AU2019340038A1

AU2019340038A1 - Methods and apparatus for aquatic ectoparasite reduction

Info

Publication number: AU2019340038A1
Application number: AU2019340038A
Authority: AU
Inventors: Ian Andrew Jamieson
Original assignee: Pulcea Ltd
Current assignee: Pulcea Ltd
Priority date: 2018-09-11
Filing date: 2019-09-11
Publication date: 2021-05-06
Also published as: EP3849301A1; GB2584166B; NO20210408A1; CA3112091A1; WO2020053311A1; CL2021000582A1; GB2584166A

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 (

Description

METHODS AND APPARATUS FOR AQUATIC ECTOPARASITE REDUCTION

Field of the invention

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

Background to the invention

The invention relates to the field of reducing the infestation of aquatic animals, such as fish, by aquatic ectoparasites. In a non-limiting example, the commonly-farmed Atlantic salmon (Sa/mo salar) is prone to infestation by sea lice of the species Lepeophtheirus sa/monis 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 can cause fish death or reduced yield. As well as the direct effect of sea lice on the fish, sea lice infestation also causes a generalised chronic stress response in the fish, which may make them susceptible to infection by other diseases and which may reduce meat yield.

WO 2018/1 15826 (Armstrong et al.) discloses a method of killing ectoparasites in environments such as fish farms, by introducing hydrogen peroxide into the water around fish and applying ultrasound. The presence of hydrogen peroxide causes 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 bubbles, and by suitable choice of the frequency of the ultrasound causes bubbles to 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 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 formation of oxygen bubbles.

The present invention seeks to improve this treatment process for example by one or 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 power consumption, improving the speed of the process and/or improving the effectiveness of the process.

Summary of the invention

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 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein the frequency spectrum (and optionally the power) of the sound waves is varied with time.

The bubbles comprise gas formed by the action on hydrogen peroxide of enzymes, such as catalase, peroxidases etc. within and on the ectoparasite. The bubbles therefore comprise (typically predominantly) oxygen.

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 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 power) of the sound waves is varied with time. The apparatus may be for killing or injuring aquatic ectoparasites, optionally on live fish. Typically, the method comprises a bubble regulation phase in which the frequency 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 power) of the sound waves is controlled to cause bubble resonance (e.g. as bubbles change in size).

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 reduced (typically monotonically and typically progressively although it may for example be reduced through a plurality of, e.g. three or more intermediate steps). The 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 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 a resonant frequency which is similar to the centre (and/or peak) frequency of the sound waves and so as the frequency of the sound waves is reduced, the bubbles are encouraged to combine and grow. Other possible factors include fish mucus acting as a surfactant which helps to drive bubble formation over time, and the sound waves then causing bubbles to coalesce.

In some conditions, and at some frequencies, it has been observed that a plurality of 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 bubble regulation phase acoustic pressure is regulated, e.g. is increased and/or decreased. Regulating the acoustic pressure can help to encourage the coalescence 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 sound. The bubble regulation phase(s) may have a duration of at least 1 second and/or the one or more descending frequency phases may have a (e.g. combined) duration of at 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) may have a duration of at least 1 minute and/or the one or more descending frequency phases may have a (e.g. combined) duration of at least 1 minute, however in some 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 (e.g. combined) duration of at least 3 minutes or at least 4 minutes. Preferably, the bubble regulation phase(s) may have a duration of less than 20 minutes, preferably 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 reduced from greater than 10 kHz to less than 5 kHz, for example from about 20 kHz to 3 kHz. Preferably, the peak and/or centre frequency will be reduced from 10 kHz ± 2 kHz to 3 kHz ± 1 kHz, or from 6 kHz ± 2 kHz to 3 kHz ± 1 kHz. In some examples the rate of frequency reduction may be non-linear. In some examples the amplitude of sound generated may alternatively or additionally be changed, in which case the rate of change of sound amplitude may be linear or may be non-linear. The rate of peak 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 ¹. Typically, after each descending frequency phase the frequency is instantaneously returned to the higher frequency that preceded the descending frequency phase before a subsequent descending frequency (where present) phase begins.

During the or each of the one or more descending frequency phases, the peak and/or 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 less than 90%. A decrease in peak and/or centre frequency of around 50%, for example, will support an approximate doubling in the diameter of bubbles (because the 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 least 2 minutes. It may be that the one or more descending frequency phases has a (e.g. combined) duration of at least 1 minute or at least 2 minutes. It may be that the one or more descending frequency phases has a (e.g. combined) duration of at least 30 seconds or that the bubble regulation phase(s) has a duration of at least 30 seconds. It may be that the peak and/or centre frequency of sound that is generated during the or each of the one or more descending frequency phases is reduced at a rate of at least 0.05 kHz/s, or preferably at least 0.1 kHz/s, or at least 0.2 kHz/s.

It may be that the method further comprises an intermission phase, subsequent to the or each of the one or more descending frequency phases, during which intermission 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 descending frequency phases will have a diameter such that they have a resonant frequency similar to the frequency of the sound at the end of the one or more descending frequency phases (optionally at the end of each of the one or more descending frequency phases). This sound is then restricted, or stopped.

This may result in bubbles becoming so large they detach from fish and/or ectoparasites and provide a time for new bubble to form (from the decomposition of hydrogen peroxide to oxygen).

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

In some embodiments, the method comprises varying the frequency spectrum (and 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 change in the frequency spectrum (and optionally the power) of the sound waves. This enables the time when the bubbles collapse to be controlled. Therefore, it may be that 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) and a subsequent bubble collapse phase, in which the frequency spectrum (and typically power) of the sound waves is controlled to cause the collapse of bubbles. The bubble regulation phase may comprise a preliminary bubble collapse phase, prior to the one or more descending frequency phases.

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 to cause the asymmetric collapse of bubbles.

By providing a separate bubble regulation phase and bubble collapse phase, the 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 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 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.

The bubble collapse phase may have a duration of less than 1 s, or less than 100 ms, or even less than 10 ms. However, the bubble regulation phase typically has a duration 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 between bubble collapse phases may be at least 10 seconds, at least 30 seconds or at least a minute. The bubble collapse phase may take place for less than 5% or less than 2% or less than 1 % of the time. Thus, again the highest power sound waves are generated during only a relatively small fraction of the treatment time.

