NO20181156A1

NO20181156A1 - System and method for reducing sea lice exposure in marine fish farming

Info

Publication number: NO20181156A1
Application number: NO20181156A
Authority: NO
Inventors: Michel André; Marta Solé; Mike Van Der Schaar
Original assignee: Seasel Solutions As
Priority date: 2018-09-05
Filing date: 2018-09-05
Publication date: 2020-03-06
Also published as: WO2020048945A1

Description

SYSTEM AND METHOD FOR REDUCING SEA LICE EXPOSURE IN MARINE FISH FARMING

The invention relates to a system and method for reducing exposure to sea-lice (Lepeophtheirus spp. and Caligus spp.) for fish in marine fish farms by inducing controlled doses of sound energy which destroy specific organs (sensory, nervous and secretory systems) of the sea-lice present in the volume of water containing the farmed fish and/or seal-lice attached onto the fish. The controlled doses of sound show no detrimental effects on the fish.

Background of the invention

Parasites and pathogens are part of the natural biology and functioning of natural ecosystems. Sea-lice are a family of parasitic copepods of which there are several species naturally occurring in seawater. Sea-lice spreads by releasing eggs which may float up to tens of kilometres in the surface region of the seawater and gradually develops into larvae. The larvae actively seek a host fish it may adhere to and develop into grown sea-lice.

Pathogens and parasites live at the expense of the host, divert host energy from growth and reproduction into disease resistance and can cause mortality. Pathogens and parasites can drastically reduce host population. Sea lice (Lepeophtheirus salmonis) in particular, affects salmon populations, causing important damage to their health, imposing potentially huge losses for fish farms. Ectoparasitic sea lice are the most important parasite problem to date for the salmon farming industry.

The salmon lice, Lepeophtheirus salmonis, is a Caligid copepod that infests both wild and farmed salmonid fish in the northern and southern hemispheres. Salmon lice are a major disease problem in the farming of Atlantic salmon, Salmo salar. Typically, impacts on the host have usually been reported in terms of pathological lesions caused by attachment and feeding of the adult stages, as well as localised mild epithelial responses to juvenile attachment. However, many studies report pathologies associated with severe infestation. Recent new studies on the hostparasite interactions of L. salmonis showed that this parasite induces stress-related responses systemically in the host skin and gills and that the stress response and immune systems are modulated.

L. salmonis is exceptional among parasite species in infecting adult wild Atlantic salmon (Salmo salar) with 100% prevalence. The infective planktonic larval phase of L. salmonis is therefore extraordinarily effective at locating and infecting wild Atlantic salmon, even in the open North Atlantic Ocean. For this reason alone, it is highly likely that L. salmonis has the potential to present a disease threat to salmonid fish.

Understanding the nature of the interactions between L. salmonis and its host is crucial to identify possible ways to resolve negative impacts of this infection. L. salmonis can detect a range of stimuli (pressure/moving water, chemicals and light) in the external environment.

However, the response thresholds to various stimuli, and the spatial and temporal scales over which they operate in the context of host location, are largely unknown. Both physical and chemical cues are important in host location. Responses of sea lice to physical cues such as light and salinity may enable them to gather in areas where host fish are likely to be found. Chemoreception by kairomones plays a defining role in host location and recognition. Mechanoreception is an important sensory modality in host location and acts by switching on specific behaviours that enable landing on a fish.

The life cycle of L. salmonis includes ten stages, three of which are pelagic. The third of these is the infective stage of the salmon lice, the copepodid, which is sensitive to low frequency water accelerations such as those produced by a swimming fish. It carries both chemosensory aesthetes and mechanosensory setae on its antennules, indicating that both mechanical and chemical signals may be important in host-finding. Adult L. salmonis present different types of sensitive setae on their antennae.

Zooplankters such as copepods and protists use external mechanosensors for sensing spatial velocity gradients generated by preys or predators. It is understood that the absence of gravity receptors (i.e. statocysts) in planktonic animals has to do with the specific gravity of the zooplankton body, which is the same or slightly higher than water. Sensing flow-induced changes in orientation, rather than flow deformation, would enable more efficient control of vertical movements. In L. salmonis these external mechanoreceptors are located on the first antenna.

The following sequence of events during copepodid settlement and attachment can be described as:

● A searching phase that includes sensory organs (primary antennae) specialised in detecting the host

● A primary attachment phase that includes the mechanical attachment to the host through secondary antennae

● A secondary filament attachment stage that includes the production of a frontal filament anchoring the larva to the host.

Prior art

It is known that imposing sound signals may affect the behaviour of and/or impose damages to aquatic species.

An academic paper [1] describes the effect of sound exposure on cephalopods using frequencies between 50 – 400 Hz using a repeated sweep.

An academic paper [2] describes the effect of sound exposure on the non-invasive species Cotylorhiza tuberculata and Rhizostoma pulmo using frequencies between 50 – 400 Hz using a repeated sweep.

Patent publication WO 94/17657 discloses a method to remove parasites from fish that are residing in or swimming through a walled chamber. It uses sound waves to stun or kill the parasite by resonation or dislodging from the fish.

Patent publication WO 2013/051725 describes use of ultrasonic sounds for the removal of parasites from fish in a tank.

Patent publication WO 2013/095153 describes a system and method for inhibiting parasites to infest and attach to aquatic animals by using an acoustic arrangement generating signals at frequencies within 9 to 100 Hz, and which preferably produces an acoustic pressure of from 1 to 14 kPa in pulses with duration of from 1 to 10 seconds. Such acoustic signals are observed to evoke a flight/attack response of copepodids (Lepeophtheirus salmonis). The document further informs of tests involving acoustic signals at frequencies from 100 to 1000 Hz which showed low effect on the flight/attack response of these copepodids.

Objective of the invention

The main objective of the invention is to provide a system and method which specifically induces lethal harm/lesions in specific organs of copepodid and/or adult stage sea-lice present in a volume of water, either free floating or attached to a host fish.

Description of the invention

The objective of the invention is obtained by the technical features of the appended patent claims.

The present invention may be considered being a reduction to practice of a discovery believed to be novel, that a minimum level of accumulated sound doses at a specific sound pressure level (intensity) and at a specific frequency range, induces deadly lesions/damages in sea-lice present in the water either attached to a host fish or free-swimming in the volume of water. The sound doses and frequencies are optimised to target sensory and other organs of the sea-lice and has as far as the inventors have observed and knows, shown no negative impact on other marine species in or near the volume of water being exposed to the sound. The sound doses are observed to be harmful at all ten of the life-cycles of the sea-lice.

Thus, in a first aspect, the present invention relates to a system,

comprising:

a sound control unit,

an electric signal generator, and

a transducer located in a volume of seawater,

where:

the sound control unit operates/controls the electric signal generator to produce a first electric signal which is transferred to the transducer,

the transducer transforms the first electric signal to a sound in the volume of water, where the sound has:

one or more frequencies in the range of from 300 to 550 Hz, and

a sound pressure level, SPL, at 1 metre distance from the transducer in the range of from 140 to 180 dB re 1µPa<2>,

and where

the sound control unit determines a cumulative sound dose produced by the transducer, and engages the electric signal generator until the cumulative sound dose is equivalent to a sound exposure level, SEL, in the volume of seawater at 1 metre distance from the transducer of at least 180 dB re 1 µPa<2>·s or higher.

In a second aspect, the present invention relates to a method for causing lesions in sea-lice present in a volume of seawater,

where the method comprises:

producing a sound in the volume of water, where the sound has:

one or more frequencies in the range of from 300 to 550 Hz, and

a sound pressure level, SPL, in the range of from 140 to 180 dB re 1 µPa<2>, and

maintaining the produced sound at least until the cumulative dose of sound in the volume of seawater is equivalent to a sound exposure level, SEL, of at least 180 dB re 1 µPa<2>·s or higher.

