Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 1.1.5.

METHODS FOR DISINFECTION OF
AQUACULTURE ESTABLISHMENTS

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A. METHODS FOR DISINFECTION OF FISH FARMS

1.	General Principles
	The choice of disinfection procedures depends on the size, type and nature of the materials and sites to be disinfected. With the exception of the skin of personnel and eggs, which must be disinfected with non-corrosive products, the surfaces to be disinfected consist of fabric or woven material (clothes, nets), hard surfaces (plastic, cement) or permeable materials (earth, gravel). Disinfection is more difficult for permeable surfaces and requires more time. Table 1 indicates the most common ingredients and the methods to be used on the basis of these criteria.
	The use of chemical products entails the implementation of measures to protect personnel and the cultured animals and to mitigate environmental effects. It is first necessary to protect the skin and eyes from contact with dangerous substances by using impermeable clothing, boots, eye protection and a hat. The respiratory tract must be protected by a mask and the operator must not touch any food without having thoroughly washed his/her hands. Finally, the products must be stored in such a way as not to present direct or indirect danger to animal/fish or human life or the environment.
	The material must be thoroughly cleaned before being disinfected. Organic material generated/ removed during the cleaning process, such as pond sludge, etc., should also be disposed of in an appropriate manner that prevents spread of disease by such material and is environmentally safe.
	Ideally, approved procedures for the use of disinfectants in aquaculture should be established. An approval scheme should consider disinfection effect against target pathogens, toxicological and ecotoxicological properties of the products.
	Following disinfection or stamping-out, the farm should be restocked from a disease-free source.

TABLE 1. DISINFECTION AND METHOD OF USE

Processes	Indications	Method of use*	Comments
Physical
Desiccation, sunlight	Fish pathogens on earthen bottoms	Dry for 3 months at an average temperature of 18°C	Drying period can be reduced by the use of a chemical disinfectant
Dry heat	Fish pathogens on concrete, stone, iron, ceramic surfaces	Flame-blower, blow-lamp
Damp heat	Fish pathogens in transportation vehicle tanks	Steam at 100°C or more for 5 minutes
Ultra-violet rays UV-C (254 nm)	Viruses and bacteria	10 mJ/cm2	Minimum lethal dose
Ultra-violet rays UV-C (254 nm)	Infectious pancreatic necrosis (IPN) and nodavirus (VNN/VER [viral nervous necrosis/viral encephalopathy and retinopathy]) in water	125-200 mJ/cm2
Chemical
Acetic acid	Infectious salmon anaemia (ISA)	0.04-0.13%
Quartenary ammonia	Virus, bacteria, hands, plastic surfaces	0.1-1 g/litre for 1-15 minutes	IPN virus resistant
Calcium oxidea	Fish pathogens on dried earth-base	0.5 kg/m2 for 4 weeks	Replace in water and empty disinfected pools keeping the effluents at pH <8.5
Calcium hypochloritea	Bacteria and viruses on all clean surfaces and in water	30 mg available chlorine/litre. Leave to inactivate for several days or neutralise with Na thiosulfate after 3 hours	Can be neutralised with sodium thiosulfate. See special recommendations
Calcium cyanamidea	Spores on earthen bottoms	3000 kg/ha on dry surfaces; leave in contact for 1 month
Chloramine T	Destroys ISA	1% for 5 minutes
Chloramine T	Destroys IPN	1% for 30 minutes
Chlorine dioxide	ISA	100 ppm for 5 minutes	In water of low organic loading
Formic acid	Ensile fish waste	pH <4 after at least 24 hours	Destroys bacterial fish pathogens and ISA butnot IPN
Formalin	Fish pathogens in sealed premises	Released from formogenic substances, generally trioxymethylene. Comply with instructions	Nodavirus resistant
Hydrogen peroxide	ISA virus	0.02-0.06%
Iodine (iodophors)	Bacteria, viruses on nets, boots and clothing	200 mg iodine/litre for a few seconds	See special recommendations
Iodine (iodophors)	Hands, smooth surfaces	>200 mg iodine/litre a few seconds

 

TABLE 1 (CONTINUED). DISINFECTION AND METHOD OF USE

Processes	Indications	Method of use*	Comments
Chemical
Ozone	Sterilisation of water, fish pathogens	0.2-1 mg/litre for 3 minutes	Costly and very toxic for fish and humans
Ozone in seawater	Surfaces, equipment	0.5-1 mg/litre TRO (total residual oxidant ) for 30-60 minutes
Poroxy compounds, e.g. Virkon	IPN virus	1% for 1 minute
Peracetic acid	ISA virus	0.08-0.25%
Sodium hydroxidea	Fish pathogens on resistant surfaces with cracks	Mixture: Sodium hydroxide, 100 g Teepol®, 10 g Calcium hydroxide, 500 g Water, 10 litres Spray, 1 litre/10 m2 Leave for 48 hours	The most active disinfectant Ca(OH)2 stains the surfaces treated; Teepol® is a tensio-active agent.
Sodium hypochloritea	Bacteria and viruses on all clean surfaces and in water	30 mg available chlorine/litre. Leave to inactivate for a few days or neutralise with Na thiosulfate after 3 hours
Sodium hypochloritea	Nets, boots and clothing	200 mg to 1 g available chlorine/litre for several minutes. Leave to inactivate for a few days or neutralise with Na thiosulfate after 3 hours
Sodium hypochloritea	Hands	Rinse with clean water or neutralise with thiosulfate

