CA3112091A1

CA3112091A1 - Methods and apparatus for aquatic ectoparasite reduction

Info

Publication number: CA3112091A1
Application number: CA3112091A
Authority: CA
Inventors: Ian Andrew Jamieson
Original assignee: Pulcea Ltd
Current assignee: Pulcea Ltd
Priority date: 2018-09-11
Filing date: 2019-09-11
Publication date: 2020-03-19
Also published as: CL2021000582A1; GB2584166A; GB2584166B; WO2020053311A1; NO20210408A1; AU2019340038A1; EP3849301A1

Abstract

Method and apparatus for killing aquatic ectoparasites by the application of hydrogen peroxide and sound waves. In the presence of hydrogen peroxide, bubbles of oxygen form in and adjacent the ectoparasites. In a bubble regulation phase, the frequency of the sound is controlled to cause bubble growth and combination. In a subsequent bubble collapse phase, the frequency of the sound is controlled to cause asymmetrical collapse of the bubbles, injuring or killing the ectoparasites. The method and apparatus is useful, for example, for killing sea lice (Lepeoptheirus salmonis) on salmon.

Description

2

3 Field of the invention

4 The invention relates to methods and apparatus for injuring or killing aquatic ectoparasites, reducing ectoparasitic infestation on aquatic animals and improving the 7 appearance, meat quality, meat quantity and growth rates of aquatic animals.

9 Background to the invention 11 The invention relates to the field of reducing the infestation of aquatic animals, such as 12 fish, by aquatic ectoparasites. In a non-limiting example, the commonly-farmed Atlantic 13 salmon (Salmo salar) is prone to infestation by sea lice of the species Lepeophtheirus 14 salmonis and embodiments of the invention act to kill or injure sea lice to reduce the significant damage to the fish (including fish death) arising from this infestation, which 16 can cause fish death or reduced yield. As well as the direct effect of sea lice on the 17 fish, sea lice infestation also causes a generalised chronic stress response in the fish, 18 which may make them susceptible to infection by other diseases and which may reduce 19 meat yield.

2018/115826 (Armstrong et al.) discloses a method of killing ectoparasites in environments such as fish farms, by introducing hydrogen peroxide into the water 23 around fish and applying ultrasound. The presence of hydrogen peroxide causes 24 bubbles of oxygen to be generated on the surface of and/or within the body of ectoparasites. The ultrasound causes alternating compression and rarefaction of the 2 bubbles, and by suitable choice of the frequency of the ultrasound causes bubbles to 3 damage and kill ectoparasites. Because the method of injuring or killing the aquatic ectoparasite is principally physical, the method is effective even when applied to aquatic ectoparasites which are resistant to chemical-only methods (such as peroxide-resistant ectoparasites). Indeed, ectoparasites which are peroxide-resistant due to the 7 presence of high levels of catalase and other peroxidase enzymes may be more vulnerable to these treatment methods because these enzymes will cause more rapid 9 formation of oxygen bubbles.
11 The present invention seeks to improve this treatment process for example by one or 12 more of reducing the impact on fish, reducing the impact (which is a function of both wavelength and power) of ultrasound transmitted into the environment, reducing the 14 power consumption, improving the speed of the process and/or improving the effectiveness of the process.

17 Summary of the invention 19 A first aspect of the invention provides a method of injuring or killing an aquatic ectoparasite comprising: exposing the aquatic ectoparasite to an aqueous solution comprising hydrogen peroxide (i.e. H202), leading to the formation of bubbles (typically predominantly of oxygen), generating sound waves having a controllable frequency 23 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein 24 the frequency spectrum (and optionally the power) of the sound waves is varied with time.

27 The bubbles comprise gas formed by the action on hydrogen peroxide of enzymes, 28 such as catalase, peroxidases etc. within and on the ectoparasite. The bubbles 29 therefore comprise (typically predominantly) oxygen.
31 The method extends in a second aspect to a method of operating an apparatus, the apparatus comprising an aquatic enclosure comprising an aqueous solution of 33 hydrogen peroxide, the solution comprising bubbles, the bubbles comprising oxygen (typically being predominantly of oxygen), and at least one sound source configured to direct sound waves at the bubbles, wherein the frequency spectrum (and optionally the 36 power) of the sound waves is varied with time. The apparatus may be for killing or 37 injuring aquatic ectoparasites, optionally on live fish.

Typically, the method comprises a bubble regulation phase in which the frequency 3 spectrum (and typically power) of the sound waves is controlled. It may be that the frequency spectrum (and typically power) of the sound waves is controlled to cause bubble growth and/or coalescence. It may be that the frequency spectrum (and typically 6 power) of the sound waves is controlled to cause bubble resonance (e.g. as bubbles 7 change in size).

9 The bubble regulation phase may comprise or consist of one or more descending frequency phases during which the centre and/or peak frequency of the sound is 11 reduced (typically monotonically and typically progressively although it may for 12 example be reduced through a plurality of, e.g. three or more intermediate steps). The 13 bubble regulation phase may comprise a plurality of (e.g. consecutive) descending frequency phases. The bubble regulation phase may comprise at least three or at least four descending frequency phases. It may be that between 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 frequency). The decreasing frequency favours the creation of large bubbles, for example through 19 smaller bubbles merging. Without wishing to be constrained by theory, the inventor believes this to arise for a number of reasons, including because sound waves (acoustic field) favour the creation or and stability of bubbles having a diameter giving 22 a resonant frequency which is similar to the centre (and/or peak) frequency of the 23 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 mucus acting as a surfactant which helps to drive bubble formation overtime, and the sound waves then 26 causing bubbles to coalesce.

28 In some conditions, and at some frequencies, it has been observed that a plurality of 29 smaller bubbles in close proximity to each other can behave in a similar way to one larger bubble when excited by sound waves. It may be that during some or all of the 31 bubble regulation phase acoustic pressure is regulated, e.g. is increased and/or decreased. Regulating the acoustic pressure can help to encourage the coalescence 33 of bubbles.

It may be that the sound is substantially monotonic although in practice it may have a significant bandwidth and so we refer to the centre (and/or peak) frequency of the 37 sound.

2 The bubble regulation phase(s) may have a duration of at least 1 second and/or the 3 one or more descending frequency phases may have a (e.g. combined) duration of at 4 least one second. The bubble regulation phase(s) may have a duration of less than 10 seconds and/or the one or more descending frequency phases may have a (e.g.

combined) duration of at least 10 seconds. Preferably, the bubble regulation phase(s) 7 may have a duration of at least 1 minute and/or the one or more descending frequency 8 phases may have a (e.g. combined) duration of at least 1 minute, however in some 9 examples the bubble regulation phase(s) may have a duration of at least 3 minutes or at least 4 minutes and/or the one or more descending frequency phases may have a 11 (e.g.
combined) duration of at least 3 minutes or at least 4 minutes. Preferably, the 12 bubble regulation phase(s) may have a duration of less than 20 minutes, preferably 13 less than 15 minutes, or less than 12 minutes and/or the one or 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 may be 16 reduced from greater than 10 kHz to less than 5 kHz, for example from about 20 kHz 17 to 3 kHz. Preferably, the peak and/or centre frequency will be reduced from 10 kHz 18 2 kHz to 3 kHz 1kHz, or from 6 kHz 2 kHz to 3 kHz 1 kHz. In some examples the 19 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 21 of change of sound amplitude may be linear or may be non-linear. The rate of peak 22 and/or centre frequency change during the (e.g. each of the) one or more descending frequency phases may for example be in the range of 0.5 to 2 kHzs'. Typically, after 24 each descending frequency phase the frequency is instantaneously returned to the higher frequency that preceded the descending frequency phase before a subsequent 26 descending frequency (where present) phase begins.

28 During the or each of the one or more descending frequency phases, the peak and/or 29 centre frequency of sound that is generated may decrease in frequency by at least 25%
or at least 40%, for example. Typically, the peak and/or centre frequency decreases by 31 less than 90%. A decrease in peak and/or centre frequency of around 50%, for 32 example, will support an approximate doubling in the diameter of bubbles (because the 33 resonant frequency is inversely proportional to bubble diameter).

It may be that the bubble regulation phase(s) has a duration of at least 1 minute or at 36 least 2 minutes. It may be that the one or more descending frequency phases has a 37 (e.g.
combined) duration of at least 1 minute or at least 2 minutes. It may be that the 1 one or more descending frequency phases has a (e.g. combined) duration of at least 2 30 seconds or that the bubble regulation phase(s) has a duration of at least 30 seconds.
3 It may be that the peak and/or centre frequency of sound that is generated during the 4 or each of the one or more descending frequency phases is reduced at a rate of at least

5 0.05 kHz/s, or preferably at least 0.1 kHz/s, or at least 0.2 kHz/s.

6

7 It may be that the method further comprises an intermission phase, subsequent to the

8 or each of the one or more descending frequency phases, during which intermission

9 phase sound waves which cause oscillation of the bubbles are restricted in intensity (for example stopped). Generally, the bubbles present at the end of the one or more 11 descending frequency phases will have a diameter such that they have a resonant 12 frequency similar to the frequency of the sound at the end of the one or more 13 descending frequency phases (optionally at the end of each of the one or more 14 descending frequency phases). This sound is then restricted, or stopped.
16 This may result in bubbles becoming so large they detach from fish and/or 17 ectoparasites and provide a time for new bubble to form (from the decomposition of 18 hydrogen peroxide to oxygen).

The method may comprise a plurality of said descending frequency phases 21 interspersed with said intermission phases. The method may comprise alternating 22 descending frequency phases and intermission phases, for example cyclically. The 23 method may comprise a plurality of descending frequency phases, followed by an 24 interval phase, followed by a further plurality of descending frequency phases (and this may be repeated). The plurality of said descending frequency phases may be the same 26 as each other although this is not essential. The plurality of intermission phases may 27 be the same as each other. However, it may be that the duration of the intermission 28 phases is varied. It may be that bubbles form during the intermission phases, and grow 29 and/or are combined during the or each of the one or more descending frequency phases (or the plurality of descending frequency phases), facilitated at least in part by 31 the descending frequency sound.

33 In some embodiments, the method comprises varying the frequency spectrum (and 34 typically power) to cause collapse of the bubbles, typically to cause the bubbles to grow, and then to collapse in response to sound waves, typically in response to a 36 change in the frequency spectrum (and optionally the power) of the sound waves. This 37 enables the time when the bubbles collapse to be controlled.

Therefore, it may be that the method comprises a bubble regulation phase (in which 3 the frequency spectrum of the sound waves is controlled to cause bubble growth and/or coalescence) and a subsequent bubble collapse phase, in which the frequency spectrum (and typically power) of the sound waves is controlled to cause the collapse 6 of bubbles. The bubble regulation phase may comprise a preliminary bubble collapse 7 phase, prior to the one or more descending frequency phases.

9 It may be that the bubble collapse phase is an asymmetric bubble collapse phase in which the frequency spectrum (and typically power) of the sound waves is controlled 11 to cause the asymmetric collapse of bubbles.

13 By providing a separate bubble regulation phase and bubble collapse phase, the 14 bubbles can be controlled to a desired size range, chosen to be effective during subsequent bubble collapse, at a moderate or low power before being caused to 16 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 regulation phase, or less than 50% of, less than 25% or less than 10% or even less 19 than 5%
of the duration of the bubble regulation phase). Thus, the highest power sound waves are generated for only a relatively short period of time.

22 The bubble collapse phase may have a duration of less than 1 s, or less than 100 ms, 23 or even less than 10 ms. However, the bubble regulation phase typically has a duration 24 of at least 3 seconds, at least 10 seconds, at least 30 seconds or at least a minute (for example to allow time for bubbles to migrate and coalesce) and/or the period of time 26 between bubble collapse phases may be at least 10 seconds, at least 30 seconds or 27 at least a minute. The bubble collapse phase may take place for less than 5% or less 28 than 2%
or less than 1% of the time. Thus, again the highest power sound waves are 29 generated during only a relatively small fraction of the treatment time.
31 The variation in the frequency (and optionally power) of the sound waves during the 32 bubble regulation phase is typically selected to favour the formation and maintenance 33 of bubbles within a predefined size range. The frequency (and optionally power) of the 34 sound waves during the bubble collapse phase is typically selected to cause the collapse (typically asymmetrical collapse) of bubbles within the said predefined size 36 range.

