WO2011101367A1

WO2011101367A1 - Product for removing sea lice

Info

Publication number: WO2011101367A1
Application number: PCT/EP2011/052272
Authority: WO
Inventors: Delone Bentz; Svenn Rommerud
Original assignee: ENVIROM PATENT HOLDING AS
Current assignee: ENVIROM PATENT HOLDING AS
Priority date: 2010-02-17
Filing date: 2011-02-16
Publication date: 2011-08-25
Anticipated expiration: 2012-08-17
Also published as: NO333012B1; NO20100234A1; WO2011101367A4

Abstract

The present invention concerns the use of different plant oils in the production of a product for removing or preventing the attachment of sea lice onto fish. The invention also concerns methods of removing or preventing the attachment of sea lice onto fish.

Description

Product for removing sea lice

Field of invention

The present invention concerns the use of environmentally safe substances for manufacturing a product for removing and/or preventing the attachment of sea lice on fish. Specifically, the invention concerns plant extracted substances useful for managing infestation of sea lice on fish in aqua culture facilities. The invention also concerns methods for removing sea lice from fish using the products of the invention.

Background of the invention

Sea lice is the term generally used for species of copepods within the order of

Siphonostomatoida and 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. Lepeophtheirus salmonis and various Caligus species are adapted to saltwater and are major ectoparasites of farmed and wild Atlantic salmon. Several antiparasitic drugs have been developed for control purposes. L. salmonis is the major sea louse of concern and has the most known biology and interactions with its salmon host. Caligus rogercresseyi has become a major parasite of concern on salmon farms in Chile, and studies are underway to gain a better

understanding of the parasite and the host-parasite interactions. 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 co-evolved 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. It is not clear whether stressed fish are particularly prone to infestation. 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. There is little evidence of host tissue responses in Atlantic salmon at the sites of feeding and attachment, regardless of the development stage. In contrast, coho and pink salmon show strong tissue responses to L. salmonis characterized by epithelial hyperplasia and inflammation. This results in rejection of the parasite within the first week of infection in these species of salmonids. Heavy infections of farmed Atlantic salmon and wild sockeye salmon (Oncorhynchus nerka) by L. salmonis can lead to deep lesions, particularly on the head region, even exposing the skull.

Control of sea lice in aqua culture

The control of sea lice in aqua culture has been reviewed in several articles (Pike, A.W. & Wadsworth, S.L. 2000. "Sealice on salmonids: their biology and control". Adv.

Parasitol. 44, 233-337, McVicar, A.H. 2004. "Management actions in relation to the controversy about salmon lice infections in fish farms as a hazard to wild salmonid populations". Aquacult. Res. 35, 751-758, and Costello, M.J. 2006. "Ecology of sea lice parasitic on farmed and wild fish". Trends Parasitol. 22, 475-483). Identification of epidemiological factors as potential risk factors for sea lice abundance with effective sea lice monitoring programs have been shown to effectively reduce sea lice levels on salmon farms (Saksida, S., Karreman, G.A., Constantine, J., & Donald, A. 2007.

"Differences in Lepeophtheirus salmonis abundance levels on Atlantic salmon farms in the Broughton Archipelago, British Columbia, Canada". J. Fish Dis. 30, 357-366).

Natural predators of sea lice include leaner fish, including five species of wrasse (Labridae), which are used on fish farms in Norway, and to a lesser extent in Scotland, Shetland and Ireland. Their potential has not been researched in other fish farming regions, such as Pacific and Atlantic Canada or Chile.

Good husbandry techniques include fallowing, removal of dead and sick fish, prevention of net fouling, etc. Bay management plans are in place in most fish farming regions to keep sea lice populations below a level that could lead to health concerns on the farm or affect wild fish in surrounding waters. These include separation of year classes, counting and recording sea lice on a prescribed basis, use of parasiticides when sea lice counts increase, and monitoring resistance to parasiticides.

