Field of the invention

This invention relates to a method of treating vertebrate aquatic animals, preferably fish, to combat infestation by multicellular ectoparasites with

exoskeletons, especially ectoparasites of the crustacean order copepod, more particularly of the genera Lepeophtheirus (especially the salmon louse,

Lepeophtheirus salmonis) and Caligus (especially Caligus elongatus).

Background

In aquaculture there is a major problem with the infestation of farmed fish, for example salmon, with sea lice. Sea lice feed on the fish and while a few lice on a large fish may not cause serious damage, large numbers of lice on that same fish, or just a couple of lice on a juvenile fish, can be harmful or fatal. Sea lice infestation can cause fin damage, skin erosion, constant bleeding, and open wounds creating a pathway for other pathogens. More damage may be caused when infected fish jump or scrape along nets in an attempt to dislodge any irritating lice.

Fish infested with sea lice also have reduced appetite, which is likely to have a direct effect on fish growth rates, and is of particular concern to fish farmers.

In addition, wholesalers and consumers would prefer to buy fish that are free from sea lice, as infested fish are not aesthetically pleasing. This is one reason why infested fish typically sell for significantly less than lice-free fish.

Generally, infested fish may be treated with a number of different agents to remove the lice, such as organophosphates, carbamates, pyrethroids, pyrethrins, synergists, oxidizing agents (such as hydrogen peroxide), insect growth regulating chemicals or avermectins.

In commercial fish farms, fish are typically contained in cages submerged in open bodies of water, such as the sea. When treating such fish to control lice populations, the cage enclosing the fish is covered with a tarpaulin during exposure of the fish to the treatment agents. Often, the floor of the cage is raised to reduce its volume, and oxygen is bubbled through to ensure the fish do not suffocate. The tarpaulin creates a sealed enclosure for the fish, preventing the treatment agents being washed away by currents. It also allows the fish farmers to control the length of time the fish are exposed to the treatment agents, something which is under strict regulatory control in most jurisdictions.

Treatment agents are typically introduced to enclosures via perforated hoses spread out over the surface of the enclosure. The treatment agents are distributed throughout the disclosure by the movement of the fish as well as by the oxygen bubbling through the enclosure to keep the water aerated.

A number of ectoparasite treatment agents have well documented toxic effects. Concerns have long been expressed about organophosphate toxicity in particular, for example in relation to farm workers. Organophosphate poisoning does not require ingestion - cutaneous absorption can lead to signs of poisoning. Pyrethroids similarly can cause adverse reactions even on dermal exposure, such as neurotoxicity, altered dopamine uptake, and dermatitis.

This is of particular concern not only in relation to the health of workers in the aquaculture industry and the health of the cultured fish, but also in relation to the release into the environment which almost inevitably occurs when fish held in cages submerged in open water are treated.

As might be expected, resistance to ectoparasite treatment agents is now being seen. In particular, large populations of organophosphate-resistant sea lice have arisen in many locations where intensive fish farming takes place. Because of the growing problem of resistance, increasingly higher doses of chemicals are needed. This is problematic as it does nothing to address the problem of resistance, and increases exposure of both fish and farmers to chemicals and worsens environmental pollution.

Increasing the dose of certain treatment agents can lead to less obvious problems arising. For example, fish generally dislike organophosphate compounds. Introducing high levels of organophosphates to water containing fish will often cause the fish to become distressed and to try to swim away from the source of the chemical. This can result in crush damage to the fish due to being pressed against the sides of the enclosure by the mass movement of fish evading the

organophosphate in a "stampede" type scenario, a problem which is only made worse by the reduced volume of the cages used during treatment.

Another issue with conventional treatment methods is that some treatment agents such as organophosphates do not work on all stages of lice development. This means that treatment by organophosphates has to be repeated several times. Repeating the sea lice treatment is undesirable for many reasons. Obviously there is the increased cost for the farmer who has to buy more chemicals, as well as the increased exposure of both the fish and the farmer to the harmful chemicals.

