area will be made and available information will be reviewed.

10. Next workshop. The next meeting of the technical subcommittee is provisionally scheduled for 7-8 September, FRS Marine laboratory. The workshop will review the results of the laboratory based validation and recommend standards.

Overview of Scheme (DVJ)

DVJ welcomed attendees and opened the workshop with a brief overview of the scheme and progress so far. The presentation covered; i) the importance of biosecurity in aquaculture, ii) the need for a disinfectant listing scheme of this kind, iii) recent progress (including a recent meeting in London between various stakeholders and subsequent agreement by Defra Fisheries II section to administer a listing scheme –see appendix 2) and iv) an overview of the CSG7 research proposal to establish the listing scheme (including milestones and deadlines).

Aim of the Workshop (DVJ)

As DVJ explained, this workshop was the first of two meetings regarding the design of the disinfectant testing standards for the aforementioned approval scheme. The objective of Workshop 1 was to decide on technical details such as the model viral and bacterial pathogens to be tested, test agents, test conditions and to discuss QA and statistical issues. Preliminary laboratory investigations will follow, the results of which will be discussed at the second workshop (FRS Aberdeen, September 2005, possibly Wed 7th and Thurs 8th – TBA).

Norwegian experience (AH)

AH gave an overview of the Norwegian aquaculture disinfectant approval scheme. Their scheme, that is based on a draft of BPD (98/8/EC). Manufactures submit an application form, containing safety (including ecotoxicity) and efficacy information about their product to the Norwegian Medicines Agency, who administer the scheme. Efficacy and ecotoxicity aspects of the application are evaluated by the Norwegian National Veterinary Institute. Efficacy test criteria, based on available European standards, are summarised below.

Test types: suspension, surface and capacity (to relate to footbaths in which there is repeated introduction of bacteria)

Temp: 4ºC (and 15ºC if requested by the manufacturer) Contact time: 30 min max (less if requested by manufacturer) Organic loading: 1% bovine serum albumin + 1% yeast extract (final concentrations)Model bacteria: Aeromonas salmonicida subsp. salmonicida (ATCC 14174), Yersinia ruckeri (ATCC 29473),

Carnobacterium piscicola (ATCC 35586) and Staphylococcus aureus (ATCC 6538).

Model virus: Infectious pancreatic necrosis virus (IPNV) Model fungi: as stated in European CEN standards (Aspergillus niger and Candida albicans)

Interest in this voluntary scheme was slow for the first few years. To date, there are 5 registered products, representing 4 different companies. While any product can be used for routine disinfection, only these 5 are currently approved for mandatory disinfection.

As AH explained, there are very few laboratories offering the disinfectant testing service in Norway. As the approving body, NVI are not permitted to perform the laboratory work. They are only permitted to offer advice. Data can, however, be generated outside of Norway.

In Norway, the typical cost for a single test (bacterial suspension test, for example) would be in the region of 100,000 NOK, equivalent to £8,300 This is an average cost estimate. Virucidal tests will probably cost less since only one virus is tested (compared to 4 bacterial species). Manufacturers do not have to run all 3 test types if they are not all applicable to the intended use. If a product is intended for wellboat disinfection, for example, the surface test alone would be sufficient. All costs are covered by a fee of 30,000 NOK (=£2,500) (per product).

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Selection of viral agents for UK testing standards (DS)

DS highlighted key issues triggering much discussion. The key aspect of this debate was the suitability of IPNV as a model viral pathogen. As SW highlighted, BS EN 14675 uses Bovine enterovirus Type 1 as a single model virus. This virus is 1)

representative of a large number of viruses, 2) relatively easy to handle and 3) fast growing in cell culture (reaching high titres). Results can be obtained within 48 hours. PD and DS agreed that IPNV would meet all of these criteria.

AH explained that several viruses were considered for the Norwegian testing protocol but the final decision was to use IPNV as a model.

NA reminded attendees that the original driver for this project was the development of a listing scheme for aquaculture disinfectants, and this is the objective of the work.

It was suggested that there could be some degree of flexibility in the virology standard, allowing companies to select the viruses against which their product is tested. After some discussion it was agreed that this could be the cause of confusion.

Concerns were voiced over the possibility of IPNV being removed from the OIE list.

Other points raised: Diluent: Salinity must be considered. DS referred to investigation into virucidal effect of disinfectants in

freshwater compared to seawater (Jun Kurita et al, 2002, Fish Pathology 37(4), 175-181). . If there were a need for a disinfectant to be used on a well boat or in any other circumstance when only seawater is available to dilute the disinfectant, then efficacy in seawater needs to be validated

DS emphasised the importance of high organic loading to mimic the natural situation as closely as possible. DS highlighted the need for “universal standards”. There is much variation between the current standards.

Selection of bacterial pathogens for UK testing standards (DVJ)

DVJ suggested a list of bacterial pathogens for use in the bacterial testing standard (see section 2). Viral and Bacterial Discussions

The workshop attendees then divided into 2 groups to discuss the technical details of the viral and bacterial testing standards. On returning to the group, DS and DVJ later provided a synopsis of these sub-meetings for general discussion.

1. Viral test (DS)To be based on BS EN 14675 (Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field).

Model virus: IPNV As PFD highlighted, stability varies considerably between the different IPNV serotypes. Serogroup B, for example, is very labile. The Norwegian approval scheme specifies Sp. This is a detail that can be decided during the preliminary laboratory investigations. Seawater and Hard water: both terms must be defined and need to be consistent with bacterial test Temp: mandatory 4ºC Contact time: mandatory 30 mins (although other times can also be included if requested by

manufacturer- all following EN 14675)The possibility of decreasing this time to 10 mins was discussed but, after much debate, it was decided to follow EN 14675 in this (and the Norwegians). Shorter contact times will be investigated in the laboratory investigations. Organic loading: 1% bovine serum albumin + 1% yeast extract Replication:

o 3 concentrations of product (1 in active range and 1 in non-active range)o 3* preparations of each concentration of product to be testedo 3 wells for each dilution in plaque assayso 8 wells for each dilution on TCID50 plates

*It was decided that triplicate preparations of product would be tested in the initial laboratory investigations and this would be increased if necessary (depending on the statistical variation of the results).

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As SW explained, from his experiences of working with BS EN 14675, some technical aspects of the testing standards (viral and bacterial) should remain relatively flexible. The group agreed that details such as the cell type and medium for virus culture, for example, will not influence the final result and should, therefore, be the decision of the testing laboratory.SW emphasised that, in their experience, flexibility with respect to the detection system used should be allowed. It was also pointed out that the EN standards are self-validating with respect to a number of measured parameters, so are amenable to a level of flexibility, without necessarily compromising the integrity of the final result.

2. Bacterial (DVJ)To be based on the European Standard EN 1656 (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field).

Model bacteria (based on list recommended by Norwegian guidelines) o Aeromonas salmonicida subsp. salmonicida (ATCC 14174),o Yersinia ruckeri (ATCC 29473)o Carnobacterium piscicola (ATCC 35586) and o Lactococcus garvieae (to replace) Staphylococcus aureus (ATCC 6538).o (to replace Staphylococcus aureus) o It was agreed that each product will be tested against all 4 model bacteria and the report will

state which concentrations have passed (greater than 5 log reduction).o Temp: 4ºC

Seawater: Sigma sea salts suggested (to be prepared in deionised water rather than glass distilled water). Filtering and autoclaving to be compared as sterilisation techniques.

It was recommended that neutralisation dilution rather than membrane filtration is used to avoid possible problems of cell aggregation, leading to potential overestimation of degree of inactivation by product under test.

The possibility of running a surface test based on EN 14349 was discussed, but was considered to be unlikely to be possible with the resources and time available. It was agreed that this is an aspect that should be included in any continuation project.

3. Parasites (EP - Ed Peeler, Epidemiologist, CEFAS Weymouth) As NA mentioned earlier in the workshop, requests are sometimes received for information on effective methods of disinfection against G. salaris, and there would be a strong requirement for such information should an outbreak of the notifiable disease occur. Action:DS agreed to forward an article on the efficacy of Virkon Aquatic against G. salaris and an informative leaflet to NA.