The variation in the frequency (and optionally power) of the sound waves during the bubble regulation phase is typically selected to favour the formation and maintenance of bubbles within a predefined size range. The frequency (and optionally power) of the sound waves during the bubble collapse phase is typically selected to cause the collapse (typically asymmetrical collapse) of bubbles within the said predefined size range. Typically, the mean power of the sound waves in the bubble 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. Typically, the peak power of the sound waves in the bubble 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 the bubble collapse phase may be selected to create an acoustic pressure of at least 50 kPa.

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 sufficient to cause cavitation (which would increase damage to the aquatic creature).

The duration of the bubble regulation phase is typically in the range 1 to 15 minutes, 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 less than 10% of the duration of the bubble regulation phase. The duration of the bubble collapse phase is typically less than 20% or less than 10% of time between bubble 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 there are both the bubbles and the aquatic ectoparasite.

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 a jet of liquid towards the surface. In bubble jetting, the bubble collapses in a manner 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 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 can observe whether this is occurring by optical microscopy.

Typically, during the bubble collapse phase the acoustic waves have an intensity such that their acoustic pressure is greater than the Blake threshold pressure of the majority, by volume, of bubbles. By the Blake threshold pressure, we refer to the bubble forcing pressure above which bubbles will grow quasistatically without bound. The Blake threshold pressure is for 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. “The Onset of Cavitation on Liquids” (Technical Memo. 12, Acoustic Research Laboratory, Cambridge, MA, Harvard University, 1949) and Louisnard, O. and Gonzalez-Garcia, J. (H. Feng et al., eds), “Acoustic Cavitation” in Ultrasound Technologies for Food and Bioprocessing, DOI 1 10.1007/978-1 -4419-7472-3_2, which can be calculated by the person skilled in the art and which is given, for an air bubble in water in ambient conditions (o=0.0725N.nr¹ p_l,e?=2,000 Pa, po=100kPa) by:

Where p„^rit is the Blake threshold pressure, p_o is liquid pressure, _veq is vapour equilibrium saturation pressure and a_s is dimensionless Laplace tension 2o/p_oRo (where Ro is the ambient radius of the bubble).

Typically, prior to the bubble collapse phase and/or during the bubble regulation phase and/or between the preliminary bubble collapse phase (where present) and the bubble 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 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) collapse of bubbles.

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.

There may be an intermission phase after the bubble regulation phase and before 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 that by having a bubble regulation phase and a subsequent bubble collapse phase, with an intermission phase therebetween, to allow some distance to develop between some of the bubbles and the aquatic ectoparasite, damage to the aquatic ectoparasite 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 bubbles exceeds retentive forces. Bubbles may also become detached due to the movement of the ectoparasite through the water, especially where the ectoparasite is attached to the surface of a fish or other aquatic animal which is swimming through 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 lost due to buoyancy).

The duration of the intermission phase may for example be at least 10 ms, at least 100 ms, or at least 1 second. Typically, it is less than 10 minutes, less than 5 minutes or less than 3 minutes. However, in some conditions it may be less than 10 seconds or less than 1 second, maybe even less than 100 ms, for example in fast flowing water (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).

It may be that the frequency (and optionally power) of the sound waves is controlled to promote bubble coalescence immediately prior to the bubble collapse phase. For 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 is then subject to sound waves at 2.6 kHz to cause collapse.

The method may comprise retaining bubbles close to or in contact with the surface of 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 the intermission 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. 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 enable the build up of a significant volume of bubble(s) close to or attached to the surface of the ectoparasite. This is significant because asymmetrical bubble collapse (such as jetting) causes significant damage to structures within 2 bubble diameters of the surface of the bubble and so ideally bubbles are retained close to the surface of the ectoparasite.

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

Bubble jetting occurs preferentially when the bubbles are spaced apart from the adjacent surface but there is a ratio of unforced bubble radius to bubble centre to adjacent surface distance of less than 1 :5, or less than 1 :2.5 or less than 1 :1 .5, e.g. 1 :1.1 to 1 :1 .3, with about 1 :1 .2 giving good results. By unforced bubble radius, we refer 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 or at least the majority by volume of bubbles which have detached from or are located 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 bubble. Bubbles which are further than double their diameter from the surface of the aquatic ectoparasite will be less effective.

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 that relatively low power, relatively low frequency sound waves are generated during the intermission phase to maintain bubbles. In this case, the power and frequency of the sound waves are typically each lower than the power and frequency of the sound 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 Blake threshold pressure. They continue to oscillate but are in a stable condition. They can still migrate by the effects of Bjerknes forces. A balance can therefore be struck between the driving acoustic force causing the bubbles to migrate towards the ectoparasite and detachment due to buoyancy and/or fluid flow.

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 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 have been grown and/or coalesced in the one or more descending frequency phases can cause substantial damage to aquatic ectoparasites. We hypothesize that this is due to the very substantial local forces caused by oscillating bubbles.

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

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

There may be a waiting phase prior to the bubble regulation phase. The waiting phase may be after the ectoparasite was brought into contact with the aqueous solution of 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 the decomposition of hydrogen peroxide to form oxygen. The waiting phase may have a duration of at least 10 seconds, or at least 30 seconds or at least 1 minute or at least 2 minutes or at least 5 minutes or at least 10 minutes. The waiting phase may be shorter than 20 minutes, or shorter than 10 minutes, or shorter than 3 minutes, for example.

The formation of bubbles can be temperature dependent, for example hydrogen peroxide may be converted to oxygen at a higher rate at higher temperature. It may be that the method comprises determining (e.g. measuring) the temperature of the aqueous solution and varying one or more of: the duration of the bubble regulation 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 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 phase (where present), the frequency (and optionally power) of sound waves during the preliminary bubble collapse phase (where present).

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

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 waves in the bubble collapse phase between cycles may vary by more than 10%, for example.

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 selected size range. Surfactants may also increase the tendency of bubbles to detach 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. It may be that exposing the aquatic ectoparasite to the aqueous solution of hydrogen peroxide comprises immersing (i.e. submerging) the aquatic ectoparasite in the 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 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 fully in the aqueous solution of hydrogen peroxide.

It may be that exposing the aquatic ectoparasite to the aqueous solution of hydrogen 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 aqueous solution) and adding hydrogen peroxide to that aquatic environment (i.e. to the water or the aqueous solution).

