WO2023131615A1

WO2023131615A1 - Method to detach ectoparasites from fish

Info

Publication number: WO2023131615A1
Application number: PCT/EP2023/050096
Authority: WO
Inventors: Lene HØGSET
Original assignee: Intervet International B.V.; Intervet Inc.
Priority date: 2022-01-05
Filing date: 2023-01-04
Publication date: 2023-07-13

Abstract

The invention is directed to a method to detach ectoparasites from fish in water. It was found that a fish anaesthetic agent in the form of an emulsion detaches the ectoparasites from the fish in the water. The method can be used on its own or it can be combined with a mechanical ectoparasite removal treatment.

Description

Method to detach ectoparasites from fish

Field of the invention

The present invention relates to the field of detachment of ectoparasites from fish. More in particular the detachment of sea lice.

Background

Infestation of fish by parasites is a major problem for commercial fish farming. Fish farmers who are confronted with a parasite problem usually suffer substantial financial losses and carry additional expenses.

Sea lice are ectoparasites that belong to the sub-class of copepoda which affect fish, particularly farmed salmonids, negatively by feeding on the mucus, skin, tissue, and blood of the fish host. Sea lice can cause significant harm (i.e., serious fin damage, skin erosion, bleeding, and open wounds) to host fish. Additionally, sea lice can cause chronic stress response in fish, which in turn can make them susceptible to other diseases. In addition, it appears that the sea lice have immunomodulatory effects on the host fish and can function as a vector in the transmission of other fish diseases. Damages due to parasitic infestations from sea lice result in considerable animal welfare issues, fish losses and increased expense. Infestation with sea lice is considered one of the most important disease problems in salmonid farming, especially Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

Infestation with sea lice can also occur in other fish species, for example, sea bass, tilapia, carp, and the like. In addition to the costs that are associated with treatment, lower classification ratings of slaughtered fish and reduced growth rate due to reduced feed intake contribute to the economic losses.

Sea lice are meanwhile widely prevalent and encountered in all fish farms. Severe infestation kills the fish. Mortality rates of over 50%, based on sea lice infestation, have been reported from Norwegian fish farms. The extent of the damage depends on the time of year and on environmental factors, for example the salinity of the water and average water temperature. In a first phase, sea lice infestation is seen in the appearance of the parasites attached to the fish and later — even more clearly — from the damage caused to skin and tissue. The most severe damage is observed in smolts which are just in the phase in which they change from fresh water to sea water. The situation is made even worse by the specific conditions in the fish farms, where salmon of different age groups but of the same weight class are kept together; where fouled nets or cages are used; where high salt concentrations are to be found; where flow through the nets and cages is minimal and the fish are kept in a very narrow space.

There are a number of treatments already on the market for controlling sea lice including bath treatments, such as organophosphates (for example, dichlorvos and azamethiphos) and pyrethroids (for example, cypermethrin and deltamethrin), and in- feed-treatments, such as avermectins (for example, ivermectin and emamectin benzoate) and growth regulators (for example, lufenuron and teflubenzuron). However, resistance to many of these treatments has been observed and therefore, a need for new treatments remains.

Also non-medical approaches have been used to improve the management of parasites, such as the use of sea lice skirts, that are mounted around the top portion of salmon pens, to act as a shield, preventing sea lice within the upper part of the water from entering a salmon farm. Bubble Curtains are made by a device placed around the bottom circumference of the salmon pen and releases a line of air bubbles that rise from the bottom to the surface of the pen. The air bubbles create a barrier or wall and sea lice are deterred from entering the salmon pen.

Thermal treatments (i.e. Thermolicer/ Optilicer) use the low tolerance of sea lice and other parasites for sudden changes in water temperature. A thermal treatment system is fitted to a boat or barge near to a salmon pen and fish are briefly passed through the system and bathed in lukewarm water. This temperature change kills the sea lice and the salmon are released back into the pen.

Flushers (i.e. Hydrolicer, Flatsetsund Flusher) can dislodge sea lice and other parasites by jets of water (like a shower). This is taken advantage of by moving salmon through a system with waterjets to flush off lice. A flusher system is fitted to a boat or barge near to salmon pens, and fish are briefly passed through the flusher. Detached lice are collected and then salmon are returned back into the pen.

Lasers are used together with a camera to detect sea lice on the salmon, and once a louse is detected a laser pulses directly at the specific louse. The louse is hit and killed by the laser - removing the louse from the salmon.

H2O2 (Hydrogen Peroxide) is known to temporarily paralyze sea lice, causing them to fall off of the salmon and reducing their ability to re-attach. Salmon are temporarily exposed to H2O2 - either in a well boat or a specifically designed tarpaulin where H2O2 is added. After this, seawater is flushed through the pen to dilute the H2O2, breaking it down rapidly into water and oxygen.

Freshwater treatments can also be used. Sea lice are sensitive to fresh water (i.e. not sea water) and generally cannot survive when water salinity is very low. Freshwater treatments exploit this and through temporarily exposing salmon to fresh water, the sea lice detach and can then be removed.

In the wild, salmon migrate to freshwater to spawn and this change in water removes any sea lice.

Sea Lice Trap is a method that is similar to a wasp catcher. By placing traps in the water around salmon farms, they attract lice away from the pens and into the traps, therefore removing them. To attract as many sea lice as possible, some traps have flashing lights which have been found to attract sea lice.

Combinations of low voltage electric fields, waterjets, and a minimal thermal treatment (i.e. from Ace Aquatec) are used to remove sea lice. These non-medical treatments involve large investments in the devices to carry out the removal of ectoparasites on fish and there is always a risk of damaging the fish. Therefore a need remains for treatment of ectoparasites in fish.

Surprisingly it has been found that when an emulsion comprising a fish anaesthetic agent is used the ectoparasites on the fish detach from the fish. The method can advantageously be performed when the fish are in the water by adding the emulsion comprising the fish anaesthetic agent to a body of water comprising the fish. Such a method allows that the fish can remain in the water and do not need to be transferred or transported or taken out of the water. After detachment, the ectoparasites can then easily be removed from the body of water by e.g. filtration of the water.

Fish anaesthetic agents are used to anaesthesize fish for treatment of diseases, such as vaccination and/or when moving or transporting fish from one location to another.

Chance et al. (J Fish Dis, Feb 2018, 41(2), 291-298 ) studied amoebic gill disease (AGD). Neoparamoeba perurans is the causitve effect of AGD. In this study it was investigated whether exposure of anaesthetic agent AQLII-S had an effect on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28-day period to AQLII-S (isoeugenol), showed no statistical difference between treated amoebae and non-treated amoebae. This infers that with repeated exposure to AQUI-S, an increased proportion of amoeba remain attached to their substrate. Recent work from Shijie et al. (2016) found that AQUI-S at varying concentrations showed no significant effect on the viability or attachment capabilities of N. perurans 2 hr after single treatment. This suggests that repeated exposure of AQUI-S has no significant effect on the viability of N. perurans.

Summary of invention

In a first aspect, the invention is directed to a fish anaesthetic agent for use in a method to detach ectoparasites from a fish wherein the fish anaesthetic agent is in the form of an emulsion.

In an embodiment of the first aspect, the fish are in a body of water and the method comprises adding a composition comprising an emulsion of the fish anaesthetic agent in the body of water comprising the fish.

In a second aspect, the invention is directed to a method to detach ectoparasites from fish comprising applying a composition comprising an emulsion of a fish anaesthetic agent.

