NO20191485A1

NO20191485A1 - Method for removing crustacean ectoparasites from farmed salmonid fish using edible additives and light

Info

Publication number: NO20191485A1
Application number: NO20191485A
Authority: NO
Inventors: Jo Klaveness; Gordon Ritchie; Øyvind Oaland
Original assignee: Mowi ASA
Priority date: 2018-12-17
Filing date: 2019-12-17
Publication date: 2020-06-18
Also published as: CA3123798A1; EP3897114A1; NO346510B1; DK202170358A1; WO2020130843A1; CL2021001608A1; EP3897114A4

Description

METHOD FOR REMOVING CRUSTACEAN ECTOPARASITES FROM FARMED SALMONID FISH USING EDIBLE ADDITIVES AND LIGHT

Field of the invention

The invention relates to a new method for removing or killing of ectoparasites from farmed fish. More particularly, the invention relates to removing or killing crustacean ectoparasites from farmed fish. The fish may be a salmonid fish, such as Atlantic salmon (Salmo salar L.) or rainbow trout (Oncorhynchus mykiss). The crustacean ectoparasite may be salmon lice (Lepeophtheirus salmonis, Caligus rogercresseyi, Caligus spp.). Even more particularly the invention relates to a method comprising treatment means where light from an artificial light source is used in combination with at least one chemical compound at toxicologically acceptable concentrations for fish, in particular salmonid fish. More specifically the method relates to the use of a combination of regulatory approved food additives or cyclodextrin complexes thereof and light.

Background

Aquaculture, also referred to as aquafarming, is the farming of aqueous organism. Aquaculture involves cultivating freshwater and saltwater populations under controlled conditions and is in contrast to commercial harvesting or fishing where the organisms are naturally present. The farmed organisms can typically be fish, crustaceans, mollusks, aquatic plants, algae. The aquaculture farms can be in the form of tanks (closed or semi closed), fish ponds, ocean cages or nets.

Diseases, especially infectious diseases, are a problem in aquaculture. Within fish aquaculture, especially salmon farming, there has been a development of prophylactic therapy in the form of vaccines and development of treatment of disease in the form of various drugs. However, salmon lice infection remains a main problem regarding salmon farming.

Relevant prior art publications and their relevant content

Currently there are several drug substances that are used to combat salmon lice infection in salmon farms.

The combination of light and drugs related to fish has been a topic in a few published scientific documents.

Photodynamic therapy is generally a term for a therapy that involves a so-called photosensitizer and light, and oxygen, where the light activates the photosensitizer to generate singlet oxygen and other reactive and toxic species. Photodynamic therapy is well described in human medicine, see for example: Kwiatkowski S et al. Photodynamic therapy - mechanisms, photosensitizers and combinations. Biomed Pharmacother.

2018 Oct; 106:1098-1107. The clinical use of photodynamic therapy is, however, limited to some specific indications; especially some skin diseases with products comprising 5-aminolevulinic acid methyl ester as photosensitizer, and age-related macular degeneration with products comprising verteporfin as photosensitizer. Very many of the publications on photodynamic therapy relates to cancer and some publications relate to infections, however, these diseases are not commonly treated by photodynamic therapy.

D.-P-Hader et al. Fighting fish parasites with photodynamically active chlorophyllin. Parasitol. Res. 2016,115 page 2277-2283, describes a study where water-soluble chlorophyll was used. The result was “In Ichthyobodo, 2 μg/mL proved sufficient with subsequent simulated solar radiation to almost quantitatively kill the parasites, while in Dactylogyrus, a concentration of about 6 μg/mL was necessary. The LD50 value for this parasite was 1.02 μg/mL. Trichodina could be almost completely eliminated at 2 μg/mL. Only in the parasitic crustacean Argulus, no killing could be achieved by a photodynamic reaction using chlorophyllin.” The chlorophyllin was prepared from spinach and does not comprise copper.

Eliana Alves et al. 2015. Potential applications of porphyrins in photodynamic inactivation beyond the medical scope. J. Photochem. and Photobiol. C: Photochemistry Reviews, 22:34-57 is a general review publication within the field on photodynamic therapy. Figure 4 on page 43 in this publication illustrates generally a treatment protocol for photodynamic treatment of infected fish. The text in page 43 refers to Elina Alves et al. Photodynamic antimicrobial chemotherapy in aquaculture; photoinactivation studies if Vibrio fischeri in PLOS One 2011, 6, 6, e20970. The light is “solar irradiation from 380 to 700 nm consisting in 13 OSRAM lamps.” See below.

Eliana Alves et al. 2011. Photodynamic Antimicrobial Chemotherapy in Aquaculture: Photoinactivation Studies of Vibrio fischeri. PLOS One, https://doi.org/10.1371/journal.pone.0020970. The photosensitizer used in these studies is a porphyrin derivative.

Peter KJ Robinson et al. A new generation of biocides for control of crustacea in fish farms. J. Photochem and Photobiol. B:Biology,95(2009)58-63. This publication relates to the field of photodynamic therapy where light and a photosensitizer are used to generate toxic species. The photosensitizer was methylene blue or nuclear fast red, the marine species as a model organism in place of sea lice was the marine copepod Acartia clause, the lamp was a 500W tungsten halogen lamp. The result showed that the marine copepod mortality with methylene blue was high when the methylene blue concentration was 1 micromolar or above at 1 hour light activation. With nuclear fast red as photosensitizer, there was not observed any mortality during 1 hour light activation.

Methylene blue is a cationic (permanent positively charged) synthetic compound and nuclear fast red is a negatively charged sulphonic acid in sea water (pH 7.5 to 8.4). Acartia clausi is a small animal (1 mm in length, 0.2 mm broad). The length of salmon lice is up to 18 mm.

All light sources used in the above discussed prior art relate to white light.

Hydrogen peroxide has for many years been used to combat sea lice infections in fish including salmon. The concentrates are diluted to a final concentration of 1500 mg hydrogen peroxide per liter. Hydrogen peroxide is toxic for salmon lice. The mechanism of action is probably related to an oxidative effect on lice components and the formation of oxygen emboli within the salmon lice.

Drug resistance is a general problem with farming of salmon. See for example Stian Mørch Aaen et al. Drug Resistance in sea lice: a threat to salmon aquaculture.

Recently it has also been reported resistance towards hydrogen peroxide in the treatment of salmon lice. See for example: K.O. Helgesen et al. 2017. Increased catalase activity-A possible mechanism in hydrogen peroxide resistant salmon lice (Lepeophtheirus salmonis). Aquaculture 468:135-140 and K.O. Helgesen et al. 2015. First report of reduced sensitivity towards hydrogen peroxide found in the salmon louse Lepeophtheirus salmonis in Norway. Aquaculture Reports, 1, 37-42.