EP referred to work in Norway with Virkon Aquatic and Chloramine T (check) against G. salaris. Disinfectant manufacturers financed this work (ref: not published, personal communication with NVI).Post meeting note: Information forwarded to EP and D. S by contacts within the Norwegian Veterinary Institute suggest that G. salaris is killed by exposure to 1% Virkon Aquatic for 10-15 sec.EP explained that a contingency plan for G. salaris is currently underway, this information will undoubtedly compliment this scheme.There are no testing standards in Norway for parasites (AH) and no standards available in the UK veterinary field either (DG). EA have highlighted a need for this info also.Action: DVJ to liase with EP and summarise current knowledge of disinfectants and G. salaris (and other parasites). EP to provide DVJ with info that has been generated as part of the contingency plan.

4. Fungi (DA)The importance of developing appropriate standards against oomycete (aquatic ‘fungal’) pathogens, such as Saprolegnia parasitica, as well as the agents of Epizootic Ulcerative Syndrome (Aphanomyces invadens) and crayfish plague (Aphanomyces astaci) was recognisedAs DA explained, 90% of apparent fungi are, in fact, not fungi but water mould (oomycete) and fungal testing standards therefore do not necessarily apply. Action: DVJ and DA will discuss and modify existing standards and circulate for comments/suggestions (DVJ to lead). Although over 20 years old, these standards have been used more recently for commercial contracts.

Quality Assurance (DVJ)DVJ emphasised the need for a QA system. In the existing DEFRA livestock disinfectant tests, both IAH and VLA are UKAS ISO 17925 accredited. As SW explained, very few laboratories are accredited for this testing. Accreditation is an expensive process that significantly increases the cost of lab tests and consequently deters the manufacturers from submitting their products for approval.

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In the Norwegian system, in comparison, AH explained that there are no specific QA schemes.After much discussion within the group it was decided that the testing laboratory would be required to perform an internal audit and attach an audit report to their application. If an internally generated audit is attached, it would be expected that the laboratory concerned adhered to an internationally recognised Quality system (e.g. ISO x or GLP). As SW mentioned earlier in the workshop, in the DEFRA veterinary disinfectant approval system random ‘off the shelf’ checks are performed on approved products. All attendees agreed that this would be a good control measure to incorporate into the aquaculture scheme. SW mentioned the need for MSDS safety data sheets to be included in application forms.Action: DVJ to discuss this idea further with Debbie Page (Quality Department, CEFAS Weymouth) and circulate a draft QA plan for comments.

Statistical Issues (AR)

AR Emphasised the need for replicates.All agreed to try 3 replicates in the lab tests and submit results to AR to look at variation and possible need for more replicates.

SW: In the FMD test (performed to BS EN 14675) a mean log reduction would normally be calculated. A 4-log reduction is considered a pass. If the mean is borderline (e.g. 3.9) this would be a fail. Variation between replicates can be up to 1 log.

DS: raised the problem of aggregation in viral suspension tests. Need to consider this as it can cause variation in results.Reference for a new method for low dose membrane bound exposure - Georgios T Papageorgiou et al (Dec 2001) Appl & Env Micro, Vol 67(12), p5844

AR: why not test a range of temps and contact times

Action: AR to review experimental designs from DVJ and PD.

Preliminary experimental workAs suggested by DS in earlier discussions, the preliminary laboratory investigations (viral and bacterial) should aim to test a representative disinfectant from each of the following groups of biocides, based on mode of action (time permitting):

Hypochlorite Iodophores Organic chlorine release (e.g. chloramines) Peroxygen compounds Peracetic acid/ hydrogen peroxide combination products Chlorine dioxide

Hypochlorites and chloramines are often grouped as chlorine-releasing agents, and probably have similar mode of action so one product from this group (e.g. Chloramine T) could tested. Similarly, as peracetic acid is a peroxygen compound this could also be grouped in the peroxygen category. Attendees agreed that this is an ideal opportunity to generate data on some of the relatively new, more environmentally friendly products.

Manufacturers will be approached for samples to test in this early work. The group agreed, that this data is for the developmental aspect of this project and must not be used for product approval at a later date, as this could be viewed unfair to other manufacturers. As NA emphasised, the support of these companies is essential for the success of this scheme.

The group discussed the design for an investigation into the effect of salinity on the efficacy of disinfectants as some groups, Iodophores for example, have been found to be less effective in seawater. PD and DVJ agreed to draft a protocol based on the following outline:

Virus + freshwater + disinfectant A Virus + seawater 1 (Sigma sea salts or a recommended formulation e.g. Lyman and Fleming) +

disinfectant A Virus + seawater 2 (Weymouth or deep sea?) + disinfectant A Seawater sterilisation by filtration was suggested, followed by cell culture testing.

Action: DVJ and PD to discuss laboratory tests further and circulate draft protocols.

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Workshop ConclusionsThe workshop raised lots of areas for potential research that are beyond the scope of this project.Action: DVJ agreed to document these areas and submit for further funding.

Other Points:PD emphasised the importance of communicating with industry and other stakeholders to inform them of this scheme. This communication should be 2-way as their feedback is crucial at the developmental stages of this project. This is a point that was raised throughout the meeting. DVJ is currently liaising with the Defra, EA, SEPA, HSE, BACS and other industry contacts. It was mentioned that there will be a stakeholder forum towards the end of the year where the details of the scheme will be communicated. It is envisaged that parallel dialogues with each group of stakeholders will have been established (post meeting note- this is indeed the case already) prior to the forum so each can feedback into the scheme’s development prior to finalisation.

DVJ: to circulate reports on the results of contacts with stakeholders to the technical committee for comments.

MA: suggested that perhaps we should run both Renibacterium salmoninarum and Corynebacterium tests to ensure this is an appropriate substitution. PD suggested selecting the most effective and the least effective conditions to minimise the workload. As Renibacterium is extremely slow growing, however, it might be important to run all the tests at once.

MA also mentioned the intracellular bacterium P. salmonis. DS agreed this is becoming a very important pathogen.

MA raised the possibility of developing a common approval scheme for Europe, particularly, if, as looks highly likely, the efficacy testing standards recommended for both the Norwegian and UK schemes are very similar.Action: PD agreed to raise this suggestion at the next European Reference Lab meeting. MA will mention it at the PANDA meeting in Greece. DVJ will also present an overview of the scheme and its progress at the upcoming EAFP meeting in Copenhagen.

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Appendix 3 Second Technical Subcommittee Workshop 11-12 October 2005

FRS Marine Laboratory, Aberdeen

Establishment of a UK Aquaculture Disinfectant listing schemeDefra sponsored project FC1169)

Present: N. Auchterlonie (NA) - DEFRA D. Graham (DG) - DARDA. Husby (AH) - Norwegian National Veterinary InstituteD. Smail (DS) - FRS AberdeenD. Fraser (DF) - FRS AberdeenT. Hastings (TH) - FRS AberdeenP. Dixon (PD) - CEFAS WeymouthC.Joiner (CJ) - CEFAS WeymouthM. James (MJ) - SARF

Chair: D. Verner-Jeffreys (DVJ) - CEFAS Weymouth

Minutes: N. Ridout (NR) - CEFAS Weymouth

Overview of Scheme (DVJ) DVJ welcomed attendees and opened the workshop with the presentation given at the last European Association of Fish Pathologists Conference, (Copenhagen ,Sept 2005). The presentation included (i) the background behind the scheme, (ii) a brief overview of the existing Norwegian scheme, (iii) details of the suggested listing mechanism and (iv) an outline of the bacterial and viral testing standards.

A few important questions were raised within the group from the presentation:

Will the scheme cover egg disinfection?This topic arose several times throughout the workshop. Attendees agreed that egg disinfection is an important and highly relevant issue that must be considered. Although egg disinfectants will not be excluded from the scheme, concerns were raised over the suitability of EN1656 and prEN 14675 and the possibility of good egg products failing. Ideally, a third testing standard would be aimed at egg products but it was agreed that this is beyond the scope of this 1-year project.

Contact timeThe 30-minute contact time for the testing standards was questioned. It was argued that 30 mins is not realistic in the field. DVJ explained that this time is merely for the generation of comparative data. After much discussion it was agreed that the contact time should remain at 30 mins as the minimal mandatory criteria. Although 30 mins is mandatory, product manufacturers may also be allowed to submit data for shorter contact times.

PrEN 14675 was due for review this summer.Action: DVJ to check if this review has taken place.

Bacterial Standard (DVJ) DVJ gave a brief outline of the bacterial assay that follows EN 1656 (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field) and presented results to date.