However, a more significant economic consideration is that the fish cannot be fed directly prior to or during exposure to any treatment agents to ensure that the surrounding water is free from food residue and faeces which may interfere with the treatment process. Usually, fish are not fed for 24 or 48 hours before treatment. Commercially farmed fish typically increase their body weight by 1 % per day. Thus, a single treatment may result in a potential 1 -2% weight "loss"; weight which the fish would otherwise have gained had it not been treated. Although this figure is small per fish, the impact over tens of thousands of fish in a single cage can be significant, and represents a significant cost to farmers through lost feeding time. This issue is only made worse if the treatments need to be repeated. There is therefore a continuing need to provide improved treatment regimes which do not require multiple treatment cycles.

Because of the growing problem of resistance, some farmers rotate the treatment agents being used to control lice populations. Such methods generally involve the use of standard doses of each of the treatment agents being used and therefore do nothing to address the problem of chemical exposure. More recent sea lice treatment methods often use combinations of treatment agents. For example, WO 2009/010755 reported that the amounts of the treatment agents may be reduced by applying an organophosphate or carbamate first and then a pyrethroid or pyrethrin some time later. This method also targets all stages of lice development, as young lice are susceptible to pyrethroids/pyrethrins.

While such time-staggered methods may reduce the amount of treatment agent required overall to achieve an acceptable kill rate and target all

developmental stages of sea lice, the very nature of a time-staggered method can present its own problems. In particular, the second treatment is optimally applied around 8 hours after the first treatment.

As noted above, during treatment, fish are held in a shallow enclosure covered with a tarpaulin which is often out at sea. These enclosures can be very large (up to 100m across) and therefore covering the enclosure with a tarpaulin is not a simple task, especially since the weather conditions and currents have to be taken into account. In strong currents, the force of the water on the tarpaulin may threaten the integrity of the enclosure. Consequently, an issue arises when trying to perform the sequential method of WO 2009/010755 if the currents change during the day. If the conditions are not safe to use a tarpaulin 8 hours later, it may not be possible to carry out the exposure to the second treatment agent as planned.

Although not optimum, very goods results can be obtained by adding the second ectoparasite treatment agent shortly after (e.g. 10-15 minutes after) the first ectoparasite treatment agent without removing the tarpaulin. However, there are strict rules which govern how long fish may be exposed to treatment agents. For example, at sea temperatures above 10°C, treatment times with azamethiphos (an organophosphate typically used as the first treatment agent in the method of WO 2009/010755) must be reduced for the safety of the fish to not more than 30 minutes. Failure to adhere to these time limits can result in farmers being fined or even having their farming licence revoked. These time limits can make it difficult to carry out sequential methods effectively as the tarpaulin needs to be removed within 30 minutes of adding the first treatment agent. Adding the second treatment agent after 15 minutes may not expose the lice to this treatment agent for long enough periods of time to have the desired effect.

As a replacement to sequential addition of treatment agents,

WO 2010/109198 proposed using synergistic combinations of certain treatment agents added simultaneously. In these methods, the treatment agents are mixed together and added simultaneously to the enclosure.

Without wishing to be bound by theory, it is believed that two lipophilic ectoparasite treatment agents can form a complex with each other when they are mixed together before being introduced to the water containing the fish. Because of the lipophilic nature of the treatment agent molecules, such complexes will not break up when introduced to treat the lice. The formation of such complexes is thought to reduce the amount of "free" treatment agent molecules which are able to target the lice, meaning that the doses of treatment agent that are required to achieve an acceptable kill rate are higher than necessary. This effect has been shown by Sevatdal et al. in a study investigating the possibility of using piperonyl butoxide as a synergist for cypermethrin and deltamethrin (see Pest Management Science, 2005, 61 , p772-8).

It is therefore desirable to find alternative methods of treating sea lice that reduce the amount of treatment agent used, that reduce the total treatment time, that avoid the need for multiple treatment cycles, that provide excellent kill rates, and that are easy to implement.