The system according to the invention is schematically presented as seen from the side in figure 1. The control unit is presented as a box with reference number 1. The control unit may be any known electronic unit comprising software and or hardware enabling the control unit to control an electric signal generator such that is produces an electric signal which when fed to a loudspeaker/transducer, converts the electric signal to a sound in the sensitivity range of 300 to 550 Hz and having a sound pressure level in the range of from 140 to 180 dB re 1 µPa<2>, and to determine the cumulative sound exposure level of the sound to regulate the operation of the electric signal accumulator. The control unit 1(typically a Raspberry Pi or similar low power SBC) passes control signals to the electric signal generator 2 (e.g. a Monacor PA-12040) which causes the signal generator 2 to produce an electric signal being fed to a transducer 3 (e.g. a Lubell LL9161) located in a volume of water 4 (indicated by the stapled line). Reference number 12 is the water line of the sea/water. The transducers may in one example embodiment be driven by power amplifiers that can reach the voltages required for these levels; i.e. typical peak voltage levels up to 100 V.

As used herein, the term “sound pressure level, SPL”, is the equivalent continuous sound level in decibel determined as the time integral of the squared instantaneous sound pressure over the squared reference pressure over a time period of 1 second interval, and is defined by the relation:

Here, p(t) is the instantaneous (unweighted, unless specified otherwise) sound pressure at time t given in unit Pa, and pois a reference sound pressure chosen to be 1 μPa. The notation for the sound pressure level as used herein, in the case of e.g. a sound intensity of 80 dB, is: SPL = 80 dB re 1 μPa<2>, where the “re 1 μPa<2>” term informs that the sound pressure level is relative to a reference pressure level po of 1 μPa.

The term “sound exposure level, SEL” as used herein is the total cumulative squared instantaneous sound pressure over a time period expressed in decibel, and is defined by the relation:

where T is the cumulative time interval in unit seconds and TO is a reference time period chosen to be 1 second. The sound exposure level is numerically equivalent to the total sound energy. For example a sound with SPL of 90 dB re 1 μPa<2>lasting 1 second would give an SEL of 90 dB re 1 μPa2-s, while an SPL of 90 dB re 1 μPa<2>lasting for 2 seconds would give an SEL of 93 dB re 1 μPa<2>·s.

The inventors have observed that when sea-lice are exposed to relatively high doses of sound, i.e. an SEL of at least 180 dB re 1 μPa<2>·s, of a sound having one or more frequencies in the range of from 300 to 550 Hz, the sound causes lesions in one or more organs of the sea-lice which incapacitate and/or kill at least 90 % of them. In alternative embodiments, the sensitivity range may advantageously be in the range of from 300 to 500 Hz, preferably of from 300 to 450 Hz, more preferably of from 300 to 400 Hz, more preferably of from 320 to 380 Hz, more preferably of from 340 to 380 Hz, and most probably of from 340 to 360 Hz.

As used herein, the term “lesions” means an induced physical change in one or more vital organs of the sea lice, in particular (i) sensory organs responsible for the perception of acoustic pressure and particle motion associated with sound, (ii) cells responsible for the frontal filament, Cells A/B and (iii) neuronal axons of the central nervous system. The induced lesions on sensory setae incapacitate or degrade the sea-lice’s ability to perceive their surrounding environment and the Cells A/B normal function and alter the general operation of the nervous central system and consequently the normal general behaviour. The above described lesions taken separately or as whole incapacitate the sea-lice to find and attach to the host and lead to the death of the sea-lice. These lesions are observed to be formed on sea-lice both in the copepodid stage and in the adult life-cycle stages. These lesions are observed to be formed on both free-swimming sea-lice/copepodids and sea-lice/-copepodids attached to a host fish.

Without being bound by theory, these observations indicate that sea lice respond differently to exposure of sound than the known response of other marine species, by sea lice being sensitive not only towards the frequency of the sound but also towards the accumulated dose of sound (i.e. the SEL level), while the other species are observed to be sensitive towards the sound intensity (i.e. the SPL-level) at specific frequency ranges (which may be different from the sea lice sensitivity range from 300 to 550 Hz). The present invention utilizes this phenomena by applying one or more transducers to produce a controlled sound in the zone of water to be treated, where the produced sound is kept at an intensity level (i.e. an SPL-level) which is known to be safe to fish and marine mammal species and maintain this sound pressure level for a time period sufficient to reach a sound dose (i.e. an SEL-level) causing these lesions in the sea lice.

Present knowledge indicate that marine species are more or less insensitive to sound intensities below an SPL of 180 dB re 1 µPa<2>, such that this sound pressure level is a natural upper limit for the SPL of the sound provided by the one or more transducers applied in the present invention. There is as far as the inventors know no natural lower limit for the sound pressure level, any sound intensity of a sound in the sensitivity range of from 300 – 550 Hz which persists sufficiently long to reach a cumulative sound dose of at least 180 dB re 1 µPa<2>·s may be applied. However, since the sound doses required to cause the lesions in the sea-lice is a relatively huge dose of sound exposure, the sound intensity may advantageously be equivalent to at least an SPL of 140 dB re 1 µPa<2>since a continuous sound of this SPL needs about three hours to accumulate to a sound dose equivalent to an SEL of 180 dB re 1 µPa<2>·s. If the sound has an SPL of 130 dB re 1 µPa<2>, it will need about 28 hours to reach an SEL of 180 dB re 1 µPa<2>·s. On the other hand, a sound with SPL of 175 dB re 1 µPa<2>, needs only 4 seconds endurance to reach an SEL of 180 dB re 1 µPa<2>·s. Thus in summary, the present invention according to the first and second aspect may advantageously apply a sound source producing a sound having one or more frequencies in the sea-lice sensitivity range of from 300 to 550 Hz and a sound pressure level, SPL, of from 140 to 180 dB re 1 µPa<2>, more preferably of from 150 to 180 dB re 1 µPa<2>, more preferably of from 160 to 180 dB re 1 µPa<2>, more preferably from 170 to 180 dB re 1 µPa<2>, and most preferably of from 170 to 175 dB re 1 µPa<2>.

The cumulative sound doses which cause these lesions in sea-lice are observed to be equivalent to a sound exposure level, SEL, of at least 180 dB re 1 µPa<2>·s. There is as far as the inventors know no upper limit for the cumulative sound dose. The invention according to the first and second aspect, may advantageously apply a sound exposure level higher than 180 dB re 1 µPa<2>·s to ensure that the intended lesions are formed. However, due to the upper limit of safe sound pressure levels (SPL) of 180 dB re 1 µPa<2>, there is a practical limit for the sound exposure level (SEL) of 230 dB re 1 µPa<2>·s. A continuous sound of SPL of 180 dB re 1 µPa<2>needs about 28 hours to reach a cumulative sound dose equivalent to an SEL of 230 dB re 1 µPa<2>·s. Thus, in summary, the cumulative sound dose being applied in the present invention is equivalent to a sound exposure level, SEL, in the range of from 180 dB re 1 µPa<2>·s or higher, preferably of from 180 to 230 dB re 1 µPa<2>·s, more preferably of from 190 to 220 dB re 1 µPa<2>·s, more preferably of from 190 to 210 dB re 1 µPa<2>·s, and most preferably of from 190 to 200 dB re 1 µPa<2>·s.

It is observed that the sound does not need to be continuous to cause these lesions. The transducer may produce the sound dose intermittently by e.g. a series of sound doses separated by intervals of silence. It is the cumulative sound dose as such (at frequencies within the sensitivity range of from 300 to 350 Hz) which provides the effect of the invention. Thus, the volume of water may be exposed to a continuous sound until the required sound dose is achieved, or as a series of sounds separated by silence until the required sound dose is achieved.

