CA2106295C - Control of sea lice in seawater fish - Google Patents

Abstract

Pyrethroid pesticide, particularly cypermethrin or alphacypermethrin, are used in the treatment of seawater fish suffering from sea lice infestation.

Description

~~~:~92/0~347~
21~fi~9~ ~'4' ~~~r~~ t~~~

CONTROL OF SEA LICE IN SEAWATER FISH
Technical Field This invention relates to the control of sea lice in salmon and other seawater fish such as seabream.
Background Art It is known commercially to treat salmon suffering from infestation with sea lice by the use of the insecticide dichlorvos. However dichlorvos is generally only effective against mature lice and is considered not to affect juvenile sea lice. Furthermore great care has to be taken with the dichlorvos dosage as the insecticide is fatal to fish at only 8 times the recommended dose for sea lice treatment.
Further there are indications that resistance to dichlorvos is developing in sea lice. It is therefore desirable to find alternative agents for treating sea lice on salmon and other fish especially materials for which the dose recommended can be very much less than the fatal (LD 50) dose.
It is known to use the pyrethroid pesticides cyper-methrin and its related compound alphacypermethrin for both the control of pests in crops and against ectoparasites in cattle and sheep including scab, lice and ked in sheep (see The Pesticide Manual, 7th Edition, page 3690 ed C R
Worthing, The British Crop Protection Council).
Cypermethrin and alphacypermethrin are thus used in crop sprays or cattle and sheep dips or sprays. However they and other pyrethroids have not been proposed for use in treating fish. Although it is stated in the Pesticide Manual that the LD 50 (96 hours) for brown trout is 2.0-2.8 microgram per litre (mcg/1), such tests are only to check that fish are not at risk from normal agricultural usage. Further data on the toxicity of cypermethrin to fish is to be found in "Environmental Health Criteria 82: Cypermethrin", published by World Health Organisation, Geneva 1989 as part of IPCS International Programme on Chemical Safety. This _, -~. ; ;-.,.f. ~ ~?~f~ce ~p. ;-~ ;~.,rinri ~.,_ a 1 . I ' ~ 1 ___ ~ _.. .. . .. ~. ~ , WO 92/16106 "~ PCT/GB92/00470 (r i ~~ ~ ~ .,.., i summarises work on the toxicity of cypermethrin and reports in Table 8 that for technical cypermethrin, dispensed in ethanol, at lOoC the LD 50 (96 hours) for atlantic salmon having a weight of 5.3g is 2-2.4 microgram active ingredient/litre. Because of these figures it has been considered that cypermethrin is too toxic for use on fish.
However, we have found that pyrethroids, particularly cypermethrin and alphacypermethrin, can be administered to salmon and other seawater fish in a manner which is highly effective in the control of sea lice in the salmon and other fish while being much less toxic to the fish themselves than dichlorvos.
s»m~ry of the Invention Accordingly the present invention provides the use of a pyrethroid pesticide, preferably cypermethrin or alpha cypermethrin, in the treatment of salmon or other sea fish suffering from sea lice infestation in a seawater environment.
Further the invention provides use of a pyrethroid pesticide, preferably cypermethrin or alphacypermethrin, for the manufacture of a composition for treatment of sea lice infestation in salmon or other sea fish in a seawater environment.
It is not fully understood why cypermethrin is not toxic to the salmon or other seawater fish in the circumstances in which it is used in this invention. This greater tolerance may be due to the presence of seawater rather than freshwater. The use of alphacypermethrin as a component of fish food for oral administration may also be an important factor. Certainly, it is particularly surprising that alphacypermethrin is highly effective when administered orally. Good results at dosage rates equivalent to 0.005mg/litre in water have been found whereas the dosage rate for dichlorvos to give equivalent results is of the 3 5 oe~gyp f~ ~el,~c~ /-~ i~ze .

