Composition and Method to Remove Parasites From Fish and to Prevent or Treat Infestation or Infection of Parasites on Fish

Abstract

A composition and a method with active agents to inactivate or kill parasites that attack fish and to loosen parasites that are fastened to the fish are described. The composition can be used for the prevention of and treatment of infestations or infections from parasites on fish.

Claims

1-33. (canceled)

34. A method for deterring parasites from attacking and fastening to fish or for loosening parasites fastened to fish or killing parasites, comprising: taking fish into a water batch; adding a composition comprising (a) one or more capsaicinoids, or analogues or salts thereof, and (b) acetic acid to the water batch, thereby deterring parasites from attacking and fastening to fish, loosening parasites fastened to fish, and killing parasites present in the water batch.

35. The method according to claim 34, wherein one or more capsaicinoids is selected from the group consisting of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin and nonivamide.

36. The method according to claim 34, wherein the composition is an aqueous composition having a pH below 5.

37. The method according to claim 36, wherein the composition has a pH below 3.

38. The method according to claim 34, wherein said one or more capsaicinoid is capsaicin.

39. The method according to claim 38, wherein the capsaicin is provided from the plant family Capsicum.

40. The method according to claim 39, wherein the capsicum is a chili pepper.

41. The method according to claim 34, wherein said one or more capsaicinoid has a concentration in said composition within an approximate range of 0.01-5% by weight.

42. The method according to claim 34, wherein said one or more capsaicinoid is present in the composition in an amount within an approximate range of 10-100 grams per liter of fluid.

43. The method according to claim 34, wherein said parasite present in the water batch is an ectoparasite.

44. The method according to claim 43, wherein said parasite present in the water batch is a Caligidae.

45. The method according to claim 44, wherein said Caligidae is selected from the group consisting of Pseudocaligus, Caligus and Lepeophtheirus.

46. The method according to claim 34, wherein said parasite present in the water batch is salmon louse, Lepeophtheirus salmonis.

47. The method according to claim 34, wherein said fish is a salmonoid (Salmonidae).

48. The method according to claim 47, wherein said salmonoid is one or more from the group consisting of Atlantic salmon, silver salmon, trout and char.

49. The method according to claim 34, wherein a residence time of the fish in the water bath is regulated and said residence time is within an approximate range of 1-3600 seconds.

50. A composition for deterring or treating an infestation or infection of parasites on fish, comprising: (a) one or more capsaicinoids, or analogues or salts thereof, and (b) acetic acid.

51. Composition for use according to claim 50, characterised in that one or more capsaicinoids are chosen from the groups consisting of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin and nonivamide.

52. The composition according to claim 50, wherein the composition is an aqueous composition having a pH below 5.

53. The composition according to claim 50, wherein a concentration of capsaicinoids within the composition is within an approximate range of 0.01-5% by weight.

Description

DETAILED DESCRIPTION

[0042] We have found that capsaicinoid effectively fights salmon lice and can therefore be used to inactivate parasites and to prevent the parasites from infestation and attaching to the fish, such as salmon.

[0043] We do not know the biological effect mechanisms, but it is assumed that receptors interacting with capsaicinoid compounds plays a part, and it is expected therefore that all parasites that have such receptors will be influenced.

[0044] The tests that are described below have been carried out with salmon lice as an example of a parasite, but the inventive concept is not limited to salmon lice as the method will also be effective against other parasites.

[0045] Furthermore, the firsts tests on fish will be carried out with salmon, but the inventive concept is not limited to salmon, as it is expected that all fish that are attacked by parasites that are sensitive for capsaicinoid will experience an effect from the method.

[0046] Furthermore, the firsts active agents against parasites have been tested in a so-called bathing treatment, i.e. that the parasites and the fish stay a given time in a watery environment to which the active agents are added.

[0047] However, it is expected that the active agent has a general effect on the parasites and other methods to administer an active agent to the parasites are therefore possible. We know of many other chemical active agents that can be indirectly administered to the parasites, for example, in that an active agent is administered to the host, for example, via the feed or by injection.

Example 1

Manufacture of the Composition Containing an Active Agent

[0048] The first introductory tests have been carried out directly on salmon lice in that an active agent is added to the fluid in which the lice are contained.

[0049] The active agent is a capsaicinoid and a fluid is presented containing such capsaicinoids. Capsaicinoids can be found in plants of the Capsicum family, such as Cayenne.

