DK202370557A1

DK202370557A1 - Apparatus and method for aquatic animals

Info

Publication number: DK202370557A1
Application number: DKPA202370557A
Authority: DK
Inventors: Jamieson Ian
Original assignee: Pulcea Ltd
Priority date: 2021-03-31
Filing date: 2023-10-31
Publication date: 2023-12-07
Also published as: CL2023002902A1; WO2022208101A1; EP4312559A1; AU2022247198A1; CA3213487A1; GB202104648D0

Abstract

The invention provides an apparatus for aquatic animals (1), the apparatus comprising an aquatic enclosure (3, 9) for retaining an aquatic animal, wherein the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide.

Description

DK 2023 70557 A1 1

1 APPARATUS AND METHOD FOR AQUATIC ANIMALS

2

3 — Field of the invention

4

— The invention relates to apparatus and methods for disturbing, injuring or killing aquatic

6 — ectoparasites, reducing ectoparasitic infestation on aquatic animals and improving the

7 appearance, meat quality, meat quantity and growth rates of aquatic animals.

8

9 Background to the invention

11 The invention relates to the field of rearing aquatic animals, such as fish.

The 12 commonly farmed Atlantic salmon (Salmo salar) is prone to infestation by sea lice of 13 the species Lepeophtheirus salmonis and embodiments of the invention act to disturb, 14 kill or injure sea lice, or remove sea lice from the fish, to reduce the significant damage to the fish (including fish death) arising from this infestation.

Such infestation can 16 — otherwise cause fish death or reduced yield.

As well as the direct effect of sea lice on 17 the fish, sea lice infestation also causes a generalised chronic stress response in the 18 fish, which may make them susceptible to infection by other diseases and which may 19 reduce meat yield.

21 — The use of hydrogen peroxide in water to generate bubbles on the surface of and/or 22 — within the body of ectoparasites can disturb, injure or kill such ectoparasites.

However, 23 hydrogen peroxide can also be harmful to other aquatic animals, such as fish. 24

DK 2023 70557 A1 2 1 The present invention seeks to provide an improved treatment process and apparatus 2 for example by one or more of reducing the impact of the process on fish, improving 3 — the speed of the process and/or improving the effectiveness of the process. 4

Summary of the invention 6 7 Afirst aspect of the invention provides an apparatus for aquatic animals, the apparatus 8 comprising an aquatic enclosure for retaining an aquatic animal (e.g. during treatment), 9 wherein the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide (i.e. H,0,), and an enzyme for catalytic decomposition of hydrogen peroxide. 11 The aquatic enclosure may retain one or more aquatic animals. The aquatic enclosure 12 may retain one or more fish. The aquatic enclosure may retain one or more Atlantic 13 salmon. The enzyme may be a peroxidase. The enzyme may be catalase. Herein the 14 aqueous solution is used to broadly indicate an aqueous composition comprising hydrogen peroxide. The aqueous solution may also comprise (e.g. contain) an enzyme 16 — for catalytic deposition of hydrogen peroxide. It will be understood that the composition 17 — may comprise (e.g. contain) further components that may not be normally considered 18 as ‘solutes’, such as sound waves, bubbles, bubble curtains, etc. In other words, it will 19 be understood that the aqueous solution may comprise (e.g. contain) one or more components beyond those which are in solution in water. 21 22 — The action on hydrogen peroxide of the enzyme in the aqueous solution causes the 23 formation of bubbles (the bubbles predominantly comprising oxygen). More bubbles 24 — form (and/or bubbles may form more quickly) where such an enzyme is also present.

Advantageously therefore, by providing an aqueous solution comprising both an 26 enzyme for catalytic decomposition of hydrogen peroxide and hydrogen peroxide, 27 lower concentrations of hydrogen peroxide can be used to achieve the same effect 28 (e.g. the formation of a similar quantity of bubbles) than would be needed if no such 29 enzyme were present. Furthermore, because the enzyme decreases the time needed for bubbles to form, the total time for which an aquatic animal must be exposed to the 31 aqueous solution in order for bubbles to take effect is reduced. Therefore, it is possible 32 to useless hydrogen peroxide, to have shorter exposure times for the aquatic animals, 33 — or both. 34

In addition the presence of an enzyme for catalytic decomposition of hydrogen 36 peroxide means that hydrogen peroxide continuously forms bubbles until 37 (substantially) no hydrogen peroxide is left behind in the aqueous solution. The use of

DK 2023 70557 A1 3

1 an aqueous solution comprising hydrogen peroxide in combination with such an

2 enzyme is therefore more environmentally friendly than the use of an aqueous solution

3 comprising hydrogen peroxide and no such enzyme.

The skilled person will appreciate

4 that the limiting factor on the rate of the reaction of hydrogen peroxide when decomposing to water and oxygen in this instance will be one of: the concentration of

6 hydrogen peroxide, the amount of catalase available, and/or the mixing of the aqueous

7 — solution comprising hydrogen peroxide and catalase.

8

9 The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising hydrogen peroxide at a concentration greater than or equal to 0.001 mg/L, or greater 11 than or equal to 0.01 mg/L, or greater than or equal to 0.1 mg/L, or greater than or 12 equal to 1 mg/L, or greater than or equal to 20 mg/L or greater than or equal to 200 13 mg/L.

The aquatic enclosure may comprise (e.g. retain) an aqueous solution 14 comprising hydrogen peroxide at a concentration greater than or equal to 50 mg/L or greater than or equal to 80 mg/L.

The aquatic enclosure may comprise (e.g. retain) an 16 aqueous solution comprising hydrogen peroxide at a concentration less than or equal 17 to 2,500 mg/L or less than or equal to 2,200 mg/L, or less than or equal to 1500 mg/L. 18 The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising 19 hydrogen peroxide at a concentration from 20 mg/L to 2,500 mg/L, inclusive, or from 200 mg/L to 2,500 mg/L, inclusive, or from 20 mg/L to 2,200 mg/L, inclusive, or from 21 200 mg/L to 2,200 mg/L, inclusive.

The aquatic enclosure may comprise (e.g. retain) 22 anaqueous solution comprising hydrogen peroxide at a concentration of approximately 23 — 1,500 mg/L (e.g. at a concentration of from 1,300 mg/L to 1,700 mg/L, inclusive). The 24 — aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising hydrogen peroxide at a concentration of at least 20 mg/L, or at least 200 mg/L, or at least 500 26 mg/L.

The aquatic enclosure may comprise (e.g. retain) an aqueous solution 27 comprising hydrogen peroxide at a concentration of 800 mg/L.

The aqueous solution 28 may comprise hydrogen peroxide at a concentration of from 500 mg/L to 1,500 mg/L 29 inclusive.

31 The aqueous solution may comprise an enzyme for catalytic decomposition of 32 — hydrogen peroxide at a concentration of 0.0001% w/w of water to 5% w/w of water, or 33 0.001% w/w of water to 5% w/w of water, or 0.05% w/w of water to 5% w/w of water, or 34 at a concentration of 0.1% w/w of water to 4% w/w of water, or 0.5% w/w of water to 3% w/w of water.

The aqueous solution may comprise an enzyme for catalytic 36 decomposition of hydrogen peroxide at a concentration of at least 0.0001% w/w of 37 — water, or atleast 0.001% w/w of water, or at least 0.01% w/w of water, or at least 0.03%

DK 2023 70557 A1 4 1 w/w of water. The aqueous solution may comprise no more than 6% w/w of 2 > enzyme/water. The aqueous solution may comprise at least 0.001 mg/L, or 0.005 mg/L, 3 or 0.05 mg/l, or 0.075 mg/L, or 0.75 mg/L, or 1 mg/L, or 1.5 mg/L of said enzyme per 4 litre of water (e.g. per litre of sea water), and optionally not more than 20 mg/L, or 15 mg/Lor 10 mg/L, or 1 mg/l, or 0.1 mg/L, or 0.01 mg/L. The weight ratio of the hydrogen 6 peroxide to enzyme may be from 100:1 to 1:1, or from 50:1 to 1:1, or from 30:1 to 1:1, 7 or from 10:1 to 2:1, or from 5:1 to 3:1. Ratios of hydrogen peroxide to enzyme as 8 described herein provide adequate reaction of hydrogen peroxide (e.g. >95% of 9 hydrogen peroxide reacted) to form bubbles. The aqueous solution may comprise 500 mg/L hydrogen peroxide. The aqueous solution may optionally comprise at least 10 11 mg/L, 50 mg/L, 200 mg/L, 300 mg/L 400 mg/L, or 500 mg/L enzyme (e.g. catalase). 12 — The skilled person may choose to use greater amounts of enzyme (e.g. catalase) such 13 that this may be present in excess amounts. 14

The aqueous solution may be an aqueous solution of sea water. The aqueous solution 16 may be an aqueous solution of freshwater. The aqueous solution may comprise saline. 17 18 The aqueous solution may be a physiologically compatible medium. The aqueous 19 solution may comprise (e.g. be) an aquaculture medium, that is to say a medium suitable for use in aquaculture (i.e. the farming of aquatic organisms such as fish, 21 crustaceans, molluscs, aquatic plants and/or algae). The aqueous solution may 22 comprise (e.g. be) a pisciculture medium, that is to say a medium suitable for use in 23 farming fish. The aquaculture or pisciculture medium may have a similar composition 24 — to either (i.e. natural) sea water or freshwater (except for the addition of hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide). 26 27 — The apparatus may comprise mixing means. The mixing means may be a mixer. The 28 mixing means may be a stirrer. For example, the apparatus may comprise one or more 29 stirrers configured to cause motion of the aqueous solution within the enclosure. 31 — The apparatus may comprise aeration means, optionally aeration means configured to 32 — cause motion of the aqueous solution within the enclosure. The aeration means may 33 comprise (e.g. be) an aerator. The mixing means may comprise (e.g. be) the aeration 34 means (e.g. the aerator). The aeration means may comprise (e.g. be) means for generating bubbles (e.g. air bubbles or oxygen bubbles). The aeration means may 36 comprise (e.g. be) a bubble generator. The mixing means may comprise (e.g. be) a 37 bubble generator, optionally a bubble curtain generator. The mixing means may

DK 2023 70557 A1

1 comprise (e.g. be) a bubble curtain.

The aeration means may comprise (e.g. be) a

2 bubble curtain (configured to provide bubbles). The apparatus may comprise an air

3 hose for introducing (e.g. configured to introduce) air bubbles, optionally oxygen

4 bubbles.

The apparatus may comprise a curtain having a plurality of holes defined

5 therein.

The curtain may be connected to a compressor for generating bubbles (e.g.

6 — air bubbles, optionally oxygen bubbles). The apparatus may comprise a bubble curtain

7 — generator (e.g. configured to provide a bubble curtain comprising bubbles).

8

9 The mixing means may comprise (e.g. be) means for directing sound waves into the aquatic enclosure, optionally wherein the means for directing sound waves comprises 11 atransducer.

The apparatus may comprise acoustic means configured to cause motion 12 of the aqueous solution within the enclosure.

The acoustic means may comprise (e.g 13 be) a transducer.

The acoustic means may comprise (e.g. be) means for generating 14 sound waves (e.g. a sound wave generator, optionally a transducer, optionally a speaker). 16 17 In an example, the mixing means may comprise one or more: stirrers, aerators, bubble 18 curtains, bubble curtain generators, and/or means for directing sound waves (e.g. 19 transducers). The provision of mixing means increases the likelihood of interactions taking place between the hydrogen peroxide and the said enzyme in the aquatic 21 — solution, thereby increasing the rate at which bubbles are formed as well as increasing 22 the quantity of bubbles that are formed.

Accordingly, with the provision of such mixing 23 means, it is possible to use less hydrogen peroxide and to have shorter exposure times 24 — for the aquatic animals.

26 —|twill be understood that while the aquatic ectoparasite(s) and/or the aquatic enclosure 27 may comprise or contain naturally-occurring enzymes, where an aqueous solution 28 comprising hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen 29 peroxide is referred to, the said enzyme for catalytic decomposition of hydrogen peroxide is not an enzyme that is naturally occurring in the aquatic ectoparasite(s) 31 — and/or the aquatic enclosure, but an additional enzyme which is introduced for the 32 — purpose of catalytic decomposition of introduced hydrogen peroxide.

Similarly, it will 33 be understood that the hydrogen peroxide referred to herein is not the trace quantities 34 — of hydrogen peroxide that may in some circumstances be present (e.g. in sea water) — but additional hydrogen peroxide. 36

DK 2023 70557 A1 6 1 The apparatus may comprise means for directing sound waves into the aquatic 2 enclosure (i.e. a source of sound waves configured to direct sound waves into the 3 aquatic enclosure). The mixing means may comprise the means for directing sound 4 — waves.

Alternatively or additionally the means for directing sound waves may be a first means for directing sound waves, and the apparatus may comprise a second means 6 for directing sound waves wherein the mixing means comprises the second means for 7 directing sound waves.

The or each means for directing sound waves may comprise 8 (e.g. be) a transducer, optionally plurality of transducers.

The means for directing 9 sound waves may be means for directing sound waves into the aquatic enclosure at a — sound pressure level of between 160 dB and 240 dB, inclusive (e.g. in the local 11 environment of the aquatic animal, i.e. in the water or aqueous solution immediately 12 — surrounding the aquatic animal). The means for directing sound waves into the aquatic 13 — enclosure (i.e. the source of sound waves configured to direct sound waves into the 14 aquatic enclosure) may be configured to direct sound waves having a frequency between 1 kHz and 100 kHz, inclusive, or between 20 kHz and 100 kHz, inclusive, or 16 between 25 kHz and 100 kHz, inclusive, into the enclosure The means for directing 17 sound waves into the aquatic enclosure may be configured to generate and direct 18 sound waves having a frequency spectrum (and optionally power) which is variable 19 with time and configured (e.g. programmed) to vary with time the frequency spectrum (and optionally power) of the sound waves which are directed into the aquatic 21 — enclosure. 22 23 — The means for directing sound waves into the aquatic enclosure may be configured to 24 generate and direct sound waves into the aquatic enclosure such that the centre and/or peak frequency of the sound waves is higher at a first depth than at a second depth 26 — within the aquatic enclosure, wherein the first depth is greater than the second depth 27 (typically differing by at least 1 metre). The centre and/or peak frequency may by higher 28 progressively with depth.

For example, the means for directing sound waves into the 29 aquatic enclosure may comprise one or more transducers located in a base region (e.g. at the base) of the aquatic enclosure and the sound waves may comprise a range of 31 frequencies.

Thus, as lower frequencies penetrate further into water, the centre and/or 32 — peak frequency of the sound waves will be lower as the depth decreases, further from 33 — the means for directing sound waves.

However, there are other ways to arrange for the 34 centre and/or peak frequency of the sound waves to be higher at a first depth than a second depth.

For example, means for directing sound waves which output sound 36 waves with different frequency spectra may be located at different depths (higher 37 centre and/or peak frequency at the greater depth). Phased transducer arrays, which

DK 2023 70557 A1 7 1 may be located at or near the surface, may also be configured to cause the centre 2 and/or peak frequency of the sound waves to be higher at the first depth than the 3 — second depth. 4

The means for directing sound waves into the aquatic enclosure may comprise an array 6 of directional transducers arranged at or near the base of the enclosure and configured 7 to direct sound waves upwards towards the water surface. Advantageously, when 8 sound waves meet the water surface they are reflected back into the enclosure. 9

Alternatively, or in addition, the frequency (peak and/or centre frequency) and/or the 11 acoustic pressure of the sound waves within the aquatic enclosure (i.e. which are 12 — generated and directed into the aquatic enclosure) may vary with depth (e.g. increasing 13 — with depth), e.g. proportional to the square root of the water pressure at a given depth. 14 The sound may be generated by transducers located in a base region of the aquatic enclosure and have a range of frequencies and/or acoustic pressures, such that the 16 peak and/or centre frequency and/or the acoustic pressure of the sound waves within 17 the aquatic enclosure increases with depth. For example, due to the increased 18 pressure and greater depths and/or due to greater attenuation of higher frequency 19 sounds with distance from the transducers. 21 The sound waves may be varied in a bubble regulation phase in which the frequency 22 spectrum of the sound waves is controlled to cause bubble growth and/or coalescence. 23 The sounds wave may be controlled in a subsequent bubble collapse phase in which 24 — the frequency spectrum of the sound waves is controlled to cause the collapse of bubbles. 26 27 The means for directing sound waves into the aquatic enclosure (i.e. the source of 28 sound waves configured to direct sound waves into the aquatic enclosure) may 29 comprise (e.g. be) one or more (i.e. electroacoustic) transducers (e.g. an array of transducers). The one or more transducers may be one or more sonic transducers (e.g. 31 an array of sonic transducers). Sonic transducers are transducers configured to 32 generate sound waves in a surrounding medium. The one or more transducers may be 33 one or more ultrasonic transducers (e.g. an array of ultrasonic transducers). Ultrasonic 34 transducers are transducers configured to generate ultrasound waves in a surrounding medium. The means for directing sound waves into the aquatic enclosure (i.e. the 36 — source of sound waves configured to direct sound waves into the aquatic enclosure)

DK 2023 70557 A1 8 1 may comprise (e.g. consist of) one or more loudspeakers (e.g. an array of 2 loudspeakers). 3 4 — The apparatus may comprise means to measure temperature in the aquatic enclosure (for example, one or more temperature sensors). The apparatus may be configured to 6 vary the frequency spectrum of the sound waves in dependence on the measured 7 temperature. The apparatus may be configured to vary the speed of the mixers in 8 dependence on the measured temperature. 9

The enclosure may comprise a conduit. Where the aquatic enclosure comprises a 11 conduit, the apparatus may be configured to vary the pressure in the conduit (for 12 example by compressing the aquatic enclosure, or the contents of the aquatic 13 — enclosure, for example by introducing a gas, such as air, above the aqueous solution 14 in the aquatic enclosure, or a pressurisable bladder adjacent to or within the aqueous — solution). The pressure in the conduit may be varied by the use of one or more pumps 16 — and/or by one or more restrictions. The conduit may be configured such that the 17 — pressure in the conduit varies along the length of the conduit, however this is not 18 required, and it may be that the pressure in the conduit is substantially constant 19 throughout the length of the conduit. Advantageously, where the pressure is reduced (e.g. through the use of one or more pumps) this increases the size of bubbles 21 produced by action of the enzyme on the hydrogen peroxide. 22 23 Furthermore, increased pressure has the advantage that because the resonant 24 — frequency of an air bubble in aqueous solution varies with liquid pressure, by raising the pressure in the aquatic enclosure, the ratio of pressure at the bottom of the aquatic 26 — enclosure to the pressure at the top of the container (e.g. enclosure) is smaller than 27 would otherwise be the case. Accordingly, the variation in the frequency of the sound 28 waves required during the bubble regulation phase and the bubble collapse phase to 29 have a desired effect (regulating the size of bubbles, causing bubble collapse) within the aquatic enclosure is reduced. This enables better control of bubble size and 31 collapse. 32 33 In some embodiments the apparatus may comprise an enclosure for retaining (e.g. 34 configured to retain) aquatic animals and an inlet for allowing (e.g. configured to allow) aquatic animals into the enclosure. The apparatus may comprise a conduit configured 36 — for allowing (e.g. configured to allow) aquatic animals out of the enclosure, optionally 37 to selectively allow aquatic animals out of the enclosure. The enclosure may comprise

DK 2023 70557 A1 9 1 atreatment region (e.g. between the inlet and the outlet). The enclosure may comprise 2 one or more filters, optionally wherein at least one filter is an ectoparasite filter for 3 filtering (e.g. configured to filter) ectoparasites out of the aqueous solution in the 4 enclosure.