Typically, the mean power of the sound waves in the bubble collapse phase is at least 2 double, or at least 5 times or at least 10 times, higher than the mean power during the 3 bubble regulation phase. Typically, the peak power of the sound waves in the bubble 4 collapse phase is at least double, or at least 5 times or at least 10 times, higher than the mean power during the bubble regulation phase. The power of the sound during 6 the bubble collapse phase may be selected to create an acoustic pressure of at least 7 50 kPa.

9 It may be that the peak power of the sound waves in the bubble collapse phase is sufficient to cause bubble non-symmetric collapse in the aquatic enclosure but not 11 sufficient to cause cavitation (which would increase damage to the aquatic creature).

13 The duration of the bubble regulation phase is typically in the range 1 to 15 minutes, 14 for example 2 to 5 minutes. The duration of the bubble collapse phase is typically less than a minute. The duration of the bubble collapse phase is typically less than 20% or 16 less than 10% of the duration of the bubble regulation phase. The duration of the bubble 17 collapse phase is typically less than 20% or less than 10% of time between bubble 18 collapse phases.

The power of the sound waves which are generated and directed at the bubbles is controlled to thereby regulate the power of the sound waves in a target volume, where 22 there are both the bubbles and the aquatic ectoparasite.

24 The sound waves generated during the bubble collapse phase are preferably selected to cause liquid jetting (during bubble collapse). This is a kind of bubble asymmetrical collapse. It is known that when gas bubble collapse adjacent a surface they may form 27 a jet of liquid towards the surface. In bubble jetting, the bubble collapses in a manner 28 where the side furthest away from an adjacent surface/target moves quickest and collapses through the bubble during the positive acoustic pressure phase. This frequently causes a fast-moving jet of water which can puncture most biological 31 surfaces including the surface of ectoparasites. The person skilled in the art can determine the necessary power and frequency of sound waves to cause jetting and 33 can observe whether this is occurring by optical microscopy.

Typically, during the bubble collapse phase the acoustic waves have an intensity such 36 that their acoustic pressure is greater than the Blake threshold pressure of the majority, 37 by volume, of bubbles.

2 By the Blake threshold pressure, we refer to the bubble forcing pressure above which 3 bubbles will grow quasistatically without bound. The Blake threshold pressure is for 4 example referred to in Akhatov et al. I, Gumerov, N., Ohl, C.D., Parlitz, U. and Lauterborn, W, "The Role of Surface Tension in Stable Single-Bubble Sonoluminescence" (Physics Review Letters, 78(2), 227-230, 1997) and Blake, F.G.
7 "The Onset of Cavitation on Liquids" (Technical Memo. 12, Acoustic Research Laboratory, Cambridge, MA, Harvard University, 1949) and Louisnard, 0. and 9 Gonzalez-Garcia, J. (H. Feng et al., eds), "Acoustic Cavitation" in Ultrasound Technologies for Food and Bioprocessing, DOI I 10.1007/978-1-4419-7472-3 2, which 11 can be calculated by the person skilled in the art and which is given, for an air bubble 12 in water in ambient conditions (a=0.0725N.m-1 p,eq=2,000 Pa, po=100kPa) by:

4 a _ 3 14 ,crit ..,,, O P,, S
Pa ¨ P + v,eq + Po, j(27 .1 + as) 16 Where Kra is the Blake threshold pressure, po is liquid pressure, pv,eq is vapour equilibrium saturation pressure and as is dimensionless Laplace tension 2o-/poRo 18 (where Ro is the ambient radius of the bubble).

Typically, prior to the bubble collapse phase and/or during the bubble regulation phase 21 and/or between the preliminary bubble collapse phase (where present) and the bubble 22 collapse phase, the frequency of the sound waves is controlled to not exceed a frequency which will cause (e.g. asymmetrical) collapse of bubbles but during the 24 bubble collapse phase the frequency of the sound exceeds the frequency which will cause (e.g. asymmetrical) collapse of bubbles and thereby causes (e.g.
asymmetrical) 26 collapse of bubbles.

28 The bubble regulation phase and bubble collapse phase may be repeated with different variations in the frequency (e.g. intensity) of sound waves with time during the bubble regulation and bubble collapse phases. This enables the procedure to target parasites 31 with different properties.

33 There may be an intermission phase after the bubble regulation phase and before 34 bubble collapse phase. During the intermission phase some bubbles will detach from the aquatic ectoparasite, due to flow of aqueous solution or buoyancy. We have found 1 that by having a bubble regulation phase and a subsequent bubble collapse phase, 2 with an intermission phase therebetween, to allow some distance to develop between 3 some of the bubbles and the aquatic ectoparasite, damage to the aquatic ectoparasite 4 during the bubble collapse phase can be increased. Bubbles may detach from the ectoparasite once they reach a size where the forces arising from the buoyancy of the 6 bubbles exceeds retentive forces. Bubbles may also become detached due to the 7 movement of the ectoparasite through the water, especially where the ectoparasite is 8 attached to the surface of a fish or other aquatic animal which is swimming through 9 water.
In some examples the frequency and/or amplitude of sound generated during the bubble regulation phase may optionally be selected such that it corresponds to the frequency and/or amplitude necessary to cause the oscillation of bubbles of a predetermined size (and/or to prevent bubbles of a said predetermined size from being 13 lost due to buoyancy).

The duration of the intermission phase may for example be at least 10 ms, at least 100 16 ms, or at least 1 second. Typically, it is less than 10 minutes, less than 5 minutes or 17 less than 3 minutes. However, in some conditions it may be less than 10 seconds or 18 less than 1 second, maybe even less than 100 ms, for example in fast flowing water 19 (e.g.
when water is flowing relative to the aquatic animal at 0.5 - 2 m/s and it is desired for a bubble of e.g. 1 mm radius to move 0.2 - 1 mm).

22 It may be that the frequency (and optionally power) of the sound waves is controlled to 23 promote bubble coalescence immediately prior to the bubble collapse phase. For 24 example, two bubbles with a diameter of 2 mm might be subject to ultrasound at 3.3 kHz, which drives them to coalesce, forming a bubble with a diameter of 2.52 mm which 26 is then subject to sound waves at 2.6 kHz to cause collapse.

28 The method may comprise retaining bubbles close to or in contact with the surface of 29 an ectoparasite by controlling the sound waves using Bjerknes forces. It may be that for at least some time during the bubble regulation phase, including potentially during 31 the intermission phase, the frequency (and optionally power) of the sound waves is 32 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 frequency (and optionally power) of the sound waves is selected to cause the detached bubble to remain close to the surface of the ectoparasite by Bjerknes forces.
This can 36 enable the build up of a significant volume of bubble(s) close to or attached to the 37 surface of the ectoparasite. This is significant because asymmetrical bubble collapse 1 (such as jetting) causes significant damage to structures within 2 bubble diameters of 2 the surface of the bubble and so ideally bubbles are retained close to the surface of 3 the ectoparasite.

Asymmetrical bubble collapse proximate a surface can lead to jetting specifically 6 towards the surface, which in this case would be a surface of the aquatic ectoparasite.
7 Some of the bubbles may however remain attached to the surface, or within the interior 8 of the aquatic ectoparasite.

10 Bubble jetting occurs preferentially when the bubbles are spaced apart from the

11 adjacent surface but there is a ratio of unforced bubble radius to bubble centre to

12 adjacent surface distance of less than 1:5, or less than 1:2.5 or less than 1:1.5, e.g.

13 1:1.1 to 1:1.3, with about 1:1.2 giving good results. By unforced bubble radius, we refer

14 to the radius which the bubble would have if not subject to ultrasound (which leads to oscillations etc).

Typically, the duration of the intermission phase is selected so that for at least 25% of 18 or at least the majority by volume of bubbles which have detached from or are located 19 on the surface of the aquatic ectoparasite, the distance between the bubble and the surface of the aquatic ectoparasite is less than double the diameter of the respective 21 bubble.
Bubbles which are further than double their diameter from the surface of the 22 aquatic ectoparasite will be less effective.

24 During the intermission phase it may be that no sound waves are generated and directed to the aquatic ectoparasite (e.g. from a sound source).
Alternatively, it may be 26 that relatively low power, relatively low frequency sound waves are generated during 27 the intermission phase to maintain bubbles. In this case, the power and frequency of 28 the sound waves are typically each lower than the power and frequency of the sound 29 waves at the end of the bubble regulation phase. To this effect, the bubbles may be stimulated with acoustic waves having an acoustic pressure which is less than the 31 Blake threshold pressure. They continue to oscillate but are in a stable condition.
They 32 can still migrate by the effects of Bjerknes forces. A balance can therefore be struck 33 between the driving acoustic force causing the bubbles to migrate towards the 34 ectoparasite and detachment due to buoyancy and/or fluid flow.
36 It may be that prior to the bubble collapse phase, sound waves are generated at a 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 2 excessively large bubbles.

Nevertheless, it may be that there is not a bubble collapse phase after the one or more descending frequency phases. We have found that the oscillation of bubbles which 6 have been grown and/or coalesced in the one or more descending frequency phases 7 can cause substantial damage to aquatic ectoparasites. We hypothesize that this is 8 due to the very substantial local forces caused by oscillating bubbles.

The bubble regulation phase may comprise a preliminary bubble collapse phase, prior 11 to the one or more descending frequency phases. During the bubble regulation phase 12 sound is typically generated at a lower frequency and lower power than during the 13 bubble collapse phase. Typically, bubble collapse during the preliminary bubble 14 collapse phase is predominantly surface bubble collapse. This has the effect of collapsing bubbles which are already present, especially on fish (where present). This 16 has the benefit of removing bubbles which are larger than a threshold size, so that the 17 range of bubble sizes at the beginning of a descending frequency phase (e.g. a one of 18 the one or more descending frequency phases) is more defined and/or removing 19 bubbles left after a previous cycle (of a bubble regulation phase followed by a bubble collapse phase). This also has the benefit of cleaning fish - causing bubbles to collapse 21 on the surface has been found to clean fish. During the preliminary bubble collapse 22 phase sound waves may be selected to target relatively flat bubbles. Partially flattened 23 (e.g.
oblate spheroid) bubbles can be formed in hydrophilic surface layers of fish etc.
24 as opposed to more spherical bubbles being formed on the waxy hydrophobic surface of the sea lice. Their oscillations can be targeted by considering their thickness along 26 their axis and selecting sound waves with a wavelength which stimulates oscillations 27 of bubbles having that thickness.

29 The preliminary bubble collapse phase typically has a duration of less than 10 seconds or less than 5 seconds.

32 There may be a waiting phase prior to the bubble regulation phase. The waiting phase 33 may be after the ectoparasite was brought into contact with the aqueous solution of 34 hydrogen peroxide or after the bubble asymmetric collapse phase of a previous cycle.
The waiting phase provides time for bubbles to start to be formed and grow, through 36 the decomposition of hydrogen peroxide to form oxygen.

1 The waiting phase may have a duration of at least 10 seconds, or at least 30 seconds 2 or at least 1 minute or at least 2 minutes or at least 5 minutes or at least 10 minutes.
3 The waiting phase may be shorter than 20 minutes, or shorter than 10 minutes, or 4 shorter than 3 minutes, for example.
6 The formation of bubbles can be temperature dependent, for example hydrogen 7 peroxide may be converted to oxygen at a higher rate at higher temperature. It may be 8 that the method comprises determining (e.g. measuring) the temperature of the 9 aqueous solution and varying one or more of: the duration of the bubble regulation phase, the frequency (and optionally power) during the bubble regulation phase, the 11 duration of the bubble collapse phase, the frequency (and optionally power) during the 12 bubble collapse phase, the duration of the intermission phase (where present), the 13 duration of the waiting phase (where present), the duration of the one or more descending frequency phases (where present), the frequency (and optionally power) of sound waves and the variation of that with time during the one or more descending frequency phases (where present), the duration of the preliminary bubble collapse 17 phase (where present), the frequency (and optionally power) of sound waves during 18 the preliminary bubble collapse phase (where present).

The bubble regulation phase and bubble collapse phase may be repeated.