In an embodiment of the second aspect, the fish are in a body of water and the composition comprising an emulsion of a fish anaesthetic agent is added to the body of water comprising the fish.

In an embodiment of the invention and/or embodiments thereof, the detached ectoparasites are collected or removed from the body of water. In an embodiment of the invention and/or embodiments thereof, the anaesthetic agent is selected from the group consisting of tricaine methanesulfonate, metomidate, phenoxyethanol, etomidate, eugenol, isoeugenol, benzocaine, linalool, and quinaldine.

In an embodiment of the invention and/or embodiments thereof, the anaesthetic agent is selected from the group consisting of eugenol and isoeugenol.

In an embodiment of the invention and/or embodiments thereof, the emulsion is an oil-in-water emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises an emulsifier and/or a cyclodextrin.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises an emulsifier and the emulsifier is selected from the group consisting of detergent, polyglycerolester, lecithin, polyethylene glycol and polyethylene glycol ester.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises an emulsifier selected from the group consisting of polyethylene glycol, polyethylene glycol ester and polyglycerol.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent has a concentration of 10-80% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises an emulsifier and the emulsifier has concentration of 20-90% of the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises cyclodextrin and the cyclodextrin has a concentration of 20-90 % in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent is used to a concentration of 1-150 ppm in the body of water.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent is used for a time of 1-600 minutes.

In an embodiment of the invention and/or embodiments thereof, the fish are salmonid.

In an embodiment of the invention and/or embodiments thereof, the ectoparasite is a sealice, preferably the parasite is from the family caligidae, preferably from the genus Lepeophtheirus or Caligus. In an embodiment of the invention and/or embodiments thereof, the method comprises a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, freshwater treatment, waterjet treatment.

In an embodiment of the invention and/or embodiments thereof, the method does not comprise a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment.

Detailed description

The present invention is directed to a method to detach ectoparasites from fish in water. It is beneficial to be able to treat the fish in the water so avoiding transporting the fish from one location to another and/or to take the fish out of the water. It was found that an emulsion comprising a fish anaesthetic agent can be used to detach ectoparasites from fish when the fish are in the water. The emulsion comprising the fish anaesthetic agent can be added to a body of water comprising fish. After a while, the ectoparasites will detach from the fish and the ectoparasites can then be collected e.g. by filtering the water. in a body of water comprising fish. Once the ectoparasites are detached they can be removed from the water. This removal or collecting can be accomplished with a filter or net. A fine mesh sock or fine mesh net is suitably used for removal of detached ectoparasites. A water separator may also be utilised to segregate the detached ectoparasites from the fish.

A body of water is an enclosure comprising water. A body of water can be natural or artificial. The body of water can be (part of) an ocean, a river, a lake, a reservoir, a lagoon, a pond, a canal, an estuary, a stream, an oceanic bay, a river bay, a swamp, a pond, a port, or a bay. Suitably the body of water is an aquaculture facility. The aquaculture facility can be water-based or land-based. Suitably aquaculture facilities are cages, pens, either inshore or off-shore, raceways, tanks, enclosures, well boat water and fish storage vessels, net pens, tarpaulins, harbours and fjords. In the present invention, the body of water is suitably an artificial enclosure of water. Suitably the body of water is a body of water enclosed on a boat, vessel, or barge, such as a well. Suitably the body of water is a well, net pen, or tarpaulin.

Crowding is when the fish are in a volume that is smaller than normal and the density of the fish is increased. Crowding of fish is when the density of the fish is more than 10 kg/m³, more than 20 kg/m³, more than 30 kg/m³, more than 40 kg/m³, more than 50 kg/m³, more than 60 kg/m³, more than 70 kg/m³’ more than 80 kg/m³, more than 90 kg/m³, more than 1000 kg/m³, more than 110 kg/m³, more than 130 kg/m³, more than 140 kg/m³, more than 150 kg/m³, more than 160 kg/m³, more than 170 kg/m³, more than 180 kg/m³, more than 190 kg/m³, more than 200 kg/m³, more than 220 kg/m³, more than 240 kg/m³, more than 260 kg/m³, more than 280 kg/m³, or even more than 300 kg/m³ Suitably the densitiy of the fish when crowding is between 30-300 kg/m³, more suitably between 40-250 kg/m³, more suitably between 50-200 kg/m³, more suitably between 60-175 kg/m³, more suitably between 70-150 kg/m³, more suitably between 80-125 kg/m³, more suitably between 90-110 kg/m³. Suitable density of fish for crowding is between 50- 100 kg/m³, or between 30-100 kg/m³ Crowding of fish is a normal procedure in fish aquiculture and can be carried out by reducing the volume of the body of water by many ways known to the skilled person. Suitable reduction of the volume of the body of water can be carried out by the use of tarpaulins, bags or containers, e.g. such as described in WO2014204319, or by lowering the volume of water e.g. in tanks.

According to the invention and/or embodiments thereof the anaesthetic agent is safe to use with fish. There are many fish anaesthetic agents that are used to sedate fish e.g for treatment of fish, such as vaccination. Suitable fish anaesthetic agents are selected from the group consisting of tricaine methanesulfonate, metomidate, phenoxyethanol, etomidate, eugenol, isoeugenol, benzocaine, linalool, and quinaldine.

Tricaine methanesulfonate, also known as metacaine or MS-222 (ethyl 3-aminobenzoate), is sold under the tradename Tricaine-S and Finquel. It comes as a white, crystalline powder that can be dissolved in water at up to an 11% solution. Suitably tricaine methanesulfonate is used in a concentration of 40-250 mg/L. Suitably concentration for salmonid fish is between 40-60 mg/L.

Benzocaine (ethyl 4-aminobenzoate), is a white crystal that is chemically similar to MS-222. Benzocaine is usually first dissolved in ethanol or acetone before it is added to water. The standard approach is to prepare a stock solution in ethanol or acetone (usually 100 g/L) that will keep for more than a year when sealed in a dark bottle. Suitably benzocaine is used in a concentration of 40-150 mg/L. Suitably concentration for salmonid fish is between 40-60 mg/L.

Linalool

can be used to sedate fish (see e.g. US20140259191). Suitably the concentration of linalool in water is between 5 ppm and 500 ppm.

Quinaldine (2-methylquinoline) is a yellowish, oily liquid with limited water solubility that is usually dissolved in acetone or alcohol before it is mixed with water. Quinaldine sulfonate is a pale yellow, water- soluble powder. The effective treatment concentration of quinaldine solutions varies with species, but is generally 10 to 100 mg/L, sometimes 50 to 1 ,000 mg/L. Suitably concentration for salmonid fish is between 25-40 mg/L.

2-Phenoxyethanol is an opaque, oily liquid. This drug is moderately soluble in water but freely soluble in ethanol. Suitable concentrations are between 100 to 600 mg/L. Suitably concentration for salmonid fish is between 100-200 mg/L. Metomidate (methyl 3-(1-phenylethyl)imidazole-4-carboxylate hydrochloride) and etomidate (ethyl-l- methylbenzyl-imidazole-5-carboxylate) have been used extensively in human medicine. It anesthetizes fish without the usual stress of an elevated heart rate. Induction is rapid — 1 to 2 minutes — and recovery is faster than with MS-222. Suitably it is used in a concentration of 2-10 mg/L. Suitably concentration for salmonid fish is between 2-6 mg/L.