Early findings suggested genetic variation in the susceptibility of Atlantic salmon to Caligus elongatus. Research then began to identify trait markers and recent studies have shown that susceptibility of Atlantic salmon to L. salmonis can be identified to specific families and that there is a link between MHC Class II and susceptibility to lice (Glover, K.A., Grimholt, U., Bakke, H.G., Nilsen, F., Storset, A. &, Skaala, 0. 2007. "Major histocompatibility complex (MHC) variation and susceptibility to the sea louse

Lepeophtheirus salmonis in Atlantic salmon Salmo salar". Dis. Aquat. Org. 76, 57-66).

These results may possibly be used in salmon breeding to control the effect of se lice in aqua culture. The range of therapeutics for farmed fish is limited, often due to regulatory processing limitations. All drugs used have been assessed for environmental impact and risks. The parasiticides are classified into bath and in-feed treatments as follows:

There are both advantages and disadvantages to using bath treatments. Bath treatments are more difficult and require more manpower to administer, requiring skirts or tarpaulins to be placed around the cages to contain the drug. Prevention of reinfection is a challenge since it is practically impossible to treat an entire bay in a short time period. Since the volume of water is imprecise, the required concentration is not guaranteed. Crowding of fish to reduce the volume of drug can also stress the fish. Recent use of well-boats containing the drugs has reduced both the concentration and environmental concerns, although transferring fish to the well boat and back to the cage can be stressful. The major advantage to bath treatments is that all the fish will be treated equally, in contrast to in-feed treatments where amount of drug ingested can vary due to a number of reasons.

Organophosphates are acetylcholinesterase inhibitors which cause excitatory paralysis leading to death of sea lice when administered as a bath treatment. Dichlorvos was used for many years in Europe and later replaced by azamethiphos, the active ingredient in Salmosan, which is safer for operators to handle. Azamethiphos is water-soluble and broken down relatively quickly in the environment. Resistance to organophosphates began to develop in Norway in the mid 1990's, apparently due to acetylcholinesterases being altered due to mutation. Use has declined considerably with the introduction of SLICE, emamectin benzoate.

Pyrethroids are direct stimulators of sodium channels in neuronal cells, inducing rapid depolarization and spastic paralysis leading to death. The effect is specific to the parasite since the drugs used are only slowly absorbed by the host and rapidly metabolized once absorbed. Cypermethrin (Excis, Betamax) and deltamethrin (Alphamax) are the two pyrethroids commonly used to control sea lice. Resistance to pyrethroids has been reported in Norway and appears to be due to a mutation leading to a structural change in the sodium channel which prevents pyrethroids from activating the channel. Use of deltamethrin has been increasing as an alternate treatment with the rise in resistance observed with emamectin benzoate.

Bathing fish with hydrogen peroxide (350-500 mg/L for 20 min) will remove mobile sea lice from fish. It is environmentally friendly since H2O2 dissociates to water and oxygen, but can be toxic to fish, depending on water temperature, as well as to operators. It appears to knock the sea lice off the fish, leaving them capable of reattaching to other fish and reinitiating an infection.

In-feed treatments are easier to administer and pose less environmental risks than bath treatments. Feed is usually coated with the drug and drug distribution to the parasite is dependent on the pharmacokinetics of the drug getting in sufficient quantity to the parasite. The drugs have high selective toxicity for the parasite, are quite lipid soluble so that there is sufficient drug to act for approximately 2 months, and any unmetabolized drug is excreted so slowly that there are little to no environmental concerns.

Avermectins belong to the family of macrocyclic lactones and are the major drugs used as in-feed treatments to kill sea lice. The first avermectin used was ivermectinat doses close to the therapeutic level and was not submitted for legal approval for use on fish by its manufacturer. Ivermectin was toxic to some fish, causing sedation and central nervous system depression due to the drug's ability to cross the blood-brain barrier. Emamectin benzoate, which is the active agent in the formulation SLICE, has been used since 1999 and has a greater safety margin on fish. It is administered at 5(^g/kg/day for 7 days and is effective for two months, killing both chalimus and mobile stages. Withdrawal times vary with jurisdiction from zero in Canada to 175 degree days in Norway. Avermectins act by opening glutamate-gated chloride channels in arthropod neuromuscular tissues, causing hyperpolarization and flaccid paralysis leading to death. Resistance has been noted in Chalimus rogercresseyi in Chile and L. salmonis on North Atlantic fish farms. The resistance is likely due to prolonged use of the drug leading to up-regulation of P- glycoprotein, similar to what has been observed in nematode resistance to macrocyclic lactones.