22 The bubble regulation phase and bubble collapse phase may be repeated with different variations in the frequency (e.g. intensity) of sound waves with time during the bubble 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 26 waves in the bubble collapse phase between cycles may vary by more than 10%, for 27 example.

29 The method may comprise exposing the ectoparasite to an aqueous mixture of hydrogen peroxide and a surfactant. Although it is counterintuitive to include a surfactant when seeking to cause bubble collapse, the surfactant will assist in stabilising the bubbles prior to the bubble collapse phase, to enable them to grow to a 33 selected size range. Surfactants may also increase the tendency of bubbles to detach 34 from the surface. Suitable food safe surfactants used in recirculating aquaculture systems to prevent foaming are suitable and known to the person skilled in the art.

1 It may be that exposing the aquatic ectoparasite to the aqueous solution of hydrogen 2 peroxide comprises immersing (i.e. submerging) the aquatic ectoparasite in the 3 aqueous solution of hydrogen peroxide. It may be that exposing the aquatic ectoparasite to the aqueous solution of hydrogen peroxide comprises immersing (i.e.
submerging) the aquatic ectoparasite at least partially in the aqueous solution of 6 hydrogen peroxide. It may be that exposing the aquatic ectoparasite to the aqueous 7 solution of hydrogen peroxide comprises immersing (i.e. submerging) the aquatic 8 ectoparasite fully in the aqueous solution of hydrogen peroxide.

It may be that exposing the aquatic ectoparasite to the aqueous solution of hydrogen 11 peroxide comprises providing the aquatic ectoparasite in an aquatic environment (i.e.

providing the aquatic ectoparasite immersed in (i.e. submerged under) water or an 13 aqueous solution) and adding hydrogen peroxide to that aquatic environment (i.e. to 14 the water or the aqueous solution).
16 It may be that exposing the aquatic ectoparasite to the sound waves comprises generating said sound waves within the aqueous solution. It may be that exposing the 18 aquatic ectoparasite to the sound waves comprises generating said sound waves 19 within the aquatic environment (i.e. in the water or the aqueous solution) in which the aquatic ectoparasite is provided. It may be that exposing the aquatic ectoparasite to 21 the sound waves comprises directing said sound waves at the aquatic ectoparasite.

23 It may be that the aquatic ectoparasite is provided inside an aquatic enclosure and that 24 exposing the aquatic ectoparasite to the sound waves comprises directing said sound waves into the aquatic enclosure.

27 The method may comprise pressurising the aqueous solution comprising hydrogen 28 peroxide and ectoparasites. This may for example be achieved by retaining the 29 aqueous solution comprising hydrogen 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 of the aquatic enclosure, for 32 example by introducing a gas, such as air, above the aqueous solution in the aquatic enclosure, or a pressurisable bladder adjacent to or within the aqueous solution). The 34 aquatic enclosure may need to be sufficiently solid to resist the efflux of aqueous solution but need not be watertight and may for example contain apertures or take the 36 form of a tube or similar.

1 This has the advantage that because the resonant frequency of an air bubble in 2 aqueous solution varies with liquid pressure, by raising the pressure in the aquatic enclosure, the ratio of pressure at the bottom of the aquatic enclosure to the pressure 4 at the top of the container is smaller than would otherwise be the case. Accordingly, the variation in the frequency of the sound waves required during the bubble regulation 6 phase and the bubble collapse phase to have a desired effect (regulating the size of 7 bubbles, causing bubble collapse) within the aquatic enclosure is reduced. This 8 enables better control of bubble size and collapse.

The pressure at the top of the aquatic medium in the aquatic enclosure may for 11 example be raised to at least 1.5 atm (i.e. 151,987 MPa) or to at least 2 atm (i.e.
12 202,650 Pa).

Increased pressure may also be obtained by providing the aqueous solution comprising hydrogen peroxide and ectoparasites under another body of aqueous 16 solution, for example under a volume of water.

18 The method may comprise reducing the pressure of the aqueous solution so that, at 19 the top (e.g. surface) of the aqueous solution, the pressure of the aqueous solution is below atmospheric pressure, for example at most 1 atm (i.e. 101,325 Pa) or at most 21 0.9 atm (i.e. 91,192 Pa) or at most 0.75 atm (i.e. 75,994 Pa) or at most 0.5 atm (i.e.
22 50,663 Pa). This has the effect of promoting larger gas bubbles. As it is the size of 23 the bubble rather than the amount of gas (predominantly oxygen) within them that determines the damaging effect of bubble collapse and jetting this can make the process more efficient.

Alternatively, or in addition, the frequency (peak and/or centre frequency) of the sound 28 waves within the aquatic enclosure (i.e. which are generated and directed into the 29 aquatic enclosure) may vary with depth (e.g. increasing with depth), e.g. proportional to the square root of the water pressure at a given depth. The sound may be generated 31 by transducers located in a base region of the aquatic enclosure and have a range of frequencies, such that the peak and/or centre frequency of the sound waves within the 33 aquatic enclosure increase with depth due to the greater attenuation of higher 34 frequency sounds with distance from the transducers.
36 The aquatic enclosure may be a flexible enclosure. The aquatic enclosure may be a 37 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 2 (e.g.
urethane-coated canvas such as tarpaulin). The aquatic enclosure may comprise 3 a net or cage at least partially surrounded by a one or more sheets of waterproof or 4 water-resistant fabric. The aquatic enclosure may be an aquarium. The aquatic enclosure may be located on a sailing vessel. The aquatic enclosure may be located 6 on (e.g.
form part of) a boat or ship. The aquatic enclosure may be located on (e.g.
7 form part of) a wellboat. The aquatic enclosure may comprise (e.g. be) a channel or a 8 barge.
The aquatic enclosure may have an inlet and an outlet. The aquatic enclosure 9 may be a treatment enclosure located on a wellboat. The treatment enclosure may 10 have an inlet in fluid communication with an external aquatic environment (i.e.
outside 11 the wellboat).

13 The wellboat may comprise one or more water flow regulators (e.g. a pump or a siphon) configured to (i.e. in use) transport (e.g. pump) water from the external aquatic

15 environment into the treatment enclosure. The wellboat may comprise one or more

16 water flow regulators (e.g. a pump or a siphon) configured to transport (e.g. pump)

17 water from the treatment enclosure into the external aquatic environment. The wellboat

18 (e.g.
the treatment enclosure, for example the water flow regulator) may be provided

19 with aquatic ectoparasite filters configured to restrict the transport of aquatic ectoparasites out of the treatment enclosure when water is transported (e.g.
pumped) 21 from the treatment enclosure to the external aquatic environment.

23 The aquatic enclosure may have one or more walls. The aquatic enclosure may be 24 located in an aquatic environment (e.g. in the sea), that is to say the aquatic enclosure may be surrounded by the aquatic environment (e.g. the sea). An interior of the aquatic enclosure may be separated from (e.g. isolated from) the surrounding aquatic environment by one or more (e.g. solid) walls. Alternatively, the aquatic enclosure may 28 be located onshore (i.e. on land, that is to say not in an aquatic environment such as 29 the sea).
31 The interior of the aquatic enclosure may be in fluid communication with the aquatic environment by way of one or more channels (e.g. pipes). Water may be transported 33 into and/or out of the aquatic enclosure through the one or more channels (e.g.
pipes).
34 The one or more channels (e.g. pipes) may be provided with aquatic ectoparasite filters configured to inhibit transport of aquatic ectoparasites between the interior of the 36 aquatic enclosure and the aquatic environment.

1 The aquatic enclosure may comprise (e.g. be) a treatment channel (e.g. a pipe) 2 provided between (e.g. connecting) first and second aquatic animal enclosures. Water containing aquatic animals to be treated may be pumped through the treatment 4 .. channel.
6 It may be that the aqueous solution comprises hydrogen peroxide at a concentration 7 greater than or equal to 20 mg/L. Concentrations of hydrogen peroxide greater than or 8 equal to

20 mg/L are typically more effective at generating bubbles, particularly when 9 the hydrogen peroxide is dissolved in fresh water.
11 It may be that the aqueous solution comprises hydrogen peroxide at a concentration 12 greater than or equal to 200 mg/L. Concentrations of hydrogen peroxide of greater than 13 or equal to 200 mg/L are typically more effective at generating bubbles, particularly 14 .. when the hydrogen peroxide is dissolved in seawater.
16 It may be that the aqueous solution comprises hydrogen peroxide at a concentration 17 less than or equal to 2500 mg/L. Concentrations of hydrogen peroxide greater than mg/L do not typically provide any additional benefit but are increasingly expensive 19 to achieve in practice and their use in aquatic environments may be restricted by .. environmental regulations in some jurisdictions.

21

22 It may be that the aqueous solution comprises hydrogen peroxide at a concentration

23 less than or equal to 2200 mg/L. In some jurisdictions, environmental regulations

24 restrict use of solutions of hydrogen peroxide having concentrations greater than 2200 mg/L.

27 It may be that the aqueous solution comprises hydrogen peroxide at a concentration 28 between 20 mg/L and 2500 mg/L, inclusive, or between 200 mg/L and 2500 mg/L, inclusive, or between 20 mg/L and 2200 mg/L, inclusive, or between 200 mg/L
and 2200 mg/L, inclusive.

32 It may be that the aqueous solution comprises hydrogen peroxide at a concentration of approximately 1500 mg/L (e.g. at a concentration of between 1300 mg/L and 1700 34 mg/L, inclusive). Aqueous solutions of hydrogen peroxide at concentrations of approximately 1500 mg/L have been approved by regulatory authorities in some jurisdictions for use in, for example, the treatment of parasitic infestations of the marine 37 phase of the Atlantic salmon. The duration of bubble growth prior to bubble collapse is 1 typically related to the hydrogen peroxide concentration and some examples are 2 described below.

4 The resonant frequency of a bubble of gas in an infinite volume of liquid is given by the Minnaert Formula A:

7 f = 13ypo 2nr\p 9 where r is the (unforced) bubble radius, y is the polytropic coefficient, Po is the ambient pressure and p is the density of the liquid. In practice, for bubbles formed in water, on 11 the surface, this formula can be approximated by Formula B:

3.26 r Where the bubbles are not at the surface, there is a depth term, giving formula C, where 16 d is depth in m.

18 f = 1 13y(p0+(100520 2nr,\

(10052 being derived from the weight of sea water, p = o-xgxh) 22 It may be that the method comprises exposing the aquatic ectoparasite to sound waves 23 having a frequency determined by the Minnaert Formula A or by the approximate 24 Minnaert Formula B or Formula C.
26 It may be that the method comprises determining the unforced radius of bubbles 27 present at the beginning of the bubble collapse phase and thereby selecting the 28 frequency of the sound waves during the bubble collapse phase based on the Minnaert 29 Formula A or the approximate Minnaert Formula B or Formula C.
31 In practice, the bubbles produced on exposure of the aquatic ectoparasite to sound 32 waves will have a range of different sizes. It may be that the method comprises 33 determining the average or peak (unforced) radius of bubbles present at the beginning 34 of the bubble collapse phase, determining the resonant frequency corresponding to the said average or peak (unforced) radius based on the Minnaert Formula A or the approximate Minnaert Formula B or Formula C, and selecting frequencies of the sound 2 waves which lie predominantly within a range of frequencies containing the said 3 resonant frequency. The range of frequencies may have a lower bound of, for example, 4 25%, or 50%, or 75% of the said resonant frequency. The range of frequencies may have an upper bound of, for example, 125%, or 150%, or 175% of the said resonant 6 frequency.

8 The method may comprise exposing the aquatic ectoparasite to the aqueous solution comprising hydrogen peroxide for at least 30 seconds, or at least 1 minute, or at least 2 minutes, prior to the bubble collapse phase.

12 The method may comprise exposing the aquatic ectoparasite to the aqueous solution comprising hydrogen peroxide for at least 3 minutes, prior to the bubble asymmetrical 14 collapse phase. The inventors have found that exposure for at least 3 minutes combined with exposure to sound waves is sufficient to form bubbles of oxygen around 16 and/or inside, and to cause observable physical damage and/or death in, isolated 17 aquatic ectoparasites.