Summary

In one embodiment, the invention provides a first lipophilic treatment agent selected from a pyrethroid, a pyrethrin, a synergist, an organophosphate, a carbamate, an avermectin, an oxidising agent and an insect growth regulating chemical for use in a method of pesticidal treatment of vertebrate aquatic animals which method comprises topically exposing vertebrate aquatic animals to a first and a second lipophilic treatment agent, characterised in that the first and a second lipophilic treatment agents are simultaneously introduced to an enclosure containing the vertebrate aquatic animals as separate treatment agent

compositions.

In another embodiment, the invention provides a pyrethroid or pyrethrin for use in a method of treating vertebrate aquatic animals to combat infestation by multicellular ectoparasites with exoskeletons, which method comprises topically exposing the vertebrate aquatic animals to the pyrethroid or pyrethrin and a second lipophilic treatment agent (preferably an organophosphate, carbamate or piperonyl butoxide), characterised in that the pyrethroid or pyrethrin and second lipophilic treatment agent are simultaneously introduced to an enclosure containing the vertebrate aquatic animals as separate treatment agent compositions, preferably at different locations.

In another embodiment, the invention provides an organophosphate, carbamate or piperonyl butoxide for use in a method of treating vertebrate aquatic animals to combat infestation by multicellular ectoparasites with exoskeletons, which method comprises topically exposing vertebrate aquatic animals to the organophosphate, carbamate or piperonyl butoxide and a second lipophilic treatment agent (preferably a pyrethroid or pyrethrin), characterised in that the organophosphate, carbamate or piperonyl butoxide and second lipophilic treatment agent are simultaneously introduced to an enclosure containing the vertebrate aquatic animals as separate treatment agent compositions, preferably at different locations.

In another embodiment the invention provides a method of treating vertebrate aquatic animals to combat infestation by multicellular ectoparasites with exoskeletons, which method comprises topically exposing vertebrate aquatic animals to a first and a second lipophilic treatment agent, characterised in that the first and the second lipophilic treatment agents are simultaneously introduced to an enclosure containing the vertebrate aquatic animals as separate treatment agent compositions, preferably at different locations.

Detailed description

By "pesticidal treatment" is meant treatment to combat multicellular ectoparasites with exoskeletons, preferably which reside on the vertebrate aquatic animal which is topically exposed in the treatment method.

The multicellular ectoparasites with exoskeletons referred to in the method of the invention are preferably ectoparasites of the crustacean order copepod, more particularly of the genera Lepeophtheirus (especially the salmon louse,

Lepeoptheirus salmonis) and Caligus (especially Caligus elongatus), hereafter generally referred to as sea lice.

The vertebrate aquatic animals are preferably fish. More preferably, the fish are salmonids such as cod, salmon etc. Most preferred are salmon.

The vertebrate aquatic animals (e.g. the fish) which are topically exposed to the treatment agents in the method of the invention are typically infested with the ectoparasites (e.g. the sea lice).

The method of the invention is particularly applicable to farmed fish.

Farmed salmon in particular are especially prone to infestation by sea lice.

Treatment of the farmed fish is topical in that the fish are introduced into an aqueous environment containing the treatment agent or caused to transit such an environment, or have the treatment agent introduced into the aqueous environment containing the fish. Thus for example, fish may be transferred into a tank for treatment or caused to pass from one holding zone, e.g. a tank or cage, into another through a conduit, e.g. a pipe or channel, containing the treatment agent.

In the method of the invention, the treatment agents are released into the cage, tank or pond containing the fish, optionally after surrounding the cage with an impervious barrier, e.g. a tarpaulin, to cause at least temporary retention of the treatment agent within the water in the cage.