[0050] 500 g of chili of the type Cayenne (Scoville scale 30 000-50 000) is washed in clean water to rinse off any bacteria or pollutants that come from the chili production. Other types of chili with another content of capsaicinoids, such as capsaicin can be used, but one must correct for the amount of capsaicinoids in the solution.

[0051] 500 g Cayenne including the stalks are chopped into a soft mass by the use of mechanical equipment.

[0052] One litre of pure water is added to the chili mass. The mixture is heated to boiling and then removed from the heat. The procedure is necessary to release as much capsaicin as possible, and also to ensure conservation by killing the bacteria that were not removed in the first washing process.

[0053] Capsaicin is not soluble in water and one litre of 35% acetic acid is mixed into the hot chili mass and blended thoroughly. Other concentrations of acetic acid can, of course, be used. Finally, one adds a further 8 litres of water. The total amount of fluid is then 10 litres+the mass of the organic mixture.

[0054] The mixture shall then be kept cool in airtight kegs, optimally around four degrees for 10-14 days. During the 14 days the mixture is blended four times. After 14 days the organic chili mass is removed by filtration. The fluid is poured into clean kegs. The result is 10 litres of fluid which contains capsaicinoids in the solution denoted “treatment fluid”. The pH of the solution is 2.8 and the solution has a dark orange colour and a pungent smell. The solution is named the treatment fluid or Lepeoid in the examples and the tables given below.

[0055] The shelf-life of the fluid with capsaicin is very good as both capsaicin and acetic acid are natural conservation agents. By storing at between 2-4 degrees Celsius it is assumed that it lasts for several years.

[0056] The most common capsaicinoid is capsaicin which has the following formula;

##STR00001##

[0057] Pure capsaicin is a colourless and odourless material with a waxy to crystalline consistency. It is very hydrophobic, but soluble in ethanol and other hydrophobic solvents. The melting point is 57-66 degrees Celsius.

[0058] Capsaicin is the molecule that can be found in chili plants and which causes the burning heat/pain one experiences when one consumes chili or get this on one's skin, in the eyes or mucosal membranes. Capsaicin binds to specific receptors, called the TRPV1 receptors which is a kind of pain receptor that registers heat. In humans, capsaicin will be able to trigger strong, but not very serious reactions when one comes into contact with a given concentration. For a long time, it was considered that fish did not have these receptors at all, but recent studies have shown that they can also be found in fish, but one is uncertain about the significance of this.

[0059] In chili, the strength of chili is determined according to the Scoville scale which is directly correlated to the amount of capsaicin in the chili fruit. The content of capsaicin can be determined with HPLC (High performance liquid chromatography).

[0060] Cayenne (which we have used in the development of the composition disclosed herein lies between 30 000-50 000 on the Scoville scale. Pure capsaicin powder has 16 000 000 which is the max value).

[0061] Capsaicin is as mentioned very hydrophobic, which means it does not dissolve easily in water. It is therefore dependent on a different solution fluid to become hydrophilic. If a person skilled in the art should find a different solvent than water it would be pertinent to use a different solvent such as, for example, ethanol, ether, benzene, methyl sulfoxide.

[0062] We have tested in experiments with lice the effect of ethanol as a solvent for the capsaicin. However, it turns out that a combination of capsaicin and ethanol has no effect on the mortality of salmon lice. To increase the solubility of capsaicin (by using ethanol instead of water or acetic acid) does not result in an increase in the effectivity of capsaicin on parasites.

[0063] Therefore, it is the specific combination of capsaicin and acetic acid which has a pronounced, positive effect on the lice. We have also tested other acids to see if it is the pH of the solution which provides the effect. If one uses citric acid one obtains the same pH (as with acetic acid) but the effect is much lower (data not show).

[0064] In addition, we have found that the combination of acetic acid and capsaicin provides a synergistic effect, and such synergistic effect is not something which a person skilled in the art would expect.

[0065] We have, as shown, used acetic acid. To use acetic acid as a solvent for Capsaicin is not an obvious solution for a person skilled in the art.

[0066] Capsaicin has an effect on parasites on its own and this effect is reinforced synergistically when one adds acetic acid to the mixture. We have tested the effect of i) acetic acid on its own and ii) capsaicin on its own and the sum of the effects on lice for each of these is considerably lower than for the combination of capsaicin and acetic acid.