The invention has for its object to remedy or to reduce at least one of the drawbacks of the prior art, or at least provide a useful alternative to prior art.

The object is achieved through features, which are specified in the description below and in the claims that follow.

Summary of the present invention

The invention relates to a new method for treatment of salmon lice (L. salmonis, C. rogercresseyi, Caligus spp.) in farmed fish such as Atlantic salmon (S. salar L.) using an artificial light source and at least one chemical substance at toxicologically acceptable concentrations for Atlantic salmon. More specifically, the present inventors have identified a novel therapy for the treatment of salmon louse in farmed Atlantic salmon populations that comprises a combination of artificial light together with regulatory approved food additives.

The fish may be a salmonid fish, such as Atlantic salmon (Salmo salar L.) or rainbow trout (Oncorhynchus mykiss). For convenience fish is referred to as Atlantic salmon in the preceding text and in the text to follow, however, without excluding rainbow trout, other salmonid species or other relevant fish species.

The invention is defined by the independent patent claims. The dependent claims define advantageous embodiments of the invention.

The most preferred artificial light sources, according to the present invention, generate blue light. In the present description blue light comprises violet light (380nm to 450 nm, blue light (450nm to 485nm) and cyan light (485nm to 500nm). Blue light as used herein covers the wavelength from 380nm to 500nm.

The additional most preferred artificial light sources, according to the present invention, generate red light. Red light as used herein covers the wavelength from 625nm to 740nm.

More specifically, the invention relates in a first aspect to a composition in the form of an aqueous solution comprising a photosensitizer for use in a method of photodynamic therapy for an external crustacean parasite infection in salmonid fish, said external crustacean parasite infection comprises an infection of salmon lice, Lepeophtheirus salmonis, Caligus rogercresseyi and Caligus spp. Said photosensitizer is curcumin or a pharmaceutical acceptable derivative thereof, and said photosensitizer is administered in a bath treatment of the salmonid fish in need of such treatment, and the salmon lice is illuminated by light.

In one embodiment said photosensitizer may be a curcumin cyclodextrin.

In one embodiment said light may be blue light. In an alternative embodiment said light may be red light.

The salmonid fish may be located in a receptacle. The receptacle may be one of a closed tank, a semi-closed tank, a chamber, a container and a fish pond. The salmonid fish may be located in one of an ocean cage and a net pen.

The blue light source may comprise of LED lamps. The LED lamps may emit light of a wavelength of 450nm to 460nm.

Said photosensitizer may be added in the water surrounding the salmonid fish. The water surrounding the salmonid fish may form the bath treatment.

In a second aspect the invention relates to use of an artificial light device emitting blue light in accordance to the first aspect of the invention.

In a third aspect the invention may relate to a composition in the form of an aqueous solution comprising a photosensitizer for use in a method of photodynamic therapy for an external crustacean parasite infection in salmonid fish, said external crustacean parasite infection comprises an infection of salmon lice, Lepeophtheirus salmonis, Caligus rogercresseyi and Caligus spp. Said photosensitizer is riboflavin or a pharmaceutical acceptable derivative thereof, and said photosensitizer is administered in a bath treatment of the salmonid fish in need of such treatment, and the salmon lice is illuminated by light.

In one embodiment said photosensitizer may be a riboflavin phosphate.

In a forth aspect the invention may relate to a composition in the form of an aqueous solution comprising a photosensitizer for use in a method of photodynamic therapy for an external crustacean parasite infection in salmonid fish, said external crustacean parasite infection comprises an infection of salmon lice, Lepeophtheirus salmonis, Caligus rogercresseyi and Caligus spp. Said photosensitizer is chlorophyllin sodium copper salt or a pharmaceutical acceptable salt or derivative thereof, and said photosensitizer is administered in a bath treatment of the salmonid fish in need of such treatment, and the salmon lice is illuminated by light.

The energy required for the artificial light source according to the present invention can vary from a few watts to several kilowatts depending upon the nature of the artificial light source and the volume of the enclosure or ocean cage or net. The intensity of the light diminishes with the distance between the light source and the target surface, e.g. the surface of the sea lice. In some cases, and for some useful photosensitizers, such as curcumin or a pharmaceutical acceptable derivative thereof, or riboflavin or a pharmaceutical acceptable derivative thereof, or chlorophyllin sodium copper salt or a pharmaceutical acceptable salt or derivative thereof, the light intensity may be too high at a too close distance, leading to a too rapid bleaching of the photosensitizer, and thereby the photosensitizer lose its effect. In some cases, and for some useful photosensitizers, the light intensity may be too low at a too far distance, leading to that photosensitizer is not activated. It is therefore within the scope of the present invention to optimize the distance between the light source and the target surface. The optimal distance is thus dependent on among other things the actual wavelength, the actual photosensitizer, and the actual specification of the lamp, e.g. output in lumen.

Surprisingly it has been found that not only do light and regulatory approved food additives together have an additional effect on salmon lice but that light from an artificial light source and regulatory approved food additives together have a synergistic effect. The exact mechanism of the combination of light together with the regulatory approved food additives on salmon lice is not studied, but it can be speculated that the light activates the regulatory approved food additives through a photochemical process and that the formed reactive species are responsible for the observed increased toxicity on the salmon lice.

Said photosensitizer may comprise a regulatory approved food additive. Said photosensitizer may be a food additive cyclodextrin complex where the food additive comprises the regulatory approved food additive.

The regulatory approved food additive may be at least one of E100 Curcumin, E101(i) Riboflavin, E101(ii) Riboflavin-5'-phosphate, E102 Tartrazine, E123 Amaranth, E127 Erythrosine, E129 Allura Red AC, E131 Patent Blue V, E132 lndigotine; Indigo Carmine, E133 Brilliant Blue FCF, E140 Chlorophylls and chlorophyllins, E141 Copper complexes of chlorophyll and chlorophyllins, E142 Green S, E151 Brilliant Black BN; Black PN, E155 Brown HT, E160a Carotenes, E160b Annatto; Bixin; Norbixin, E160c Paprika extract; Capsanthian; Capsorubin, E160d Lycopene, E160e Beta-apo-8'-carotenal (C30), E161b Lutein, E161g Canthaxanthin, E162 Beetroot Red; Betanin, E163 Anthocyanins and E180 Litholrubine BK.