The invention may apply sinusoidal sounds, either at a single frequency or a sound composed of two or more sinusoidal sounds superimposed onto each other. The produced sound may in one example embodiment be in the form of a series of two or more sequential sinusoidal sounds having different frequencies which are provided either attacca (following each other without periods of silence) or intermittently. In the latter example embodiment, the sound control unit may determine the sound dose provided by each of the sinusoidal sounds separately and terminate the sinusoidal sounds as each of them reach the required minimum cumulative sound dose or alternatively, determine the sum of the sound doses provided by these sequential sinusoidal sounds and terminate the sound when the sum reaches the required cumulative sound dose. In a further example embodiment, the sound produced by the transducer may have a continuous frequency in a selected third octave band within the sensitivity range of from 300 to 550 Hz.

Producing a sound within a specified range of frequencies and with an intended sound intensity and to establish the accumulated dose of sound provided by this sound, is a well-established technology known to the person skilled in the art. The invention may apply any known way of determining the cumulative sound dose from a sound of a specific intensity. In one example embodiment, the control unit may determine the sound exposure level in the volume of seawater by keeping track of the time endurance of the sound signal. If for example, the transducer is known or tuned to produce a sound having an SPL of 140 dB re 1 µPa<2>, the control unit may keep track of the time the sound is produced and terminate after e.g. 3 hours (since the required minimum SEL will be achieved after two hours and 47 minutes).

Alternatively, in one example embodiment, the transducer may be calibrated to ensure that it produces sound with a known SPL. The calibration may e.g. be obtained by measuring the resultant SPL of the produced sound as a function of the peak voltage of the electric signal being fed to the transducer. In this example embodiment, the sound control unit may apply the peak voltage of the electric signal being provided to the transducer to determine the SPL of the produced sound and maintain this sound (either continuous or intermittently) until the required minimum SEL is achieved).

In one example embodiment of the invention, the sound is produced by one or more calibrated underwater transducers capable of producing sound having frequencies covering all or part of the sensitivity range for the target organism of from 350 to 500 Hz and where the one or more transducers produces a sound with a sound pressure level of at least up to 140 dB re 1 µPa<2>at 1 m distance for individual frequencies, and 180 dB re 1µPa<2>at 1 m for selected third octave bands within this sensitivity range.

In a further example embodiment, the system may additionally comprise a hydrophone located in the volume of seawater and which continuously measures the instantaneous sound pressure at a location within the volume of water and transfers this information to the sound control unit. In this example embodiment, the sound control unit may advantageously comprise a logical control unit having a processor loaded with hardware or software which when executed, applies the measured (unweighted) instantaneous sound pressures from the hydrophone to calculate the sound exposure level according to relation (2) above. Alternatively, the logical control unit may be present in the hydrophone such that the sound control unit is provided with a more or less continuous update of the measured sound exposure level, SEL, as determined by the hydrophone. The hydrophones may in one example embodiment, be calibrated hydrophones able to record the acoustic pressure in a given frequency range with maximum sound pressure levels at least up to 180 dB re 1µPa<2>without saturation. The hydrophone system may in one example embodiment be arranged to include a preamplifier and an analogue to digital converter (preferably sampling at least at 40 kHz with 16-bit resolution) providing digitized data to a sound exposure control system.

The invention according to the first and second aspect of the invention may apply more than one transducer to enable exposing relatively large volumes of water for the required sound doses to incapacitate sea-lice being present in the water. The distance between adjacent transducers may advantageously be from 20 to 100 metres. The transducers may be positioned/distributed in the volume of water to be treated in any pattern/manner. Likewise, the system may apply more than one hydrophone to control/keep track of the sound exposure level at one or more local regions inside the volume of water being exposed to the sound.

The invention according to the first and second aspect is especially suited for use in cage type ocean fish farms where sea lice are difficult to prevent due to being carried into the cages by natural seawater flowing through the cages. One or more transducers placed in the cage or just outside the cage may expose the volume of water inside the cage to a sound within the sensitivity range of the sea lice at a sound pressure level providing the required cumulative sound dose to cause lesions in all, or at least a major fraction of, sea lice present inside a cage in a matter of four hours or less. The sound exposure to the water in the cage may in one example embodiment be provided 24-7 as sequential series of sinusoidal sound signals of duration of from 1 to 4 hours separated by 1 to 4 hours of silence in the season (time of year) when naturally occurring sea lice/copepodids are abundant and actively seeking a host fish to avoid fish in the farm from being infected with sea lice. In this example embodiment, the sound pressure level imposed to the water inside the fish cage may advantageously be sufficiently high to reach the required sound exposure levels to incapacitate sea lice in this water within an exposure time of four hours or less, i.e. having an SPL of from 170 to 180 dB re 1 µPa<2>to enable exposing a major portion of sea lice/copepodids being inside the cage(s) to a cumulative sound dose of at least 180 dB re 1 µPa<2>·s before they attach to the farmed fish.

In a third aspect the invention relates to a fish farm for salmonids, comprising a fish cage (10) submerged into seawater and containing one or more salmonids to be farmed, and

a system according to the first aspect of the invention where the transducer or transducers is/are located inside the cage (10) or in the seawater at the outside of the cage (10) within a distance to the nearest part of the fish cage (10) of less than 5 metres.

An example embodiment of the invention according to the third aspect is shown schematically as seen from the side in figure 2. The figure shows a fish cage 10, here exemplified as a cubic metal cage with outer walls defined by a net submerged a distance into the sea and which contains a number of salmonids. A transducer 3 is located in the centre of the fish cage 10. Otherwise, this example embodiment is similar to the example embodiment shown in figure 1 and described above.

In another example embodiment, shown schematically as seen from the side in figure 3, the fish cage 10 is in the form of a fish net closed in the bottom and suspended from a floating ring-shaped structure 11 where the transducer is located at the centre inside fish net. Otherwise this example embodiment is identical to the example embodiment shown in figure 2.

In yet another example embodiment, shown schematically as seen from the side in figure 4, the sound is provided by two transducers 3 located opposite each other on the outside of the suspended fish net forming the fish cage 10. Both transducers receive the electric signal provided by the electric signal generator 2. This example embodiment comprises further a hydrophone 5 located at the centre of the fish cage 10 for measuring the instantaneous sound pressure, p(t), provided by the transducers 3. The sound control unit 1 is in this example embodiment in communication with the hydrophone 5. The hydrophone may feed the measured instantaneous sound pressure, p(t) to the sound control unit. In this example embodiment, the control unit 1 determines the cumulative dose of sound by applying the measured instantaneous sound pressures to calculate a measured sound exposure level (by relation 2) and apply the determined cumulative sound dose to control the electric signal unit 2. Alternatively, the hydrophone may be equipped with a logical control unit adapted to calculate a sound exposure level from the measured instantaneous sound pressure and feed this information to the control unit 1.

The use of two or more transducers distributed along the periphery of the zone of water to be exposed and having one or more hydrophones in the centre of the zone of water to measure and regulate the cumulative sound dose, is especially suited for floating fish net type ocean fish farms having a diameter above 20 metres.

In one example embodiment of the invention according to any aspect, the fish being farmed is Atlantic salmon (Salmo salar) and the sea-lice being targeted is Lepeophtheirus salmonis.

In a fourth aspect, the invention relates to use of the system according to the first aspect for preventing and/or treating fish affected by sea-lice. The system may be used for this purpose in any of the varieties and example embodiments described above for the first aspect.

Thus, in a fourth aspect, the invention relates to a system for use in preventing and/or treating fish affected by sea lice, the system comprising

a sound control unit (1),

an electric signal generator (2), and

a transducer (3) located in a volume of seawater (4),

where:

the transducer transforms the first electric signal to a sound in the volume of water, and where the sea lice attached to the fish or being present in the water surrounding the fish is subjected to a sound, where:

one or more frequencies is/are in the range of from 300 to 550 Hz, and a sound pressure level, SPL, at 1 metre distance from the transducer in the range of from 140 to 180 dB re 1µPa<2>,

and where

the sound control unit determines a cumulative sound dose produced by the transducer, and engages the electric signal generator until the cumulative sound dose is equivalent to a sound exposure level, SEL, in the volume of seawater of at least 180 dB re 1 µPa<2>·s or higher.