 

a	Dangerous - See precautions indicated in general recommendations
*	The concentrations indicated are those for the active substance. NB: The chemicals must be approved for the prescribed use and used according to the manufacturer's specifications.
2.	Neutralisation of Halogens
	Chlorine and iodine are highly toxic for aquatic animals and, in order to prevent serious accidents that could result from a manipulation error, it is recommended to neutralise these products with sodium thiosulfate - five moles of thiosulfate neutralise four moles of chlorine. The molecular proportions are the same for iodine.
	Accordingly, in order to inactivate chlorine, the amount of thiosulfate should be 2.85 times the amount of chlorine (in grams):
		Number of grams of thiosulfate = 2.85 x number of grams of chlorine.
	For iodine, the amount of thiosulfate should be 0.78 times the amount of iodine in grams:
		Number of grams of thiosulfate = 0.78 x number of grams of iodine.
	It is also possible to prepare a thiosulfate solution at 1% by weight, in which case the neutralising volumes will be as follows (in ml):
	1.	for chlorine:
		28.5 x [number of litres of the disinfecting solution x concentration mg/litre] / 100
	2.	for iodine:
		it is necessary to multiply by 7.8 instead of by 28.5.

B. METHODS FOR DISINFECTION OF MOLLUSCS FARMS

1.	General Principles
	The general principles pertaining to disinfection of mollusc farms (hatcheries, holding facilities) involve the application of chemical treatments in sufficient concentrations, and for sufficient periods, to kill all pathogenic organisms that would otherwise gain access to surrounding water systems. As the inherent toxicity of disinfectants prohibits safe use in open water, or open water systems, disinfection can only reasonably be applied to hatcheries and tank holding facilities, and, as a rule, all disinfectants must be neutralised before release into the surrounding environment. In addition, as mollusc farms are generally seawater based, compounds produced during seawater disinfection (residual oxidants) must also be disposed of carefully.
	Disinfection of eggs and larval stages is not considered practical for most molluscan systems. In addition, there is little information on specific disinfection procedures for pathogens of molluscs (i.e. Marteilia spp., Haplosporidium spp., Bonamia spp., Perkinsus spp., iridovirus and pathogenic levels of marine microbes) or seawater. Therefore, disinfectants and concentrations are based on related pathogens or seawater sterilisation. Three stages of disinfection can be applied to hatcheries:
	a)	pretreatment of influent water, e.g. filters (1.0 and 0.22 µm) or chemical disinfection (see Section B.2.) = protection of stocks of molluscs;
	b)	treatment within the facilities (especially recycling systems) = protection of stocks of molluscs;
	c)	treatment of effluent water = protection of the environment.
2.	Disinfectants* - Pipelines and Tanks
	(*The products specified have proven satisfactory for the purposes indicated; this does not mean that other products may not be equally satisfactory.)
	Routine disinfection of pipelines and tanks is highly recommended; the frequency of disinfection will vary according to the turnover of stocks of molluscs. High concentrations of molluscs should be rotated between disinfected tanks as often as practical and/or kept in seawater that has been disinfected with ozone (see Section B.3.a) or chlorine (see Section B.3.b) and subsequently neutralised. Each new batch of molluscs introduced into a facility should be placed in predisinfected tanks.
	As the presence of organic matter will reduce the disinfection capacity of most disinfectants, filtering influent water (see Section B.1.a) is recommended. In addition, all surfaces must be thoroughly cleaned prior to disinfection. The detergent used must be compatible with the disinfectant and both must be compatible with the surface being treated (e.g. iodophor solutions are generally acidic so cannot be used on concrete, which is alkaline). Ensure that the waste produced from washing is disinfected before disposal. Complete coverage of the surfaces is required, e.g. using a high pressure spray or soak. Wear appropriate protective clothing when working with any disinfectant (see Section A.1).
	Regular air- or heat-drying of pipelines (daily), tanks and other equipment (e.g. algal culture carboys), in addition to disinfection of their surfaces, is also recommended (especially for disease outbreaks of unidentified aetiology).