The enclosure may comprise mixing means (e.g. as described herein) and the mixing means may be positioned between the inlet and the treatment region. 6 7 — The means for directing sound waves into the aquatic enclosure (i.e. the source of 8 sound waves configured to direct sound waves into the aquatic enclosure) may be 9 configured to direct sound waves having a variable frequency (and optionally also a — variable power level). The frequency may be greater than or equal to 1 kHz, or greater 11 than or equal to 20 kHz, or greater than or equal to 25 kHz.

The means for directing 12 — sound waves into the aquatic enclosure (i.e. the source of sound waves configured to 13 direct sound waves into the aquatic enclosure) may be configured to direct sound 14 waves having a frequency less than or equal to 100 kHz into the enclosure.

16 It may be that the means for directing sound waves into the aquatic enclosure (i.e. the 17 — source of sound waves configured to direct sound waves into the aquatic enclosure) is 18 configured to direct soundwaves having a sound pressure level greater than or equal 19 to 160 dB into the aquatic enclosure.

It may be that the means for directing sound waves into the aquatic enclosure (i.e. the source of sound waves configured to direct 21 sound waves into the aquatic enclosure) is configured to direct sound waves having a 22 sound pressure level less than or equal to 240 dB into the aquatic enclosure. 23 24 It may be that the means for directing sound waves into the aquatic enclosure (i.e. the source of sound waves configured to direct sound waves into the aquatic enclosure) is 26 configured to direct soundwaves into the aquatic enclosure to generate a local energy 27 intensity level of between 0.001 W/cm? and 0.01 W/cm?, inclusive. 28 29 It may be that the means for directing sound waves into the aquatic enclosure (i.e. the — source of sound waves configured to direct sound waves into the aquatic enclosure) is 31 configured to direct sound waves into the aquatic enclosure for a continuous period of 32 atleast 30 seconds, or at least 1 minute, or at least 2 minutes, or at least 3 minutes, or 33 atleast 4 minutes, or at least 5 minutes, or at least 10 minutes, or at least 15 minutes, 34 — or atleast 20 minutes.

36 The aquatic enclosure may comprise a cage (e.g. a net), the cage being at least 37 partially surrounded by one or more sheets of fabric.

The one or more sheets of fabric

DK 2023 70557 A1 10 1 — may be waterproof or water-resistant fabric.

The one or more sheets of fabric may 2 comprise (e.g. be) tarpaulin.

The aquatic enclosure may be an aquarium. 3 4 — The cage may be a first cage and the aquatic enclosure may comprise at least one further cage.

In other words, the aquatic enclosure may comprise at least two cases. 6 The aquatic enclosure may comprise a conduit extending between the at least two 7 cages, the conduit being sized and shaped such that an aquatic animal (e.g. a fish) 8 may travel between the cages via the conduit.

A mixing means (optionally the mixing 9 means (e.g. the means for directing sound waves)) may be positioned within the conduit.

A bubble curtain generator may be positioned within the conduit.

The bubble 11 curtain generator may comprise (e.g. be) the mixing means. 12 13 — The aquatic enclosure may be a flexible enclosure.

The aquatic enclosure may be a 14 — fabric enclosure (i.e. an enclosure formed by one or more sheets of fabric). The aquatic enclosure may be formed by one or more sheets of waterproof or water-resistant fabric 16 (e.g. urethane-coated canvas such as tarpaulin). 17 18 — The aqueous solution may comprise sound waves.

The apparatus may comprise one 19 or more water and/or sound permeable shields configured to keep fish within the aquatic enclosure away from the means for directing sound waves into the aquatic 21 — enclosure.

This shields fish from potential damage cause by excessive sound intensity 22 adjacent the means for directing sound waves into the aquatic enclosure.

The one or 23 — more water and sound permeable shields may be located intermediate the means for 24 directing sound waves into the aquatic enclosure and a main fish-receiving chamber of the aquatic enclosure. 26 27 The apparatus may comprise one or more sound absorbing barriers and/or reflecting 28 medium.

The apparatus may comprise means for generating one or more (e.g. sound 29 absorbing) bubble curtains (e.g. one or more bubble generators or bubble curtain generators). The aqueous solution may comprise (e.g. retain) a bubble curtain, 31 optionally a bubble curtain defining a tubular shape, optionally a toroidal shape.

The 32 one or more sound absorbing barriers and/or reflecting medium and/or means for 33 generating one or more sound absorbing bubble curtains may be located around the 34 periphery of (at or beyond the periphery) of the aquatic enclosure.

This enables greater intensity sounds to be generated while minimising noise pollution and reduces the 36 — power of sound escaping from the enclosure (e.g. into the sea). 37

DK 2023 70557 A1 11 1 The means for generating the one or more (e.g. sound absorbing) bubble curtains may 2 be a mixing means (e.g. a mixer or stirrer, optionally a transducer) arranged to cause 3 — mixing of the aqueous solution (e.g. including water, hydrogen peroxide, and enzyme). 4

The bubble curtain may be a tubular bubble curtain, optionally a toroidal bubble curtain. 6 The bubble curtain may define a tubular shape, optionally a toroidal shape. For 7 example, the bubble curtain may define a region of the enclosure comprising a 8 relatively high density of bubbles (e.g. relative to a lower density of bubbles in other 9 regions of the enclosure) wherein bubbles are introduced into the enclosure in a lower — central region of the enclosure, such that the bubbles rise under buoyancy, travel 11 across an upper region of the enclosure towards the peripheries of the enclosure, and 12 subsequently travel downwards towards the lower region of the enclosure, due to the 13 current caused by the introduction of further bubbles and/or the mixing means. 14

A tubular (e.g. toroidal) bubble curtain will be understood to be an arrangement of 16 bubbles comprising: 17 a (e.g. generally) vertical portion extending from a lower region of the enclosure 18 to an upper region of the enclosure (e.g. formed as a result of bubbles rising due to 19 buoyancy, and/or a current generated by the introduction of further bubbles and/or a mixing means); 21 a first (e.g. generally) planar horizontal portion arranged at or close to (e.g. 22 within 10 centimetres of, e.g. within 5 centimetres of, e.g. within 1 centimetre of) the 23 — surface of the aqueous solution and extending from the vertical portion to the periphery 24 — ofthe enclosure (e.g. formed as a result of a current generated by the introduction of further bubbles and/or a mixing means); 26 a (e.g. generally) cylindrical portion arranged around the periphery of the 27 enclosure (e.g. formed as a result of a current generated by the introduction of further 28 bubbles and/or a mixing means); and optionally 29 a second (e.g. generally) planar horizontal portion arranged at or close to (e.g. — within 10 centimetres of, e.g. within 5 centimetres of, e.g. within 1 centimetre of) the 31 lower limit of the enclosure (e.g. formed as a result of a current generated by the 32 introduction of further bubbles and/or a mixing means). 33 34 In other words, the tubular (e.g. toroidal) bubble curtain may have a tubular (e.g. toroidal) shape which extends throughout the enclosure, with internal sections of the 36 — tube (e.g. toroid) (e.g. other than the vertical, cylindrical and planar portions) within the 37 — spaces defined by the vertical, cylindrical and planar portions. The internal sections

DK 2023 70557 A1 12 1 may contain relatively few (e.g. no) bubbles.

A 1 litre volume of the tubular (e.g. 2 toroidal) bubble curtain may contain 100 times more bubbles, e.g. 1,000 times more 3 bubbles, e.g. 10,000 times more bubbles, e.g. 100,000 times more bubbles than a 1 4 litre volume of the internal sections.

6 The apparatus may comprise a tubular (e.g. toroidal) bubble curtain generator 7 configured to generate a tubular (e.g. toroidal) bubble curtain.

The tubular (e.g. 8 toroidal) bubble curtain generator may be located in a lower central region of the 9 aquatic enclosure.

The tubular (e.g. toroidal) bubble curtain generator may comprise (e.g. be provided by) one or more mixing means (e.g. as described herein) and the one 11 or more mixing means may be positioned in a lower central region of the aquatic 12 — enclosure. 13 14 The bubble curtain (e.g. the tubular bubble curtain, optionally the toroidal bubble curtain) may also cause motion of the aqueous solution (e.g. mixing) within the 16 — enclosure.

Bubbles from the bubble curtain may cause mixing and, accordingly, it 17 should be understood that the bubble curtain (e.g. the tubular bubble curtain, optionally 18 the toroidal bubble curtain) may be the mixing means.

The mixing (optionally by said 19 bubble curtain) may last at least 10 seconds per one minute, and optionally no more than 20 seconds per one minute.

However, in some embodiments the mixing 21 (optionally by said bubble curtain) may last for at least 50%, e.g. at least 75%, e.g. at 22 least 90%, optionally 100% of a treatment period.

For example, the mixing (optionally 23 by said bubble curtain) may last for at least 5 minutes, e.g. at least 10 minutes, e.g. at 24 — least 20 minutes.

The mixing (optionally by said bubble curtain) typically lasts for less than 2 hours, e.g. for less than 1 hour, optionally for less than 45 minutes. 26 27 Mixing of the aqueous solution of water, hydrogen peroxide, and an enzyme, has been 28 found particularly beneficial because this improves the rate at which the hydrogen 29 peroxide is broken down and thus the concentration of bubbles produced.

As a result, it is possible to use lower concentrations of hydrogen peroxide, and less hydrogen 31 peroxide in total, as well as to reduce the exposure times of the aquatic animals to the 32 — hydrogen peroxide. 33 34 — The bubble curtain generator (e.g. the tubular bubble curtain generator, optionally the toroidal bubble curtain generator) may comprise a mixing means in an aquatic 36 — enclosure for retaining an aquatic animal, wherein the aquatic enclosure retains an 37 aqueous solution comprising hydrogen peroxide and an enzyme for catalytic

DK 2023 70557 A1 13 1 decomposition of hydrogen peroxide.

It has been found that the use of such a mixing 2 means in an aqueous solution comprising hydrogen peroxide and the enzyme results 3 in a surprisingly large quantity of small bubbles being produced in a short space of 4 — time.

This has the effect of causing the aqueous solution in the region of the mixing means to become less transparent, e.g. translucent, and in some cases opaque. 6 — Without wishing to be bound by any theory, it is believed that such a bubble curtain 7 generator (e.g. as defined by a mixing means introduced into an aqueous solution 8 comprising hydrogen peroxide and the enzyme) produces a liquid comprising a high 9 density of bubbles with suitable sizes such that light and sound transmission through the solution is at least partially blocked. 11 12 Accordingly, an aspect of the invention provides an apparatus for aquatic animals, the 13 apparatus comprising an aquatic enclosure for retaining an aquatic animal, wherein 14 the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide, 16 — and wherein the aquatic enclosure further comprises a mixing means (optionally one 17 — or more: stirrers, mixers, paddles, means for directing sound waves, transducers, 18 bubble generators, bubble curtain generators, and/or aerators, etc) configured to cause 19 mixing of the aqueous solution (e.g. to thereby increase the rate of decomposition of the hydrogen peroxide, e.g. by the enzyme). 21 22 It may be that the mixing of the aqueous solution is sufficient to increase the rate of 23 decomposition of hydrogen peroxide (e.g. by the enzyme) to thereby generate 24 — sufficient bubbles in the aqueous solution to reduce light transmission through a distance (e.g. at least 10 centimetres, at least 20 centimetres, at least 50 centimetres) 26 of the solution by at least 50%, or at least 70%, or at least 90%, e.g. as compared to 27 the distance (e.g. at least 10 centimetres, at least 20 centimetres, at least 50 28 centimetres) through the (same) aqueous solution (e.g. under the same conditions) 29 — without bubbles and/or without mixing.

The mixing of the aqueous solution may be sufficient to increase the rate of decomposition of hydrogen peroxide (e.g. by the 31 enzyme) to thereby generate sufficient bubbles (of appropriate size) in the aqueous 32 — solution such that the light transmission of the aqueous solution is less than 70%, or 33 less than 50%, or less than 30%, or less than 10%, or less than 1%, (e.g. as compared 34 to the same aqueous solution (e.g. under the same conditions) without bubbles and/or without mixing). 36

DK 2023 70557 A1 14 1 The aqueous solution in the region (e.g. within 10 centimetres, e.g. within 50 2 centimetres, e.g. within 1 meter) of the mixing means may become less transparent, 3 e.g. translucent, and in some cases opaque when the mixing means mix the aqueous 4 — solution. In other words, the aqueous solution may be at least translucent or opaque in part (e.g. inthe region (e.g. within 10 centimetres, e.g. within 50 centimetres, e.g. within 6 1 meter) of the mixing means when the mixing means mix the aqueous solution). 7 Alternatively, the aqueous solution may be translucent or opaque (e.g. in the region 8 (e.g. within 10 centimetres, e.g. within 50 centimetres, e.g. within 1 meter) of the mixing 9 means when the mixing means mix the aqueous solution). The translucency of an aqueous solution may be measured by any known and effective technique, for 11 example, by placing a sample solution of standardised thickness in the light path of a 12 spectrophotometer and measuring transmittance, as a percentage of light transmitted 13 — through the solution. 14

Accordingly, a test for translucence may be performed as follows, using a 16 — spectrophotometer (e.g. a dual-beam Perkin Elmer Lambda 40 spectrophotometer). 17 pour into a suitable container of poly(methylmethacrylate) (PMMA) allowing the 18 solution to equilibrate to an ambient temperature of 20 to 25 °C. Said container may be 19 arranged to allow light to travel through at least 10 centimetres (or at least 20 centimetres, or at least 50 centimetres) thickness of the solution, for example, e 21 container may have an internal diameter of at least 10 centimetres (or at least 20 22 centimetres, or at least 50 centimetres). Measurement of light transmission may be 23 carried out in the visible part of the light spectrum (e.g. approximately 410 to 800 24 nanometres, e.g. at 580 nanometres), with an identical but empty container in the reference beam of the spectrophotometer. A transmittance measured at any 26 temperature in the range from 20 to 25 °C is usually adequately accurate to reflect the 27 light transmission, but measurement is typically made at 25 °C if more precision is 28 required. ‘Translucent’ or ‘opaque’ as used in this context means an aqueous solution 29 according to the invention (i.e. an aqueous solution comprising sufficient bubbles of a suitable size) with the light transmission as described herein. The test of measuring 31 the light transmission described herein may be referred to as ‘Light Transmission Test’. 32 It will be understood by a skilled worker that the light transmission can be determined 33 by the ‘Light Transmission Test’, or by any other tests that are suitable, provided that 34 — the same measurement conditions are used to measure the aqueous solution with sufficient bubbles and the solution without bubbles (optionally without mixing). 36

DK 2023 70557 A1 15 1 The average (e.g. mean, optionally median) bubble size (e.g. diameter) may be not 2 more than 1 mm, or not more than 500 um, or not more than 100 um, or not more than 3 10 pum, or not more than 1 um, or not more than 0.1 um.

For example, the average (e.g. 4 — mean, optionally median) bubble size (e.g. diameter) may be from 0.1 mm to 0.4 mm, or from 0.1 mm to 0.8 mm.

Diameter refers to the longest length measurable in any 6 dimension in the event the bubble is not a perfect sphere.

The bubble size can be 7 measured by any known and effective techniques, for example, by dynamic light 8 scattering (DLS). Suitable instrument may include but is not limited to, for example, 9 Zetasizer from Malvern Instruments.

The measurement temperature may be between 20 and 25 °C, optionally 25 °C. 11 12 — The sound absorbing barriers and/or reflecting medium may comprise one or more 13 layers of material on or in a solid or flexible wall defining the aquatic enclosure.

The 14 layers may be selected to cause reflection of sound waves back into the aquatic enclosure.

For example, there may be first and third layers with an intermediate second 16 — layer, where the first and third layers are more dense, or heavier than the second layer. 17 18 The sound absorbing and/or reflecting medium may comprise a layer of bubbles 19 around the side of at least some of the aquatic enclosure, formed using one or more air bubble generators and/or bubble curtain generators.

The air bubble generator 21 and/or bubble curtain generator which may comprise an air pump and air stones.

The 22 air bubble generator and/or bubble curtain generator may comprise one or more 23 perforated belts, for example.

The air bubble generator and/or bubble curtain generator 24 — may comprise (e.g. be provided by) a mixing means (e.g. a mixing means as described herein). 26 27 The apparatus may comprise noise cancellation apparatus, comprising one or more 28 sound generators (e.g. acoustic transducers, loudspeakers) arranged to generate 29 cancelling sound in antiphase with the said sound generated and directed into the aquatic enclosure. 31 32 — The aquatic enclosure may be located on a sailing vessel.

The aquatic enclosure may 33 be located on (e.g. form part of) a boat or ship.

The aquatic enclosure may be located 34 on (e.g. form part of) a wellboat.

The aquatic enclosure may be a treatment enclosure located on a wellboat.

The treatment enclosure may have an (e.g. aquatic animal) inlet 36 in fluid communication with an external aquatic environment (i.e. outside the wellboat), 37 optionally through which the aquatic animal may be transported from the external

DK 2023 70557 A1 16 1 aquatic environment into the treatment enclosure. Wellboats are a particularly 2 convenient way of providing an aquatic enclosure. The aquatic enclosure may 3 comprise a tarpaulin. The aquatic enclosure may comprise at least part of a tarpaulin. 4

The wellboat (or tarpaulin) as described herein, may comprise one or more water flow 6 regulators (e.g. a pump or a siphon) configured to (i.e. in use) transport (e.g. pump) 7 — water from the external aquatic environment into the treatment enclosure. The wellboat 8 (or tarpaulin) may comprise one or more water flow regulators (e.g. a pump or a siphon) 9 configured to transport (e.g. pump) water from the treatment enclosure into the external aquatic environment. Transporting (e.g. pumping) water from the external aquatic 11 environment into the treatment enclosure may also comprise transporting the aquatic 12 animal into the treatment enclosure. 13 14 The aquatic enclosure may comprise at least two cages with a conduit therebetween, the conduit being sized and shaped such that aquatic animals (e.g. fish) may travel 16 — between the cages via the conduit. The conduit may comprise one or more dosing 17 — points, for the introduction of hydrogen peroxide and an enzyme for catalytic 18 decomposition of hydrogen peroxide, or for the introduction of an aqueous solution 19 comprising hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide. The conduit may retain water flowing in a direction along the length of the 21 conduit from one of the at least two cages to another of the at least two cages. The 22 conduit may be of a length that is sufficiently great that in the time it takes for an aquatic 23 animal (e.g. fish) to travel across the full length of the conduit, bubbles will have formed 24 — on at least ten percent of the surface of any aquatic ectoparasite on the surface of the said aquatic animal (e.g. fish). The conduit may be at least 50 meters in length, or at 26 least 150 meters in length, or at least 200 meters in length. The conduit may be 27 — between 200 and 300 meters in length. The conduit may be no more than 500 meters 28 inlength. Advantageously, aquatic animals (e.g. fish) travelling between the two cages 29 via the conduit are therefore conveniently exposed to the aqueous solution comprising hydrogen peroxide and the enzyme. The skilled person will appreciate that aquatic 31 — animals (for example fish) may swim with or against the direction of water flow. 32 33 The wellboat (or tarpaulin) (e.g. the treatment enclosure, for example the one or more 34 — water flow regulators) may be provided with aquatic ectoparasite filters configured to restrict the transport of aquatic ectoparasites out of the treatment enclosure when water 36 — is transported (e.g. pumped) from the treatment enclosure to the external aquatic 37 environment.

DK 2023 70557 A1 17 1 2 The aquatic enclosure may have one or more walls.

The aquatic enclosure may be 3 located in an aquatic environment (e.g. in the sea), that is to say the aquatic enclosure 4 — may be surrounded by the aquatic environment (e.g. the sea). An interior of the aquatic enclosure may be separated from (e.g. at least partially isolated from) the surrounding 6 aquatic environment by one or more (e.g. solid) walls.