The system according to the fourth aspect may be used with one or more frequencies is/are in the range of from 300 to 500 Hz, preferably of from 300 to 450 Hz, more preferably of from 300 to 400 Hz, more preferably of from 320 to 380 Hz, more preferably of from 340 to 380 Hz, and most probably of from 340 to 360 Hz, and with a sound pressure level, SPL, is in the range of from 150 to 180 dB re 1 µPa<2>, preferably of from 160 to 180 dB re 1 µPa<2>, more preferably from 170 to 180 dB re 1 µPa<2>, and most preferably of from 170 to 175 dB re 1 µPa<2>, and with a cumulative sound dose is equivalent to a sound exposure level, SEL, in the range of from 180 to 230 dB re 1 µPa<2>·s, preferably of from 190 to 220 dB re 1 µPa<2>·s, more preferably of from 190 to 210 dB re 1 µPa<2>·s, and most preferably of from 190 to 200 dB re 1 µPa<2>·s.

List of figures

Figure 1 is a schematically drawing as seen from the side of an example embodiment of the system according to the invention.

Figure 2 is a schematically drawing as seen from the side of an example embodiment of a fish farm according to the invention.

Figure 3 is a schematically drawing as seen from the side of another example embodiment of a fish farm according to the invention.

Figure 4 is a schematically drawing as seen from the side of another example embodiment of a fish farm according to the invention.

Figure 5 is a graph showing attachment counts of sea-lice in experiment 1 with sealice not exposed to the sound of the present invention.

Figure 6 show light microscopy images of healthy sea-lice not exposed to the sound attachment. A: Arrows point to some sea lice attached to the Salmon dorsal fin. B: Sea lice attached to Salmon skin. C: Attached sea lice. The antenna is visible. Red coloration is due to some blood after salmon sacrifice. D: Arrow points to a sealice attached near the salmon eye. E, F: Detail of D. Bigger magnification allows seeing the sea lice body structure.

Figure 7 show light microscopy images of the morphology of healthy sea-lice. LM (A, B, H-J) and SEM (C-G). External morphology of L. salmonis. A: Dorsal and B: Ventral view of the head of an adult sea-lice. C: Ventral view of the whole body of an adult L. salmonis. D: Ventral view of the head of sea lice. E: Ventral view of an adult L. salmonis showing the mouth, maxilas and abdominal arms. F: Detail of the ventral cavity showing the mouth and the three maxilas. G: String of sea lice eggs. In the low part of the string, two larvae are being extruding of the eggs. H: Nauplius just hatched of the egg. I: Copepodid of sea lice.

Figure 8 is a graph showing attachment counts of sea-lice in experiment 2 with sealice exposed to one or two doses of the sound of the present invention.

Figure 9 show SEM-photographs of Lepeophtheirus salmonis copepodids sensory setae morphology of control animals, where; A: Dorsal and B: Ventral view of a L. salmonis copepodid. C: Cephalotorax dorsal view showing some paired setae distributed along the body. D: Detail from C shows the structure of a birrame setae. E: Dorsal view of the abdomen showing some paired setae. F: Mouth of the copepodid. G: Ventral arms showing pinnate setae. H: Caudal ramus showing the distal setae. I: First antenna. J-L: Detail of the first antenna setae showing their irregularly branching tips.

Figure 10 show SEM-photographs of Lepeophtheirus salmonis morphology of control animals, where A: Dorsal and B: Ventral view of a L. salmonis copepodid. C: Copepodid head ventral view showing some paired setae on the first antenna. D: Detail of the mouth of the copepodid showing the powerful teeth.

Figure 11 is SEM-photographs of Setae on distal segment of first antenna L-salmonis copepodids, where A: Ventral view of the two antennas on an animal sacrificed 48h after sound exposure. B-F: Normal setae on control animal. The tips on the setae distal segments are totally free (not fused). G-J: Different views of animals sacrificed 0h after sound exposure showing fusion (arrowheads) on the basal segment of the setae on the distal segment of the first antenna, but the distal tips are less fused. K-N: Different views of animals sacrificed 24h after sound exposure showing fusion (arrowheads) on the basal segment of the setae on the distal segment of the first antenna but presenting less fusion on the distal tips. O-R: Different views of animals sacrificed 48h after sound exposure showing almost totally fused (arrowheads) distal segment of the first antenna. S-V: Different views of animals sacrificed 48h after sound exposure showing totally fused distal segment of the first antenna.

Figure 12 is SEM-photographs of Setae on ventral arms and caudal ramus of L-salmonis copepodids, where: A-C: Normal distribution f the pinnate setae of the ventral arms on control animals. D: Ventral arms have lost some of the pinnate setae (arrowheads) on a copepodid sacrificed 48h after sound exposure. E: Fusion of the pinnate setae on ventral arms (arrowheads) on a copepodid sacrificed 48h after sound exposure. F: Totally fused pinnate setae of the ventral arms on a copepodid sacrificed 72h after sound exposure. G: Normal distribution of the pinnate setae on the caudal ramus in a control animal. H: Caudal ramus has lost some of the distal pinnate setae (arrowheads) on a copepodid sacrificed 48h after sound exposure. I: Fusion of the pinnate setae (arrowheads) of the caudal ramus on a copepodid sacrificed 72h after sound exposure.

Figure 13 is a graphical representation of obtained Setae fusion on sea-lice first antenna (%) as function of sound exposure time and sacrifice time after exposure. The optimal time of sound exposure was 4 h, that achieve maximum setae fusion on the sea lice first antenna (95.24 %) and, 0 h after sound exposure was the optimal time for sacrifice sea-lice and process samples for the posterior analysis (as indicated by the red arrow).

Figure 14 is a graphical representation of obtained Setae fusion on sea lice first antenna (%) as function of frequency. 350 Hz achieve the maximum percentage of setae fusion. Between 350 Hz and 550 Hz the fusion percentage is higher than 90%.

Figure 15 is a graphical representation of obtained Setae fusion on sea lice first antenna (%) in function of frequency combinations. 350 – 450 Hz and 350 - 550 Hz are the combinations that achieve the maximum percentage of setae fusion (95.2%).

Figure 16 is a graphical representation of obtained attachment count as function of sound exposure duration in experiment 5.

Figure 17 is TEM-photographs showing Sagittal section of the copepodid anterior cephalotorax before and after exposure to the sound according to the invention.

Figure 18 is TEM-photographs showing sagital section of the copepodid anterior cephalotorax showig CElls a & B involved in the frontal filament production before and after exposure to the sound according to the invention.

Verification of the invention

The invention will be explained in further details by verification experiments to verify the effect of the invention. A series of experiments were conducted on copepodids, pre-adults and adults in both laboratory and sea conditions to determine i) the acoustic parameters that would trigger the expected lesions on the exposed individuals and ii) the effects of the methods on the fish health.

Experiment 1 - Validation and standardization of healthy copepodids attachment rate & proportion

The objective of experiment 1 is to determine the speed and proportion of healthy (i.e. not exposed to the sound) copepodids attachment to the host in a laboratory installation to form basis for experiments verifying the effect of the invention.

Sea lice specimens

One thousand (n = 1.000) copepodids from L. salmonis were kept (until they were required for the experiments) in a closed system of natural seawater (at 7-10 °C, salinity 35 ‰) consisting of a plastic tank with a capacity of 20 L. An extra supplement of oxygen was delivered with an air pump in order to facilitate the respiration and the copepodid movements in the water column.

Healthy sea lice attachment rates

The copepodids (n=1000) were introduced into the plastic tank in presence of 10 healthy salmons (n = 10) at time T = 0 hours, then a first salmon was retrieved (and sacrificed) at time T= 1 hour, a second salmon at time T= 2 hours, and so on until all 10 salmons were retrieved (at T= 10 hours). The number of sea-lice attached to each salmon was counted and is shown graphically in figure 5.