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 aquatic ectoparasite to the sound waves comprises generating said sound waves 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 the sound waves comprises directing said sound waves at the aquatic ectoparasite.

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

The method may comprise pressurising the aqueous solution comprising hydrogen peroxide and ectoparasites. This may for example be achieved by retaining the 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 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 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 form of a tube or similar. 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 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 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 enables better control of bubble size and collapse.

The pressure at the top of the aquatic medium in the aquatic enclosure may for example be raised to at least 1 .5 atm (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 solution, for example under a volume of water.

The method may comprise reducing the pressure of the aqueous solution so that, at 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 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. < 50,663 Pa). This has the effect of promoting larger gas bubbles. As it is the size of the bubble rather than the amount of gas (predominantly oxygen) within them that 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 waves within the aquatic enclosure (i.e. which are generated and directed into the 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 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 increase with depth due to the greater attenuation of higher frequency sounds with distance from the transducers.

The aquatic enclosure may be a flexible enclosure. The aquatic enclosure may be a 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 (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 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 on (e.g. form part of) a boat or ship. The aquatic enclosure may be located on (e.g. form part of) a wellboat. The aquatic enclosure may comprise (e.g. be) a channel or a barge. The aquatic enclosure may have an inlet and an outlet. The aquatic enclosure may be a treatment enclosure located on a wellboat. The treatment enclosure may have an inlet in fluid communication with an external aquatic environment (i.e. outside the wellboat).

The wellboat may comprise one or more waterflow 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. 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 aquatic environment. The wellboat (e.g. the treatment enclosure, for example the water flow regulator) 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 environment.

The aquatic enclosure may have one or more walls. The aquatic enclosure may be 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 be located onshore (i.e. on land, that is to say not in an aquatic environment such as the sea).

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). 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 aquatic enclosure and the aquatic environment. The aquatic enclosure may comprise (e.g. be) a treatment channel (e.g. a pipe) provided between (e.g. connecting) first and second aquatic animal enclosures. Water containing aquatic animals to be treated may be pumped through the treatment channel.

It may be that the aqueous solution comprises hydrogen peroxide at a concentration greater than or equal to 20 mg/L. Concentrations of hydrogen peroxide greater than or equal to 20 mg/L are typically more effective at generating bubbles, particularly when the hydrogen peroxide is dissolved in fresh water.

It may be that the aqueous solution comprises hydrogen peroxide at a concentration greater than or equal to 200 mg/L. Concentrations of hydrogen peroxide of greater than or equal to 200 mg/L are typically more effective at generating bubbles, particularly when the hydrogen peroxide is dissolved in seawater.

It may be that the aqueous solution comprises hydrogen peroxide at a concentration less than or equal to 2500 mg/L. Concentrations of hydrogen peroxide greater than 2500 mg/L do not typically provide any additional benefit but are increasingly expensive to achieve in practice and their use in aquatic environments may be restricted by environmental regulations in some jurisdictions.

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

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

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 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 phase of the Atlantic salmon. The duration of bubble growth prior to bubble collapse is typically related to the hydrogen peroxide concentration and some examples are described below.

The resonant frequency of a bubble of gas in an infinite volume of liquid is given by the Minnaert Formula A: where r is the (unforced) bubble radius, g is the polytropic coefficient, p₀ is the ambient pressure and p is the density of the liquid. In practice, for bubbles formed in water, on the surface, this formula can be approximated by Formula B:

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

(10052 being derived from the weight of sea water, p = oxgxh)

It may be that the method comprises exposing the aquatic ectoparasite to sound waves having a frequency determined by the Minnaert Formula A or by the approximate Minnaert Formula B or Formula C.

It may be that the method comprises determining the unforced radius of bubbles present at the beginning of the bubble collapse phase and thereby selecting the frequency of the sound waves during the bubble collapse phase based on the Minnaert Formula A or the approximate Minnaert Formula B or Formula C.

In practice, the bubbles produced on exposure of the aquatic ectoparasite to sound waves will have a range of different sizes. It may be that the method comprises determining the average or peak (unforced) radius of bubbles present at the beginning 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 waves which lie predominantly within a range of frequencies containing the said resonant frequency. The range of frequencies may have a lower bound of, for example, 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 frequency.

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.

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 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 and/or inside, and to cause observable physical damage and/or death in, isolated aquatic ectoparasites.

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 15 minutes, or at least 20 minutes, prior to the bubble collapse phase. The longer that 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 are formed (until they are caused to collapse).

It may be that the hydrogen peroxide has a concentration of 1500 mg/L ± 50% (or ± 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 and then causing bubble collapse is 12 minutes ± 50%. It may be that the hydrogen peroxide has a concentration of 375 mg/L ± 50% (or ± 25%) and the duration of a cycle 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 ± 25%). It may be that the duration of a cycle of forming bubbles and then causing bubble collapse is 24 minutes ± 50% (or ± 25%). It will be understood that the term ectoparasite refers to a parasite which lives on the 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, Ca/igus. The aquatic ectoparasite typically belongs to one of the following species: Lepeophtheirus sa Imonis, Ca/igus c/emensi, Ca/igus rogercresseyi, Ca/igus e/ongatus.

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

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 solution may comprise a solution of hydrogen peroxide in fresh water.

The aqueous solution may be a physiologically compatible medium. The aqueous 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 comprise (e.g. be) a pisciculture medium, that is to say a medium suitable for use in 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).

A third aspect of the invention provides a non-therapeutic method of improving the appearance, meat quality, meat quantity and/or growth rate of an aquatic animal 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 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein 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 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 of the sound waves is controlled to cause the collapse of bubbles.

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 to the formation of bubbles (typically predominantly of oxygen), generating sound waves having a controllable frequency spectrum (from a sound source) and directing the sound waves at the bubbles, wherein 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 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 power) of the sound waves is controlled to cause bubble resonance (e.g. as bubbles change in size).

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 (e.g. ectoparasitosis) on an aquatic animal, or in a method of killing ectoparasites, wherein the aquatic animal, or the ectoparasites, are exposed both to an aqueous 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.