Particularly preferably, the treatment agents are released into the water within a cage, e.g. a sea cage, over an extended period so as to ensure exposure of the fish to the treatment agent before the agent is flushed out of the cage by the flow of surrounding water. Where the agents are to be released into a sea cage, the sea-cage net will typically be raised to a depth of 5-15 (e.g. 10) metres and then surrounded by impervious tarpaulins to isolate the cage to be treated. Typically, the depth of enclosed water is often such that there will be some space (e.g. about 0.5- 1 m) between the net bottom and the tarpaulin.

Thus, the method of the invention is preferably performed on fish held within a sea cage.

By introducing the two lipophilic treatment agents as separate treatment agent compositions, the actual concentrations of the treatment agents when they come into contact is so low that the likelihood of forming a complex is significantly reduced. This means that there are more "free" (i.e. non-complexed) treatment agent molecules that can target the lice.

By "separately" is meant that the first and second treatment agents are added as compositions comprising only one lipophilic treatment agent (i.e. only the first or only the second treatment agent), such that the first and second treatment agents only come into contact with one another in the enclosure containing the vertebrate aquatic animals.

As used herein, the term "treatment agent composition" is meant a composition, typically an aqueous solution, containing a lipophilic treatment agent. Such compositions are typically made by dispersing or dissolving the lipophilic treatment agent (or diluting a concentrate containing the lipophilic treatment agent) in water. Typically, the water in which the aquatic vertebrate animals to be treated is used, such as sea water.

As used herein, the term "simultaneously" is intended to mean "at the same time". However, in some embodiments of the invention, the first and second lipophilic treatment agents used in the invention may be introduced to the enclosure sequentially such that the second treatment agent is added after a delay, wherein the delay is shorter than the time taken to completely add the first treatment agent. For example, if it takes 10 minutes to completely add the first treatment agent, the second treatment agent may only be added after 5 minutes.

Alternatively, the first and second treatment agent compositions may be introduced at the same time, but it may take less time to introduce one composition than the other. For example, the first treatment agent composition may take 10 minutes to fully introduce into the enclosure, whereas the second treatment agent composition may only take 5 minutes to fully introduce. If both were added at the same time, the last 5 minutes would involve only adding one treatment agent composition, but would still fall within the scope of the invention provided that both treatment agent compositions are being added at the same time for at least part of the method.

Thus, "simultaneously introduced" as used herein encompasses adding one treatment agent at a time, providing that for at least part of the addition the other treatment agent is also being introduced such that during the time of adding the lipophilic treatment agents, at least some of the time both lipophilic treatment agents are being added at the same time.

Preferably, there is no delay between the introduction of the first and second lipophilic treatment agents.

The exposure to the treatment agents is preferably for a period of up to about 100 minutes, preferably up to about 60 minutes, more preferably up to about 45 minutes, more preferably up to about 40 minutes, even more preferably up to about 30 minutes, even more preferably up to about 20 minutes.

If the water temperature is above 10°C, the treatment time is preferably up to about 30 minutes.

In some embodiments, the treatment agent compositions are added to different locations of the enclosure.

As used herein, the term "location" is intended to mean any two or three dimensional point of entry for adding the treatment agents to water (e.g. by applying to the surface of the enclosure or by pumping the treatment agent composition through a nozzle submerged in the enclosure).

In one embodiment, the treatment agents are added via pumps at different locations under the water. If treatment agents are to be added below the water surface, they should typically be added where the highest density of fish is. If a sea cage is used in the method of the invention, this is typically at least 1 m, preferably 1 -5 m, e.g. 2-3 m below the water surface.

In another embodiment, the treatment agents are added via perforated hoses spread over the surface of the water. In another embodiment, the treatment agents are added via a sprinkler-type system to the surface of the water.

Preferably, the treatment agents are added via perforated hoses or sprinkler systems, particularly preferably via perforated hoses. It is not necessary for the method of the invention to use solely surface application or solely underwater application. Thus, in one embodiment, one treatment agent is added to the surface of the water, while the other is added underneath the water surface.

By "different locations" is meant locations which are different from one another and do not overlap or in any way coincide.