~~~~9 2 / 004~7~1 21~~~~~ 24_ The active ingredients used according to the present invention are preferably administered to the salmon or other fish in their feed but they could be added as a water or bath treatment to the fish.
The active ingredient can be used in suspension or emulsified concentrate form or as a solid formulation (e. g.
powder or granules) of a particle size typically in the range 10 to 103 microns. The range of suspension concentrate formulations is suitably 10 - 250 g active ingredient per litre, and powder or granular formulations typically being 1.0 to 2.5~ w/w pre-mixes added to finished feed.
Accordingly the invention also provides salmon feeds and treatment baths containing an active ingredient accord-ing to the present invention and a method of treatment using such active ingredient.
Suitably the active ingredient is administered orally, for example in feed to achieve a dose range of~between 0.025 - 5:0 mg/kg fish/daily preferably 0.8-1.5 mg/kg, more preferably about 1.0 mg/kg for salmon, and is added to baths at a range between 0.001 and 0.5 ppm.
It is believed that the active ingredient when administered orally is taken up by the fish and passes through to the skin where the lice exist as topical ectoparasites. Because of the residual concentration of pyrethroids in the fish it is desirable to leave sufficient time after the last application for the concentration of pyrethroid to have fallen to an acceptable level before killing and consuming the fish.
Brief Description of the Drawincrs The invention is further illustrated by way of example with reference to the following Examples and the accompanying drawings, in which Figures 1 to 8 are graphs illustrating the Examples.
-'t~Y~t L:~i;C2 ~~~~....~.a~~ . ....
r "~'tOtl ~v a : ~ -,., y ? _ .';. ~:r". s ... .. . _.. ,_ r~,.'C~.;