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

Typically, the method comprises a bubble regulation phase in which the frequency 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 power) of the sound waves is controlled to cause bubble resonance (e.g. as bubbles change in size). 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) 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 aquatic enclosure retains an aqueous solution comprising hydrogen peroxide, and the means for directing sound waves into the aquatic enclosure is configured to generate and direct sound waves having a frequency spectrum (and optionally power) which is 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 enclosure.

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 by excessive sound intensity adjacent the means for directing sound waves into the aquatic enclosure. The one or more water and sound permeable shields are typically located intermediate the means for directing sound waves into the aquatic enclosure and a main fish-receiving chamber of the aquatic enclosure.

The means for directing sound waves into the aquatic enclosure may be configured to generate and direct sound waves into the aquatic enclosure such that the centre and/or peak frequency of the sound waves is higher at a first depth than a second depth within 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 aquatic enclosure may comprise one or more transducers located in a base region (e.g. at the base) of the aquatic enclosure and the sound waves may comprise a range of 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 the means for directing sound waves. However, there are other ways to arrange for the centre and/or peak frequency of the sound waves to be higher at a first depth than a second depth. For example, means for directing sound waves which output sound 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 may be located at or near the surface, may also be configured to cause the centre and/or peak frequency of the sound waves to be higher at the first depth than the 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 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 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 frequency sounds with distance from the transducers.

The means for directing sound waves into the aquatic enclosure may be configured to 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 aquatic enclosure, wherein the first depth is greater than the second depth (typically 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 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 by transducers located in a base region of the aquatic enclosure and the sound within the enclosure may have a 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 depths.

The apparatus may further comprise one or more sound absorbing barriers and/or 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 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 phase in which the frequency spectrum of the sound waves is controlled to cause the collapse of bubbles.

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 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 (e.g. an array of sonic transducers). Sonic transducers are transducers configured to 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 medium.

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

The apparatus may comprise means to measure temperature in the aquatic enclosure (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. For example, the apparatus may be configured to vary one or more of: the duration of 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), 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 phase (where present), the frequency (and optionally power) of sound waves during the preliminary bubble collapse phase (where present).

The apparatus may be configured to raise the pressure in the aquatic enclosure (for 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 aqueous solution but need not be watertight and may for example contain apertures or take the form of a tube or similar.

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 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 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 enables better control of bubble size and collapse.

The apparatus may comprise means to raise the pressure at the top (e.g. surface) of 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 enclosure.

The pressure in the aquatic enclosure may for example be raised to at least 1 .5 atm (i.e. > 151 ,987 MPa) or to at least 2 atm (i.e. > 202,650 Pa).

The apparatus may be configured to reduce the pressure of the aqueous solution so that, at 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 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. < 50,663 Pa). This has the effect of promoting larger gas bubbles. As it is the size of the bubble rather than the amount of gas (predominantly oxygen) within them that determines the damaging effect of bubble collapse and jetting this can make the process more efficient. The apparatus may comprise pressure reducing means, for example a pump.

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 be configured to direct sound waves having a variable frequency (and typically also a variable power level). The frequency may be greater than or equal to 1 kHz, or greater than or equal to 20 kHz, or greater than or equal to 25 kHz into the enclosure. 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 be configured 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 source of sound waves configured to direct sound waves into the aquatic enclosure) may be configured to direct sound waves having a frequency between 1 kHz and 100 kHz, inclusive, or between 20 kHz and 100 kHz, inclusive, or between 25 kHz and 100 kHz, inclusive, into the enclosure.

The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising hydrogen peroxide at a concentration greater than or equal to 20 mg/L or greater than or equal to 200 mg/L. The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising hydrogen peroxide at a concentration less than or equal to 2500 mg/L or less than or equal to 2200 mg/L. The aquatic enclosure may comprise (e.g. retain) an aqueous solution comprising hydrogen peroxide at a concentration 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. 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 a concentration of between 1300 mg/L and 1700 mg/L, inclusive).

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 having a sound pressure level greater than or equal to 160 dB into the aquatic enclosure.

It may be that the means for directing sound waves into the aquatic enclosure (i.e. the source of sound waves configured to direct sound waves into the aquatic enclosure) is configured to direct sound waves having a sound pressure level less than or equal to 240 dB into the aquatic enclosure.

It may be that the means for directing sound waves into the aquatic enclosure (i.e. the source of sound waves configured to direct sound waves into the aquatic enclosure) is configured to direct soundwaves into the aquatic enclosure to generate a local energy intensity level of between 0.001 W/cm² and 0.01 W/cm², inclusive. 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 achieve a sound pressure level of between 160 dB and 240 dB, inclusive, in the local environment of the aquatic animal (i.e. in the water or aqueous solution immediately surrounding the aquatic animal).

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 at least 30 seconds, or at least 1 minute, or at least 2 minutes, or at least 3 minutes, or at least 4 minutes, or at least 5 minutes, or at least 10 minutes, or at least 15 minutes, or at least 20 minutes.

The aquatic enclosure may be a flexible enclosure. The aquatic enclosure may be a 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 (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 water-resistant fabric.

The aquatic enclosure may comprise a sound absorbing and/or reflecting medium, to 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 aquatic enclosure.

The sound absorbing and/or reflecting medium may comprise a plurality of layers 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 the first and third layers are more dense, or heavier than the second layer.

The sound absorbing and/or reflecting medium may comprise a layer of bubbles 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 example. The apparatus may comprise noise cancellation apparatus, comprising one or more sound generators (e.g. acoustic transducers, loudspeakers) arranged to generate cancelling sound in antiphase with the said sound generated and directed into the aquatic enclosure.

The aquatic enclosure may be an aquarium.

The aquatic enclosure may be located on a sailing vessel. The aquatic enclosure may 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.

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

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 external aquatic environment (i.e. outside the wellboat), through which the aquatic animal may be transported from the external aquatic environment into the treatment enclosure.

The wellboat may comprise one or more waterflow 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 external aquatic environment into the treatment enclosure may also comprise transporting the aquatic animal into the treatment enclosure.

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

The aquatic enclosure may have one or more walls.

The aquatic enclosure may be 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 be located onshore (i.e. on land, that is to say not in an aquatic environment such as the sea).

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). 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 aquatic enclosure and the aquatic environment.

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

A seventh aspect of the invention provides a method of injuring or killing a pathogenic 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 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein the frequency spectrum (and optionally power) of the sound waves is varied with time.