Teflubenzuron, the active agent in the formulation Calicide, is a chitin synthesis inhibitor and prevents moulting. It thus prevents further development of larval stages of sea lice, but has no effect on adults. It has been used only sparingly in sea lice control, largely due to concerns that it may affect the moult cycle of non-target crustaceans, although this has not been shown at the concentrations recommended.

A number of studies are underway to examine various antigens, particularly from the gastrointestinal tract and reproductive endocrine pathways, as vaccine targets, but no vaccine against sea lice is currently on the market.

As of today, evolved resistance towards the different marketed sea lice control compositions, compounds and formulations is a huge challenge. The salmon aqua culture industry in Chile is experiencing considerable financial losses, and the same situation is rapidly becoming a problem in Norway. The considerable infestation of ever growing populations of sea lice in the Norwegian fiords also greatly impacts the wild salmon populations, which are declining rapidly year by year. Thus, there is a great need in the aqua culture industry for novel approaches to meet the challenge posed by the ever growing resistance in sea lice populations towards the known formulations.

Thus, the present invention is directed to find solutions to this problem, and it provides solutions which are environmentally safe and sustainable.

Summary of the invention

The present invention provides the use of certain plant oils in the management of sea lice infestation in aqua culture facilities. More specifically, the invention concerns the use of one or more plant oils for manufacturing a product for removing and/or preventing the attachment of sea lice on fish.

The invention also provides methods of preventing or removing sea lice from fish, wherein a product comprising one or more plant oils is administered.

The present invention is based on the surprising observation that certain plant oils, especially when administered directly to the skin of fish infested with sea lice, are able to paralyze and cause the sea lice to detach themselves from the skin of the treated fish.

Detailed description of the invention

The inventors of the present invention found that certain plant oils may be used to remove sea lice from Atlantic salmon. Specifically, tests were performed with clove oil from the clove plant, Syzygium aromaticum, which proved that plant oils could be potent new substances in the management of sea lice infestation. Although purified eugenol from cloves has been known to affect certain terrestrial insects, it was quite surprising and rather unexpected that clove oil would have a measurable effect on crustaceans like sea lice attached to salmon in an aquatic environment, because of the dilution effect when the clove oil is added directly to water, and because clove oil is poorly soluble in water. In addition, clove oil has not been demonstrated to have an effect on other species than terrestrial insects, and only when administered in a dry environment. Thus, to find that the administration of clove oil under water actually had an effect on totally unrelated aquatic organisms such as sea lice was surprising.

The experiments performed and described in this application show that clove oil administered as a bath treatment has an effect on the sea lice, however, when the clove oil is administered directly to the skin of salmon infested with sea lice, the effect is significantly higher. The administration of pure clove oil without any additional excipients showed promising results. Thus, the present invention provides the use of plant oils, and especially plant oils containing the compounds eugenol and/or thymol, as a new approach to prevent sea lice from infesting fish, or remove sea lice from fish.

Clove oil is an essential oil from the clove plant, Syzygium aromaticum. It is a natural analgaesic and antiseptic used primarily in dentistry for its main ingredient eugenol. It can also be purchased in pharmacies over the counter, as a home remedy for dental pain relief, mainly toothache. The oil produced by cloves can be used in many things from flavouring medicine to remedies for bronchitis, the common cold, a cough, fever, sore throat and tending to infections. Eugenol is an allyl chain-substituted guaiacol. Eugenol is a member of the phenylpropanoid class of chemical compounds. It is a clear to pale yellow oily liquid extracted from certain essential oils especially from clove oil, nutmeg, cinnamon, basil and bay leaf, e.g. from Syzygium aromaticum, Cinnamomum verum, Cinnamomum tamala, Myristica fragrans, Ocimum basilicum, Melaleuca leucadendra and Illicium anisatum. Eugenol is also present in oils from the plant species of Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria, Cinnamomum camphora, Cinnamomum cassia, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota, Echinophora tenuifolia, Elettaria cardamomum, Eucalyptus,

Hyssopus officinalis, Illicium verum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba, Myrtus communis, Ocimum gratissimum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa,

Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, and Trachyspermum ammi.