[0067] Acetic acid can have several functions which we are investigating in tests. Acetic acid can have a conserving effect on the treatment fluid. Furthermore, acetic acid reduces the pH of the solution and the aqueous composition which results has a pH of 2.8. Acetic acid is also an irritant which can contribute to the effect on salmon lice.

Example 2

[0068] Bathing Treatment of Parasites with a Fluid Comprised of Capsaicinoid and Acetic Acid

[0069] A series of tests has been carried out with salmon lice (Lepeophteirus salmonis) for the following stages of development: small movers, large movers and sexually mature salmon lice. The aim of the tests was to find out which concentration of capsaicinoid was appropriate to get a quick effect on salmon lice and their ability to suck on to the wall of the container at the same time before the salmon lice died.

[0070] Clean, white containers were filled with 0.5 litres of seawater (pH 8.9) at a temperature of 16.6 degrees Celsius. Five to eight live salmon lice at different stages of development where placed in each container.

[0071] Before pouring in the mixture as given in example 1 at different concentrations, the water in the tank was stirred gently so that the fluid should mix swiftly with the content in the container.

[0072] How much fluid with an active agent which was added to the seawater can be found in table 1 given below.

TABLE-US-00001 TABLE 1 Results for various concentrations of treatment methods and lethality in salmon lice. Time to Temper- Time to death all Concentration pH* ature reaction stages Comments 10 ml treatment 4.48 16.7 Direct Approx. 5 Large movers fluid per liter minutes die after 3 min sea water 15 ml treatment 4.12 16.6 Direct Approx. 4 fluid per liter minutes sea water 20 ml treatment 3.99 16.6 Direct 1 min and fluid per liter 20 sec sea water *pH in treatment bath (mixture of fluid with active ingredients and sea water)

Example 3

Reaction Pattern and Lethality for Salmon Lice

[0073] In this experiment we considered fish welfare. Many studies have been carried out with salmon and its ability to tolerate pH changes but most of the studies are associated with low pH and increased aluminium levels over time. It is known that a pH down to 4.0 is acceptable to salmon over long periods, but at pH values below 4.0 the stress level for the salmon being tested increases. Therefore, we wanted to focus on concentration levels of the treatment fluid which are as effective as possible without affecting the welfare of the salmon.

TABLE-US-00002 TABLE 2 Result of reaction pattern and lethality in salmon lice by addition of 20 ml treatment fluid per liter sea water Time to Release Trial number pH reaction time* 1 4.14 Direct 30 sec 2 4.08 Direct 24 sec 3 4.0 Direct 14 sec 4 4.02 Direct 14 sec 5 3.98 Direct 12 sec 6 4.12 Direct 35 sec 7 4.01 Direct 14 sec 8 3.98 Direct 17 sec 9 4.02 Direct 14 sec 10 4.0 Direct 15 sec *Time it takes from addition of treatment fluid to the salmon lice starts to detach from the container wall.

[0074] In repeated experiments with 20 ml treatment fluid per litre seawater, we found that an average time from added treatment fluid to the salmon lice reacting to the situation is immediate. Jerks and contractions are observed in the salmon lice. After an average time of 18 seconds, the salmon lice attempt to flee from the ambient conditions. They let go of the container wall and try to swim away. A short time afterwards (seconds) they turn onto their back and fall towards the bottom of the container. They can make several attempts to swim away. Alternatively, they just loosen themselves from the container wall and sink to the bottom. The majority of the salmon lice were dead after 1 minute and 20 seconds.

[0075] In the experiments there was a trend that large sexually mature salmon lice died first, followed by the large movers and finally the small movers.

Example 4

The Effect of Increasing Concentration and Temperature on Copepodites

[0076] The tables 3-6 show the effect of increasing concentration and temperature on movement (time until movements stop). The tables show that increasing concentration and increasing temperature reduced the time for the movement of the parasites stop.

TABLE-US-00003 TABLE 3 Results of the response of copepodites (time to stop moving in seconds) to Lepeoid concentrations (5, 10, 20 mL/L), tested the first time at 12° C. Bioassays were run in triplicates (test 1-3) with ~20-30 copepodites. 12° C._1 Stop moving Concentration Test Response (s) Notes 20 mL/L 1 Direct (*) 94 3 move after 60 s 2 Direct (*) 161 4 move after 90 s 3 Direct (*) 92 4 move after 60 s 10 mL/L 1 Direct (**) 96 3 move after 50 s 2 Direct (**) 114 2 move after 60 s 3 Direct (**) 137 2 move after 60 s  5 mL/L 1 ca. 30 s 209 6 move after 60 s 2 ca. 30 s 495 6 move after 60 s 3 ca. 30 s 459 8 move after 60 s Notes for tables 2 to 5 (*) Direct response, some show extreme high activity, some directly stop moving. (**) Direct response, but less extreme than with 20 mL/L.