The pathogenic amoeba is typically a pathogenic amoeba which colonises aquatic animals. The aquatic animals are typically fish. The aquatic animals may be salmonids. The aquatic animals may belong to the family Sa/monidae. The aquatic animals may belong to one of the following genera: Sa/mo, Oncorhynchus. The aquatic animals may belong to one of the following species: Sa/mo sa/ar, Oncorhynchus tshawytscha, Oncorhynchus keta, Oncorhynchus kisutch, Oncorhynchus gorbuscha, Oncorhynchus nerka, Oncorhynchus masou, Oncorhynchus mykiss.

Additionally or alternatively, the aquatic animals may belong to one of the following families: Arripidae, Carangidae, Poiynemidae, Cich/idae, Cyprinidae. The aquatic animals may belong to one of the following genera: Arripis, E/agatis, E/eutheronema, Hucho, Dicentrarchus, Sparus, Rachycentron, Lates, Serio/a, Ti/apia, Cyprinus. The aquatic animals may belong to one of the following species: Hucho hucho, Arripis trutta, E/agatis bipinnuiata, Eieutheronema tetradactyium, Dicentrarchus iabrax, Sparus aurata, Rachycentron canadum, Lates caicarifer, Serio/a iaiandi, Cyprinus carpio, Tiiapia baioni, Tiiapia guinasana, Tiiapia ruweti, Tiiapia sparrmanii.

Additionally or alternatively, the aquatic animals may belong to one of the following orders: Si/uriformes or Nematognathi The aquatic animals may be catfish.

Additionally or alternatively, the aquatic animals may belong to one of the following groups: Caridea, Dendrobranchiata. The aquatic animals may be shrimp or prawns.

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 genus Neoparamoeba. The pathogenic amoeba may belong to the species Neoparamoeba perurans.

The bubble collapse phase is typically regulated so that the collapsing bubbles damages groups of amoeba.

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 spectrum (from a sound source) and directing the sound waves at the bubbles, wherein 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 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 of the sound waves is controlled to cause the collapse of bubbles.

Amoebic infection of the aquatic animal typically comprises infection of the aquatic animal by pathogenic amoeba. The aquatic animal may be a fish. The aquatic animal may be a salmonid. The aquatic animal may belong to the family Sa/monidae. The aquatic animal may belong to one of the following genera: Sa/mo, Oncorhynchus. The aquatic animal may belong to one of the following species: Sa/mo sa/ar, Oncorhynchus tshawytscha, Oncorhynchus keta, Oncorhynchus kisutch, Oncorhynchus gorbuscha, Oncorhynchus nerka, Oncorhynchus masou, Oncorhynchus mykiss.

Additionally or alternatively, the aquatic animal may belong to one of the following families: Arripidae, Carangidae, Poiynemidae, Cich/idae, Cyprinidae. The aquatic animal may belong to one of the following genera: Arripis, E/agatis, E/eutheronema, Hucho, Dicentrarchus, Sparus, Rachycentron, Lates, Serio/a, Ti/apia, Cyprinus. The aquatic animal may belong to one of the following species: Hucho hucho, Arripis trutta, E/agatis bipinnuiata, Eieutheronema tetradactyium, Dicentrarchus iabrax, Sparus aurata, Rachycentron canadum, Lates caicarifer, Serio/a iaiandi, Cyprinus carpio, Tiiapia baioni, Tiiapia guinasana, Tiiapia ruweti, Tiiapia sparrmanii.

Additionally or alternatively, the aquatic animal may belong to one of the following orders: Si/uriformes or Nematognathi The aquatic animal may be a catfish.

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

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 (i.e. H₂0₂), leading to the formation of bubbles (typically predominantly of oxygen), generating sound waves having a controllable frequency spectrum (from a sound source) and directing the sound waves at the fish, wherein the frequency spectrum (and optionally the power) of the sound waves is varied with time.

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 first, second, third, fourth, fifth and sixth aspects of the invention may be optional features of the seventh, eighth or ninth aspects of the invention, replacing the words “aquatic ectoparasite” with “pathogenic amoeba” and the words “ectoparasitic infestation” with “amoebic infection”, or in the case of the seventh aspect of the invention, replacing the words“ectoparasitic infestation” with“amoebic gill disease” and the word“aquatic animal” with“fish”.

Description of the Drawings

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

Figure 1 shows an Atlantic salmon infested with sea lice;

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

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

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

Figures 5A to 5D are a series plots of example frequency and amplitude output by an ultrasound generator with time, during an example treatment cycle according to the second example procedure;

Figures 6is a flow chart of a first example procedure for the ultrasound treatment of the sea lice; Figure 7 is a flow chart of a second example procedure for the ultrasound treatment of the sea lice;

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

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

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

Figure 1 1 shows sea lice detached from the Atlantic salmon and caught in a lice filter of the wellboat of Figure 9;

Figure 12 shows the Atlantic salmon of Figure 9 having been returned to the undersea cage; and

Figure 13 shows a wellboat adapted to pressurise an enclosure.

Detailed Description of a First Example Embodiment

Figure 1 shows an Atlantic salmon 1 belonging to the species Sa/mosa/ar. The salmon 1 is infested with sea lice 2A and 2B belonging to the species Lepeophtheirus sa/monis. The sea lice 2A and 2B are parasites which cling to and feed off the salmon, causing damage to the salmon’s skin and fins and creating open wounds which permit other 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.

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

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. The tarpaulin enclosure 9 is waterproof and completely encircles the cage 3. Water can flow between the interior of the cage 3 and the space enclosed between the cage 3 and the tarpaulin enclosure 9 but water cannot flow beyond the tarpaulin enclosure 9. In Figure 3, an array of underwater ultrasonic transducers 1 1 has also been introduced into the space enclosed between the cage 3 and the tarpaulin enclosure 9. The array of underwater ultrasonic transducers 1 1 is tethered to the float 10 which also 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 body of the enclosure to protect fish from excessive sound in use.

The apparatus shown in Figure 3 is used to treat the salmon in order to injure or kill the salmon lice and reduce the parasitic infestation. In use, hydrogen peroxide is added to the water enclosed within the tarpaulin enclosure 9. In an example, sufficient hydrogen 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, the hydrogen peroxide begins to decompose in the water and generates bubbles 12 of oxygen around the surface of the salmon. Bubbles are preferentially formed on the surface of, and inside, the sea lice attached to the salmon.