As seen from figure 5, the number of attached copepodids was relatively slowly increasing with time until about 9 hours and then increased rapidly. The sea-lice were showing a consistent attachment proportion after 8 hours, being even greater after 10 hours.

Conclusion experiment 1: The sea-lice were showing a consistent attachment to the salmon after 8 hours (n>15) in these tank conditions.

Experiment 2 - Determination of the attachment of copepodids after exposure to sounds

The objective of experiment 2 was to determine the copepodid capacity to attach to the host after being exposed to sound. It aimed at providing a ‘lesion threshold’ which would maximize the effects while minimizing the sound exposure time.

Sea lice specimens

Five sets, each of five hundred (n=500) copepodids from L. salmonis, were kept (until required for the experiments) in closed plastic tanks with natural seawater at the same conditions as in experiment 1 (at 7-10 °C, salinity 35 ‰). An extra supplement of oxygen was delivered with an air pump in order to facilitate the respiration and the copepodid movements in the water column. The copepodids were maintained in the tanks until exposure. Specimens used as controls were kept in the same conditions as the specimens exposed to noise.

Sound Exposure Protocol

Four of the five sets of copepodids where exposed to the sound and one set was applied as control.

The sound exposure consisted of a 50-400 Hz sinusoidal wave sweeps with 100% duty cycle and a 1-second sweep period for two hours. The sweep was produced and amplified through an in-air loudspeaker while the level received was measured by a calibrated B&K 8106 hydrophone (SPL=157±5 dB re 1 μPa<2>with peak levels up to SPL = 175 dB re 1μPa<2>).

Sound exposed sea lice attachment

Based on the results from experiment 1, the sea-lice were allowed 8 hours to locate and attach to a fish, such that the first salmon was extracted 8 hours after exposure which time is considered being T0 in experiment 1, the second salmon after 9 hours of exposure which is considered T1, and so on until all five salmons are extracted and sacrificed. The sacrificing process was identical for controls and exposed animals.

The average count of attached sea lice at T = 8, 9 and 10 in experiment 1 is applied in experiment 2 as a reference (control) attachment rate for comparison with the attachment rates after sound exposure. The reference attachment rate is thus set to be 21 sea-lice on each fish.

One set of copepodids (n= 500) was used as control, i.e. not exposed to sounds other than the background noise of the environment. This set was applied to confirm that the sea-lice were alive along the experimental process. This control set was used to check, after all experiments with exposed lice had ended, that their attachment rate was still superior than the sound exposed ones, demonstrating that the low rates of attachment found in the exposed lice was due to the sound exposure and not to a natural mortality process of the lice.

One set of copepodids (n= 500) was transferred to a tank with healthy salmons immediately after exposure to sound. The first salmon was extracted (and sacrificed to count the copepodids) after 8 hours. The second salmon was extracted one later (after 9 hours), and so on every hour until 12 hours after exposure.

One set of copepodids (n=500) was transferred to a tank with 5 healthy salmons 24 hours after exposure to the sound. The same sequence as above was followed, i.e., first salmon extracted after 8 hours and so on every hour, until 12 hours after exposure.

One set of copepodids (n=500) was transferred to a tank with 5 healthy salmons 48 hours after the first exposure to the sound and immediately after the second exposure to the sound (produced by the same sound protocol as the first sound). The same sequence as above was followed, i.e., first salmon extracted after 8 hours and so on every hour, until 12 hours after exposure.

One set of copepodids (n=500) was transferred to a tank with 5 healthy salmons 72 hours after the first sound exposure and 24 hours after the second sound exposure. The same sequence as above was followed, i.e., first salmon extracted after 8 hours and so on every hour, until 12 hours after exposure.

Imaging Techniques

Individuals were processed according to routine SEM procedures.

Light microscopy (LM)

Previous to the samples treatment by routine SEM procedures some light microscopy images of individuals not exposed to the sound were taken to clarify the morphology and location of the sensory setae, and the attachment process of the sea-lice to the salmons. The photographs are shown in figure 6.

A set of light microscopy images of healthy sea-lice not attached to a host fish are shown in figure 7.

Scanning electron microscopy

Ten (n=10) L. salmonis copepodids were extracted from every set (control and every treatment of exposed sea lice (0 h, 24 h, 48 h, and 72 h after sound exposure) before introducing the rest of the animals in the tanks in presence of salmons, and were used to analyse the lesions after sound exposure.

Fixation was performed in glutaraldehyde 2.5 % for 24-48h at 4 ºC. Samples were dehydrated in graded alcohol solutions and critical-point dried with liquid carbon dioxide in a Leica EmCPD030 unit (Leica Microsystems, Austria). The dried specimens were mounted on specimen stubs with double-sided tape. The mounted samples were gold-palladium coated with a Polaron SC500 sputter coated unit (Quorum Technologies, Ltd.) and viewed with a variable pressure Hitachi S3500N scanning electron microscope (Hitachi High-Technologies Co., Ltd, Japan) at an accelerating voltage of 5 kV in the Institute of Marine Sciences of the Spanish Research Council (CSIC) facilities.

Lepeophtheirus salmonis copepodids sensory setae morphology of control animals Copepodids (length 0, 7 ± 0,01; width 0,2 ± 0,01) (Figures 9, 10) present 10 pairs of setules arranged symmetrically about medial longitudinal axis (6 pairs of simple setules, 4 pairs bifurcates) on the dorsal shield of Cephalothorax and 2 simple setules near the base of Rostrum.

The first antenna (Figure 9, 10) presents a proximal segment with 3 unramed setae and a distal segment with 5 setae with irregularly branching tips, 7 unramed setae and 1 aesthete. The second antenna exhibits 3 segments with a spiniform process.

The 3 thoracic legs (Figure 9, 10) present plumose setae, semipinnate setae, pinnate setae, spines, spiniform process and fine setules. Caudal ramus (Figure 9, 10) shows short and long pinnate setae and aesthete.

Ultrastructural analysis of the setae of L. salmonis Copepodids after noise exposure Some ultrastructural changes took place on setae copepodids of L. salmonis following acoustic exposure. All exposed individuals presented lesions in the setae and incremental effects versus time. From 0 h until 72 h after sound exposure, all the copepodids presented different grade of fusion of the irregularly branching tips of the setae on the distal segment of the first antenna (Figure 11).

In control animals, the first antenna presented completely free setae with irregularly branching tips on the distal segment (Figure 9J-L, 11A-F). At 0 h and 24 h after sound exposure the fusion of the irregular tips of the setae had started but was incomplete (~ 40% of the distal segments of the sensory setae were fused, Figure 11 G-N), eliciting an initial (0h after sound exposure) decrease in the number of attachments to salmons, but showing a slight increase at 24 h after sound exposure.

This suggested to apply a second dose of sound, as it was previously explained. After this second exposition, all observed sea lice presented an almost total fusion (48 h after the first exposure, Figure 11 O-R, ~ 80% of the distal segments of the sensory setae were fused) and totally fused distal segments of the first antenna (72 h after the first exposure, Figure 11 S-V, 100%).

This fusion explains why the sea lice could not find the salmons and these severe lesions represent the cause of the final decrease of the attachments.

Additionally, to the effects on the setae of the distal segment of the first antenna, the copepodids showed loss or fusion of some distal pinnate setae on the caudal ramus and disorganized distribution, loss or fusion of the pinnate setae of the ventral arms (Figure 12). These abnormalities could provoke a disorganized displacement of the sea lice, which could also contribute to the decrease in the number of attachments to the salmons.