Bxam~les illustrating. the Invention Bxam~le 1 In vitro acute toxicit at different concentrations to sea lice Sea lice for these trials were collected from Marine Harvest's Strontian site, Loch Sunart, Scotland.
The lice were collected from freshly killed salmon.
Salmon were killed with a blow to the cranium and were not to be subjected to anaesthesia (e.g. benzocaine.C02) or any such compounds as they may affect the lice. The lice were removed from the salmon with forceps and placed in plastic bags of fresh sea water (FSW) and transported in closed insulated containers immediately to the experimental facility.
The lice were separated into three groups of fifteen into glass 200 ml containers each containing 150 ml of the appropriate working solution to be tested. Concentrations will depend on the solution tested, in addition to a control. Sea water used for the trial was drawn from Loch Ailort, Scotland. Once in the containers the lice are placed into an incubator at 10°C.
After 1 hour the mortality of the lice was recorded and the solution changed with FSW. Mortality of lice was recorded according to the following scale:
A - Alive, ability to swim M - Moribund) inability to swim, twitching/response to stimulus.
D - Dead, no movement or response to stimulus.
After observations on the lice after 1 hour have been made samples are placed back into the incubator and mor-tality was recorded at hourly intervals until approximately ~~Q% of the controls were dead or moribund, both being considered as respondent to the test and grouped together for analysis.
Solutions of 0.001, 0.01, 0.1, 1 and 10 ppm of cyper-methrin (PH006) and alphacypermethrin (PH007) in sea water were freshly prepared and used immediately in this test.
The solutions were prepared by adding 1 ml of a 10% w/w emulsified concentrate of each of PH006 or PH007 to 24 ml absolute ethyl alcohol and mixing with 975 ml sea water to produce a 100 ml stock solution. The stock solutions were diluted 1 to 10 (100 ml stock solution to 900 ml sea water) to produce: 10, 1.0, 0.1, 0.01 and 0.001 ppm working solutions Temperatures and salinities during the trials were 11.5°C and 16-18 ppt (parts per thousand). It was noted that the salinity of the water from the harvest site was 12.5 ppt at the time of lice collection.
It was found that 30% or more of the controls were dead within 8 hours. The results for the response of the lice after 1 hour after treatment are shown in Figure 1 and Figures 2 and 3 summarize the acute toxicities for the individual compounds over time.
B~ ale 2 In vitro toxicity test to different states of sea lice Sea lice for these trials were selected from McConnell ~ 25 Salmon's Laga Bay site, Loch Sunart, Scotland using the method described in Example 1.
The lice were separated into g-~oups used in triplicate (PH006, PH007 and a control) and each group consisted of 15 adult females, 15 adult males and 15 preadult 1 females.
The lice were subjected to a one hour bath treatment _ 6 _ using a 0.01 ppm solution (prepared using the method of Example 1) of each of PH006 and PH007 and a control solution as described in Example 1 and then followed by a recovery period in fresh salt water.. Temperatures and salinities during the trials were 11.5°C and 18.5 ppt respectively.
As in Example 1 responses to the treatment were recorded at hourly intervals until 30~ or more of the control samples were either dead or moribund.
The results are summarized in Figure 4. In all groups male survival was poorest followed by preadult 1 females.
Compound PH007 was found to be marginally more toxic than compound PH006. Both compounds were found to have sig-nificant toxicity to the smaller stages of lice.
Bxamvle 3 In vivo toxicity to sea lice Two trials were conducted to assess the toxicities of PH006 and PH007 to sea lice in vivo.
A total of 150 juvenile salmon (Salmon salary were obtained from Marine Harvest's Inver Ailort hatchery, Loch Ailort, Scotland. The fish were transferred to fibreglass tanks (measuring 145 cm x 145 cm) with a water depth of 30 cm and given a 24 hour acclimatisation period. All tanks were supplied with flow-through sea water and were aerated.
These fish were maintained as stock and used for the trials listed below.
Trial 1 Adult and pre adult (male and female) sea lice were collected from Marine Harvest's Strontian site, as in Example 1. The lice were collected from harvested fish as above. The lice were given a 2 hour acclimatisation period at the sea water facility prior to being released and _ 7 _ allowed to infect salmon.
Salmon were infected with lice by anaesthetizing the fish (0.075% benzocaine solution) and soaking them in a small mesh lined container with large numbers of lice. The fish were removed from the infection bath when a minimum of lice could be seen attached to them. All fish were placed into a single tank (as above) and allowed 24 hours to recover.
Concentrations tested for bath treatments were: 0.01, 10 0.05, 0.1 ppm of PH006 and of PH007 in addition to two control groups (containing absolute alcohol but no active ingredient).
Individual groups of fish were randomly selected and placed into separate tanks (105 cm x 105 cm with adjustable depth) supplied with flow-through sea water and an ap-propriate amount of compound stock solution (either 100 ppm stock solution prepared by adding 100 ml of 10% w/w concentrate to 100 ml absolute alcohol or 1000 ppm stock solution prepared by adding 1 ml of 10% w/w concentrate to 99 ml absolute alcohol) added to achieve the desired treatment concentration. During treatments, water supply to the tanks was shut off and a constant volume maintained.
Aeration was used during all treatments. Treatments were one hour in duration after which the water in the tanks was flushed out and sea water supplies reinstated. Two control groups were used, a pre treatment control and a treatment control. Nine fish were used in the pre treatment group and eight fish were used, per group, for the remaining groups.
The pre treatment control was enumerated for lice before the beginning of the experiment to establish the parasite load 24 hours following infection. The second or treatment control was enumerated along with the experimental groups (48 hours after infection) for parasite load to determine the number of lice lost due to handling or to poor water quality. Mean numbers of parasite per fish for the different groups were compared with a students t-test.