19 The method may comprise exposing the aquatic ectoparasite to the aqueous solution comprising hydrogen peroxide for at least 5 minutes, or at least 10 minutes, or at least minutes, or at least 20 minutes, prior to the bubble collapse phase. The longer that 22 the aquatic ectoparasite is exposed to the aqueous solution comprising hydrogen peroxide, the greater the number of bubbles that are formed (until they are caused to coalesce). The longer that the aquatic ectoparasite is exposed to the aqueous solution comprising hydrogen peroxide, also typically the greater the size of the bubbles that 26 are formed (until they are caused to collapse).

28 It may be that the hydrogen peroxide has a concentration of 1500 mg/L 50% (or 29 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 concentration of 750 mg/L 50% (or 25%) the duration of a cycle of forming bubbles 32 and then causing bubble collapse is 12 minutes 50%. It may be that the hydrogen 33 peroxide has a concentration of 375 mg/L 50% (or 25%) and the duration of a cycle 34 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 36 25%). It may be that the duration of a cycle of forming bubbles and then causing bubble 37 collapse is 24 minutes 50% (or 25%).

2 It will be understood that the term ectoparasite refers to a parasite which lives on the 3 outside of its host animal (e.g. on the skin, scales or fins of a fish).

The aquatic ectoparasite typically belongs to the family Caligidae. The aquatic ectoparasite typically belongs to one of the following genera: Lepeophtheirus, Caligus.
7 The aquatic ectoparasite typically belongs to one of the following species:

Lepeophtheirus salmonis, Caligus clemensi, Caligus rogercresseyi, Caligus elongatus.

The aquatic ectoparasite may be a marine ectoparasite (i.e. an ectoparasite adapted 11 for life in marine environments, e.g. the ocean). The aqueous solution may comprise a 12 solution of hydrogen peroxide in sea water.

14 The aquatic ectoparasite may be a freshwater ectoparasite (i.e. an ectoparasite adapted for life in freshwater environments, e.g. in rivers or lakes). The aqueous 16 solution may comprise a solution of hydrogen peroxide in fresh water.

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, 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 typically has a similar composition to either (i.e. natural) sea water or fresh water (except for the addition of hydrogen peroxide).

26

27 A third aspect of the invention provides a non-therapeutic method of improving the

28 appearance, meat quality, meat quantity and/or growth rate of an aquatic animal

29 comprising: exposing the amoeba to an aqueous solution comprising hydrogen peroxide (i.e. H202), leading to the formation of bubbles (typically predominantly of 31 oxygen), generating sound waves having a controllable frequency spectrum (from a 32 sound source) and directing the sound waves at the bubbles, wherein the frequency 33 spectrum (and optionally the power) of the sound waves is varied with time.

Typically, the method comprises a bubble regulation phase in which the frequency 36 spectrum of the sound waves is controlled to cause bubble growth and/or coalescence 1 and a subsequent bubble collapse phase in which the frequency spectrum of the sound 2 waves is controlled to cause the collapse of bubbles.

4 A fourth aspect of the invention provides a method of reducing aquatic ectoparasitic infestation (e.g. ectoparasitosis) on an aquatic animal comprising: exposing the ectoparasite to an aqueous solution comprising hydrogen peroxide (i.e. H202), leading 7 to the formation of bubbles (typically predominantly of oxygen), generating sound 8 waves having a controllable frequency spectrum (from a sound source) and directing 9 the sound waves at the bubbles, wherein the frequency spectrum (and optionally the 10 power) of the sound waves is varied with time.

Typically, the method comprises a bubble regulation phase in which the frequency 13 spectrum (and typically power) of the sound waves is controlled. It may be that the frequency spectrum (and typically power) of the sound waves is controlled to cause 15 bubble growth and/or coalescence. It may be that the frequency spectrum (and typically 16 power) of the sound waves is controlled to cause bubble resonance (e.g. as bubbles 17 change in size).

19 A fifth aspect of the invention provides hydrogen peroxide, or an aqueous solution comprising hydrogen peroxide, for use in a method of reducing ectoparasitic infestation 21 (e.g.
ectoparasitosis) on an aquatic animal, or in a method of killing ectoparasites, 22 wherein the aquatic animal, or the ectoparasites, are exposed both to an aqueous 23 solution comprising said hydrogen peroxide (and typically also bubbles of gas, typically predominantly of oxygen) and to sound waves, and wherein the frequency spectrum (and optionally the power) of the sound waves is varied with time.

27 The aqueous solution comprising hydrogen peroxide, which is provided, may be an 28 aqueous solution comprising hydrogen peroxide and bubbles of gas (typically 29 predominantly of oxygen).

Typically, the method comprises a bubble regulation phase in which the frequency 32 spectrum (and typically power) of the sound waves is controlled. It may be that the frequency spectrum (and typically power) of the sound waves is controlled to cause 34 bubble growth and/or coalescence. It may be that the frequency spectrum (and typically power) of the sound waves is controlled to cause bubble resonance (e.g. as bubbles 36 change in size).

1 A sixth aspect of the invention provides apparatus for use in reducing aquatic ectoparasitic infestation (i.e. ectoparasitosis) on an aquatic animal, the apparatus comprising an aquatic enclosure for retaining the aquatic animal (i.e. during treatment) 4 and means for directing sound waves into the aquatic enclosure (i.e. a source of sound waves configured to direct sound waves into the aquatic enclosure), wherein the 6 aquatic enclosure retains an aqueous solution comprising hydrogen peroxide, and the 7 means for directing sound waves into the aquatic enclosure is configured to generate 8 and direct sound waves having a frequency spectrum (and optionally power) which is 9 variable 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 11 enclosure.

13 The apparatus may comprise one or more water and sound permeable shields configured to keep fish within the aquatic enclosure away from the means for directing sound waves into the aquatic enclosure. This shields fish from potential damage cause 16 by excessive sound intensity adjacent the means for directing sound waves into the 17 aquatic enclosure. The one or more water and sound permeable shields are typically 18 located intermediate the means for directing sound waves into the aquatic enclosure 19 and a main fish-receiving chamber of the aquatic enclosure.
21 The means for directing sound waves into the aquatic enclosure may be configured to 22 generate and direct sound waves into the aquatic enclosure such that the centre and/or 23 peak frequency of the sound waves is higher at a first depth than a second depth within 24 the aquatic enclosure, wherein the first depth is greater than the second depth (typically differing by at least 1 metre). The centre and/or peak frequency may by higher progressively with depth. For example, the means for directing sound waves into the 27 aquatic enclosure may comprise one or more transducers located in a base region (e.g.
28 at the base) of the aquatic enclosure and the sound waves may comprise a range of frequencies. Thus, as lower frequencies penetrate further into water, the centre and/or peak frequency of the sound waves will be lower as the depth decreases, further from 31 the means for directing sound waves. However, there are other ways to arrange for the 32 centre and/or peak frequency of the sound waves to be higher at a first depth than a 33 second depth. For example, means for directing sound waves which output sound 34 waves with different frequency spectra may be located at different depths (higher centre and/or peak frequency at the greater depth). Phased transducer arrays, which 36 may be located at or near the surface, may also be configured to cause the centre 1 and/or peak frequency of the sound waves to be higher at the first depth than the 2 second depth.

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 6 aquatic enclosure) may vary with depth (e.g. increasing with depth), e.g. proportional 7 to the square root of the water pressure at a given depth. The sound may be generated 8 by transducers located in a base region of the aquatic enclosure and have a range of frequencies, such that the peak and/or centre frequency of the sound waves within the aquatic enclosure increases with depth due to the greater attenuation of higher 11 frequency sounds with distance from the transducers.

13 The means for directing sound waves into the aquatic enclosure may be configured to 14 generate and direct sound waves into the aquatic enclosure such that the acoustic pressure of the sound waves is higher at a first depth than a second depth within the 16 aquatic enclosure, wherein the first depth is greater than the second depth (typically 17 differing by at least 1 metre).

Alternatively, or in addition, the acoustic pressure of the sound waves within the aquatic enclosure (i.e. which are generated and directed into the aquatic enclosure) may vary 21 with depth (e.g. increasing with depth), e.g. proportional to the square root of the water 22 pressure at a given depth. The sound may be generated by transducers located in a 23 base region of the aquatic enclosure and the sound within the enclosure may have a 24 range of acoustic pressures, e.g. such that the acoustic pressure of sound waves within the aquatic enclosure increases with depth due to the increased pressure at increased 26 depths.

28 The apparatus may further comprise one or more sound absorbing barriers and/or 29 means for generating one or more sound absorbing bubble curtains (e.g. one or more bubble generators). The one or more sound absorbing barriers and/or means for generating one or more sound absorbing bubble curtains may be located around the periphery of (at or beyond the periphery) of the aquatic enclosure. This enables greater 33 intensity sounds to be generated while minimising noise pollution.

Typically, the sound waves are varied in a bubble regulation phase in which the frequency spectrum of the sound waves is controlled to cause bubble growth and/or coalescence. The sounds wave may be controlled in a subsequent bubble collapse 1 phase in which the frequency spectrum of the sound waves is controlled to cause the 2 collapse of bubbles.

4 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) may 6 comprise (e.g. be) one or more (i.e. electroacoustic) transducers (e.g. an array of transducers). The one or more transducers are typically one or more sonic transducers 8 (e.g. an array of sonic transducers). Sonic transducers are transducers configured to 9 generate sound waves in a surrounding medium. The one or more transducers may be one or more ultrasonic transducers (e.g. an array of ultrasonic transducers).
Ultrasonic transducers are transducers configured to generate ultrasound waves in a surrounding 12 medium.

14 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) may 16 comprise (e.g. consist of) one or more loudspeakers (e.g. an array of loudspeakers).

18 The apparatus may comprise means to measure temperature in the aquatic enclosure 19 (for example, one or more temperature sensors) and be configured to vary the frequency spectrum of the sound waves in dependence on the measured temperature.
21 For example, the apparatus may be configured to vary one or more of: the duration of 22 the bubble regulation phase, the frequency (and optionally power) during the bubble regulation phase, the duration of the bubble collapse phase, the frequency (and optionally power) during the bubble collapse phase, the duration of the intermission phase (where present), the duration of the waiting phase (where present), the (e.g.

combined) duration of the one or more descending frequency phases (where present), 27 the frequency (and optionally power) of sound waves and the variation of that with time 28 during the one or more descending frequency phases (where present), the duration of 29 the preliminary bubble collapse phase (where present), the frequency (and optionally power) of sound waves during the preliminary bubble collapse phase (where present).

32 The apparatus may be configured to raise the pressure in the aquatic enclosure (for 33 example by compressing the aquatic enclosure, or the contents of the aquatic enclosure, for example by introducing a gas, such as air, above the aqueous solution in the aquatic enclosure, or a pressurisable bladder adjacent to or within the aqueous solution). The aquatic enclosure will need to be sufficiently solid to resist the efflux of 1 aqueous solution but need not be watertight and may for example contain apertures or 2 take the form of a tube or similar.

4 This has the advantage that because the resonant 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 enclosure to the pressure 7 at the top of the container is smaller than would otherwise be the case. Accordingly, 8 the variation in the frequency of the sound waves required during the bubble regulation 9 phase and the bubble collapse phase to have a desired effect (regulating the size of bubbles, causing bubble collapse) within the aquatic enclosure is reduced.
This 11 enables better control of bubble size and collapse.

13 The apparatus may comprise means to raise the pressure at the top (e.g. surface) of 14 the aqueous solution in the aquatic enclosure. The apparatus may comprise a pressure gauge. The apparatus may comprise a pump arranged to pressurise the aquatic 16 enclosure.

18 The pressure in the aquatic enclosure may for example be raised to at least 1.5 atm 19 (i.e. 151,987 MPa) or to at least 2 atm (i.e. 202,650 Pa).

Increased pressure may also be obtained by providing the aqueous solution comprising hydrogen peroxide and ectoparasites under another body of aqueous 23 solution, for example under a volume of water.

The apparatus may be configured to reduce the pressure of the aqueous solution so 26 that, at the top (e.g. surface) of the aqueous solution, the pressure of the aqueous 27 solution is below atmospheric pressure, for example at most 1 atm (i.e. 101,325 Pa) 28 or at most 0.9 atm (i.e. 91,192 Pa) or at most 0.75 atm (i.e. 75,994 Pa) or at most 29 0.5 atm (i.e. 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 31 them that determines the damaging effect of bubble collapse and jetting this can make 32 the process more efficient. The apparatus may comprise pressure reducing means, for 33 example a pump.