	a)	Chlorine is usually applied as sodium hypochlorite (Chlorox®, household bleach, etc.). Fill all pipelines with 50 mg chlorine/litre (= 50 parts per million [ppm]). Allow an exposure time of at least 30 minutes before flushing with clean seawater. This solution is effective against most microbial agents as well as labyrinthulid protozoans. Chlorinated seawater must be neutralised prior to release from the holding facility. Optimal neutralisation is achieved by passage through activated charcoal (removes excess chlorine and chloramines). Reducing agents such as sodium thiosulfate or aeration (which do not remove toxic chloramines) may also be used.
	b)	Iodophors are generally applied as alkaline solutions (Wescodyne®, Betadine®) at 200-250 mg iodine/litre (ppm) with a contact time of at least 10 minutes.
	NOTE: Iodophors are not effective against certain protozoans in suspension, e.g. over 1000 mg iodine/litre is tolerated by Labyrinthuloides haliotidis of abalone. Iodophors may be effective against protozoan parasites following air or heat drying of tank surfaces and pipelines.
3.	Disinfectants - Effluent Water
	a)	Ozone has been used successfully in controlling the microbial content of effluent water from quarantine facilities. Residual compounds, formed as a result of the interaction of ozone with seawater (residual oxidants), at levels of 0.08-1.0 mg/litre are considered sufficient to significantly reduce live microbes (principally bacteria).
		NOTE: The measurement of residual ozone in seawater is problematic due to the rapid and continuous formation of oxidant products in seawater. Residuals formed between ozone and seawater (hypobromite, bromine or hypobromous acid) are toxic to oyster larvae (and possibly other mollusc larvae) and should be removed using a charcoal filter before passing through/out of the mollusc facility. UV treatment of seawater post-ozonation may be required for complete sterilisation, e.g. for quarantine.
	b)	Chlorine administered as sodium hypochlorite at a concentration of 25 mg chlorine/litre is effective against certain protozoans (L. haliotidis); however, 50 mg chlorine/litre is recommended for complete microbial sterilisation (as for pipelines and tanks - see Section B.2.a). Higher concentrations may be used under certain conditions (e.g. quarantine); however, these require proportionately greater neutralisation treatments and exhaust systems to deal with the toxic fumes produced.
	c)	Iodophors are not as effective as the above two treatments for killing protozoans.
4.	Disinfectants - Clothing and Equipment
	Clean surfaces with detergent and disinfectants prior to proper disinfection.
	a)	Iodophors (e.g. Wescodyne®, Betadine®) at 200-250 mg iodine/litre can be used as a footbath. NOTE: Iodophors will stain clothing.
	b)	Chlorine (household bleach solution at 50 mg chlorine/litre) is also an effective footbath or equipment wash.
	c)	Sodium hydroxide (1% NaOH + 0.1% Teepol® or other detergent) makes an effective footbath for rubber boots. NOTE: Do NOT use for dress shoes/boots.
5.	Special Recommendations
	a)	Both chlorine and ozone produce long-lived residual oxidant compounds in seawater. Seawater at 35 parts per thousand (ppt) salinity contains 60 ppm bromide ion, which produces hypobromite in the presence of ozone. Disinfected artificial seawater, at the same salinity, produces bromine and hypobromous acid. As these, along with other residual compounds, are toxic to larval oysters (and possibly other molluscs), treated seawater must be passed through an activated charcoal filter before being used for live mollusc larvae.
		Alternative protocols for halogen neutralisation involve treatment with sodium or potassium thiosulfate (see Section A.2).
	b)	Monitoring of residual oxidants must be carried out regularly, especially where temperature fluctuations occur. As residual ozone cannot be measured accurately in seawater, alternative monitoring protocols must be installed, such as a feedback loop.
		Exhaust systems should also be in place to remove toxic fumes (produced during disinfection) from enclosed work areas. Ensure compliance with local atmospheric regulations when discharging toxic fumes.
	c)	The following management practices can be used to reduce opportunistic pathogen proliferation within a mollusc hatchery or holding facility:
		i)	maintain pathogen-free algal stocks and cultures;
		ii)	use appropriate water filtration, regular disinfection of tanks, pipes and equipment, and footbaths, and water changes;
		iii)	isolate infected stocks and associated equipment at the first sign of disease;
		iv)	discard infected stock and sterilise equipment;
		v)	identify the source of infection within the holding facility to prevent further infection (algal stocks, seawater influent system, broodstock, larval stock).