Eugenol has been used to anaesthetize fish and is a component of clove oil (70 to 90 percent by weight). It is an effective anesthesia at doses of 2 - 120 mg/L. Suitably it is used in a concentration of 40-120 mg/L. Suitably concentration for salmonid fish is between 10-50 mg/L.

Isoeugenol is an aesthetic agent used in fish at doses of 10 to 250 mg/L. Suitable concentration for salmonid fish is between 2 and 20 mg/L.

Isoeugenol can exist as two geometric isomers, trans and cis, shown below.

CH₃ “

/raw-isomer cis-isomer

Preferably the trans-isomer of isoeugenol is used, or a mixed form wherein more trans isomer is present than cis-isomer. Suitably, 60% or more of the isoeugenol is in the trans form, more suitably 65% of the isoeugenol is in the trans form, more suitably 70% of the isoeugenol is in the trans form, more suitably 75% of the isoeugenol is in the trans form, more suitably 80% of the isoeugenol is in the trans form, more suitably 85% of the isoeugenol is in the trans form, more suitably 90% of the isoeugenol is in the trans form, more suitably 95% of the isoeugenol is in the trans form. Most suitably, the isoeugenol is substantially free of the cis isomer. Preferably, 2% or less of the cis isomer present. Preferably the trans isomer is provided in a form which is greater than 99% pure, ie with less than 1% of the cis isomer present and more preferably greater than 99.5% pure. The presence of the cis isomer may be detected using either nuclear magnetic resonance (NMR) spectroscopy or gas liquid chromatography (GLC) . Proton NMR is a preferred method for detecting the presence of the cis isomer. Isoeugenol in the form of the trans isomer substantially free of the cis isomer may be obtained from a mixture of the cis and trans isomers using methods known in the art, for example as described by Boedecker, Fr. And Volk, H., Her., 64B, 61 (1931), and by Bruckner- Wilhelms, A. Ann. Univ. Sci. Budapest, Rolando Eotvos Nominatae, Sect Chim., 13, pp 101-5 (1972); and in Chemical Abstracts 80, 95415C (1974). These methods involve separating out the trans isomer as its sodium salt, acidifying, dissolving the product in solvent and distilling to obtain the pure isomer. Isoeugenol is commercially available as AQUI-S and ICTYOCLOVE.

The fish anaesthetic agent is used in the form of an emulsion. Although in principle the fish anaesthetic agent can be used as such or dissolved in alcohol, it was found that the fish anaesthetic agent in the form of an emulsion had superior properties. Suitably the emulsion is an oil-in-water emulsion. Suitably, the emulsion comprises an emulsifier and/or a cyclodextrin.

Suitably, the emulsion comprises an emulsifier. Suitably, this emulsifier can be any commercially available emulsifier that stabilizes an oil-in-water emulsion. Emulsifier for oil in water emulsions; O/W emulsions are made with more water-soluble emulsifiers (HLB 8 - 18) that can either be nonionic, anionic or cationic. Nonionic emulsifiers are by far the most widely used O/W emulsifiers as they are unaffected by pH changes and are available in a large variety and also made with natural components. Suitably, the emulsifier is selected from the group consisting of detergent, polyglycerolester, lecithin, and polyethylene glycol. More suitably, the emulsifier is polyethylene glycol, polyethylene glycol ester, or polyglycerol ester.

The emulsion may comprise a polyethylene glycol (PEG). Polyethylene glycol with an average molecular weight of 200-700 are liquid, whereas others of higher molecular weights are liquefiable. Polyethylene glycol with an average molecular weight of 200-700 are preferred.

Commercially available polyethylene glycol include PEG 200, PEG 300, PEG 400 and PEG 600. Suitably the polyethylene glycol is PEG 400.

Suitable polyethylene glycol ester is polyethylene glycol sorbitan ester with a fatty acid. An example of a polyethylene glycol sorbitan esters with a fatty acid is Polysorbate. Examples of polysorbates are:

• Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate)

• Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate)

• Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate)

• Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate)

Polysorbates are commercially available under the brand names Kolliphor.Scattics, Alkest, Canarcel, and

Tween. Particularly suitable is polyethylene oxide sorbitan mono-oleate (polysorbate 80). This surfactant is commercially available under the trade name Polysorbate 80, Alkest TW 80, Scattics, Canarcel, Poegasorb 80, Montanox 80, Tween 80.

Polyglycerol fatty acid esters are suitable as emulsifier for the emulsion. Polyglycerol fatty acid esters are usually a mixture of reaction products formed by the esterification of polyglycerols with fats and oils or with fatty acids. Polyglycerol esters of fatty acids (PEFA) (E 475) are commercially available.

Suitably the emulsion comprises a cyclodextrin. Cyclodextrins constitute a group of natural cyclic oligosaccharides derived from starch with six, seven, or eight glucose molecules bound by a(1-4) glycosidic bonds forming a cylindrical-shaped structure referred to as a-, p. and y-cyclodextrins, respectively. The central cavity of these molecules is hydrophobic, whereas the outer edges of the walls surrounding this cavity are hydrophilic. This hydrophobic cavity forms inclusion complexes with a wide range of organic host molecules. Suitably the fish anaesthetic agent forms a water-soluble inclusion complex with -cyclodextrins, preferably p-cyclodextrins. The proportion of the fish anaesthetic agent in the inclusion complex is comprised between 5 and 30% of the total mass of the complex, preferably between 10 and 20%, and the proportion of cyclodextrins in the inclusion complex is comprised between 70 and 95% of the total mass of the complex, preferably between 80 and 90%.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: A fish anesthetic agent and an emulsifier, or a fish anesthetic agent and cyclodextrin, or a fish anesthetic agent, an emulsifier and a cyclodextrin.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: A fish anesthetic agent and polyethylene glycol ester, or a fish anesthetic agent and p-cyclodextrin, or a fish anesthetic agent, a polyethylene glycol ester and a p-cyclodextrin, or a fish anesthetic agent, a polyethylene glycol ester and a polyethylene glycol with an average molecular weight between 200 and 700.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: A fish anesthetic agent selected from eugenol or isoeugenol and an emulsifier, or a fish anesthetic agent selected from eugenol or isoeugenol and cyclodextrin, or a fish anesthetic agent selected from eugenol or isoeugenol, an emulsifier and a cyclodextrin..

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: A fish anesthetic agent selected from eugenol or isoeugenol and a polyethylene glycol ester, or a fish anesthetic agent selected from eugenol or isoeugenol and p-cyclodextrin, or a fish anesthetic agent selected from eugenol or isoeugenol, a polyethylene glycol ester and a p- cyclodextrin, or a fish anesthetic agent selected from eugenol or isoeugenol, a polyethylene glycol ester and polyethylene glycol with an average molecular weight between 200 and 700.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise:

A fish anesthetic agent and polysorbate, or a fish anesthetic agent and p-cyclodextrin, or a fish anesthetic agent, a polysorbate and a p-cyclodextrin, or a fish anesthetic agent, a polysorbate and a PEG 400.

