Journal of Marine Science and Engineering Article Farmed Salmon Show No Pathological Alterations When Exposed to Acoustic Treatment for Sea Lice Infestation Marta Solé 1, * , Maria Constenla 2 , Francesc Padrós 2 , Antoni Lombarte 3 , José-Manuel Fortuño 3 , Mike van der Schaar 1 and Michel André 1, * 1 Laboratory of Applied Bioacoustics, Technical University of Catalonia-Barcelona TECH, 08800 Barcelona, Spain; mike.vanderschaar@upc.edu 2 Fish Diseases Diagnostic Service, Veterinary School, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; Maria.Constenla@uab.cat (M.C.); francesc.padros@uab.cat (F.P.) 3 Institut de Ciències del Mar, Spanish National Research Council (CSIC), 08003 Barcelona, Spain; toni@icm.csic.es (A.L.); jmanuel@icm.csic.es (J.-M.F.) * Correspondence: marta.sole@upc.edu (M.S.); michel.andre@upc.edu (M.A.) Abstract: The use of bioacoustic methods to address sea lice infestation in salmonid farming is a promising innovative method but implies an exposure to sound that could affect the fish. An assessment of the effects of these techniques related to the salmon’s welfare is presented here. The fish were repeatedly exposed to 350 Hz and 500 Hz tones in three- to four-hour exposure sessions, reaching received sound pressure levels of 140 to 150 dB re 1 µPa2 , with the goal of reaching total  sound exposure levels above 190 dB re 1 µPa2 s. Gross pathology and histopathological analysis  performed on exposed salmons’ organs did not reveal any lesions that could be associated to sound Citation: Solé, M.; Constenla, M.; exposure. The analysis of their otoliths through electron microscopy imaging confirmed that the Padrós, F.; Lombarte, A.; Fortuño, sound dose that was used to impair the lice had no effects on the fish auditory organs. J.-M.; van der Schaar, M.; André, M. Farmed Salmon Show No Keywords: salmon; Salmo salar; acoustic trauma; scanning electron microcopy; otolith organ; lateral Pathological Alterations When Exposed to Acoustic Treatment for line; histopathology; vaterite; neuromast Sea Lice Infestation. J. Mar. Sci. Eng. 2021, 9, 1114. https://doi.org/ 10.3390/jmse9101114 1. Introduction Academic Editor: Sea lice infection is still one of the most devastating diseases in the salmon industry [1] Francesco Tiralongo and many different methodologies and strategies to prevent or reduce the impact of the disease have been developed [2]. The efficiency of these strategies in the field has Received: 21 September 2021 been shown to be variable due to different environmental and husbandry factors but, Accepted: 11 October 2021 together with these treatments, salmon welfare in response to the use of certain preventive Published: 14 October 2021 and delousing methods has become more relevant. This is the reason why nowadays the implementation of these strategies must be based on good efficacy scores but also Publisher’s Note: MDPI stays neutral well-balanced safety procedures. Recent findings on the use of acoustic treatments have with regard to jurisdictional claims in demonstrated the potential of this approach to address lice infestation [3] but, together published maps and institutional affil- with these very encouraging results, it was also necessary to conduct studies to ensure that iations. the risk to the acoustically treated salmon was negligible. Fishes are indeed able to detect and respond to a wide range of sounds. Experi- mental studies can investigate the range of frequencies that a fish could detect, and then determine the lowest levels of the detected sound at each frequency (the ‘threshold’, or Copyright: © 2021 by the authors. lowest signal that an animal will detect in some statistically determined percentage of Licensee MDPI, Basel, Switzerland. signal presentations—most often 50%). However, for most commercial species there is This article is an open access article no audiogram available in the literature to assess their sensitivity to noise. Salmo salar distributed under the terms and audiograms based on mean (±SE) minimum received levels (dB re 1 µPa) that elicited a conditions of the Creative Commons characteristic auditory evoked potential (electrical response that is produced anytime a Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ sound is perceived and that can be recorded out of the brain from electrodes) revealed that 4.0/). salmon are most sensitive to 200 Hz frequency sounds [4]. J. Mar. Sci. Eng. 2021, 9, 1114. https://doi.org/10.3390/jmse9101114 https://www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2021, 9, 1114 2 of 20 As most fish can hear sounds, a series of published experiments and studies have looked at the effects of intense sound sources on the otolith organ that is responsible for sound reception. Nine months of exposure to broadband noise in an aquaculture facility did not induce hearing loss in two species of fish (Nile tilapia, Oreochromis niloticus and bluegill sunfish, Lepomis macrochirus) [5]. Other studies showed that salmonids did not trigger temporary threshold shifts (equivalent to a temporary acoustic trauma), whereas northern pike (Esox lucius) and sandbar shark (Carcharhinus plumbeus) suffered from hearing loss that recovered within 24 h after exposure [6,7]. Several animals examined after a post-exposure period up to 52 days showed 2–7% sensory cell loss after sound exposure [8]. Other studies analysed the relationship between auditory hair cell damage and hearing loss [9,10]. Experimental studies have examined the effects of pile driving and airguns on fish. Although the associated source levels did not lead to mortality in any of the exposed animals, and despite no effects being found on external and internal anatomy or damage on sensory hair cells of the otolith organs [11,12], more recent studies suggest that damage to the sensory hairs of fish inner ear tissues are likely to occur at levels considerably higher than those inducing other physiological effects, such as swim bladder ruptures [13]. Teleost fishes present an inner ear that contains three calcareous structures (otoliths), overlaying the sensory epithelia that enable their capacity for hearing and balance. The sagitta, the largest otholith, is usually composed of calcium carbonate crystals in the form of aragonite. A deformity, extremely common in farmed fish, where the aragonite is replaced by vaterite (a clearer crystallised form of the calcium carbonate), heavily affects the farmed salmon [14]. Some studies looked at the effects of anthropogenic sounds on the fish behaviour, sowing minor behavioural and startle responses of fish maintained in cages at the start of the air-gun exposure, responses which appeared to decline at subsequent air-gun emissions, but this sound level did not appear to elicit a decline in catch [15–18]. In addition there were no permanent changes in the behaviour of the fish or invertebrates throughout the course of the studies, and animals did not appear to leave the exposed area [16]. Contrary to these findings, more recent studies reported alarm responses, flight reactions, aggressive behaviours, changes in antipredator defence behaviour and reproduction related behaviour (courtship vocal activity, spawning), alterations on schooling behaviour [19] and, in some cases, effects in larval development in addition to these behavioural changes [20]. Fish physiological responses to noise exposure, like stress, were also measured. Com- plementary to other health indicators, corticosteroid levels are considered a measure of stress. Several studies that looked at stress levels in different fish species concluded they were not influenced by noise exposure [5,21]. Nevertheless, it is fair to mention that these experimental approaches were undertaken in cages where fish could not avoid the exposed areas, making it difficult to definitively conclude on the short- and long-term physiological impact of noise exposure [22]. In the context of a project that aimed at addressing the problem of sea lice Lepeoph- theirus salmonis infestation on salmon (Salmo salar) by using acoustic and bioacoustic techniques (SEASEL SOLUTIONS AS. Project: An acoustic and bioacoustic solution to sea lice infestation on salmon- P.O.BOX 93 N-6282 BRATTVÅG. Norway, [3]) and given the in- conclusive results of previous studies, an evaluation of the possible effects on salmon after sound exposure was necessary. Here, we proceeded with a series of controlled exposure experiments to determine the salmon sensitivity to the sounds that would be used in our method against lice infestation, under laboratory and field conditions. Given the industrial nature of this project, this assessment was absolutely necessary in order to ensure that the risk for the commercial caged salmon was negligible and would not result in an economic burden for the companies using the method. J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 3 of 21 J. Mar. Sci. Eng. 2021, 9, 1114 that the risk for the commercial caged salmon was negligible and would not result in 3 ofan 20 economic burden for the companies using the method. 