C. METHODS FOR DISINFECTION OF CRUSTACEAN FARMS

1.	General Principles
	The choice of a disinfection method for use in a crustacean farm depends on many factors, which may include: the reason(s) for disinfection, whether the 'farm' is a broodstock facility, hatchery, or growout farms; and the type of growout farm. Because the penaeid shrimp are the hosts for all but one of the crustacean diseases currently listed in the Aquatic Code, this Section will focus on penaeid shrimp.
2.	Reason(s) for Disinfection
	Disinfection is employed as a common disease management tool in shrimp farming. It may be used as a routine practice in biosecurity programmes designed to exclude specific diseases, as well as a routine sanitary measure employed to reduce disease incidence within farms, or it may be used in disease eradication (stamping out) efforts. The specific reason for disinfection, will determine the disinfection strategy used and how it is applied.
3.	Occurrence of Listed Diseases
	When an OIE listed disease, or an important but unlisted emerging disease occurs for the first time at a particular farm, at a particular site (i.e. at a quarantine facility), or within a region or country previously believed to be free of that disease, it may be advisable, if not required, to eradicate the disease by depopulating the facility and performing a thorough disinfection of all or part of the facility. Fallowing of the affected facility for a defined period of time may be warranted in some situations (see Chapter 1.7.1, Guidelines for fallowing in aquaculture in the Aquatic Code).
4.	Prevention of Disease Spread to Wild Populations
	The direct disposal of diseased populations of live shrimp (any life stage; i.e. fertilised or unfertilised eggs, larvae, postlarvae, juveniles, or adults) or waste products derived from them (i.e. processing plant wastes such as shells, broken shrimp pieces, etc.) into receiving waters (i.e. creeks, rivers, estuaries, bays, littoral areas) is a dangerous practice that facilitates the spread of disease from farmed populations to wild crustacean stocks or to neighbouring farms that use the same water supply, and it should not be permitted to occur. With cultured stocks, when the decision is made to discard a population (i.e. that is being cultivated in a hatchery tank or growout pond) due to the presence of disease (or poor culture performance which may be due to an undiagnosed disease), the stock in the tank or pond should be harvested and/or humanely killed in the tank or pond. The water in the tank or pond should be disinfected (see specific subsections on disinfection of tanks and growout ponds in the Section 5) prior to discharge. The emptied tank or pond should be disinfected prior to restocking.
5.	Routine Sanitation and Biosecurity
	Many crustacean farms, especially those cultivating penaeid shrimp, employ measures that use a number of disinfection methods for disease prevention and control. These measures may be part of a farm's routine biosecurity programme that may be designed for exclusion of specific diseases as well as serving as general pest and disease exclusion measures.
	5.1.	Disinfectants
		The following list comprises the disinfectants recommended for use in shrimp farms (the appropriate disinfectant regimes for each specific application are discussed in the appropriate subsection):
		-	chlorine (as calcium hypochlorite, HTHT or a bleach solution containing a sufficient concentration of hypochlorite);
		-	formaldehyde gas (from sublimated paraformaldehyde or concentrated formalin/potassium permanganate reaction);
		-	iodine (as contained in iodophors);
		-	lime (as calcium oxide or calcium hydroxide);
		-	UV light (from natural sunlight);
		-	ozone;
		-	steam;
		-	hot water (60ºC);
		-	concentrated acids;
		-	desiccation;
		-	detergents (for general cleaning, with some degree of disinfection capability for many products).
	5.2.	Hatcheries and broodstock rearing/holding facilities
		Virtually all penaeid shrimp hatcheries and broodstock holding/rearing facilities use seawater that has been disinfected to remove potential pathogens, pests, and disease-carrying agents via mechanical filtration, UV irradiation, and/or chemical disinfection. This may be by passive source water filtration (i.e. by the use of seawater wells or well points) or by mechanical filtration using high pressure pumps and a variety of water filtration devices and pore sizes. Some facilities use filtration coupled with UV light disinfection of source water, while others use chemical disinfection methods, using either chlorination and de-chlorination or high doses of ozone and subsequent removal of residual oxidants. Chemical disinfection of source water typically requires the use of one or more water storage reservoirs in which the water is treated and detoxified before use in the shrimp hatchery or broodstock facility. Numerous manuals are available that provide specifics on hatchery and broodstock facility design and operation for shrimp culture, and in which details on source water disinfection are provided.
		a)	Disinfection of eggs and larvae in penaeid shrimp hatcheries
			Certain penaeid shrimp viral diseases (i.e. Spherical baculovirosis, Tetrahedral baculovirosis, Hepatopancreatic parvovirus infections) are transmitted by faecal contamination of spawned eggs. These diseases, as well as infections due to certain other shrimp viruses such as White spot disease virus, and certain bacterial and fungal disease agents, can be eliminated or have their incidence reduced through the routine use of disinfection protocols when used to surface disinfect eggs and/or recently hatched nauplii. A widely used method is given below:
			For fertilised eggs*
			(*Fertilised eggs are more sensitive than nauplii to formalin.)
			Collect fertilised eggs. Rinse with running seawater for 1-2 minutes. Fully immerse eggs in 100 ppm (parts per million) formalin for 1 minute. Fully immerse eggs in iodophor (0.1 ppm iodine) for 1 minute. Rinse in running seawater 3-5 minutes. Transfer to disinfected larval rearing tanks.
			For nauplii*
			(*Nauplii are much easier to collect than are fertilised eggs in hatcheries.)
			Using phototaxic response to light, collect nauplii with netting or screen. Rinse with running seawater for 1-2 minutes. Fully immerse nauplii in 400 ppm formalin for 30-60 seconds. Fully immerse nauplii in iodophor (0.1 ppm iodine) for 1 minute. Rinse in running seawater 3-5 minutes. Transfer to disinfected larval rearing tanks.
		b)	Disinfection of tanks, equipment, pipes, air stones, etc.
			For routine sanitation, hatchery and broodstock tanks (i.e. tanks for broodstock maturation, matting, spawning, larval rearing and indoor nursery) should be cleaned, disinfected and dried between use. Tanks used for the above-named purposes in crustacean (especially shrimp) hatcheries are typically precast fiberglass tanks or they are constructed of concrete or wood and either coated or painted with resin-based coatings (e.g. epoxy or fiberglass resin) or lined with plastic liners manufactured for that purpose. After harvest of the stock from the tank, all loose objects and large-sized organic debris such as algae, faeces and left-over feed should be removed. With relatively small tanks, it is advisable after harvest of the stock to fill the tank to capacity, immerse all nonporous corrosion resistant equipment (i.e. airlines, air stones, stand pipes, screens, sampling containers, etc.) in the tank, and then add calcium hypochlorite to provide a minimum of 200 ppm of free chlorine. This should be allowed to set overnight. After the proper chlorinated soak-time, the tank can be drained and freshwater rinsed. Before draining the system, the treated water should be dechlorinated (see specific subsections on chlorination described in this Section), unless appropriate effluent collection and treatment systems are in place. After the tank has been rinsed it should be allowed to completely dry. In the case of large tanks, an initial cleaning to remove loose debris should be followed by disinfection with a concentrated (~1600 ppm as chlorine) solution of calcium hypochlorite. All inside and outside surfaces should then be sprayed with this chlorine solution. The tank should then be allowed to set for several hours and then rinsed, filled and flushed. Surfaces should then be scrubbed free of all remaining material. After disinfection with chlorine, small or large tanks should be rinsed with clean water, then filled and flushed to ensure that no chlorine residues remain before the tank is restocked for another crop.
	5.3.	Disinfection of growout ponds
		Following the routine harvest of a crop from a growout pond (or from a large tank or raceway used for growout of a crop), the pond (tank) bottom should be inspected. Large deposits of organic debris should be treated or removed. This is easily accomplished in lined tanks, raceways, or ponds (i.e. by flushing with a high pressure hose), but poses more of a challenge in large earth bottom ponds. Many methods of pond bottom disinfection and treatment between crops are practiced. These methods are given in detail in a number of shrimp farming manuals, and some will be listed here only with minimal details:
		a)	Chlorination
			This disinfectant may be used for routine treatment of ponds between crops or when disease eradication is the goal. After draining the pond, remove (and dispose of [see section on carcass disposal in Section C.6]) as many animals from the system as is possible (this may be difficult in pond systems where the removal of large numbers of dead shrimp would not be practical). Partially refill the pond (or fill to capacity if required), discontinue the addition of new water, stop the discharge of effluent water, and remove any internal or external sources of aeration or aeration devices, which might be subject to corrosion. Then evenly distribute sufficient granulated calcium hypochlorite (such as Olin HTHT) to provide a minimum residual free chlorine concentration of 10 ppm within the entire system s water. (NB: The person(s) applying the chlorine should wear waterproof outer ware to protect their skin, an approved chlorine mask, and goggles or a face shield for eye protection.) Redistribute additional calcium hypochlorite as often as required to maintain the residual concentration at near or 10 ppm. Allow the system to set for a minimum of 24-48 hours (especially if applied to large systems) at this minimal chlorine concentration. The chlorine will kill all shrimp and most, if not all, of the other organisms occupying the water column or resident in the pond. After the pond has been treated with chlorine for the required minimum time and before any water is discharged, neutralise the chlorine either passively by exposure to sunlight and air for approximately an additional 48 hours (without the addition of new chlorine) or by the addition of sodium thiosulphate at a rate of five (5) molecules of sodium thiosulphate for each four (4) molecules of chlorine (or the weight of sodium thiosulphate being 2.85 times the weight of chlorine in the water, see example table below).