Alternatively, the aquatic 7 enclosure may be located onshore (i.e. on land, that is to say not in an aquatic 8 environment such as the sea). 9 — The interior of the aquatic enclosure may be in fluid communication with the aquatic 11 environment by way of one or more channels (e.g. pipes). Water may be transported 12 — into and/or out of the aquatic enclosure through the one or more channels (e.g. pipes). 13 — The one or more channels (e.g. pipes) may be provided with aquatic ectoparasite filters 14 configured to inhibit transport of aquatic ectoparasites between the interior of the aquatic enclosure and the aquatic environment. 16 17 The aquatic enclosure may comprise (e.g. be) a treatment channel (e.g. a pipe) 18 provided between (e.g. connecting) first and second aquatic animal enclosures.

Water 19 containing aquatic animals to be treated may be pumped through the treatment channel.

A mixing means may be provided in the treatment channel (e.g. a mixing 21 — means as described herein). 22 23 The apparatus may comprise a nozzle for introducing the aqueous solution into the 24 — aquatic enclosure.

The nozzle may be a venturi nozzle, optionally a dual-input venturi nozzle.

Such a nozzle provides a convenient way of mixing hydrogen peroxide and the 26 enzyme (e.g. with water, e.g. to thereby form an aqueous solution of hydrogen peroxide 27 andthe enzyme) for (e.g. subsequent) introduction into an aquatic enclosure. 28 29 The aqueous solution may comprise 100% to 700% (saturation) dissolved oxygen.

For example, the aqueous solution may comprise 110% to 500% (saturation) dissolved 31 oxygen, or 150% to 300% (saturation) dissolved oxygen.

The aqueous solution may 32 comprise at least 70% dissolved oxygen, or at least 90% (saturation) dissolved oxygen. 33 The aqueous solution may comprise no more than 800% (saturation) dissolved 34 — oxygen.

The aqueous solution may optional comprise more than 400% (saturation) dissolved oxygen, or more than 500% (saturation), or more than 550% (saturation), or 36 — more than 560% (saturation), or more than 600% (saturation). Herein, the percentages 37 of dissolved oxygen comprised in the aqueous solution will be understood as based on

DK 2023 70557 A1 18 1 the level of oxygen saturation of said solution.

Advantageously, where an aqueous 2 solution comprises dissolved oxygen, it is possible to use lower concentrations of 3 — hydrogen peroxide and/or enzyme in order to produce bubbles in the aqueous solution. 4 Furthermore, dissolved oxygen levels of 700% and less are safer for aquatic animals than oxygen levels in excess of 700%. In addition, without wishing to be bound by 6 theory, where oxygen saturation levels are 100% or higher, the decomposition of 7 hydrogen peroxide into water and oxygen leads to the formation of more bubbles than 8 where the oxygen saturation levels are less than 100% because it is less likely that the 9 oxygen will be dissolved in the water rather than forming bubbles (in particular, there is a transition to rapid bubble nucleation in the region of 560% dissolved oxygen). The 11 aqueous solution may comprise at least 300% dissolved oxygen.

This beneficially aids 12 — the formation of a relatively large quantity of relatively small bubbles, such that the 13 — aqueous solution contains a region having a relatively high density of bubbles, as a 14 result.

16 Indeed, the use of an aqueous solution comprising dissolved oxygen as described 17 herein may also be used as an alternative to or additionally to an aqueous solution 18 comprising hydrogen peroxide and an enzyme for the decomposition of hydrogen 19 peroxide.

Accordingly, an aspect of the invention provides an apparatus for aquatic animals, the apparatus comprising an aquatic enclosure for retaining an aquatic 21 — animal, wherein the aquatic enclosure retains an aqueous solution comprising at least 22 70% dissolved oxygen (optionally 100% to 700% dissolved oxygen). 23 24 It will be understood that the skilled person will be able to determine the effective amount of enzyme required for the decomposition of the hydrogen peroxide to take 26 — place. 27 28 The aqueous solution may comprise 10 millilitres of enzyme per 1 litre of water (e.g. of 29 <33% w/w% catalase having a Micrococcus lysodeikticus source, commercially available as Catalase - L12051 from Phillip Harris Manufacturing Limiting, Weston- 31 super-Mare, UK). The aqueous solution may comprise at least 3 millilitres of enzyme 32 — per 1 litre of water, or at least 5 millilitres of enzyme per 1 litre of water.

The aqueous 33 — solution may comprise no more than at least 100 millilitres of enzyme per 1 litre of 34 — water, or no more than 50 millilitres of enzyme per 1 litre of water.

For example, the aqueous solution may comprise between 8 and 20 millilitres of enzyme per 1 litre of 36 — water. 37

DK 2023 70557 A1 19 1 The aqueous solution may optionally comprise 0.5 pg or more of enzyme (e.g. 2 catalase) per 500 mg of hydrogen peroxide, or at least 2 ug, or at least 5 ug, or at least 3 50 pug.

The aqueous solution may optionally comprise no more than 200 pg of enzyme 4 (e.g. catalase) per 500 mg of hydrogen peroxide, or no more than 500 pg, or no more than 1,000 pg.

For example, the aqueous solution may optionally comprise between 2 6 pg and 100 pg of enzyme (e.g. catalase) per 500 mg of hydrogen peroxide.

The 7 aqueous solution may optionally comprise 0.5 ug of enzyme (e.g. catalase) per litre of 8 water, or at least 2 ug, or at least 5 ug, or at least 50 pg.

The aqueous solution may 9 optionally comprise no more than 200 pg of enzyme (e.g. catalase) per litre of water, or no more than 500 ug, or no more than 1,000 pg.

For example, the aqueous solution 11 may optionally comprise between 2 pg and 100 pg of enzyme (e.g. catalase) per litre 12 of water.

The aqueous solution may optionally comprise an excess of enzyme, for 13 — example, the aqueous solution may optionally comprise more catalase than is needed 14 to cause the catalytic decomposition of all of the hydrogen peroxide present in the aqueous solution within 1 minute, or within 5 minutes, or within 10 minutes, or within 16 20 minutes, or within 1 hour. 17 18 — The skilled person will appreciate that the amount of catalase needed will depend on 19 (among other things) the rate of mixing of the aqueous solution.

Accordingly, the aqueous solution may optionally comprise 0.5 mg or more of enzyme (e.g. catalase) 21 per 500 mg of hydrogen peroxide, or at least 2 mg, or at least 5 mg, or at least 50 mg. 22 — The aqueous solution may optionally comprise no more than 200 mg of enzyme (e.g. 23 catalase) per 500 mg of hydrogen peroxide, or no more than 500 mg, or no more than 24 — 1,000 mg.

For example, the aqueous solution may optionally comprise between 2 mg and 100 mg of enzyme (e.g. catalase) per 500 mg of hydrogen peroxide.

The aqueous 26 — solution optionally may comprise 0.5 mg of enzyme (e.g. catalase) per litre of water, or 27 atleast 2 mg, or at least 5 mg, or at least 50 mg.

The aqueous solution may optionally 28 comprise no more than 200 mg of enzyme (e.g. catalase) per litre of water, or no more 29 than 500 mg, or no more than 1,000 mg.

For example, the aqueous solution may optionally comprise between 2 mg and 100 mg of enzyme (e.g. catalase) per litre of 31 — water.

The aqueous solution may optionally comprise at least 10 mg/L, 50 mg/L, 200 32 mg/L, 300 mg/L 400 mg/L, or 500 mg/L enzyme (e.g. catalase) per litre of water.

The 33 aqueous solution may optionally comprise at least 10 mg/L, 50 mg/L, 200 mg/L, 300 34 — mg/L 400 mg/L, or 500 mg/L enzyme (e.g. catalase) per 500 mg of hydrogen peroxide.

36 — The apparatus may comprise mixing means (e.g. one or more mixers, stirrers, paddles, 37 — bubble streams, bubble curtains, bubble curtain generators, means for directing sound

DK 2023 70557 A1 20 1 — waves, transducers, or other suitable mixing means) configured to cause motion of the 2 aqueous solution. The method may comprise mixing the aqueous solution. The method 3 may comprise introducing the enzyme (e.g. catalase) at the top of the aquatic 4 — enclosure, e.g. at a surface of the aqueous solution. 6 The aqueous solution may comprise between 1 kU and 100 kU of enzyme (e.g. 7 catalase) per 1 litre of water. The aqueous solution may comprise at least 5 kU of 8 enzyme (e.g. catalase) per 1 litre of water, or at least 10 kU, or at least 20 kU. The 9 aqueous solution may comprise no more than 150 kU of enzyme (e.g. catalase) per 1 litre of water, or no more than 170 kU, or no more than 200 kU, or no more than 500 11 kU. It will be understood that 1 U (1 enzyme unit) of enzyme (e.g. catalase) is the 12 amount which decomposes 1 umole of hydrogen peroxide per minute at pH 7.0 and 25 13 °C (e.g. while the hydrogen peroxide concentration falls from 10.3 mM to 9.2 mM), and 14 that 1 kU is 1,000 times this amount. 16 Advantageously, the use of an aqueous solution comprising hydrogen peroxide and an 17 enzyme for catalytic decomposition of hydrogen peroxide means that more bubbles 18 are formed (and/or or bubbles are formed more quickly) than is the case where an 19 aqueous solution comprises hydrogen peroxide and no (or very little of) such enzyme.

The amount of bubbles produced in total depends on the concentration of hydrogen 21 peroxide (e.g. the quantity of hydrogen peroxide) used. As bubbles preferentially form 22 on hydrophobic surfaces, such as the waxy surfaces of ectoparasites, and bubble 23 formation on ectoparasites leads to the parasites being detached from the aquatic 24 — animal host (and often injured and/or killed), the inventors have surprisingly found that itis possible to use lower concentrations of hydrogen peroxide than have been used 26 — previously. The use of an enzyme means that the concentrations can be reduced 27 — further, while an effect is still obtained. In addition, because the enzyme increases the 28 speed at which bubbles are produced, it is possible to detach ectoparasites (and/or 29 injure or kill) ectoparasites in shorter time periods, meaning that the aquatic animal does not have to be exposed to the aqueous solution for as long. 31 32 It has been surprisingly found that the formation of bubbles on the surfaces of 33 ectoparasites disturbs the ectoparasites. The ectoparasites have been observed to 34 — move (e.g. swimming in tight circles) before re-settling near their original position. In moving in this way the ectoparasites can sometimes dislodge some of the bubbles on 36 their surfaces, however sometimes they cannot, making it more likely that they are 37 unable to stay with the aquatic animal host. As such, formation of bubbles with an

DK 2023 70557 A1 21 1 aqueous solution comprising hydrogen peroxide and an enzyme for catalytic 2 decomposition of hydrogen peroxide is a convenient way to dislodge parasites from 3 aquatic animals. It has been found that the bubbles are effective at removing female 4 ectoparasites from the aquatic animal. To expand on this, it has been found that when — an ectoparasite is buoyantly removed from an aquatic animal as the result of bubbles 6 on the ectoparasite, the ectoparasite swims in small circles, apparently in an attempt 7 to reattach itself to the aquatic animal. This swimming motion tends to cause some of 8 the bubbles to become detached. However, as female ectoparasites are typically larger 9 than male ectoparasites, the circles in which they swim are also relatively larger. This — has the result that fewer bubbles become detached from female ectoparasites as they 11 swim, meaning that they are affected by increased buoyancy due to the bubbles for a 12 — longer period, which in turn causes them to move further from the aquatic animal. As a 13 — result, the invention is particularly effective at removing female ectoparasites from 14 aquatic animals. 16 The enzyme may be peroxidase. The enzyme may be catalase. The catalase may be 17 sourced from Micrococcus lysodeikticus. However, the skilled person will appreciate 18 that other sources of catalase may be used. For example, the catalase may be sourced 19 from Aspergillus niger. The catalase may be sourced from Micrococcus luteus. The catalase may be sourced from Ipomoea batatas. 21 22 The apparatus may be apparatus for use in a method of reducing ectoparasitic 23 infestation of an aquatic animal, wherein the method comprises: 24 - providing an aquatic enclosure for retaining an aquatic animal - combining hydrogen peroxide and an enzyme for catalytic decomposition of 26 hydrogen peroxide with water to thereby form an aqueous solution (e.g. 27 comprising hydrogen peroxide at a concentration between 500 mg/L and 1,500 28 mg/L, inclusive) 29 - introducing the aqueous solution into the aquatic enclosure; - introducing one or more aquatic animals into the aquatic enclosure to thereby 31 expose the aquatic animal to the aqueous solution; 32 - keeping the one or more aquatic animals in the aquatic enclosure (e.g. for at 33 least 10 seconds and for less than 30 minutes). 34

The hydrogen peroxide and enzyme for catalytic decomposition of hydrogen peroxide 36 may be introduced into the aquatic enclosure and subsequently mixed to form an 37 aqueous solution of hydrogen peroxide. The method may comprise first introducing the

DK 2023 70557 A1 22 1 hydrogen peroxide and enzyme for catalytic decomposition of hydrogen peroxide into 2 the aquatic enclosure and subsequently mixing the hydrogen peroxide, enzyme, and 3 — water, to form an aqueous solution of hydrogen peroxide. 4

The apparatus may comprise one or more cages. The method may comprise 6 combining hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen 7 peroxide with water to form an aqueous solution comprising hydrogen peroxide at a 8 concentration between 500 mg/L and 1,500 mg/L, in one or more cages. The method 9 may comprise combining hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide with water to form an aqueous solution 11 comprising hydrogen peroxide at a concentration between 500 mg/L and 1,500 mg/L, 12 outside of the one or more cages and wherein the method further comprises 13 introducing the aqueous solution into the one or more cages at the surface of the or 14 — each cage. 16 The method may comprise keeping the aquatic animals in the aquatic enclosure for at 17 least 30 seconds, or for at least 2 minutes, or for at least 5 minutes. The method may 18 comprise keeping the aquatic animals in the aquatic enclosure for up to 15 minutes, or 19 upto 20 minutes, or up to 25 minutes. For example, the method may comprise keeping the aquatic animals in the aquatic enclosure for between 3 and 25 minutes, or between 21 4 and 22 minutes, or between 5 and 20 minutes. The method may comprise exposing 22 the aquatic animals to the aqueous solution for an exposure period of at least 30 23 seconds, or for at least 2 minutes, or for at least 5 minutes, optionally for up to 15 24 — minutes, or up to 20 minutes, or up to 25 minutes. For example, the method may comprise exposing the aquatic animals to the aqueous solution for an exposure period 26 — of between 3 and 25 minutes, or between 4 and 22 minutes, or between 5 and 20 27 — minutes. The method may comprise mixing (e.g. causing the mixing means to mix) the 28 aqueous solution during (e.g. at least 30% of, e.g. at least 50% of, optionally at least 29 75% of, optionally all of) the exposure period. 31 The one or more cages may comprise at least one dosing point configured to allow 32 introduction of hydrogen peroxide and an enzyme for catalytic decomposition of 33 — hydrogen peroxide (optionally an aqueous solution comprising hydrogen peroxide and 34 such an enzyme) into the said cage. The at least one dosing point may be at the top of the said cage, for example at a water surface. The at least one dosing point may be at 36 the base of the said cage. The one or more cages may comprise a mixing means (e.g. 37 a mixer, aerator, bubble curtain generator, bubble curtain, means for directing sound

DK 2023 70557 A1 23 1 waves, transducer, etc.) and the mixing means may be located at the base of the cage. 2 For example, the one or more cages may comprise an aerator at the base of the cage, 3 wherein the aerator is configured to introduce a stream of bubbles into the cage from 4 — below.

Advantageously, such a mixing means (e.g. a stream of bubbles) causes mixing of the aqueous solution. 6 7 According to a further aspect of the invention there is provided a method of reducing 8 ectoparasitic infestation of an aquatic animal, wherein the method comprises: 9 - providing an aquatic enclosure for retaining an aquatic animal - combining hydrogen peroxide and an enzyme for catalytic decomposition of 11 hydrogen peroxide with water to thereby form an aqueous solution (e.g. 12 comprising hydrogen peroxide at a concentration between 500 mg/L and 1,500 13 mg/L, inclusive) (e.g. and said enzyme); 14 - introducing the aqueous solution into the aquatic enclosure; - introducing one or more aquatic animals into the aquatic enclosure to thereby 16 expose the aquatic animal to the aqueous solution; 17 - keeping the one or more aquatic animals in the aquatic enclosure (e.g. for at 18 least 10 seconds and for less than (e.g. no more than) 30 minutes). 19 The method may comprise combining hydrogen peroxide and the said enzyme in a nozzle.

The method may comprise causing a mixing means to mix the aqueous 21 — solution. 22 23 According to an aspect of the invention there is provided a method of reducing 24 — ectoparasitic infestation on aquatic animals, wherein the said method comprises administering an aqueous solution comprising 100% to 700% dissolved oxygen to an 26 — aquatic enclosure comprising aquatic animals.

According to an aspect of the invention 27 there is provided a method of reducing ectoparasitic infestation on aquatic animals, 28 wherein said method comprises administering hydrogen peroxide and an enzyme for 29 catalytic decomposition of hydrogen peroxide to an aquatic enclosure comprising aquatic animals. 31 32 The method may be a method of preparing an apparatus for aquatic animals, 33 comprising the steps of: 34 providing an aquatic enclosure for retaining an aquatic animal; combining hydrogen peroxide and an enzyme for catalytic decomposition of 36 — hydrogen peroxide with water to thereby form an aqueous solution (e.g. comprising 37 — hydrogen peroxide at a concentration from 500 mg/L to 1500 mg/L); and

DK 2023 70557 A1 24 1 introducing the aqueous solution into the aquatic enclosure. 2 3 The method may be a method of use of the apparatus to disturb an aquatic 4 — ectoparasite. The method may be a method of use of the apparatus to injure an aquatic — ectoparasite. The method may be a use of the apparatus to kill an aquatic ectoparasite. 6 The apparatus may be used to disturb an aquatic ectoparasite. The apparatus may be 7 used to injure an aquatic ectoparasite. The apparatus may be used to kill an aquatic 8 ectoparasites. The characteristics of said apparatus may be as described herein. 9

Combining hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen 11 peroxide with water may take place substantially at the same time as introducing the 12 — aqueous solution into the aquatic enclosure. The hydrogen peroxide and enzyme for 13 catalytic decomposition of hydrogen peroxide may be introduced into the aquatic 14 enclosure and subsequently mixed to form an aqueous solution of hydrogen peroxide.

The method may comprise first introducing the hydrogen peroxide and enzyme for 16 catalytic decomposition of hydrogen peroxide into the aquatic enclosure and 17 subsequently mixing the hydrogen peroxide, enzyme, and water, to form an aqueous 18 — solution of hydrogen peroxide. 19

The method may comprise keeping the aquatic animals in the aquatic enclosure for at 21 least 1 minute, or for at least 2 minutes, or for at least 5 minutes. The method may 22 comprise keeping the aquatic animals in the aquatic enclosure for up to 20 minutes, or 23 — upto30 minutes, or up to 45 minutes. 24

The method may comprise mixing the aqueous solution in the aquatic enclosure. 26 Mixing the aqueous solution increases the likelihood of interactions taking place 27 between the hydrogen peroxide and the enzyme in the aquatic solution, thereby 28 increasing the rate at which bubbles are formed (i.e. due to the decomposition of 29 hydrogen peroxide by the enzyme) as well as increasing the quantity of bubbles that are formed. 31 32 — The method may comprise varying the concentration (e.g. mass per litre) of hydrogen 33 peroxide in dependence on a measured temperature. The method may comprise 34 — varying the concentration (e.g. mass per litre) of the enzyme in dependence on a measured temperature. The method may comprise varying the speed of mixing of the 36 aqueous solution in the aquatic enclosure in dependence on a measured temperature. 37

DK 2023 70557 A1 25

1 The method may comprise exposing the aquatic animal to sound waves.

The method

2 may comprise exposing the aquatic animal to ultrasound waves.