2. Methods 2. Methods 2.1. Laboratory Experiments 2.1. Laboratory Experiments After determining the effective combination of sound parameters (frequency, time of After determining the effective combination of sound parameters (frequency, time of exposure and amplitude) on the sea lice L. salmonis through controlled exposure experi- exposure and amplitude) on the sea lice L. salmonis through controlled exposure experi- ments (CEE) [3], we repeated these experiments on salmon in the same configuration and ments (CEE) [3], we repeated these experiments on salmon in the same configuration and assessed the potential alterations and lesions that the method could induce with respect assessed the potential alterations and lesions that the method could induce with respect to to behavioural levels, the vestibular system, the lateral line and also in the fish organs after behavioural levels, the vestibular system, the lateral line and also in the fish organs after sound sound exposure. exposure. 2.1.1. 2.1.1. Salmon Salmon Specimens Specimens Maintenance Maintenance and and Health Health Assessment Assessment AA set set of of salmon salmon (S.(S. salar) salar) (n (n == 50; 50; weight weight204.6 204.6± ± 25.5 g; total 25.5 g; total length length26.6 26.6 ±± 2.5 2.5 cm), cm), was was received received and kept in and kept in continuous continuousobservation observationatatthetheLAB LABinfrastructure infrastructure inin a closed a closed system system of of recirculating natural seawater (at ◦ 7–10 °C, salinity 35‰ and natural recirculating natural seawater (at 7–10 C, salinity 35‰ and natural oxygenation) consistingoxygenation) con- sisting of two of two mechanically mechanically filteredfiltered (physicochemical (physicochemical self-filtration self-filtration system system with activated with activated carbon carbon and sand,anddriven sand, driven by a circulation by a circulation pump pump and refrigeration and refrigeration system) system) fiberglass-rein- fiberglass-reinforced forced plasticplastic tanks tanks with with a capacity a capacity of 2000 of 2000 L each L each andandconnected connectedtotoeacheach other (Figure (Figure S1, S1, Supplemental Supplemental Material). The The fish fish stock stock was was then then progressively progressively acclimatised acclimatised to to the the test conditions conditions forfor two two weeks. weeks. FishFish were were regularly regularly fed fed ad ad libitum libitum with with commercial commercial food and feeding feeding rates were also also monitored. monitored.After Afterthese thesetwo twoweeks, weeks,1010fishfishwere were sampled sampled in in order order to proceed to proceed with a complete with a completehistopathological histopathologicalexamination examinationto guarantee an adequate to guarantee health an adequate status status health for thefortest. theThese fish were test. These takentaken fish were as controls and and as controls sampled sampledand and processed processedin the in same the wayway same as fish exposed as fish exposedto sound to sound(Figure 1). 1). (Figure Figure 1. Sound exposure exposureprotocol, protocol,sampling samplingcollection, collection, and and analysis. analysis. (1) (1) After After 2 weeks 2 weeks of acclimation, of acclimation, 10 salmon 10 salmon were were taken taken for sample analysis as controls previous to the sound exposure. (2) Forty salmon were exposed to sound for sample analysis as controls previous to the sound exposure. (2) Forty salmon were exposed to sound (Weeks 1, 2, 3:2 (Weeks 1, 2, 3:2 cycles [350 Hz (65 V-2 h) and 500 Hz (65 V-2 h)], daily with 2 h rest in between two cycles. Week 4:3 cycles [350 cycles [350 Hz (65 V-2 h) and 500 Hz (65 V-2 h)], daily with 2 h rest in between two cycles. Week 4:3 cycles [350 Hz (65 V-2 h) Hz (65 V-2 h) and 500 Hz (65 V-2 h)], daily with 2 h rest in between two cycles). (3) Samples of exposed salmon were sequen- and 500 Hz (65 V-2 h)], daily with 2 h rest in between two cycles). (3) Samples of exposed salmon were sequentially taken tially taken for analysis after exposure (week 1 to 4). for analysis after exposure (week 1 to 4). 2.1.2. Sound Controlled Exposure Experiment (CEE) Protocol 2.1.2. Sound Controlled Exposure Experiment (CEE) Protocol A A set setofof40 40salmon salmonwere wereexposed daily exposed to sounds daily during to sounds 4 weeks during (Figure 4 weeks 1). The (Figure 1). CEE The protocol consisted of a cycle of 350 Hz (65 V-2 h) and 500 Hz (65 V-2 h) exposure, CEE protocol consisted of a cycle of 350 Hz (65 V-2 h) and 500 Hz (65 V-2 h) exposure, twice twice daily with a 2 h period of rest in between the two cycles, during the first three weeks (see [3] for the description of the selection of these sound exposure levels in concurrence J. Mar. Sci. Eng. 2021, 9, 1114 4 of 20 with the sea lice exposure experiments). In the last week of the experiments we added another cycle of exposure (i.e., three times 4 h exposure a day) to increase the sound dose received by the fish. The transducer used was the Lubell LL-1424HP with the capacity to reach levels of at least 180 dB re 1 µPa at the frequencies of interest, although it was driven at 65 vrms, well below its maximum rating. The transducer was driven by a Monacor PA-12040. The sound production system was calibrated as a whole and for each individual frequency. A cali- brated hydrophone (B&K 8106 with Nexus signal conditioner and IOtech WaveBook/516 ADC) was used to make spot measurements in the exposure and control tanks to verify the levels and then further used to monitor the exposure experiments. The hydrophone system was arranged to provide its digitized data to a sound exposure control system that was driving the transducer. The received sound pressure levels were estimated to be 152 dB re 1 µPa2 at 350 Hz and 155 dB re 1 µPa2 at 500 Hz. With the animals moving around the tank the levels would vary. The exposure target was to reach a sound exposure level dose of at least 190 dB re 1 µPa2 s. The estimated SEL for the frequency and duration combination used above was 195 dB re 1 µPa2 s. 2.1.3. Sample Collection Ten exposed salmon were euthanised by bath immersion in 2-phenoxyethanol (2- PE) each week during 4 weeks. All salmon (both control and exposed individuals) were equally treated: samples from otoliths, inner ear and internal organs were processed for histopathological and SEM analysis. 