For example, a preferred method of adding the treatment agents involves using two or more perforated hoses, with each hose applying a solution containing one treatment agent to a location of the surface of the enclosure, such that the two or more locations are separate from one another.

Preferably, the treatment agents are added to the surface of the water containing the vertebrate aquatic animals. In such embodiments, the first treatment agent is added to a first location and the second treatment agent is added to a second location, wherein the first and second locations do not overlap. Preferably, the first and second locations are separated by an intervening area of the surface of the enclosure. Preferably, the intervening area is at least 1 m, preferably at least 2m, more preferably at least 5m in its smallest dimension.

In a preferred embodiment, the first treatment agent is added to a first location that is at the surface of the water and the second treatment agent is added to a second location underneath the water surface. An advantage of applying the two treatment agent compositions at different locations is that mixing of the treatment agent molecules will occur more slowly than if the two agents were applied at the same location, meaning that when they do come into contact they will be at very low concentrations, minimising the possibility of a complex being formed.

The first and second lipophilic treatment agents used in the invention are not limited. Suitable treatment agents include organophosphates such as

azamethiphos or dichlorvos; carbamates such as carbaryl; pyrethroids such as cypermethrin or deltamethrin; pyrethrins such as pyrethrum; oxidizing agents; insect growth regulating chemicals such as diflubenzuron or teflurbenzuron; avermectins such as ivermectin or emamectin benzoate; or synergists such as piperonyl butoxide (PBO).

By "synergist" is meant a treatment agent that may have little or no activity against sea lice when used alone, but which increases the potency of the other treatment agent being used. For example, PBO is known to increase the potency of pyrethrins and pyrethroids when used to control sea lice. By "lipophilic" is meant treatment agents containing groups which interact primarily via van der Waals interactions with other molecules. Such groups are unlikely to have strong interactions with water, such that they have a tendency to associate with other lipophilic groups when in an aqueous environment to reduce the overall amount of contact with the surrounding water molecules.

While certain treatment agents disclosed herein contain groups capable of hydrogen bonding (such as azamethiphos), as a whole these compounds are classed as lipophilic within the meaning of the present invention as they also contain some groups which cannot form strong interactions with water and thus will favour the formation of a complex.

Preferably, the first lipophilic treatment agent is an organophosphate or carbamate. Preferably, the second lipophilic treatment agent is a pyrethroid or pyrethrin. Particularly preferably, the first lipophilic treatment agent is an organophosphate or carbamate and the second lipophilic treatment agent is a pyrethroid or pyrethrin.

If the first and second lipophilic treatment agents are introduced to the enclosure sequentially, it is preferred that the first lipophilic treatment agent is an organophosphate or carbamate. More preferably, if the first lipophilic treatment agent is an organophosphate or carbamate, the second lipophilic treatment agent is a pyrethroid or pyrethrin.

Other preferred combinations include the first lipophilic treatment agent being a piperonyl butoxide and the second treatment agent being pyrethroid or pyrethrin.

The organophosphates or carbamates used in the invention may be selected from malathion, parathion, dichlorvos, azamethiphos, chlorpyrifos, chlorthion, trichlorphon, methyl parathion, azinphos methyl, tetrachlorvinphos, phosmet, diazinon, coumaphos, bromophos, dioxathion, ethion, dicrotophos, isophenphos, fenchlorphos and carbaryl.

Preferred organophosphates include parathion, dichlorvos, azamethiphos, chlorthion, trichlorphon, methyl parathion, azinphos methyl, tetrachlorvinphos, phosmet, diazinon, coumaphos, bromophos, dioxathion, dicrotophos, isophenphos and fenchlorphos. The use of azamethiphos or dichlorvos however is preferred, with azamethiphos being the most preferred.

A preferred carbamate is carbaryl. Pyrethroids or pyrethrins used in the invention may be selected from permethrin, phenothrin, cypermethrin, pyrethrin, bifenthrin, resmethrin, sumithrin, tetramethrin, cyfluthrin, transfluthrin, imiprothrin, tau-fluvalinate, fluvalinate, fenpropathrin and deltamethrin. The use of deltamethrin or cypermethrin however is preferred.