TABLE-US-00004 TABLE 4 Results of the response of copepodites (time to stop moving in seconds) to Lepeoid-concentrations (5, 10, 20 mL/L), tested the second time at 12° C. Bioassays were run in triplicates (test 1-3) with ~20-30 copepodites. 12° C._2 Stop moving Concentration Test Response (s) Notes 20 mL/L 1 Direct (*) 90 4 move after 40 s 2 Direct (*) 86 2 move after 30 s 3 Direct (*) 116 3 move after 30 s, 1 quiver after 90 s 10 mL/L 1 Direct (**) 390 3 move after 110 s, 1 after 195 s 2 Direct (**) 224 3 move after 90 s, 1 after 150 s 3 Direct (**) 254 5 move after 60 s, 1 after 190 s  5 mL/L 1 ca. 10 s 826 1 move after 9.5 min 2 ca. 10 s 1131 2 move after 15.5 min 3 ca. 10 s 1210 2 move after 18.5 min

TABLE-US-00005 TABLE 5 Results of the response of copepodites (time to stop moving in seconds) to Lepeoid concentrations (5, 10, 20 mL/L), tested at 20° C. Bioassays were run in triplicates (test 1-3) with ~20-30 copepodites. 20° C. Stop moving Concentration Test Response (s) Notes 20 mL/L 1 Direct (*) 67 5 move after 20 s; 1 after 50 s 2 Direct (*) 45 1 moves after 30 s 3 Direct (*) 51 2 move after 30 s 10 mL/L 1 Direct (**) 84 3 move after 50 s 2 Direct (**) 70 1 moves after 50 s 3 Direct (**) 67 1 moves after 55 s  5 mL/L 1 ca. 10 s 250 2 move after 60 s 2 ca. 10 s 248 2 move after 120 s 3 Direct (**) 295 5 move after 110 s

TABLE-US-00006 TABLE 6 Results of the response of copepodites (time to stop moving in seconds) to Lepeoid concentrations (5, 10, 20 mL/L), tested at 7° C. Bioassays were run in triplicates (test 1-3) with ~20-30 copepodites. 7° C. Stop moving Concentration Test Response (s) Notes 20 mL/L 1 Direct (*) 130 2 move after 60 s; ‘quivering’ after 90 s 2 Direct (*) 119 3 move after 60 s; ‘quivering’ after 90 s 3 Direct (*) 115 3 move after 60 s; ‘quivering’ after 86 s 10 mL/L 1 Direct (**) 234 3 move after 120 s 2 Direct (**) 214 2 move after 150 s 3 Direct (**) 194 3 move after 140 s  5 mL/L 1 Direct (**) 1275 2 move after 20 min. 2 Direct (**) 1200 3 move after 15.5 min 3 Direct (**) 1290 3 move after 15 min

Example 5—Effect of Increasing Concentration and Temperature on Adult Lice

[0077] When the active composition was introduced to adult lice the initial response was, in the main, that male lice spin round for a short period (seconds). The temperature and concentration influence the time until all movements have stopped (tables 7-9).

TABLE-US-00007 TABLE 7 Results of the response of adult lice and time to stop moving to Lepeoid-concentrations (10, 20 mL/L), tested the first time at 12° C. Bioassays were run in triplicates (test 1-3) with ~5 adult lice, except for the 20 mL/L, which was run 5 times. 12° C. Stop moving Concentration Test M/F Response (s) Notes 20 mL/L 1 3/2 Direct, but less extreme 450 5 min. lice start to bend as with copepodites 2 2/3 No ‘panicky’ responce 342 4.75 min all bended 3 3/2 226 1 min all move. 3 min. 1 male moves 4 4/1 1 directly moves, others 440 4.3 min 1 male moves stop being active 5 4/1 Direct 490 90 s all move. 5.5 min 3 males move 10 mL/L 1 3/2 1 M and 1 F started to 1350 6.5 min all move. swim directly 2 5/0 1200 7.5 min all move 3 0/5 1020 5 min. 4 stuck to the size Notes for this and following tables: M/F = male/female numbers w/prov = with provocation, slight tail pinch Response was noted when different from general response of males (see text).