Products testedFour commercially available disinfectants are being used in the validation exercises (both bacterial and viral standard testing).

The first product tested was a leading peroxygen based disinfectant,

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The second product was a leading iodophore based disinfectant. This contained 2.85% active iodine and other cleaning agents, including sulphuric acid).

The third product was a Chloramine T based disinfectant The fourth product was a hydrogen peroxide and peracetic acid based disinfectant.

All products were supplied by the manufacturers to Cefas for use in the validation exercise. It should be strongly emphasised that the data discussed at the workshop was very preliminary and should not be taken as evidence of a particular product’s suitability for use as an aquaculture disinfectant.

Isolate storage3 cryopreservation methods have been used for the 4 test bacteria: Protect™ as used in CEFAS bacterial culture collection method and 2 methods recommended in EN12353. All isolates were successfully resurrected 7 wks post-storage (all methods).

Plating techniqueNo significant differences have been detected between the spread and pour plate methods with all four test bacteria (P=0.838, ANOVA). Some problems were encountered with satellite colonies when using the former.

Neutralisation dilution The standard specifies that, if possible, product test mixtures should be inactivated at the end of the contact time by neutralisation dilution. Suitable neutralisers were identified for all products tested, suggesting that this is an appropriate inactivation method for the modified standard (e.g. the neutralisers tested were not toxic to any of the organisms substituted and demonstrably inactivated the disinfectants tested).

Chloramine T containing product resultsA Chloramine T containing product has been tested at 10 -10,000 ppm. MIC appears to be between 500 and 800 ppm for A. salmonicida, Y. ruckeri and L. garvieae and around 1000 ppm for C. piscicola.

Iodophore containing product results The Iodophore containing product has been tested at 1600 – 16,000 ppm (2.85% active iodine). MIC

appears to be between 1600 and 2800 ppm for A. salmonicida and Y. ruckeri, and between 2800 and 4000 ppm for C. piscicola and L. garvieae.

Virkon Aquatic& Proxitane® 5:23These products have yet to be tested by EN1656 but a preliminary study has identified suitable neutralisers for each product. Post-meeting note: both products have now been tested successfully at least once.

RepetitionSome repeat tests have been performed for the iodophore containing and Chloramine T containing disinfectants (on separate days, not alongside). Variations are evident in the data (even pass/fail differences) but, as DVJ explained, this appears to be around the MIC and is not necessarily an indication of test errors or poor reproducibility. AH confirmed similar findings in the Norwegian test repeats.It was suggested that 3 replicates of product should be prepared on the same day and run in the same test. DVJ explained that this is not practical at this stage when dealing with 4 bacteria and several product concentrations. AH agreed and explained that they run repeats on separate days also. Action: DVJ to discuss repeat data with CEFAS statistician, Alan Reese.

Seawater investigationFiltered Weymouth seawater was used to prepare the Iodophore containing disinfectant at 4 and 1.6 ml/l. When run alongside equivalent hard water preparations, no significant differences were detected, although 4ml/l caused total clearance and 1.6ml/l showed no reduction (A.sal tested only). Action: It was suggested that, time permitting, this test will be repeated using FAM30 at concentrations between those tested previously to confirm this result.Action: It was also agreed that a similar investigation should be carried out with the viral standard.

Attendees agreed that a standard seawater is needed and should be clearly specified. After much discussion it was suggested that one such formulation could be Lyman & Fleming’s synthetic seawater. (Ref: Lyman, J. and Fleming R.H. (1940) Composition of seawater. J. Mar. Res. 3, 134-146.

Product preparationEN 1656 states that the test products must be used within 1 hour of preparation, but the viral standard stipulates 2 hours. Attendees agreed that these times should ideally be consistent.Action: DVJ/PD to discuss with Stuart Williams

Timing

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EN 1656 stipulates timings +/- 10 seconds. This specification is difficult to meet when dealing with several product concentrations and 4 test bacteria.Action: DVJ to discuss with Ian McLaren

Action: Amended EN 1656 to be drafted and circulated for comment.

Viral Standard (CJ)CJ gave an outline of the viral assay described in prEN 14675 (Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field), presented results and highlighted a number of technical difficulties experienced with this standard so far. Peroxygen-based disinfectant resultsThe Peroxygen-based disinfectant passed the 4-log reduction criterion at 1/200, 1/350 and 1/500 but failed at 1/800. No problems have been experienced with cytotoxicity and dilution neutralisation has proven effective.

Iodophore containing product resultsThe iodophore containing product has been tested up to 880ppm (2.85% active iodine). Preliminary cytotoxicity tests suggest 220-880ppm are cytotoxic. Dilution neutralisation appears effective but titres are difficult to read due to this cytotoxicity. Up to 220ppm, viral titres are similar to those of controls. At 440ppm the viral titre is clearly decreased but a 4-log drop cannot be demonstrated due to the cytotoxicity problems.

Chloramine T containing product resultsAt the strongest concentration tested so far (2%) The Chloramine T containing product demonstrated a 1.5 Log reduction in titre. Precipitation of proteins is posing a problem but no cytotoxicity has been encountered. Dilution neutralisation has proven effective.Action: CJ to test prEN 14675 with higher concentrations of the Chloramine T containing product (e.g. 3%). CytotoxicityIt was agreed that cytotoxicity investigations should continue to be run as a pre-test rather than alongside the main test as stated in prEN 14675.

DS described the use of microdialysis chambers to overcome the cytotoxicity experienced with the particular iodophore containing product tested (that contains other potentially cytotoxic constituents, such as sulphuric acid). Dialysis has proven highly effective in previous studies with both plaque and TCID50 methods.

It was debated whether the final viral standard should (i) include this dialysis technique (or similar) for all tests or (ii) suggest possible techniques that can be employed where necessary to overcome cytotoxicity problems.All agreed that if such a step were to be included in the standard it would need to be validated before the final standard is issued and there would need to be a control incorporated into the standard to ensure no virus has been lost during the dialysis step.

Action: It was suggested that CJ tests prEN 14675 with a different iodophore (e.g. PVP-Iodine) as the cytotoxicity problems encountered with the particular product tested may have been caused by some of the other constituents in the biocide than the complexed iodine (e.g. sulphuric acid). CJ also to test cytotoxicity and neutralisation with sodium thiosulphate as a neutraliser for FAM30 before attempting dialysis.Action: DVJ to check remaining staff time to contract.

PrecipitationThe precipitation problem experienced with the Chloramine T containing product was discussed. As CJ explained, precipitation is a common problem with chlorine-based products. As DS highlighted, live virus may be trapped in the precipitate. Such trapping will also be true in the field unless a pre-cleaning step is incorporated to remove organic matter. Action: CJ to test the Chloramine T containing product with low level or no soiling.

NeutralisationUnlike the bacterial standard, PrEN 14675 does not include neutralisation validation steps. CJ described pre-tests that ensure the neutraliser is (i) effective and (ii) not cytotoxic. Such tests are to be included in the final standard.

Unlike EN 1656, prEN 14675 does not list suitable neutralisers or suggest alternative neutralisation methods for cases where dilution is ineffective. AH explained that in the Norwegian scheme dilution neutralisation is the preferred method but, if technical difficulties arise, alternative methods will be considered. As time is limited in this study, it is not possible to list a number of suitable alternatives (e.g. sodium thiosulphate) as each will need to be validated. It was proposed that the flexibility practised by the Norwegian scheme should be adopted but suggestions should be included in the modified standard.

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Action: PD and DS to draft a clause regarding neutralisation and circulate for review.

Sodium hydroxide controlThe relevance of the test virus inactivation test was debated. PD previously asked Stuart Williams about this and was informed that it originated on the German protocol but was argued when the UK terrestrial animals scheme was set up. AH said the step is included to help identify day-to-day and inter - and intra-laboratory sensitivity variability (a test organism sensitivity control).

After much discussion it was agreed that this step will be omitted.

Action: Amended prEN 14675 to be drafted and circulated for comment.

Quality Assurance (DVJ)It was agreed that the testing lab submitting data for inclusion on the list should adhere to a widely recognised quality system such as UKAS or GLP. It was suggested that a minimum standard, such as ISO 9001, is stated as acceptable.