The artificial light source may comprise of LED lamps. The wavelength of the emitted light from the artificial light source may be between 200 and 800 nm. The wavelength of the emitted light may be between 400 and 800 nm. The wavelength of the emitted light may be between 200 and 400 nm. The wavelength of the emitted light may be between 200 and 300 nm.

Said artificial light source may be a part of a device for automatic localization of salmon lice. Said artificial light source may be a light source used in cultivation or farming of salmonid fish.

Said photosensitizer may be added in a food pellet for feeding the salmonid fish. Said photosensitizer may be added in the water surrounding the salmonid fish.

An artificial light device may be used according to the methods described above.

In one aspect the present invention may relate to a treatment of salmon louse in farmed fish populations that comprises a combination of regulatory approved food additives as cyclodextrin complexes and artificial light.

In one aspect the present invention may relate to a protocol for the present treatment. Suitably, the food additive is administered prior to, after or at approximately the same time as the artificial light.

In one aspect the present invention may relate to a protocol for the present treatment. The present treatment can be performed when the Atlantic salmon is in receptacles or enclosures, e.g. closed tanks or semi-closed tanks, pipes, chambers, containers, fish ponds, ocean cages or net pens or a combination of such enclosures.

In one aspect the present invention may relate to the artificial light source. The light source according to the present invention can be a light source positioned in the air or placed in water. The light source can optionally be a plurality of light sources. The light source should generate light with wave lengths from 200 to 800 nm. The light source might also generate electromagnetic radiation that is outside this range. The light source might be in the form of a laser. The light source or light systems may comprise halogen lamps, mercury lamps, light emitting electrodes (LEDs) or other suitable light systems. The light source can be a stationary light source or a movable light source.

In one aspect the present invention may relate to a drug treatment protocol. Optionally, according to the present invention other drug substances or drug like compounds might, in addition to regulatory approved food additives, be useful for the treatment according to the present invention. These drugs include other drugs that have been shown to be toxic to salmon lice including commercial products that are approved for salmon lice infections in Atlantic salmon.

In one aspect the present invention may relate to the dose or concentration of the regulatory approved food additive in the clinical situation.

In one aspect the present invention may relate to the exposure time of the treatment process. The time necessary to perform the present process varies from minutes to hours depending upon several factors like for example nature of the salmon lice infection, number of Atlantic salmon per cubic meter of water, concentration of regulatory approved food additive in the water and optionally other drugs and drug like compounds and nature of the artificial light source.

The present invention may relate to resistance. The method according to the present invention is new for treatment of Atlantic salmon infected by salmon lice and has advantages related to treatment of Atlantic salmon infected by resistant salmon lice and also related to generation of new forms of resistance. The possible mechanism of action related to the present invention is biologically not a typical process where resistance processes are generated.

The advantages by a combination of light together with food additives according to the present invention versus state-of-the-art treatment of salmon lice relate to one or more of the following topics: reduced amount of drugs, improved efficacy on salmon lice, reduced toxicity to the Atlantic salmon, resistance issues, cost and time.

Detailed description of the present invention

Surprisingly it has been found a new method for treatment of salmon lice (L. salmonis and C. rogercresseyi) in farmed fish such as Atlantic salmon (S. salar L.) using an artificial light source and at least one chemical substance at toxicologically acceptable concentrations for Atlantic salmon.

The present invention relates to the selection of food additives. Approved food additives are listed with E-numbers. See for example: https://www.food.gov.uk/businessguidance/eu-approved-additives-and-e-numbers

Relevant food additives with E-numbers used together with light from an artificial light source for treatment of salmon lice (L. salmonis and C. rogercresseyi) in farmed Atlantic salmon according to the present invention are:

E100 Curcumin

E101(i) Riboflavin ,(ii) Riboflavin-5'-phosphate

E102 Tartrazine

E104 Quinoline yellow

E110 Sunset Yellow FCF; Orange Yellow S

E120 Cochineal; Carminic acid; Carmines

E122 Azorubine; Carmoisine

E123 Amaranth

E124 Ponceau 4R; Cochineal Red A

E127 Erythrosine

E129 Allura Red AC

E131 Patent Blue V

E132 lndigotine; Indigo Carmine

E133 Brilliant Blue FCF

E140 Chlorophylls and chlorophyllins

E141 Copper complexes of chlorophyll and chlorophyllins

E142 Green S

E150a Plain caramel

E150b Caustic sulphite caramel

E150c Ammonia caramel

E150d Sulphite ammonia caramel

E151 Brilliant Black BN; Black PN

E153 Vegetable carbon

E155 Brown HT

E160a Carotenes

E160b Annatto; Bixin; Norbixin

E160c Paprika extract; Capsanthian; Capsorubin

E160d Lycopene

E160e Beta-apo-8'-carotenal (C30)

E161b Lutein

E161g Canthaxanthin

E162 Beetroot Red; Betanin

E163 Anthocyanins

E170 Calcium carbonate

E171 Titanium dioxide

E172Iron oxides and hydroxides

E173 Aluminium

E174 Silver

E180 Litholrubine BK

The preferred compounds according to the present invention are:

E100 Curcumin, E101(i) Riboflavin; (ii) Riboflavin-5'-phosphate, E102 Tartrazine, E123 Amaranth, E127 Erythrosine, E129 Allura Red AC, E131 Patent Blue V, E132 lndigotine; Indigo Carmine, E133 Brilliant Blue FCF, E140 Chlorophylls and chlorophyllins, E141 Copper complexes of chlorophyll and chlorophyllins, E142 Green S, E151 Brilliant Black BN; Black PN; E155 Brown HT, E160a Carotenes, E160b Annatto; Bixin; Norbixin, E160c Paprika extract; Capsanthian; Capsorubin, E160d Lycopene, E160e Betaapo-8'-carotenal (C30), E161b Lutein, E161g Canthaxanthin, E162 Beetroot Red; Betanin, E163 Anthocyanins and E180 Litholrubine BK.

The most preferred compounds according to the present invention are:

E100 Curcumin, E101(i) Riboflavin; (ii) Riboflavin-5'-phosphate, E102 Tartrazine, E123 Amaranth ,E127 Erythrosine ,E129 Allura Red AC,E131 Patent Blue V, E132 lndigotine; Indigo Carmine ,E133 Brilliant Blue FCF, E140 Chlorophylls and chlorophyllins, E141 Copper complexes of chlorophyll and chlorophyllins, E142 Green S, E160a Carotenes E160c Paprika extract; Capsanthian; Capsorubin, E160d Lycopene, E161b Lutein, E161g Canthaxanthin, E162 Beetroot Red; Betanin and E163 Anthocyanins.