Pond size   Average depth   Volume   Chlorine dose   Chlorine required   HTH (65% active Cl)   Thiosulphate required   1 hectare   1 m   10,000 m3   10 ppm   100 kg   154 kg   285 kg

			Periodic testing should be done for residual chlorine; water should not be discharged until it has reached 0 ppm. Once the chlorine levels have been ascertained to be at 0 ppm, the system water can be safely dumped into the farm's effluent system. In some culture systems, in particular raceways, tanks and small lined ponds (i.e. those systems in which the majority of the shrimp were not removed prior to disinfection), the dead shrimp should be collected for proper disposal (see section on carcass disposal in Section C.6).
		b)	Liming
			The lime, as calcium oxide or calcium hydroxide, should be applied to a very moist bottom at a rate of 5000 kg/ha or 1500 kg/ha, respectively. Great care should be taken to assure that the lime is spread evenly over the soil surface. The pond should then be allowed to set for at least a week, or at least until the soil has dried to the point of cracking to a depth of approximately 10-20 cm. Additional lime may be applied after ploughing (see below) at a rate of 50% of that normally prescribed. The pond should again be dried for at least a week, depending on the weather.
		c)	Drying and ploughing
			Whether or not a pond is treated by chlorination or liming or left to dry untreated, ploughing is a commonly used method of treating a pond bottom to reduce its organic content, improve nutrient recycling, buffer pH, eliminate pests, and achieve disinfection through a combination of microbial degradation, exposure to sunlight, aeration, and desiccation. In some regions, drying and ploughing of dry pond bottoms may only be possible during the 'dry season'. When pond drying is an option, the pond bottom should be allowed to dry until the surface has cracked to a depth of approximately 10 cm. Once this level of drying has been reached, the soil should be broken up to a depth of approximately 20 cm with a plough, tiller, disk harrow, tine harrow or other similar farm implement. Ponds treated in this manner should be left for at least a week before being refilled and restocked.
	5.4.	Disinfection of source water
		Because several of the listed diseases of shrimp listed in the Aquatic Code, as well as a number of other important diseases, can be introduced into shrimp farms with source water when it contains vectors or carriers (i.e. wild infected crab or shrimp larvae), most farms operate with biosecurity plans that include provisions for the disinfection of source water. This may be accomplished by a variety of means which may include one or some combination of the following strategies:
		a)	Filtration of source water - source water is pumped into a supply/settling canal where it first passes through coarse bar screens to remove large aquatic animals and debris. Then the water is passed through a series of progressively finer screens, and final filtration is accomplished by passing source water through a fine mesh (150-250 µm mesh size) bag screen before being introduced into a culture pond or storage reservoir.
		b)	Instead of using mesh nets, some farms place filtration structures in the supply canal system. A series of compartments within these structures are filled with filter matrixes, beginning with coarse gravel for initial removal of large aquatic animals and debris, an intermediate section which contains a finer matrix of sand and gravel, and the end section which contains fine sand.
		c)	Chlorination and de-chlorination - source water is pumped to a supply canal or directly into culture ponds or reservoirs (with or without filtration) and treated with sufficient chlorine to kill any potential vectors or carriers in the source water.
		d)	'Zero' or reduced water exchange: Some farms use supplemental aeration and re-circulation of water in culture ponds and within the supply and discharge systems of the shrimp farm to reduce source water requirements. This reduces the volume of source water that must be disinfected before use, as well as reducing nutrient loss from farms with effluent.
6.	Disease Eradication and Total Facility Clean-up
	This action may be necessary for disease control when significant, untreatable diseases occur at sites where eradication is an option. The confirmed diagnosis of an listed diseases, or of an important but unlisted emerging disease occurring for the first time at a particular farm, at a particular site (i.e. at a quarantine facility), or within a region or country previously believed to be free of that disease, are events wherein it may be advisable, if not required, to eradicate the disease by depopulating the affected facility and performing a thorough disinfection of all or part of the facility.
	Fallowing of the affected facility for a defined period of time may be warranted in some situations (see Chapter 1.7.1, Guidelines for fallowing in aquaculture in the Aquatic Code).
	The following steps/actions may be used to achieve eradication of a disease through a total facility clean-up (TCU):
	6.1.	Depopulate all living shrimp stocks from the affected facility
		a)	Discontinue stocking of the facility.
		b)	Harvest and sell (if permitted) marketable stocks through normal market channels. In some circumstances cooking the product before marketing may be advisable. Cooking, either by steam or boiling water, will kill or deactivate all known disease agents of shrimp.
		c)	For unmarketable stocks the following are options for disposable after harvest:
			i)	Incineration: burn collected shrimp in a government approved (if required) incinerator. The limitations to this procedure are inherent to the disposal of shrimp. That is, shrimp contain large amounts of water and therefore this procedure may only be feasible for small quantities of shrimp or to larger quantities if the shrimp have been dried prior to incineration.
			ii)	Burial: although this technique should be applicable to a greater number of instances, it still has its limitations. The shrimp should be placed in a pit of sufficient depth to accommodate all of the shrimp and still provide for at least 50 cm of fill covering the shrimp. The pit should be located some distance from the facility undergoing TCU and a comparable distance from any other facility culturing shrimp. Drainage from the pit area should not be into the aquifer from which the TCU site (or any shrimp culture site) may pump its source water or into the area (estuary or beach) from which source water is drawn. Once a proper site has been selected, then the actual burial can take place. The bottom of the pit should be covered with calcium oxide (quicklime) at a rate of approximately 500 g/m2 (5000 kg/ha) or with calcium hydroxide (slaked or hydrated lime) at a rate of approximately 150 g/m2 (1500 kg/ha). The shrimp should be placed in the pit in layers of approximately 10 cm depth, each covered by a quantity of slaked lime or quicklime sufficient to completely cover the layer (equivalent to approximately 33-100% of the weight of the shrimp). The entire pit, including the top layer of shrimp carcasses, should then be overlaid with a minimum of 50 cm of fill dirt. In some locations, local environmental, public health and zoning officials should be consulted before the shrimp burial pit(s) is (are) dug.