3

4 — The method extends in a further aspect to a method of operating an apparatus, the apparatus comprising an aquatic enclosure comprising an aqueous solution of

6 hydrogen peroxide, the solution comprising bubbles, the bubbles comprising oxygen

7 (typically being predominantly of oxygen).

8

9 It may be that exposing the aquatic animal to sound waves comprises generating said — sound waves within the aqueous solution (e.g. within the aquatic enclosure). It may be 11 — that exposing the aquatic animal to the sound waves comprises directing said sound 12 — waves at the aquatic ectoparasite.

It may be that the aquatic ectoparasite is provided 13 inside an aquatic enclosure and that exposing the aquatic ectoparasite to the sound 14 waves comprises directing said sound waves into the aquatic enclosure.

16 The method may be a non-therapeutic method of improving the appearance, meat 17 quality, meat quantity and/or growth rate of an aquatic animal.

The method may be a 18 method of reducing aquatic ectoparasitic infestation (e.g. ectoparasitosis) on an 19 aquatic animal.

21 The method may comprise a bubble regulation phase in which the frequency spectrum 22 (and optionally power) of the sound waves is controlled, e.g. to cause bubble growth 23 and/or coalescence.

It may be that the frequency spectrum (and optionally power) of 24 — the sound waves is controlled to cause bubble resonance (e.g. as bubbles change in size). 26 27 The bubble regulation phase may comprise or consist of one or more descending 28 frequency phases during which the centre and/or peak frequency of the sound is 29 reduced (optionally monotonically and optionally progressively although it may for example be reduced through a plurality of, e.g. two, three, or more intermediate steps). 31 — The bubble regulation phase may comprise a plurality of (e.g. consecutive) descending 32 frequency phases.

The bubble regulation phase may comprise at least two, at least 33 three, or at least four descending frequency phases.

It may be that between 34 consecutive descending frequency phases the (centre and/or peak) frequency is (e.g. instantaneously) increased again, e.g. to the initial frequency (optionally to a different 36 frequency). The decreasing frequency favours the creation of large bubbles, for 37 example through smaller bubbles merging.

Without wishing to be constrained by

DK 2023 70557 A1 26 1 theory, the inventor believes this to arise for a number of reasons, including because 2 sound waves (acoustic field) favour the creation or and stability of bubbles having a 3 diameter giving a resonant frequency which is similar to the centre (and/or peak) 4 — frequency of the sound waves and so as the frequency of the sound waves is reduced, the bubbles are encouraged to combine and grow. Other possible factors include fish 6 mucus acting as a surfactant which helps to drive bubble formation over time, and the 7 sound waves then causing bubbles to coalesce. 8 9 Furthermore, the inventor has found that there is a synergistic effect associated with — using sound waves both to regulate and stabilise (or to cause coalescence of bubbles) 11 — andto mix the aqueous solution, at least because such mixing of the aqueous solution 12 — also enhances the rate at which hydrogen peroxide is broken down by the enzyme to 13 thereby form bubbles. 14

In some conditions, and at some frequencies, it has been observed that a plurality of 16 smaller bubbles in close proximity to each other can behave in a similar way to one 17 larger bubble when excited by sound waves. It may be that during some or all of the 18 bubble regulation phase acoustic pressure is regulated, e.g. is increased and/or 19 decreased. Regulating the acoustic pressure can help to encourage the coalescence of bubbles. 21 22 The method may comprise mixing (e.g. causing the mixing means to mix) the aqueous 23 — solution to thereby cause coalescence of bubbles. 24

It may be that the sound is substantially monotonic although in practice it may have a 26 significant bandwidth and so we refer to the centre (and/or peak) frequency of the 27 sound. 28 29 The bubble regulation phase(s) may have a duration of at least 1 second and/or the one or more descending frequency phases may have a (e.g. combined) duration of at 31 least one second. The bubble regulation phase(s) may have a duration of less than 10 32 seconds and/or the one or more descending frequency phases may have a (e.g. 33 — combined) duration of at least 10 seconds. Preferably, the bubble regulation phase(s) 34 may have a duration of at least 30 seconds and/or the one or more descending frequency phases may have a (e.g. combined) duration of at least 30 seconds, 36 however in some examples the bubble regulation phase(s) may have a duration of at 37 least 1 minute or at least 2 minutes and/or the one or more descending frequency

DK 2023 70557 A1 27 1 phases may have a (e.g. combined) duration of at least 1 minutes or at least 2 minutes. 2 Preferably, the bubble regulation phase(s) may have a duration of less than 20 3 — minutes, preferably less than 15 minutes, or less than 12 minutes and/or the one or 4 — more descending frequency phases may have a (e.g. combined) duration of less than 20 minutes, preferably less than 15 minutes or less than 12 minutes. The frequency 6 may be reduced from greater than 10 kHz to less than 5 kHz, for example from about 7 — 20 kHz to 3 kHz. Preferably, the peak and/or centre frequency will be reduced from 10 8 > kHz+2kHzto3kHz+ 1kHz, or from 6 kHz + 2 kHz to 3 kHz + 1 kHz. In some examples 9 the rate of frequency reduction may be non-linear. In some examples the amplitude of sound generated may alternatively or additionally be changed, in which case the rate 11 of change of sound amplitude may be linear or may be non-linear. The rate of peak 12 — and/or centre frequency change during the (e.g. each of the) one or more descending 13 frequency phases may for example be in the range of 0.5 to 2 kHzs. Optionally, after 14 each descending frequency phase the frequency is instantaneously returned to the higher frequency that preceded the descending frequency phase before a subsequent 16 descending frequency phase begins. 17 18 During the or each of the one or more descending frequency phases, the peak and/or 19 centre frequency of sound that is generated may decrease in frequency by at least 25% or atleast 40%, for example. The peak and/or centre frequency may decrease by less 21 than 90%. A decrease in peak and/or centre frequency of around 50%, for example, 22 — will support an approximate doubling in the diameter of bubbles (because the resonant 23 frequency is inversely proportional to bubble diameter). 24

It may be that the bubble regulation phase(s) has a duration of at least 15 seconds or 26 atleast 30 seconds. It may be that the one or more descending frequency phases has 27 a(e.g. combined) duration of at least 15 seconds or at least 30 seconds. It may be that 28 the one or more descending frequency phases has a (e.g. combined) duration of at 29 least 5 seconds or that the bubble regulation phase(s) has a duration of at least 5 seconds. It may be that the peak and/or centre frequency of sound that is generated 31 — during the or each of the one or more descending frequency phases is reduced at a 32 rate of at least 0.05 kHz/s, or preferably at least 0.1 kHz/s, or at least 0.2 kHz/s. 33 34 This may result in bubbles becoming so large they detach from fish and/or ectoparasites and provide a time for new bubble to form (from the decomposition of 36 — hydrogen peroxide to oxygen). 37

DK 2023 70557 A1 28 1 The method may comprise a plurality of said descending frequency phases 2 interspersed with said intermission phases. The method may comprise alternating 3 descending frequency phases and intermission phases, for example cyclically. The 4 — method may comprise a plurality of descending frequency phases, followed by an interval phase, followed by a further plurality of descending frequency phases (and this 6 may be repeated). The plurality of said descending frequency phases may be the same 7 — as each other although this is not essential. The plurality of intermission phases may 8 be the same as each other. However, it may be that the duration of the intermission 9 phases is varied. It may be that bubbles form during the intermission phases and grow — and/or are combined during the or each of the one or more descending frequency 11 phases (or the plurality of descending frequency phases), facilitated at least in part by 12 — the descending frequency sound. 13 14 In some embodiments, the method comprises varying the frequency spectrum (and optionally power) to cause collapse of the bubbles, e.g. to cause the bubbles to grow, 16 — and then to collapse in response to sound waves, e.g. in response to a change in the 17 — frequency spectrum (and optionally the power) of the sound waves. This enables the 18 time when the bubbles collapse to be controlled. 19

Therefore, it may be that the method comprises a bubble regulation phase (in which 21 the frequency spectrum of the sound waves is controlled to cause bubble growth and/or 22 coalescence) and a subsequent bubble collapse phase, in which the frequency 23 spectrum (and optionally power) of the sound waves is controlled to cause the collapse 24 — of bubbles. The bubble regulation phase may comprise a preliminary bubble collapse phase, prior to the one or more descending frequency phases. 26 27 It may be that the bubble collapse phase is an asymmetric bubble collapse phase in 28 which the frequency spectrum (and optionally power) of the sound waves is controlled 29 to cause the asymmetric collapse of bubbles. 31 By providing a separate bubble regulation phase and bubble collapse phase, the 32 bubbles can be controlled to a desired size range, chosen to be effective during 33 subsequent bubble collapse, at a moderate or low power before being caused to 34 collapse at a relatively high power (e.g. higher than at any time during the bubble regulation phase) for a short period of time (e.g. less than the duration of the bubble 36 — regulation phase, or less than 50% of, less than 25% or less than 10% or even less

DK 2023 70557 A1 29 1 — than5% of the duration of the bubble regulation phase). Thus, the highest power sound 2 > waves are generated for only a relatively short period of time. 3 4 — The bubble collapse phase may have a duration of less than 1 s, or less than 100 ms, — orless than 10 ms, or less than 1 ms.

However, the bubble regulation phase may have 6 — aduration of at least 1 seconds, at least 3 seconds, at least 10 seconds or at least 30 7 seconds (for example to allow time for bubbles to migrate and coalesce) and/or the 8 period of time between bubble collapse phases may be at least 10 seconds, at least 9 30 seconds or at least a minute.

The bubble collapse phase may take place for less than 5% or less than 2% or less than 1% of the time.

Thus, again the highest power 11 sound waves are generated during only a relatively small fraction of the treatment time. 12 13 — The variation in the frequency (and optionally power) of the sound waves during the 14 bubble regulation phase may be selected to favour the formation and maintenance of bubbles within a predefined size range.

The frequency (and optionally power) of the 16 sound waves during the bubble collapse phase may be selected to cause the collapse 17 e.g. asymmetrical collapse) of bubbles within the said predefined size range. 18 19 The mean power of the sound waves in the bubble collapse phase may be at least double, or at least 5 times or at least 10 times, higher than the mean power during the 21 bubble regulation phase.

The peak power of the sound waves in the bubble collapse 22 phase may be at least double, or at least 5 times or at least 10 times, higher than the 23 mean power during the bubble regulation phase.

The power of the sound during the 24 — bubble collapse phase may be selected to create an acoustic pressure of at least 50 kPa. 26 27 It may be that the peak power of the sound waves in the bubble collapse phase is 28 sufficient to cause bubble non-symmetric collapse in the aquatic enclosure but not 29 sufficient to cause cavitation (which would increase damage to the aquatic creature).

31 — The duration of the bubble regulation phase may be in the range 1 to 60 seconds, for 32 example 5 to 20 seconds.

The duration of the bubble collapse phase may be less than 33 aminute.

The duration of the bubble collapse phase may be less than 20% or less than 34 — 10% of the duration of the bubble regulation phase.

The duration of the bubble collapse phase may be less than 20% or less than 10% of time between bubble collapse phases. 36

DK 2023 70557 A1 30 1 The power of the sound waves which are generated and directed at the bubbles is 2 controlled to thereby regulate the power of the sound waves in a target volume, where 3 there are both the bubbles and the aquatic ectoparasite. 4

The sound waves generated during the bubble collapse phase are preferably selected 6 to cause liquid jetting (during bubble collapse). This is a kind of bubble asymmetrical 7 collapse. It is known that when gas bubbles collapse adjacent a surface they may form 8 ajet of liquid towards the surface. In bubble jetting, the bubble collapses in a manner 9 where the side furthest away from an adjacent surface/target moves quickest and collapses through the bubble during the positive acoustic pressure phase. This 11 frequently causes a fast-moving jet of water which can puncture most biological 12 surfaces including the surface of ectoparasites. The person skilled in the art can 13 determine the necessary power and frequency of sound waves to cause jetting and 14 can observe whether this is occurring by optical microscopy. 16 During the bubble collapse phase the acoustic waves may have an intensity such that 17 — their acoustic pressure is greater than the Blake threshold pressure of the majority, by 18 — volume, of bubbles. 19

By the Blake threshold pressure, we refer to the bubble forcing pressure above which 21 bubbles will grow quasistatically without bound. The Blake threshold pressure is for 22 example referred to in Akhatov et al. I, Gumerov, N., Ohl, C.D., Parlitz, U. and 23 Lauterborn, W, “The Role of Surface Tension in Stable Single-Bubble 24 Sonoluminescence” (Physics Review Letters, 78(2), 227-230, 1997) and Blake, F.G. “The Onset of Cavitation on Liquids” (Technical Memo. 12, Acoustic Research 26 Laboratory, Cambridge, MA, Harvard University, 1949) and Louisnard, O. and 2/ — Gonzalez-Garcia, J. (H. Feng et al., eds), "Acoustic Cavitation” in Ultrasound 28 Technologies for Food and Bioprocessing, DOI | 10.1007/978-1-4419-7472-3 2, which 29 can be calculated by the person skilled in the art and which is given, for an air bubble in water in ambient conditions (0 = 0.0725 N.m" Pv,cq = 2,000 Pa, po = 100 kPa) by: 31 32 På" = Po — Py, eg + Po (5) ; 27 1+ ag 33

DK 2023 70557 A1 31 1 Where p?" js the Blake threshold pressure, p, is liquid pressure, is vapour equilibrium 2 saturation pressure and a, is dimensionless Laplace tension 20 /py Ry (where R, is the 3 ambient radius of the bubble). 4

Prior to the bubble collapse phase and/or during the bubble regulation phase and/or 6 between the preliminary bubble collapse phase and the bubble collapse phase, the 7 — frequency of the sound waves may be controlled to not exceed a frequency which will 8 cause (e.g. asymmetrical) collapse of bubbles but during the bubble collapse phase 9 the frequency of the sound exceeds the frequency which will cause (e.g. asymmetrical) collapse of bubbles and thereby causes (e.g. asymmetrical) collapse of bubbles. 11 12 The bubble regulation phase and bubble collapse phase may be repeated with different 13 variations in the frequency (e.g. intensity) of sound waves with time during the bubble 14 regulation and bubble collapse phases. This enables the procedure to target parasites with different properties. 16 17 — The method may comprise an intermission phase subsequent to the or each of the one 18 — or more descending frequency phases, during which intermission phase sound waves 19 — which cause oscillation of the bubbles are restricted in intensity (for example stopped).

There may be an intermission phase after the bubble regulation phase and before 21 bubble collapse phase. Generally, the bubbles present at the end of the one or more 22 descending frequency phases will have a diameter such that they have a resonant 23 frequency similar to the frequency of the sound at the end of the one or more 24 descending frequency phases (optionally at the end of each of the one or more descending frequency phases). This sound is then restricted or stopped. 26 27 During the intermission phase some bubbles will detach from the aquatic ectoparasite, 28 due to flow of aqueous solution or buoyancy. We have found that by having a bubble 29 regulation phase and a subsequent bubble collapse phase, with an intermission phase therebetween, to allow some distance to develop between some of the bubbles and 31 the aquatic ectoparasite, damage to the aquatic ectoparasite during the bubble 32 collapse phase can be increased. Bubbles may detach from the ectoparasite once they 33 reach a size where the forces arising from the buoyancy of the bubbles exceeds 34 retentive forces. Bubbles may also become detached due to the movement of the — ectoparasite through the water, especially where the ectoparasite is attached to the 36 surface of a fish or other aquatic animal which is swimming through water. In some 37 examples the frequency and/or amplitude of sound generated during the bubble

DK 2023 70557 A1 32 1 regulation phase may optionally be selected such that it corresponds to the frequency 2 and/or amplitude necessary to cause the oscillation of bubbles of a predetermined size 3 (and/or to prevent bubbles of a said predetermined size from being lost due to 4 buoyancy). 6 The duration of the intermission phase may for example be at least 0.1 ms, at least 7 0.25 ms, at least 10 ms, at least 100 ms, or at least 1 second. The duration of the 8 intermission phase may be less than 2 minutes, less than 1 minute, or less than 30 9 seconds. However, in some conditions it may be less than 1 second, or less than 0.5 seconds, maybe even less than 30 ms, for example in fast flowing water (e.g. when 11 water is flowing relative to the aquatic animal at 0.5- 2 m/s and it is desired for a bubble 12 of e.g. 1 mm radius to move 0.2 - 1 mm). 13 14 It may be that the frequency (and optionally power) of the sound waves is controlled to promote bubble coalescence immediately prior to the bubble collapse phase. For 16 — example, two bubbles with a diameter of 2 mm might be subject to ultrasound at 3.3 17 — kHz, which drives them to coalesce, forming a bubble with a diameter of 2.52 mm which 18 is then subject to sound waves at 2.6 kHz to cause collapse. 19

The method may comprise retaining bubbles close to or in contact with the surface of 21 — an ectoparasite by controlling the sound waves using Bjerknes forces. It may be that 22 for at least some time during the bubble regulation phase, including potentially during 23 the intermission phase, the frequency (and optionally power) of the sound waves is 24 selected to cause the detached bubble to remain close to the surface of the — ectoparasite by Bjerknes forces. It may be that prior to the bubble collapse phase, the 26 frequency (and optionally power) of the sound waves is selected to cause the detached 27 bubble to remain close to the surface of the ectoparasite by Bjerknes forces. This can 28 enable the build-up of a significant volume of bubble(s) close to or attached to the 29 surface of the ectoparasite. This is significant because asymmetrical bubble collapse (such as jetting) causes significant damage to structures within 2 bubble diameters of 31 — the surface of the bubble and so ideally bubbles are retained close to the surface of 32 the ectoparasite. 33 34 Asymmetrical bubble collapse proximate a surface can lead to jetting specifically towards the surface, which in this case would be a surface of the aquatic ectoparasite. 36 — Some of the bubbles may however remain attached to the surface, or within the interior 37 of the aquatic ectoparasite.

DK 2023 70557 A1 33 1 2 Bubble jetting occurs preferentially when the bubbles are spaced apart from the 3 — adjacent surface but there is a ratio of unforced bubble radius to bubble centre to 4 — adjacent surface distance of less than 1:5, or less than 1:2.5 or less than 1:1.5, e.g.

T1:1.1to 1:1.3, with about 1:1.2 giving good results. By unforced bubble radius, we refer 6 to the radius which the bubble would have if not subject to ultrasound (which leads to 7 oscillations etc). 8 9 The duration of the intermission phase may be selected so that for at least 25% of or atleast the majority by volume of bubbles which have detached from or are located on 11 — the surface of the aquatic ectoparasite, the distance between the bubble and the 12 — surface of the aquatic ectoparasite is less than double the diameter of the respective 13 bubble. Bubbles which are further than double their diameter from the surface of the 14 aquatic ectoparasite will be less effective. 16 During the intermission phase it may be that no sound waves are generated and 17 directed to the aquatic ectoparasite (e.g. from a sound source). Alternatively, it may be 18 that relatively low power, relatively low frequency sound waves are generated during 19 the intermission phase to maintain bubbles. In this case, the power and frequency of the sound waves may be each lower than the power and frequency of the sound waves 21 atthe end of the bubble regulation phase. To this effect, the bubbles may be stimulated 22 with acoustic waves having an acoustic pressure which is less than the Blake threshold 23 — pressure. They continue to oscillate but are in a stable condition. They can still migrate 24 by the effects of Bjerknes forces. A balance can therefore be struck between the driving acoustic force causing the bubbles to migrate towards the ectoparasite and 26 detachment due to buoyancy and/or fluid flow. 27 28 It may be that prior to the bubble collapse phase, sound waves are generated at a 29 frequency which is sufficiently low to cause collapse of bubbles in excess of a size threshold, or to cause such bubbles to become detached. This can be used to remove 31 excessively large bubbles. 32 33 Nevertheless, it may be that there is not a bubble collapse phase after the one or more 34 descending frequency phases. We have found that the oscillation of bubbles which have been grown and/or coalesced in the one or more descending frequency phases 36 — can cause substantial damage to aquatic ectoparasites. We hypothesize that this is 37 due to the very substantial local forces caused by oscillating bubbles.