The even most preferred compound is curcumin.

The concentration of the food additive varies depending upon the various parameters like for example choice of food additive, temperature, disease stage, light source and the density of Atlantic salmon. Typical concentration range for the food additives according to the present invention is in the range 0.1 micromolar (1 time 10<-7 >molar (M)) to 400 micromolar (400 times 10<-6 >molar(M)). More preferred the concentration range for the food additive cyclodextrin complex according to the present invention is in the range 0.5 micromolar to 300 micromolar.

The present invention relates to treatment of salmon louse in farmed fish populations that comprises a combination of regulatory approved food additives as cyclodextrin complexes and light from an artificial light source.

Cyclodextrins (CDs) are cyclic oligosaccharides what are known to form complexes with various chemical and drugs. Drug complexes with CDs are currently used in human medicine. The potential advantages by using drug CD complexes relate to chemical stability, aqueous solubility and oral bioavailability.

The chemical group called CDs include alpha-CD which is a 6-membered sugar ring molecule, beta-CD which is a 7-membered sugar ring molecule, gamma-CD which is a 7-membered sugar ring molecule and an almost unlimited number of chemical derivatives with various degree of substitution on these three different sugar ring molecules.

The preferred and the most preferred food additives also relate to the food additive cyclodextrin complexes.

Regarding the cyclodextrin part of the food additive cyclodextrin complexes are the three unsubstituted cyclodextrins, methyl cyclodextrin and 2-hydroxypropyl cyclodextrins the preferred cyclodextrins.

Regarding the cyclodextrin part of the food additive cyclodextrin complexes is betacyclodextrin the most preferred cyclodextrin.

This means that typically curcumin beta-cyclodextrin, chlorophyll beta-cyclodextrin and chlorophyllin beta-cyclodextrin are among the most preferred food additive cyclodextrin complexes to be used together with artificial light for treatment of salmon louse in farmed fish according to the present invention.

The food additive cyclodextrin complexes according to the present invention can be prepared by standard methods for preparation of such complexes. These methods include co-evaporation, spray-drying, freeze drying and kneading. One simple method is to carefully mix the food additive with the cyclodextrin (typically molar ratio 1 to 5) with small amounts of water forming a very thick paste for 10 minutes using a mortar and pestle, dry the paste in an oven and prepare a powder of the dry material using the mortar and pestle.

The concentration of the food additive cyclodextrin complex varies depending upon the various parameters like for example choice of food additive, temperature, disease stage, light source and the density of Atlantic salmon. Typical concentration range for the food additive cyclodextrin complex according to the present invention is in the range 0.1 micromolar (1 time 10<-7 >molar (M)) to 400 micromolar (400 times 10<-6 >molar(M)). More preferred the concentration range for the food additive cyclodextrin complex according to the present invention is in the range 0.5 micromolar to 300 micromolar.

The present invention relates to a protocol for the present treatment. Suitably, food additive is administered prior to, after or at approximately the same time as the light. The light might be present after the food additive concentration around the Atlantic salmon is reduced. Any protocol using food additive and light is within the scope of the present invention. It is up to the skilled person on salmon lice treatment to select the best suited protocol for each treatment.

A first preferred protocol is to apply the regulatory approved food additive some time prior to the light treatment. In this case the light and the regulatory approved food additive are present partly together or the light is present only after the aqueous concentration of hydrogen peroxide is reduced but still present within the salmon louse.

A second preferred protocol is to apply the regulatory approved food additive and together and optionally continue to use light after the aqueous concentration of the regulatory approved food additive is reduced.

A third preferred protocol is to apply light after the aqueous concentration of the regulatory approved food additive is reduced but the regulatory approved food additive still is present within the salmon lice.

The most preferred protocol is to apply light, at least partly, when the concentration of the regulatory approved food additive still is quite high in the water surrounding the Atlantic salmon and the salmon lice.

The present invention relates to a protocol for the present treatment. The present treatment can be performed when the Atlantic salmon is in receptacles or enclosures, e.g. closed tanks or semi-closed tanks, pipes, chambers, containers, fish ponds, ocean cages or net pens or a combination of such enclosures.

The preferred methods of the present invention relate to treatment of Atlantic salmon with salmon lice within a tank/boat or ocean cages or net pens.

The absolutely most preferred methods of the present invention relate to treatment of Atlantic salmon infected with salmon lice within an ocean cage or a net pen.

The present invention relates to the position of the artificial light source. One preferred position of the light source is in the air above the water. Another preferred position of the light source is on the walls or the bottom of a receptacle or enclosure, e.g. closed tank or semi-closed tank, pipe, chamber, container, or fish ponds. Another preferred position of the light source is under water within the receptacle or the ocean cage or net by mechanical arrangements and/or floating devices. The most preferred position of the light source in a large ocean cage or net is under water.

The artificial light source should generate light that has wave lengths from 200nm to 800nm. One preferred artificial light source according to the present invention is that the artificial light source generates visible light within the wavelength band from 400nm to 800nm.

One preferred artificial light source according to the present invention is that the artificial light source generates light within one of the following colors: red, yellow, green, blue or white.

Another preferred light source according to the present invention is that the artificial light source generates UV light within the wavelength band from 200nm to 400nm. Another preferred artificial light source according to the present invention is that the artificial light source generates UVC light within the wavelength band from 200nm to 300nm.

The artificial light source might also generate electromagnetic radiation that is outside these wavelength bands. If so, the artificial light source must also generate some light within referred wave lengths or colors.

The artificial light source can be one single artificial light source or a plurality of artificial light sources. If the volume is huge, like in an ocean cage or net, it is according to the present invention preferred to use a plurality of artificial light sources.

One artificial preferred light source according to the present invention is that the artificial light source is based on laser.

Another preferred artificial light source according to the present invention is that the artificial light source is based on LEDs, halogen lamp or mercury lamp. The most preferred type of lamps among these lamps are LED lamps.

The most preferred artificial light sources according to the present invention are LED lamps generating blue light; typically, in the wavelength range from 380nm to 500nm. Examples of such lamps are optionally dimmable, underwater lamps currently commercially available and used in aquaculture for other purposes.

The light from the artificial light source may be stationary, i.e. same light in the same area over time, or the artificial light source may be movable.

The present invention relates to a drug treatment protocol.

Optionally, according to the present invention, other drug substances or drug like compounds may, in addition to the regulatory approved food additive, be useful for the treatment according to the present invention.

One preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds are selected among other drugs that are regulatory approved for use to treat Atlantic salmon infected by salmon lice.