A fish anesthetic agent selected from eugenol or isoeugenol and polysorbate, or a fish anesthetic agent selected from eugenol or isoeugenol and p-cyclodextrin, or a fish anesthetic agent selected from eugenol or isoeugenol, a polysorbate and a p-cyclodextrin, or a fish anesthetic agent selected from eugenol or isoeugenol, a polysorbate and PEG 400.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent has a concentration of 1 %-95% in the emulsion. More preferably, the fish anaesthetic agent has a concentration of 10%-80% in the emulsion. More preferably, the fish anaesthetic agent has a concentration of 15%-75% in the emulsion, more preferably, the fish anaesthetic agent has a concentration of 20%-70% in the emulsion, more preferably, the fish anaesthetic agent has a concentration of 25%-65% in the emulsion, more preferably, the fish anaesthetic agent has a concentration of 30%-60% in the emulsion, more preferably, the fish anaesthetic agent has a concentration of 35%-55% in the emulsion, more preferably, the fish anaesthetic agent has a concentration of 40%-50% in the emulsion.

In the present invention and/or embodiments thereof, % are % per weight unless otherwise indicated.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises an emulsifier and the emulsifier has concentration of 20-90% of the emulsion. More preferably, the emulsifier has a concentration of 25%-85% in the emulsion, more preferably, the emulsifier has a concentration of 30%- 80% in the emulsion, more preferably, the emulsifier has a concentration of 35%-75% in the emulsion, more preferably, the emulsifier has a concentration of 40%-70% in the emulsion, more preferably, the emulsifier has a concentration of 45%-65% in the emulsion, more preferably, the emulsifier has a concentration of 50%-60% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises cyclodextrin and the cyclodextrin has a concentration of 20-90 % in the emulsion. More preferably, the cyclodextrin has a concentration of 25%-85% in the emulsion, more preferably, the cyclodextrin has a concentration of 30%- 80% in the emulsion, more preferably, the cyclodextrin has a concentration of 35%-75% in the emulsion, more preferably, the cyclodextrin has a concentration of 40%-70% in the emulsion, more preferably, the cyclodextrin has a concentration of 45%-65% in the emulsion, more preferably, the cyclodextrin has a concentration of 50%-60% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises a polyethylene glycol and the polyethylene glycol has a concentration of 1-95% in the emulsion, More preferably the polyethylene glycol has a concentration of 5%-90% in the emulsion, more preferably the polyethylene glycol has a concentration of 10%-85% in the emulsion, more preferably the polyethylene glycol has a concentration of 15%-80% in the emulsion, more preferably the polyethylene glycol has a concentration of 20%-75% in the emulsion, more preferably the polyethylene glycol has a concentration of 25%-70% in the emulsion, more preferably the polyethylene glycol has a concentration of 30%-65% in the emulsion, more preferably the polyethylene glycol has a concentration of 35%-60% in the emulsion, more preferably the polyethylene glycol has a concentration of 40%-55% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises a polyethylene glycol and the polyethylene glycol has a concentration of 1-50% in the emulsion, More preferably the polyethylene glycol has a concentration of 2%-45% in the emulsion, more preferably the polyethylene glycol has a concentration of 4%-40% in the emulsion, more preferably the polyethylene glycol has a concentration of 5%-35% in the emulsion, more preferably the polyethylene glycol has a concentration of 6%-30% in the emulsion, more preferably the polyethylene glycol has a concentration of 7%-25% in the emulsion, more preferably the polyethylene glycol has a concentration of 8%-20% in the emulsion, more preferably the polyethylene glycol has a concentration of 9%-15% in the emulsion, more preferably the polyethylene glycol has a concentration of 10%-12% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises a polyethylene glycol and a further emulsifier, and the polyethylene glycol has a concentration of 1 %-20% and the further emulsifier has a concentration of 20%-70% in the emulsion, more preferably the polyethylene glycol has a concentration of 2%-18% and the further emulsifier has a concentration of 25%-65% in the emulsion, more preferably the polyethylene glycol has a concentration of 5%-15% and the further emulsifier has a concentration of 30%-60% in the emulsion, more preferably the polyethylene glycol has a concentration of 7%-12% and the further emulsifier has a concentration of 40%-55% in the emulsion, more preferably the polyethylene glycol has a concentration of 8%-10% and the further emulsifier has a concentration of 45%-50% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises a polysorbate and the polysorbate has a concentration of 1-95% in the emulsion, More preferably the polysorbate has a concentration of 5%-90% in the emulsion, more preferably the polysorbate has a concentration of 10%- 85% in the emulsion, more preferably the polysorbate has a concentration of 15%-80% in the emulsion, more preferably the polysorbate has a concentration of 20%-75% in the emulsion, more preferably the polysorbate has a concentration of 25%-70% in the emulsion, more preferably the polysorbate has a concentration of 30%-65% in the emulsion, more preferably the polysorbate has a concentration of 35%- 60% in the emulsion, more preferably the polysorbate has a concentration of 40%-55% in the emulsion. In an embodiment of the invention and/or embodiments thereof, the emulsion comprises a polysorbate and the polysorbate has a concentration of 1-50% in the emulsion, More preferably the polysorbate has a concentration of 2%-45% in the emulsion, more preferably the polysorbate has a concentration of 4%- 40% in the emulsion, more preferably the polysorbate has a concentration of 5%-35% in the emulsion, more preferably the polysorbate has a concentration of 6%-30% in the emulsion, more preferably the polysorbate has a concentration of 7%-25% in the emulsion, more preferably the polysorbate has a concentration of 8%-20% in the emulsion, more preferably the polysorbate has a concentration of 9%- 15% in the emulsion, more preferably the polysorbate has a concentration of 10%-12% in the emulsion.

In an embodiment of the invention and/or embodiments thereof, the emulsion comprises a polysorbate and a further emulsifier, and the polysorbate has a concentration of 1%-20% and the further emulsifier has a concentration of 20%-70% in the emulsion, more preferably the polysorbate has a concentration of 2%-18% and the further emulsifier has a concentration of 25%-65% in the emulsion, more preferably the polysorbate has a concentration of 5%-15% and the further emulsifier has a concentration of 30%-60% in the emulsion, more preferably the polysorbate has a concentration of 7%-12% and the further emulsifier has a concentration of 40%-55% in the emulsion, more preferably the polysorbate has a concentration of 8%-10% and the further emulsifier has a concentration of 45%-50% in the emulsion.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: 10%-80% fish anesthetic agent and 20%-90% emulsifier, or 10%-80% fish anesthetic agent and 20%-90% cyclodextrin, or 10%-80% fish anesthetic agent, 20%-70% emulsifier and 20%-70% cyclodextrin, or 10%-80% fish anesthetic agent, 20%-70% emulsifier and 1%-20% polyethylene glycol.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: 10%-80% fish anesthetic agent and 20%-90% polysorbate, or 10%-80% fish anesthetic agent and 20%-90% p-cyclodextrin, or 10%-80% fish anesthetic agent, 20%-70% polysorbate and 20%-70% p-cyclodextrin, or 10%-80% fish anesthetic agent, 20%-70% polysorbate and 1%-20% polyethylene glycol with an average molecular weight between 200 and 700.