The means for directing sound waves into the aquatic enclosure (i.e. the source of 36 sound waves configured to direct sound waves into the aquatic enclosure) may be configured to direct sound waves having a variable frequency (and typically also a 1 variable power level). The frequency may be greater than or equal to 1 kHz, or greater 2 than or equal to 20 kHz, or greater than or equal to 25 kHz into the enclosure. The 3 means for directing sound waves into the aquatic enclosure (i.e. the source of sound 4 waves configured to direct sound waves into the aquatic enclosure) may be configured 5 to direct sound waves having a frequency less than or equal to 100 kHz into the enclosure. The means for directing sound waves into the aquatic enclosure (i.e. the 7 source of sound waves configured to direct sound waves into the aquatic enclosure) 8 may be configured to direct sound waves having a frequency between 1 kHz and 100 9 kHz, inclusive, or between 20 kHz and 100 kHz, inclusive, or between 25 kHz and 100 10 kHz, inclusive, into the enclosure.

12 The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising 13 hydrogen peroxide at a concentration greater than or equal to 20 mg/L or greater than 14 or equal to 200 mg/L. The aquatic enclosure may comprise (e.g. retain) an aqueous 15 solution comprising hydrogen peroxide at a concentration less than or equal to 2500 16 mg/L or less than or equal to 2200 mg/L. The aquatic enclosure may comprise (e.g.
17 retain) an aqueous solution comprising hydrogen peroxide at a concentration between 18 20 mg/L
and 2500 mg/L, inclusive, or between 200 mg/L and 2500 mg/L, inclusive, or 19 between 20 mg/L and 2200 mg/L, inclusive, or between 200 mg/L and 2200 mg/L, inclusive. The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising hydrogen peroxide at a concentration of approximately 1500 mg/L
(e.g. at 22 a concentration of between 1300 mg/L and 1700 mg/L, inclusive).

24 It may be that the means for directing sound waves into the aquatic enclosure (i.e.
the 25 source of sound waves configured to direct sound waves into the aquatic enclosure) is configured to direct soundwaves having a sound pressure level greater than or equal 27 to 160 dB into the aquatic enclosure.

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 configured to direct sound waves having a sound pressure level less than or equal to 32 240 dB into the aquatic enclosure.

34 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 configured to direct soundwaves into the aquatic enclosure to generate a local energy 37 intensity level of between 0.001 W/cm2 and 0.01 W/cm2, inclusive.

2 It may be that the means for directing sound waves into the aquatic enclosure (i.e.
the 3 source of sound waves configured to direct sound waves into the aquatic enclosure) is configured to direct soundwaves into the aquatic enclosure to achieve a sound pressure level of between 160 dB and 240 dB, inclusive, in the local environment of 6 the aquatic animal (i.e. in the water or aqueous solution immediately surrounding the 7 aquatic animal).

9 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 configured to direct sound waves into the aquatic enclosure for a continuous period of 12 at least

30 seconds, or at least 1 minute, or at least 2 minutes, or at least 3 minutes, or 13 at least 4 minutes, or at least 5 minutes, or at least 10 minutes, or at least 15 minutes, 14 or at least 20 minutes.
16 The aquatic enclosure may be a flexible enclosure. The aquatic enclosure may be a 17 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 19 (e.g.
urethane-coated canvas such as tarpaulin). The aquatic enclosure may comprise a net or cage at least partially surrounded by a one or more sheets of waterproof or 21 water-resistant fabric.

23 The aquatic enclosure may comprise a sound absorbing and/or reflecting medium, to 24 reduce the power of sound escaping from the enclosure (e.g. into the sea). This may comprise one or more layers of material on or in a solid or flexible wall defining the 26 aquatic enclosure.

28 The sound absorbing and/or reflecting medium may comprise a plurality of layers 29 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 layer, where

31 the first and third layers are more dense, or heavier than the second layer.

32

33 The sound absorbing and/or reflecting medium may comprise a layer of bubbles

34 around the side of at least some of the aquatic enclosure, formed using air bubble generators which may comprise an air pump and air stones or perforated belts, for 36 example.

1 The apparatus may comprise noise cancellation apparatus, comprising one or more 2 sound generators (e.g. acoustic transducers, loudspeakers) arranged to generate cancelling sound in antiphase with the said sound generated and directed into the 4 aquatic enclosure.
6 The aquatic enclosure may be an aquarium.

8 The aquatic enclosure may be located on a sailing vessel. The aquatic enclosure may 9 be located on (e.g. form part of) a boat or ship. The aquatic enclosure may be located on (e.g. form part of) a wellboat.

12 The aquatic enclosure may comprise (e.g. be) a channel or a barge (i.e. through which 13 the aquatic animal is moved during treatment). The aquatic enclosure may have an 14 inlet and an outlet, wherein the aquatic animal may travel through the aquatic enclosure from the inlet to the outlet (i.e. during treatment).

17 The aquatic enclosure may be a treatment enclosure located on a wellboat. The treatment enclosure may have an aquatic animal inlet in fluid communication with an 19 external aquatic environment (i.e. outside the wellboat), through which the aquatic animal may be transported from the external aquatic environment into the treatment 21 enclosure.

23 The wellboat may comprise one or more water flow regulators (e.g. a pump or a siphon) configured to (i.e. in use) transport (e.g. pump) water from the external aquatic environment into the treatment enclosure. Transporting (e.g. pumping) water from the 26 external aquatic environment into the treatment enclosure may also comprise 27 transporting the aquatic animal into the treatment enclosure.

29 The wellboat may comprise one or more water flow regulators (e.g. a pump or a siphon) configured to transport (e.g. pump) water from the treatment enclosure into the external 31 aquatic environment. Transporting (e.g. pumping) water from the treatment enclosure 32 to the external aquatic environment may also comprise transporting the aquatic animal 33 from the treatment enclosure to the external aquatic environment.

The wellboat (e.g. the treatment enclosure, for example the one or more 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 is transported (e.g. pumped) from the treatment enclosure to the external aquatic 2 environment.

4 The aquatic enclosure may have one or more walls.
6 The aquatic enclosure may be located in an aquatic environment (e.g. in the sea), that 7 is to say the aquatic enclosure may be surrounded by the aquatic environment (e.g.
8 the sea). An interior of the aquatic enclosure may be separated from (e.g.
isolated from) 9 the surrounding aquatic environment by one or more (e.g. solid) walls.
Alternatively, the aquatic enclosure may be located onshore (i.e. on land, that is to say not in an 11 aquatic environment such as the sea).

13 The interior of the aquatic enclosure may be in fluid communication with the aquatic environment by way of one or more channels (e.g. pipes). Water may be transported into and/or out of the aquatic enclosure through the one or more channels (e.g. pipes).
16 The one or more channels (e.g. pipes) may be provided with aquatic ectoparasite filters configured to inhibit transport of aquatic ectoparasites between the interior of the 18 aquatic enclosure and the aquatic environment.

The aquatic enclosure may comprise (e.g. be) a treatment channel (e.g. a pipe) 21 provided between (e.g. connecting) first and second aquatic animal enclosures.

seventh aspect of the invention provides a method of injuring or killing a pathogenic 24 amoeba comprising: exposing the amoeba to an aqueous solution comprising hydrogen peroxide (i.e. H202), leading to the formation of bubbles (typically predominantly of oxygen), generating sound waves having a controllable frequency 27 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein 28 the frequency spectrum (and optionally power) of the sound waves is varied with time.

Typically, the method comprises a bubble regulation phase in which the frequency 31 spectrum of the sound waves is controlled to cause bubble growth and/or coalescence 32 and a subsequent bubble collapse phase in which the frequency spectrum of the sound 33 waves is controlled to cause the collapse of bubbles.

The pathogenic amoeba is typically a pathogenic amoeba which colonises aquatic 36 animals.
The aquatic animals are typically fish. The aquatic animals may be salmonids.
37 The aquatic animals may belong to the family Salmonidae. The aquatic animals may 1 belong to one of the following genera: Salmo, Oncorhynchus. The aquatic animals may 2 belong to one of the following species: Salmo salar, Oncorhynchus tshawytscha, Oncorhynchus keta, Oncorhynchus Idsutch, Oncorhynchus gorbuscha, Oncorhynchus 4 nerka, Oncorhynchus masou, Oncorhynchus mykiss.

Additionally or alternatively, the aquatic animals may belong to one of the following 7 families: Arripidae, Carangidae, Polynemidae, Cichlidae, Cyprinidae. The aquatic 8 animals may belong to one of the following genera: Arripis, Elagatis, Eleutheronema, 9 Hucho, Dicentrarchus, Sparus, Rachycentron, Lates, Serb/a, Tilapia, Cyprinus. The aquatic animals may belong to one of the following species: Hucho hucho, ArripLs trutta, 11 Elagatis bipinnulata, Eleutheronema tetradactylum, Dicentrarchus labrax, Sparus 12 aurata, Rachycentron canadum, Lates calcanfer, Seriola lalandi, Cyprinus carpi , 13 Tilapia baloni, Tilapia guinasana, Tilapia ruweti, Tilapia sparrmanil Additionally or alternatively, the aquatic animals may belong to one of the following 16 orders: Sllunformes or Nematognathi. The aquatic animals may be catfish.

Additionally or alternatively, the aquatic animals may belong to one of the following 19 groups: Candea, Dendrobranchiata. The aquatic animals may be shrimp or prawns.
21 The pathogenic amoeba may be a pathogenic amoeba which causes amoebic gill 22 disease (AGD) in fish such as salmonids. The pathogenic amoeba may belong to the 23 genus Neoparamoeba. The pathogenic amoeba may belong to the species 24 Neoparamoeba perurans.
26 The bubble collapse phase is typically regulated so that the collapsing bubbles 27 damages groups of amoeba.

29 An eighth aspect of the invention provides a method of reducing amoebic infection in an aquatic animal comprising: exposing the aquatic animal to an aqueous solution comprising hydrogen peroxide (i.e. H202), leading to the formation of bubbles (typically predominantly of oxygen), generating sound waves having a controllable frequency 33 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein 34 the frequency spectrum (and optionally the power) of the sound waves is varied with time.

Typically, the method comprises a bubble regulation phase in which the frequency 2 spectrum of the sound waves is controlled to cause bubble growth and/or coalescence 3 and a subsequent bubble collapse phase in which the frequency spectrum of the sound 4 waves is controlled to cause the collapse of bubbles.

6 Amoebic infection of the aquatic animal typically comprises infection of the aquatic 7 animal by pathogenic amoeba. The aquatic animal may be a fish. The aquatic animal 8 may be a salmonid. The aquatic animal may belong to the family Salmonidae. The 9 aquatic animal may belong to one of the following genera: Salmo, Oncorhynchus. The 10 aquatic animal may belong to one of the following species: Salmo salar, Oncorhynchus tshawytscha, Oncorhynchus keta, Oncorhynchus kisutch, Oncorhynchus gorbuscha, 12 Oncorhynchus nerka, Oncorhynchus masou, Oncorhynchus mykiss.

Additionally or alternatively, the aquatic animal may belong to one of the following 15 families: Arripidae, Carangidae, Polynemidae, Cichlidae, Cyprinidae. The aquatic 16 animal may belong to one of the following genera: Arripis, Elagatis, Eleutheronema, 17 Hucho, Dicentrarchus, Sparus, Rachycentron, Lates, Serb/a, Tllapia, Cyprinus. The 18 aquatic animal may belong to one of the following species: Hucho hucho, ArnOS trutta, 19 Elagatis bipinnulata, Eleutheronema tetradactylum, Dicentrarchus labrax, Sparus 20 aurata, Rachycentron canadum, Lates calcanfer, Seriola lalandi, Cyprinus carpi , 21 Tllapia baloni, Tllapia guinasana, Tilapia ruweti, Tllapia sparrmanil Additionally or alternatively, the aquatic animal may belong to one of the following 24 orders: Sllunformes or NematognathZ The aquatic animal may be a catfish.

Additionally or alternatively, the aquatic animal may belong to one of the following 27 groups: Candea, Dendrobranchiata. The aquatic animal may be a shrimp or a prawn.