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

2H₂0₂ ® 2H₂0 + 0₂

Hydrogen peroxide is thermodynamically unstable and can decompose spontaneously to form oxygen and water. It may be that the bubbles are formed predominantly on the surface of the aquatic ectoparasite. However, bubbles may also be formed inside (i.e. 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 ectoparasites. This may provide a mechanism for bubble formation inside and on the surface of (e.g. adjacent pores of) the aquatic ectoparasite.

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 on the ectoparasites.

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

Initially, hydrogen peroxide is added 50 to the aquatic enclosure containing fish (or 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 order of a few seconds through to around 2 minutes is provided to give sufficient time for a significant amount of hydrogen peroxide to be decomposed to form oxygen and thereby form bubbles. The person skilled in the art will appreciate that the waiting phase may have longer 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 to 6 minutes. The waiting phase may be selected to be longer when the water temperature is lower, for example).

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 (in this example linearly) and the amplitude is kept constant. 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 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 centre) frequency) is selected to facilitate the growth of bubbles which are close to the size at which they will predominantly buoyantly detach from the ectoparasites, for example they may grow to about 1 -2 mm.

In the example indicated in the plot of figure 5A, each bubble collapse phase comprises one descending frequency phase and each bubble collapse phase last for 1 minute and the descending frequency phase is from an initial frequency 60 of about 13 kHz to a final frequency 62 of about 3 kHz. In the example indicated in the plot of figure 5C, each bubble collapse phase comprises a set of four descending frequency phases, each set of four lasting for 1 minute and, the sets of four descending frequency phases 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 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 for new bubbles to form from the decomposition of hydrogen peroxide. In this example, the intermission phase lasts about 4 minutes.

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

Patterns of bubble regulation phases and intermission phases (i.e. following an initial 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 and 25 minutes, more preferably between 18 and 22 minutes.

A first pattern (as described above) may, for example, include:

an initial 4-minute waiting phase;

a 1 -minute bubble regulation phase;

a 4-minute intermission phase;

a 1 -minute bubble regulation phase;

a 4-minute intermission phase;

a 1 -minute bubble regulation phase;

a 4-minute intermission phase; and

a 1 -minute bubble regulation phase.

The above first pattern would therefore include a total of 4 minutes of waiting phase, 4 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 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 or, more preferably, through a continuous sweep-through of the frequencies (e.g. at 0.2 kHz/s) and this reduction of frequency may be repeated several times (in the example indicated in Figure 5C, with a reduction in frequency from 6 kHz to 3 kHz at a rate of 0.2 kHz/s, the reduction of frequency would be repeated four times during each 1 -minute bubble regulation phase).

It will be appreciated that other patterns of bubble regulation phases and intermission phases (i.e. following an initial waiting phase) may also be suitable. Accordingly, a second pattern may for example include:

an initial 4-minute waiting phase;

a 2-minute bubble regulation phase;

a 4-minute intermission phase;

a 2-minute bubble regulation phase;

a 4-minute intermission phase; and

a 4-minute bubble regulation phase.

The above second pattern would therefore include a total of 4 minutes of waiting phase, 8 minutes of bubble regulation phases (during each of which sound is generated, as described above), and 8 minutes of intermission phases. Here, the bubble regulation phases include sound generation wherein the frequency of sound generated either begins at 6 kHz and is reduced to 3 kHz, or begins at 10 kHz and is reduced to 3 kHz, and is reduced either in discrete frequency steps or, more preferably, through a 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 lead to the following, third pattern of bubble regulation phases and intermission phases (i.e. following the said longer initial waiting phase):

a 5-minute waiting phase;

a 1 -minute bubble regulation phase;

a 5-minute intermission phase;

a 1 -minute bubble regulation phase;

a 5-minute intermission phase; and

a 3-minute bubble regulation phase.

A fourth pattern of bubble regulation phases and intermission phases (i.e. following a longer initial waiting phase as suitable for lower water temperatures) may be as follows: a 6-minute waiting phase; a 4-minute bubble regulation phase;

a 6-minute intermission phase; and

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 spectra of the or each previous bubble regulation phase. This provides the advantage of encouraging the coalescence of any smaller bubbles which have not coalesced during the or each previous bubble regulation phase. Alternatively or additionally, some examples may include bubble regulations phases in which the frequency is changed through a wider frequency band.

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 of between 170 and 250 dB, e.g. between 201 and 217 dB, the inventor has observed this effect to occur at around 3.5 kHz (e.g. for 1 pPa at a distance of 1 m from the sound source). However, this effect may occur at other frequencies in different conditions 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) at a rate of approximately 5 seconds per kHz, as this rate both causes coalescence of bubbles but also allows for bubbles to oscillate for a period of time (e.g. several seconds) before they reach a size at which they are lost due to their buoyancy. This appears to be particularly effective in damaging and/or removing sea lice.

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 phase, as described above, provides the further advantage of continuing to cause the 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 bubbles are held close to surfaces due to the Bjerknes force).

Figure 7 is a flow chart of a second example of an ultrasound treatment procedure 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 8B illustrates the peak frequency (8A), and amplitude (8B), of ultrasound generated over time, during 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 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 time for a significant amount of hydrogen peroxide to be decomposed to form oxygen and thereby form bubbles.

In the second phase 104 (the preliminary bubble collapse phase), ultrasound is generated at a relatively high frequency and moderately high amplitude, selected to cause bubbles which are present on fish and ectoparasites to collapse symmetrically. This removes bubbles from previous cycles of this procedure and can be useful to clean fish and treat open wounds. This phase is also useful as a step of the first example procedure described above.

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 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 also assists growth. The bubbles become sufficiently large that some will detach from the surface of the ectoparasite.

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 bubbles are within a defined size range during the later bubble asymmetric collapse phase. The defined size range is typically reasonably narrow, e.g. a range of less than 1 mm of diameter, or less than 0.5 mm of diameter, or less than ±50%, with the centre 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 distance from the surface of the ectoparasite, carried by the flow of liquid or due to the 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 would be less than 2 mm. In an example bubbles have a radius of about 1 mm and move about 0.5 to 2 mm for example, from the surface of the ectoparasite. In the fifth phase 1 10 (the bubble asymmetric collapse phase), ultrasound is generated at a lower frequency than in previous steps and with a relatively high amplitude. This 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 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 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 phase) but sufficient to cause oscillations. The sounds waves might be generated with a power of <190 dB during this phase (at least during each of the one or more descending frequency phases).