DVJ proposed that Debbie Page (Quality Department, CEFAS Weymouth) audits the bacterial and viral tests, determines study critical points and produces a QA checklist to be completed by the testing lab during internal audit. Data submitted for consideration must have an internal QA audit report attached. All agreed this process. Action: DVJ to liaise with Debbie Page

It was emphasised that DEFRA will reserve the right to perform checks on testing laboratories and, as discussed in the first workshop, products may be tested at random from the shelf. Questions were raised regarding the funding of such testing. E.g. should the estimated costs of such QA tests be subsumed into the original costs to be met by manufacturers?

RepeatsAfter much discussion it was agreed that the final testing standards (viral and bacterial) should ideally stipulate that all 3 concentrations of the test product are to be repeat tested 3 times, not just the marginal product concentration. However, it was also recognised that this would potentially entail a great deal of work for a testing lab.

It was debated whether it would be acceptable to only do the repeat testing on the most resistant test bacteria identified in the first round of testing under the modified EN1656.It was agreed, however, that the cytotoxicity and neutralisation pre-tests for the viral standard do not require repeats. For the bacterial standard, all agreed that 3 parallel dilution series should ideally be performed for the plate counts.

If repeat tests are to be performed on the same day, it was agreed that separate batches of product should be prepared.

Action: AH to forward statistical evaluation of EN1040 test results to DVJ as an example.

Action: DVJ to discuss with Alan Reece a practical route forward, in terms of recommending appropriate levels of standard replication, to give Defra confidence that submitted data in an application form is a reliable indication of in vitro activity at the particular test concentrations indicated.

Finalising schemeAll agreed on the importance of holding a stakeholder meeting to discuss (i) the technical details and (ii) the administrative aspect of the scheme with the industry, giving them the final chance to raise concerns and put forward suggestions immediately prior to scheme finalisation. Representatives from Defra Aquaculture, Salmon, Freshwater Fisheries and Whaling (ASFFW) Division should also be present at the stakeholders meeting.DVJ mentioned that he has been communicating with a number of different stakeholders already about the scheme and no major concerns had been forwarded to date Biocide manufacturing companies he had been in contact with were generally very positive about the scheme, seeing potential marketing advantages in having a product listed. They also pointed out the global marketing dimensions. Aquaculture production worldwide is a very significant sector. a Defra listing would also be very useful to them for marketing products to producers in countries other then just the UK. This has been their experience with obtaining listings on the Defra terrestrial diseases scheme. DVJ is hoping to attend the next British Association of Chemical Specialists (BACS).microbiology subgroup meeting, where he will update members about the scheme To be arranged mid-week, early Feb 2006, Defra venue (London).Defra ASFFW need to be brought up to speed with scheme developments with a view to their taking ownership and rolling it out the by the end of this FY (April 06).

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Actions:DVJ to further discuss mechanisms of scheme with ASFFW before the meeting.DVJ to find out date of BACS meeting. NA to arrange meeting room and provide preliminary briefing to ASFFW.NA agreed to give an update on the project at the CARD meeting, Nov 2005 (DVJ to forward presentation)

ParasitesAll agreed that the efficacy of disinfectants against parasites, such as Gyrodactylus salaris, is of high importance and an area that requires more research. However, due to insufficient resources, such investigations are outside the scope of this project. DF emphasised that this is an issue that will undoubtedly come up at the stakeholders meeting, for which we must have clear answers.

Suggestions for European or CARD funding for future work were proposed, along with the possibility of collaboration with Norway. Such work could also include fungi.

Collaboration with Ellen Ariel (EU Reference Laboratory) was suggested.

Concerns were raised over bringing G. salaris into the UK for this work. NS explained that G.derjaviny has previously provided an effective G. salaris model.

Actions:TH to speak to Gyrodactylus task force about this issue.TH to speak to David Wyman about the possibility of a 2007/2008 project.

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Appendix 4 Record of Meeting to Discuss Disinfectants Listing scheme. Room 5.04 in Whitehall Place London, 14:00 – 16:00,

10/8/06

Present: D. Verner – Jeffreys (DVJ; Cefas)I. Wellman (IW; Defra EDPCD)N. Auchterlonie (NA; Defra VEROD)T. Miles (TM; Defra VEROD)

The purpose of the meeting was to discuss how Defra could progress with implementation of the proposed Aquaculture disinfectant listing scheme (Project F1169).

The meeting started with DVJ summarising progress that Cefas had made in progressing the scheme, which included the following:

- Convening two workshops composed of aquaculture health professionals and other invited experts to discuss appropriate testing standards

- Testing the proposed standards in the laboratory- Developing guidelines that can be given to companies wishing to have their products listed detailing how

the tests could be carried out.- Developing Quality Assurance checklists that testing labs can use to help validate, for Defra’s benefit,

that reported tests were performed to the recommended standards- Discussing the proposed scheme with a variety of different stakeholders

The following issues were then raised at the meeting.

1. Defra needs to now implement the scheme. This will require administrative input as well as the TVA advice that TM can provide.

2. Before scheme implementation, a consultation document detailing the proposed scheme, that can be circulated to the different stakeholders for comment, needs to be prepared.

3. DVJ said he was willing to prepare a document that could then be reviewed by Defra detailing how the scheme may potentially be administered. Defra could then review this document as a starting point for producing the required consultation document for circulation to stakeholders. The process of drafting this draft consultation document could also be used to identify areas where Defra will require further advice prior to implementation of a scheme.

4. .A team within Defra will need to administer the scheme, this needs to be identified.5. The proposed scheme is different in many ways from the EDPCD administered terrestrial scheme.

a. It is proposed as a voluntary scheme (like the terrestrial scheme), but with no legislative framework. IW mentioned that various Orders require the use of an approved disinfectant. Manufacturers apply to have their products approved under the terrestrial scheme on a voluntary basis. It is the administration of the approval mechanism that is provided for by the Diseases of Animals (Approved Disinfectants) Order 1978, which is itself made under the Animal Health Act. IW suggested that a legal framework may be necessary for the administration of the aquaculture scheme and asked if legal advice had been sought.

b. Testing of disinfectants will not be done internally by Defra nominated labs, instead data will be provided to Defra, by the companies, detailing the performance of their products under the test conditions specified by Defra.

6. It was suggested that IW’s team could potentially be well placed to administer the scheme, on a cost recovery basis (even if the aquaculture disinfectant listing scheme is not linked to the terrestrial scheme). IW referred back to previous discussions about the Animal Health Act (which provides power to administer the terrestrial scheme) expressly excluding fish diseases. A ‘fish health’ team in, or coming to, the Animal Health and Welfare Directorate as identified as another potential team to administer the aquaculture scheme.

7. .DVJ noted that the administrative burden should be light. Detailed guidelines and an application forms will be prepared that can be forwarded to companies wishing to have their products listed. The completed application forms can then be forwarded directly to a list of recognised experts in the UK government fisheries laboratories for review. The experts can then decide if a particular product ahs passed the required tests and, communicate that back to Defra. Products that are demonstrated, to the external experts satisfaction, to pass required testing guidelines can then be listed.

8. To make the review process straightforward for the external experts, checklists have been prepared that the testing laboratories will need to complete as part of the reports detailing their findings.

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9. IW noted that a minimum of three months must elapse during the consultation process, thus the scheme cannot come into operation at the earliest 90 days post distribution of the consultation documents detailing the proposed scheme.

10. DVJ noted that he wishes to complete FC1169 and prepare a final report which has already had no cost extension of 4 months. Production of the consultation documents could form an appropriate point to terminate the project. Cefas would continue to assist with implementation of the scheme through their normal advice functions to Defra after this point.

11. IW also noted that she encountered a number of issues that may have direct bearing on the scheme during her attempts to amend and update the Terrestrial scheme. In particular, any attempt by Defra to charge fees may need to be approved through a Fees order.

12. In general it was recognised that the experience IW has gained in implementing changes to the terrestrial scheme may be very valuable to helping the aquaculture disinfectant scheme become established.

13. It was discussed how to ensure that recommended products on the list were not only efficacious but also safe. Following the proposed changes in the terrestrial scheme model, it is recommended that products can only be listed if they already comply with the relevant section s of The Biocidal Products Directive (BPD; 98/9/EC). Such products and their active ingredients will have been required to satisfy strict safety criteria to comply with BPD.

14. IT requirements were also briefly discussed. Defra will need a mechanism and format to list the products and their suppliers on the web.