2.1.4. Analysis of Salmon Otolith Organs by Scanning Electron Microscopy Otolith organs epithelia from individual fishes were inspected with SEM imaging techniques to detect any possible alteration of the sensory epithelia. The samples were processed with routine SEM procedures. Fixation was performed in glutaraldehyde 2.5% for 24–48 h at 4 ◦ C. Samples were dehydrated in graded ethanol solutions and critical-point dried with liquid carbon dioxide in a Bal-Tec CPD030 unit (Leica Mycrosystems, Vienna, Austria). The dried specimens were mounted on specimen stubs with double-sided tape. The mounted samples were gold coated with a Quorum Q150R S sputter coated unit (Quorum Technologies, Laughton, East Sussex, UK) and viewed with a variable pressure Hitachi S-3500N scanning electron microscope (Hitachi High-Technologies Co., Tokyo, Japan) at an accelerating voltage of 5 kV in the Institute of Marine Sciences of the Spanish Research Council (CSIC) facilities. 2.1.5. Quantification and Data Analysis For quantification of lesions, the region comprising the whole sensory area of the saccule, utricle and lagena was considered. The length of the sensory epithelium areas comprising hair cells was determined for each sample, and 2500 µm2 (50 µm × 50 µm) sampling squares were placed along the centre length of the area at 5%, 25%, 50%, 75% and 95% of the length axe of the macula statica princeps (Figure 2). Numbers of hair cell bundles were counted in sampling squares of both saccules, utricles and lagenas of each fish. In order to identify whether there were lesions due to the acoustic exposure the samples were treated as follows: 1. All controls taken at different times were grouped together into a single control group. 2. The two samples that were taken for each animal were combined by summing both the intact hair cells and the extruded or missing hair cells over the samples. 3. The hair cells were summed over all the regions, obtaining a single intact hair cells count and single extruded/missing count per animal. 4. The extruded/missing count was divided by the intact hair cell count. J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 5 of 21 J. Mar. Sci. Eng. 2021, 9, 1114 3. The hair cells were summed over all the regions, obtaining a single intact hair5cells of 20 count and single extruded/missing count per animal. 4. The extruded/missing count was divided by the intact hair cell count. Figure 2. SEM. Figure 2. SEM. S. S. salar salar macula macula of of saccule saccule (A), (A), utricle (B) and utricle (B) and lagena lagena (C). (C). Hair Hair cell cell bundle bundle count count locations locations on on macula. macula. Hair Hair cell counts were sampled at five predetermined locations: 5, 25, 50, 75 and 95% of the total macular length. cell counts were sampled at five predetermined locations: 5, 25, 50, 75 and 95% of the total macular length. A 2500 A 2500 µm2µm 2 box box was placed at each sampling area and hair cells were counted within each box. Scale bar (A) = 1 mm. (B,C) = 500 µm. was placed at each sampling area and hair cells were counted within each box. Scale bar (A) = 1 mm. (B,C) = 500 µm. For For each each experiment experiment this this resulted resulted in in aa series series or or damaged damaged hair hair cell cell ratios ratios for for the the control control group (10) and each sampled group over time (after weeks 1, 2 and group (10) and each sampled group over time (after weeks 1, 2 and 3 in the LAB 3 in the LAB with 10 with samples each), and for each organ that was analysed (lagena, saccule, utricle). 10 samples each), and for each organ that was analysed (lagena, saccule, utricle). A A Kruskal– Wallis test was test Kruskal–Wallis performed for each for was performed organ eachand each organ experiment and to identify each experiment if thereifwas to identify a there difference in median was a difference hair cell in median damage hair between cell damage the control between group group the control and any andofany the of time- the sampled groups. time-sampled All calculations groups. were were All calculations performed with Matlab performed R2019a. with Matlab R2019a. 2.1.6. Salmon 2.1.6. Salmon Gross Gross Pathology Pathology andand Histopathological Analysis Salmon were Salmon were anaesthetised anaesthetised and sacrificed with an overdose overdose of 2-phenoxyethanol 2-phenoxyethanol and spinal severance spinal severance and and were weresubjected subjectedtotoa agross grosspathology pathology examination examination after a standardised after a standard- necropsy procedure in order to identify potential external lesions or alterations. ised necropsy procedure in order to identify potential external lesions or alterations. Particular Par- attention was paid to identify lesions such as haemorrhages or other vascular ticular attention was paid to identify lesions such as haemorrhages or other vascular dis- disturbances, and mainlyand turbances, in the swim mainly in bladder, the swimasbladder, these are asthe most these arefrequently described described the most frequently lesions in previously lesions papers. papers. in previously Immediately after Immediately after examination, examination, samples samples of of different different tissues tissues (liver, (liver, digestive digestive system, system, swim bladder, spleen, kidney, gonads and skeletal musculature) were fixed swim bladder, spleen, kidney, gonads and skeletal musculature) were fixed in 10% buff- in 10% buffered formalin. ered Fixed formalin. samples Fixed werewere samples processed processedfor routine histological for routine studies histological by progressive studies by progres- sive dehydration, clearing, embedding in paraffin, block sectioning, staininghaematoxylin dehydration, clearing, embedding in paraffin, block sectioning, staining with with haema- and eosin toxylin and(H/E) eosinand examined (H/E) under the and examined undermicroscope. the microscope. 2.1.7. Behavioural Observations 2.1.7. Behavioural Observations Salmon behaviour was monitored before, during and after the sound exposure, for Salmon behaviour was monitored before, during and after the sound exposure, for a a period of 10 min each time, in order to determine behavioural alterations (expected period of 10 min each time, in order to determine behavioural alterations (expected be- behavioural reactions were jumps, rolls and twitches). Jumps were defined as fast accelera- havioural reactions were jumps, rolls and twitches). Jumps were defined as fast accelera- tions in swimming speeds that ended in a jump, rolls involved turning 90◦ in the horizontal tions in swimming speeds that ended in a jump, rolls involved turning 90° in the horizon- or vertical plane, and twitches were defined as rapid spasmodic contractions of the body tal or vertical of the salmon. plane, and twitches were defined as rapid spasmodic contractions of the body of the salmon. 2.2. Sea Trial Experiments 2.2. SeaSound 2.2.1. Trial Experiments Exposure 2.2.1.Hardware. Sound Exposure Under the sea trial protocol (Figures 3 and 4; see complete description in [3]), the system Hardware. and method Under the sea included producing trial protocol (Figuresthe3sounds and 4; using calibrated see complete transducers description in capable [3]), of reproducing the system sound and method covering included from 300 producing theHz to 600using sounds Hz. calibrated The transducers used transducers were Lubell capable LL916C projectors, of reproducing installed sound covering at 300 from theHz centre of Hz. to 600 each cage. The They were transducers driven used were by a Monacor Lubell LL916C PA-12040. projectors,A controlat installed system consisting the centre of eachof an HTI-99-HF cage. hydrophone They were driven con- by a Mon- nected acor to an MCCDAQ PA-12040. A control USB-1608G whichof system consisting wasan connected HTI-99-HFto a Raspberry hydrophone Pi monitored connected to an correct functioning of the system, the exposure time periods, and the accumulated sound exposure levels. J.J.Mar. Mar.Sci. Sci.Eng. Eng.2021, 2021,9,9,xxFOR FORPEER PEERREVIEW REVIEW 66 ofof 21 21 J. Mar. Sci. Eng. 2021, 9, 1114 MCCDAQUSB-1608G MCCDAQ USB-1608Gwhich whichwas wasconnected connectedto toaaRaspberry RaspberryPiPimonitored monitoredcorrect correct6func- func- of 20 tioningof tioning ofthe thesystem, system,the theexposure exposuretime timeperiods, periods,and andthe theaccumulated