In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise: 10%-80% fish anesthetic agent selected from eugenol or isoeugenol and 20%-90% emulsifier, or 10%-80% fish anesthetic agent selected from eugenol or isoeugenol and 20%-90% cyclodextrin, or 10%-80% fish anesthetic agent selected from eugenol or isoeugenol, 20%-70% emulsifier and 20%-70% cyclodextrin, or 10%-80% fish anesthetic agent selected from eugenol or isoeugenol, 20%-70% emulsifier and 1%-20% polyethylene glycol. In preferred embodiments of the invention and/or embodiments thereof, the emulsion may comprise:

10%-80% fish anesthetic agent selected from eugenol or isoeugenol and 20%-90% polysorbate, or

10%-80% fish anesthetic agent selected from eugenol or isoeugenol and 20%-90% p- cyclodextrin, or

10%-80% fish anesthetic agent selected from eugenol or isoeugenol, 20%-70% polysorbate and

20%-70% p-cyclodextrin, or

10%-80% fish anesthetic agent selected from eugenol or isoeugenol, 20%-70% polysorbate and 1%-20% polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

30-60 % fish anaesthetic agent ;

25-65 % polysorbate; and

5-15 % polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

35-55 % fish anaesthetic agent ;

30-60 % polysorbate; and

8-12 % polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

40-50 % fish anaesthetic agent ;

35-55 % polysorbate; and

9-11 % polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

50% fish anaesthetic agent ;

40% polysorbate; and

10% polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

2-20% fish anaesthetic agent ;

2-30 % polysorbate; and

50- 96% polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

5-15% fish anaesthetic agent ;

5-25 % polysorbate; and

60-90% polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise 7-15% fish anaesthetic agent ;

7-20 % polysorbate; and

75-86 % polyethylene glycol with an average molecular weight between 200 and 700.

Suitable emulsions of the present invention and/or embodiments thereof comprise

5% fish anaesthetic agent ;

10% polysorbate; and

85% polyethylene glycol with an average molecular weight between 200 and 700.

A preferred emulsion comprises about 50% isoeugenol, preferably wherein the isoeugenol contains greater than 99% of the trans isomer; about 40% by weight Polysorbate 80; and about 10% by weight PEG 400.

In another preferred embodiment, the composition contains: about 50% isoeugenol, preferably wherein the isoeugenol contains greater than 99% by weight of the trans isomer; and about 50% by weight Polysorbate 80.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent is used to a concentration of 1-150 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 2-100 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 3-80 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 4-60 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 5-40 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 6-30 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 7-25 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 8-20 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 9-15 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 1-20 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 2-18 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 3-16 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 4-14 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 5-12 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 6-10 ppm in the body of water. Preferably the fish anaesthetic agent is used to a concentration of 7-8 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 1-17 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 2-16 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 3-15 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 4-14 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 5-13 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 6-12 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 7-11 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 8-10 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 7-9 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 9-11 ppm in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 10-17 ppm in the body of water.

Different fish anaesthetic agent may be used in different concentration. It will also depend on the fish to be treated. A skilled person is well aware of suitable concentration of the fish anaesthetic agent in the body of water.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent is used to a concentration of 1-30 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 2-28 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 3-26 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 4-25 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 5-24 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 6-22 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 7-20 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 8-18 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 9-17 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 10-16 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 11-15 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 12-14 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 1-17 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 2-16 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 3-15 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 4-14 mg/L the body of water. Preferably the fish anaesthetic agent is used to a concentration of 5-13 mg/L the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 6-12 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 7-11 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 8-10 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 7-9 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 9-11 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 10-17 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 4-12 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 5-11 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 6-10 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 2-10 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 3-9 mg/L in the body of water. Suitably, the fish anaesthetic agent is used in a concentration of 4-8 mg/L in the body of water. Suitably, where the fish anaesthetic agent employed is eugenol, it is preferred that the amount of eugenol used is from 2-12.5 mg/L, more preferably from 3-10 mg/L, most preferably from 6-8 mg/L.

Suitably, where the compound employed is isoeugenol, it is preferred that the amount of isoeugenol used is from 1-20 mg/L, more preferably from 3-15 mg/L, more preferably from 5-10 mg/L, most preferably from 5-8.5 mg/L.

In an embodiment of the invention and/or embodiments thereof, the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 1-600 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 2-400 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 3-300 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 4-250 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 5-200 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 6-180 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 7-150 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 8-120 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 9-100 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 10-90 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 12-80 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 14-70 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 15-60 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 16-50 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 18-45 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 20-40 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 22-35 minutes. More preferably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 24-30 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 10-500 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 15-450 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 20-400 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 30-350 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 40-300 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 50-290 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 55-280 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 60-280 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 65-275 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 70-270 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 75-265 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 80-260 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 85-255 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 90-250 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 95-245 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 100-240 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 105-235 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 110-230 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 115-225 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 120-220 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 125-215 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 130-210 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 135-205 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 140-200 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 145-195 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 150-190 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 155-185 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 160-180 minutes. Suitably the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 165-175 minutes.

According to the invention and/or embodiments thereof fish are treated by adding an emulsion to a body of water comprising the fish. Suitably, fish are organisms belonging to the class Pisces such as salmon, trout, char, ayu, carp, crucian carp, goldfish, roach, whitebait, eel, conger eel, sardine, flying fish, sea bass, sea bream, parrot bass, snapper, mackerel, horse mackerel, tuna, bonito, yellowtail, rockfish, fluke, sole, flounder, blowfish, filefish. Preferred fish are salmonid, such as salmon and trout. More preferably, the fish is salmon.

The present invention and/or embodiments thereof provide for the detachment of ectoparasite from fish. Ectoparasites of fish may comprises species from the group consisting of Salmonicola species, Caligus species, Ergasilis spp., Sparycotile species, Sciaenocotyle species, Zeuxapta seriolae, Cerathotoa species, Lermnathropus species, Argulus species, Bomolochus species, Chondracanthus species, Lerneae cyprinacea, Lernaeocera species, Lernaaeenicus species, Lepeophterius species, Nerocila orbignyi, and Anilocra physodes. Suitably ectoparasites of fish may comprises species from the group consisting of Salmonicola species, Caligus species, Ergasilis spp., Sparycotile species, Sciaenocotyle species, Zeuxapta seriolae, Cerathotoa species, Lermnathropus species, Argulus species, and Lepeophterius species. Suitably ectoparasites of fish may comprises species from the group consisting of Caligus species, Cerathotoa species, Lermnathropus species, Argulus species, and Lepeophterius species.

Suitably the ectoparasite is a parasite from the family caligidae, preferably from the genus Lepeophtheirus or Caligus. Preferably the ectoparasite is a sea louse. Sea lice is the term generally used for species of copepods within the family Caligidae. There are 36 genera within this family which include approximately 42 Lepeophtheirus and 300 Caligus species. Sea lice are marine ectoparasites ( external parasites) which feed on the mucus, epidermal tissue, and blood of host marine fishes. The genera Lepeophtheirus and Caligus parasitize marine fishes and have been recorded on farmed salmon, halibut, cod, herring. Lepeophtheirus salmonis and various Caligus species are adapted to saltwater and are major ectoparasites of farmed and wild Atlantic salmon. L. salmonis is generally the major sea louse of concern. Caligus rogercresseyi has become a major parasite of concern on salmon farms in Chile. Recent evidence is also emerging that L. salmonis in the Atlantic has sufficient genetic differences from L. salmonis from the Pacific, suggesting that Atlantic and Pacific L. salmonis may have independently coevolved with Atlantic and Pacific salmonids, respectively. Sea lice cause physical and enzymatic damage at their sites of attachment and feeding which results in abrasion-like lesions that vary in their nature and severity depending upon a number of factors. These include host species, age and general health of the fish. Sea lice infection itself causes a generalized chronic stress response in fish since feeding and attachment cause changes in the mucus consistency and damage the epithelium resulting in loss of blood and fluids, electrolyte changes, and cortisol release. This can decrease salmon immune responses and make them susceptible to other diseases and reduces growth and performance. The degree of damage is also dependent on the species of sea lice, the developmental stages that are present, and the number of sea lice on a fish.