W'O 92/16106 ~ PCT/GB92/0047(1 v _ 8 _ Temperatures and salinities during treatments were 11°C and 17.5 ppt respectively.
After 24 hours, fish were enumerated for mortalities and sacrificed with a blow to the cranium; weighed, and the number of lice per fish was recorded. Feed was withheld from the fish throughout the entire experiment.
A total of 65 fish were used for the trial with a mean weight of 457.01 gms (std. dev. - 83.04). None of the fish died during any of the treatments, except one fish in the PH007 0.01 ppm group which had jumped out of its holding tank overnight. In the PH007 0.01 ppm group, some of the fish showed signs of stress for a couple of hours after treatment (see Example 4 for characterisation of stress behaviours). The mean number of lice per fish in the pre treatment control group was 26.78 (std. dev. - 15.79). The mean number of lice per fish in the treatment control group was 14.88 (std. dev. - 7.10). The means were found not to be significantly different (alpha - 0.05; t statistic -1.96). In the three PH006 treatments only one louse was found (0.01 pgm treatment). There were no live lice found in any of the PH007 treatments.
Trial 2 The same protocols were used as in the previous trial using bath treatment concentrations of 0.001, 0.005 and 0.01 ppm. The lice used for the trial were collected from the McConnell Salmon's Laga Bay site, Loch Sunart. Eight fish were used in the pre treatment group, nine fish were used in the treatment control and seven fish were used in each of the experimental groups. Temperatures and salinities during the trials were 11°C and 22.5-24.0 ppt (27.0 ppt at the lice collection site).
A total of fifty nine fish were used for the trial with a mean weight of 483 . 83 gms ( std. dev . - 68 . 21 ) . None of the fish died during any of the treatments except one fish WO 92/16106 ~ ~ ~ ~, PC'T/GB92/00470 _ g _ in the PH007 0.005 group which had jumped out of its holding tank overnight. In the PH007 0.01 ppm group, some of the fish showed signs of stress for a couple of hours after treatment (see Example 4 for a characterisation of stress behaviours). The mean number of lice per fish in the pre treatment control group was 26.88 (std. dev. - 11.38). The mean number of lice per fish in the treatment control group was 26.67 (std. dev. - 8.5). The means between the two control groups were not different (alpha = 0.05; t statis-tic - 0.043). Lice were only found in the 0.01 ppm treat-ment groups with a mean number of lice per fish of 2.14 (std. dev. - 1.86) for PH006 and 1.14 (std. dev. - 0.89) for PH007. The results for this trial are summarized in Figure 5.
Examy~le 4 Three groups of eight fish taken from the same stock as for Example 3 were placed into three separated tanks and given three 1 hour treatments with compounds PH006 and PH007 at 0.5 ppm at 24 hour intervals. The first treatment was in 105 x 105 cm tanks. For the second and third treatment fish were moved to 145 cm square tanks. The same treatment protocols were used as in the in vivo trials of Example 3.
Results for this trial are summarized in Figure 6. The mean weights for the fish used in the trial were not recorded but are estimated at 469.77 gms as calculated from the combined weights (n - 124) of fish from the first two trials of Example 3 which represent 84$ of the stock fish which were used for all. three trials. No control fish were lost during the entire experiment. In the PH006 one fish died of ter the third treatment. Ir. the PH007 group one fish died after each of the first two treatments. Although very few fish were lost in each of the treatments, they were notably stressed by the treatment displaying a repeated recovery behavioural pattern. During treatment fish would i~