The essential oil of thyme species (Thymus vulgaris/hyemalis/zygis) contains the compound thymol. Thymol is an antiseptic, and a phenyl derivative like eugenol. Before the advent of modern antibiotics, it was used i.e. to medicate bandages. Thymol, which is also known as 2-isopropyl-5-methylphenol (IPMP), is a monoterpene phenol derivative of cymene, C10H14O, isomeric with carvacrol.

Thus, in a first embodiment, the present invention provides the use of one or more plant oils for manufacturing a product for removing and/or preventing the attachment of sea lice on fish.

In a more specific embodiment, the one ore more plant oils contain eugenol and/or thymol, and in an even more specific embodiment the one ore more plant oils contain eugenol.

In the embodiments above, the one or more plant oils could be obtained from plants selected from the group consisting of, but not limited to, Syzygium aromaticum, Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria, Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota,

Echinophora tenuifolia, Elettaria cardamomum, Eucalyptus, Hyssopus officinalis, Illicium verum, Illicium anisatum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba, Myristica fragrans, Myrtus communis,

Ocimum gratissimum, Ocimum basilicum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa, Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, Trachyspermum ammi, Thymus vulgaris, T.hyemalis, and T.zygis. In another embodiment, the one or more plant oils could be obtained from plants selected from the group consisting of Syzygium aromaticum, Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria, Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota, Echinophora tenuifolia,

Elettaria cardamomum, Eucalyptus, Hyssopus officinalis, Illicium verum, Illicium anisatum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba, Myristica fragrans, Myrtus communis, Ocimum gratissimum, Ocimum basilicum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa, Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, and Trachyspermum ammi.

In an even more specific embodiment, the one or more plant oils could be obtained from plants selected from the group consisting of Syzygium aromaticum,

Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Melaleuca alternifolia, Melaleuca bracteata, and Melaleuca Leucadendron.

In another even more specific embodiment of the invention, the plant oil could be obtained from Syzygium aromaticum. In another embodiment, the one or more plant oils are selected from clove oil, melalecuca oil, cinnamon oil and/or thyme oil.

In a more specific embodiment of the invention, the plant oil is clove oil.

In an additional embodiment, the invention concerns a method of preventing or removing sea lice from fish, wherein a product comprising one or more plant oils is administered as a bath treatment.

In a related embodiment, the invention concerns a method of preventing or removing sea lice from fish, wherein a product comprising one or more plant oils is administered directly to the skin of the fish.

In a more specific embodiment, in any of the methods in the above embodiments, the one or more plant oils contain eugenol and/or thymol.

In an even more specific embodiment, in any of the methods of the embodiments above, the one or more plant oils are obtained from plants selected from the group consisting of, but not limited to, Syzygium aromaticum, Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria, Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota, Echinophora tenuifolia,

Elettaria cardamomum, Eucalyptus, Hyssopus officinalis, Illicium verum, Illicium anisatum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba, Myristica fragrans, Myrtus communis, Ocimum gratissimum, Ocimum basilicum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa, Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, Trachyspermum ammi, Thymus vulgaris, T.hyemalis, and T.zygis or any combination of two ore more thereof.

In an even more specific embodiment, in any of the methods of the embodiments above, the one or more plant oils are obtained from the group consisting of

Syzygium aromaticum, Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria, Cinnamomum camphora, Cinnamomum cassia,

Cinnamomum verum, Cinnamomum tamala, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota, Echinophora tenuifolia, Elettaria cardamomum,

Eucalyptus, Hyssopus officinalis, Illicium verum, Illicium anisatum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba,

Myristica fragrans, Myrtus communis, Ocimum gratissimum, Ocimum basilicum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa, Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, and Trachyspermum ammi.