DK 2023 70557 A1 34 1 2 The bubble regulation phase may comprise a preliminary bubble collapse phase, prior 3 tothe one or more descending frequency phases.

During the bubble regulation phase 4 — sound may be generated at a lower frequency and lower power than during the bubble collapse phase.

Bubble collapse during the preliminary bubble collapse phase may be 6 predominantly surface bubble collapse.

This has the effect of collapsing bubbles which 7 — are already present, especially on fish.

This has the benefit of removing bubbles which 8 are larger than a threshold size, so that the range of bubble sizes at the beginning of a 9 descending frequency phase (e.g. a one of the one or more descending frequency phases) is more defined and/or removing bubbles left after a previous cycle (of a 11 bubble regulation phase followed by a bubble collapse phase). This also has the 12 benefit of cleaning fish - causing bubbles to collapse on the surface has been found to 13 — clean fish.

During the preliminary bubble collapse phase sound waves may be selected 14 to target relatively flat bubbles.

Partially flattened (e.g. oblate spheroid) bubbles can be formed in hydrophilic surface layers of fish etc. as opposed to more spherical bubbles 16 — being formed on the waxy hydrophobic surface of the sea lice.

It has been surprisingly 17 — found that, in general, bubbles form preferentially on the surface of the lice rather than 18 — on the surface of fish.

Their oscillations can be targeted by considering their thickness 19 along their axis and selecting sound waves with a wavelength which stimulates oscillations of bubbles having that thickness. 21 22 — The preliminary bubble collapse phase may have a duration of less than 10 seconds 23 — orless than 5 seconds.

The preliminary bubble collapse phase may have a duration of 24 less than 1 second.

26 There may be a waiting phase prior to the bubble regulation phase.

The waiting phase 27 may be after the ectoparasite was brought into contact with the aqueous solution of 28 hydrogen peroxide or after the bubble asymmetric collapse phase of a previous cycle. 29 The waiting phase provides time for bubbles to start to be formed and grow, through the decomposition of hydrogen peroxide to form oxygen. 31 32 The waiting phase may have a duration of at least 3 seconds, or at least 10 seconds 33 — or atleast 30 seconds or at least 45 seconds or at least 1 minute or at least 2 minutes. 34 The waiting phase may be shorter than 1 minutes, or shorter than 5 minutes, or shorter than 3 minutes, for example. 36

DK 2023 70557 A1 35 1 The formation of bubbles can be temperature dependent, for example hydrogen 2 peroxide may be converted to oxygen (e.g. by action of an enzyme for catalytic 3 decomposition of hydrogen peroxide) at a higher rate at higher temperature. It may be 4 — that the method comprises determining (e.g. measuring) the temperature of the aqueous solution and varying one or more of: the duration of the bubble regulation 6 phase, the frequency (and optionally power) during the bubble regulation phase, the 7 duration of the bubble collapse phase, the frequency (and optionally power) during the 8 bubble collapse phase, the duration of the intermission phase, the duration of the 9 waiting phase, the duration of the one or more descending frequency phases, the frequency (and optionally power) of sound waves and the variation of that with time 11 during the one or more descending frequency phases, the duration of the preliminary 12 — bubble collapse phase, the frequency (and optionally power) of sound waves during 13 — the preliminary bubble collapse phase. 14 — The bubble regulation phase and bubble collapse phase may be repeated. 16 17 The bubble regulation phase and bubble collapse phase may be repeated with different 18 variations in the frequency (e.g. intensity) of sound waves with time during the bubble 19 regulation and bubble collapse phases. This enables the procedure to target parasites with different properties. The difference in centre (and/or peak) frequency of the sound 21 waves in the bubble collapse phase between cycles may vary by more than 10%, for 22 example. 23 24 The aqueous solution may comprise one or more (e.g. dissolved) salts. The aqueous solution may comprise sodium chloride. The aqueous solution may comprise 26 potassium chloride. 27 28 According to a further aspect of the invention, there is provided apparatus for aquatic 29 animals, the apparatus comprising an aquatic enclosure for retaining a freshwater aquatic animal, wherein the aquatic enclosure retains an aqueous solution comprising 31 hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide, 32 wherein the aqueous solution comprises hydrogen peroxide at a concentration 33 — between 50 mg/L and 80 mg/L inclusive. 34

The apparatus may be apparatus for use in a method of reducing ectoparasitic 36 infestation of a freshwater aquatic animal, wherein the method comprises: 37 - providing an aquatic enclosure for retaining a freshwater aquatic animal

DK 2023 70557 A1 36 1 - combining hydrogen peroxide and an enzyme for catalytic decomposition of 2 hydrogen peroxide with water to thereby form an aqueous solution comprising 3 hydrogen peroxide; 4 - introducing the aqueous solution into the aquatic enclosure; - introducing one or more freshwater aquatic animals into the aquatic enclosure 6 to thereby expose the said freshwater aquatic animal to the aqueous solution); 7 - keeping the one or more aquatic animals in the aquatic enclosure (e.g. for at 8 least 1 minute and for less than 10 hours (e.g. less than 5 hours, less than 4 hours, 9 less than 3 hours, less than 60 minutes, optionally between 3 and 4 hours)). 11 According to a further aspect of the invention, there is provided a method of reducing 12 ectoparasitic infestation of a freshwater aquatic animal, wherein the method comprises: 13 - providing an aquatic enclosure for retaining a freshwater aquatic animal 14 - combining hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide with water to thereby form an aqueous solution comprising 16 — hydrogen peroxide; 17 - introducing the aqueous solution into the aquatic enclosure; 18 - introducing one or more freshwater aquatic animals into the aquatic enclosure 19 to thereby expose the said freshwater aquatic animal to the aqueous solution; - keeping the one or more aquatic animals in the aquatic enclosure (e.g. with the 21 aqueous solution) (e.g. for at least 1 minute and for less than 10 hours (e.g. less than 22 m—5 hours, less than 4 hours, less than 3 hours, less than 60 minutes, optionally between 23 3 and 4 hours)). 24

The method may comprise keeping the aquatic animals in the aquatic enclosure (e.g. 26 with the aqueous solution) for at least 1 minute, or for at least 2 minutes, or for at least 27 5 minutes. The method may comprise keeping the aquatic animals in the aquatic 28 enclosure (e.g. with the aqueous solution) for up to 20 minutes, or up to 30 minutes, or 29 up to 45 minutes. The method may comprise exposing the aquatic animals to the aqueous solution for at least 1 minute, or for at least 2 minutes, or for at least 5 minutes. 31 — The method may comprise keeping the aquatic animals in the aquatic enclosure (e.g. 32 — with the aqueous solution) for up to 20 minutes, or up to 30 minutes, or up to 45 33 minutes. 34

The method may comprise combining hydrogen peroxide and the enzyme (i.e. an 36 enzyme for catalytic decomposition of hydrogen peroxide) with water to form an 37 aqueous solution comprising hydrogen peroxide (and said enzyme), and introducing

DK 2023 70557 A1 37 1 the aqueous solution into the aquatic enclosure. The skilled person will appreciate 2 volume of water and any input for the aqueous solution should be taken into account 3 — when determining quantities and concentrations of hydrogen peroxide and/or enzyme. 4

The hydrogen peroxide and enzyme for catalytic decomposition of hydrogen peroxide 6 may be introduced into the aquatic enclosure and subsequently mixed to form an 7 aqueous solution of hydrogen peroxide. The method may comprise first introducing the 8 hydrogen peroxide and enzyme for catalytic decomposition of hydrogen peroxide into 9 the aquatic enclosure and subsequently mixing the hydrogen peroxide, enzyme, and — water, to form an aqueous solution of hydrogen peroxide. 11 12 Combining hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen 13 peroxide with water leads to the formation of bubbles (typically predominantly of 14 oxygen). The method may comprise generating sound waves having a controllable frequency spectrum (from a sound source e.g. a means for producing sound waves) 16 — and directing the sound waves at the bubbles, wherein the frequency spectrum (and 17 optionally power) of the sound waves is varied with time. 18 19 According to a further aspect of the invention there is provided use of an enzyme which catalyses the decomposition of hydrogen peroxide, to thereby enhance the efficacy of 21 amethod of reducing ectoparasitic infestation on aquatic animals, wherein said method 22 comprises administering hydrogen peroxide and the enzyme to an aquatic enclosure 23 comprising aquatic animals. The method may comprise directing sound into the aquatic 24 — enclosure. The method may comprise mixing the hydrogen peroxide and the enzyme (e.g. mixing the aqueous solution). 26 27 According to a further aspect of the invention, there is provided an enzyme which 28 catalyses the decomposition of hydrogen peroxide, for use in enhancing the efficacy of 29 amethod of reducing ectoparasitic infestation on aquatic animals, wherein said method comprises administering hydrogen peroxide and the enzyme to an aquatic enclosure 31 comprising aquatic animals. The method may comprise directing sound waves into the 32 aquatic enclosure. The method may comprise mixing the hydrogen peroxide and the 33 enzyme (e.g. mixing the aqueous solution). 34

The method may be a method of injuring or killing a pathogenic amoeba. The method 36 — may be a method of reducing amoebic infection in an aquatic animal. The method may 37 be a method of treating amoebic gill disease in fish. The apparatus may be an

DK 2023 70557 A1 38 1 apparatus for use in reducing aquatic ectoparasitic infestation (i.e. ectoparasitosis) on 2 an aquatic animal. The method may be a method of cleaning an aquatic animal (e.g. a 3 fish). 4

The apparatus may be configured to vary one or more of: the duration of the bubble 6 regulation phase, the frequency (and optionally power) during the bubble regulation 7 — phase, the duration of the bubble collapse phase, the frequency (and optionally power) 8 during the bubble collapse phase, the duration of the intermission phase, the duration 9 of the waiting phase, the (e.g. combined) duration of the one or more descending frequency phases, the frequency (and optionally power) of sound waves and the 11 variation of that with time during the one or more descending frequency phases, the 12 duration of the preliminary bubble collapse phase, the frequency (and optionally power) 13 — of sound waves during the preliminary bubble collapse phase. 14

The method may comprise pressurising the aqueous solution comprising hydrogen 16 peroxide and enzyme for catalytic decomposition of hydrogen peroxide and 17 ectoparasites. This may for example be achieved by retaining the aqueous solution 18 comprising hydrogen peroxide and enzyme for catalytic decomposition of hydrogen 19 peroxide and ectoparasites in a said aquatic enclosure and raising the pressure in the aquatic enclosure (for example by compressing the aquatic enclosure, or the contents 21 — of the aquatic enclosure, for example by introducing a gas, such as air, above the 22 aqueous solution in the aquatic enclosure, or a pressurisable bladder adjacent to or 23 — within the aqueous solution). The aquatic enclosure may need to be sufficiently solid 24 — toresist the efflux of aqueous solution but need not be watertight and may for example contain apertures or take the form of a tube or similar. 26 27 This has the advantage that because the resonant frequency of an air bubble in 28 aqueous solution varies with liquid pressure, by raising the pressure in the aquatic 29 enclosure, the ratio of pressure at the bottom of the aquatic enclosure to the pressure at the top of the container is smaller than would otherwise be the case. Accordingly, 31 — the variation in the frequency of the sound waves required during the bubble regulation 32 phase and the bubble collapse phase to have a desired effect (regulating the size of 33 bubbles, causing bubble collapse) within the aquatic enclosure is reduced. This 34 — enables better control of bubble size and collapse.

DK 2023 70557 A1 39 1 The pressure at the top of the aquatic medium in the aquatic enclosure may for 2 example be raised to at least 1.5 atm (i.e. 2 151,987 MPa) or to at least 2 atm (i.e. 2 3 202,650 Pa). 4

Increased pressure may also be obtained by providing the aqueous solution 6 comprising hydrogen peroxide and enzyme for catalytic decomposition of hydrogen 7 peroxide and ectoparasites under another body of aqueous solution, for example under 8 avolume of water. 9

The method may comprise reducing the pressure of the aqueous solution so that, at 11 the top (e.g. surface) of the aqueous solution, the pressure of the aqueous solution is 12 — below atmospheric pressure, for example at most 1 atm (i.e. < 101,325 Pa) or at most 13 0.9atm (i.e. < 91,192 Pa) or at most 0.75 atm (i.e. < 75,994 Pa) or at most 0.5 atm (i.e. 14 < 50,663 Pa). This has the effect of promoting larger gas bubbles. As it is the size of the bubble rather than the amount of gas (predominantly oxygen) within them that 16 determines the damaging effect of bubble collapse and jetting this can make the 17 — process more efficient. 18 19 Alternatively, or in addition, the frequency (peak and/or centre frequency) of the sound waves within the aquatic enclosure (i.e. which are generated and directed into the 21 aquatic enclosure) may vary with depth (e.g. increasing with depth), e.g. proportional 22 tothe square root of the water pressure at a given depth. The sound may be generated 23 by transducers located in a base region of the aquatic enclosure and have a range of 24 frequencies, such that the peak and/or centre frequency of the sound waves within the aquatic enclosure increase with depth due to the greater attenuation of higher 26 frequency sounds with distance from the transducers. The sound may be generated by 27 transducers located in a base region of the aquatic enclosure and the (e.g. peak and/or 28 centre) frequency of the sound may be varied over time. For example, the transducers 29 may generate a chirp, e.g. a descending chirp. 31 It may be that the aqueous solution comprises hydrogen peroxide at a concentration 32 greater than or equal to 20 mg/L. Concentrations of hydrogen peroxide greater than or 33 equal to 20 mg/L are typically more effective at generating bubbles, particularly when 34 — the hydrogen peroxide is dissolved in fresh water. 36 It may be that the aqueous solution comprises hydrogen peroxide at a concentration 37 — greater than or equal to 200 mg/L. Concentrations of hydrogen peroxide of greater than

DK 2023 70557 A1 40 1 — or equal to 200 mg/L are typically more effective at generating bubbles, particularly 2 when the hydrogen peroxide is dissolved in seawater. 3 4 It may be that the aqueous solution comprises hydrogen peroxide at a concentration less than or equal to 2500 mg/L. Concentrations of hydrogen peroxide greater than 6 2500 mg/L do not typically provide any additional benefit but are increasingly expensive 7 to achieve in practice and their use in aquatic environments may be restricted by 8 environmental regulations in some jurisdictions. 9

It may be that the aqueous solution comprises hydrogen peroxide at a concentration 11 less than or equal to 2200 mg/L. In some jurisdictions, environmental regulations 12 — restrict use of solutions of hydrogen peroxide having concentrations greater than 2200 13 mg/L. 14

It may be that the aqueous solution comprises hydrogen peroxide at a concentration 16 between 20 mg/L and 2500 mg/L, inclusive, or between 200 mg/L and 2500 mg/L, 17 inclusive, or between 20 mg/L and 2200 mg/L, inclusive, or between 200 mg/L and 18 — 2200 mg/L, inclusive. 19

It may be that the aqueous solution comprises hydrogen peroxide at a concentration of 21 approximately 1500 mg/L (e.g. at a concentration of between 1300 mg/L and 1700 22 mg/L, inclusive). Aqueous solutions of hydrogen peroxide at concentrations of 23 approximately 1500 mg/L have been approved by regulatory authorities in some 24 jurisdictions for use in, for example, the treatment of parasitic infestations of the marine phase of the Atlantic salmon. The duration of bubble growth prior to bubble collapse is 26 typically related to the hydrogen peroxide concentration and some examples are 27 — described below. 28 29 The resonant frequency of a bubble of gas in an infinite volume of liquid is given by the

Minnaert Formula A: 31 32 f= = 33 34 — where r is the (unforced) bubble radius, y is the polytropic coefficient, p, is the ambient pressure and p is the density of the liquid. In practice, for bubbles formed in water, on 36 — the surface, this formula can be approximated by Formula B:

DK 2023 70557 A1 41 1 2 f= 3 — Where the bubbles are not at the surface, there is a depth term, giving formula C, where 4 dis depthinm. 7 8 (10052 being derived from the weight of sea water, p =o x g x h) 9

It may be that the method comprises exposing the aquatic ectoparasite to sound waves 11 having a frequency determined by the Minnaert Formula A or by the approximate 12 Minnaert Formula B or Formula C. 13 14 It may be that the method comprises determining the unforced radius of bubbles present at the beginning of the bubble collapse phase and thereby selecting the 16 — frequency of the sound waves during the bubble collapse phase based on the Minnaert 17 Formula A or the approximate Minnaert Formula B or Formula C. 18 19 In practice, the bubbles produced on exposure of the aquatic ectoparasite to sound waves will have a range of different sizes. It may be that the method comprises 21 determining the average or peak (unforced) radius of bubbles present at the beginning 22 ofthe bubble collapse phase, determining the resonant frequency corresponding to the 23 said average or peak (unforced) radius based on the Minnaert Formula A or the 24 — approximate Minnaert Formula B or Formula C, and selecting frequencies of the sound waves which lie predominantly within a range of frequencies containing the said 26 resonant frequency. The range of frequencies may have a lower bound of, for example, 27 25%, or 50%, or 75% of the said resonant frequency. The range of frequencies may 28 have an upper bound of, for example, 125%, or 150%, or 175% of the said resonant 29 frequency. 31 — The method may comprise exposing the aquatic ectoparasite to the aqueous solution 32 comprising hydrogen peroxide and enzyme for catalytic decomposition of hydrogen 33 peroxide for at least 30 seconds, or at least 1 minute, or at least 2 minutes, prior to the 34 — bubble collapse phase.

DK 2023 70557 A1 42 1 The method may comprise exposing the aquatic ectoparasite to the aqueous solution 2 for at least 30 seconds, or at least 1 minute (e.g. 1 to 3 minutes), prior to the bubble 3 collapse phase. The inventors have found that exposure for at least 30 seconds 4 — combined with exposure to sound waves is sufficient to form bubbles of oxygen around and/or inside, and to cause observable physical damage and/or death in, isolated 6 aquatic ectoparasites. 7 8 The method may comprise exposing the aquatic ectoparasite to the aqueous solution 9 for at least 5 minutes, or at least 10 minutes, or at least 15 minutes, or at least 20 — minutes, prior to the bubble collapse phase. The longer that the aquatic ectoparasite 11 is exposed to the aqueous solution, the greater the number of bubbles that are formed 12 — (until they are caused to coalesce). The longer that the aquatic ectoparasite is exposed 13 to the aqueous solution, also typically the greater the size of the bubbles that are 14 formed (until they are caused to collapse). Use of longer periods prior to exposing the — ectoparasite to the aqueous solution can be more effective where lower concentrations 16 — of hydrogen peroxide are used. 17 18 It may be that the hydrogen peroxide has a concentration of 1500 mg/L + 50% (or + 19 25%) and the duration of a cycle of forming bubbles and then causing bubble collapse is 6 minutes + 50% (or + 25%). It may be that the hydrogen peroxide has a 21 concentration of 750 mg/L + 50% (or + 25%) the duration of a cycle of forming bubbles 22 and then causing bubble collapse is 12 minutes + 50%. It may be that the hydrogen 23 peroxide has a concentration of 375 mg/L + 50% (or + 25%) and the duration of a cycle 24 of forming bubbles and then causing bubble collapse is 12 minutes + 50% (or + 25%).