In an embodiment of the invention and/or embodiments thereof, the method comprises a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment either with freshwater or salt water, fresh-water treatment, waterjet treatment, exposure to H2O2 (hydrogenperoxide). The detachment of the ectoparasite may be enhanced by a removal technique. Suitable the fish anaesthetic agent is first allowed to contact the ectoparasite for 1-360 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 2-330 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 3-300 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 4-270 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 5-240 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 6-210 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 7-180 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 8-150 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 9-120 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 10-90 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 12-80 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 14-75 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 16-70 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 18-60 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 20-50 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 22-45 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 24-40 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 26-35 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 28-30 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 1-60 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 2-55 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 3-50 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 4-45 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 5-40 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 6-35 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 7-30 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 8-25 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 9-20 minutes and then a removal treatment is used. More suitably the fish anaesthetic agent is first allowed to contact the ectoparasite for 10-18 minutes and then a removal treatment is used. The combination of the fish anaesthetic agent and a removal treatment may reduce the treatment time significantly.

In an embodiment of the invention and/or embodiments thereof, the method does not comprise a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and exposure to H2O2 (hydrogen peroxide). The detachment of the ectoparasites of the present invention by the use of the emulsion comprising a fish anaesthetic agent can also be used as a stand-alone technique. The emulsion is an easy-to-use product whereas the removal techniques require large apparatus that are expensive.

In an embodiment of the invention and/or embodiments thereof, the method comprises crowding the fish. Suitably the densitiy of the fish when crowding is between 30-300 kg/m³ more suitably between 40-250 kg/m³, more suitably between 50-200 kg/m³, more suitably between 60-175 kg/m³, more suitably between 70-150 kg/m³, more suitably between 80-125 kg/m³, more suitably between 90-110 kg/m³. Suitable density of fish for crowding is between 50-100 kg/m³, or between 30-100 kg/m³

In an embodiment of the invention and/or embodiments thereof, the invention is directed to a method of delousing fish comprising the steps of a) adding a composition comprising an emulsion of a fish anaesthetic agent in a body of water comprising the fish b) crowding the fish to a density of at least 30 kg/m³ c) optionally removing lice that are detached.

Suitably, the fish are crowded for at least 10 minutes, suitably for at least 15 minutes, suitably for at least 20 minutes, suitably for at least 30 minutes, suitably for at least 45 minutes, suitably for at least 60 minutes, suitably for at least 75 minutes, suitably for at least 90 minutes. Suitably the fish are crowded from 10 minutes to 24 hours, suitably from 15 minutes to 20 hours, suitably from 20 minutes to 18 hours, suitably from 25 minutes to 16 hours, suitably from 30 minutes to 15 hours, suitably from 45 minutes to 14 hours, suitably from 60 minutes to 13 hours, suitably from 90 minutes to 12 hours, suitably from 2 hours to 11 hours, suitably from 2.5 hours to 10 hours, suitably from 3 hours, to 9 hours, suitably from 3.5 hours to 8 hours, suitably from 4 hours to 7.5 hours, suitably from 4.5 hours to 7 hours, suitably from 5 hours to 6.5 hours, suitably from 5.5 hours to 6 hours.

All embodiments described above regarding the emulsion of a fish anaesthetic agent are envisioned in this method. Suitably, the method comprises a step consisting of a fish ectoparasite removal treatment. Suitably the fish ectoparasite removal treatment is selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and exposure to hydrogen peroxide.

In a first embodiment the invention is related to a fish anaesthetic agent for use in a method to detach ectoparasites from fish.

A second embodiment the invention is related to a fish anaesthetic agent for use according to embodiment 1 wherein the fish are in a body of water and the method comprises adding a composition comprising an emulsion of the fish anaesthetic agent in a body of water comprising the fish.

In a third embodiment the invention is related to a a fish anaesthetic agent for use according to any embodiment 1 or 2 wherein the method further comprises collecting or removing detached ectoparasites.

In a fourth embodiment the invention is related to a fish anaesthetic agent for use according to any embodiment 1 to 3 wherein the anaesthetic agent is selected from the group consisting of tricaine methanesulfonate, metomidate, phenoxyethanol, etomidate, eugenol, isoeugenol, benzocaine, linalool, and quinaldine.

In a fifth embodiment the invention is related to a fish anaesthetic agent for use according to any embodiment 1 to 4 wherein the anaesthetic agent is selected from the group consisting of eugenol and isoeugenol.

In a sixth embodiment the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 5 wherein the emulsion is an oil-in-water emulsion.

In a seventh embodiment the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 6 wherein the emulsion comprises an emulsifier and/or a cyclodextrin.

In embodiment 8, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 7 wherein the emulsion comprises an emulsifier and the emulsifier is selected from the group consisting of detergent, polyglycerol ester, lecithin, polyethylene glycol and polyethylene glycol ester. In embodiment 9, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 8 wherein the emulsion comprises an emulsifier selected from the group consisting of polyethylene glycol, polyethylene glycol ester, and polyglycerol.

In embodiment 10, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 9 wherein the emulsion comprises an polyethylene glycol and/or polyethylene glycol ester.

In embodiment 11 , the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 10 wherein the emulsion comprises polyethylene glycol ester and the polyethylene glycol ester is polysorbate.

In embodiment 12, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 11 wherein the fish are salmonid.

In embodiment 13, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 12 wherein the ectoparasite is a sea louse, preferably a parasite from the family caligidae, preferably from the genus Lepeophtheirus or Caligus.

In embodiment 14, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 13 wherein the fish anaesthetic agent has a concentration of 10-80% in the emulsion.

In embodiment 15, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 14 wherein the emulsion comprises an emulsifier and the emulsifier has concentration of 20-90% of the emulsion.

In embodiment 16, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 15 wherein the emulsion comprises cyclodextrin and the cyclodextrin has a concentration of 20-90% in the emulsion.

In embodiment 17, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 16 wherein the fish anaesthetic agent is used to a concentration of 1-150 ppm in the body of water.

In embodiment 18, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 17 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of 1-600 minutes.

In embodiment 19, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 18 wherein the method comprises a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, and waterjet treatment and exposure to hydrogenperoxide.

In embodiment 20, the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 19 wherein the method comprises a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, and waterjet treatment.

In embodiment 21 , the invention is related to a fish anaesthetic agent for use according to any of embodiment 1 to 18 wherein the method does not comprise a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and exposure to hydrogenperoxide.

In embodiment 22, the invention is related to a method to detach ectoparasites from fish comprising applying a composition comprising an emulsion of a fish anaesthetic agent.

In embodiment 23 the invention is related to a method according to embodiment 22 wherein the fish are in a body of water and the composition comprising an emulsion of a fish anaesthetic agent is added to the body of water comprising the fish.

In embodiment 24 the invention is related to a method according to any of embodiment 22 to 23 wherein the detached ectoparasites are collected or removed.

In embodiment 25, the invention is related to a method according to any of embodiment 22 to 24 wherein the fish anaesthetic agent is selected from the group consisting of tricaine methanesulfonate, metomidate, phenoxyethanol, etomidate, eugenol, isoeugenol, benzocaine, linalool, and quinaldine.