29 The pathogenic amoeba may be a pathogenic amoeba which causes amoebic gill disease (AGD) in fish such as salmonids. The pathogenic amoeba may belong to the 31 genus Neoparamoeba. The pathogenic amoeba may belong to the species 32 Neoparamoeba perurans.

34 A ninth aspect of the invention provides a method treating amoebic gill disease in a fish comprising: exposing the fish to an aqueous solution comprising hydrogen peroxide 36 (i.e.
H202), leading to the formation of bubbles (typically predominantly of oxygen), generating sound waves having a controllable frequency spectrum (from a sound 1 source) and directing the sound waves at the fish, wherein the frequency spectrum 2 (and optionally the power) of the sound waves is varied with time.

4 Typically, the method comprises a bubble regulation phase in which the frequency spectrum of the sound waves is controlled to cause bubble growth and/or coalescence 6 and a subsequent bubble collapse phase in which the frequency spectrum of the sound 7 waves is controlled to cause the collapse of bubbles.

9 Optional and preferred features of any one aspect of the invention are optional features of any other aspect of the invention. In particular: optional and preferred features of the 11 first, second, third, fourth, fifth and sixth aspects of the invention may be optional 12 features of the seventh, eighth or ninth aspects of the invention, replacing the words 13 "aquatic ectoparasite" with "pathogenic amoeba" and the words "ectoparasitic 14 infestation" with "amoebic infection", or in the case of the seventh aspect of the invention, replacing the words "ectoparasitic infestation" with "amoebic gill disease"
16 and the word "aquatic animal" with "fish".

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

23 Figure 1 shows an Atlantic salmon infested with sea lice;

Figure 2 shows a plurality of infested Atlantic salmon retained in an undersea cage;

27 Figure 3 shows the undersea cage of Figure 2 surrounded by a tarpaulin enclosure and 28 an array of ultrasonic transducers, before treatment has commenced;

Figure 4 shows the treatment apparatus of Figure 4 during treatment;

32 Figures 5A to 5D are a series plots of example frequency and amplitude output by an 33 ultrasound generator with time, during an example treatment cycle according to the 34 second example procedure;
36 Figures 6is a flow chart of a first example procedure for the ultrasound treatment of the 37 sea lice;

2 Figure 7 is a flow chart of a second example procedure for the ultrasound treatment of 3 the sea lice;

Figures 8A and 8B respectively show the frequency and amplitude of ultrasound output 6 by the ultrasound generator with time, during a treatment cycle according to the second 7 example procedure;

9 Figure 9 shows a wellboat being loaded with infested Atlantic salmon from an undersea cage;

12 Figure 10 shows Atlantic salmon during treatment with hydrogen peroxide and 13 exposure to ultrasound on the wellboat of Figure 9;

Figure 11 shows sea lice detached from the Atlantic salmon and caught in a lice filter 16 of the wellboat of Figure 9;

18 Figure 12 shows the Atlantic salmon of Figure 9 having been returned to the undersea 19 cage; and 21 Figure 13 shows a wellboat adapted to pressurise an enclosure.

23 Detailed Description of a First Example Embodiment Figure 1 shows an Atlantic salmon 1 belonging to the species Salmo salar. The salmon 26 1 is infested with sea lice 2A and 2B belonging to the species Lepeophtheirus salmon/s.
27 The sea lice 2A and 2B are parasites which cling to and feed off the salmon, causing 28 damage to the salmon's skin and fins and creating open wounds which permit other 29 pathogens to enter the fish. Sea lice infestation is a particular problem in salmon farms where many salmon are reared together in a caged environment.

32 Figure 2 shows several salmon 1 retained within a floating cage 3 in the sea 4. The 33 cage 3 is tethered to a floating platform 5. The cage 3 is generally cylindrical in shape, 34 having one continuous, generally cylindrical wall 6 and a base 7. The cage 3 is open at the surface of the sea 8. The wall 6 and base 7 of the cage are formed from a nylon 36 mesh (or a mesh made of any other suitable plastics material) having openings which 1 are sufficiently small that the salmon cannot escape from the cage, but water is still 2 able to flow freely through the cage wall and base.

4 As shown in Figure 3, in order to treat the salmon to remove the sea lice, the cage 3 is surrounded by a tarpaulin enclosure 9 tethered to the floating platform 5 and a float 10.
6 The tarpaulin enclosure 9 is waterproof and completely encircles the cage 3. Water 7 can flow between the interior of the cage 3 and the space enclosed between the cage 8 3 and the tarpaulin enclosure 9 but water cannot flow beyond the tarpaulin enclosure 9 9. In Figure 3, an array of underwater ultrasonic transducers 11 has also been introduced into the space enclosed between the cage 3 and the tarpaulin enclosure 9.
11 The array of underwater ultrasonic transducers 11 is tethered to the float 10 which also 12 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 14 body of the enclosure to protect fish from excessive sound in use.
16 The apparatus shown in Figure 3 is used to treat the salmon in order to injure or kill the 17 salmon lice and reduce the parasitic infestation. In use, hydrogen peroxide is added to 18 the water enclosed within the tarpaulin enclosure 9. In an example, sufficient hydrogen 19 peroxide is added to form an aqueous solution within the enclosure 9 having a hydrogen peroxide concentration of approximately 1500 mg/L. As shown in Figure 4, 21 the hydrogen peroxide begins to decompose in the water and generates bubbles 12 of 22 oxygen around the surface of the salmon. Bubbles are preferentially formed on the 23 surface of, and inside, the sea lice attached to the salmon.

Bubbles are typically formed by decomposition of hydrogen peroxide to form oxygen 26 and water according the following chemical equation:

28 2H202 2H20 + 02 Hydrogen peroxide is thermodynamically unstable and can decompose spontaneously 31 to form oxygen and water. It may be that the bubbles are formed predominantly on the 32 surface of the aquatic ectoparasite. However, bubbles may also be formed inside (i.e.
33 inside the body of) the aquatic ectoparasite. Hydrogen peroxide may be decomposed biologically by the enzyme catalase (or other antioxidant enzymes such as glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, superoxide reductase, glutathione reductase and thioredoxin), commonly present within the body of aquatic 1 ectoparasites. This may provide a mechanism for bubble formation inside and on the surface 2 of (e.g. adjacent pores of) the aquatic ectoparasite.

4 The ultrasonic transducers are switched on and the transducers generate ultrasonic waves 13 which propagate through the water enclosed within the tarpaulin enclosure 9 and are incident 6 on the ectoparasites.

8 Figures 5A to 5D illustrate the peak (and/or centre) frequency (5A, 5C), and amplitude (5B, 9 5D), of ultrasound generated overtime, during five phases (separated by dashed lines). Figure 6 is a flow chart of a first example of an ultrasound treatment procedure which, in this example, 11 is divided into three phases.

13 Initially, hydrogen peroxide is added 50 to the aquatic enclosure containing fish (or 14 alternatively the fish can be brought into an aquatic enclosure which already comprises hydrogen peroxide. In the first phase 52 (the waiting phase), a period of time, typically of the 16 order of a few seconds through to around 2 minutes is provided to give sufficient time for a 17 significant amount of hydrogen peroxide to be decomposed to form oxygen and thereby form 18 bubbles. The person skilled in the art will appreciate that the waiting phase may have longer 19 or shorter periods of time, depending on the conditions. For example, it may be preferable for a waiting phase to have a duration of less than 10 minutes, or 2 to 8 minutes, or preferably 4 21 to 6 minutes. The waiting phase may be selected to be longer when the water temperature is 22 lower, for example).

24 The second phase 54, is a bubble regulation phase comprising at least one descending frequency phase. The peak (and/or centre) frequency of the ultrasound is gradually reduced 26 (in this example linearly) and the amplitude is kept constant. This promotes the formation of 27 larger bubbles, in particular it promotes bubbles coalescing with each other, at an ever-greater 28 size as the frequency of the ultrasound is reduced. Generally gaseous oxygen will continue to 29 be generated with time which also assists growth. The bubbles become sufficiently large that some will detach from the surface of the ectoparasite. The final frequency (lowest peak (and/or 31 centre) frequency) is selected to facilitate the growth of bubbles which are close to the size at 32 which they will predominantly buoyantly detach from the ectoparasites, for example they may 33 grow to about 1-2 mm.

In the example indicated in the plot of figure 5A, each bubble collapse phase comprises one 36 descending frequency phase and each bubble collapse phase last for 1 minute RECTIFIED SHEET (RULE 91) ISA/EP

1 and the descending frequency phase is from an initial frequency 60 of about 13 kHz to 2 a final frequency 62 of about 3 kHz. In the example indicated in the plot of figure 50, 3 each bubble collapse phase comprises a set of four descending frequency phases, 4 each set of four lasting for 1 minute and, the sets of four descending frequency phases 5 being interspersed with intermission phases. Again, each of the four descending frequency phases is from an initial frequency 60 of about 13 kHz to a final frequency 7 62 of about 3 kHz.

Thereafter, in the third phase 56 (the intermission phase), there is a pause in ultrasound generation. This provides time for some of the larger bubbles to detach and 11 for new bubbles to form from the decomposition of hydrogen peroxide. In this example, 12 the intermission phase lasts about 4 minutes.

14 This process is repeated several times, for example 4 times.
16 Patterns of bubble regulation phases and intermission phases (i.e. following an initial 17 waiting phase) may be determined depending upon the conditions (e.g. water temperature). A full cycle of such a pattern of bubble regulation phases and intermission phases (i.e. including an initial waiting phase) is typically anticipated to have a duration of between 10 minutes and 30 minutes, preferably between 15 minutes 21 and 25 minutes, more preferably between 18 and 22 minutes.

23 A first pattern (as described above) may, for example, include:
24 - an initial 4-minute waiting phase;
- a 1-minute bubble regulation phase;
26 - a 4-minute intermission phase;
27 - a 1-minute bubble regulation phase;
28 - a 4-minute intermission phase;
29 - a 1-minute bubble regulation phase;
- a 4-minute intermission phase; and 31 - a 1-minute bubble regulation phase.

33 The above first pattern would therefore include a total of 4 minutes of waiting phase, 4 34 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 1-minute 36 bubble regulation phases include sound generation wherein the frequency of sound generated begins at 6 kHz and is reduced to 3 kHz, either in discrete frequency steps 1 or, more preferably, through a continuous sweep-through of the frequencies (e.g. at 2 0.2 kHz/s) and this reduction of frequency may be repeated several times (in the 3 example indicated in Figure 50, with a reduction in frequency from 6 kHz to 3 kHz at a 4 rate of 0.2 kHz/s, the reduction of frequency would be repeated four times during each 1-minute bubble regulation phase).

7 It will be appreciated that other patterns of bubble regulation phases and intermission 8 phases (i.e. following an initial waiting phase) may also be suitable.
Accordingly, a 9 second pattern may for example include:
- an initial 4-minute waiting phase;
11 - a 2-minute bubble regulation phase;
12 - a 4-minute intermission phase;
13 - a 2-minute bubble regulation phase;
14 - a 4-minute intermission phase; and - a 4-minute bubble regulation phase.

17 The above second pattern would therefore include a total of 4 minutes of waiting phase, 18 8 minutes of bubble regulation phases (during each of which sound is generated, as 19 described above), and 8 minutes of intermission phases. Here, the bubble regulation phases include sound generation wherein the frequency of sound generated either 21 begins at 6 kHz and is reduced to 3 kHz, or begins at 10 kHz and is reduced to 3 kHz, 22 and is reduced either in discrete frequency steps or, more preferably, through a 23 continuous sweep-through of the frequencies (e.g. at between 0.2 kHz/s and 1 kHz/s).

If the water temperature is lower, a longer initial waiting phase is required.
This may 26 lead to the following, third pattern of bubble regulation phases and intermission phases 27 (i.e. following the said longer initial waiting phase):
28 - a 5-minute waiting phase;
29 - a 1-minute bubble regulation phase;
- a 5-minute intermission phase;
31 - a 1-minute bubble regulation phase;
32 - a 5-minute intermission phase; and 33 - a 3-minute bubble regulation phase.