It may be that the hydrogen peroxide has a concentration of 200 mg/L + 50% (or + 26 25%). It may be that the duration of a cycle of forming bubbles and then causing bubble 27 collapse is 24 minutes + 50% (or + 25%). 28 29 A further aspect of the invention provides hydrogen peroxide, at a concentration of between 500 mg/L and 1,500 mg/L inclusive, and an enzyme for catalytic 31 decomposition of hydrogen peroxide, or an aqueous solution comprising hydrogen 32 peroxide at a concentration of between 500 mg/L and 1,500 mg/L inclusive, and an 33 enzyme for catalytic decomposition of hydrogen peroxide, for use in a method of 34 reducing ectoparasitic infestation (e.g. ectoparasitosis) on an aquatic animal, or in a method of killing ectoparasites, wherein the aquatic animal, or the ectoparasites, are 36 exposed both to an aqueous solution comprising said hydrogen peroxide (and typically 37 — also bubbles of gas, typically predominantly of oxygen). Optionally the method may

DK 2023 70557 A1 43 1 comprise exposing aquatic animal, or the ectoparasites to sound waves. Optionally the 2 method may comprise varying the frequency spectrum (and optionally the power) of 3 — the sound waves with time. 4

The aquatic animals are typically fish. The aquatic animals may be salmonids. The 6 aquatic animals may belong to the family Salmonidae. The aquatic animals may belong 7 to one of the following genera: Salmo, Oncorhynchus. The aquatic animals may belong 8 to one of the following species: Salmo salar, Oncorhynchus tshawytscha, 9 Oncorhynchus keta, Oncorhynchus kisutch, Oncorhynchus gorbuscha, Oncorhynchus nerka, Oncorhynchus masou, Oncorhynchus mykiss. 11 12 Additionally or alternatively, the aquatic animals may belong to one of the following 13 families: Arripidae, Carangidae, Polynemidae, Cichlidae, Cyprinidae. The aquatic 14 — animals may belong to one of the following genera: Arripis, Elagatis, Eleutheronema,

Hucho, Dicentrarchus, Sparus, Rachycentron, Lates, Seriola, Tilapia, Cyprinus. The 16 — aquatic animals may belong to one of the following species: Hucho hucho, Arripis trutta, 17 Elagatis bipinnulata, Eleutheronema tetradactylum, Dicentrarchus labrax, Sparus 18 — aurata, Rachycentron canadum, Lates calcarifer, Seriola lalandi, Cyprinus carpio, 19 Tilapia baloni, Tilapia guinasana, Tilapia ruweti, Tilapia sparrmanii. 21 Additionally or alternatively, the aquatic animals may belong to one of the following 22 orders: Siluriformes or Nematognathi. The aquatic animals may be catfish. 23 24 — Additionally or alternatively, the aquatic animals may belong to one of the following groups: Caridea, Dendrobranchiata. The aquatic animals may be shrimp or prawns. 26 27 The aquatic ectoparasite may be a marine ectoparasite (i.e. an ectoparasite adapted 28 for life in marine environments, e.g. the ocean). The aqueous solution may comprise a 29 solution of hydrogen peroxide (e.g. and an enzyme for catalytic decomposition of hydrogen peroxide) in sea water. 31 32 The aquatic ectoparasite may be a freshwater ectoparasite (i.e. an ectoparasite 33 adapted for life in freshwater environments, e.g. in rivers or lakes). The aqueous 34 — solution may comprise a solution of hydrogen peroxide (e.g. and an enzyme for catalytic decomposition of hydrogen peroxide) in fresh water. It will be understood that 36 the term ectoparasite refers to a parasite which lives on the outside of its host animal 37 (e.g. on the skin, scales, or fins of a fish).

DK 2023 70557 A1 44 1 2 The aquatic ectoparasite typically belongs to the family Caligidae. The aquatic 3 — ectoparasite typically belongs to one of the following genera: Lepeophtheirus, Caligus. 4 The aquatic ectoparasite typically belongs to one of the following species: — Lepeophtheirus salmonis, Caligus clemensi, Caligus rogercresseyi, Caligus elongatus. 6 The pathogenic amoeba is typically a pathogenic amoeba which colonises aquatic 7 — animals. The pathogenic amoeba may be a pathogenic amoeba which causes amoebic 8 gill disease (AGD) in fish such as salmonids. The pathogenic amoeba may belong to 9 the genus Neoparamoeba. The pathogenic amoeba may belong to the species

Neoparamoeba perurans. 11 12 Amoebic infection of the aquatic animal typically comprises infection of the aquatic 13 — animal by pathogenic amoeba. 14

The aqueous solution may be an aqueous solution for disturbing (e.g. configured to 16 disturb) aquatic ectoparasites. The aqueous solution may be an aqueous solution for 17 — stunning (e.g. configured to stun) the aquatic ectoparasites. The aqueous solution may 18 be an aqueous solution for to injuring (e.g. configured to injure) aquatic ectoparasites. 19 The aqueous solution may be an aqueous solution for killing (e.g. configured to kill) aquatic ectoparasites. In a further aspect of the invention, there is provided an aqueous 21 solution as described herein for use to reduce ectoparasitic infestation of an aquatic 22 animal. The ectoparasites may be female ectoparasites. The ectoparasites may be 23 female ectoparasites of the family Caligidae. 24

The apparatus or the aqueous solution may comprise bubbles. The bubbles may be 26 from (e.g. may be provided by) a bubble curtain, optionally a tubular (e.g. toroidal) 27 — bubble curtain. Said bubbles may be bubbles for disturbing ectoparasites. The bubbles 28 may be bubbles for injuring ectoparasites. The bubbles may be bubbles for killing 29 ectoparasites. The apparatus or the aqueous solution may be configured to cause bubbles to form on ectoparasites to thereby (e.g. buoyantly) detach them from an 31 aquatic animal. The bubbles may form on ectoparasites to thereby detach them from 32 — an aquatic animal. The bubbles may form on the ectoparasites to thereby prevent the 33 ectoparasites from resettling on the aquatic animal. The apparatus or the aqueous 34 — solution may be configured to cause bubbles to form on ectoparasites to thereby limit the extent to which the ectoparasites are able to resettle on the aquatic animal. It will 36 — be understood that the bubbles provide buoyancy to the ectoparasites and thus can 37 buoyantly dislodge them from an aquatic animal host.

DK 2023 70557 A1 45 1 2 An aspect of the invention provides a method of disturbing an aquatic ectoparasite. 3 — The method may be a method of injuring an aquatic ectoparasite.

The method may be 4 a method of killing an aquatic ectoparasite.

The method may comprise a step of exposing the ectoparasite to an aqueous solution comprised in as herein described. 6 The method may comprise exposing the ectoparasite to bubbles.

In other words, the 7 — method may be a method carried out on an ectoparasite.

It will be understood that the 8 presence of a host for the ectoparasite (e.g. an aquatic animal such as a fish) is not 9 necessary and that the invention is equally applicable whether or not a host is present.

11 The bubbles may be provided by a bubble curtain.

The bubble curtain may have (e.g. 12 — may define) a tubular (e.g. toroidal) shape. 13 14 Optional and preferred features of any one aspect of the invention are optional features of any other aspect of the invention. 16 17 It will be appreciated that the steps of any of the above-described methods may be 18 — completed in any order unless inherently incompatible.

In some examples, one or more 19 of the steps of any of the above-described methods may be completed simultaneously.

21 Description of the Drawings 22 23 An example embodiment of the present invention will now be illustrated with reference 24 to the following Figures in which:

26 Figure 1 shows an Atlantic salmon infested with sea lice; 27 28 Figure 2 shows a plurality of infested Atlantic salmon retained in an undersea cage; 29 — Figure 3 shows the undersea cage of Figure 2 surrounded by a tarpaulin enclosure and 31 — an array of ultrasonic transducers, before treatment has commenced; 32 33 — Figure 4 shows the treatment apparatus of Figure 4 during treatment; 34 — Figures 5A to 5D are a series plots of example frequency and amplitude output by an 36 ultrasound generator with time, during an example treatment cycle according to the 37 second example procedure;

DK 2023 70557 A1 46 1 2 Figures 6 is a flow chart of a first example procedure for the ultrasound treatment of 3 the sealice; 4

Figure 7 is a flow chart of a second example procedure for the ultrasound treatment of 6 the seal lice; 7 8 Figures 8A and 8B respectively show the frequency and amplitude of ultrasound output 9 by the ultrasound generator with time, during a treatment cycle according to the second example procedure; 11 12 Figure 9 shows a wellboat being loaded with infested Atlantic salmon from an undersea 13 cage; 14

Figure 10 shows Atlantic salmon during treatment with hydrogen peroxide and 16 exposure to ultrasound on the wellboat of Figure 9; 17 18 Figure 11 shows sea lice detached from the Atlantic salmon and caught in a lice filter 19 of the wellboat of Figure 9; 21 — Figure 12 shows the Atlantic salmon of Figure 9 having been returned to the undersea 22 cage; 23 24 — Figure 13 shows a wellboat adapted to pressurise an enclosure; 26 Figure 14 is a photograph of bubbles on two different surfaces; 27 28 Figure 15 is photograph of bubbles forming on a male sea louse; 29

Figure 16 is a photograph of bubbles forming on a female sea louse; 31 32 Figure 17 is a diagram of the movement of water containing a high density of bubbles 33 in an example embodiment of a toroidal bubble curtain; 34

Figure 18 is a diagram of a cross sectional view of the movement of water containing 36 a high density of bubbles in a further example embodiment; and 37

DK 2023 70557 A1 47 1 Figure 19 is a diagram of a cross sectional view of an enclosure for use to treat 2 — ectoparasitic infestation, containing an acoustic device. 3 4 — Detailed Description of a First Example Embodiment 6 It will be understood by those skilled in the art that any dimensions and relative 7 orientations such as lower and higher, above and below, and directions such as 8 vertical, horizontal, upper, lower, longitudinal, axial, radial, lateral, circumferential, etc. 9 referred to in this description refer to, and are within expected structural tolerances and limits for, the technical field and the apparatus described, and these should be 11 interpreted with this in mind. 12 13 — Figure 1 shows an Atlantic salmon 1 belonging to the species Salmo salar. The salmon 14 1isinfested with sea lice 2A and 2B belonging to the species Lepeophtheirus salmonis.

The sea lice 2A and 2B are parasites which cling to and feed off the salmon, causing 16 damage to the salmon's skin and fins and creating open wounds which permit other 17 pathogens to enter the fish. Sea lice infestation is a particular problem in salmon farms 18 — where many salmon are reared together in a caged environment. 19

Figure 2 shows several salmon 1 retained within a floating cage 3 in the sea 4. The 21 cage 3 is tethered to a floating platform 5. The cage 3 is generally cylindrical in shape, 22 having one continuous, generally cylindrical wall 6 and a base 7. The cage 3 is open 23 atthe surface of the sea 8. The wall 6 and base 7 of the cage are formed from a nylon 24 — mesh (or a mesh made of any other suitable plastics material) having openings which are sufficiently small that the salmon cannot escape from the cage, but water is still 26 able to flow freely through the cage wall and base. 27 28 The cage 3 further has a mixer 8 at its base, the mixer 8 having two paddles 24a, 24b 29 — which are rotatable around a central axis to cause motion of the surrounding aqueous solution in the cage 3. In addition, the cage 3 has a dosing point 18 at its base, from 31 which various substances and/or chemicals may be introduced. For example, an 32 aqueous solution of hydrogen peroxide and an enzyme for catalytic decomposition of 33 — hydrogen peroxide, such as peroxidase or catalase, can be introduced into the cage 3 34 — either via the dosing point 18 at the base, or at the surface of the water at the top of the caged. 36

DK 2023 70557 A1 48 1 As shown in Figure 3, in order to treat the salmon to remove the sea lice, the cage 3 is 2 surrounded by a tarpaulin enclosure 9 tethered to the floating platform 5 and a float 10. 3 — The tarpaulin enclosure 9 is waterproof and completely encircles the cage 3. Water 4 — can flow between the interior of the cage 3 and the space enclosed between the cage 3 and the tarpaulin enclosure 9 but water cannot flow beyond the tarpaulin enclosure 6 9. In Figure 3, an array of underwater ultrasonic transducers 11 has also been 7 introduced into the space enclosed between the cage 3 and the tarpaulin enclosure 9. 8 The array of underwater ultrasonic transducers 11 is tethered to the float 10 which also 9 supports a power source for the transducers (not shown). A water and sound permeable barrier 15 (e.g. a mesh) is provided between the transducers and the main 11 body of the enclosure to protect fish from excessive sound in use. 12 13 — The apparatus shown in Figure 3 is used injure or kill the salmon lice on the salmon 14 (and reduce the parasitic infestation of the salmon). In use, hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide is added to the water 16 enclosed within the tarpaulin enclosure 9. In an example, sufficient hydrogen peroxide 17 is added to form an aqueous solution within the enclosure 9 having a hydrogen 18 peroxide concentration of approximately 1,000 mg/L and an enzyme for catalytic 19 decomposition of hydrogen peroxide (in this case, catalase) is also added to the aqueous solution.

As shown in Figure 4, the hydrogen peroxide begins to decompose 21 in the water as is acted upon by the catalase and generates bubbles 12 of oxygen 22 around the surface of the salmon.

Bubbles are preferentially formed on hydrophobic 23 surfaces, such as waxy surfaces.

It has been surprisingly found that the extent of this 24 preferential formation is much greater than would have been expected, with large numbers of bubbles forming very close together on more hydrophobic surfaces and 26 almost no bubbles forming on surfaces which are less hydrophobic, even where these 27 surfaces are positioned side-by-side in the same aqueous solution.

As such, bubbles 28 preferentially form on the surface of (and occasionally inside) the sea lice attached to 29 the salmon and less preferentially form on the surfaces of the salmon themselves.

This effect is shown in Figure 14, where it can be seen that bubbles preferentially form on 31 hydrophobic surface 202 (this surface being coated with wax) and that relatively few 32 bubbles form on a non-hydrophobic surface 204. Figures 15 and 16 show bubbles 210 33 forming on the surfaces of male 206 and female 208 sea lice, respectively.

When 34 — sufficient bubbles form on the lice 206, 208 the lice are buoyantly removed from the — surface of the host aquatic animal. 36

DK 2023 70557 A1 49

1 Bubbles are typically formed by decomposition of hydrogen peroxide by an enzyme

2 (here catalase) to form oxygen and water according to the following chemical equation:

3

4 2H,0, —25 2H,0 + 0,

6 Hydrogen peroxide is thermodynamically unstable and can decompose spontaneously

7 to form oxygen and water, however enzymes such as catalase accelerate this process.

8 It may be that the bubbles are formed predominantly on the surface of the aquatic

9 — ectoparasite.

However, bubbles may also be formed inside (i.e. inside the body of) the aquatic ectoparasite.

Hydrogen peroxide is decomposed biologically by the enzyme in 11 — the aqueous solution as well as by catalase (or other antioxidant enzymes such as 12 glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, superoxide 13 reductase, glutathione reductase and thioredoxin), commonly present within the body 14 — of aquatic ectoparasites.

This may provide a mechanism for bubble formation inside and on the surface of (e.g. adjacent pores of) the aquatic ectoparasite.

It will be 16 — understood that while the aquatic ectoparasite(s) and/or the aquatic enclosure may 17 comprise or contain naturally-occurring enzymes, where an aqueous solution 18 comprising hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen 19 peroxide is referred to, the said enzyme for catalytic decomposition of hydrogen peroxide is not an enzyme that is naturally occurring in the aquatic ectoparasite(s) 21 and/or the aquatic enclosure, but an additional enzyme which is introduced for the 22 — purpose of catalytic decomposition of introduced hydrogen peroxide. 23 24 — The ultrasonic transducers are switched on and the transducers generate ultrasonic waves 13 which propagate through the water enclosed within the tarpaulin enclosure 26 > 9and are incident on the ectoparasites. 27 28 Figures 5A to 5D illustrate the peak (and/or centre) frequency (5A, 5C), and amplitude 29 (5B, 5D), of ultrasound generated over time, during five phases (separated by dashed lines). Figure 6 is a flow chart of a first example of an ultrasound treatment procedure 31 — which, in this example, is divided into three phases. 32 33 Initially, hydrogen peroxide and catalase are added 50 to the aquatic enclosure 34 containing fish (or alternatively the fish can be brought into an aquatic enclosure which already comprises hydrogen peroxide and catalase.

In the first phase 52 (the waiting 36 — phase), a period of time, typically of the order of a few seconds through to around 1 37 minute is provided to give sufficient time for a significant amount of hydrogen peroxide

DK 2023 70557 A1 50 1 to be decomposed (e.g. under action of the enzyme) to form oxygen and thereby form 2 bubbles. The person skilled in the art will appreciate that the waiting phase may have 3 a duration of a longer or shorter period of time, depending on the conditions. For 4 — example, it may be preferable for a waiting phase to have a duration of less than 10 seconds, or 20 to 30 seconds, or preferably 30 seconds to 2 minutes. The waiting 6 phase may be selected to be longer when the water temperature is lower, for example. 7 8 The second phase 54, is a bubble regulation phase comprising at least one descending 9 frequency phase. The peak (and/or centre) frequency of the ultrasound is gradually — reduced (in this example linearly) and the amplitude is kept constant. This promotes 11 the formation of larger bubbles. In particular it promotes bubbles coalescing with each 12 — other, at an ever-greater size, as the frequency of the ultrasound is reduced. Generally, 13 gaseous oxygen will continue to be generated with time which also assists growth. The 14 bubbles become sufficiently large that some will detach from the surface of the ectoparasite. The final frequency (lowest peak (and/or centre) frequency) is selected 16 — to facilitate the growth of bubbles which are close to the size at which they will 17 predominantly buoyantly detach from the ectoparasites, for example they may grow to 18 about 1to 2 mm. 19

Inthe example indicated in the plot of figure 5A, each bubble collapse phase comprises 21 one descending frequency phase, each bubble collapse phase lasts for 1 minute, and 22 the descending frequency phase is from an initial frequency 60 of about 13 kHz to a 23 — final frequency 62 of about 3 kHz. In the example indicated in the plot of figure 5C, 24 — each bubble collapse phase comprises a set of four descending frequency phases, — each set of four lasting for 1 minute, and the sets of four descending frequency phases 26 being interspersed with intermission phases. Again, each of the four descending 27 frequency phases is from an initial frequency 60 of about 13 kHz to a final frequency 28 62 of about 3 kHz. 29

Thereafter, in the third phase 56 (the intermission phase), there is a pause in 31 ultrasound generation. This provides time for some of the larger bubbles to detach and 32 — for new bubbles to form from the decomposition of hydrogen peroxide. In this example, 33 — the intermission phase lasts about 4 minutes. 34

This process is repeated several times, for example 4 times. 36

DK 2023 70557 A1 51 1 Patterns of bubble regulation phases and intermission phases (i.e. following an initial 2 waiting phase) may be determined depending upon the conditions (e.g. water 3 temperature). A full cycle of such a pattern of bubble regulation phases and 4 intermission phases (i.e. including an initial waiting phase) is typically anticipated to have a duration of between 3 minutes and 30 minutes, preferably between 5 minutes 6 and 25 minutes, more preferably between 10 and 20 minutes. 7 8 > A first pattern (as described above) may, for example, include: 9 - an initial 1-minute waiting phase; - —232-minute bubble regulation phase; 11 - —24-minute intermission phase; 12 - —232-minute bubble regulation phase; 13 - 0234-minute intermission phase; and 14 - optionally a further 2-minute bubble regulation phase; - optionally a further 4-minute intermission phase; and 16 - optionally a further 2-minute bubble regulation phase. 17 18 — The above first pattern would therefore include a total of 1 minute of waiting phase, 8 19 minutes of bubble regulation phases (during each of which sound is generated, as described above), and 12 minutes of intermission phases.