become lethargic until the tank was flushed and filled with FSW. Immediately after treatment and during the period when tanks were filling, fish displayed a marked loss of co-ordination with occasional convulsive fits as marked by sideways shaking of the head. Alternatively, fish would lie on the bottom of the tank slowly ventilating or would swim slowly with mouths wide open. In several instances several fish were observed lying on the bottom of the tanks motionless) slowly ventilating, for several hours eventually recovering by the next day. The control fish did not display any of these behaviours.
Consistent with in vitro tests, compound PH007 was found to be marginally more toxic than compound PH006 to both lice and salmon in vivo. Both compounds showed good efficacy in removing lice from salmon at a concentration of 0.005 ppm. Compound PH006 was the most efficacious since salmon could tolerate repeated doses at 100 times the dose rate required to remove all the lice from infected fish.
Low salinities of the sea water at the facility may have stressed the lice as indicated by a slight decrease in the number of lice between the two control groups in the first experiment than in the second where salinities were higher.
It was interesting to note that higher salinities during Trial 2 of Example 3, which should have reduced the en-vironmental stress on the lice, did not reduce the toxicity of the compounds.
R~cam~le 5 Two separate trials were run and are referred to as Trial 1 and Trial 2.
Atlantic salmon smolts were obtained from Marine Aquaculture's loch Fyne grow-out site and transferred to holding tanks at the Sea Life Centre. Fish were held for 4 weeks during which time they were offered pelleted salmon feed ad libitum. Prior to each of the trials, fish were divided into several groups of thirteen (depending on the WO 92/16106 ~ PCT/GB92/00470 concentrations tested) and infected with sea lice collected at the Kerrera fisheries grow-out site near Oban.
Techniques used for collecting lice are those outlined in previous Examples. To infect fish, approximately 225-300 lice were added to each of the experimental tanks, where the volume had been temporarily reduced to 200 litres, and allowed to stand static for four hours, of ter which water supplies were reinstated.
Twenty four hours following infection, three fish were removed from each of the groups, sacrificed, weighed and enumerated for lice. Based on the average weight of the fish, fish were fed a medicated diet containing compound PH007 at dose rates of: 0.025, 0.05 and 0.1 (Trial 1)and 0.1. 0.5 and 1.0 (Trial 2) mg/kg for three consecutive days.
Separate control groups were used for each of the trials.
The compound was incorporated into the feed in a precooled (<40oC) 5% gelatin solution added to the feed at a rate of lml/12.5g of feed. Fish were fed the medicated feed at a rate of 1.5% body weight/day for three days and then fed unmedicated feed ad libitum for the remainder of the experimental period. The compound used for the experimental groups (ref PH/RDL/007H) was a 5% w/w fine beige powder.
Controls were given doses, corresponding to the highest doses used in the experimental groups, with a placebo (ref PH/RDL/007A) which was also a fine beige powder.
Fish were sampled for lice 4, 7 and 14 days following the last medicated feed treatment. To enumerate fish for lice, fish were sedated in a mild benzocaine solution (2 ml 10%, in acetone, per 1 litre FSW) and the number of pre/adult stages of lice recorded. On the third and last sampling fish were sacrificed) weight and number of lice recorded and then destroyed.
Results Temperatures and salinities throughout the experimental period were 12.0 + 1.OOC and 31 - 36 ppt. The mean weights i.

and lice burdens (number lice/fish) in both trials for the pre treatment counts are given in Tables 1 and 2. Mean numbers of lice following treatments for each of the experimental groups for Trials 1 and 2 are given in Figures 7 and 8 respectively.
There were no fish mortalities in either of the trials throughout the experiment and no unusual behaviours or reactions were noted in any of the fish. However, fish were notably agitated following infection as indicated by a significant increase in jumping and flashing activity in the tanks. It is likely that lice were knocked off fish as they came into contact with objects such as screening material covering the tanks and the standpipes.
Table 1. Trial 1 - Mean weights and lice burdens in pretreatment samples (N=three fish in each group).
Grou Mean weight (g) Mean No Lice (mg/~g) (std.dev.) (std.dev.) 0.0 162.3 (40.1) 10.3 (2.9) 0.025 160.7 (36.1) 15.0 (3.0) 0.05 146.7 (38.7) 15.3 (5.1) 0.1 159.3 (22.1) 13.0 (7.0) Table 2. Trial 2 - Mean weiqhts and lice burdens in pretreatment samples (N=three fish in each group).
Grou Mean weight (g) Mean No Lice (mg/~g) (std.dev.) (std.dev.) 0.0 147.0 (45.9) 20.3 (10.0) 0.1 210.0 (42.6) 2?.7 (4.2) 0.5 167.7 (4.5) 34.0 (16.1) 1.0 169.0 (66.3) 29.3 (23.2) Conclusions Although these results are based on artificial laboratory conditions, they clearly indicate that compound 2~~~~9~

