NO20190379A1

NO20190379A1 - Treatment composition for a marine ectoparasite and a method for preparing the treatment composition

Info

Publication number: NO20190379A1
Application number: NO20190379A
Authority: NO
Inventors: Joseph Gibbons; Gordon Ritchie
Original assignee: Mowi ASA
Priority date: 2019-03-21
Filing date: 2019-03-21
Publication date: 2020-09-22
Also published as: WO2020190151A1; NO20200335A1

Description

TREATMENT COMPOSITION FOR A MARINE ECTOPARASITE AND A METHOD FOR PREPARING THE TREATMENT COMPOSITION

This invention concerns a bath treatment of fish infected with an ectoparasite. In particular the invention concerns a topical treatment of farmed fish. Said farmed fish may be a marine fish or a salmonoid fish. The ectoparasite may be a crustacean ectoparasite such as a sea louse or an amoeba. The amoeba may cause Amoebic Gill Disease. The invention concerns in particular a hypersaline composition of sea water to which either a NaCl-salt, a brine or a blend of sea water salts has been added such that the total salinity of the composition is larger than in sea water. The hypersaline composition may comprise fresh water to which brine or a blend of sea water salts has been added. The invention also concerns a method for making the hypersaline composition. The invention concerns further to treat the fish in a first bath that may be a fresh water bath, and subsequently in a second bath that may be a hypersaline bath. The treatment may be carried out in the same receptacle by adding sea water salts to the fresh water to gradually increase the salinity. The invention also concerns use of a carefully selected range of hypersalinity combined with a carefully selected treatment time.

Sea lice is a well-known parasitic problem in fish farming, especially in farming of Atlantic salmon in Norway, Scotland, Ireland, Faroes, Canada and Chile. There are several species of the sea lice. Especially important in the Atlantic is the species Lepeophtheirus salmonis.

Several treatment methods are known for treatment of sea lice:

Bath treatment: organophosphates, pyrethroids and hydrogen peroxide and fresh water In-feed treatment: ivermectins and acylureas

In addition it is known to use so called cleaner fish which will pick sea lice from the skin of infected fish.

Sea lice have over the years built resistance and / or tolerance towards several of the known treatments, especially against several of the bath treatment therapeutics and infeed therapeutics.

There is a need for new active components against sea lice. In addition to be an effective therapeutic, there are environmental constraints to consider in the choice of the active compound. The commercial value of each individual (fish) is relatively low, which means that price of the active compound is an issue.

The therapeutic margin must be satisfactory. Although the value of each individual is relatively low, the number of individuals in each pen is large. The total commercial value of the population within one pen is therefore considerable. Treatment of farmed fish is a flock treatment either as a bath treatment or as an in-feed treatment.

Another ectoparasite that affects farmed fish is an amoeba causing the disease Amoebic Gill Disease. The causative agent is Paramoeba perurans. This amoeba infects several different species of marine fish, both wild fish and farmed fish. This includes sea bream, turbot, ayu and mackerel.

Patent application WO 2017/123096 discloses a method and device for removing parasites from anadromous fish. A first container is provided with freshwater and a second container is provided with brine having a salinity higher than sea water. The fish is guided from a net cage to the first container and the fish is kept there for a first period. Then, the fish is transferred from the first container to the second container and the fish is kept there for a second period. Thereafter the fish is returned to the net cage. The application discloses that this treatment can be performed in the opposite order, i.e. first a saline bath followed by a freshwater bath. WO 2017/123096 discloses further that the fish stay in each container for at least one hour such as from one hour to ten hours. In working examples WO 2017/123096 discloses to use a brine having a salinity of 62 – 66 g/l. Best effect was observed by first bathing the fish for four hours in freshwater followed by bathing for one hour in brine and repeating this sequence once. A reduction of 100% of sea lice was reported.

Patent application WO 2017/150988 discloses to add a potassium compound, such as potassium chloride to sea water to make up a treatment solution. The fish were exposed to the solution for up to five hours. Only a small portion of the sea lice had fallen off after one hour.

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.