Another preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds that are cholinesterase inhibitors, synthetic pyrethroids, chitin synthase inhibitors or glutamate-based chlorine ion channel regulators.

Another preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds are selected among compounds that are photosensitizers.

Another preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds are selected among compounds that are phototoxic.

The present invention relates to the dose or concentration of the regulatory approved food additive in the clinical situation.

The present invention relates to the timing of the treatment process. The time necessary to perform the present process varies from minutes to hours depending upon several factors like for example nature of the salmon lice infection, number of Atlantic salmons per cubic meter, the nature and the concentration of the regulatory approved food additive and optionally other drugs and drug like compounds, and nature of the light source.

The present invention relates to resistance. The method according to the present invention is new for treatment of Atlantic salmon infected by salmon lice and has advantages related to treatment of Atlantic salmon infected by resistant salmon lice and also relates to generation of new forms of resistance. The possible mechanism of action related to the present invention is biologically not a typical process where resistance processes are generated.

The advantages by a combination of light together with the regulatory approved food additive according to the present invention relate to one or more of the following topics: reduced amount of drugs, improved efficacy on salmon lice, and reduced toxicity to the Atlantic salmon.

Experimental

Preparation of compounds

Compound 1

Chlorophyllin sodium copper salt 2-Hydroxypropyl-β-cyclodextrin complex (2:3) Chlorophyllin sodium copper salt (1.412 gram, 2mmol) and 2-Hydroxypropyl-βcyclodextrin (Average Mw ~1,460) (4.38 gram, 3mmol) were mixed in a mortar with a pestle. Water (3 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 60 degrees centigrade in dark overnight. The product was in the form of a green powder. The product comprised 24.4% chlorophyllin sodium copper salt.

Compound 2

Chlorophyllin sodium copper salt gamma-cyclodextrin complex (2:3) Chlorophyllin sodium copper salt (1.412 gram, 2mmol) and gamma-cyclodextrin (3.89 gram, 3mmol) were mixed in a mortar with a pestle. Water (3 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 60 degrees centigrade in dark overnight. The product was in the form of a green powder. The product comprised 26.6% chlorophyllin sodium copper salt.

Compound 3

Curcumin 2-Hydroxypropyl-β-cyclodextrin complex (2:3)

Curcumin (0.736 gram, 2mmol) and 2-Hydroxypropyl-β-cyclodextrin (Average Mw ~1,460) (4.38 gram, 3 mmol) were mixed in a mortar with a pestle. Water (3 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 14.4 % curcumin.

Compound 4

Curcumin gamma-cyclodextrin complex (2:3)

Curcumin (0.736 gram, 2mmol) and gamma-cyclodextrin (3.89 gram, 3mmol) were mixed in a mortar with a pestle. Water (5 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 60 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 15.9 % curcumin.

Compound 5

Methylene Blue 2-Hydroxypropyl-β-cyclodextrin complex (2:3)

Methylene Blue hydrate (640 mg, 2mmol) and 2-Hydroxypropyl-β-cyclodextrin (Average Mw ~1,460) (4.38 gram, 3mmol) were mixed in a mortar with a pestle. Water (3 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a blue powder. The product comprised 12.8% methylene blue.

Compound 6

Methylene Blue gamma-cyclodextrin complex (2:3)

Methylene Blue hydrate (640 mg, 2mmol) and gamma-cyclodextrin (3.89 gram, 3mmol) were mixed in a mortar with a pestle. Water (3 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a green powder. The product comprised 14.1% methylene blue.

Compound 7

Curcumin-β-cyclodextrin complex (1:4)

Curcumin (0.368 gram, 1mmol) and β-cyclodextrin (4.54 gram, 4mmol) were mixed in a mortar with a pestle. Water (5 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 7.8% curcumin.

Compound 8

Curcumin-β-cyclodextrin complex (1:4) Polysorbate 80

Curcumin (0.368 gram, 1mmol), Polysorbate 80 (250mg) and β-cyclodextrin (4.54 gram, 4mmol) were mixed in a mortar with a pestle. Water (5 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 7.4% curcumin.

Compound 9

Curcumin-β-cyclodextrin complex (1:4)

A mixture of curcumin (0.368 gram, 1mmol) and β-cyclodextrin (4.54 gram, 4mmol) and absolute ethanol (30 ml) was stirred in a beaker for 20 hours at room temperature. The mixture was headed to 70 degrees centigrade for 2 hours. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow fluffy powder. The product comprised 7.8% curcumin.

Compound 10

Curcumin-β-cyclodextrin complex (1:4) Polysorbate 80

A mixture of curcumin (0.368 gram, 1mmol), Polysorbate 80 (0.25g) and βcyclodextrin (4.54 gram, 4mmol) and water (10 ml) was stirred in a beaker for 24 hours at room temperature. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 7.4% curcumin.

Compound 11

Curcumin-β-cyclodextrin complex (1:4)

A mixture of curcumin (0.368 gram, 1mmol), Polysorbate 80 (0.25g) and βcyclodextrin (4.54 gram, 4mmol) and water (10 ml) was stirred in a beaker for 24 hours at room temperature. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 7.8% curcumin.

Compound 12

Curcumin-β-cyclodextrin complex (1:8) Polysorbate 80

A mixture of curcumin (0.368 gram, 1mmol), Polysorbate 80 (0.25g) and βcyclodextrin (9.08 gram, 8mmol) and water (10 ml) was stirred in a beaker for 24 hours at room temperature. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 3.8% curcumin.

Compound 13

Curcumin-β-cyclodextrin complex (1:8)

A mixture of curcumin (0.368 gram, 1mmol) and β-cyclodextrin (9.08 gram, 8mmol) and water (10 ml) was stirred in a beaker for 24 hours at room temperature. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 3.9% curcumin.

Compound 14

Riboflavin 2-Hydroxypropyl-β-cyclodextrin complex (2:3)

Riboflavin (0.752 gram, 2mmol) and 2-Hydroxypropyl-β-cyclodextrin (Average Mw ~1,460) (4.38 gram, 3 mmol) were mixed in a mortar with a pestle. Water (3 ml) was added and the formed paste was mixed in the mortar for 5 minutes. The product was dried at 65 degrees centigrade in dark overnight. The product was in the form of a yellow powder. The product comprised 14.7% riboflavin.

Example 1

In vivo testing on Salmon louse (white light and red light, low concentrations

Salmon louse (Lepeophtheirus salmonis) were collected from a salmon farming facility in Norway.

LABORATORY SETUP

The following three treatment compounds were tested in this study:

Control, seawater control.