As a result, in comparison to the generation of continuous ultrasound in the presence of hydrogen peroxide, the invention enables the high intensity ultrasound pulse to cause increased damages to the ectoparasites in a short period of time. This avoids generating sustained high intensity ultrasound and thereby mitigates potential effects 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.

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 sa/monis and similar parasites has a surface layer of a hydrophobic wax like substance. Bubbles forming on this surface have a high contact angle, where the bubble is more spherical and the centre further away from the surface than would be the case without the hydrophobic surface layer. The bubbles on the lice are more readily collapsed as a jet being nominally created at a distance of 0.9 - 1 .0 bubble radii from the surface which could be grown to 1 .2. Where the bubbles are smaller than this and located on the surface of the fish they are more prone to shear wave collapse.

The duration of each phase can be predetermined or may be determined using measurements of bubble size, for example using optical sensors.

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 sa/monis and similar parasites have a surface layer of a hydrophobic wax like substance. Bubbles forming on this surface have a high contact angle, where the bubble is more spherical and the centre further away from the surface than would be the case without the hydrophobic surface layer. The bubbles on the lice are more readily collapsed as a jet being nominally created at a distance of 0.9 - 1 .0 bubble radii from the surface which could be grown to 1 .2. Where the bubbles are smaller than this and located on the surface of the fish they are more prone to shear wave collapse.

In general, the ultrasound intensity during the bubble regulation phase (preliminary bubble collapse phase and the or each of the one or more descending frequency phases) is kept such that the acoustic pressure which stimulates the bubble is below 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 or more descending frequency phases can be determined experimentally. In an example, sound has a frequency of 20 kHz during the preliminary bubble collapse phase and then this is reduced progressively to 3 kHz at the rate of 1 kHzs ¹. The cycles of sound treatment (bubble regulation phase and collapse phase plus waiting/intermission phases) are typically repeated.

The concentration of hydrogen peroxide can be varied over a reasonably wide range. It may be reduced below 1500 mg/L, which is environmentally advantageous, by allowing a longer period of time for bubbles to form prior to collapse. In an example, the concentration of hydrogen peroxide is 1500 mg/L and a cycle of forming bubbles 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 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 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 then causing bubble collapse has a duration of 12 minutes.

In some embodiments, the ultrasound treatment is applied in a wellboat. Figure 9 shows a treatment wellboat 14 adjacent the floating cage 3 in the sea 4. The wellboat 14 contains a treatment enclosure 15 configured to retain a body of water. An array of underwater ultrasonic transducers 16 is provided at one end of the treatment enclosure 15. A vent 17 connects the treatment enclosure 15 to the surrounding sea water 4 by way of a sea lice filter 18.

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 treatment enclosure 15 from the cage 3 by way of a siphon 20.

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

Hydrogen peroxide is added to the water in the treatment enclosure 15 until the hydrogen peroxide concentration of the water reaches approximately 1500 mg/L. The 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.

The array of ultrasonic transducers are switched on and the transducers emit ultrasonic 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.

After the treatment is finished, the ultrasonic transducers are switched off and, as shown in Figure 1 1 , the vent 17 is opened to allow the treatment water to disperse into 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 cage 3 by way of the siphon 20. The salmon in the cage have been effectively deloused, as shown in Figure 12.

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 solid walls, or at least walls with a relatively small cross-sectional area of apertures. The pressure within the enclosure is then increased by, for example, adding pressurised air, or inflating a bladder within the cage, or bringing the enclosure into 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 top of the water in the enclosure to above atmospheric pressure. This has the effect of raising the pressure at the upper surface of the water. This reduces the ratio of the pressure between the bottom of the enclosure and the top. As can be seen from the Minnaert Formulas above, the resonant frequency of bubbles is a function of pressure (roughly proportional to the square root of pressure).

Accordingly, by reducing the ratio of the pressure between the water at the bottom and at the top of the enclosure ultrasound may be generated which is optimised to cause 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 and collapse.

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 pump 27 as a vacuum pump to evacuate air from the air space 26. This promotes rapid bubble growth.

Nevertheless, there will still generally be a significant variation in pressure within the aquatic enclosure. For example, in a well boat treatment with a well boat which is up to 8m deep, there will be a variation in the frequency of the resonant frequency of bubbles 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 will have a bandwidth selected to provide a balance between broad enough to regulate 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.

The frequency of sound which is generated and directed into the aquatic enclosure may also be varied with depth, especially in embodiments where sound is transmitted 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 waves increases with depth. This reduces or avoids a tendency for smaller bubbles to be formed at greater depths (e.g. due to the increased pressure at greater depths) as the bubble diameter for a given resonant frequency decreases as pressure increases. At greater depths an increased acoustic pressure is also required to cause oscillation (again, due to the increased pressure at greater depths). Preferably, the acoustic 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 internal pressure of the bubble, or between 10% and 70% of the internal pressure of the bubble. The advantage of providing an acoustic pressure which is similar to the 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 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 depth decreases. Similarly, this allows higher acoustic field strengths to be generated at greater depths, and (as the sound waves are attenuated as they travel upwards) the acoustic field strength to be reduced at shallower depths.

Pressure variation is of less concern in embodiments where the aquatic enclosure is 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 and aquatic animals. Optimisation includes taking into account the effect of hydrogen peroxide on the aquatic animals and in particular determining the hydrogen peroxide concentration and treatment time taking into account the tolerance of hydrogen 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 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 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 through experiment to obtain a target bubble size. The duration of the intermission phase can also be determined experimentally, bearing in mind that the jetting effect requires the bubbles to be within about 2 bubble diameters of the surface of the endoparasite.

Although the above example has predominantly focused on asymmetric bubble collapse, which we have found to be particularly effective in killing or injuring ectoparasites, in some applications the sound wave properties are selected so that the bubble collapse will be symmetric. This can be useful for example when killing amoeba.