Each of the four 2-minute 21 bubble regulation phases include sound generation wherein the frequency of sound 22 generated begins at 6 kHz and is reduced to 3 kHz, either in discrete frequency steps 23 — or, more preferably, through a continuous sweep-through of the frequencies (e.g. at 24 0.2 kHz/s) and this reduction of frequency may be repeated several times (in the example indicated in Figure 5C, with a reduction in frequency from 6 kHz to 3 kHz at a 26 rate of 0.2 kHz/s, the reduction of frequency would be repeated four times during each 27 2-minute bubble regulation phase). 28 29 It will be appreciated that other patterns of bubble regulation phases and intermission phases (i.e. following an initial waiting phase) may also be suitable.

Accordingly, a 31 — second pattern may for example include: 32 - an initial 30-second waiting phase; 33 - —232-minute bubble regulation phase; 34 - 0234-minute intermission phase; - —232-minute bubble regulation phase; 36 - 0234-minute intermission phase; and 37 - 024-minute bubble regulation phase.

DK 2023 70557 A1 52 1 2 The above second pattern would therefore include a total of 30 seconds of waiting 3 phase, 8 minutes of bubble regulation phases (during each of which sound is 4 — generated, as described above), and 8 minutes of intermission phases.

Here, the bubble regulation phases include sound generation wherein the frequency of sound 6 generated either begins at 6 kHz and is reduced to 3 kHz, or begins at 10 kHz and is 7 reduced to 3 kHz, and is reduced either in discrete frequency steps or, more preferably, 8 through a continuous sweep-through of the frequencies (e.g. at between 0.2 kHz/s and 9 1kHz/s).

11 If the water temperature is lower, a longer initial waiting phase is required.

This may 12 lead to the following, third pattern of bubble regulation phases and intermission phases 13 (i.e. following the said longer initial waiting phase): 14 - a Z-minute waiting phase; - a 1-minute bubble regulation phase; 16 - a b-minute intermission phase; 17 - a 1-minute bubble regulation phase; 18 - a b5-minute intermission phase; and 19 - a 3-minute bubble regulation phase.

21 Afourth pattern of bubble regulation phases and intermission phases (i.e. following a 22 longer initial waiting phase as suitable for lower water temperatures) may be as follows: 23 - a 3-minute waiting phase; 24 - a4-minute bubble regulation phase; - 02a6-minute intermission phase; and 26 - 024-minute bubble regulation phase. 27 28 In some patterns, the final bubble regulation phase of each pattern may include 29 generating sound having a higher frequency spectrum compared to the frequency spectra of the or each previous bubble regulation phase.

This provides the advantage 31 of encouraging the coalescence of any smaller bubbles which have not coalesced 32 — during the or each previous bubble regulation phase.

Alternatively or additionally, some 33 examples may include bubble regulations phases in which the frequency is changed 34 — through a wider frequency band.

36 — As bubbles coalesce the total size of a given bubble increases which also increases its 37 buoyancy.

Eventually, the buoyancy of the bubble is great enough that the Bjerknes

DK 2023 70557 A1 53 1 forceis overcome, and the bubble is buoyantly lost. At typical acoustic field strength of 2 > between 170 and 250 dB, e.g. between 201 and 217 dB, the inventor has observed 3 — this effect to occur at around 3.5 kHz (e.g. for 1 uPa at a distance of 1 m from the sound 4 — source). However, this effect may occur at other frequencies in different conditions and/or when using different sound sources. Accordingly, it is advantageous to 6 decrease from an initial high frequency (e.g. 6 kHz) to a lower frequency (e.g. 3 kHz) 7 — atarate of approximately 5 seconds per kHz (optionally 1 second per kHz, or less than 8 => 1second per kHz), as this rate both causes coalescence of bubbles but also allows for 9 bubbles to oscillate for a period of time (e.g. several seconds) before they reach a size at which they are lost due to their buoyancy. This appears to be particularly effective in 11 damaging and/or removing sea lice. 12 13 As the size of the bubbles changes so does the resonant frequency of the bubbles. 14 Therefore, varying the frequency of sound produced during the bubble regulation phase, as described above, provides the further advantage of continuing to cause the 16 bubbles to oscillate as they increase in size. Oscillation of the bubbles due to the sound 17 — waves can also help to cause the bubbles to stick to the lice and/or to the fish (i.e. the 18 bubbles are held close to surfaces due to the Bjerknes force). 19

Figure 7 is a flow chart of a second example of an ultrasound treatment procedure 21 which, in this example, is divided into five phases. In contrast to the first example, the 22 second example includes a bubble collapse phase. Figures 8A and 8B illustrate the 23 peak frequency (8A), and amplitude (8B), of ultrasound generated over time, during 24 — the five phases (separated by dashed lines). 26 Initially, hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen 27 peroxide (in this case, catalase) are added 100 to the aquatic enclosure containing fish 28 (or alternatively the fish can be brought into an aquatic enclosure which already 29 comprises hydrogen peroxide and the enzyme (in this case, catalase)). 31 In the second phase 104 (the preliminary bubble collapse phase), ultrasound is 32 generated at a relatively high frequency and moderately high amplitude, selected to 33 — cause bubbles which are present on fish and ectoparasites to collapse symmetrically. 34 — This removes bubbles from previous cycles of this procedure and can be useful to clean fish and treat open wounds. This phase is also useful as a step of the first 36 example procedure described above. 37

DK 2023 70557 A1 54

1 Inthe third phase 106 (the one or more descending frequency phases), as before, the

2 frequency of the ultrasound is gradually reduced, and the amplitude is kept relatively

3 low.

This promotes the formation of larger bubbles, in particular it promotes bubbles

4 coalescing with each other, at an ever-greater size, as the frequency of the ultrasound is reduced.

Generally, gaseous oxygen will continue to be generated with time which

6 also assists growth.

The bubbles become sufficiently large that some will detach from

7 — the surface of the ectoparasite.

8

9 The second and third phases together function as the bubble regulation phase.

Their — purpose is to regulate the size of bubbles with the aim that an effective proportion of 11 bubbles are within a defined size range during the later bubble asymmetric collapse 12 — phase.

The defined size range is typically reasonably narrow, e.g. a range of less than 13 1 mm of diameter, or less than 0.5 mm of diameter, or less than +50%, with the centre 14 diameter of the size range being in the range 0.2 - 2 mm, for example.

16 Thereafter, in the fourth phase 108 (the intermission phase), there is a pause in 17 ultrasound generation.

This provides time for some of the bubbles to move a short 18 distance from the surface of the ectoparasite, carried by the flow of liquid or due to the 19 buoyancy of the bubbles.

The distance should be less than 2 bubble diameters.

If the bubbles have a diameter of 1 mm at the end of the intermission phase the distance 21 would be less than 2 mm.

In an example, bubbles have a radius of about 1 mm and 22 move about 0.5 to 2 mm for example, from the surface of the ectoparasite. 23 24 In the fifth phase 110 (the bubble asymmetric collapse phase), ultrasound is generated at a lower frequency than in previous steps and with a relatively high amplitude.

This 26 causes bubbles to collapse and create micro-jets directed at the surface of the 27 — ectoparasite (or within the body of the ectoparasite if they remain within the 28 — ectoparasite). The pulse can have relatively short duration and can cause substantial 29 damage to the ectoparasites.

The high-power level is acceptable due to the short duration.

In this phase the sound wave might for example be generated with a power 31 of > 210 dB.

The bubble regulation phase is typically carried out at a power which is 32 insufficient to cause bubble collapse (except briefly in the preliminary bubble collapse 33 — phase) but sufficient to cause oscillations.

The sounds waves might be generated with 34 a power of <190 dB during this phase (at least during each of the one or more descending frequency phases). 36

DK 2023 70557 A1 55 1 Asaa result, in comparison to the generation of continuous ultrasound in the presence 2 of hydrogen peroxide, the invention enables the high intensity ultrasound pulse to 3 cause increased damage to the ectoparasites in a short period of time. This avoids 4 generating sustained high intensity ultrasound and thereby mitigates potential effects ofthat ultrasound on the environment and/or on fish. Furthermore, it can reduce overall 6 power consumption as the high amplitude phase is relatively short. 7 8 It has been found that bubble jetting is effective in damaging ectoparasites, while 9 minimising damage to fish, especially where the bubble has detached from the ectoparasite surface but is within two bubble radii of the ectoparasite surface. 11 Lepeophtheirus salmonis and similar parasites has a surface layer of a hydrophobic 12 wax like substance. Bubbles forming on this surface have a high contact angle, where 13 — the bubble is more spherical and the centre further away from the surface than would 14 be the case without the hydrophobic surface layer. The bubbles on the lice are more readily collapsed as a jet being nominally created at a distance of 0.9 - 1.0 bubble radii 16 — from the surface which could be grown to 1.2. Where the bubbles are smaller than this 17 — and located on the surface of the fish, they are more prone to shear wave collapse. 18 19 The duration of each phase can be predetermined or may be determined using measurements of bubble size, for example using optical sensors. 21 22 It has been found that bubble jetting is effective in damaging ectoparasites, while 23 minimising damage to fish, especially where the bubble has detached from the 24 ectoparasite surface but is within two bubble radii of the ectoparasite surface.

Lepeophtheirus salmonis and similar parasites have a surface layer of a hydrophobic 26 wax like substance. Bubbles forming on this surface have a high contact angle, where 27 the bubble is more spherical and the centre further away from the surface than would 28 be the case without the hydrophobic surface layer. The bubbles on the lice are more 29 readily collapsed as a jet being nominally created at a distance of 0.9 - 1.0 bubble radii from the surface which could be grown to 1.2. Where the bubbles are smaller than this 31 and located on the surface of the fish, they are more prone to shear wave collapse. 32 33 In general, the ultrasound intensity during the bubble regulation phase (preliminary 34 — bubble collapse phase and the or each of the one or more descending frequency phases) is kept such that the acoustic pressure which stimulates the bubble is below 36 — the Blake threshold pressure (usually by a factor of at least 1.5), but above the Blake 37 threshold pressure (usually by a factor of at least 1.5) during the collapse phase. The

DK 2023 70557 A1 56 1 frequency of sound waves during the preliminary bubble collapse phase and the one 2 or more descending frequency phases can be determined experimentally. In an 3 example, sound has a frequency of 20 kHz during the preliminary bubble collapse 4 phase and then this is reduced progressively to 3 kHz at the rate of 1 kHzs"!. The cycles of sound treatment (bubble regulation phase and collapse phase plus intermission 6 phases) are typically repeated. 7 8 The concentration of hydrogen peroxide can be varied over a reasonably wide range. 9 It may be reduced below 1,500 mg/L, which is environmentally advantageous, by allowing a longer period of time for bubbles to form prior to collapse. In an example, 11 — the concentration of hydrogen peroxide is 1,500 mg/L and a cycle of forming bubbles 12 — and then causing bubble collapse has a duration of less than 30 seconds. In another 13 example, the concentration of hydrogen peroxide is 750 mg/L and a cycle of forming 14 bubbles and then causing bubble collapse has a duration of 8 to 10 minutes. In a further example, the concentration of hydrogen peroxide is 375 mg/L and a cycle of forming 16 bubbles and then causing bubble collapse has a duration of 9 to 11 minutes. In a still 17 — further example, the concentration of hydrogen peroxide is 200 mg/L and a cycle of 18 forming bubbles and then causing bubble collapse has a duration of 12 minutes. 19

In some embodiments, the ultrasound treatment is applied in a wellboat. Figure 9 21 shows a treatment wellboat 14 adjacent the floating cage 3 in the sea 4. The wellboat 22 14 contains a treatment enclosure 15 configured to retain a body of water. An array of 23 underwater ultrasonic transducers 16 is provided at one end of the treatment enclosure 24 15. Avent 17 connects the treatment enclosure 15 to the surrounding sea water 4 by way of a sea lice filter 18. 26 27 In use, the vent 17 is closed so that the treatment enclosure 15 is isolated from the 28 surrounding sea water. Salmon 19, which are infested with sea lice, are drawn into the 29 treatment enclosure 15 from the cage 3 by way of a siphon 20. 31 As shown in Figure 10, once transported from the cage 3 into the treatment enclosure 32 15, the salmon may be treated for sea lice infestation by exposure to hydrogen 33 peroxide, an enzyme for catalytic decomposition of hydrogen peroxide (in this case, 34 catalase), and ultrasound. 36 — Hydrogen peroxide and catalase are added to the water in the treatment enclosure 15 37 until the hydrogen peroxide concentration of the water reaches approximately 1,000

DK 2023 70557 A1 57 1 mg/L. The hydrogen peroxide decomposes to form bubbles of oxygen 21 around the 2 salmon and, preferentially on the surface of, and inside, the sea lice attached to the 3 salmon. 4

The array of ultrasonic transducers is switched on and the transducers emit ultrasonic 6 sound waves 22 which propagate through the water enclosed within the treatment 7 — enclosure 15. The frequency spectrum of the generated ultrasound is varied with time 8 as set out above with respect to Figures 5 and 6 or with respect to Figures 7 and 8. 9

After the treatment is finished, the ultrasonic transducers are switched off and, as 11 shown in Figure 11, the vent 17 is opened to allow the treatment water to disperse into 12 the surrounding sea 4. Sea lice 23 which have detached from the salmon 19 are 13 — trapped by the sea lice filter 18. The salmon 19 may then be transferred back into the 14 cage 3 by way of the siphon 20. The salmon in the cage have been effectively deloused, as shown in Figure 12. 16 17 — In some embodiments, the enclosure (e.g. cage, tank, or pipe) within which treatment 18 — takes place is sufficiently solid to retain water under pressure. For example, it may have 19 solid walls, or at least walls with a relatively small cross-sectional area of apertures.

The pressure within the enclosure is then increased by, for example, adding 21 pressurised air, or inflating a bladder within the cage, or bringing the enclosure into 22 — contact with higher pressure water (e.g. a hydrostatic head). In an example shown in 23 — Figure 13, enclosure 15 is sealed with a cover 25 above an air space 26 and air is 24 — introduced continuously by a pump 27, through pipe 28, to increase the pressure at the top of the water in the enclosure to above atmospheric pressure. This has the effect of 26 raising the pressure at the upper surface of the water. This reduces the ratio of the 27 — pressure between the bottom of the enclosure and the top. As can be seen from the 28 > Minnaert Formulas above, the resonant frequency of bubbles is a function of pressure 29 (roughly proportional to the square root of pressure). 31 Accordingly, by reducing the ratio of the pressure between the water at the bottom and 32 at the top of the enclosure ultrasound may be generated which is optimised to cause 33 — the desired effect in both the bubble regulation phase and the bubble collapse phases 34 — throughout a greater volume of the enclosure. In practice, the ultrasound which is generated may be at a range of frequencies and this approach may allow the 36 bandwidth of the ultrasound to be reduced, enabling greater control of bubble growth 37 and collapse.

DK 2023 70557 A1 58 1 2 — Another way to increase the pressure is by fluidically connecting the sealed tank to a 3 raised tank, to thereby increase the head pressure at the surface. In alternative 4 embodiments the pressure at the surface of the water can be reduced, e.g. by running pump 27 as a vacuum pump to evacuate air from the air space 26. This promotes rapid 6 — bubble growth. 7 8 Nevertheless, there will still generally be a significant variation in pressure within the 9 aquatic enclosure. For example, in a well boat treatment with a well boat which is up to 8m deep, there will be a variation in the frequency of the resonant frequency of bubbles 11 of a given size of 33%, and for a tarpaulin treatment with an enclosure formed of a 12 tarpaulin which is 10 m deep, there will be a variation in the resonant frequency of 13 bubbles of any given size of 42%. Pressurisation would reduce these frequency 14 variations. However, there will still be a variation in the optimal frequency for bubble formation, control, and collapse with depth. Accordingly, the sound which is generated 16 — will have a bandwidth selected to provide a balance between broad enough to regulate 17 and collapse bubbles at a range of depths and narrow enough to avoid excessively 18 disrupting the formation and collapse of bubbles at a range of depths. 19

The frequency of sound which is generated and directed into the aquatic enclosure 21 may also be varied with depth, especially in embodiments where sound is transmitted 22 using transducers which are vertically spaced within the aquatic enclosure. 23 Transducers can be arranged so that the peak (and/or centre) frequency of the sound 24 — waves increases with depth. This reduces or avoids a tendency for smaller bubbles to be formed at greater depths (e.g. due to the increased pressure at greater depths) as 26 the bubble diameter for a given resonant frequency decreases as pressure increases. 27 At greater depths, an increased acoustic pressure is also required to cause oscillation 28 (again, due to the increased pressure at greater depths). Preferably, the acoustic 29 pressure should be a significant proportion of the internal pressure of a bubble, for example, the acoustic pressure generated might be between 5% and 95% of the 31 internal pressure of the bubble, or between 10% and 70% of the internal pressure of 32 the bubble. The advantage of providing an acoustic pressure which is similar to the 33 internal pressure of a bubble is that this encourages bubble movement, oscillation, and 34 coalescence. In an example, the transducers are located at the bottom of the aquatic enclosure and sound with a range of frequencies is directed upwards. Accordingly, as 36 — lower frequency sounds penetrate further in water, the peak frequency and/or centre 37 frequency of the sounds waves increases with distance from the transducers, i.e. as

DK 2023 70557 A1 59 1 depth decreases. Similarly, this allows higher acoustic field strengths to be generated 2 atgreater depths, and (as the sound waves are attenuated as they travel upwards) the 3 acoustic field strength to be reduced at shallower depths. 4

Pressure variation is of less concern in embodiments where the aquatic enclosure is 6 relatively shallow, for example where the aquatic enclosure is a pipe or shallow tray. 7 8 Figure 17 is a diagram of the flow of water containing a high density of bubbles in an 9 example embodiment of a toroidal bubble curtain (this being an example of a tubular — bubble curtain). Such a bubble curtain can be used in the apparatus. In detail more, 11 — the curtain is generated by a mixer 8 in a lower central region of the aquatic enclosure 12 6. The mixer mixes the water, hydrogen peroxide and catalase, which causes an 13 accelerated rate of the breakdown of hydrogen peroxide, leading to a large number of 14 small bubbles forming (e.g. at least 1,000 bubbles per litre of aqueous solution, the bubbles having an average (e.g. mean, optionally median) diameter of 100 um). These 16 bubbles rise under buoyancy towards the top of the enclosure 6 and from there move 17 outwards towards the periphery of the enclosure 6, and subsequently back down 18 — towards the base of the enclosure, as a result of the currents caused by the movement 19 of the mixer and the introduction of additional bubbles. 21 — The resulting toroidal bubble curtain is beneficial for reducing the sound escaping from 22 the enclosure, thereby minimising noise pollution. 23 24 — Figure 18 is a diagram of a cross sectional view of a further example of the flow of water containing a high density of bubbles, in which the bubbles define a bubble curtain 26 219. In this example embodiment, a weighted pipe (or hose) 217 with a plurality of 27 perforations (e.g. holes, not shown) defined therein is disposed in a lower region 28 (preferably at the bottom) of the enclosure 6. Said pipe 217 is configured to be disposed 29 around the periphery of the enclosure 6. The bubbles are introduced to the enclosure 6 viathe perforations. A large number of small bubbles as described in Fig. 17 may be 31 introduced. These bubbles rise under buoyancy towards the top of the enclosure 6, 32 travel around the periphery of the enclosure (see directions of flow indicated by 215), 33 and subsequently back down towards the base of the enclosure as a result of 34 introduction of additional bubbles from the pipe 217. In this way, a bubble curtain 219 is provided around the periphery of the enclosure. This bubble curtain provides a 36 source of bubbles and also reduces transmission of the sound waves from the 37 enclosure into the surrounding environment (which may be the sea, for example). Thus,