In this context, sea water is used in its ordinary meaning, and defined as a water with a salinity from 20 to 40 ppt. The ratios of different ions / salts in the sea water are assumed to be constant. Sea water salts are defined as a mixture of salts made up of the dominant salts found in sea water and at inclusion levels of the different salts that correspond to the ratios found in sea water. Commercially available sea water salt blends are known as instant ocean salt. Sea water Hypersaline sea water is a water with a salinity above 40 ppt. A brine is made by concentration of sea water. The brine may be produced by a reverse osmosis process. The ratios of different ions / salts in the brine are assumed to be similar to the ratios in sea water, i.e. no selective removal or concentration of any ions /salts. A brine is in this context regarded as a sea water salt solution.

The inventors have found that a bath treatment with hypersaline composition makes at least the preadult and adult stages of the sea lice to drop of the fish after some time of exposure. The preadult and adult stages are the free stages of sea lice.

The hypersaline composition is in one embodiment made up by adding NaCl salt to sea water of ordinary salinity and composition. In another embodiment the hypersaline composition is made up by adding a brine to the ordinary sea water.

Hypersaline sea water according to the invention may be filled into a tank of a well boat. In an alternative embodiment of the invention, the hypersaline sea water may be filled into a closed cage floating in the sea. The closed cage may have a rigid wall. The closed cage may have a flexible wall. Bath treatment or topical treatment of fish in a treatment receptacle on board a well boat or within a treatment receptacle formed by a closed cage is known to the skilled person. The skilled person will know how to administer fish into the treatment receptacle, and how to administer the fish out of the treatment receptacle. The skilled person will also know how to observe the fish during the treatment and make proper action if the fish show sign of any discomfort.

In a first aspect the invention relates more particularly to a hypersaline composition for therapeutic, topical treatment of a fish infected with an ectoparasite,

where the hypersaline composition comprises one of:

- sea water salts and freshwater;

- sea water salts and sea water; and

- a NaCl solution and sea water,

and the total salinity of the hypersaline composition is from and including 50 ppt and up to and including 60 ppt, and

where the fish is treated in one of the sequences:

- transfer of the fish from a first sea water container to a first receptacle containing a solution consisting of sea water, increase the salinity of the solution in the first receptacle by adding one of a sea water salt solution and a NaCl solution to make the hypersaline composition, keep the fish in the hypersaline composition for from and including 30 minutes up to and including 60 minutes, transfer of the fish to a second receptacle containing fresh water, keep the fish in the fresh water for from and including 30 minutes up to and including 60 minutes, and transfer of the fish to a second sea water container, where the second sea water container may be the same as the first sea water container;

- transfer of the fish from a first sea water container to a first receptacle containing a solution consisting of fresh water, keep the fish in the solution for from and including 30 minutes up to and including 60 minutes, increase the salinity of the solution in the first receptacle by adding one of a sea water salt solution and a NaCl solution to make the hypersaline composition, keep the fish in the hypersaline composition for from and including 30 minutes up to and including 60 minutes, and transfer of the fish to a second sea water container, where the second sea water container may be the same as the first sea water container.

The total salinity of the composition may be from and including 55 ppt and up to and including 60 ppt. The total salinity of the composition may be from and including 57 ppt and up to and including 60 ppt. The total salinity of the composition may be from and including 50 ppt and up to and including 57 ppt. The total salinity of the composition may be from and including 50 ppt and up to and including 55 ppt. The total salinity of the composition may be from and including 55 ppt and up to and including 57 ppt.

The fish may be a salmonoid fish. The fish may be Atlantic salmon (Salmo salar). The fish may be Rainbow trout (Oncorhynchus mykiss). The fish may be a Pacific salmon (Oncorhynchus spp.). The ectoparasite may be a crustacean ectoparasite. The crustacean ectoparasite may be a member of the family Caligidae. The crustacean ectoparasite may be a member of the genus Lepeophtheirus. The crustacean ectoparasite may be a member of the genus Caligus.

The ectoparasite may be an amoeba. The amoeba may be the species Paramoeba perurans. This amoeba infects several different species of marine fish, both wild fish and farmed fish. This includes sea bream, turbot, ayu and mackerel in addition to salmonoid fish.