Immediate Actions arising:1. DVJ to draft potential scope of scheme for review by TM and IW.2. TM to discuss with Richard Drummond how the scheme can be progressed (source necessary

administrative input).3. IW to forward most recent copy of consultation documents detailing proposed changes to the

terrestrial scheme (Revision of the Diseases of Animals (Approved Disinfectants) Order 1978) to DVJ. DVJ to use these documents to assist in preparation of draft consultation documents re aquaculture disinfectant scheme.

4. IW to discuss scope of scheme with her Head of Branch.

Post meeting note: In order for this work to go forward in the most effective way the correct team within Defra needs to be identified and DVJ will prepare a scoping paper to facilitate this. IW spoke with her Head of Branch who confirmed that it is more appropriate for this issue to be considered by the head of branch who will have responsibility for the terrestrial mechanism following the reorganisation of the Division; IW will arrange this when the scoping paper is received and the re-organisation is complete (new teams expected to be in place by end Sept.) so that a full discussion can be held with interested parties.

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Appendix 5 Scope of required work to implement schemeDefra Aquaculture Disinfectant Listing Scheme

IntroductionUse of safe, effective disinfectants is very important for the implementation of effective biosecurity in intensive aquaculture, as for other farming operations. Important uses include treatment of equipment (e.g. tanks, nets or transportation containers) and effluent, limiting disease spread both within and between sitesDisinfectants are also required for decontamination of premises where there have been outbreaks of notifiable diseases (e.g. Infectious Salmon Anaemia (ISA), Spring Viraemia of Carp (SVC) or Viral Haemorrhagic Septicaemia (VHS)).

Under provisions of the Animal Health Act 1981, Defra maintain a list of disinfectants that are approved for use in the control of notifiable diseases of terrestrial animals and birds. This system of approval does not extend to the pathogens of fish and shellfish.Defra is proposing to develop a new scheme that will give disinfectant manufactures the opportunity to prove that their products are effective against relevant aquaculture pathogens, as well terrestrial diseases. Products that satisfy Defra criteria will be placed on a list, which will be freely accessible to farmers and aquaculture health professionals, aiding them in the selection of effective products.The scheme will have UK-wide relevance and is being developed in consultation with representatives from FRS Marine Laboratory, Aberdeen (an agency of the Scottish Executive Environment and Rural Affairs Department), and the Department of Agriculture and Rural Development, Northern Ireland (DARD). The agencies responsible for the testing of disinfectants within the terrestrial scheme have also been consulted.Defra have funded a Cefas led R& D project (FC 1169) to develop applicable aquaculture disinfectant testing standards and to assist Defra with implementation of the scheme. The purpose of this document is to scope out the work still required for Defra to be in a position to implement the scheme.

Disinfectant Testing StandardsWe have undertaken a laboratory testing exercise, sponsored by Defra (FC 1169), to develop aquaculture disinfectant testing standards. The standards were developed in consultation with relevant experts from across the UK and Norway. (The Norwegian Medicines Authority administers an aquaculture disinfectant approvals scheme under a draft of the Biocides Directive BPD 98/8/EC.) This portion of the work is complete, with two standards recommended for demonstrating a products efficacy against bacterial pathogens (a modified EN 1656) and viral agents (a modification of prEN, substituting Infectious Pancreatic Necrosis virus IPNv for bovine enterovirus).

Guidelines for manufacturersAs part of the process to develop the testing standards, explanatory documents for forwarding to companies wishing to have their products listed have been drafted. These guidelines detail what data is required by Defra to support a product listing and also includes detailing the supporting Quality Assurance.

Further work is required, by Defra, before the scheme can be implemented. Below is presented a possible model listing scheme, including, where relevant, areas that need attention.

Suggested listing mechanism:1. A company applies to Defra to have a product listed (section within Defra to handle applications needs to

be identified)2. Defra send out application forms to the company that detail the information required by Defra to enable a

product listing (these need to be drafted- draft forms are attached).3. The company completes these forms and returns them to Defra.4. The company also pays Defra a fee to cover the costs of handling their application (these costs need to

be determined).5. Unlike the terrestrial scheme, companies do not send products to Defra to test. Instead, companies need

to supply the data required to support of a listing. The application form will detail the tests that need to be carried out and how the results should be reported. Also included will be a detailed Quality Assurance statement that needs to be completed by the laboratory that undertook the testing confirming that the tests were done as specified by the relevant testing standard.

6. Defra reviews the returned documentation. After firstly checking that the form has been correctly completed, the technical aspects of the application form are then reviewed by experts identified by Defra

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(an outcome of the technical workshops held under FC1169 was to identify a panel of experts in UK government fish health laboratories that would be competent to review application forms).

7. If the application form passes Defra scrutiny, the product can then be listed.8. Listing entails both Defra writing to the company confirming their product, at the specified dilutions, is

efficacious against bacterial and viral pathogens and listing the product on the Defra website.9. The listing will detail the product name, the address of the manufacturer and the dilution rates effective

against (i) bacterial pathogens of aquaculture relevance ii) viral agents of aquaculture relevance.10. It is proposed that supplementary information can also be added, at the manufacturer’s discretion, about

the products efficacy against other aquaculture pathogens and under different contact times and temperature conditions than those specified in the obligatory tests. Defra recommends that suitable modifications of the recommended bacterial (EN1656) and viral (14675) standards used for the mandatory testing be employed with manufacturers strongly recommended to consult with Defra as to what tests they wish to employ before completing the application form. (Defra will refer such queries to the appropriate Aquaculture health experts for comment)

11. Listing will be subject to certain conditions, for example, the composition, manufacturer and plant must remain unchanged from the time of testing and when tested, the product must be efficacious for use under the testing standard for which it is listed. A change in these conditions results in the revocation of listing for the product and its trade name products. It is proposed that a listing will be granted for a period of 2 years. At the end of each 2-year period manufacturers will be required to reconfirm compliance with the conditions of the listing

12. Defra reserves the right to check test products to ensure that marketed products efficacious for use under the testing standard for which they are listed.

Various actions are required before such a scheme can be implemented:

1. A unit within Defra needs to be identified to develop the listing scheme2. A unit within Defra needs to be identified who will administer the scheme once it is established3. Applications forms need to be drafted for forwarding to companies wishing to have their products

listed, these will probably need to detail (suggestion):a. the product to be listed

i. physical form under which the disinfectant is to be tested and soldii. product composition including percentage of each ingredient and CAS numbers for

active ingredient b. MSDS for productc. Statement from the manufacturer that the product is compliant with the requirements of

Biocidal Products Directive and Regulationsd. Listing applied for (control of bacterial diseases and/or control of viral diseases) –mandatory

testing criteriae. Other supplementary information about the performance of dilutions of the product against

other aquaculture pathogens and/or under different contact times and temperatures (using appropriate modifications of EN1656 and EN14675 as appropriate).-optional testing criteria.

f. The dilution rates for the respective listingsg. Forms for filling out by the laboratory commissioned to test the products detailing the

products performance when tested using the recommended standards (modified EN 1656 and EN14675)

h. The QA checklists that needs to be completed and signed by the testing laboratories asserting that the testing was done as recommended by the relevant testing standards.

4. Fees need to be set to allow those administering the scheme to recover their costs.5. Before the scheme can be implemented there will likely need to be a consultation period with

interested parties. This will require preparation of consultation documents detailing the proposed scheme to interested parties (including manufacturers, other interested governmental bodies, farmers and aquaculture health professionals). It is my understanding that Cabinet Office guidelines recommend a minimum of 12 weeks for written consultation should be allowed at least once during development of policy. (Defra needs to determine whether a ‘policy’ being developed here).

6. There may be a requirement for a regulatory assessment exercise to be carried out 7. Relevant government departments include the devolved administrations in Scotland, Northern Ireland

and Wales, the agencies responsible for discharge of chemicals into watercourses in the UK (Environment Agency, SEPA and xx), as well as the Health and Safety Executive. All these bodies have been apprised of the scheme during its conception.

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Appendix 6 Guideline for listing a product as effective against bacterial diseases of aquaculture relevance.

In support of listing a product dilution as being effective against bacterial diseases of aquaculture relevance, the product shall be tested using a modification of EN standard 1656, (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field) as described below. The results, demonstrating that the reported effective dilution passed the aforementioned test, shall then be reported to Defra.