	6.2.	Disinfection of culture tanks and ponds
		For methods see appropriate subsections in Section C.5.
	6.3.	Clean-up procedures for facility components other than culture areas
		In order for a TCU to be effective, it may be necessary to disinfect the entire facility after all the shrimp have either been harvested or disposed of in some other manner. After depopulation of the facility, every possible animate and inanimate carrier of the disease agent must be identified and either removed from the facility or thoroughly disinfected. The movement of disease agents between live shrimp or dead numerous shrimp can be easily understood, while the same can not be said for their movement via inanimate components. Hence, all areas, units, subunits or components which are contaminated or potentially contaminated must go through a cleaning and disinfection process. See Table 1 in Section A and Section C.5.1 for a list of disinfectants and their methods of application.
		a)	Buildings
			The disinfection regime used should be building-specific and dependent upon the use-pattern of that particular building.
			i)	Office buildings: these buildings would most often be subject only to foot traffic from people who have been in contaminated buildings or culture areas. Because of this, the greatest focus of attention should be the floors and cold storage units in the building. Floors should be thoroughly cleaned (if they are non-porous) with standard detergents and cleaning solutions, followed by a thorough drying. If the floors are carpeted, they should be vacuumed and cleaned with a detergent appropriate for carpets, or 'steam' cleaned. All other areas within these buildings, such as walls, bathrooms, desks, refrigerators, freezers, etc. should be examined for possibly contaminated materials (i.e. frozen shrimp in freezers) and any such item found and its container should be cleaned and disinfected or disposed of in a sanitary manner.
			ii)	Culture buildings: it must be assumed that these buildings have had direct contact with the disease agents and will therefore be handled in a different manner from that of the office buildings. The disinfection regime for these buildings will consist of two steps. First, the building should be thoroughly swept and/or vacuumed (where appropriate) to remove as much large-sized organic and inorganic debris as possible. This should be followed with the second step, treatment with chlorine. Chlorine solution (~1600 ppm) should be applied (by spraying) to all surfaces which are not prone to the corrosive actions of chlorine. Those surfaces which should not be chlorinated, can first be sponged with a iodophor solution minimally providing 200 ppm of free iodine. These can then be covered with plastic or any other protective material. Floor surfaces can be soak-chlorinated to a depth of 5 cm with a 200 ppm chlorine solution. This should be allowed to set for a minimum of 48 hours. If many of the sprayed surfaces are somewhat susceptible to corrosion by chlorine, those surfaces can be freshwater-rinsed after the 48-hour treatment.
				In buildings where disinfection with chlorine is not practical, fumigation with formaldehyde gas should be considered. After a general cleaning, fumigation of a sealable building can be initiated. The entire process, from the time the building is first gassed until it can be occupied again, should take a minimum of 36-60 hours. The entire building must be totally sealed off during the actual fumigation; there should be no means by which the gas can escape once it is placed in the building. If possible, the electrical service for the building should be turned off. The required environment for formaldehyde gas disinfection is a minimum temperature of 18ºC with a high relative humidity (at saturation is best, i.e. floors should be wet, etc.). Generation of formaldehyde gas is accomplished by the addition of 17.5 g potassium permanganate to each 35 ml of 100% formalin (a 37-39% aqueous solution of formaldehyde gas) for each 2.83 m3 (100 ft3) of space. Ideally, each room in the structure should have its own source of formaldehyde gas to assure that all areas of the building are uniformly treated. The correct amount of each compound (potassium permanganate and formalin) should be weighed out into separate containers, the formalin should be placed in a non-plastic container that is at least 10 times the combined volume of both the formalin and the potassium permanganate. (The person applying a formaldehyde gas fumigation should wear waterproof outer ware to protect their skin, an approved formaldehyde gas mask, and goggles or a face shield for eye protection.) The containers with the proper amounts of the two reagents should then be placed on the floor in the centre of the room, on a large disposable protective (plastic) mat. The formalin and potassium permanganate should not be mixed at this time. Once all rooms have the correct amounts of the two compounds, the building has been completely sealed and the environment modified as necessary, the actual fumigation can begin. The mixing of the two compounds must be done very rapidly and carefully as the reaction is immediate and somewhat violent as formaldehyde gas is emitted. Starting with the room farthest from the exterior door, add the permanganate to the formalin and proceed to the next room. After all rooms have been completed, lock the exterior door and seal it from the outside with tape. The building should be allowed to set for a minimum of 12 hours. After this disinfection period the building should be flushed with clean air for 24-48 hours. There should be no detectable odour of formaldehyde when people are allowed to reoccupy the building.
				An alternate method for the generation of formaldehyde gas is the sublimation of powdered paraformaldehyde. For each 2.83 m3 (100 ft3) of space, approximately 28 g paraformaldehyde should be used. It can be sublimated by being placed in an electric fry pan, which has been set on high. This procedure is somewhat more dangerous, because formaldehyde is flammable and a spark from such a heating device could theoretically ignite the gas. The same procedures noted above for the formalin/permanganate mixture in regards to venting, etc. should also be followed for the use of paraformaldehyde.
			iii)	Processing buildings: these buildings are typically constructed to permit routine disinfection. For the most part, the procedures followed in the routine operation of such buildings are appropriate for a TCU, provided that the building, its cold rooms, and its freezers are also disinfected and thoroughly dried. If considered necessary, fumigation with formaldehyde gas may be done to insure destruction of the disease agent(s) of concern.