Results

The attachment count in experiment 2 is shown graphically in figure 8. The figure shows a clear decrease in the attachment rate compared to the control. At T24 after exposure, the attachment rate showed an increment although the rate remained inferior to the control. It was decided to expose the remaining sets of sea-lice to a second dose of sound equal to the first dose of sound. At T0 after the second exposure (T48 after the first one), the sea-lice showed a significant decrease of the attachment rate, reaching the same numbers as at T0 after the first exposure. This decrease became dramatic at T24 (T72 after the first exposure).

Conclusion experiment 2: Exposure to sound impairs L. salmonis sensory perception and prevent them to detect and attach to their host. We determined that two exposure cycles were required to trigger the lesions impairing the lice to attach to the salmons: at 0H and 24H after the exposure, incomplete fusion (~ 40% of the distal segments of the sensory setae are fused); at 48H after exposure, almost complete fusion (~ 80%); at 72H, total fusion (100%). These results show that a two cycle-exposure was triggering enough level of lesions to prevent the lice to attach to the salmon. 2.000 sound-exposed lice were sequentially introduced to 20 salmons. Only less than 2 (two) lice were found to attach to a single salmon. In proportion: 2 % of the introduced lice attached to the salmon after sound exposure.

This result clearly shows that the method of exposing copepodids to sounds significantly prevents them to find their host. It has to be noted that the density of copepodids in such a small enclosure is much greater to what is found in open waters. As for the sound level used during the exposure (~150 dB re 1 µPa<2>), which is about 30 dB lower than what is considered as a threshold level that could harm wildlife.

Our study shows ultrastructural images, which characterize pathological changes in copepodid L. salmonis sensory setae of their first antenna after sound exposure. An increased damage severity was observed with time after sound exposure. Damaged setae were bent, flaccid and fused sensory hairs. The mechanism by which sound induced this trauma has yet to be determined but it directly affects the main L. salmonis sensory organ. These lesions suppose an immediate cancellation of eating and breeding of the exposed individuals because they are not able to attach to the host, thus challenge the lice survival in the water column.

These findings are consistent with the initial hypothesis of this study: exposure to sound impairs L. salmonis sensory perception and prevent them to detect and attach to their host.

Experiment 3- Determination of sound exposure parameters to trigger lesions that prevent sea lice attachment; effects of sound exposure on salmon; detachment rate of lice after exposing salmon to noise

The objectives of the third experiment are determination of the time, frequency range and amplitude of the optimum exposure signal

Experiment 3-1: Evolution of copepodid lesions vs. time

Five sets, each of five hundred (n=500) copepodids from L. salmonis, were kept (until required for the experiments) in closed plastic tanks with natural seawater at the same conditions as in experiment 1 (at 7-10 °C, salinity 35 ‰). An extra supplement of oxygen was delivered with an air pump in order to facilitate the respiration and the copepodid movements in the water column. The copepodids were maintained in the tanks until exposure. Specimens used as controls were kept in the same conditions as the specimens exposed to noise

Sound Exposure Protocol

Sequential Controlled Exposure Experiments (CEE) were conducted on copepodids (16 x 500) of L. salmonis. Four additional set of copepodids (4 x 500) was used as a control. Individuals were maintained in the tank system (tank A) until the time of exposure. The same sound exposure parameters were applied as in experiment 2.

Table 1

Imaging Techniques

The same imaging techniques were used as in experiment 2. Individuals were processed according to routine SEM procedures.

Scanning electron microscopy

All sets of L. salmonis copepodids: control and exposed sea lice (0 h, 24 h, 48 h and 72 h after sound exposure) were used for the analysis.

Results

The resulting Saeta fusions obtained in experiment 3-1 is shown graphically in figure 13.

Conclusion experiment 3-1: After the sound exposure and the analysis of the setae fusion on the first antenna of the sea lice, we determined that the optimal time of exposure in our conditions was 4 h. The optimum time of sacrifice after sound exposure for the samples collection and analysis was 0 h after sound exposure (see Figure 13).

Experiment 3-2: Exposure parameters vs. lesions: Copepodids

Experiment 3-2-1: Frequency and frequency combinations

Objective of experiment 3-2: Determination of the signal frequency that triggers lesions in copepodids.

Sea lice specimens

Fifty (50) sets of five hundred (n=500) copepodids from L. salmonis were kept (until required for the experiments) in a closed system of natural seawater (at 7-10 °C, salinity 35 ‰) consisting of a plastic tank with a capacity of 20 L. An extra supplement of oxygen was delivered with an air pump in order to facilitate the respiration and the copepodid movements in the water column.

Individuals were maintained in the tank system until exposure. Several specimens (see table 2 below) were used as controls and were kept in the same conditions as the experimental animals until they were exposed to noise.

Sound Exposure Protocol

Sequential Controlled Exposure Experiments (CEE) were conducted on copepodids (25 x 500) of L. salmonis. A same number of copepodids (25 x 500) was used as a control. The same setting for the sound exposure was used as in experiment 3-1. Individuals were maintained in the tank system (tank A) until exposure.

Based on the output of experiment 1 (time of attachment), experiment 2 (proportion of lesions incompatible with the attachment) and experiment 3-1 (time of exposure and time after exposure required to reach the proportion of lesions), a series of discrete frequencies, ranging from 100 to 1.000 Hz, as well as combinations were tested to identify the ones that trigger major damage.

Imaging Techniques

The same imaging techniques were used as previously described. Individuals were processed according to routine SEM procedures.

Scanning electron microscopy

All sets of L. salmonis copepodids (control and exposed sea lice) as shown in Table 2 were used for the analysis.

Table 2

Results

The obtained Setae fusion on sea-lice first antenna (%) as function of single frequencies is shown graphically in figure 14, and in figure 15 as function of frequency combinations.

Conclusion experiment 3-2: After the sound exposure and the analysis of the setae first antenna of the sea lice, we found maximum fusion at 350 Hz (95.5 %) but on the frequencies between 300 Hz and 550 Hz we achieved a percentage of setae fusion higher than 90 % (Figure 14). After the sound exposure that combined the frequencies that previously have achieved the maximum fusion and after the analysis of the setae first antenna of the sea lice, we found 350 Hz-450 Hz and 350 Hz. 550 Hz were the combination that achieved the maximum percentage of setae fusion (95.2 %) (Figure 15). There was not a significant increase on the level of fusion with the combination of two frequencies.

Experiment 3-2-2: Amplitude

Determination of the minimum amplitude needed to trigger lesions

Sea lice specimens

Fifty (10) sets of five hundred (n=500) copepodids from L. salmonis were were kept (until required for the experiments) in a closed system of natural seawater (at 7-10 °C, salinity 35 ‰) consisting of a plastic tank with a capacity of 20 L. An extra supplement of oxygen was delivered with an air pump in order to facilitate the respiration and the copepodid movements in the water column.

Individuals were maintained in the tank system until exposure. Several specimens (see below) were used as controls and were kept in the same conditions as the experimental animals until they were exposed to noise.

Sound Exposure Protocol

Sequential Controlled Exposure Experiments (CEE) were conducted on copepodids (5 x 500) of L. salmonis. Five additional sets of copepodids (5 x 500) were used as control. The same sound exposure used as in experiment 3-2-1 was followed.

Individuals were maintained in the tank system (tank A) until the time of exposure.

The output of experiment 2 and experiment 3-2-1 determined the required time of exposure and the best 5 frequencies (or combination of frequencies) that trigger the lesions. It was aimed at testing different amplitudes (A) to choose the rights levels that would ensure the required SEL to induce lesions to the sea-lice.

The tested combinations are given in table 3:

Table 3

Imaging Techniques

Scanning electron microscopy

All sets of L. salmonis copepodids (control and treatments of exposed sea lice) were used to analyze the lesions after sound exposure.