Modifications of EN1656 to be followed1. The test organisms recommended in the standard (Section 5.2.1. and all references thereafter) should be

substituted for: Aeromonas salmonicida subsp. salmonicida (ATCC 14174), Yersinia ruckeri (ATCC 29473), Carnobacterium piscicola (ATCC 35586) and Lactococcus garvieae (NCIMB 702927).

2. The recommended temperature at which test plates are incubated prior to enumeration of viable colonies shall be changed from 36 or 37°C to 22°C 1°C (Section 5.3.2.3 and all references thereafter). Alternative incubation temperatures may also be selected, although in this case validation data should accompany the test result, justifying the employment of the alternative incubation temperature, for review by Defra.

3. Choice of experimental conditions (Section 5.5.1) mandatory conditions:a. For all dilutions, the temperature to be tested is 4 °C 1 °C (not 10 °C)b. For all dilutions, the contact time to be tested is 30 min 10 sec *c. For all dilutions, the Interfering substance to be tested is as described for dirty conditions (10g/l

yeast extract plus 10g/l bovine serum albumin solution) 4. Optional data (Section 5.5.1)

a. The product performance at 20 °C can also be reported to Defra.b. The product performance at a contact time of 5 minutes 10 sec * can also be reported to Defra.

5. * Although the standard specifies contact times should be 10 sec, and testing laboratories should endeavour to adhere to this requirement, reported deviations of 30 sec will not be regarded as an invalidation of the reported test results. Deviations > 30 seconds will invalidate a reported test result.

Quality Assurance:1. Testing Laboratory. The laboratory performing the testing shall adhere to a recognised International

laboratory quality system (e.g. ISO 17025, Good Laboratory Practice or Good Manufacturing Practice).2. Audited data. In support of the product dilution that is claimed to be effective against aquaculture

pathogens, test results shall be accompanied by an audit report affirming that the test was done to the conditions specified in the standard. To assist this process, a Quality Assurance checklist is supplied with this guideline ( Appendix 2).

ReportingTest results shall be reported to Defra as suggested (Section 5.8), with the dilution that the company concerned wishes to report as being an effective concentration, and one other concentration, shown to pass the test under the recommended conditions in the test report (demonstrate a 105 or more reduction in viability within 30 min at 4 °C in the presence of 10g/l yeast extract plus 10g/l bovine serum albumin solution) for all four of the test organisms (Section 1 above). Test results for at least one concentration that failed the test should also to be included. It should be noted that failure to report results using the format suggested (EN 1656 Section 5.8), including the results of required validation exercises, may result in the application not being accepted by Defra.

Listing.Defra will list on their website:

the product concerned the dilution that the company wishes to have listed as being effective against aquaculture relevant

bacterial pathogens under the mandatory testing conditions -other concentrations reported to Defra as being effective against aquaculture relevant bacterial

pathogens, under the mandatory testing conditions -Dilutions of the product demonstrated to be effective against aquaculture relevant bacterial pathogens

under the optional testing conditions -Links to product relevant information supplied by the manufacturers (note, this will include a disclaimer

emphasizing that supplying a Link through the Defra website does not mean Defra approves or condones any claims made by the site referred to).

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Appendix 7 Modified EN 1656 Quality Assurance Checklist

Quality Assurance Checklist for Modification of EN1656 (Evaluation of Bactericidal activity of chemical disinfectants and antiseptics used in veterinary field).

Reference RequirementSatisfactory

CommentY NExperimental conditions:EN 1656 5.5 modification 3a

Temperature to be tested is 4oC +/- 1oC

EN 1656 5.5 modification 4a

Optional additional performance data at 20oC may be reported

EN 1656 5.5 modification 5

Contact time to be tested is 30 min +/- 30 secs

EN 1656 5.5 modification 4b

Optional data for 5 minute contact time may be reported

EN 1656 5.5 modification 3a

Test suspension maintained at 4oC+/-1oC

EN1656 modification 3c Interfering substance as for high level soiling (10g/l yeast extract+10g/l bovine albumin) used for all dilutions

Test Organisms:

EN1656 modification 1

A. salmonicida ATCC 14174Y. ruckeri ATCC29473Carnobacterium piscicola ATCC 29473Lactococcus garviae (NCIMB 702927)(Stored frozen at –80 on cryopreservative beads)

EN16565.4.1.2

24 hour second or third subcultures from the stock culture must be used. A third subculture may be prepared from a 48 hour second subculture if necessary.

EN16565.4.1.3

Cell suspensions prepared to between 1.5 x 108 and 5 x 108 cfu/ml

EN16565.4.1.3

Prepared suspensions maintained in waterbath at 20oC +/- 1oC and used within 2 hours

EN1656 modification 2 Dilutions prepared to 10-6 and 10-7 for counting and pour plates incubated at 22oC +/-1oC

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CommentY NTest substance:Nominal Active Ingredient concentrationBatch NumberManufacture DateLegal StatusExpiry DateStorageHandling and SafetyEN16565.4.2

Product test solutions prepared freshly in standardized hard water and used within 60 minutes.

Culture Media and Reagents:EN16565.2.2.1

Reagents appropriate for microbiological purposes

EN16565.2.2.2

Water either glass distilled or adhering to criteria water for injectable preparations (European Pharmacopoeia)

EN16565.2.2.3

Media used for maintenance and viable counts is Tryptone soy agar

EN16565.2.2.4

Diluent used is Tryptone Sodium Chloride solution.

EN16565.2.2.5

Neutralizer validated in accordance with annex A

EN16565.2.2.7

Standardized Hard water freshly prepared on the day

Test procedureValidation of dilution-neutralization method

Done for each test strain according to EN1656:2000 annex A

EN1656:2000 annex A c) (modification 3a)

1 ml interfering substance, 1 ml diluent and 8 ml strongest product dilution placed in sterile tube and transferred to waterbath at 4oC

EN1656:2000 annex A c)

1ml of the mixture placed into tube containing 8 ml neutralizer in waterbath at 20oC and left for 5 min +/- 10 secs

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CommentY NEN1656:2000 annex A c)

Add 1 ml 10-5 dilution of bacterial suspension and immediately start stopwatch


Vortex and return to 20oC water bath for 30min +/- 1 min.


Vortex immediately before end of contact time


Take 1 ml of sample in duplicate and prepare plates in duplicate using pour plate method.

EN1656:2000 annex A modification 2

Incubate at 22 +/1o C for 24 hours

EN1656:2000 annex A modification 2

Count then Incubate at 22 +/1o C for a further 24 hours and re-count.


Use highest number of cfu/plate to calculate viable count using method given in EN16565.6.1.2

Dilution-Neutralization MethodEN16565.5.2.2.2

Equilibrate water and neutraliser to 20oC and all other reagents to test temperature

EN16565.5.2.2.2

Pipette 1ml interfering substance and 1ml bacterial suspension into test tube

EN16565.5.2.2.2

Avoid touching upper part of test tube sides when adding bacteria

EN16565.5.2.2.2

Immediately start stopwatch, mix and place in water bath at 4oC+/1oC

EN16565.5.2.2.2

After 2mins+/- 10 secs add 8ml of product test solution

EN16565.5.2.2.2

Restart stopwatch, mix and return to waterbath for 30 mins +/- 30 secs

EN16565.5.2.2.2

Mix just before end of contact time

EN16565.5.2.2.2

At end of contact time pipette 1ml mixture into tube containing 8 ml neutralizer and 1 ml water in a waterbath at 20oC

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CommentY NEN16565.5.2.2.2

After 5 mins +/-10s transfer 2 x 1ml neutralized mixture into Petri dishes

EN16565.5.2.2.2

Quickly add 12 – 15 ml melted TSA cooled to 45oC+/-1oC (in exceptional cases neutralizer can be added to TSA)

EN16565.5.2.2.3

Incubate at 22oC+/-1oC for 24 hours

EN16565.5.2.2.3

Determine cfu for each plate and incubate for a further 24 hours

EN16565.5.2.2.3

Recount only plates showing well separated colonies

EN16565.5.2.2.3

Using highest number of colonies for each plate calculate cfu/ml in test mixture according to EN1656 5.6.1.2

Calculation and expression of results:EN1656 5.6.1.1 Only colony counts of

less than 300 to be used for calculation of viable count

EN1656 5.6.1.1 At least one of each plate set must have more than 15 colonies

EN1656 5.6.3 For each organism and product test concentration reduction in viability calculated using formula given in EN1656 5.6.3

Conclusion and test report

The product is deemed to have passed the test if it demonstrates a 105 or more reduction in viability within 30 min or less using the specified organisms and test conditions

EN1656 5.8 The test report shall refer to the EN1656 standard and shall include the information specified at EN1656 5.8 using the format suggested in that annex.