			iv)	Other buildings: buildings (feed storage, maintenance, tool rooms, etc.) should be treated somewhat like the office building. Care should be taken to remove all the large-sized debris, which would normally be found in relative abundance within these types of buildings. Potentially contaminated surfaces within such buildings should next be spray-chlorinated and allowed to set for 24-48 hours. This should be followed by a freshwater rinse. All equipment, which should not be exposed to the corrosive action of chlorine, should be removed before the spraying, and they should be disinfected by surface disinfection with 200 ppm of iodophor. Once the equipment has been disinfected, it can be brought back into the building. Fumigation with formaldehyde gas is another option for this type of building.
		b)	Culture support equipment and systems
			These are operational units of the shrimp culture facility which may be housed in a building.
			i)	Artemia systems: all Artemia decapsulation and cyst hatching units and tanks should be treated in the same manner as other tanks. They should be cleaned of all large debris, then filled to the top with clean water and calcium hypochlorite added to achieve a final concentration of 200 ppm (free Cl2). Chlorination should be allowed to continue for 24-48 hours. The outside of such tanks may be spray-chlorinated (1600 ppm chlorine). Treated tanks can then be dechlorinated with sodium thiosulphate, drained, freshwater rinsed, and allowed to dry for a minimum of one week. Unopened containers of Artemia cysts at the facility can be retained. These should, however, be surface disinfected with chlorine (200 ppm) or iodophor (200 ppm).
			ii)	Algae systems: containers, tanks, incubators and rooms used to produce algae for feeding the larval stages of shrimp may be handled and disinfected in nearly the same way as other tanks systems. The only major difference being that special care must be taken to assure that all chlorine residues have been rinsed from the units before they are used again. In the case of the culture tubes, flasks, carboys, and flasks used to culture algae, a combination of acid (10% HCl) rinse or steam sterilisation can be used in lieu of disinfection with chlorine or idophor.
				Disinfection of stock cultures of living algae is not possible. The use of disinfection is clearly out of the question; any compound which would kill the disease agent would likewise kill the algae. Hence, there are two basic methods of minimising the chance of a disease agent being present in the stock cultures.
			-	Dilution: all stock cultures can be cloned from the existing stocks. Each culture should be diluted either by means of serial dilutions (for broth cultures) or streaked for single colonies (agar cultures). All dilutions must be performed using strict aseptic techniques with all media being properly autoclaved. Passages from the stock cultures should not occur until the algae culture room has itself been disinfected as per the above building procedures. Once a culture has been diluted and cloned by either of these methods, to the point where there remains only one cell of the original culture, the risk is negligible that a (shrimp) disease agent may be present.
			-	New Stock Cultures: If existing stock culture are discarded in a TCU, new stocks should be purchased from algae supply laboratories, or obtained from other sources where contamination with (shrimp) disease agents is unlikely, such as isolating desired species from wild populations of algae. New stock cultures should not be obtained from any facility that also cultures shrimp and may be contaminated with (shrimp) disease agents of concern.
			iii)	Farm equipment: nets, seines, porous air-line tubing, etc. which are relatively inexpensive and easily obtainable should be discarded and removed from the facility during a TCU rather than being disinfected as they are not readily disinfected and chlorine is likely to damage them and shorten their useful life.
				Non-expendable equipment such as large size flexible plastic tubing, pumps and pipes, transfer tanks, cages, harvest cages, harvest tables, Secchi disks, laboratory glassware, etc. should be soak-chlorinated in 200 ppm solutions for 24-48 hours. This is most easily accomplished by placing these objects in the tanks that are filled with 200 ppm solutions of chlorine. Care should be taken to have all items completely submerged (use heavy items to weigh-down more buoyant objects). A good guide is to place everything (except those that are to be thrown away) that is loose or can be unsecured from its point of attachment, into the 200 ppm chlorine solution in their respective tanks.
				In the case of those similar type items which are associated with ponds, they should be placed in a special series of tanks set up near their respective ponds. These tanks should be filled with 200 ppm chlorine solutions. Following soak-chlorination, these items should be allowed to dry and be exposed to natural UV (sunlight) sterilisation. They should be turned at least once to expose all areas of the items to direct sunlight.
				Tools and machinery, such as tractors, trucks, portable and stationary power tools, etc., should be thoroughly cleaned with standard cleaning solutions. All traces of mud, shrimp feed, etc. must be removed from these items. Following this, disinfection of surfaces likely to have been contaminated in normal use should be rinsed off with an iodophor solution (at a concentration of 200 ppm) or cleaned with steam.
				Small tools and instruments such as, scales and balances, test instruments, small power tools, etc., should be gently sponged off with 200 ppm of chlorine solutions if they are inert plastic or 200 ppm of iodophor if they are otherwise. These should then be placed back in their respective buildings during the formaldehyde fumigation. High precision electronic test equipment should not be subjected to the fumigation, especially if there has been little chance that it was ever contaminated.
			iv)	'New-Water' Plumbing: all new-water plumbing which is contained within buildings, especially those which have blind ends or terminate in manifolds, should be filled with a minimum 200 ppm chlorine solution. The chlorine solution should be held in the lines for 24-48 hours minimum, followed by clean water rinsing. Pipes may also be disinfected by recirculating hot water (>60ºC) through them for several hours.
			v)	Uniforms, boots, etc.: all items worn or used by employees should be either disposed of or thoroughly washed and disinfected. In the case of clothing, such as coveralls, normal washing which incorporates a chlorine bleach is sufficient, especially if accompanied by sun drying. Other items, such as boots, gloves and other non-cloth items can be safely soak-chlorinated in a 200 ppm chlorine solution. This should be followed by a freshwater rinse. These items should also be contained within their respective buildings during formaldehyde fumigation.
			vi)	Feed items: all feed items, such as prepared feeds, fresh feeds (i.e. squid, bloodworms, frozen Artemia, bivalve molluscs, etc.) should be removed from the facility and replaced with new feeds from sources known to be free of contamination by shrimp disease-causing agents.
7.	Re-stocking of Disinfected Farms
	Following a TCU, restocking of the disinfected facilities or farms should be accomplished only with stocks known to be free of the diseases listed in the Aquatic Code or other emerging or significant diseases of concern.

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