In a second aspect the invention relates more particularly to a method for making the hypersaline composition as described above. The hypersaline composition is made by the steps:

a) choose one of a first starting liquid and a second starting liquid, said first starting liquid comprises fresh water and said second starting liquid comprises natural sea water; b) make one of a first hypersaline master batch and a second hypersaline master batch, said first hypersaline master batch is one of brine, dry sea water salt blend dissolved in fresh water and dry sea water salt blend dissolved in natural sea water, and said second hypersaline master batch is dry NaCl dissolved in natural sea water;

and one of step c) and d), where

c) make the hypersaline composition by pouring the first hypersaline master batch into one of the first starting liquid and the second starting liquid until a target salinity is achieved, and

d) make the hypersaline composition by pouring the second hypersaline master batch into the second starting liquid until a target salinity is achieved.

The brine may be produced by a reverse osmosis process. The brine may be of a salinity between 100 and 150 ppt.

The dry sea water salt blend may be a blend of salts in the ratios found in natural sea water. The target salinity may be from and including 50 ppt and up to and including 60 ppt. The target salinity may be from and including 50 ppt and up to and including 57 ppt. The target salinity may be from and including 50 ppt and up to and including 55 ppt. The target salinity may be from and including 55 ppt and up to and including 60 ppt. The target salinity may be from and including 55 ppt and up to and including 57 ppt. The target salinity may be from and including 57 ppt and up to and including 60 ppt.

The reverse osmosis process may be carried out on board a vessel and the brine may be collected in a receptacle prior to mixing with sea water or mixing with fresh water.

The vessel may be a well boat. Sea water may be filled in at least one of the well boat’s tanks and the brine may be transported from the receptacle to the at least one tank. In another embodiment the brine is transported from the reverse osmosis apparatus to at least one tank of a well boat and mixed with sea water in the at least one tank.

Example 1

In vitro bioassay

Adult female, adult male and preadult sea lice (L. salmonis) were collected from Atlantic salmon in a commercial fish farm. For each treatment sea lice were exposed to the treatment condition.

Table 1 Moribund sea lice (%) after exposure to different solutions

Table 2 Moribund sea lice (%) after exposure to a combination of different solutions

Example 2

In vitro bioassay

Mixed sex adult sea lice were collected from a commercial aquaculture site and placed in to 36 modified 50 ml capacity falcon tubes, which had multiple 2mm diameter drilled holes. Tubes were held vertically in a rack fully submerged in sea water (30ppt) which was

kept on ice to maintain a temperature of 10-14°C during collection. When several lice had been placed in to a tube, a lid was placed on it until all tubes were deemed to have sufficient lice numbers. At this point tubes were shaken in sea water and lids removed so unbound lice were not in any tube. Numbers of bound sea lice were counted in tubes prior to being submerged into designated salinities for allocated length of time. Each salinity x time combination comprised of three replicate tubes. Time of assessment was the end point, with tubes which were shaken prior to the counting of bound lice then placed in to sea water (30ppt). Temperature was maintained during bioassays at 12 (±1) °C. Two assays were performed using SW (30 ppt) control, 45 ppt, 50 ppt and 55 ppt with 15, 30-and 60-minute immersion times. A third assay was performed using SW (30 ppt), 50 ppt, 55 ppt and 60 ppt with 15, 30- and 60-minute immersion times.

Numbers of lice in each tube that were knocked down were used to calculate percentage of initial tube population still attached for each.

All hyper-saline treatments were created from the mixing of 25ppt salt water well water and supplementation to desired salinity through the addition of instant ocean salt.

Table 3 Mean percent attached (unaffected) sea lice (%) after exposure to different saline solutions

The two in vitro studies show that ordinary sea water has a small effect on sea lice. This is as expected as sea lice is fully adapted to live in sea water. Any observed effect is due to the experimental set up. It is further observed that hypersaline water of 40 ppt and 45 ppt has only a minor effect within the observation period up to 60 minutes. It is also observed that an immersion time of 15 minutes appear to be too short to obtain a reliable reduction in the number of sea lice that are able to attach themselves to a wall.

Example 3

In vivo study

Part A

A hypersaline master batch solution was made by mixing a commercially available marine salt composition (NaCl 66.1%, MgSO416.3%, MgCl212.7%, CaCl23.3%, KCl 1.6%) without any anti-caking agents with fresh water in a mixing vessel. Final salinity was >100ppt.