DK 2023 70557 A1 60 1 noise pollution is reduced. Furthermore, in this example embodiment wherein the pipe 2 217 is arranged around the periphery of the base of the enclosure 6, this 3 advantageously allows for the provision of a centrally located acoustic device (e.g. a 4 — transducer or loudspeaker, not shown) without interfering with transmission of the — soundwaves generated therefrom. 6 7 Figure 19 is a diagram of a cross sectional view of an example embodiment of an 8 enclosure 6 for use to treat ectoparasitic infestation, containing an acoustic device, 9 herein the form of a loudspeaker 216. The enclosure 6 retains an outer tarpaulin layer 218 and an inner tarpaulin layer 220. The acoustic device 216 is disposed in a lower 11 region of the enclosure 6, within the inner tarpaulin layer 220. The acoustic device 216 12 generates sound waves 223. A liquid contained in the outer tarpaulin 218 can 13 effectively blocks further, undesirable sound transmission. In other words, the liquid 14 limits noise pollution which would otherwise be cause by the sound waves 223 from the acoustic device 216 being transmitted outside of the enclosure 6. The liquid may 16 be the aqueous solution described herein (i.e. an aqueous solution comprising 17 — hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide, 18 optionally comprising dissolved oxygen) and may comprise a relatively high density of 19 relatively small bubbles. The enclosure may contain additional mixing means (e.g. a stirrer, not shown) to help maintain homogeneity of the fluid, thereby compensating any 21 gas loss. The mixing by the mixing means may encourage the creation of bubbles (e.g. 22 by decomposition of hydrogen peroxide by the enzyme) and/or the coalescence of 23 — smaller bubbles into larger bubbles. 24

The process parameters can be set by experiment and adapted for different parasites 26 and aquatic animals. Optimisation includes taking into account the effect of hydrogen 27 peroxide on the aquatic animals and in particular determining the hydrogen peroxide 28 concentration and treatment time taking into account the tolerance of hydrogen 29 peroxide of the aquatic animal. The ultrasound power required for the bubble collapse phase can be determined initially by calculation of the sound power density and the 31 Blake threshold pressure in the conditions which will be experienced in use and then 32 optimised. Optical microscopy and video can be employed to monitor bubble size and 33 also to view damage to endoparasites. The ultrasound power and frequency for the 34 preliminary bubble collapse phase can be determined through experiment. The frequency and power levels during the bubble regulation phase can also be determined 36 through experiment to obtain a target bubble size. The duration of the intermission 37 phase can also be determined experimentally, bearing in mind that the jetting effect

DK 2023 70557 A1 61 1 requires the bubbles to be within about 2 bubble diameters of the surface of the 2 —>oendoparasite. The concentration of hydrogen peroxide can be determined 3 experimentally. The amount of enzyme (e.g. catalase) can be determined 4 experimentally. The rate of disappearance of hydrogen peroxide may be followed by observing the rate of decrease in the absorbance at 240 nm to thereby determine the 6 enzyme units (U) present. The speed and duration of mixing can also be varied and/or 7 — determined experimentally. 8 9 The enzyme units (U) may be determined using standard protocols, as will be — understood by the person skilled in the art, and such protocols will vary in dependence 11 on the enzyme used. For example, where the enzyme is catalase, a UV 12 spectrophotometric assay may be carried out to measure the decrease in hydrogen 13 peroxide as the following reaction takes place: 14 2H,0, 22% 21,0 + 0, 16 17 — For example, in determining the enzyme units of catalase, the following steps may be 18 — used, however the skilled person will appreciate that other methods may be suitable: 19 Using ultrapure water (218 MQxcm resistivity at 25 °C) for the preparation of reagents, prepare a 50 mM potassium phosphate buffer for pH 7.0 at 25 C. To prepare 200 ml: 21 - Add 6.15 ml of 1.0 M Potassium phosphate dibasic solution (e.g. Sigma Aldritch 22 Catalog Number P8584) into a beaker; 23 - Add 3.85 ml of 1.0 M Potassium phosphate monobasic solution (e.g. Sigma 24 Aldritch Catalog Number P8709); - Make up the final volume to 200 ml using ultrapure water. 26 - Adjust the pH to 7.0 at 25 °C using 1 M KOH or HCI. 27 28 > Prepare a Hydrogen Peroxide Solution at 0.036% (w/w) 29 - Prepare in Phosphate Buffer using hydrogen peroxide (30% (w/w), e.g. Sigma

Aldritch Catalog Number H1009); 31 - Determine the A24, of this solution using Phosphate Buffer as a blank. The Az40 32 must be between 0.550 and 0.520 absorbance units; 33 - If necessary, add hydrogen peroxide to increase the absorbance of Phosphate; 34 - Buffer to decrease the absorbance; - Keep solution chilled on ice during assay. 36

DK 2023 70557 A1 62 1 Prepare a Catalase Solution with Catalase supplied as a crystalline suspension, such 2 as Sigma Aldritch Catalog Number C30. 3 a. Incubate product at 37 °C for ~1 hour to obtain complete dissolution. 4 b. Using a positive displacement pipette, prepare an initial dilution of "1,000 units/ml in 37 °C Phosphate Buffer. 6 c. Incubate initial dilution for "1 hour at 37 °C until dissolution is achieved (no swirls). 7 d. Immediately before use, perform a secondary dilution to ~100 units/ml in 37 °C 8 Phosphate Buffer. The secondary dilution must be prepared fresh each time. 9 -In a 3.00 ml reaction mix, the final concentrations are “50 mM potassium phosphate, 11 0.036% (w/w) hydrogen peroxide, and ~10 units of catalase. 12 13 1. Using a suitable thermostatted spectrophotometer set at A4, and 25 °C, blank 14 the instrument versus a quartz cuvette containing Phosphate Buffer; 2. Pipette 2.90 ml of Hydrogen Peroxide Solution into a quartz cuvette (Note: Run 16 only one Test at a time); 17 3. Place the cuvette in the spectrophotometer and allow the substrate to 18 equilibrate to 25 °C; 19 4. Add 0.10 ml of Catalase Solution to the cuvette, immediately mix by inversion and monitor the decrease in absorbance by taking one reading per second for 21 ~180 seconds; 22 5. Record the time required for the Az4) to decrease from 0.45 to 0.40 absorbance 23 units; 24 6. Perform steps 1to 5 three times. 26 — The following calculation may then be used to determine the number of enzyme units: 27 08 Units _ (3.45)(df) ml enzyme (time)(0.1) 29 — Where: 3.45 corresponds to the decomposition of 3.45 umoles of hydrogen peroxide 31 in a 3.0 ml reaction mixture producing a decrease in the Ax from 0.45 to 0.40; df is 32 dilution factor; time is minutes required for the Az to decrease from 0.45 to 0.40 33 absorbance units, and 0.1 is millilitre of enzyme added to the cuvette. 34

Nevertheless, the skilled person will appreciate that the rate of the reaction in which 36 — hydrogen peroxide decomposes into water and oxygen will be limited by one or more

DK 2023 70557 A1 63

1 of: the concentration of hydrogen peroxide, the amount of enzyme (e.g. catalase) and

2 the rate of mixing of the aqueous solution.

3

4 — Although the above examples have predominantly focused on asymmetric bubble collapse, which we have found to be particularly effective in killing or injuring

6 ectoparasites, in some applications the sound wave properties are selected so that the

7 — bubble collapse will be symmetric.

This can be useful for example when killing amoeba.

8

9 In summary, there is provided an apparatus for aquatic animals (1), the apparatus comprising an aquatic enclosure (3, 9) for retaining an aquatic animal, wherein the 11 aquatic enclosure retains an aqueous solution comprising hydrogen peroxide and an 12 enzyme for catalytic decomposition of hydrogen peroxide. 13 14 Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not 16 — intended to and do not exclude other components, integers, or steps.

Throughout the 17 — description and claims of this specification, the singular encompasses the plural unless 18 the context otherwise requires.

In particular, where the indefinite article is used, the 19 specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, or groups 21 described in conjunction with a particular aspect, embodiment, or example of the 22 invention are to be understood to be applicable to any other aspect, embodiment or 23 — example described herein unless incompatible therewith.

All of the features disclosed 24 — in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any 26 combination, except combinations where at least some of such features and/or steps 27 — are mutually exclusive.

The invention is not restricted to the details of any foregoing 28 embodiments.

The invention extends to any novel one, or any novel combination, of 29 the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of 31 any method or process so disclosed.

Numerical ranges expressed in the format ‘from 32 x toy and ‘between x and y' are understood to include x and y, unless specified 33 otherwise.

When for a specific feature multiple optional ranges are described, it is 34 — understood that all ranges combining the different endpoints are also contemplated.

Claims

DK 2023 70557 A1 64 1 Claims 2

3 1. Apparatus for aquatic animals, the apparatus comprising an aquatic enclosure 4 for retaining an aquatic animal, wherein the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide and an enzyme for catalytic 6 decomposition of hydrogen peroxide. 7

8 2. Apparatus according to claim 1, wherein the apparatus comprises a mixing 9 means. 11 3. Apparatus according to claim 2, wherein the mixing means comprises means 12 for directing sound waves into the aquatic enclosure, optionally wherein the 13 means for directing sound waves comprises a transducer. 14 4, Apparatus according to any one preceding claim, wherein the apparatus 16 comprises means for directing sound waves into the aquatic enclosure, and 17 wherein the means for directing sound waves are means for directing sound 18 waves at a sound pressure level of between 160 dB and 240 dB, the sound 19 waves having a frequency of between 1 kHz and 100 kHz, inclusive. 21 5. Apparatus according to any one preceding claim, wherein the aquatic enclosure 22 comprises a cage, the cage being surrounded by one or more sheets of fabric. 23 24 6. Apparatus according to claim any one preceding claim, wherein the aquatic enclosure is located on a wellboat. 26 27 7. Apparatus according to any one preceding claim, wherein the aquatic enclosure 28 comprises at least two cages with a conduit therebetween, the conduit being 29 sized and shaped such that an aquatic animal may travel between the cages via the conduit. 31 32 8. Apparatus according to claim 7, wherein a mixing means is positioned within 33 the conduit. 34

9. Apparatus for aquatic animals, the apparatus comprising:

DK 2023 70557 A1 65 1 an aquatic enclosure for retaining an aquatic animal, wherein the 2 aquatic enclosure retains an aqueous solution comprising hydrogen peroxide 3 and an enzyme for catalytic decomposition of hydrogen peroxide; and 4 a mixing means configured to cause mixing of the aqueous solution to thereby increase the rate of decomposition of the hydrogen peroxide by the 6 enzyme. 7 8 10. Apparatus according to claim 9, wherein the mixing means is configured to 9 cause sufficient mixing to increase the rate of decomposition of hydrogen peroxide to thereby generate sufficient bubbles in the aqueous solution to 11 reduce light transmission through 10 centimetres of the aqueous solution by at 12 least 50%. 13 14 11. Apparatus according to any one preceding claim, wherein the apparatus comprises a nozzle for introducing the aqueous solution into the aquatic 16 enclosure. 17 18 12. Apparatus according to any one preceding claim, comprising means for 19 generating one or more bubble curtains, the or each bubble curtain defining a tubular shape. 21 22 13. Apparatus according to any one preceding claim, wherein the aqueous solution 23 further comprises a bubble curtain, the bubble curtain defining a tubular shape. 24

14. Apparatus according to any one preceding claim, wherein the aqueous solution 26 further comprises sound waves, the sound waves having a sound pressure 27 level of between 160 dB and 240 dB and a frequency of between 1 kHz and 28 100 k Hz, inclusive. 29

15. Apparatus according to any one preceding claim, wherein the aqueous solution 31 comprises 100% to 700% dissolved oxygen. 32 33 16. Apparatus for aquatic animals, the apparatus comprising an aquatic enclosure 34 for retaining an aquatic animal, wherein the aquatic enclosure retains an aqueous solution comprising 100% to 700% dissolved oxygen. 36 37

DK 2023 70557 A1 66 1 17. Apparatus for use in a method of reducing ectoparasitic infestation of an aquatic 2 animal, wherein the method comprises: 3 providing an aquatic enclosure for retaining an aquatic animal; 4 combining hydrogen peroxide and an enzyme for catalytic decomposition of hydrogen peroxide with water to thereby form an aqueous 6 solution; 7 introducing the aqueous solution into the aquatic enclosure; 8 introducing one or more aquatic animals into the aquatic enclosure to 9 thereby expose the aquatic animal to the aqueous solution; and keeping the one or more aquatic animals in the aquatic enclosure. 11 12 18. A method of reducing ectoparasitic infestation on aquatic animals, wherein said 13 method comprises administering hydrogen peroxide and an enzyme for 14 catalytic decomposition of hydrogen peroxide to an aquatic enclosure comprising aquatic animals. 16 17 19. A method of reducing ectoparasitic infestation on aquatic animals, wherein the 18 said method comprises administering an aqueous solution comprising 100% to 19 700% dissolved oxygen to an aquatic enclosure comprising aquatic animals.

21 20. A method according to claim 18 or claim 19, further comprising directing sound 22 waves into the aquatic enclosure. 23 24 21. A method according to any one of claims 18 to 20, further comprising mixing the hydrogen peroxide and the enzyme, or mixing the aqueous solution. 26 27 22. Use of an enzyme which catalyses the decomposition of hydrogen peroxide, to 28 enhance the efficacy of a method of reducing ectoparasitic infestation on 29 aquatic animals, wherein said method comprises administering hydrogen peroxide and the enzyme to an aquatic enclosure comprising aquatic animals. 31 32 23. Use of an enzyme for catalytic decomposition of hydrogen peroxide to enhance 33 the efficacy of a method according to any one of claims 18, 20 or 21, wherein 34 the method further comprises directing sound waves into the aquatic enclosure.

36 24. An enzyme which catalyses the decomposition of hydrogen peroxide, for use 37 in enhancing the efficacy of a method of reducing ectoparasitic infestation on

DK 2023 70557 A1 67 1 aquatic animals, wherein said method comprises administering hydrogen 2 peroxide and the enzyme to an aquatic enclosure comprising aquatic animals. 3 4 25. An enzyme which catalyses the decomposition of hydrogen peroxide, for use in enhancing the efficacy of the method according to claim any one of claims 6 18, 20, or 21, wherein the method further comprises directing sound waves into 7 the aquatic enclosure. 8 9 26. A method of reducing ectoparasitic infestation of an aquatic animal, wherein the method comprises: 11 providing an aquatic enclosure for retaining an aquatic animal; 12 combining hydrogen peroxide and an enzyme for catalytic 13 decomposition of hydrogen peroxide with water to thereby form an aqueous 14 solution comprising hydrogen peroxide and said enzyme; introducing the aqueous solution into the aquatic enclosure; 16 introducing one or more aquatic animals into the aquatic enclosure to 17 thereby expose the aquatic animal to the aqueous solution; and 18 keeping the one or more aquatic animals in the aquatic enclosure. 19

27. A method according to claim 26, wherein the method comprises mixing the 21 aqueous Solution in the aquatic enclosure. 22 23 28. A method according to claim 26 or claim 27, wherein the method comprises 24 exposing the aquatic animal to sound waves. 26 29. Apparatus according to claim 17 or a method according to any one of claims 18 27 to 27, wherein the apparatus comprises a nozzle for introducing the aqueous 28 solution into the aquatic enclosure, 29 or wherein the method comprises combining hydrogen peroxide and the enzyme with water to form an aqueous solution comprising hydrogen peroxide, 31 and introducing the aqueous solution into the aquatic enclosure. 32 33 30. Apparatus for aquatic animals, the apparatus comprising an aquatic enclosure 34 for retaining a freshwater aquatic animal, wherein the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide and an enzyme for catalytic 36 decomposition of hydrogen peroxide, and wherein the aqueous solution 37 comprises hydrogen peroxide.

DK 2023 70557 A1 68 1 2 31. Apparatus according to claim 29 or claim 30, for use in a method of reducing 3 ectoparasitic infestation of a freshwater aquatic animal, wherein the method 4 comprises: providing an aquatic enclosure for retaining a freshwater aquatic 6 animal; 7 combining hydrogen peroxide and an enzyme for catalytic 8 decomposition of hydrogen peroxide with water to thereby form an aqueous 9 solution comprising hydrogen peroxide; introducing the aqueous solution into the aquatic enclosure; 11 introducing one or more freshwater aquatic animals into the aquatic 12 enclosure to thereby expose the said freshwater aquatic animal to the aqueous 13 solution; and 14 keeping the one or more aquatic animals in the aquatic enclosure. 16 32. A method of reducing ectoparasitic infestation of a freshwater aquatic animal, 17 wherein the method comprises: 18 providing an aquatic enclosure for retaining a freshwater aquatic 19 animal; combining hydrogen peroxide and an enzyme for catalytic 21 decomposition of hydrogen peroxide with water to thereby form an aqueous 22 solution comprising hydrogen peroxide; 23 introducing the aqueous solution into the aquatic enclosure; 24 introducing one or more freshwater aquatic animals into the aquatic enclosure to thereby expose the said freshwater aquatic animal to the aqueous 26 solution; and 27 keeping the one or more aquatic animals in the aquatic enclosure 28 29 33. A method according to claim 32, wherein the method comprises exposing the aquatic animal to sound waves. 31 32 34. An apparatus, method, use, or product as claimed in any preceding claim, 33 wherein the enzyme is catalase. 34

35. An apparatus, method, use, or product according to claim 34, wherein the 36 aqueous solution comprises 0.5 pg or more of enzyme per 500 mg of hydrogen 37 peroxide.

DK 2023 70557 A1 69 1 2 36. An apparatus according to any one of claims 1to 17, 29 to 31, 34 or 35, wherein 3 the apparatus comprises one or more aquatic animals. 4

37. Use of an apparatus, or an aqueous solution, according to any one of claims 1 6 to 17, 29 to 31, 34 or 35, to disturb aquatic ectoparasites. 7 8 38. Use according to claim 37, wherein the apparatus or aqueous solution 9 comprises bubbles to disturb aquatic ectoparasites. 11 39. An aqueous solution comprising hydrogen peroxide and an enzyme for catalytic 12 decomposition of hydrogen peroxide, for use to reduce ectoparasitic infestation 13 of an aquatic animal. 14

40. An aqueous solution according to claim 39, wherein the aqueous solution 16 comprises bubbles, such that the bubbles form on ectoparasites to thereby 17 detach them from an aquatic animal or, 18 wherein the bubbles form on ectoparasites to thereby prevent the ectoparasites 19 from resettling on the aquatic animal. 21 41. An aqueous solution comprising: hydrogen peroxide; an enzyme for the 22 catalytic decomposition of hydrogen peroxide; and bubbles, for use in a method 23 of treating ectoparasitic infestation of an aquatic animal, wherein the aquatic 24 animal is exposed both to the aqueous solution and the bubbles. 26 42. Use according to claim 37 or 38 or an aqueous solution according to any one 27 of claims 39 to 41, wherein the ectoparasites are female ectoparasites. 28 29 43. Use according to claim 37 or claim 38, or an aqueous solution according to any one of claims 39 to 42, wherein the aqueous solution comprises a bubble 31 curtain configured to provide bubbles, wherein the bubble curtain defines a 32 tubular shape. 33 34 44, A kit of parts comprising apparatus for use in reducing ectoparasitic infestation of an aquatic animal, wherein the apparatus is according to any one of claims 36 1to 17, 29 to 31, 34, 35, or 36. 37

DK 2023 70557 A1 70 1 45. A method of disturbing, injuring or killing an aquatic ectoparasite, comprising a 2 step of exposing the ectoparasite to an aqueous solution comprised in 3 apparatus according to any one of claims 1to 17, 29 to 31, 34 or 35, particularly 4 to the bubbles.