accumulatedsound soundexposure exposure levels. levels. Figure Figure 3.3.Sound Figure3. Soundexposure Sound exposuresystem exposure system[3]. system [3]. [3]. Figure Figure4. Drawing Drawingof the experimental setup. Note that thethe depth of the structure thatthat holds the the loudloud speakers was was modified Figure 4.4.Drawing ofofthe the experimental experimental setup. setup. Note Note that that depth the depth ofofthe the structure structure holds that holds the loud speakers speakers modi- was modi- along the fiedalong fied duration alongthe of thedurationthe experiments. durationofofthe M9 theexperiments.loud experiments.M9 speakers M9loud were loudspeakers lowered speakerswere to werelowered − 5 m [3]. loweredtoto−5 −5mm[3]. [3]. Acoustic and time parameters: salmon were exposed to continuous exposure Acousticand Acoustic andtime timeparameters: parameters:salmon salmonwere wereexposed exposedto tocontinuous continuousexposure exposure(Fig- (Fig- (Figures 3 and 4; see complete description in [3]), to individual 350 Hz and 500 Hz signals ures33and ures and4;4;see seecomplete completedescription descriptionin in[3]), [3]),to toindividual individual350350Hz Hzandand500500HzHzsignals signals during, respectively, a cumulative cycle of 1 h and 2 h, and this combination was initially during,respectively, during, respectively,aacumulative cumulativecycle cycleofof11hhand and22h,h,and andthis thiscombination combinationwas wasinitially initially played back every 4 h. The received sound pressure levels were estimated conservatively playedback played backevery every44h.h.The Thereceived receivedsound soundpressure pressurelevels levelswere wereestimated estimatedconservatively conservatively (as the measurement point was fixed while the cages provided a lot of space for the fish to (asthe (as themeasurement measurementpoint pointwas wasfixed fixed while while the the cages cages provided provided aa lot lot of of space space forthe for thefish fish move around) to be 139 dB re 1 µPa2 at 350 Hz and 142 dB re 1 µPa2 at 500 Hz. The received tomove to movearound) around)to tobe be139 139dBdBrere11µPa µPa2 2atat350 350HzHzand and142 142dBdBre re11µPa µPa2 2atat500 500Hz. Hz.TheThe levels at the monitoring hydrophone could vary by 5 dB between exposure windows. receivedlevels received levelsatatthe themonitoring monitoringhydrophone hydrophonecould couldvary varybyby55dB dBbetween betweenexposure exposurewin-win- The estimated SEL for the frequency and duration combination used above and one 3-h dows.The dows. Theestimated estimatedSELSELfor forthe thefrequency frequencyand andduration durationcombination combinationused usedabove aboveandandone one exposure session was 179 dB re 1 µPa2 s. In order to be able to more rapidly reach a target SEL similar to those obtained during the laboratory experiments (195 dB re 1 µPa s) it was 2 J. Mar. Sci. Eng. 2021, 9, 1114 7 of 20 decided to increase the received level by 10 dB for both frequencies at one of the cages, playing both frequencies for 2 h each continuously. 2.2.2. Sample Collection Every 3 weeks (in weeks 3, 6, 9 and 12 after sound exposure) samples from salmon were collected: 1. Week 3 and week 6: three salmon from each cage (taken from the two exposed cages and from the four control cages). Total: six exposed individuals and 12 control individuals per week. 2. Week 9 and week 12: three salmon (from the two exposed cages and from the two control cages). Total: six exposed individuals and six control individuals per week. All collected salmon were used to assess salmon health status. External surfaces, mouth and internal organs of each individual were checked macroscopically for gross pathologies. Then, an anterior and a posterior body wedge, which included skeletal muscle (including white and red muscle), head and trunk kidney, swimbladder, stomach, intestine, liver, perivisceral fat (with pancreatic tissue) and gonads were extracted and processed for histological analysis at the Pathological Diagnostic Service in Fish (SDPP) of the Autonomous University of Barcelona (UAB). A portion of the body containing lateral lines as well as the whole head of the salmon were taken and processed to assess possible lesions in the inner ear structures (otolith organ) and lateral lines at the LAB (Laboratory of Applied Bioacoustics). 2.2.3. Analysis of Salmon Otolith Organs by Scanning Electron Microscopy Otolith organs epithelia from individual fishes were observed by SEM imaging tech- niques to detect any possible alteration of the sensory epithelia. The samples were pro- cessed by routine SEM procedures. Fixation was performed in glutaraldehyde 2.5% for 24–48 h at 4 ◦ C. Samples were dehydrated in graded ethanol solutions and critical-point dried with liquid carbon dioxide in a Bal-Tec CPD030 unit (Leica Mycrosystems, Vienna, Austria). The dried specimens were mounted on specimen stubs with double-sided tape. The mounted samples were gold coated with a Quorum Q150R S sputter coated unit (Quorum Technologies, Laughton, East Sussex, UK) and viewed with a variable pressure Hitachi S-3500N scanning electron microscope (Hitachi High-Technologies Co., Tokyo, Japan) at an accelerating voltage of 5 kV in the Institute of Marine Sciences of the Spanish Research Council (CSIC) facilities. 2.2.4. Quantification and Data Analysis We considered for the quantification the region comprising the whole sensory area of the saccule, utricle and lagena. The length of the sensory epithelium areas comprising hair cells was determined for each sample, and 2500 µm2 (50 × 50 µm) sampling squares were placed along the centre length of the area at 5, 25, 50, 75 and 95% of the length axe of the macula statica princeps (Figure 2). Numbers of hair cell bundles were counted in sampling squares of both saccules, utricles and lagenas of each fish. In order to identify whether there were lesions due to the acoustic exposure, the samples were treated as follows: 1. All controls taken at different times were grouped together into a single control group. 2. The two samples that were taken for each animal were combined by summing both the intact hair cells and the extruded or missing hair cells over the samples. 3. The hair cells were summed over all the regions, obtaining a single intact hair cells count and single extruded/missing count per animal. 4. The extruded/missing count was divided by the intact hair cell count. For each experiment this resulted in a series of damaged hair cell ratios for the control group (12) and each sampled group over time (after weeks 3, 6, and 12 with six samples each), and for each organ that was analysed (lagena, saccule, utricle). A Kruskal–Wallis test was performed for each organ and each experiment to identify if there was a difference in J. Mar. Sci. Eng. 2021, 9, 1114 8 of 20 median hair cell damage between the control group and any of the time-sampled groups. All calculations were performed with Matlab R2019a. 2.2.5. Analysis of Presence of Vaterite in Salmon Otolith Sagittal otoliths are primary hearing structures in the inner ear of all teleost (bony) fishes and are normally composed of aragonite, though abnormal vaterite replacement is sometimes seen. Additional to the analysis of salmon otolith epithelia by scanning electron microscopy, the evaluation and quantification of the presence of vaterite in otoliths were per- formed by optic microscopy. The proportion of otoliths presenting vaterite was quantified. 