First and second plastic tanks of 1 m<3 >were used as treatment receptacles. Each group of fish comprised ten healthy Atlantic salmon (Salmo salar). The fish had no history of particular illnesses and were showing no signs of diseases. In particular there were no signs of gills diseases. Average fish weight was 5.5 kg.

The fish were naturally infected by sea lice. On average each fish was infected by 4.9 females with egg strings (i.e. gravid females), 4.7 adult female, 5.3 adult male, 10.6 preadults. Chalimus stages were not counted.

All treatments were done in triplicate.

Atlantic salmon (n=10) were gently transferred with a hand net to the first plastic tank / first treatment receptacle containing sea water. Salinity in the first plastic tank was gradually increased by pouring hypersaline solution from the master batch slowly in discrete water jets from the mixing vessel. Salinity in the first tank was measured by a refractometer. Salinity was increased until a target level of 60 ppt was achieved. The Atlantic salmon were exposed to the 60 ppt hypersaline solution for one hour. Thereafter the Atlantic salmon were gently transferred with a hand net from the first plastic tank to the second plastic tank / second treatment receptacle. The second plastic tank contained fresh water. The Atlantic salmon were exposed to fresh water for one hour. Finally, the Atlantic salmon were gently transferred with a hand net to a holding vessel where the Atlantic salmon were closely monitored for mortality post treatment.

A control group of Atlantic salmon (n=10) were gently transferred with a hand net from a pen floating in sea water to the first plastic tank / first treatment receptacle containing sea water. The Atlantic salmon were exposed to sea water for one hour in the first tank.

Thereafter the Atlantic salmon were gently transferred with a hand net from the first plastic tank to the second plastic tank / second treatment receptacle. The second plastic tank contained sea water. The Atlantic salmon were exposed to sea water for one hour in the second tank. Finally, the Atlantic salmon were gently transferred with a hand net to a holding vessel where the Atlantic salmon were closely monitored for mortality post treatment.

Table 4 Clearance (reduction in %) 60 ppt / 1h bath followed by freshwater / 1h bath

Part B

Part B was performed similar to Part A. The fish were from the same group and had the same infectious load. The hypersaline master batch solution was made up as described in Part A.

Atlantic salmon (n=10) were gently transferred with a hand net to the first plastic tank / first treatment receptacle containing freshwater. The Atlantic salmon were exposed to fresh water for one hour. Thereafter salinity in the first plastic tank was gradually increased by pouring hypersaline master batch solution slowly in discrete water jets from the mixing vessel. Salinity in the first tank was measured by a refractometer. Salinity was increased until the target level of 60 ppt was achieved. The Atlantic salmon were exposed to the 60 ppt solution for one hour. Finally, the Atlantic salmon were gently transferred with a hand net to a holding vessel where the Atlantic salmon were closely monitored for mortality post treatment.

Table 5 Clearance (reduction in %) freshwater / 1h bath followed by 60 ppt / 1h bath

Table 6 Average clearance from Part A and Part B

As seen from Table 4, the control test shows that sea lice are effectively removed by the handling procedure. However, it should be noted that fish becomes slower and easier to be caught (re-captured) when they have been exposed to 60 ppt salinity for one hour. In contrast the fish at the equivalent procedural step in the control group are active when recaptured. By this, probably more sea lice would detach from the Atlantic salmon during the capture than they would do on treatment.

It was observed that the fish behaved calmly during the periods of gradually and slowly increasing the salinity of the treatment to the intended 60 ppt solution. This was observed for the gradual increase in salinity from fresh water to 60 ppt salinity, and from sea water to 60 ppt salinity.

Example 4

Animal safety study

This study investigated the safety and welfare impacts of administering one-hour hypersaline treatments at either 55ppt, 57ppt, and 60ppt using both ca.1500g and <150g Atlantic salmon held in 5°C and 15°C water temperature.

It was found that a behavioral assessment scoring system was a good indicator of animal safety during a hypersaline bath treatment regime. Behavioral assessment was evaluated according to “activity”, “positioning” and “orientation”. Scoring was according to table 7.