In a more specific embodiment, in any of the methods in the above embodiments, the one or more plant oils could be obtained from plants selected from the group consisting of Syzygium aromaticum, Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron. In a more specific embodiment, in any of the methods in the above embodiments, the plant oil could be obtained from Syzygium aromaticum.

In a specific embodiment, in any of the methods in the above embodiments, the one or more plant oils are selected from clove oil, melalecuca oil, cinnamon oil, and/or thyme oil In a more specific embodiment, in any of the methods in the above embodiments, the plant oil is clove oil.

The invention also concerns a method of preventing or removing sea lice from fish, wherein a product containing one or more plant oils is administered as a bath treatment, wherein the one or more plant oils are selected from plant oils of any of the above embodiments.

The invention also concerns a method of preventing or removing sea lice from fish, wherein a product containing one or more plant oils are administered directly to the skin of the fish, wherein the one or more plant oils are selected from plant oils of any of the above embodiments.

Examples

Examples 1-4 were performed to investigate the effects of clove oil applied as bath treatment on sea lice (Lepeophtheirus salmonis) in bioassays. The sea lice (all mobile stages) were obtained from Atlantic salmon in the field, and transported to VESO Vikan, Norway in three 5 1 buckets with seawater. Egg strings were removed from female lice and placed in a hatching system to hatch the eggs in order to produce copepodids for testing and infestation of Atlantic salmon. The tests were performed with water at 12 °C. The temperature of the water in the observation period (the period between exposure and evaluation) in Example 4 was kept at 12 °C, by placing the bioassay-boxes in 12 °C seawater.

Substance D is pure clove oil purchased from BERJE Inc., Fema No. 2325, Cas No. 8080-34-8.

Example 1 :

Adult males (5) were placed in 50 ml seawater in petri-dishes. One hundred (100) μΐ of substance D was added to each petri-dish. The effect was observed by

investigation of the lice capabilities to move or suck themselves to the wall of the dish. The results are given in tables 1 and 2 below.

Table 1. Average time from adding substance to inactivation.

Substance Stage and Average time to

sex inactivation

D Adult male 6 min Table 2. Time from adding substance to inactivation

Example 2:

The effect of substance D (highest effect of 100 μΐ in 50 ml seawater in Example 1), were further investigated on 10 lice, 5 adult females in one petri-dish and 5 adult males in another petri-dish. The effects were observed by investigation the lice capability to move or suck itself to the wall of the dish. The results are given in tables 3 and 4 below.

Table 3. Average time from adding substance to inactivation.

Table 4. Time from adding substance to inactivation

Example 3 :

A dose titration study with the same substance as in Example 2 were performed. Ten (10) preadult/adult males were placed in each bioassay-box, and exposed for 30 minutes to the following doses: 0, 1 , 10, 50 and 100 ppm. The effect was evaluated 22 h. post end of exposure. All 10 sea lice exposed to 100 ppm of substance D were inactivated after 30 minutes of exposure. However, these sea lice recovered during the 22 hours observation period (period from end of exposure to evaluation). At evaluation 22 hours post end of exposure, only 2 lice in different groups were found to be inactivated. No dose response was found at evaluation. The results are given in table 6 below.

Table 5. Dilutions of substance

Table 6. Results from dose-titration study

Example 4:

A standard bioassay was performed on the sampled lice to determine the sensitivity to the pyrethroid deltamethrin (AlphaMax; Pharmaq AS, Norway). The data were analysed by Probit analysis (PoloPlus, LeOra Software Inc. Berkeley, Ca, USA) for estimation of ECso to deltamethrin. (EC₅₀ - The concentration that immobilize 50 % of the target organism (moribund + dead)).

Due to low response it was not possible to estimate the EC₅₀ of deltametrin to this strain of sea lice. The response in the highest concentration (3 ppb) was only 3 inactivated of totally 13 lice. This indicates that the sea lice had reduced sensitivity or resistance to deltamethrin.