Results and conclusion experiment 3-2-1

In this experiment, different combinations of times, frequencies and levels of exposure were tried in order to determine the amplitude associated to a frequency that achieved maximum level of setae fusion with less time. After the sound exposure and the analysis of the setae first antenna of the sea lice, it was found maximum setae fusion with the combination of 350Hz-2h- SEL of 194 dB re 1 µPa<2>·s and 500Hz-2h- SELof 194 dB re 1 µPa<2>·s, SPL of ~155dB re 1 µPa<2>(93.02 %) other combinations (e.g., 350Hz-2h- and 500Hz-1h SEL of 194 dB re 1 µPa<2>·s , 350Hz-3h- SEL of 192 dB re 1 µPa<2>·s) achieved a percentage of setae fusion higher than 90 % (as shown in Table 4):

Table 4 Setae fusion on sea lice first antenna (%) as a function of frequency, time and level of exposure in the tank conditions.

These values were used to determine the sound exposure parameters that will be used in experiment 4 and during the field test.

Experiment 4: Effects of noise exposure on Salmons

Objective: Determining if the salmons, after being exposed to noise, show behavioural or pathological alterations, or on the contrary, habituation to sound.

Previous literature review looked at the effects on fish after exposure to sound. The review evidenced that the levels used in previous experiments far exceeded the levels that our method uses and yet did not elicit any pathology.

Salmon specimens

A set of healthy salmons (n=140), were acclimated to the LAB infrastructures (time = fifteen days), regular food intake and growth were monitored to determine the normal growth patterns. After that period of time, ten (10) salmons (5 of tank A and 5 of tank B) were taken from the tanks to be measured and checked and samples of its inner organs and otolith organs were processed to be used as a control samples.

Sound exposure protocol

A set of forty (40) salmons were exposed daily over four weeks to look at possible lesions on fishes after sound exposure. An additional set of salmons (n=10) was used as a control.

Sequential Controlled Exposure Experiments (CEE) were conducted on salmons. The exposure consisted on a cycle of 350 Hz for 2 hours and 500 Hz for 2 hours (SEL of 195 dB re 1 µPa<2>·s) exposure, twice daily with a period of rest of 2 h between the two cycles, during three weeks. The last group of 10 salmons were exposed three times a day for one week. The sound was produced and amplified through an underwater speaker while the level received was measured by a calibrated B&K 8106 hydrophone (R (same conditions than sea lice).

Exposed salmons were sacrificed weekly (after 1 week, 2 weeks, 3 weeks and 4 weeks of sound exposure) to assess the possible lesions on their tissues. The analysis to be performed is as follows:

Scanning electron microscopy

Otolith organs epithelia from individual fishes were observed by SEM imaging techniques to detect any possible alteration of the sensory epithelia.

Gross pathology and Histological analysis

In addition to the analysis of the salmon otolith organ by SEM, the anaesthetised (with 2-phenoxyethanol) salmons were subjected to a supplementary gross pathology and histological analysis to assess possible lesions in other tissues.

Samples of different tissues of both the exposed and control individuals were analysed and the swim bladder were assessed through gross anatomical dissection to determine a possible rupture caused by the effect of sound.

The Pathological Diagnostic Service in Fish (SDPP) of Autonomous University of Barcelona (UAB) performed the histological analysis. Histological analysis of different tissues samples of both the exposed and control individuals were performed. Samples of salmon (Salmo salar) submitted for general histopathological examination (H/E stain): Anterior body wedge which include skeletal muscle, trunk kidney, swim bladder, stomach, pyloric caeca, liver, adipose tissue, pancreas and gonad; posterior body wedge which include skeletal muscle, posterior kidney, swim bladder, intestine, spleen and adipose tissue.

Behavioural observations

Salmon behavioural were visually observed before, during and after the sound exposure, for a period of 10 min each time, in order to determine behavioural alterations (expected behavioural reactions were jumps, rolls and twitches). Jumps were defined as fast acceleration in swimming speed that ended in a jump, rolls involved turning 90º on horizontal or vertical plane, and twitches were defined as rapid spasmodic contractions of the body) of salmons.

Results

No acoustic trauma (hearing loss) was found in fish after exposing them to sound sources that far exceed the levels used in our method. No lesions were found on the otolith three sensory epithelia (saccule, utricle & lagena). No gross pathology & histopathological alterations associated to sound exposure were found in the analysed samples. Despite the lack of evidence of acoustic-related pathology in the literature, we proceeded with our own experiments to determine the salmon sensitivity to the sound dose that would be used in our method.

Conclusion experiment 4: Otolith organs epithelia observed by SEM imaging techniques did not show any alteration of the sensory epithelia. The gross pathology and histological analysis on salmons did not show any lesion that could be associated to sound exposure. No salmon behavioural reactions were observed before, during and after the sound exposure.

Based on the literature survey described above and on the results of the experiments performed under the same conditions as the ones that will be conducted in sea conditions, we conclude that no effect on salmon, nor on another species of local fish is expected to happen when using our method. Both the range of frequencies, which do not correspond to a region where fish are acoustically most sensitive to, as well as the sound levels that will be used to produce a sufficient Sound Exposure Level (SEL) so lice cannot detect the fish, represent enough guarantee to safely apply the method in real conditions.

Note: the gross pathology and histopathological analysis of salmon inner organs were performed by an independent agency: The Pathological Diagnostic Service in Fish (SDPP), Autonomous University of Barcelona (UAB, Spain)

Experiment 5: Detachment of sea lice copepodids

Determination of the proportion of sea lice copepodids detaching from the host after exposure to sound.

Salmon specimens and sound exposure

Healthy (non-infested by lice) salmons (n=80) were introduced with sea lice copepodids in the LAB tanks for a period of 8 h (this time corresponds to the results obtained in experiment 1). After 8 hours, the salmons were exposed to sounds for 4 hours, twice a day, leaving a non-exposure period of two hours between two consecutive exposures. Salmons (n=10) were sequentially extracted to count lice after each exposure period. This protocol was applied for 4 consecutive days. The effects of the exposure were measured through the quantification of attached sea lice over time.

Results

The obtained attachment count as function of sound exposure duration is given graphically in figure 16.

Conclusion experiment 5: After exposure to sounds (counts were sequentially made after each 4-hour exposure time), eight times less copepodids were counted on the salmons compared to control animals, i.e. a clear decrease in the attachment rate. This shows a significant effect of sound exposure triggering sea lice to detach from salmon.

Experiment 6: Determination of ultrastructural lesions on inner tissues of sea lice copepodids

Observation of the nervous system and Cells A & B alterations after sound exposure that can affect the anchoring to the host in the secondary attachment stage.

A set of 2.000 copepodids were exposed for 8 hours to the sound stimuli defined in experiment 3, being 350 Hz (for 4 hours) and 500 Hz (for 4 hours).

The same imaging techniques as in previous experiments were used to process and observe the samples. A particular attention was given to inner structures like cells A and B and the associated central nervous system.

Conclusion experiment 6: Pathological changes in copepodid L. salmonis inner tissues (Cells A & B and nervous system) were characterised before and after sound exposure as shown in e.g. figures 17 and 18. Sound exposure affected Cells A/B responsible from the precursor secretions of the frontal filament, preventing the firm anchorage to the host (fish). Sound exposure permanently affects the central nervous system of the copepodids, altering their normal behaviour and challenging their chance of survival.

Experiment 7: Sea experiments, determination of effects of sound exposure on salmon; detachment rate of lice after exposing salmon to noise

Two cages (5 x 5 m) were used in sea conditions where fish (n=250) were introduced at T0 of the experiments. A set of transducers (n=4) was introduced in the exposed cage while the second cage was used as control. The control cage was located at 300m from the exposed cage.

The sound exposure sequence determined in experiment 3 was played continuously to the fish in the exposed cage with one hour of signal off between sequences.

Experiment 7-1: Effects of sound exposure on fish

Objective: Determining if the salmons, after being exposed to noise in sea conditions, show behavioural or pathological alterations, or on the contrary, habituation to sound.