Table 7 Behavioral assessment scoring system

Orientation scoring, i.e. loss of balance, was found to be the best behavioural indicator of stress during treatments, where fish will begin tilting in the water column before observations of fish lying on their flanks occurred.

Fish of an appropriate size were distributed across six tanks (30 fish per tank) with each of three treatment groups being represented in duplicate. After a minimum of seven days acclimation, feed was withheld for 48 hours prior to welfare assessment. During immersion in hypersaline water, behaviour of populations was monitored as stipulated in table 7. Whole tank populations were immersed in designated treatments and after one hour rehoused in original host tanks. Behavioural assessments were executed and recorded every 10 minutes during immersion bath, commencing after 10 minutes of the last fish entering the bath. Assessments monitored population activity, positioning, and orientation using a semi-quantitative scoring system as shown in table 7.

Table 8 Observed orientation scores during hypersaline immersion of Atlantic salmon

<1>One dead fish during immersion period

<4>Four dead fishes during immersion period

<a>One moribund fish after immersion period

†All fishes dead

The effects of one-hour hypersaline treatments on Atlantic salmon were dependent on sampling time, water temperature, fish size and treatment duration. These variables, along with time post-smolt, i.e. small fish, and salt concentration will be important considerations for treatment administration and post-treatment monitoring and handling in the field. Overall, hypersaline treatments of 57 ppt for 1 hr are efficacious and can be administered safely under certain guidelines.

Throughout all phases, loss of balance (i.e. orientation) was the best predictor of physiological stress and increased with treatment duration. Although not directly comparable, orientation scores were generally higher in warm water when compared to cold water treatments, coinciding with higher rates of mortality (table 8). Loss of orientation was therefore the most important marker for monitoring treatment safety and recovery. When a control bath (25 ppt) was used, there were no observations of anomalous orientation which suggests that these observations are due to hypersaline stress alone and not fish handling. Conversely, scores for positioning in the water column and level of salmon activity are not likely to be useful markers for field application due to the effects of handling and variability, respectively.

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. Hypersaline composition for therapeutic, topical treatment of a fish infected with an ectoparasite,

where the hypersaline composition comprises one of:

- sea water salts and freshwater;

- sea water salts and sea water; and

- a NaCl solution and sea water,

where the fish is treated in one of the sequences:

- transfer of the fish from a first sea water container to a first receptacle containing a solution consisting of fresh water, keep the fish in the fresh water for from and including 30 minutes up to and including 60 minutes, increase the salinity of the solution in the first receptacle by adding one of a sea water salt solution and a NaCl solution to make the hypersaline composition, keep the fish in the hypersaline composition for from and including 30 minutes up to and including 60 minutes, and transfer of the fish to a second sea water container, where the second sea water container may be the same as the first sea water container.

2. Composition according to claim 1, wherein the total salinity of the composition is from and including 50 ppt and up to and including 57 ppt.

3. Composition according to claim 1, where the fish is a salmonoid fish.

4. Composition according to claim 1, where the ectoparasite is a crustacean ectoparasite.

5. Composition according to claim 4, where the crustacean ectoparasite is a member of the family Caligidae.

6. Composition according to claim 1, where the ectoparasite is an amoeba.

7. Method for making the hypersaline composition according to any one of claim 1 and 2, c h a r a c t e r i s e d i n that the hypersaline composition is made by the steps:

a) choose one of a first starting liquid and a second starting liquid, said first starting liquid comprises fresh water and said second starting liquid comprises natural sea water;

b) make one of a first hypersaline master batch and a second hypersaline master batch, said first hypersaline master batch is one of brine, dry sea water salt blend dissolved in fresh water and dry sea water salt blend dissolved in natural sea water, and said second hypersaline master batch is dry NaCl dissolved in natural sea water;

and one of step c) and d), where

8. The method according to claim 7, wherein the brine is produced by a reverse osmosis process.

9. The method according to claim 7, wherein the dry sea water salt blend is a blend of salts in the ratios found in natural sea water.

10. The method according to claim 7, wherein the target salinity is from and including 50 ppt and up to and including 60 ppt.

11. The method according to claim 10, wherein the target salinity is from and including 50 ppt and up to and including 57 ppt.