Table 7. Result from bioassay with AlphaMax.

Conclusion, examples 1-4

Exposure of adult male sea lice to 100 μΐ substance D in 50 ml seawater resulted in inactivation after 6 minutes. All 10 sea lice exposed to 100 ppm of substance D were inactivated after 30 minutes of exposure, but recovered during 22 hours post end of exposure.

There were not registered any significant difference between time to inactivation between adult males and adult females in Example 2. Example 5 :

Effect of substance D on Atlantic salmon (Salmo salar) infected with sea lice (Lepeophtheirus salmonis).

The objective of this test was to investigate the effect of substance (D) on Atlantic salmon with sea lice. The study was a "proof of concept" study based on the observed effect of substance D on sea lice in petri-dishes and dose-response bioassay

Sea lice, adult females with egg strings, were sampled in the field. The egg strings were hatched and nauplii larvae developed to copepodids. Sea water adapted Atlantic salmon (n=120, average weight 61 g) were infested with copepodids. The fish were divided in 4 groups after 17 days. Each group of 30 fish was placed in separate 0.6 m tanks with seawater. The tanks were treated with either: 0, 100, 250 and 500 ppm of substance D for 30 minutes, one concentration in each tank, 20 days after infestation. Seawater 10-12 °C with a salinity ranging from 30 - 32 o/oo, were used in the study.

The fish were anaesthetized by benzocaine, washed with clean seawater before it is put together with sea lice copepodids for about 2 minutes in a white container with seawater.

Prior to exposure, the fish were divided in 4 groups with 30 fish in each, and placed in separate 0.6 m tanks. The water flow to each tank were stopped before exposure and the volume in each tank was adjusted to 100 1. The correct amount of substance D was added to each tank to obtain the concentrations described in table 8. The tanks were aerated during exposure. The tanks 1 (0 ppm), 2 (100 ppm) and 3 (250 ppm) were exposed for 30 minutes, while exposure in tank 4 (500 ppm) was stopped after 10 minutes because the fish was affected by the substance. After exposure, the water with substance D were flushed out and replaced with clean seawater.

Table 8 Tanks and concentrations

One fish from the same infested group were anaesthetized with benzocaine. The fish, and the sea lice on it, were treated directly with substance D with a painting brush.

The fish and sea lice were then observed in clean seawater. The fish were tagged by clipping of the adipose fin, and placed in tank 2 (100 ppm)

The effect on the fish was observed during exposure, and each day by registration of dead fish until evaluation of sea lice.

The effect was evaluated for sea lice 6 days post exposure by sampling all the fish from each tank/ exposure group. The fish were sacrificed with a blow to the head and evaluated for sea lice.

Results and discussion:

The effect on fish.

The exposed salmon in the 100 ppm group showed somewhat slower swimming capability during exposure. All fish recovered quickly in clean seawater, and all fish survived until evaluation. The reaction of the fish in the 250 ppm group was the same as in the 100 ppm group, but they were heavier affected. All fish recovered quickly in clean seawater, and all fish survived until evaluation. The reaction in the 500 ppm group was severe, and the exposure was stopped after 10 minutes. The effect looked similar to anaesthetic, i.e., the fish were lying with the ventral side facing up. The fish recovered in a short time after the exposure bath was replaced by clean seawater. All fish survived until evaluation.

The fish that was treated with substance D with a painting brush recovered as normal from being anaesthetized. The fish was found in tank 2 (100) at evaluation, i.e., it survived the treatment.

The effect on sea lice

At evaluation it was found 30 fish in the control group, 32 fish in the 100 ppm group, 30 in I the 250 ppm and in the 500 ppm group. Since one extra fish was added to the 100 ppm group (one fish painted with substance D), the original number of fish in this tank must have been 31.

It was found a total of 95 lice in the control group (average 3.2 per fish), 70 in the 100 ppm group (average 2.2 per fish), 89 in the 250 ppm group (average 3.0) and 92 in the 500 ppm group (average 3.1).