2.2.6. Analysis of Superficial Neuromasts of the Salmon Lateral Line by Scanning Electron Microscopy A portion of the body containing lateral lines of all the fishes (48, control and exposed) were collected during the three weeks of sampling. Superficial neuromasts of the lateral line from individual fishes were observed by SEM imaging techniques to detect any possible alteration of the sensory epithelia. The samples were processed by routine SEM procedures. Fixation was performed in glutaraldehyde 2.5% for 24–48 h at 4 ◦ C. Samples were dehydrated in graded ethanol solutions and critical-point dried with liquid carbon dioxide in a Bal-Tec CPD030 unit (Leica Mycrosystems, Vienna, Austria). The dried specimens were mounted on specimen stubs with double-sided tape. The mounted samples were gold coated with a Quorum Q150R S sputter coated unit (Quorum Technologies, Laughton, East Sussex, UK) and viewed with a variable pressure Hitachi S-3500N scanning electron microscope (Hitachi High-Technologies Co., Tokio, Japan) at an accelerating voltage of 5 kV in the Institute of Marine Sciences of the Spanish Research Council (CSIC) facilities. 2.2.7. Salmon Gross Pathology and Histopathological Analysis The salmon were subjected to a gross pathology and histological analysis to assess possible lesions in internal organs. The same procedure as in LAB experiments was followed to collect and analyse of the samples. Salmon were anaesthetised and sacrificed with an overdose of 2-phenoxyethanol and spinal severance and were subjected to a gross pathology examination after a standardised necropsy procedure in order to identify potential external lesions or alterations. Particular attention was paid to identify lesions such as haemorrhages or other vascular disturbances and mainly in the swim bladder as these were the most frequently described lesions in previous papers. Immediately after examination, samples of different tissues (liver, digestive system, swim bladder, spleen, kidney, gonads and skeletal musculature) were fixed in 10% buffered formalin. Fixed samples were processed for routine histological studies by progressive dehydration, clearing, embedding in paraffin, block sectioning, staining with haematoxylin and eosin (H/E) and examined under the microscope. 3. Results 3.1. Laboratory Experiments 3.1.1. Analysis of Salmon Otolith Organs by Scanning Electron Microscopy The fish basic inner ear structure consists of three semicircular canals and their sensory epithelia, the cristae and three otolithic end organs (utricle, saccule, lagena) including their maculae with respective sensory epithelia, the macula saccule, the macula utricle and the macula lagena (Figure 5). Otolith organs epithelia from individual fishes were observed through SEM imaging techniques to detect any possible alteration of the sensory epithelia. No effects were detected in any of the exposed animals nor in control animals. Figures 6–8 show a healthy aspect of hair cells on the three epithelia from the different times of sample collection. their maculae with respective sensory epithelia, the macula saccule, the macula utricle and the macula J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW lagena (Figure 5). 9 of 21 J. Mar. Sci. Eng. 2021, 9, 1114 9 of 20 their maculae with respective sensory epithelia, the macula saccule, the macula utricle and the macula lagena (Figure 5). Figure 5. (A) LM (light microscopy). (B–D): SEM (scanning electron microscopy). Salmon inner ear. (A) The location of the three sensory epithelia and the three semicircular channels in the salmon inner ear is visible. (B) White line encloses the saccule sensory epithelium. (C) White line encloses the utricle sensory epithelium. (D) White line encloses the lagena sen- sory epithelium. Scale bar: (B) = 1 mm. (C,D) = 500 µm. Figure Figure 5. 5. (A) (A) LM LM (light (lightmicroscopy). microscopy). (B–D): (B–D): Otolith SEM SEM(scanning organs (scanning epithelia electron electron from microscopy). microscopy). individual Salmon fishesSalmon wereinner ear.ear. inner observed (A)through TheThe (A) location SEMofimaging location the of three sensory epithelia and the three sensory epithelia and the three semicircular the threeto channels semicircular in the salmon inner ear is visible. (B) White line encloses the techniques detect anychannels possibleinalteration the salmonofinner the ear is visible. sensory (B) White epithelia. No line encloses effects were de- saccule sensory the saccule epithelium. sensory (C) White epithelium. (C)in line encloses White the utricle sensory epithelium. (D) White line encloses the lagena sen- tected any line encloses of the the utricle exposed animals sensory epithelium. nor in (D) White control animals. line encloses Figures the lagena 6–8 show a healthy sory epithelium. Scale bar: (B) = 1 mm. (C,D) = 500 µm. sensory epithelium. Scale bar: (B) =of aspect 1 mm. (C,D)on hair cells = 500 theµm. three epithelia from the different times of sample collection. Otolith organs epithelia from individual fishes were observed through SEM imaging techniques to detect any possible alteration of the sensory epithelia. No effects were de- tected in any of the exposed animals nor in control animals. Figures 6–8 show a healthy aspect of hair cells on the three epithelia from the different times of sample collection. Figure 6. SEM (scanning electron microscopy). Saccule sensory epithelium. (A,B) Exposed animals. Control. (C–F) By comparison with control animals, the images of the saccule epithelium show healthy sensory hair cells in all cases of exposed animals (1 week (C), 2 weeks (D), 3 weeks (E) and 4 weeks (F) of sound exposure). Scale bar: E = 30 µm. (A,C) = 20 µm. (F) = 10 µm. (D) = 5 µm. (B) = 3 µm. J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 10 of 21 J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 10 of 21 Figure 6. SEM (scanning electron microscopy). Saccule sensory epithelium. (A,B) Exposed animals. Control. (C–F) By Figure Sci. Eng.6.2021, J. Mar.comparison SEM 9, (scanning with 1114 electron microscopy). control animals, the images ofSaccule sensory the saccule epithelium. epithelium show(A,B) Exposed healthy animals. sensory Control. hair cells in all(C–F) cases By 10 of 20 of comparison with (1 exposed animals control animals, week (C), the (D), 2 weeks images of the 3 weeks (E)saccule epithelium and 4 weeks (F) ofshow soundhealthy sensory exposure). Scalehair bar:cells in µm. E = 30 all cases (A,C)of = exposed 20 animals µm. (F) (1 week = 10 µm. (D) = 5(C), µm. 2 weeks (B) = 3 (D), µm. 3 weeks (E) and 4 weeks (F) of sound exposure). Scale bar: E = 30 µm. (A,C) = 20 µm. (F) = 10 µm. (D) = 5 µm. (B) = 3 µm. Figure Figure 7. 7. SEM SEM(scanning (scanningelectron electronmicroscopy). microscopy).Utricle saccule Utricle sensory saccule epithelium. sensory (A,B)(A,B) epithelium. Exposed animals. Exposed Control. animals. (C–F) Control. Figure By 7. SEM (scanning comparison with electron control microscopy). animals, the imagesUtricle of thesaccule sacculesensory epithelium. epithelium show (A,B) Exposed healthy sensory animals. hair cellsControl. in all (C–F) cases of (C–F) By comparison with control animals, the images of the saccule epithelium show healthy sensory hair cells in all cases By comparison with control animals, the images of the saccule epithelium show healthy sensory hair cells in exposed animals (1 week (C), 2 weeks (D), 3 weeks (E) and 4 weeks (F) of sound exposure). Scale bar: (A) = 30 µm. (B,D)all cases of of exposed exposed animals animals (1 (1 week week (C),(C), 2 2 weeks weeks (D),(D), 3 3 weeks weeks (E) (E) and and 4 4 weeks weeks (F) (F) of of sound sound exposure). exposure). Scale Scale bar: bar: (A) = (A)µm. 30 = 30 µm. (B,D) = 10 µm. (C,E,F) = 5 µm. (B,D) = 10(C,E,F) = 10 µm. µm. (C,E,F) = 5 µm.