Compound 3

Curcumin 2-Hydroxypropyl-β-cyclodextrin complex (2:3), concentration in seawater: 7.4 mg curcumin per litre (curcumin solution; 20 micromolar).

Chlorophyllin sodium copper salt (Sigma C6003-25G), concentration in seawater: 4 mg per litre (chlorophyll solution; 5.5 micromolar).

5-Aminolevulinic acid hydrochloride (5-ALA) (Sigma-Aldrich). Concentration in seawater: 168 mg per litre (5-ALA solution; 1 millimolar).

This pilot study involved exposing four treatment compounds to three different light sources for 24 h. In this study they were placed in the following 3 groups:

Group 1: White LED light (240V, 50W, 3500lm)

Group 2: Red light. 100 red LED lamp array (10 x 10) mounted on a heat sink. Specifications: 620-625nm, DC32-36V, 3500mA, 1000-1500lm

Group 3: Dark

In the case of group 1 and 2, a water bath (10°C) provided with a light source was set up for each group. Group 3 was placed in a sealed temperature-controlled cabinet. In addition, each treatment received additional aeration throughout the exposure period. This provided aeration and ensures that the treatment solution remains in suspension, allowing for even distribution of the treatment compound.

BIOASSAY PROTOCOL

Nine glass flasks were filled with 300 ml seawater. Five lice were placed in each flask. The flasks were then randomly distributed among the three different treatment groups, resulting in 4 containers in each group. Each flask was gently stirred to ensure that all lice were attached, the contents were emptied over a sieve, after which 300 ml treatment compound was then introduced into the flask. The flasks were then placed in their respective water bath or dark cabinet.

Group 3 was placed in the darkened cabinet immediately following exposure to the treatment compound. Sixty minutes after the exposure period began a count of the number of affected/unaffected lice was conducted.

Groups 1 and 2 were exposed to treatment compound for 1 hour, after which the lights were turned on and they were then exposed to a combination of treatment compound and light source. One hour after the start of light exposure a count of the number of affected/unaffected lice was conducted.

Additional counts of affected/unaffected lice in all 3 groups was conducted 4 hours into the exposure period. A final count of affected/unaffected lice was conducted at 24 h.

RESULTS

Results from group 1, white light treatment (Table 1), indicate that there was no treatment effect until conclusion of the 24 h exposure period. The curcumin solution (Compound 3) resulted in 60% parasites affected by the treatment. The chlorophyll solution (chlorophyllin sodium copper salt) treatment group showed no treatment effect, in fact, a number of egg string had hatched in the chlorophyll solution, all nauplii present were alive and swimming. The 5-ALA solution showed no treatment effect. The concentration of 5-ALA was 1 millimolar which was 50 times higher than the concentration of curcumin which was 20 micromolar.

Table 1. Percentage (%) affected/unaffected parasites after 1 h, 4 h, and 24 h exposure to white LED light in combination with 3 treatment solutions.

Group 2, red light treatment (Table 2), resulted in no affected lice until the 24 h count. At the 24 h count, no affected parasites were observed in the seawater control solution. In comparison, 20% of the lice were affected in chlorophyll solution and 40% in the curcumin solution. The 5-ALA solution showed no treatment effect. The concentration of 5-ALA was 1 millimolar which was 50 times higher than the concentration of curcumin which was 20 micromolar.

Table 2. Percentage (%) affected/unaffected parasites after 1 h, 4 h, and 24 h exposure to red LED light in combination with 3 treatment solutions.

Group 3, dark/no light treatment (Table 3), resulted in 20% affected lice at the 1 h count in the curcumin solution. However, at the conclusion of the bioassay all lice exposed to this solution were determined to be unaffected.

Table 3. Percentage (%) affected/unaffected parasites after 1 h, 4 h, and 24 h exposure to dark/no light in combination with 3 treatment compounds.

Example 2 In vivo testing on Salmon louse (white light, red light and blue light, higher concentrations

LABORATORY SETUP

The following three treatment compounds were tested in this study:

Control (seawater)

Treatment I: Curcumin 2-hydroxypropyl-beta-cyclodextrin (2:3) (labelled MH-103) (Compound 3). Concentration: 80 micromolar (29.6 mg curcumin (204 mg complex) per liter).

Treatment II: Curcumin beta-cyclodextrin labelled MH-107 (1:4) (Compound 7). Concentration: 640 micromolar (22.7 mg curcumin (304 mg complex) per liter).

Treatment III: Copper complex of chlorophyllin sodium salt 2-hydroxypropyl-betacyclodextrin complex (2:3) (Compound 1). Concentration: 22 micromolar (16 mg copper complex of chlorophyllin sodium salt (64 mg complex) per liter).

In this study the four treatment compounds were exposed to the following light sources for 24 h:

White LED light.(240V, 50W, 3500lm)

Red light. 100 red LED lamp array (10 x 10) mounted on a heat sink. Specifications: 620-625nm, DC32-36V, 3500mA, 1000-1500lm). (The power supply did not function optimally in these experiments resulting in somewhat reduced intensity of the red light (only 30 V, 0.51 A)).

Blue light: 100 blue LED lamp array (10 x 10) mounted on a heat sink. Specifications: 380-500 nm, DC32-36V, 3500mA (100W). Luminous flux: 5000-6000lm.

Two water baths were set up with a constant water temperature of 12°C. Solid barriers were placed in the water baths to separate the three light groups . Each treatment container received additional aeration throughout the exposure period. This not only provided aeration, but also ensured that the treatment solution remained in suspension, allowing for even distribution of the treatment compound.

BIOASSAY PROTOCOL

Eleven glass flasks were filled with 300 ml seawater. Ten lice were transferred, using forceps, into each flask. The flasks were randomly labelled and distributed among the 3 light groups. Due to limited lice numbers only 2 control flasks were available, so only 2 treatment groups had controls. Each flask was gently stirred to ensure that all lice were attached, the contents were emptied over a sieve, after which 300 ml treatment compound was then introduced into the flask. The flasks were then placed in their respective waterbath. Sixty minutes after the exposure period began a count of the number of affected/unaffected lice was conducted. All groups were exposed to the treatment compound for 30 min, after which the lights were turned on and they were then exposed to a combination of treatment compound and light source. One hour after the start of light exposure a count of the number of affected/unaffected lice was conducted. A final count of affected/unaffected lice was conducted at 24 h.