Thus, it is a possible effect of the 100 ppm treatment. However, since the number of sea lice were higher in the 250 ppm group, i.e. no dose response, the lower number of sea lice in the 100 ppm group is probably due to random variation of numbers of sea lice on the fish.

The single fish that was treated with substance D with a painting brush had 4 sea lice attached prior to treatment. Three sea lice were hit by the substance. They fell off the fish in less than two minutes after treatment. The data were not evaluated statistically. The lower number of sea lice in the 100 ppm group could not be the effect of the substance, since no dose response was demonstrated, i.e. the number and distribution of sea lice in the group treated with 250 ppm were almost equal to the control group. Conclusion

The study demonstrated an effect of substance D as bath treatment similar to anaesthetic, with dose response to Atlantic salmon. The study showed a possible effect on sea lice of 100 ppm substance D as a bath treatment. However, no dose response was shown, i.e., not increased effect by a higher dose. However, the study demonstrated that substance D was effective in treatment of Atlantic salmon with sea lice, when it was administered directly on the fish with a painting brush.

Claims

PATENT CLAIMS

1. The use of one or more plant oils for manufacturing a product for removing and/or preventing the attachment of sea lice on fish.

2. The use of claim 1 , wherein the one ore more plant oils contain eugenol and/or thymol.

3. The use of claims 1-2, wherein the one or more plant oils are obtained from plants selected from the group consisting of Syzygium aromaticum, Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria, Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota, Echinophora tenuifolia,

Elettaria cardamomum, Eucalyptus, Hyssopus officinalis, Illicium verum, Illicium anisatum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba, Myristica fragrans, Myrtus communis, Ocimum gratissimum, Ocimum basilicum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa, Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, Trachyspermum ammi, Thymus vulgaris, T.hyemalis, and T.zygis.

4. The use of any of the preceding claims, wherein the one or more plant oils are selected from clove oil, melalecuca oil, cinnamon oil and/or thyme oil.

5. The use of any of the preceding claims, wherein the plant oil is clove oil

6. Method of preventing or removing sea lice from fish, wherein a product containing one or more plant oils is administered as a bath treatment.

7. Method of preventing or removing sea lice from fish, wherein a product containing one or more plant oils are administered directly to the skin of the fish.

8. The method of any of claims 6-7, wherein the one or more plant oils contain eugenol and/or thymol.

9. The method of any of claims 6-8, wherein the one or more plant oils are obtained from plants selected from the group consisting of Syzygium aromaticum, Acorus calamus, Anasarum canadense, Aniba rosaedora, Artemisia dracunculus, Canarium indicum, Canarium lucozonium, Cananga odorata, Croton elutaria,

Cinnamomum camphora, Cinnamomum cassia, Cinnamomum verum, Cinnamomum tamala, Citrus paradisi, Citrus sinensis, Cymbopogon citratus, Cymbopogon nardus, Cymbopogon winterianus, Dacrydium franklinii, Daucus carota, Echinophora tenuifolia, Elettaria cardamomum, Eucalyptus, Hyssopus officinalis, Illicium verum, Illicium anisatum, Laurus nobilis, Levisticum officianale, Lippia citriodora, Magnolia magnolia, Melaleuca alternifolia, Melaleuca bracteata, Melaleuca Leucadendron, Michelia alba, Myristica fragrans, Myrtus communis, Ocimum gratissimum, Ocimum basilicum, Ocotea pretiosa, Peumus boldus, Pimenta dioica, Pimenta racemosa, Pimpinella anisum, Ravensara aromatica, Rosa centifolia, Rosa damascena, Rosa rugosa, Rosmarinus officinalis, Satureia hortensis, Satureia montana, Tagetes minuta, Trachyspermum ammi, Thymus vulgaris, T.hyemalis, and T.zygis or any combination of two ore more thereof.

10. The method of any of claims 6-9, wherein the plant oil is obtained from Syzygium aromaticum.

1 1. The method of any of claims 6-8, wherein the one or more plant oils are selected from clove oil, melalecuca oil, cinnamon oil, and/or thyme oil.

12. The method of any of claims 6-8, wherein the plant oil is clove oil.