Preferred organophosphate and pyrethroid combinations include:

azamethiphos and deltamethrin; azamethiphos and cypermethrin; dichlorvos and deltamethrin; dichlorvos and cypermethrin. The most preferred combinations are azamethiphos and deltamethrin; and azamethiphos and cypermethrin.

In a particularly preferred embodiment, the first treatment agent is an organophosphate or carbamate and is added to a first location that is below the surface of the water and the second treatment agent is a pyrethroid or pyrethrin and is added to a second location at the water surface. Preferably, said first treatment agent is an organophosphate and said second treatment agent is a pyrethroid. More preferably, said first treatment agent is azamethiphos and said second treatment agent is either cypermethrin or deltamethrin.

In some embodiments, the temperature of the water containing the fish is above 10°C. Preferably, one of the lipophilic treatment agents is azamethiphos and the temperature of the water containing the fish is above 10°C.The activity of treatment agents can vary significantly. The skilled person would be aware of the concentrations of treatment agents that should be used, based on published studies or from documents associated regulatory approval for that treatment agent. If necessary, suitable concentrations can be determined by experiment.

Generally speaking, the pyrethroid/pyrethrin concentration when added to the water is preferably up to about 30 ppb (by wt), especially about 0.5 to 20 ppb, particularly about 1 to 15 ppb, most preferably about 2 to 7 ppb. The

organophosphate (e.g. azamethiphos) concentration, in the water, is preferably below about 1000 ppb, especially 0.5 to 100 ppb. Typically, organophosphates such as azamethiphos can be used at levels of 5 to 20 ppb, but in more resistant lice populations higher levels such as 5 to 75 ppb, preferably 5 to 50 ppb may need to be used. The carbamates may be used in the dosages and ratios set out herein for organophosphates.

More specifically, the preferred concentration for deltamethrin is 0.5 to 3 ppb, preferably 1 to 2 ppb, e.g. about 2 ppb, while that for cypermethrin is 5 to 20 ppb, preferably about 5-15 ppb. In one embodiment of the invention, the weight of pyrethroid to organophosphate biocide is about 1 :20 to 10:1 , particularly about 1 :10 to 5:1 , especially about 1 :5 to 5:1 or 1 :3 to 3:1 .

These values represent the concentration of treatment agent in the water containing the vertebrate aquatic animals (i.e. the actual levels needed to control the sea lice population). The concentrations are achieved by adding a

concentrated solution (typically having ppm levels of treatment agent) to the enclosure containing the vertebrate aquatic animals. The skilled person is able to determine how much should be added from knowledge of the volume of the enclosure containing the vertebrate aquatic animals.

Because of the lipophilic natures of the treatment agents used in the invention, use of metal equipment is preferred. As treatment agents are typically added to enclosures via perforated hoses, metal, particularly aluminium, hoses are preferred. Lipophilic molecules can cling to plastic surfaces, reducing the amount of treatment agent passing through a plastic or rubber hose.

However, plastics that have been specially formulated to reduce their lipophilicity are also suitable, or materials that have been coated with such plastics. For example, the invention can suitably be carried out using equipment (e.g. tanks, hoses) coated with polytetrafluoroethylene (PTFE) such as Teflon®. More generally, any plastic having a low lipophilicity may be used, either for a coating or for the equipment itself.Another embodiment the invention is directed to a kit comprising in separate containers a first lipophilic treatment agent composition (for example comprising a carbamate or organophosphate) and a second topical lipophilic treatment agent composition (for example comprising a pyrethroid or pyrethrin), and preferably also instructions for the use of said compositions in the method of the invention.