A fourth pattern of bubble regulation phases and intermission phases (i.e.
following a 36 longer initial waiting phase as suitable for lower water temperatures) may be as follows:
37 - a 6-minute waiting phase;

1 - a 4-minute bubble regulation phase;
2 - a 6-minute intermission phase; and 3 - a 4-minute bubble regulation phase.

In some patterns, the final bubble regulation phase of each pattern may include generating sound having a higher frequency spectrum compared to the frequency 7 spectra of the or each previous bubble regulation phase. This provides the advantage 8 of encouraging the coalescence of any smaller bubbles which have not coalesced 9 during the or each previous bubble regulation phase. Alternatively or additionally, some examples may include bubble regulations phases in which the frequency is changed 11 through a wider frequency band.

13 As bubbles coalesce the total size of a given bubble increases which also increases its buoyancy. Eventually, the buoyancy of the bubble is great enough that the Bjerknes force is overcome and the bubble is buoyantly lost. At typical acoustic field strengths 16 of between 170 and 250 dB, e.g. between 201 and 217 dB, the inventor has observed 17 this effect to occur at around 3.5 kHz (e.g. for 1 Pa at a distance of 1 m from the sound 18 source).
However, this effect may occur at other frequencies in different conditions 19 and/or when using different sound sources. Accordingly, it is advantageous to decrease from an initial high frequency (e.g. 6 kHz) to a lower frequency (e.g. 3 kHz) 21 at a rate of approximately 5 seconds per kHz, as this rate both causes coalescence of 22 bubbles but also allows for bubbles to oscillate for a period of time (e.g. several 23 seconds) before they reach a size at which they are lost due to their buoyancy. This 24 appears to be particularly effective in damaging and/or removing sea lice.
26 As the size of the bubbles changes so does the resonant frequency of the bubbles.

Therefore, varying the frequency of sound produced during the bubble regulation 28 phase, as described above, provides the further advantage of continuing to cause the 29 bubbles to oscillate as they increase in size. Oscillation of the bubbles due to the sound waves can also help to cause the bubbles to stick to the lice and/or to the fish (i.e. the 31 bubbles are held close to surfaces due to the Bjerknes force).

33 Figure 7 is a flow chart of a second example of an ultrasound treatment procedure 34 which, in this example, is divided into five phases. In contrast to the first example, the second example includes a bubble collapse phase. Figures 8A and 86 illustrates the 36 peak frequency (8A), and amplitude (86), of ultrasound generated over time, during 37 the five phases (separated by dashed lines).

Initially, hydrogen peroxide is added 100 to the aquatic enclosure containing fish (or alternatively the fish can be brought into an aquatic enclosure which already comprises 4 hydrogen peroxide. In the first phase 102 (the waiting phase), a period of time, typically of the order of a few seconds through to around 2 minutes is provided to give sufficient 6 time for a significant amount of hydrogen peroxide to be decomposed to form oxygen 7 and thereby form bubbles.

9 In the second phase 104 (the preliminary bubble collapse phase), ultrasound is generated at a relatively high frequency and moderately high amplitude, selected to 11 cause bubbles which are present on fish and ectoparasites to collapse symmetrically.
12 This removes bubbles from previous cycles of this procedure and can be useful to 13 clean fish and treat open wounds. This phase is also useful as a step of the first 14 example procedure described above.
16 In the third phase 106 (the one or more descending frequency phases), as before, the frequency of the ultrasound is gradually reduced and the amplitude is kept relatively 18 low.
This promotes the formation of larger bubbles, in particular it promotes bubbles 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 21 also assists growth. The bubbles become sufficiently large that some will detach from 22 the surface of the ectoparasite.

24 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 26 bubbles are within a defined size range during the later bubble asymmetric collapse 27 phase.
The defined size range is typically reasonably narrow, e.g. a range of less than 28 1 mm of diameter, or less than 0.5 mm of diameter, or less than 50%, with the centre 29 diameter of the size range being in the range 0.2 -2 mm, for example.

Thereafter, in the fourth phase 108 (the intermission phase), there is a pause in ultrasound generation. This provides time for some of the bubbles to move a short 33 distance from the surface of the ectoparasite, carried by the flow of liquid or due to the 34 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 36 would be less than 2 mm. In an example bubbles have a radius of about 1 mm and 37 move about 0.5 to 2 mm for example, from the surface of the ectoparasite.

2 In the fifth phase 110 (the bubble asymmetric collapse phase), ultrasound is generated 3 at a lower frequency than in previous steps and with a relatively high amplitude.
This 4 causes bubbles to collapse and create micro-jets directed at the surface of the ectoparasite (or within the body of the ectoparasite if they remain within the ectoparasite). The pulse can have relatively short duration and cause substantial 7 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 9 of > 210 dB. The bubble regulation phase is typically carried out at a power which is insufficient to cause bubble collapse (except briefly in the preliminary bubble collapse 11 phase) but sufficient to cause oscillations. The sounds waves might be generated with 12 a power of <190 dB during this phase (at least during each of the one or more 13 descending frequency phases).

As a result, in comparison to the generation of continuous ultrasound in the presence 16 of hydrogen peroxide, the invention enables the high intensity ultrasound pulse to 17 cause increased damages to the ectoparasites in a short period of time. This avoids generating sustained high intensity ultrasound and thereby mitigates potential effects 19 of that ultrasound on the environment and/or on fish. Furthermore, it can reduce overall power consumption as the high amplitude phase is relatively short.

22 We have found that bubble jetting is effective in damaging ectoparasites, while minimising damage to fish, especially where the bubble has detached from the ectoparasite surface but is within two bubble radii of the ectoparasite surface.
Lepeophtheirus salmoniS and similar parasites has 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.

33 The duration of each phase can be predetermined or may be determined using 34 measurements of bubble size, for example using optical sensors.
36 We have found that bubble jetting is effective in damaging ectoparasites, while minimising damage to fish, especially where the bubble has detached from the ectoparasite surface but is within two bubble radii of the ectoparasite surface.

Lepeophtheirus salmonis and similar parasites have a surface layer of a hydrophobic 3 wax like substance. Bubbles forming on this surface have a high contact angle, where 4 the bubble is more spherical and the centre further away from the surface than would 5 be the case without the hydrophobic surface layer. The bubbles on the lice are more 6 readily collapsed as a jet being nominally created at a distance of 0.9 - 1.0 bubble radii 7 from the surface which could be grown to 1.2. Where the bubbles are smaller than this 8 and located on the surface of the fish they are more prone to shear wave collapse.

10 In general, the ultrasound intensity during the bubble regulation phase (preliminary 11 bubble collapse phase and the or each of the one or more descending frequency 12 phases) is kept such that the acoustic pressure which stimulates the bubble is below 13 the Blake threshold pressure (usually by a factor of at least 1.5), but above the Blake threshold pressure (usually by a factor of at least 1.5) during the collapse phase. The frequency of sound waves during the preliminary bubble collapse phase and the one 16 or more descending frequency phases can be determined experimentally. In an 17 example, sound has a frequency of 20 kHz during the preliminary bubble collapse 18 phase and then this is reduced progressively to 3 kHz at the rate of 1 kHzs-1. The cycles 19 of sound treatment (bubble regulation phase and collapse phase plus 20 waiting/intermission phases) are typically repeated.

22 The concentration of hydrogen peroxide can be varied over a reasonably wide range.
23 It may be reduced below 1500 mg/L, which is environmentally advantageous, by 24 allowing a longer period of time for bubbles to form prior to collapse. In an example, 25 the concentration of hydrogen peroxide is 1500 mg/L and a cycle of forming bubbles 26 and then causing bubble collapse has a duration of 3 minutes. In another example, the concentration of hydrogen peroxide is 750 mg/L and a cycle of forming bubbles and 28 then causing bubble collapse has a duration of 6 minutes. In a further example, the concentration of hydrogen peroxide is 375 mg/L and a cycle of forming bubbles and 30 then causing bubble collapse has a duration of 3 minutes. In a still further example, the concentration of hydrogen peroxide is 200 mg/L and a cycle of forming bubbles and 32 then causing bubble collapse has a duration of 12 minutes.

34 In some embodiments, the ultrasound treatment is applied in a wellboat. Figure 9

35 shows a treatment wellboat 14 adjacent the floating cage 3 in the sea 4. The wellboat

36 14 contains a treatment enclosure 15 configured to retain a body of water. An array of

37 underwater ultrasonic transducers 16 is provided at one end of the treatment enclosure 1 15. A
vent 17 connects the treatment enclosure 15 to the surrounding sea water 4 by 2 way of a sea lice filter 18.

4 In use, the vent 17 is closed so that the treatment enclosure 15 is isolated from the surrounding sea water. Salmon 19, which are infested with sea lice, are drawn into the 6 treatment enclosure 15 from the cage 3 by way of a siphon 20.

8 As shown in Figure 10, once transported from the cage 3 into the treatment enclosure 9 15, the salmon may be treated for sea lice infestation by exposure to hydrogen peroxide and ultrasound.

12 Hydrogen peroxide is added to the water in the treatment enclosure 15 until the 13 hydrogen peroxide concentration of the water reaches approximately 1500 mg/L. The 14 hydrogen peroxide decomposes to form bubbles of oxygen 21 around the salmon and, preferentially on the surface of, and inside, the sea lice attached to the salmon.

17 The array of ultrasonic transducers are switched on and the transducers emit ultrasonic 18 sound waves 22 which propagate through the water enclosed within the treatment enclosure 15. The frequency spectrum of the generated ultrasound is varied with time as set out above with respect to Figures 5 and 6 or with respect to Figures 7 and 8.

22 After the treatment is finished, the ultrasonic transducers are switched off and, as 23 shown in Figure 11, the vent 17 is opened to allow the treatment water to disperse into 24 the surrounding sea 4. Sea lice 23 which have detached from the salmon 19 are trapped by the sea lice filter 18. The salmon 19 may then be transferred back into the 26 cage 3 by way of the siphon 20. The salmon in the cage have been effectively 27 deloused, as shown in Figure 12.

29 In some embodiments, the enclosure (e.g. cage, tank or pipe) within which treatment takes place is sufficiently solid to retain water under pressure. For example, it may have 31 solid walls, or at least walls with a relatively small cross-sectional area of apertures.
32 The pressure within the enclosure is then increased by, for example, adding pressurised air, or inflating a bladder within the cage, or bringing the enclosure into 34 contact with higher pressure water (e.g. a hydrostatic head). In an example shown in Figure 13, enclosure 15 is sealed with a cover 25 above an air space 26 and air is introduced continuously by a pump 27, through pipe 28, to increase the pressure at the 37 top of the water in the enclosure to above atmospheric pressure. This has the effect of 1 raising the pressure at the upper surface of the water. This reduces the ratio of the 2 pressure between the bottom of the enclosure and the top. As can be seen from the 3 Minnaert Formulas above, the resonant frequency of bubbles is a function of pressure 4 (roughly proportional to the square root of pressure).

Accordingly, by reducing the ratio of the pressure between the water at the bottom and 7 at the top of the enclosure ultrasound may be generated which is optimised to cause 8 the desired effect in both the bubble regulation phase and the bubble collapse phases 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 bandwidth of the ultrasound to be reduced, enabling greater control of bubble growth 12 and collapse.

14 Another way to increase the pressure is by fluidically connecting the sealed tank to a raised tank, to thereby increase the head pressure at the surface. In alternative embodiments the pressure at the surface of the water can be reduced, e.g. by running 17 pump 27 as a vacuum pump to evacuate air from the air space 26. This promotes rapid 18 bubble growth.

Nevertheless, there will still generally be a significant variation in pressure within the 21 aquatic enclosure. For example, in a well boat treatment with a well boat which is up to 22 8m deep, there will be a variation in the frequency of the resonant frequency of bubbles 23 of a given size of 33%, and for a tarpaulin treatment with an enclosure formed of a tarpaulin which is 10 m deep, there will be a variation in the resonant frequency of bubbles of any given size of 42%. Pressurisation would reduce these frequency 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 28 will have a bandwidth selected to provide a balance between broad enough to regulate 29 and collapse bubbles at a range of depths and narrow enough to avoid excessively disrupting the formation and collapse of bubbles at a range of depths.