= 5 µm. Figure 8. SEM (scanning electron microscopy). Lagena sensory epithelium. (A,B) Exposed animals. Control. (C–F) By com- Figure Figure 8. parison SEM 8.with SEM (scanning control (scanning electron animals, themicroscopy). electron images of theLagena microscopy). sensory saccule Lagena epithelium. epithelium sensory (A,B) show healthy epithelium. Exposed sensory (A,B) animals. cellsControl. hairanimals. Exposed (C–F) in allControl. cases By com- of(C–F) exposed By parison animals with (1 control week (C), animals, 2 weeks the (D), images 3 of weeks the (E) saccule and 4 epithelium weeks (F) of show sound healthy sensory exposure). Scale hair bar: cells (A) = in 30 all µm. cases of (C,D) exposed = comparison with control animals, the images of the saccule epithelium show healthy sensory hair cells in all cases of exposed10 µm. animals=(1 (B,E,F) week (C), 2 weeks (D), 3 weeks (E) and 4 weeks (F) of sound exposure). Scale bar: (A) = 30 µm. (C,D) = 10 µm. 5 µm. animals (1 week (C), 2 weeks (D), 3 weeks (E) and 4 weeks (F) of sound exposure). Scale bar: (A) = 30 µm. (C,D) = 10 µm. (B,E,F) = 5 µm. (B,E,F) = 5 µm. 3.1.2. Quantification and Data Analysis 3.1.2. Quantification 3.1.2.No Quantification and and Data Data Analysis Analysis significant differences were found between hair cell assessment in control and No significant No animals exposed significant differences differences during were were found between between hair hair cell found(Kruskal-Wallis LAB experiments cell assessment (Figure 9). in test)assessment control and in control and exposed animals during LAB experiments (Kruskal-Wallis test) (Figure (Kruskal-Wallis test) (Figure 9). 9). 3.1.3. Gross Pathology and Histological Analysis The gross morphological (Figure 10) and histopathological analysis (Figures 11–14) on sampled salmon did not show any lesions that could be associated to sound exposure. All dissected salmon were presenting a completely normal aspect. J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 11 of 21 J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 11 of 21 J. Mar. Sci. Eng. 2021, 9, 1114 11 of 20 Figure 9. Overview of the damaged hair cell ratio from data collected at the LAB experiments. (A) lagena, (B) saccule and (C) Figure utricle.9.The Overview red lineofisthe thedamaged hair cell median with the ratio boxesfrom data by defined collected the 25 at the75 and LAB experiments. percentiles. The (A) lagena,are whiskers (B)defined saccule and by 1.5 (C)the times utricle. The red line interquartile is the and distance median with(+) outliers thebeyond boxes defined by the that range. No25significant and 75 percentiles. Thewere differences whiskers arebetween found defined controls by 1.5 andtimes the interquartile exposed distance(A) salmons (p-values: and0.21, outliers (+) beyond (B) 0.92, that range. No significant differences were found between controls (C) 0.54). and exposed salmons (p-values: (A) 0.21, (B) 0.92, (C) 0.54). 3.1.3. Gross Pathology and Histological Analysis 3.1.3. Gross Figure 9. Overview of the damaged hairPathology anddata cell ratio from Histological Analysis collected at the LAB experiments. (A) lagena, (B) saccule Figure 9. Overview of the damaged The gross hair morphological cell with ratio from data (Figure at collected 10)the and LABhistopathological experiments. (A) analysis (Figures lagena, (B) saccule11–14) and and (C) utricle. The red line is theThe median the boxes defined by gross morphological (Figure 10) andthe 25 and histopathological 75 percentiles. The whiskers analysis are defined11–14) (Figures (C) utricle. The red line is theon sampled median withsalmon the boxes did not defined show any by the 25lesions and 75 that could percentiles. be Theassociated whiskers to aresound definedexposure. by 1.5 by 1.5 times the interquartileon distance sampledandsalmon outliers did (+) beyond not show thatany range. No significant lesions that could differences were found be associated between to sound exposure. times the interquartile distanceAlland outlierssalmon dissected (+) beyond werethat range. No asignificant presenting completely differences normal were found between controls aspect. controls and exposed salmons All(p-values: dissected(A)salmon 0.21, (B)were 0.92, presenting (C) 0.54). a completely normal aspect. and exposed salmons (p-values: (A) 0.21, (B) 0.92, (C) 0.54). 3.1.3. Gross Pathology and Histological Analysis The gross morphological (Figure 10) and histopathological analysis (Figures 11–14) on sampled salmon did not show any lesions that could be associated to sound exposure. All dissected salmon were presenting a completely normal aspect. Figure Figure Figure 10. 10.10. Dissected Dissected Dissected salmon salmon salmon showing showing showing the the the inner inner inner organs organs organs with with normal normal with aspect. aspect. normal No No aspect. No internal internal or orexternal or external internal haemorrhages haemorrhages external oror le-le- or lesions, haemorrhages sions, sions, nor nor nor alterations alterations alterations in inswim in the the swim the swim bladder bladder bladder were detected. were detected. were detected. Figure 10. Dissected salmon showing the inner organs with normal aspect. No internal or external haemorrhages or le- sions, nor alterations in the swim bladder were detected. Figure 11. Swim bladder with normal aspect of salmon at different samplings times. (A) Control. (B) 1 week after exposure. Figure (C) 211. Figure Swim weeks 11. Swimbladder after with exposure. bladder withnormal (D) aspect 3 weeks normal ofofsalmon after aspect salmonat exposure. different at(E) 4 weeks different samplings times.(A) after exposure. samplings times. (A)Control. ScaleControl. bar: 100 (B) (B) 1 1week µm. week after after exposure. exposure. (C)(C) 2 weeks after exposure. (D) 3 weeks after exposure. (E) 4 weeks after exposure. Scale bar: 100 2 weeks after exposure. (D) 3 weeks after exposure. (E) 4 weeks after exposure. Scale bar: 100 µm. µm. 3.1.4. Behavioural Observations No behavioural reaction was observed before, during and after the sound exposure. No jumps, rolls and twitches were observed during the 10 min of observation on each period of analysis. A major proportion of salmon acquired a perpendicular position near to the transducer when it was placed in the tank and maintained this position during the Figure 11. Swim bladder with normal aspect of salmon entire experiment. Theat different rest of the samplings times. fishes placed (A) Control. themselves (B) 1 week counter after current in exposure. the tank (C) 2 weeks after exposure. (D)while 3 weeks after exposure. (E) 4 weeks after exposure. Scale bar: 100 µm. the sound exposure was performed. All salmon were observed eating consistently over the whole experimental period (Figure 15). J. Mar. Sci. Eng. 2021, 9, 1114 12 of 20 J. J. Mar. Mar. Sci. Sci. Eng. Eng. 2021, 2021, 9, 9, xx FOR FOR PEER PEER REVIEW REVIEW 12 12 of of 21 21 Figure 12. Head Figure 12. Head kidney kidney (first line) and (first line) and posterior posterior kidney kidney (second (second line) line) of of salmon salmon at at different different sampling sampling times. times. (A) (A) Control. (A) Control. Control. (B) (B) 11 week week week after after exposure. exposure. (C) (C) 2 2 weeks weeks after after exposure. exposure. (D) (D) 3 3 weeks weeks after after exposure. exposure. (E) (E) 4 4 weeks weeks after after exposure. exposure. exposure. (C) 2 weeks after exposure. (D) 3 weeks after exposure. (E) 4 weeks after exposure. ScaleScale Scale bar: bar: 100 100 µm. µm. bar: 100 µm. Figure 13. 