TABLE-US-00008 TABLE 8 Results of the response of adult lice and time to stop moving to Lepeoid-concentrations (10, 20 mL/L), tested the first time at 20° C. Bioassays were run in triplicates (test 1-3) with ~5 adult lice. 20° C. Stop moving Concentration Test M/F Response (s) Notes 20 mL/L 1 2/3 425 50 s 1 F bends, 80 s all move w/prov; 200 s all bended 2 3/2 Male repond directly 430 80 s 1 F does not respond w/prov, for some seconds 110 s bending starts 3 1/4 Male responds directly 310 100 s 1 F moves, 4 min. Male move w/prov. 10 mL/L 1 2/3 Direct response male 750 60 s 2 M/1 F move & 2 F w/prov, 5.5 min. Bending start, move w/prov 2 4/2 764 60 s all move w/prov, 9.5 min only 1 M moves w/prov 3 2/3 indirect response 630 60 s all move, 120 s males move, females w/prov, 9.5 min 1 F bends, 1 M w/prov

TABLE-US-00009 TABLE 9 Results of the response of adult lice and time to stop moving to Lepeoid concentrations (10, 20 mL/L), tested the first time at 7° C. Bioassays were run in triplicates (test 1-3) with ~5 adult lice. 7° C. Stop moving Concentration Test M/F Response (s) Notes 20 mL/L 1 2/4 Strong response all 875 5 min all bended, 7.5 min move w/prov 2 1/4 821 90 s all active w/prov, 9 min all bended 3 1/4 920 100 s all move, 7 min 1 M and 1 F move a little 10 mL/L  1* 2/4 1800 120 s all move, 6 min. started to bend, 22 min. 2 F lifeless  2* 4/1 1800 7 min all move, after 30 min. 1 F moves  3* 3/4 1920 5 in. all move.; 23 min. 1 F stopped responding to trigger *Tests 1 and 2 were kept in Lepeiod solution, 3 was transferred to clean seawater, in the afternoon, no movement was observed in tests 1 and 2.

Capsaicin

[0078] Not much can be found in the literature about capsaicin in a marine environment. Previous descriptions relate to capsaicin being used in bottom lubrication agents used on boats and other submersible surfaces. Capsaicin is described as a natural, not poisonous ingredient. It is a stable, organic alkaloid. The challenge is the poor water solubility of capsaicin. It is said that to use capsaicin as an active ingredient in bottom lubricants on ships leads to a relatively small risk for the marine environment. Capsaicin is biologically broken down on land. It is broken down by bacteria and one half will be broken down in two to eight days. There is little risk for leaks to the groundwater. It does not evaporate. A strong smell and taste means that animals stay away.

[0079] Capsaicin is not absorbed through the skin but the stomach. It is broken down in the liver. There is no information about the effect during pregnancy or on breastmilk. Capsaicin can influence resistance in air passages. Therefore, one can assume that people with obstructive lung diseases can be sensitive to capsaicin. Capsaicin can irritate skin and the mucosal membranes.

Acetic Acid

[0080] Acidification of the ocean is a global problem. Emission of CO.sub.2 contributes to 30% of the acidification. In the fjords the environment is dynamic and changes according to the temperature of the water and the currents. Acetic acid in large concentrations will be damaging to the marine environment. The treatment fluid will have a concentration of acetic acid of 3.5% which is lower than ordinary household vinegar. Furthermore, after treatment in the bathing fluid, the fish will be transferred to the net cage with seawater and the treatment fluid does not need to be discharged into the sea.

Analogues

[0081] The biological effect-mechanisms of the active compounds disclosed herein are not known, but as indicated above it is possible that they work via a receptor family called vanilloide receptors, subtype 1 (TRPV 1). All capsaicinoid compounds that work via this receptor family are considered to be capable of inactivating or killing parasites.

[0082] In the preferred embodiments of the innovation, such capsaicinoid compounds will be manufactured from plants that contain such compounds, such as Cayenne pepper. In other embodiments the capsaicinoid compounds will be manufactured synthetically, or they can be manufactured using bacteriological processes. Furthermore, analogues of the capsaicinoid compounds are within the scope of this invention disclosure. Such analogues have the same base structure as capsaicinoid but have different substituents in non-essential places in the molecule.

[0083] The inventive embodiments also include different salt forms of the capsaicinoid compounds.