In another embodiment the invention is directed to the use of a first lipophilic treatment agent (such as an organophosphate or carbamate) and a second lipophilic treatment agent (such as a pyrethroid or pyrethrin) for the preparation of treatment agent compositions for application to a vertebrate aquatic animals to combat infestation by multicellular ectoparasites with exoskeletons, characterised in that said administration comprises simultaneously introducing the first and second treatment agents to an enclosure containing the vertebrate aquatic animals as separate treatment agent compositions, preferably at different locations.

In another embodiment the invention is directed to a combination of a first and a second lipophilic treatment agent for use in a method of treating vertebrate aquatic animals to combat infestation by multicellular ectoparasites with

exoskeletons, which method comprises topically exposing farmed fish to a first and a second lipophilic treatment agent, characterised in that the first and a second lipophilic treatment agents are introduced separately to an enclosure containing the vertebrate aquatic animals, preferably to different locations.

The lipophilic treatment agents used in the method of the invention may take any convenient application form, e.g. solution, dispersion, powder, etc. Since they will generally be diluted on or before application, their concentrations and formulations are not critical. Commercially available compositions may be used in the method of the invention.

Preferred features of the invention may be combined in any manner. Thus certain features which are, for clarity, described herein in the context of separate embodiments, may be combined in any manner. Conversely, various features that are, for brevity, described in the context of a single preferred feature, may also be provided separately or in any subcombination. Further, reference to values stated in ranges include each and every value within that range.

Examples The following non-limiting Examples further illustrate the present invention. Commercial Trial 1

Method: To a large sea cage (~160m diameter) holding salmon in coastal waters off Gunnaraya, Snillfjord, Norway was added -2.5 kg Salmosan (azamethiphos ) via a tube under the surface of the water, at a depth of 1 -3 m. At the same time,

Alphamax (deltamethrin) was added to the surface of the water. The final concentration of azamethiphos (the active ingredient in Salmosan) in the water in which the fish were held was 50 ppb, and the final concentration of deltamethrin (the active ingredient in Alphamax) in the water in which the fish was held was 2 ppb. The concentration of azamethiphos (50 ppb) is half the recommended dosage. The fish were exposed to the treatment agents for 40 minutes.

The method resulted in 96% lice removal. Repeat Trials

The protocol described above was repeated at numerous commercial salmon farms along the Norwegian coast. The trials took place during the Autumn/Winter/Spring months, such that the temperature of the sea water in which the fish were held was below 10°C.

Three sizes of sea cages were used, the largest of which has a diameter of approximately 160 m and holds 700-850 tonnes of fish (as per the example above). Medium sized cages (approx. 120 m in diameter) hold 300-400 tonnes of fish.

Small sea cages hold around 100 tonnes of fish and have an approximate size of 20 m x 20 m. The size of the sea cage is not relevant for the efficacy of the claimed method. For smaller sea cages, the amount of Salmosan/Alphamax was adjusted such that the final concentrations of treatment agents in the water remained the same. Total exposure time was again held at 40 minutes for all cage sizes.

Following numerous trials, it was found that the methodology consistently gave commercially acceptable results, with typically around 95% lice removal.

In locations where poor results were obtained, the protocol was repeated with equivalent to 5 Kg Salmosan for a 160 m cage (i.e. 100 ppb azamethiphos, the recommended dose). Poorer results were attributed to resistant sea lice populations.

Alternative Protocol

The above protocol can be adapted to use cypermethrin in place of deltamethrin, such that the final concentration of cypermethrin in the water in which the fish are held is 15 ppm. Comparison with the Sequential Method of WO 2009/010755

Salmon held in a sea cage were treated in accordance with the method of WO 2009/010755 with 500 g Salmosan (10% of the recommended dose, 10 ppb final concentration in water), followed 15 minutes later by a full dose of Alphamax (i.e. 2 ppb final concentration in water). The fish were held in the sea cage with the treatment agents for 60-65 minutes. The method was repeated at various locations and was found to be between 70 and 75% effective (actual results: 70%, 73%, 75%).