13. Stomach Figure 13. Stomach (first line), line), intestine intestine (second line), line), liver liver (third line) and spleen spleen (fourth line) of salmon at different Figure Stomach (first (first line), intestine (second (second line), liver (third line) and (third line) and spleen (fourth line) (fourth line) of of salmon salmon at at different different sampling times. (A) Control. (B) 1 week after exposure. (C) 2 weeks after exposure. (D) 3 weeks after exposure. (E) 4 weeks sampling times. (A) Control. (B) 1 week after exposure. (C) 2 weeks after exposure. (D) 3 weeks after exposure. (E) 44 weeks sampling times. (A) Control. (B) 1 week after exposure. (C) 2 weeks after exposure. (D) 3 weeks after exposure. (E) weeks after exposure. Scale bar: 100 µm. after exposure. Scale bar: 100 µm. after exposure. Scale bar: 100 µm. J. Mar. Sci. Eng. 2021, 9, 1114 13 of 20 J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 13 of 21 Figure 14. Pancreas and adipose tissue (visceral fat) (first line) and gonad (second line) of salmon at different sampling times. (A) Control. (B) 1 week after exposure. (C) 2 weeks after exposure. (D) 3 weeks after exposure. (E) 4 weeks after exposure. Scale bar: 100 µm. 3.1.4. Behavioural Observations No behavioural reaction was observed before, during and after the sound exposure. No jumps, rolls and twitches were observed during the 10 min of observation on each period of analysis. A major proportion of salmon acquired a perpendicular position near to the transducer when it was placed in the tank and maintained this position during the Figure 14. Figure 14. Pancreas Pancreas and and adipose adipose entiretissue tissue (visceral (visceral fat) experiment. The(first fat) (first line) restline) andfishes and of the gonadplaced gonad (secondthemselves (second line) of line) of salmon salmon at different at different counter sampling sampling current in the tank times. (A) Control. (B) 1 week times. (A) Control. (B) 1 week after after exposure. exposure. (C) (C) 22 weeks weeks after after exposure. exposure. (D) (D) 3 3 weeks weeks after after exposure. exposure. (E) (E) 4 4 weeks weeks after after while the sound exposure was performed. All salmon were observed eating consistently exposure. Scale bar: 100 µm. exposure. Scale bar: 100 µm.over the whole experimental period (Figure 15). 3.1.4. Behavioural Observations No behavioural reaction was observed before, during and after the sound exposure. No jumps, rolls and twitches were observed during the 10 min of observation on each period of analysis. A major proportion of salmon acquired a perpendicular position near to the transducer when it was placed in the tank and maintained this position during the entire experiment. The rest of the fishes placed themselves counter current in the tank while the sound exposure was performed. All salmon were observed eating consistently over the whole experimental period (Figure 15). Figure15. Figure 15. Salmon Salmon swimming swimming in inthe thetanks tanksand andshowing showingnormal normalbehaviour. behaviour. 3.2. 3.2. Sea Sea Trial TrialExperiments Experiments 3.2.1. 3.2.1. Analysis of Analysis of Salmon Salmon Otolith Otolith Organs Organsby byScanning ScanningElectron ElectronMicroscopy Microscopy Otolith Otolith organs observed by organs observed by SEM SEMimaging imagingtechniques techniquesdid didnot notshow showanyany alteration alteration on on the sensory epithelia. Figures 16–18 show a healthy aspect of hair cells on the the sensory epithelia. Figures 16–18 show a healthy aspect of hair cells on the three epi- three epithelia at different thelia at different times times of of sample sample collection collection at at seasea trials. trials. 3.2.2. Quantification and Data Analysis Figure No15.significant Salmon swimming in thewere differences tanksfound and showing normal between hairbehaviour. cell assessment during control and exposed animals at sea trial experiments (Kruskal–Wallis test). (Figure 19). 3.2. Sea Trial Experiments 3.2.1. Analysis of Salmon Otolith Organs by Scanning Electron Microscopy Otolith organs observed by SEM imaging techniques did not show any alteration on the sensory epithelia. Figures 16–18 show a healthy aspect of hair cells on the three epi- thelia at different times of sample collection at sea trials. J. Mar. Sci. Eng. 2021, 9, 1114 14 of 20 J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 14 of 21 FigureFigure 16. SEM.16. SEM. Salmon Salmon inner inner ear.ear. Saccule Saccule sensoryepithelium. sensory epithelium. (A) (A) Control. Control.(B) 3 weeks (B) 3 weeks after sound after exposure. sound (C) 6 (C) exposure. weeks 6 weeks after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals, the images of the saccule after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals, the images of the saccule J. Mar.epithelium show Sci. Eng. 2021, healthy 9, x FOR PEERsensory REVIEW hair cells in all cases of exposed animals. Scale bar: (B) = 20 µm. (A,D) = 10 µm. (C) =155 of 21 epithelium µm. show healthy sensory hair cells in all cases of exposed animals. Scale bar: (B) = 20 µm. (A,D) = 10 µm. (C) = 5 µm. FigureFigure 17. SEM. 17. SEM. Salmon Salmon inner inner ear.ear. Utricle Utricle sensoryepithelium. sensory epithelium. (A) (A) Control. Control.(B) 3 weeks (B) 3 weeksafter sound after exposure. sound (C) 6 (C) exposure. weeks 6 weeks after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals, the images of the saccule after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals, the images of the saccule epithelium show healthy sensory hair cells in all cases of exposed animals. Scale bar: (A) = 30 µm. (B–D) = 10 µm. epithelium show healthy sensory hair cells in all cases of exposed animals. Scale bar: (A) = 30 µm. (B–D) = 10 µm. J. Mar.Figure Sci. Eng. 17.2021, SEM.9, 1114 15 of 20 Salmon inner ear. Utricle sensory epithelium. (A) Control. (B) 3 weeks after sound exposure. (C) 6 weeks after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals, the images of the saccule epithelium show healthy sensory hair cells in all cases of exposed animals. Scale bar: (A) = 30 µm. (B–D) = 10 µm. J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW 16 of 21 Figure 18. SEM. Salmon inner ear. Lagena sensory epithelium. (A) Control. (B) 3 weeks after sound exposure. (C) 6 weeks after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals (A), the images of the saccule epithelium show healthy sensory hair cells in all cases of exposed animals (B–D). Scale bar: (C) = 30 µm. (A) = 20 µm. (B) = 10 µm. (D) = 5 µm. . Figure 18. SEM. Salmon inner ear. Lagena sensory epithelium. (A) Control. (B) 3 weeks after sound exposure. (C) 6 weeks 3.2.2. Quantification and Data Analysis after sound exposure. (D) 12 weeks after sound exposure. By comparison with control animals (A), the images of the saccule epithelium show healthy sensory No significant hair cells in alldifferences wereanimals cases of exposed found between hair (B–D). Scale cell(C)assessment bar: during = 30 µm. (A) control = 20 µm. (B) = 10 µm. (D) = 5 µm. and exposed animals at sea trial experiments (Kruskal–Wallis test). (Figure 19). Figure 19. Overview of the damaged hair cell ratio from data collected at sea experiments. (A) Lagena, (B) saccule, (C) utricle. The red line is the median with the boxes defined by the 25th and 75th percentiles. The whiskers are defined Figure 19. Overview of the damaged hair cell ratio from data collected at sea experiments. (A) Lagena, (B) saccule, (C) by 1.5 times utricle. the interquartile The red distance line is the median and with theoutliers (+) beyond boxes defined that25th by the range. andNo significant 75th differences percentiles. were found The whiskers between are defined by controls and exposed salmons (p-values (A) 0.29, (B) 0.38, (C) 0.78). 1.5 times the interquartile distance and outliers (+) beyond that range. No significant differences were found between controls and exposed salmons (p-values (A) 0.29, (B) 0.38, (C) 0.78). 3.2.3. Analysis of Salmon Otolith 3.2.3.Vaterite Analysis of Salmon incidence onOtolith salmon otoliths (Figure 20) did not present a linear pattern over time Vaterite (Table 1).incidence The percentage of fish on salmon affected otoliths increased (Figure in week 20) did 3 (75%)aand not present week linear 6 (83, over pattern 3%), and decreased in the week 12 (16, 6%) (Figure S2A,B, Supplemental Material). time (Table 1). The percentage of fish affected increased in week 3 (75%) and week 6 (83, Considering the total 3%), andincidence decreasedofinvaterite the week in the sampled 12 (16, fishes, S2A,B, 6%) (Figure there were more otoliths Supplemental affectedCon- Material). (65, 6%) than otoliths with no vaterite (34, 3%) (Figure S2C). Right otoliths were sidering the total incidence of vaterite in the sampled fishes, there were more otoliths af- more likely to be vateritic fected (65, 6%)than thanleft otoliths, otoliths with with no52% and 48% vaterite (34, of 3%)otoliths (Figureshowing vaterite S2C). Right formation. otoliths were Out of all fish sampled, 9, 4% had two vateritic otoliths (Figure S2D,E). Considering more likely to be vateritic than left otoliths, with 52% and 48% of otoliths showing vaterite the total incidence of vaterite on the sampled fishes, the proportion otolith affected formation. Out of all fish sampled, 9, 4% had two vateritic otoliths (Figure S2D,E). Con- by vaterite sidering the total incidence of vaterite on the sampled fishes, the proportion otolith af- fected by vaterite in control cages (75%) was higher than in exposed cages (50%) (Figure S2F). The analysis of the vaterite presence in otoliths was not related to sound exposure, but to a deficiency in nutrition associated to captivity (Table 1). 