RESULTS

When looking at the white LED group (Table 4), treatment I had 30% affected parasites as compared to the other treatments with no affected parasites. The treatments were more effective at the 24 h count, with treatment I being most effective with 100% affected lice, treatment II had 80% affected lice, and treatment III had 30% affected lice (Table 4). At the 24 h count it was observed that treatment I contained a lot of precipitate, whereas the other groups had none. Due to low numbers of parasites, the red LED group did not include a control. Treatment efficacy at the 1 h control was low with treatment I resulting in 20% affected lice and treatment III with 10% affected lice (Table 5). By the 24 h mark treatment III resulted in 0% affected lice. However treatment I resulted in 80% affected lice, and treatment II had 50% affected lice (Table 5). Again, there were issues with the presence of a high amount of precipitate in treatment I. The blue LED group resulted in no affected lice in either the control or treatment III for both the 1 h and 24 h counts (Table 6). Treatment I was the most effective with 70% affected lice at the 1 h count and 90% affected lice at the 24 h count. Treatment II resulted in 20% affected lice after 1 h and 30% affected lice after 24 h of exposure. In the blue light group at the 24 h count, treatment III had large amounts of precipitate out of solution with lice observed moving through it (Table 5).

Table 4. Percentage (%) affected/unaffected parasites after 1 h and 24 h exposure to white LED light in combination to four treatment solutions.

Table 5. Percentage (%) affected/unaffected parasites after 1 h and 24 h exposure to red LED light in combination to 3 treatment solutions.

Table 6. Percentage (%) affected/unaffected parasites after 1 h and 24 h exposure to blue LED light in combination to 4 treatment solutions.

Example 3 In vivo testing on Salmon louse

LABORATORY SETUP

The following three treatment compounds and in different concentrations were tested in this study:

Treatment I (T1): Curcumin 2-hydroxypropyl-beta-cyclodextrin (MH-103). Concentration: 160 micromolar curcumin (408 mg complex per liter).

Treatment II (T2): Riboflavin 5’-phosphate sodium Ph. Eur. Concentration: 100 micromolar (47.8 mg per liter).

Treatment III (T3): Riboflavin 5 Ph. Eur. Concentration: 100 micromolar (37.6 mg per liter).

Treatment IV (T4): Riboflavin 5 Ph. Eur. Concentration: 300 micromolar (112.8 mg per liter).

Treatment V (T5): Riboflavin 5’-phosphate sodium Ph. Eur. Concentration: 300 micromolar (143.4 mg per liter).

In this study the three treatment compounds were exposed to the following light sources:

White LED light. (100-240V, 50W, Luminous flux: 3500lm)

Blue light. 100 blue LED lamp array (10 x 10) mounted on a heat sink. Specifications: 450-460nm, DC32-36V, 3500mA (100W), Luminous flux: 1000-1500lm).

Dark (temperature-controlled cabinet)

BIOASSAY PROTOCOL

Light regimens

Light regimen 1:

0-30 minutes: No light

30- 120 minutes: Light

120-240 minutes: No light

Light regimen 2:

0-120 minutes: Light

120-240 minutes: No light

The following protocol was followed for each of the two light regimen. Twelve glass flasks were filled with 300 ml seawater. Ten preadult 2 stage lice were transferred, using forceps, into each flask. The flasks were randomly labelled and distributed among the 3 light groups, resulting in 4 flasks per light treatment. Each flask was gently stirred to ensure that all lice were healthy and able to attach, the contents were emptied over a sieve, after which 300 ml treatment compound was then introduced into the flask. The flasks were then placed in their respective water bath/darkened cabinet and exposed to the respective lights as described below. Observations of the number of affected/unaffected lice were conducted at 10, 30, 40, 50, 60, 90, 120, 180, 240 min. At the completion of each round (240 min), the lice were rinsed in seawater, dried of excess fluid, placed in marked vials and frozen at -80°C for further chemical analysis.

RESULTS

Light regimen 1

Treatment I, curcumin cyclodextrin (160 micromolar) was found to be potent. The percentage of active salmon lice after 40 minutes was zero using blue light. The treatment I solution was also active using white light with 50% active lice after 40 minutes and 20% active lice after 90 minutes.

The treatment II, riboflavin-5-phosphate solution and the treatment III, riboflavin solution were not active using the high intensity lights (Table 7). HPLC analysis of sea water solutions of both riboflavin and riboflavin phosphate showed extensive degradation of both compounds after irradiation with the intensive 100W blue LED light after 10 minutes. Sea water solutions of both riboflavin and riboflavin phosphate showed no degradation in dark during 120 minutes. In a real aquaculture situation, the average light intensity will be much lower than in this example. The inventors expect that the lower blue light intensity will not degrade the riboflavin molecules and thereby these compounds will work as photosensitizers for treatment of salmon infected by salmon lice.

Table 7 Percentage active salmon lice recorded during nine observations during a 4h exposure to three different compounds and three different light sources / conditions

Light regimen 2

Exposure to the light regimen 2 confirmed that the riboplavin-5-phosphate solution (treatment IV) and riboflavin solution (treatment V) were not active in higher concentrations (300 micromolar) (Table 8).

Table 8 Percentage active salmon lice recorded during nine observations during a 4h exposure to two different compounds and three different light sources / conditions

Example 4

SOURCING PARASITES

The following lice strains were used. The first was sourced from iLab, Bergen, where they cultured, collected, and shipped the lice to the laboratory based at NMBU (Norwegian University of Life Sciences, Adamstuen, Oslo). The lice were shipped in a single 2.5L container filled with oxygenated seawater.. The second was sourced from the University of Bergen (UiB), where they cultured, collected, and shipped the lice to the laboratory based at NMBU. The lice were shipped in four 2.5L plastic bottles, filled with oxygenated seawater. All surviving lice were placed in a temperature-controlled cabinet (12°C) with added air supply until commencement of the bioassays.

LABORATORY SETUP

All bioassays were conducted at a constant water temperature of 12°C using two water baths and a temperature-controlled cabinet and included the following three light treatments: white LED, blue LED and no light (Table 1). The bioassays were conducted in two rounds, one round for each parasite strain using the same treatment compounds being used as shown in Table 2.

In this study the salmon lice were exposed to the following light sources:

White LED light. (100-240V, 50W, Luminous flux: 3500lm)

Blue light. 100 blue LED lamp array (10 x 10) mounted on a heat sink and cooled by an external fan. Specifications: 450-460nm, DC32-36V, 3500mA (100W), Luminous flux: 1000-1500lm).