PH007 has potential as an orally administered sea lice chemotherapeutant. Overall reductions in the number of lice present, as seen in the differences between pretreatment and the successive control samples were probably caused by the high activity of the fish following infection. Maximum efficacy was achieved with four days from treatment with no further, significant, reduction in lice numbers 1 or 2 weeks following treatment. However, the dose rate of 1.0 mg/kg may not necessarily be indicative of a true therapeutic dose due to the small sample sizes of the groups and the artificial system used. In addition, the variable feeding response in the experimental system may explain the difference in efficacy between Trial 1 & 2 in the 0.1 mg/kg groups. However, reduced efficacy in the second experiment may have, in part been due to slightly lower water temperatures. Overall it was established that the compound achieved good efficacy without any ill effects to the treated fish.
A number of other pyrethroid pesticides have been tested in accordance with the invention and the following table gives the % mortality in sea lice following 1 hour bath treatments in-vitro with 6 different pyrethroid compounds each administered at 2 different concentrations.
Hours post Treatment 2 5 COI~OUND 1 6 Negative control 0 0 Solvent (ABS) control 10 10 Cypermethrin 0.1 ppm 0 70 Cypermethrin 0.01 ppm Deltametrin 0.1 ppm 20 90 Deltametrin 0.01 ppm 20 Resmethrin 0.6 ppm 50 70 Resmethrin 0.06 ppm 50 50 Permethrin 0.5 ppm 0 30 Permethrin 0.05 ppm 0 10 Cyhalothrin 0.2 ppm 60 40 Cyhalothrin 0.02 ppm 0 10 Tetramethrin 0.8 ppm 10 100 Tetramethrin 0.08 ppm 0 70

Claims (15)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Use of a pyrethroid pesticide for he manufacture of a composition for the treatment of sea lice infestation in seawater fish in a seawater environment.

2. Use according to claim 1 wherein the pyrethroid pesticide is cypermethrin or alphacypermethrin.

3. Use according to claim 1 or 2 wherein the composition containing the pesticide is a composition to be administered orally.

4. Use according to any of claims 1 to 3 wherein the seawater fish is salmon.

5. Use according to claim 3, wherein the composition containing the cypermethrin or alphacypermethrin is to be administered orally at a dosage rate of 0.025 to 5 mg/kg of fish body weight.

6. Use of a pyrethroid pesticide in water for the manufacture of a treatment suspension for salmon suffering from sea lice infestation.

7. Use according to claim 6 wherein the pyrethroid pesticide is cypermethrin or alphacypermethrin.

8. Use according to claim 6 or 7 wherein the concentration of the pyrethroid pesticide in the treatment suspension is in a range between 0.001 and 0.5 ppm.

9. The use of a treatment suspension comprising 0.001 to 0.5 ppm pyrethroid pesticide suspended in water for treatment of salmon suffering from sea lice infestation by administering said suspension to the salmon externally.

10. A composition when used for controlling sea lice infestation in salmon which comprises a pyrethroid pesticide suspended in water.

11. A composition according to claim 10 wherein the pyrethroid pesticide is cypermethrin or alphacypermethrin.

12. The use of a composition according to claim 10 or 11 for controlling sea lice infestation in salmon by externally administered said composition to the salmon.

13. A food composition for seafish, characterized in that in addition to food ingredients it contains a pyrethroid pesticide.

14. A food composition according to claim 13 characterized in that it contains cypermethrin or alphacypermethrin as the pyrethroid pesticide.

15. A food composition according to claim 13 or 14, characterized in that it contains the pyrethroid pesticide in an amount to provide a dosage of 0.025 to 5 mg pyrethroid per kg body weight of seafish.