In embodiment 26, the invention is related to a method according to any of embodiment 22 to 25 wherein the fish anaesthetic agent is selected from the group consisting of eugenol and isoeugenol.

In embodiment 27, the invention is related to a method according to any of embodiment 22 or 26 wherein the emulsion is an oil in water emulsion.

In embodiment 28, the invention is related to a method according to any of embodiment 22 to 27 wherein the emulsion comprises an emulsifier and/or a cyclodextrin.

In embodiment 29, the invention is related to a method according to any of embodiment 22 to 28 wherein the emulsion comprises an emulsifier and the emulsifier is selected from the group consisting of detergent, polyglycerolester, polyethylene glycol, polyethylene glycol ester, and lecithin. In embodiment 30, the invention is related to a method according to any of embodiment 22 to 29 wherein the emulsion comprises an emulsifier selected from the group consisting of polyethylene glycol, polyethylene glycol ester, and polyglycerol.

In embodiment 31 , the invention is related to a method according to any of embodiment 22 to 30 wherein the emulsion comprises polyethylene glycol and/or polyethylene glycol ester.

In embodiment 32, the invention is related to a method according to any of embodiment 22 to 31 wherein the emulsion comprises polyethylene glycol ester and preferably the polyethylene glycol ester is polysorbate.

In embodiment 33, the invention is related to a method according to any of embodiment 22 to 32 wherein the fish are salmonid.

In embodiment 34, the invention is related to a method according to any of embodiment 22 to 33 wherein the ectoparasite is sea louse, preferably a parasite from the family caligidae, preferably from the genus Lepeophtheirus or Caligus.

In embodiment 35, the invention is related to a method according to any of embodiment 22 to 34 wherein the fish aneasthetic is used to a concentration of 1-150 ppm in the body of water.

In embodiment 36, the invention is related to a method according to any of embodiment 22 to 35 wherein the fish aneasthetic is used in a concentration of 1-20 ppm in the body of water.

In embodiment 37, the invention is related to a method according to any of embodiment 22 to 36 wherein the fish aneasthetic is used in a concentration of 1-20 mg/L in the body of water.

In embodiment 38, the invention is related to a method according to any of embodiment 22 to 37 wherein the fish aneasthetic is used in a concentration of 2-15 mg/L in the body of water.

In embodiment 39, the invention is related to a method according to any of embodiment 22 to 38 wherein the fish aneasthetic is used in a concentration of 2-10 mg/L in the body of water.

In embodiment 40, the invention is related to a method according to any of embodiment 22 to 39 wherein the fish aneasthetic is used in a concentration of 4-8 mg/L in the body of water.

In embodiment 41 , the invention is related to a method according to any of embodiment 22 to 40 wherein the fish anaesthetic agent has a concentration of 10-80% in the emulsion.

In embodiment 42, the invention is related to a method according to any of embodiment 22 to 41 wherein the emulsion comprises an emulsifier and the emulsifier has concentration of 20-90% of the emulsion. In embodiment 43, the invention is related to a method according to any of embodiment 22 to 42 wherein the emulsion comprises cyclodextrin and the cyclodextrin has a concentration of 20-90 % in the emulsion.

In embodiment 44, the invention is related to a method according to any of embodiment 22 to 43 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 1 and 600 minutes.

In embodiment 45, the invention is related to a method according to any of embodiment 22 to 44 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 30 and 300 minutes.

In embodiment 46, the invention is related to a method according to any of embodiment 22 to 45 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 45 and 280 minutes.

In embodiment 47, the invention is related to a method according to any of embodiment 22 to 46 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 60 and 250 minutes.

In embodiment 48, the invention is related to a method according to any of embodiment 22 to 47 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 75 and 230 minutes.

In embodiment 49, the invention is related to a method according to any of embodiment 22 to 48 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 90 and 200 minutes.

In embodiment 50, the invention is related to a method according to any of embodiment 22 to 49 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 100 and 180 minutes.

In embodiment 51 , the invention is related to a method according to any of embodiment 22 to 50 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 120 and 150 minutes.

In embodiment 52, the invention is related to a method according to any of embodiment 22 to 51 wherein the method comprises a step consisting of an ectoparasite removal treatment selected from the group consisting of thermal treatment, freshwater treatment, waterjet treatment and exposure to hydrogen peroxide. In embodiment 53, the invention is related to a method according to any of embodiment 22 to 52 wherein the method comprises a step consisting of an ectoparasite removal treatment selected from the group consisting of thermal treatment, freshwater treatment, and waterjet treatment.

In embodiment 54, the invention is related to a method according to any of embodiment 22 to 53 wherein the method does not comprise a step consisting of an ectoparasite removal treatment selected from the group consisting of thermal treatment, freshwater treatment, waterjet treatment and hydrogen peroxide.

In embodiment 55, the invention is related to a method according to any of embodiment 22 to 54 wherein the method comprises a step of crowding the fish.

In embodiment 56, the invention is related to a method of delousing fish comprising the steps of a) adding a composition comprising an emulsion of a fish anaesthetic agent in a body of water comprising the fish, wherein the emulsion of a fish anaesthetic agent is according to any of the embodiments described herein; b) crowding the fish

In embodiment 57, the invention is related to a method according to embodiment 56 wherein the fish density is at least 30 kg/m³.

In embodiment 58, the invention is related to a method according to any of embodiment 56-57 comprising removing lice that are detached.

In embodiment 59, the invention is related to a method according to any of embodiment 56-58 comprising a step consisting of a fish ectoparasite removal treatment.

In embodiment 60, the invention is related to a method according to embodiment 59 wherein the fish ectoparasite removal treatment is selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and exposure to hydrogen peroxide.

In embodiment 61 , the invention is related to a method according to any of embodiment 56-60, the fish are crowded for at least 10 minutes.

In embodiment 62, the invention is related to a method according to any of embodiment 56-61 , the fish are crowded for at least 15 minutes, up until 24 hours. The invention will now be further described by the following, non-limiting, examples.

Example 1

A minimum of 30 sea lice was used in a pre-adult and adult stage of which about 10 were preadult stage, 10 adult males and 10 adult females. Three different concentrations of 2, 3 and 4 ppm of Aqui-S were used. Aqui-S consist of 50% isoeugenol and 50% polysorbate 80. The sea lice were kept in boxes that are permeable for water without letting the sea lice out and are stored in sea water. After acclimatation, the sea lice were exposed to Aqui-S for 90 minutes. Examination of lice were done immediately after exposure, and after one day of acclimatization in clean seawater.

Each louse was inspected individually with a forceps. Lice are classified as live when one of the following criteria was fulfilled:

• Fast swimming

• Reaction when touched

• Capable of sticking to the wall

• Wagging the tail before sticking to the wall

The lice were categorized as “living attached”, “living detached” and moribund.

Discussion

Figure 1 shows the results after 24 hours when the lice were exposed for 90 minutes with 2 and 3 ppm of Aqui-S which corresponds to 1 and 1 .5 ppm of isoeugenol. When compared to the control (no exposure to Aqui-S) at 2 ppm 27% of the sea lice were still detached and at 3 ppm 42% of the sea lice were still detached whereas in the control group only 6% was detached.