Dark (temperature-controlled cabinet)

Treatment I (T1): Riboflavin 2-hydroxypropyl-beta-cyclodextrin. (Compound 14). Concentration: 300 micromolar

Treatment II (T2): Riboflavin phosphate. Concentration: 300 micromolar

Treatment III (T3): Sea water control

Treatment IV (T4): 10 minutes: Curcumin 2-hydroxypropyl-beta-cyclodextrin. Concentration 160 micromolar (408 mg complex per liter)

Treatment V (T5): 20 minutes: Curcumin 2-hydroxypropyl-beta-cyclodextrin. Concentration 160 micromolar (408 mg complex per liter)

Treatment VI (T6): 30 minutes: Curcumin 2-hydroxypropyl-beta-cyclodextrin. Concentration 160 micromolar (408 mg complex per liter)

BIOASSAY PROTOCOL

The following protocol was followed for each round of bioassays. Eighteen glass flasks were filled with 300 ml seawater. Ten preadult 2 stage lice were transferred, using forceps, into each flask. The flasks were randomly labelled and distributed among the 3 light groups, resulting in 4 flasks per light treatment. Each flask was gently stirred to ensure that all lice were healthy and able to attach, the contents were emptied over a sieve, after which 300 ml treatment compound was then introduced into the flask. Control group (T3) were then placed in their respective water bath/darkened cabinet and exposed to the respective lights for four hours. Treatment I (T1) received a change of solution every every hour, treatment II (T2) received one solution change at the 2-hour mark. Treatment IV (T4) was exposed to the treatment solution for 10 minutes, treatment V (T5) was exposed for 20 minutes, and treatment VI (T6) for 30 minutes; after which they were rinsed with seawater, 300ml clean seawater was placed in the blue light treatment. Observations of the number of affected/unaffected lice were conducted at 30, 60, 120, 180, and 240 min, due to lack of visibility counts were made for group 4 and 5 when the treatment solutions were replaced.

RESULTS

The first round of experiments was conducted using lice supplied by iLab. A malfunction occurred in the water bath containing the blue light treatments with 2 containers being lost during the treatment. They have been marked with (x) in Table 9. At the conclusion of the bioassay the control groups (T3) had 100% survival in all light treatments (Table 9). The riboflavin treatment (T1) had no discernable effect on survival of the salmon lice. Curcumin cyclodextrin treatments (T4-T6) were the most effective with treatment IV dropping to 30% from the 30min observation onwards. Treatment V had 10% active lice until the final count where all lice were affected. Treatment VI was lost at the 120min observation, however the percentage active lice had dropped to 10% at the 60min observation.

HPLC analysis of sea water solutions of both riboflavin and riboflavin phosphate showed extensive degradation of both compounds after irradiation with the intensive 100W blue LED light after 10 minutes. Sea water solutions of both riboflavin and riboflavin phosphate showed no degradation in dark during 120 minutes. In a real aquaculture situation, the average light intensity will be much lower than in this example. The inventors expect that the lower blue light intensity will not degrade the riboflavin molecules and thereby these comounds will work as photosensitizers for treatment of salmon infected by salmon lice.

The second round of bioassays used lice from UiB. At the conclusion of the bioassay the control groups (T3) had 100% survival in all light treatments (Table 10). The riboflavin treatment (T2) had no discernable effect on survival of the salmon lice. All the curcumin cyclodextrin treatments (T4-T6) were effective at reducing survival of the lice, with the majority affected within 60min of exposure to blue light (Table10).

Table 9. Percentage active salmon lice recorded during five observations during a 4h exposure to eight different treatment compounds and three different light sources / conditions

Table 10. Percentage active salmon lice recorded during five observations during a 4h exposure to eight different treatment compounds and three different light sources / conditions

Example 5 Seawater solubility and light stability of curcumin cyclodextrins

Solubility

8 mg of curcumin, 55.5 mg of compound 3 ([2-hydroxypropyl] beta-cyclodextrin curcumin) and 82.8 mg of compound 7 (beta-cyclodextrin curcumin) were each dissolved in 12 mL sea water separately using vortex mixer at room temperature. The solutions were saturated solutions. Samples were taken out from the solutions, centrifuged and analysed by HPLC. The peak area for compounds compound 3 and compound 7 was almost 29 and 11 times more than the peak area for curcumin, respectively (including all impurities in all three compounds).

Conclusion: 2-HP beta-cyclodextrin curcumin was 29 times more soluble than curcumin in sea water. Beta-cyclodextrin curcumin was 11 times more soluble than curcumin in seawater.

Blue light stability

40 micromolar solutions of curcumin, compound 3 ([2-hydroxypropyl] betacyclodextrin curcumin) (MH-103) and compound 7 (beta-cyclodextrin curcumin) (MH-107) in sea water/acetonitrile were prepared (100mL sea water and 1mL acetonitrile). From each solution 2 samples were taken, one kept in dark and one kept under blue light. Aliquots of each sample were analysed by HPLC after 10 min, 20 min, 40 min, 80 min and 120 min. Results are shown in Table 11.

Table 11. HPLC analysis (peak area) of curcumin, compound MH-103 and compound MH-107 over time when not exposed to light and exposed to blue light.

Conclusion: Blue light is very effective in degradation of curcumin. The curcumin cyclodextrin complexes are much more stable than curcumin in presence of blue light. Compound 7 (MH-107) comprising beta-cyclodextrin is much more stable than compound 3 (MH-103) comprising 2-hydroxypropyl-beta-cyclodextrin in presence of blue light.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

Claims

C l a i m s

1. Composition in the form of an aqueous solution comprising a photosensitizer for use in a method of photodynamic therapy for an external crustacean parasite infection in salmonid fish, said external crustacean parasite infection comprises an infection of salmon lice, Lepeophtheirus salmonis, Caligus rogercresseyi and Caligus spp., wherein said photosensitizer is curcumin or a pharmaceutical acceptable derivative thereof, and said photosensitizer is administered in a bath treatment of the salmonid fish in need of such treatment, and the salmon lice is illuminated by light.

2. A method according to claim 2, where said photosensitizer is a curcumin cyclodextrin.

3. A method according to any one of claim 1 or 2, where said light is blue light.

4. A method according to any one of claim 1 or 2, where said light is red light. A method according to any of the preceding claims, wherein the salmonid fish is located in a receptacle.

5. A method according to any one of claims 1 to 4, wherein the salmonid fish is located in an ocean cage or a net pen.

6. A method according to any one of the preceding claims, wherein the blue light source comprises of LED lamps.

7. A method according to any one of the preceding claims, wherein said photosensitizer is added in the water surrounding the salmonid fish.

8. Use of an artificial light device emitting blue light in accordance to any one of the preceding claims.