32 The frequency of sound which is generated and directed into the aquatic enclosure 33 may also be varied with depth, especially in embodiments where sound is transmitted 34 using transducers which are vertically spaced within the aquatic enclosure.
Transducers can be arranged so that the peak (and/or centre) frequency of the sound 36 waves increases with depth. This reduces or avoids a tendency for smaller bubbles to 37 be formed at greater depths (e.g. due to the increased pressure at greater depths) as 1 the bubble diameter for a given resonant frequency decreases as pressure increases.
2 At greater depths an increased acoustic pressure is also required to cause oscillation 3 (again, due to the increased pressure at greater depths). Preferably, the acoustic 4 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 6 internal pressure of the bubble, or between 10% and 70% of the internal pressure of 7 the bubble. The advantage of providing an acoustic pressure which is similar to the 8 internal pressure of a bubble is that this encourages bubble movement, oscillation and 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 11 lower frequency sounds penetrate further in water, the peak frequency and/or centre frequency of the sounds waves increases with distance from the transducers, i.e. as 13 depth decreases. Similarly, this allows higher acoustic field strengths to be generated 14 at greater depths, and (as the sound waves are attenuated as they travel upwards) the acoustic field strength to be reduced at shallower depths.

17 Pressure variation is of less concern in embodiments where the aquatic enclosure is 18 relatively shallow, for example where the aquatic enclosure is a pipe or shallow tray.

The process parameters can be set by experiment and adapted for different parasites 21 and aquatic animals. Optimisation includes taking into account the effect of hydrogen 22 peroxide on the aquatic animals and in particular determining the hydrogen peroxide concentration and treatment time taking into account the tolerance of hydrogen 24 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 26 Blake threshold pressure in the conditions which will be experienced in use and then optimised. Optical microscopy and video can be employed to monitor bubble size and 28 also to view damage to endoparasites. The ultrasound power and frequency for the preliminary bubble collapse phase can be determined through experiment. The frequency and power levels during the bubble regulation phase can also be determined 31 through experiment to obtain a target bubble size. The duration of the intermission 32 phase can also be determined experimentally, bearing in mind that the jetting effect 33 requires the bubbles to be within about 2 bubble diameters of the surface of the 34 endoparasite.
36 Although the above example has predominantly focused on asymmetric bubble collapse, which we have found to be particularly effective in killing or injuring 1 ectoparasites, in some applications the sound wave properties are selected so that the 2 bubble collapse will be symmetric. This can be useful for example when killing amoeba.

Claims

PCT/EP 2019/074 283 - 12.10.2020 1 Claims 3 1. A method of injuring or killing an aquatic ectoparasite comprising: exposing the 4 aquatic ectoparasite to an aqueous solution comprising hydrogen peroxide, 5 leading to the formation of bubbles, generating sound waves having a 6 controllable frequency spectrum and directing the sound waves at the bubbles, 7 wherein the frequency spectrum of the sound waves is varied with time.

9 2. A method of operating an apparatus, the apparatus comprising an aquatic 10 enclosure comprising an aqueous solution of hydrogen peroxide, the solution 11 comprising bubbles, the bubbles comprising oxygen, and at least one sound 12 source configured to direct sound waves at the bubbles, wherein the frequency 13 spectrum of the sound waves is varied with time.

15 3. A method according to claim 1 or claim 2, comprising a bubble regulation phase 16 in which the frequency spectrum of the sound waves is controlled, wherein the 17 bubble regulation phase comprises one or more descending frequency phases 18 during which the centre and/or peak frequency of the sound is reduced to 19 thereby cause bubble growth and/or coalescence.
21 4. A method according to claim 3, wherein during the or each of the one or more 22 descending frequency phases the centre and/or peak frequency of sound that 23 is generated decreases in frequency by at least 40% and less than 90%.

5. A method according to any one preceding claim, wherein the aqueous solution 26 comprises hydrogen peroxide at a concentration greater than or equal to 27 mg/L, optionally wherein the peak and/or centre frequency of the sound waves 28 is reduced from 10 kHz 2 kHz to 3 kHz 1 kHz, or from 6 kHz 2 kHz to 3 29 kHz 1 kHz.
31 6. A method according to any one of claims 3 to 5, wherein the bubble regulation 32 phase has a duration of at least 1 minute and, during the or each of the said 33 one or more descending frequency phases, the centre and/or peak frequency 34 of sound that is generated is reduced at a rate of at least 0.1 kHz/s.
AMENDED SHEET

PCT/EP 2019/074 283 - 12.10.2020 1 7. A method according to any one of claims 3 to 6, wherein the bubble regulation 2 phase comprises a plurality of descending frequency phases during which the 3 centre and/or peak frequency of the sound is reduced.

8. A method according to any one of claims 3 to 7, further comprising an 6 intermission phase, subsequent to the or each of the one or more descending 7 frequency phases, during which intermission phase sound waves restricted in 8 intensity.

9. A method according to claim 7 or claim 8, wherein the method comprises a 11 plurality of said descending frequency phases interspersed with said 12 intermission phases.

14 10. A method according to any one of claims 3 to 9, comprising a bubble regulation phase and a subsequent bubble collapse phase.

17 11. A method according to claim 10, further comprising an intermission phase after 18 the bubble regulation phase and before the bubble collapse phase.

12. A method according to claim 10 or claim 11, wherein the bubble collapse phase 21 has a duration of less than 1 s; and the bubble regulation phase has a duration 22 of at least 10 seconds and/or the period of time between bubble collapse 23 phases is at least 10 seconds.

13. A method according to any one of claims 3 to 12, wherein the bubble regulation 26 phase comprises a preliminary bubble collapse phase, prior to the one or more 27 descending frequency phases.

29 14. A method according to any one of claims 3 to 13, comprising a waiting phase prior to the bubble regulation phase.

32 15. A method according to any one of claims 3 to 14, comprising determining the 33 temperature of the aqueous solution and varying one or more of the following 34 parameters in dependence on the temperature: the duration of the bubble regulation phase, the frequency during the bubble regulation phase, the 36 duration of the bubble collapse phase, the frequency during the bubble collapse 37 phase, the duration of the intermission phase (where present), the duration of AMENDED SHEET

PCT/EP 2019/074 283 - 12.10.2020 1 the waiting phase (where present), the duration of the one or more descending 2 frequency phases (where present), the frequency of sound waves and the 3 variation of that with time during the one or more descending frequency phases 4 (where present), the duration of the preliminary bubble collapse phase (where present), the frequency of sound waves during the preliminary bubble collapse 6 phase (where present).

8 16. A method according to any one preceding claim, wherein the ectoparasite is 9 exposed to an aqueous mixture of hydrogen peroxide and a surfactant.
11 17. A method according to any one preceding claim, comprising pressurising the 12 aqueous solution comprising hydrogen peroxide and ectoparasites or 13 reducing the pressure of the aqueous solution so that, at the top of the aqueous 14 solution, the pressure of the aqueous solution is below atmospheric pressure.
16 18. A method according to any one preceding claim, wherein the aquatic 17 ectoparasite belongs to the family Caligidae.

19 19. A method according to any one preceding claim wherein the peak and/or centre frequency of the sound waves within the aquatic enclosure increases with depth 21 within the aquatic enclosure.

23 20. A non-therapeutic method of improving the appearance, meat quality, meat 24 quantity and/or growth rate of an aquatic animal comprising: exposing the amoeba to an aqueous solution comprising hydrogen peroxide, leading to the 26 formation of bubbles, generating sound having a controllable frequency 27 spectrum and directing the sound at the bubbles, wherein the frequency 28 spectrum of the sound waves is varied with time.

21. A method of reducing aquatic ectoparasitic infestation on an aquatic animal 31 comprising: exposing the ectoparasite to an aqueous solution comprising 32 hydrogen peroxide, leading to the formation of bubbles, generating sound 33 having a controllable frequency spectrum and directing the sound at the 34 bubbles, wherein the frequency spectrum of the sound waves is varied with time.

AMENDED SHEET

PCT/EP 2019/074 283 - 12.10.2020 1 22. Apparatus for use in reducing aquatic ectoparasitic infestation on an aquatic 2 animal, the apparatus comprising an aquatic enclosure for retaining the aquatic 3 animal and means for directing sound waves into the aquatic enclosure, 4 wherein the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide, and the means for directing sound waves into the aquatic 6 enclosure is configured to generate and direct sound waves having a frequency 7 spectrum which is variable with time and configured to vary with time the 8 frequency spectrum of the sound waves which are directed into the aquatic 9 enclosure.
11 23. Apparatus according to claim 22, comprising one or more water and sound 12 permeable shields configured to keep fish within the aquatic enclosure away 13 from the means for directing sound waves into the aquatic enclosure.

24. Apparatus according to claim 22 or claim 23, wherein the aqueous solution 16 comprises hydrogen peroxide at a concentration greater than or equal to 17 mg/L, optionally wherein the means for directing sound waves is configured to 18 reduce the peak and/or centre frequency of the sound waves is from 10 kHz 19 2 kHz to 3 kHz 1 kHz, or from 6 kHz 2 kHz to 3 kHz 1 kHz.
21 25. Apparatus according to any one of claims 22 to 24, wherein the apparatus is 22 configured to raise the pressure in the aquatic enclosure or to reduce the 23 pressure of the aqueous solution so that, at the top of the aqueous solution, the 24 pressure of the aqueous solution is below atmospheric pressure 26 26. Apparatus according to any one of claims 22 to 25, comprising means to 27 measure temperature in the aquatic enclosure and wherein the apparatus is 28 configured to vary the frequency spectrum of the sound waves in dependence 29 on the measured temperature.
31 27. Apparatus according to any one of claims 22 to 26, wherein the means for 32 directing sound waves into the aquatic enclosure are configured to generate 33 and direct sound waves into the aquatic enclosure such that the centre and/or 34 peak frequency of the sound waves is higher at a first depth than a second depth within the aquatic enclosure, wherein the first depth is greater than the 36 second depth.

AMENDED SHEET

PCT/EP 2019/074 283 - 12.10.2020 1 28. Apparatus according to any one of claims 22 to 27, wherein the means for 2 directing sound waves into the aquatic enclosure comprises one or more 3 transducers located in a base region of the aquatic enclosure and the sound 4 waves comprise a range of frequencies, so that the centre and/or peak frequency of the sound waves will be lower as the depth decreases within the 6 aquatic enclosure.

8 29. Apparatus according to any one of claims 22 to 28, wherein the means for 9 directing sound waves into the aquatic enclosure are configured to generate and direct sound waves into the aquatic enclosure such that the acoustic 11 pressure of the sound waves is higher at a first depth than a second depth within 12 the aquatic enclosure, wherein the first depth is greater than the second depth.

14 30. Apparatus according to any one of claims 22 to 29, wherein the means for directing sound waves into the aquatic enclosure comprises one or more 16 transducers located in a base region of the aquatic enclosure, such that the 17 acoustic pressure of the sound waves will be higher as the depth decreases 18 within the aquatic enclosure.

31. Apparatus according to any one of claims 22 to 30, further comprising one or 21 more sound absorbing barriers and/or means for generating one or more sound 22 absorbing bubble curtains.

24 32. A method of reducing amoebic infection in an aquatic animal comprising:
exposing the aquatic animal to an aqueous solution comprising hydrogen 26 peroxide, leading to the formation of bubbles, generating sound waves having 27 a controllable frequency spectrum and directing the sound waves at the 28 bubbles, wherein the frequency spectrum of the sound waves is varied with 29 time.
31 33. Hydrogen peroxide, or an aqueous solution comprising hydrogen peroxide, for 32 use in a method of reducing ectoparasitic infestation on an aquatic animal, or 33 in a method of killing ectoparasites, wherein the aquatic animal, or the 34 ectoparasites, are exposed both to an aqueous solution comprising said hydrogen peroxide and to sound waves, and wherein the frequency spectrum 36 of the sound waves is varied with time.

AMENDED SHEET

PCT/EP 2019/074 283 - 12.10.2020 1 34. A method of treating amoebic gill disease in a fish comprising:
exposing the fish 2 to an aqueous solution comprising hydrogen peroxide, leading to the formation 3 of bubbles, generating sound waves having a controllable frequency spectrum 4 and directing the sound waves at the fish, wherein the frequency spectrum of 5 the sound waves is varied with time.

AMENDED SHEET