Example 2

Aqui-S treatment is tested on salmon infested with sea lice. As comparison clove oil is also tested. Aqui- S is an emulsion comprising 50% isoegenol and 50% polysorbate 80. Clove oil is not an emulsion. 10 salmon per group are tested accordig to the below schedule:

Fish were checked directly after exposure and 2-4 hours after exposure to check for detached sea lice. After 4 hours a mechanical delousing treatment is carried out and the removed sea lice are counted.

Example 3

Salmon in two different cages were crowded together in a tarpaulin and pumped into a delouse barge. The fish in one cage were sedated with Aqui-S and the fish in the second cage were not sedated and represented the control group. In total 4000 fish were used in the treatment group and 4000 fish were used in the control group.

Aqui-S was added to the tarpaulin at a dose of 2.7 ml per cubic meter with a holding time of 15 minutes. The control group was not sedated. The fish were pumped from the tarpaulin in the cage to the delouse barge, but without undergoing the delousing process. Fish were crowded to approx. 40 kg salmon per 1000 liter of water. After crowding and pumping, the fish were returned to the cage via a halfpipe. The number of lice attached to the fish were counted before sedation and after the fish came out of the barge, as well as four days afterwards. The lice filter of the lice barge was not used in the experiment.

The number of lice attached to the fish were counted before sedation and after the fish came out of the barge.

Before sedation:

A total of 20 fish were caught and then anesthetized in a conventional manner with Finquel Vet and lice were counted.

After handling:

In one cage Aqui-S was added and dispersed for the holding time. After this was reached, the pumps were started to flow the fish to the barge. Again 20 salmon were caught and anesthetized with Finquel and the number of salmon lice were counted on these 20 salmon.

The control group was treated in the same way except there was no Aqui-S added to the cage.

Result:

As can be seen, the number of motile lice in the test cage (Aqui S) was reduced from 0.75 to only 0.1 , whereas this number remained high (reduced from 1 .5 to 0.65) for the non-treated salmon.

The results demonstrate that motile lice were reduced from 0.75/fish before sedation to 0.1 /fish after sedation, which represents a reduction of 87%. For adult female lice, there was a reduction of 33%. In the untreated control group, which were crowded and pumped only, there was a reduction of 46% for motile lice and 0% for adult female lice.

Example 4

Fish at an average weight of 165 gram, infested with pre-adult and adult sea lice at an average of 9.8 lice per fish (50.4% males, 49.6% females), underwent bath treatments with Aqui-S (50% isoeugenol and 50% polysorbate 80) or clove oil, or salt water and canola oil as respective controls, at different doses and exposure times.

After treatments were completed, fish were anesthetized in tricaine methanesulfonate (TMS; 40 mg/L), and attached lice were counted prior to euthanizing the fish. Detached lice were counted in the treatment baths, nets used for transfer of fish from treatment bath to TMS, and TMS bath.

(iso)eugenol concentration of 0, 4, and 8 mg/L were used for a bath duration time of 120 and 240 minutes.

Aqui-S treatment baths were prepared as per manufacturer’s instructions, by diluting a 10% (v/v) stock solution in 50 L water of similar salinity and temperature to that of the holding tanks, to the desired isoeugenol concentration. Water baths without Aqui-S were used as controls.

Clove oil treatment baths were prepared by diluting the clove oil in canola oil to the desired eugenol concentration and layering 500 mL of the diluted clove oil onto a 50L water bath. Canola oil without clove oil was used as control. Fish were passed individually through the layer of oil, and after all fish were in the bath, the oil layer was removed.

Result

Lice detachment in fish treated with Aqui-S at 8 mg/L isoeugenol for 120 or 240 minutes was

45.1% and 43.1% respectively, which was significantly higher than in the salt water control (Fisher exact test, p<0.0001). Since no added benefit was observed from the 240 minute treatment, only 120 minute treatment baths were tested for clove oil at 8 mg/L eugenol concentration, and canola oil control. Aqui-S treatment efficacy was significantly higher than both (Fisher exact test, p<0.0001), and clove oil did not show any efficacy compared to the canola oil control.

Claims

1. A fish anaesthetic agent for use in a method to detach ectoparasites from fish wherein the fish anaesthetic is in the form of an emulsion.

2. A fish anaesthetic agent for use according to claim 1 wherein the fish are in water and the method comprises adding a composition comprising an emulsion of the fish anaesthetic agent in a body of water comprising the fish.

3. A fish anaesthetic agent for use according to any of claim 1 or 2 wherein the method further comprises collecting or removing detached ectoparasites.

4. A fish anaesthetic agent for use according to any of claim 1 to 3 wherein the anaesthetic agent is selected from the group consisting of tricaine methanesulfonate, metomidate, phenoxyethanol, etomidate, eugenol, isoeugenol, benzocaine, linalool, and quinaldine.

5. A fish anaesthetic agent for use according to any of claim 1 to 4 wherein the emulsion is an oil-in- water emulsion.

6. A fish anaesthetic agent for use according to any of claim 1 to 5 wherein the emulsion comprises an emulsifier and/or a cyclodextrin.

7. A fish anaesthetic agent for use according to any of claim 1 to 6 wherein the emulsion comprises an emulsifier and the emulsifier is selected from the group consisting of detergent, polyglycerolester, polyethylene glycol, polyethylene glycol ester and lecithin.

8. A fish anaesthetic agent for use according to any of claim 1 to 7 wherein the fish are salmonid.

9. A fish anaesthetic agent for use according to any of claim 1 to 8 wherein the ectoparasite is a parasite from the family caligidae, preferably from the genus Lepeophtheirus or Caligus.

10. A fish anaesthetic agent for use according to any of claim 1 to 9 wherein the fish anaesthetic agent has a concentration of 10-80% in the emulsion.

11 . A fish anaesthetic agent for use according to any of claim 1 to 10 wherein the emulsion comprises an emulsifier and the emulsifier has concentration of 20-90% of the emulsion.

12. A fish anaesthetic agent for use according to any of claim 1 to 11 wherein the emulsion comprises cyclodextrin and the cyclodextrin has a concentration of 20-90 % in the emulsion.

13. A fish anaesthetic agent for use according to any of claim 1 to 12 wherein the fish anaesthetic agent is used to a concentration of 1-150 ppm in the body of water, preferably 1-50 ppm, more preferably 1- 25 ppm, more preferably 2-10 ppm.

14. A fish anaesthetic agent for use according to any of claim 1 to 13 wherein the fish anaesthetic agent is allowed to contact the ectoparasite for a time of between 1 and 600 minutes, preferably 60-300 minutes, more preferably 90-240 minutes, more preferably 120-180 minutes .

15. A fish anaesthetic agent for use according to any of claim 1 to 14 wherein the method comprises a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and exposure to hydrogen peroxide.

16. A fish anaesthetic agent for use according to any of claim 1 to 14 wherein the method does not comprise a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and hydrogen peroxide.

17. A fish anaesthetic agent for use according to any of claim 1 to 15 wherein the method comprises a step of crowding the fish.

18. A method of delousing fish comprising the steps of a) adding a composition comprising an emulsion of a fish anaesthetic agent in a body of water comprising the fish b) crowding the fish to a density of at least 30 kg/m³ c) optionally removing lice that are detached.

19. A method according to claim 18 comprises a step consisting of a fish ectoparasite removal treatment selected from the group consisting of thermal treatment, fresh-water treatment, waterjet treatment and exposure to hydrogen peroxide.

20. A method according to anyone of claims 18-19 wherein the crowding is for at least 15 minutes.