At the same locations, the fish were treated in accordance with the method described above, i.e. 2.5 kg Salmosan (50% of the recommended dose, 50 ppb final concentration in water) was added under the water at a depth of 1 -3 m, whilst a full dose of Alphamax (i.e. 2 ppb final concentration in water) was added at the surface of the water. The fish were held in the sea cage with the treatment agents for 40 minutes. The method was found to be consistently more effective than the method described above, with commercially acceptable results obtained in each case, i.e. about 95% lice removal. The method of WO 2009/010755 discloses a sequential method of treating sea lice with a first sea lice treatment agent that is an organophosphate or carbamate, followed by treatment with a second sea lice treatment agent that is a pyrethroid or pyrethrin. The advantage of the sequential method is that it allows very low dosages of treatment agents to be used. However, the method of the present invention allows the treatment times to be reduced by roughly 1/3 to only 40 minutes. This is desirable since with reduced treatment times, there is a reduced risk of oxygen levels dropping dangerously low during treatment, risking suffocation of the fish. There is a further advantage that reduced treatment times allow the method of the present invention to be used in warmer seas, since when sea temperatures are above 10°C, azamethiphos use is restricted to 30 minutes for the health of the fish. Further embodiments of the invention include:

Embodiment 1 : A method of treating vertebrate aquatic animals to combat infestation by multicellular ectoparasites with exoskeletons, which method comprises topically exposing vertebrate aquatic animals to a first and a second lipophilic treatment agent, characterised in that the first and the second lipophilic treatment agents are simultaneously introduced to an enclosure containing the vertebrate aquatic animals as separate treatment agent compositions. Embodiment 2: A method as in Embodiment 1 wherein said multicellular ectoparasites with exoskeletons are sea lice.

Embodiment 3: The method of either of Embodiments 1 or 2 wherein said vertebrate aquatic animals are fish.

Embodiment 4: The method of any one of Embodiments 1 to 3, wherein the first and second lipophilic treatment agents are introduced to different locations of the enclosure. Embodiment 5: The method of any one of Embodiments 1 to 4, wherein said first and a second lipophilic treatment agents are introduced to the surface of the water of the enclosure.

Embodiment 6: The method of Embodiment 6 wherein said first lipophilic treatment agent is introduced to a first area on the surface of the water and a second lipophilic treatment agent is introduced to a second area on the surface of the water, wherein the first and second areas do not overlap.

Embodiment 7: The method of any one of Embodiments 1 to 4 wherein said first lipophilic treatment agent is introduced underneath the water of the enclosure and a second lipophilic treatment agent is introduced at the surface of the water in the enclosure.

Embodiment 8: The method of any one of Embodiments 1 to 7 wherein said first lipophilic treatment agent is a carbamate, an organophosphate or piperonyl butoxide and wherein said second lipophilic treatment agent is a pyrethroid or a pyrethrin.

Embodiment 9: The method of Embodiment 8 wherein said pyrethroid is selected from permethrin, phenothrin, cypermethrin, pyrethrin, bifenthrin, resmethrin, sumithrin, tetramethrin, cyfluthrin, transfluthrin, imiprothrin, tau-fluvalinate, fluvalinate, fenpropathrin and deltamethrin.

Embodiment 10: The method of Embodiment 8 wherein said organophosphate is selected from malathion, parathion, dichlorvos, azamethiphos, chlorpyrifos, chlorthion, trichlorphon, methyl parathion, azinphos methyl, tetrachlorvinphos, phosmet, diazinon, coumaphos, bromophos, dioxathion, ethion, dicrotophos, isophenphos and fenchlorphos. Embodiment 1 1 : The method of any preceding Embodiment wherein the first and second lipophilic treatment agents are selected from:

azamethiphos and deltamethrin;

azamethiphos and cypermethrin;

dichlorvos and deltamethrin; and

dichlorvos and cypermethrin.

Embodiment 12: The method of any preceding Embodiment wherein the vertebrate aquatic animals are exposed to said first and a second lipophilic treatment agents for up to 60 minutes.