Complete all boxes as appropriate and add full comments for any non-satisfactory marking.

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Note, if the membrane filtration, rather then neutralization dilution, method is employed the checklist shall be modified as appropriate to show the additional required steps and test procedures and all references to neutralization dilution marked N/A (not appropriate).

Any operation not included in the standard as well as any incident which may affect the results shall be reported in full.

Test conducted on: (Date) By: (Tester signature)

Technical Manager signature: Date:

Quality Assurance Statement

This test was/was not conducted in accordance with the conditions specified in the standard/guidelines attached.

Quality Assurance Auditor signature: Date:

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APPENDIX 8

Listing a product as effective against viral diseases of aquaculture relevance.

In support of listing a product dilution as being effective against viral diseases of aquaculture relevance, the product shall be tested using a modification of EN standard 14675, (Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field) as described below. The results, demonstrating that the reported effective dilution passed the aforementioned test, shall then be reported to Defra.

Modifications of EN14675 to be followed

1. The test organisms recommended in the standard (Section 4/5.2.1 and all references thereafter) should be substituted for: Infectious pancreatic necrosis virus (IPNV), serogroup A2, Spjarup (SP) isolate (ATCC).

2. Choice of experimental conditions (Section 5.5.1.1) mandatory conditions:a. For all dilutions, the temperature to be tested is 4 °C 1 °C (not 10 °C)

b. For all dilutions, the contact time to be tested is 30 min 10 sec *c. For all dilutions, the Interfering substance to be tested is as described for dirty conditions (10g/l

yeast extract plus 10g/l bovine serum albumin solution)

* Although the standard specifies contact times should be 10 sec, and testing laboratories should endeavour to adhere to this requirement, reported deviations of 30 sec will not be regarded as an invalidation of the reported test results. Deviations > 30 seconds will invalidate a reported test result.

As stated in the standard, additional or alternative specific virucidal activity can be determined applying other contact times, temperatures and test organisms in accordance with 5.2 and 5.5.1.1 in order to take into account intended specific use conditions, although in this case validation data should accompany the test result, justifying the employment of the alternative conditions, for review by Defra.

. Section 5.5.1.2. As routine production of > 107.5 TCID50 IPNV can be problematical, lower concentrations can be used in the assay providing a 4 log reduction can be derived from the results.

3. The preparation of hard water should be amended if ANALAR grade chemicals are used.

4. MDBK cells, used to multiply the virus (Section 5.5.1.2), should be replaced by Chinook salmon embryo (CHSE-214) cells or alternative cell lines dependent on the virus to be tested.

5. Cells may be attached to the plate as a confluent monolayer of cells rather than in suspension (Section 5.5.1.3). If this is the case cell culture techniques may differ.

6. Virus titrations/dilutions may be made on the microtitre plate if plates contain a monolayer of cells (rather than in separate containers then transferred to the plate as in section 5.6). When this is the case the neutralising dilutions will not be made in ice cold diluent (Section 5.1) and the time between removing the test mix from the water bath and neutralising dilution should be 30s

7. Titrations may be made with 6 wells per dilution (Standard states 8 wells per dilution section 5.6.2 and 5.8).

8. Dilution is used to stop the action of the test product therefore a control test is needed to confirm this works. Dilute the product 1/10 and 1/100 then add the virus for 30 minutes at 4 °C before titration. The titre of the virus should not decrease as the product should have been inactivated by the dilution before the virus is added. If the product fails this test alternative neutralisers will have to be tested.

9. Titration of test virus suspension (Annex A.3) states ‘1 ml of hard water is to be used instead of the product test solution’. 8 ml of product test solution is used throughout the tests therefore it should be 8 ml used in this test.

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10. The test virus reference inactivation test using formic acid (Annex A.2) is not necessary. Delete also 3.9 (under terms and definitions).

11. Treatment of cells (Annex A.1) in the standard the control uses PBS but maintenance medium should be used.

12. The level of CPE recorded (section 6.1) is not necessary, only record + if CPE is present or – if no CPE.

13. Test virus suspension shall not be less than 107.5 TCID50 per ml? I used lower and you could still read it

14. 5.3.2.3 substitute 15 C 1C for 37 C (temperature for incubating CHSE-214 cells and all other references to incubating CHSE-214 cells).

15. 5.3.2.11 Suggest 6-well multiplates are used here instead of petri dishes

16. 5.6.3 Plaque assay. Substitute 20 C 1C for virus absorption temperature and 15 C for all subsequent incubation temperature. Low gelling temperature agarose should also be used. NBF can be used for fixation.

If any problems arise following this standard (e.g. cell cytotoxicity) Defra should be contacted for advice.

Quality Assurance:

17. Testing Laboratory. The laboratory performing the testing shall adhere to a recognised International laboratory quality system (ISO 17025, Good Laboratory Practice or Good Manufacturing Practice).

18. Audited data. In support of the product dilution that is claimed to be effective against aquaculture pathogens, test results shall be accompanied by an audit report affirming that the test was done to the conditions specified in the standard. To assist this process, a Quality Assurance checklist is supplied with this guideline.

ReportingTest results shall be reported to Defra as suggested (Section 5.8), with the dilution that the company concerned wishes to report as being an effective concentration, and one other concentration, shown to pass the test under the recommended conditions in the test report (demonstrate a 104 or more reduction in viability within 30 min at 4 °C in the presence of 10g/l yeast extract plus 10g/l bovine serum albumin solution) for all four of the test organisms (Section 1 above). Test results for at least one concentration that failed the test are also to be included.

Listing.Defra will list on their website:

The product concerned The dilution that the company wishes to have listed as being effective against aquaculture relevant viral

pathogens under the mandatory testing conditions -Other concentrations reported to Defra as being effective against aquaculture relevant viral pathogens,

under the mandatory testing conditions -Dilutions of the product demonstrated to be effective against aquaculture relevant viral pathogens under

the optional testing conditions

PRODUCT: Reference Requirement Satisfactory CommentName Y NNominal Active Ingredient concentrationBatch NumberManufacture DateLegal StatusExpiry DateStorage

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PRODUCT: Reference Requirement Satisfactory CommentHandling and SafetyConcentration PrEN

14675:2003 draft

Shall be 1.25 times the desired test concentration

Test solutions PrEN 14675:2003 draft

Prepared freshly in Hard water (water for ready to use product).

Test solutions PrEN 14675:2003 draft

Used in the test within 2 hours

Experimental conditions:Temperature P0360 4oCContact time P0360 30 minutes +-30secsInterfering substance(High level soiling only)

P0360 10g/l bovine albumin plus 10g/l yeast extract

Test organism P0360 IPNV A2 SparjupAdditional test organismWater (for culture media)

Produced by reverse osmosis

Hard water (for dilution of product)

P0360 ANALAR

Cell culture medium P0360 CHSE-214Preparation of Microtitre plates

P0360 Cell suspensions prepared by trypsinising with.25% trypsin (EDTA) solution

Preparation of Microtitre plates

P0360 Cells split at 1:3 ratio in L-15 +15%FCS to enable formation of monolayer in two days

Viral suspension dilutions/titration

Done in the plate, 6 wells per dilution

Pre-test for cytotoxicity PrEN 14675:2003 A.1 Solutions prepared as for

the test using water instead of virus and inoculated into monolayer cell cultures

PrEN 14675:2003 A.1 Comparative virus titrations performed on cells that have not been treated with disinfectants

PrEN 14675:2003 A.1 Only dilutions showing a low degree of cell destructions or produce virus titre reduction log<0.5 used for determination of residual infectivityRecord +ve results as + and wells without CPE as -

Test MethodP0360 10.0 Vortex just before end of

contact timePrEN 14675:2003 draft

5.1 0.5ml virus/disinfectant taken after 30 minutes (+/- 30 secs.?) contact time

P0360 10.0 Add .5ml of test mixture to 4.5ml MEM + 5% FCS at 4+-1oC

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PRODUCT: Reference Requirement Satisfactory CommentP0360 10.0 Prepare dilutions to 10-8 in