Claims

1. A method of injuring or killing an aquatic ectoparasite comprising: exposing the aquatic ectoparasite to an aqueous solution comprising hydrogen peroxide, leading to the formation of bubbles, generating sound waves having a controllable frequency spectrum and directing the sound waves at the bubbles, wherein the frequency spectrum of the sound waves is varied with time.

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

3. A method according to claim 1 or claim 2, comprising a bubble regulation phase in which the frequency spectrum of the sound waves is controlled to cause bubble growth and/or coalescence, wherein the bubble regulation phase comprises one or more descending frequency phases during which the centre and/or peak frequency of the sound is reduced.

4. A method according to any one preceding claim, wherein during the or each of the one or more descending frequency phases the centre and/or peak frequency of sound that is generated decreases in frequency by at least 40% and less than 90%.

5. A method according to any one preceding claim, the bubble regulation phase has a duration of at least 1 minute and, during the or each of the said one or more descending frequency phases, the centre and/or peak frequency of sound that is generated is reduced at a rate of at least 0.1 kHz/s.

6. A method according to any one preceding claim, further comprising an intermission phase, subsequent to the or each of the one or more descending frequency phases, during which intermission phase sound waves which cause oscillation of the bubbles are restricted in intensity.

7. A method according to claim 6, wherein the method comprises a plurality of said descending frequency phases interspersed with said intermission phases.

8. A method according to any one preceding claim, comprising a bubble regulation phase in which the frequency spectrum of the sound waves is controlled to cause bubble growth and/or coalescence, and a subsequent bubble collapse phase.

9. A method according to claim 8, further comprising an intermission phase after the bubble regulation phase and before the bubble collapse phase.

10. A method according to claim 8 or claim 9, wherein the bubble collapse phase has a duration of less than 1 s; and the bubble regulation phase has a duration of at least 10 seconds and/or the period of time between bubble collapse phases is at least 10 seconds.

1 1. A method according to any one preceding claim wherein the bubble regulation phase comprises a preliminary bubble collapse phase, prior to the one or more descending frequency phases.

12. A method according to any one preceding claim comprising a waiting phase prior to the bubble regulation phase.

13. A method according to any one preceding claim, comprising determining the temperature of the aqueous solution and varying one or more of the following parameters in dependence on the temperature: the duration of the bubble regulation phase, the frequency during the bubble regulation phase, the duration of the bubble collapse phase, the frequency during the bubble collapse phase, the duration of the intermission phase (where present), the duration of the waiting phase (where present), the duration of the one or more descending frequency phases (where present), the frequency 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 phase (where present), the frequency of sound waves during the preliminary bubble collapse phase (where present).

14. A method according to any one preceding claim, wherein the ectoparasite is exposed to an aqueous mixture of hydrogen peroxide and a surfactant.

15. A method according to any one preceding claim, comprising pressurising the aqueous solution comprising hydrogen peroxide and ectoparasites or reducing the pressure of the aqueous solution so that, at the top of the aqueous solution, the pressure of the aqueous solution is below atmospheric pressure.

16. A method according to any one preceding claim, wherein the aquatic ectoparasite belongs to the family Caligidae.

17. 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 within the aquatic enclosure.

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

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

20. Apparatus for use in reducing aquatic ectoparasitic infestation on an aquatic animal, the apparatus comprising an aquatic enclosure for retaining the aquatic animal and means for directing sound waves into the aquatic enclosure, wherein the aquatic enclosure retains an aqueous solution comprising hydrogen peroxide, and the means for directing sound waves into the aquatic enclosure is configured to generate and direct sound waves having a frequency spectrum which is variable with time and configured to vary with time the frequency spectrum of the sound waves which are directed into the aquatic enclosure.

21. Apparatus according to claim 20, comprising 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.

22. Apparatus according to claim 20 or claim 21 , wherein the apparatus is configured to raise the pressure in the aquatic enclosure or to reduce the pressure of the aqueous solution so that, at the top of the aqueous solution, the pressure of the aqueous solution is below atmospheric pressure

23. Apparatus according to any one of claims 20 to 22, comprising means to measure temperature in the aquatic enclosure and configured to vary the frequency spectrum of the sound waves in dependence on the measured temperature.

24. Apparatus according to any one of claims 20 to 23, wherein the means for directing sound waves into the aquatic enclosure are configured to generate and direct sound waves into the aquatic enclosure such that the centre and/or peak frequency of the sound waves is higher at a first depth than a second depth within the aquatic enclosure, wherein the first depth is greater than the second depth.

25. Apparatus according to any one of claims 20 to 24, wherein the means for directing sound waves into the aquatic enclosure comprises one or more transducers located in a base region of the aquatic enclosure and the sound 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 aquatic enclosure.

26. Apparatus according to any one of claims 20 to 25, wherein the means for directing sound waves into the aquatic enclosure are configured to 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 aquatic enclosure, wherein the first depth is greater than the second depth.

27. Apparatus according to any one of claims 20 to 26, wherein the means for directing sound waves into the aquatic enclosure comprises one or more transducers located in a base region of the aquatic enclosure, such that the acoustic pressure of the sound waves will be higher as the depth decreases within the aquatic enclosure.

28. Apparatus according to any one of claims 19 to 27, further comprising one or more sound absorbing barriers and/or means for generating one or more sound absorbing bubble curtains.

29. A method of reducing amoebic infection in an aquatic animal comprising: exposing the aquatic animal to an aqueous solution comprising hydrogen peroxide, leading to the formation of bubbles, generating sound waves having a controllable frequency spectrum and directing the sound waves at the bubbles, wherein the frequency spectrum of the sound waves is varied with time.

30. Hydrogen peroxide, or an aqueous solution comprising hydrogen peroxide, for use in a method of reducing ectoparasitic infestation on an aquatic animal, or in a method of killing ectoparasites, wherein the aquatic animal, or the ectoparasites, are exposed both to an aqueous solution comprising said hydrogen peroxide and to sound waves, and wherein the frequency spectrum of the sound waves is varied with time.

31. A method of treating amoebic gill disease in a fish comprising: exposing the fish to an aqueous solution comprising hydrogen peroxide, leading to the formation of bubbles, generating sound waves having a controllable frequency spectrum and directing the sound waves at the fish, wherein the frequency spectrum of the sound waves is varied with time.