10 fish were taken from the exposed and controlled cages at T0, T1 (one week after) and T2 (two weeks after T0) and the same organs as in experiment 3 were extracted to look for possible lesions. The same protocol for fixation and observation was followed as well.

Conclusion experiment 7-1: Control and exposed fish were compared. Otolith organs epithelia did not show any alteration of the sensory epithelia. The gross pathology and histological analysis on salmons did not show any lesion that could be associated to sound exposure. No behavioural reactions were observed before, during and after the sound exposure.

Experiment 7-2: Determination of ultrastructural lesions on inner tissues of preadult and adult sea lice after exposure to sound in sea conditions

Objective: Observation of the nervous system and Cells A & B alterations after sound exposure that can affect the permanence of pre-adult and adult sea lice on the fish.

Samples of pre-adult and adult of sea lice were processed at T0 (controls), T1 and T2, to observe the nervous system and Cells A & B possible alterations after sound exposure that can affect the permanence of pre-adult and adult sea lice on the fish.

Conclusion experiment 7-2: We expect to characterize pathological changes in pre-adult and adult L. salmonis inner tissues (Cells A & B and nervous system) after sound exposure in the same way as we found in copepodids. Sound exposure should affect Cells A/B responsible from the precursor secretions of the frontal filament that happens at every moult, inducing the detachment from the host. Sound exposure should permanently affect the central nervous system of the pre-adult and adult lice, permanently altering their normal behaviour and inducing their death.

References

1. André et al., “Low-frequency sounds induce acoustic trauma in cephalopods”, Front Ecol Environ, 2011, (https://doi.org/10.1890/100124)

2. Solé et al., “Evidence of Cnidarians sensitivity to sound after exposure to low frequency noise underwater sources”, Nature Scientific Reports 6, 2016, (DOI: 10.1038/srep37979)

Claims

CLAIMS 1. A system, comprising: a sound control unit (1), an electric signal generator (2), and a transducer (3) located in a volume of seawater (4), where: the sound control unit operates/controls the electric signal generator to produce a first electric signal which is transferred to the transducer, the transducer transforms the first electric signal to a sound in the volume of water, where the sound has: one or more frequencies is/are in the range of from 300 to 550 Hz, and a sound pressure level, SPL, at 1 metre distance from the transducer in the range of from 140 to 180 dB re 1µPa<2>, and where the sound control unit determines a cumulative sound dose produced by the transducer, and engages the electric signal generator until the cumulative sound dose is equivalent to a sound exposure level, SEL, in the volume of seawater of at least 180 dB re 1 µPa<2>·s or higher.
2. A system according to claim 1, wherein the one or more frequencies is/are in the range of from 300 to 500 Hz, preferably of from 300 to 450 Hz, more preferably of from 300 to 400 Hz, more preferably of from 320 to 380 Hz, more preferably of from 340 to 380 Hz, and most probably of from 340 to 360 Hz.
3. A system according to claim 1 or 2, where the sound pressure level, SPL, is in the range of from 150 to 180 dB re 1 µPa<2>, preferably of from 160 to 180 dB re 1 µPa<2>, more preferably from 170 to 180 dB re 1 µPa<2>, and most preferably of from 170 to 175 dB re 1 µPa<2>.
4. A system according to any of the preceding claims, where the cumulative sound dose is equivalent to a sound exposure level, SEL, in the range of from 180 to 230 dB re 1 µPa<2>·s, preferably of from 190 to 220 dB re 1 µPa<2>·s, more preferably of from 190 to 210 dB re 1 µPa<2>·s, and most preferably of from 190 to 200 dB re 1 µPa<2>·s.
5. A system according to any of the preceding claims, where the sound control unit (1) determines the cumulative sound dose by: either: applying a pre-set value for the sound pressure level provided by the transducer to calculate the minimum endurance of the produced sound to reach the cumulative sound dose, applying a calibrated transducer with a known sound pressure level to calculate the minimum endurance of the produced sound to reach the cumulative sound dose, or applying a hydrophone located in the volume of water (4) to measure the instantaneous sound pressure, p(t), in the water, and applying these measurements to determine the cumulative sound dose.
6. A system according to any of the preceding claims, wherein the produced sound is a single frequency sinusoidal sound having a sound pressure level of 140 dB re 1 μPa<2>which is produced continuously for periods of 1 to 2 hours followed by periods of silence of 1 to 2 hours, and where this cycle of sound followed by silence is repeated one or more times.
7. A system according to any of the preceding claims, wherein the produced sound has a continuous frequency in a selected third octave band within the range of from 300 to 550 Hz.
8. A system according to any of the preceding claims, where the sound pressure level, SPL, is determined by the relation:

where p(t) is an unweighted instantaneous sound pressure at time t given in unit Pa, and p0is a reference sound pressure chosen to be 1 μPa.
9. A system according to any of the preceding claims, where the sound exposure level, SEL, is determined by the relation:

where p(t) is an unweighted instantaneous sound pressure at time t given in unit Pa, and pois a reference sound pressure chosen to be 1 μPa, T is a cumulative time interval in unit seconds, and To is a reference time period chosen to be 1 second.
10. A system according to any of the preceding claims, where the sound exposure level, SEL, is at 1 metre distance from the transducer.
11. A fish farm for salmonids, comprising a fish cage (10) submerged into seawater and containing one or more salmonids to be farmed, and a system according to any of claims 1 to 10, and applying one or more transducers (3) either inside the cage (10) or in the seawater at the outside of the cage (10) within a distance to the nearest part of the fish cage (10) of less than 5 metres.
12. A fish farm according to claim 11, wherein the fish cage (10) is in the form of a fish net closed in the bottom and suspended from a floating ring-shaped structure (11), and where the transducer (3) is located at the centre inside fish cage.
13. A fish farm according to claim 11, wherein the fish cage (10) is in the form of a fish net closed in the bottom and suspended from a floating ring-shaped structure (11), and where it is applied one or more transducers (3) distributed along and located in the sea at the outside of the suspended fish net, and where a hydrophone (5) in communication with the control unit (1) is located in the water at the centre of the fish cage (10) and measures the instantaneous sound pressure, p(t), at this location in the water, and feeds information of the measured sound pressure, either as the instantaneous sound pressure, p(t), or as a sound exposure level calculated from the measured instantaneous sound pressure, p(t), to the control unit (1).
14. A method for causing lesions in sea-lice present in a volume of seawater, where the method comprises: producing a sound in the volume of water, where the sound has: one or more frequencies in the range of from 300 to 550 Hz, and a sound pressure level, SPL, in the range of from 140 to 190 dB re 1 µPa<2>, and maintaining the produced sound at least until the cumulative dose of sound in the volume of seawater is equivalent to a sound exposure level, SEL, of at least 180 dB re 1 µPa<2>·s or higher.
15. A method according to claim 14, wherein the one or more frequencies is/are in the range of from 300 to 500 Hz, preferably of from 300 to 450 Hz, more preferably of from 300 to 400 Hz, more preferably of from 320 to 380 Hz, more preferably of from 340 to 380 Hz, and most probably of from 340 to 360 Hz.
16. A method according to claim 14 or 15, where the sound pressure level, SPL, is in the range of from 150 to 180 dB re 1 µPa<2>, preferably of from 160 to 180 dB re 1 µPa<2>, more preferably from 170 to 180 dB re 1 µPa<2>, and most preferably of from 170 to 175 dB re 1 µPa<2>.
17. A method according to any of claims 14 - 16, where the cumulative sound dose is equivalent to a sound exposure level, SEL, in the range of from 180 to 230 dB re 1 µPa<2>·s, preferably of from 190 to 220 dB re 1 µPa<2>·s, more preferably of from 190 to 210 dB re 1 µPa<2>·s, and most preferably of from 190 to 200 dB re 1 µPa<2>·s.
18. A sound producing system for use in preventing and/or treating fish affected by sea lice, the sound producing system comprising the system according to any of claims 1 to 10.