MEM + 2%FCS and keep and 4oC

P0360 10.0 >= 6 wells of confluent monolayer of cells in micro titre plates inoculated with each dilution

P0360 10.0 Titre of infectivity calculated after incubation by formula described by Karber 1931

VerificationPrEN 14675:2003 draft

6.4 a) Test is valid if log reduction of 4 after treatment

PrEN 14675:2003 draft

6.4 b) Difference between logarithmic titre of the virus control minus the logarithmic titre of the virus in the reference inactivation test is between log –2.5 and log – 2.5 after 30 minutes


6.4 c) The cytotoxicity of the product solution does not affect cell morphology or virus susceptibility so as to compromise demonstration of log 4 reduction in virus titre


6.4 d) Comparative virus titration on cells inoculated with test mixture or virus dilutions result in a difference of log< 1 of virus titre


7 Product deemed to have passed if demonstrates log 4 or more reduction in titre within 30 minutes at 4oC under the defined test conditions

Reporting of ResultsPrEN 14675:2003 draft

6.6 Results tabulated as raw data and also expressed as negative logarithmic values of TCID50


6.6 No virus multiplication in highest concentration expressed as < and virus multiplication in all dilutions expressed as >

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PRODUCT: Reference Requirement Satisfactory CommentThe report shall include:

a) ID of laboratory

b) Identification of the sample :name of the product ;batch number and - if available - expiry date; manufacturer ;date of delivery ;storage conditions ;product diluent recommended by the manufacturer for use ; active substance(s) and its/their concentration(s) (optional) ;appearance of the productc) Experimental conditions :date(s) of test (period of analysis);diluent used for product test solution;product test concentrations;appearance of product dilutions;contact times ;test temperatures ;diluent to stop action of product.d) Test results :validation of test results ;titre of virus suspension ;maximum detectable virus inactivation ;virus inactivation of the reference virus inactivation test after 30 minutes.e) Conclusionf)Locality, date and identified signature

Complete all boxes as appropriate and add full comments for any non-satisfactory marking.

Any operation not included in the standard, as well as any incident which may affect the results, shall be reported in full.

Test conducted on: (Date) By: (Tester signature)

Technical Manager signature: Date:

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

(1) Department for Environment Food and Rural affairs. Explanatory note on the approval of disinfectants for the purposes of the animal health act 1981.

(2) Alderman, D. (1985) Iodophore disinfectants: a code of practice for their use in fish farming. Ministry of agriculture Fisheries and Food Directorate of Fisheries Research Fisheries Notice Number 74 (8pp)

(3) Finlay, J. (1978) Disinfectants in Fish Farming. Ministry of agriculture Fisheries and Food Directorate of Fisheries Research Fisheries Notice Number 59 (7pp)

(4) Torgersen I (1998) Physical and chemical inactivation of the infectious salmon anaemia (ISA) virus. pp. 44-53, In: Workshop on ISA. Annex 5. St Andrews, New Brunswick, 1998

(5) Anon. (2002) Final Report of the Joint Industry Working Group on ISA. FRS marine laboratory Aberdeen. 136p.

(6) Fisheries Research Services, Marine Laboratory Aberdeen. 2000 Field guide for disinfection with regard to ISA virus, version 2. Aberdeen Fisheries Research Services, 7pp

(7) Ahne W. (1982) Vergleichende Untersuchungen über die Stabilität von vier fischpathogen Viren (VHSV, PRF, SVCV, IPNV) Zentralbl. Veterinärmed. (B) 29:457-476.

(8) Amend DF & Pietsch JP (1972) Virucidal activity of two iodophors to salmonid viruses. J. Fish. res. Bd. Canada 29:61-65.

(9) Dorson M & Michel C. (1987) Evaluation de l'efficacite de cinq ammoniums quaternaires sur les principaux virus et bacteries pathogenes pour les salmonides. Bull. Fr. Peche Piscic. 305:61-66.

(10) Wedemeyer, GA, Nelson NC, Smith CA (1978) Survival of the salmonid viruses infectious hematopoietic necrosis (IHNV) and infectious pancreatic necrosis (IPNV) in ozonated, chorinated and untreated waters. J. Fish. Res. Bd Can. 35(6). 875-879.

(11) Elliott DG & Amend DF (1978) Efficacy of certain disinfectants against infectious pancreatic necrosis virus. J. Fish Biol. 12:277-286.

(12) Antec website http://www.antecint.co.uk/main/virkaqua.htm(13) Alpharma Animal Health / AKZO Nobel (Chloramine T trade info)(14) Kasai H, Ishikawa A, Hori Y, Watanabe K & Yoshimizu M (2000) Disinfectant effects of

electrolyzed salt water on fish pathogenic bacteria and viruses. Nippon Suisan Gakkaishi 66 (6) 1020-1025.

(15) Smail DA, Huntly PJ & Munro ALS (1993) Fate of four fish pathogens after exposure to fish silage containing fish farm mortalities and conditions for the inactivation of infectious pancreatic necrosis virus. Aquaculture 113, 173-181.

(16) Pascho RJ, Landolt ML & Ongerth JE (1995) Inactivation of Renibacterium salmoninarum by free chlorine. Aquaculture 131, 165-175.

(17) FRS Marine Laboratory (2002). Ref no. FC1183 ROAME Final Report 5pp(18) Smail DA, Grant R, Simpson D, Bain N & Hastings TS (2004) Disinfectants against

cultured infectious salmon anaemia (ISA) virus: the virucidal effect of 3 iodophors, chloramine T, chlorine dioxide and peracetic acid/hydrogen peroxide/acetic acid mixture. Aquaculture 240, 29-38.

(19) Silcox, A. (2000) Disinfectants in aquaculture. CEFAS Aston University Professional Training Year Final Report 167pp

SID 5 (Rev. 3/06) of 58

http://www.antecint.co.uk/main/virkaqua.htm

(20) Holden L.J. (2002) Investigations into the effect of organic material on the efficacy of disinfectants commonly used in aquaculture. CEFAS Aston University Professional Training Year Final Report 84pp

(21) Dunbar, S. (2002) the use of disinfectants in aquaculture. CEFAS Aston University Professional Training Year Final Report 93pp

(22) BS EN 14675 chemical disinfectants and antiseptics – quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field – Test method and requirements (phase 2, step 1).

(23) Anon PR(24) Anon EN 14349 chemical disinfectants and antiseptics – quantitative suspension test for the

evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field on non-porous surface without mechanical action – Test method and requirements (phase 2, step 1).

(25) Anon BS EN 14675 chemical disinfectants and antiseptics – quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field – Test method and requirements (phase 2, step 1).

(26) Anon EN1656 chemical disinfectants and antiseptics – quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field – Test method and requirements (phase 2, step 1).(26)

(27) Anon EN 1040:1997 Chemical disinfectants and antiseptics – basic bactericidal activity – test method and requirements (phase 1)

(28) Norwegian Regulations 20th February 1997 No. 194 concerning the cleaning and disinfection of aquaculture sites etc.

(29) Guidelines for Distributors of Disinfectants for Technical Use in Aquaculture Systems etc. Prepared by Norwegian Medicines Agency and Norwegian National Veterinary Institute (NVI) 30pp.

(30) Anon. (2003) The Report of the aquaculture Health Joint Working Group on Infectious Pancreatic Necrosis in Scotland. FRS marine laboratory Aberdeen. 90p

(31) Barnes, A.C., Hastings, T.S. and Amyes, S.G.B. (1995) Aquaculture antibacterials are antagonized by seawater cations. J. Fish Diseases 18, 463-465.

(32) Scottish Executive (1998) Final Report of the Joint Government/Industry Working Group on infectious Salmon Anaemia (ISA) in Scotland.

Presentations

Verner – Jeffreys, D. W., Dixon, P. F. Graham, D.A. Fraser and Smail, D.A. (2005) Use of ‘approved’ disinfectants in UK aquaculture. Presentation given at European Association of Fish Pathologists Conference Copenhagen. Also presented to British Association of Chemical Specialists and Defra chaired Committee for Aquaculture Health

Publications

Verner – Jeffreys, D. W. and Ridout, N.J (2006) Regulations governing the use of disinfectants in UK aquaculture and the development of a Defra aquaculture disinfectants listing scheme. Finfish News 1, 22-25

Further publications in the aquaculture trade press and a peer-reviewed publication are planned.

SID 5 (Rev. 3/06) of 58

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