3%), and decreased in the week 12 (16, 6%) (Figure S2A,B, Supplemental Material). Con- sidering the total incidence of vaterite in the sampled fishes, there were more otoliths af- fected (65, 6%) than otoliths with no vaterite (34, 3%) (Figure S2C). Right otoliths were J. Mar. Sci. Eng. 2021, 9, 1114 more likely to be vateritic than left otoliths, with 52% and 48% of otoliths showing 16 ofvaterite 20 formation. Out of all fish sampled, 9, 4% had two vateritic otoliths (Figure S2D,E). Con- sidering the total incidence of vaterite on the sampled fishes, the proportion otolith af- fected by vaterite in control cages (75%) was higher than in exposed cages (50%) (Figure in control cages (75%) was higher than in exposed cages (50%) (Figure S2F). The analysis of S2F). The analysis of the vaterite presence in otoliths was not related to sound exposure, the vaterite presence in otoliths was not related to sound exposure, but to a deficiency in butnutrition to a deficiency in to associated nutrition captivityassociated (Table 1). to captivity (Table 1). FigureFigure 20. LM. 20. (A) LM.Left andand (A) Left right otolith right composed otolith composedentirely entirely of aragonite.(B) of aragonite. (B)Right Right otolith otolith composed composed basically basically of vaterite. of vaterite. Table 1. Vaterite incidence rate on the salmon otoliths (%). Time Vaterite Incidence Rate (%) Week 3 75 Week 6 83.3 Week 12 16.6 Total Incidence - Vateritic otolith 65.6 No vateritic otolith 34.3 Location - Right otolith 52 Left otolith 48 Cages - Control 75 Exposed 50 3.2.4. Analysis of Superficial Neuromast of the Salmon Lateral Line by Scanning Electron Microscopy Neuromasts are either contained in canals or are located on the epithelium of the head, trunk and tail. Each lateral line scale has a single canal neuromast and three groups of superficial neuromasts (Figure 21). In comparison with control animals the lateral line superficial neuromasts observed by SEM imaging techniques did not show any alteration of the sensory epithelia (Figure 22) in exposed salmon. 3.2.5. Gross Pathology and Histological Analysis No macroscopic pathology nor histopathological alteration were observed in any of the tissue analysed that could be associated to sound exposure, exactly in the same way to those observed in LAB experiments. See Figure S3 (Supplementary Material) as an example of the normal aspect of the swim bladder of salmon (the most sensitive organ to sound exposure) throughout the samplings of the sea trial. 3.2.4. Analysis of Superficial Neuromast of the Salmon Lateral Line by Scanning Electron Microscopy Neuromasts are either contained in canals or are located on the epithelium of the head, trunk and tail. Each lateral line scale has a single canal neuromast and three groups J. Mar. Sci. Eng. 2021, 9, 1114 of superficial neuromasts (Figure 21). In comparison with control animals the lateral17 ofline 20 superficial neuromasts observed by SEM imaging techniques did not show any alteration of the sensory epithelia (Figure 22) in exposed salmon. Figure21. Figure 21. SEM. SEM. (A) (A) Light Light microscopy microscopy (LM). (LM). (B–D): (B–D):SEM. SEM.(A) (A)Lateral Lateralline linesystem systemlocated locatedon onthe the salmonscales salmon scalesof oftrunk trunk(c(csigns signsthe thelateral lateralline linechannel). channel).(B) (B)Lateral Lateralsystem systemlocated locatedon onthe theepithelium epithelium J. Mar. Sci. Eng. 2021, 9, x FOR PEER REVIEW of the head (arrows). (C) Holes (arrows) of the channel of the lateral line system on the trunk. 18 of(D) 21 of the head (arrows). (C) Holes (arrows) of the channel of the lateral line system on the trunk. Upper channel view on the scales (c). Scale bar: (B,C) = 3 mm. (D) = 2 mm. (D) Upper channel view on the scales (c). Scale bar: (B,C) = 3 mm. (D) = 2 mm. Figure 22. Figure 22. SEM. SEM. Superficial Superficial neuromast. neuromast. (A) (A)Upper Upperview viewofofa asuperficial superficialneuromast. (B)(B) neuromast. Normal Normalas- pect of hair cell bundles on superficial neuromast of a control salmon. (C,D) Hair cell bundles aspect of hair cell bundles on superficial neuromast of a control salmon. (C,D) Hair cell bundles on on superficial neuromast of a 12-week exposed salmon. No lesions are visible. Scale bar: (A) = 200 µm. superficial neuromast of a 12-week exposed salmon. No lesions are visible. Scale bar: (A) = 200 µm. (B) = 30 µm. (C) = 5 µm. (D) = 3 µm. (B) = 30 µm. (C) = 5 µm. (D) = 3 µm. 3.2.5. 4. Gross Pathology and Histological Analysis Discussion No macroscopic The use of acousticpathology methods as nor anhistopathological alteration effective new approach were observed to address in any of sea lice infestation thesalmonis) (L. tissue analysed should that first could ensurebethat associated to sound these methods doexposure, exactlyaffect not negatively in the same the fish.way In to those this observed context, in LABresults the present experiments. showedSee Figure that S3 (Supplementary salmon exposed to sound Material) with theas an ex- same ample of the normal characteristics aspectlice as previous of the swim bladder exposure of salmon experiments [3] did(the notmost sensitive present organin any lesion to sound exposure) throughout the samplings of the sea trial. 4. Discussion The use of acoustic methods as an effective new approach to address sea lice infesta- J. Mar. Sci. Eng. 2021, 9, 1114 18 of 20 internal organs, nor in the otolith and lateral line sensory epithelia and they also did not induce behavioural alterations. We did not find an increase in mortality due to sound exposure, which is consistent with some previous experiments of sound exposure where much higher sound levels (e.g., pile driving and airgun) [11,12,15–17] did not show an effect on fish survival. Although some authors reported sensory epithelia cell loss in the otolith organ after sound exposure [8,10,23], our analysis did not reveal any damage in auditory tissues. We proceeded to analyse the vaterite proportion of the otoliths. Sagittal otoliths are essential components of the sensory organs that are composed of calcium carbonate. In abnormal otoliths, aragonite (the normal crystal form) is replaced with vaterite that decreases hearing sensitivity, reducing growth rates [14]. In some Chinook salmon studies vateritic sagittae were bigger and less dense than the aragonitic form, and vaterite presence was associated with moderately altered saccular epithelia and a significant decrease in auditory sensi- tivity [24]. The functional cause of the degradation remains speculative and a variety of physiological factors may be involved. Among other hypotheses the most likely explana- tion is that hearing loss is caused by the lower density of the vaterite otoliths, which could induce a decrease in the differential movement between the saccular epithelium and its otolith. It would require a higher force to stimulate the sensory epithelium and trigger a neural response and, therefore, auditory sensitivity would be reduced [24]. After assessment (in our samples the otolith proportion that was affected by vaterite in control cages was higher than in exposed cages) we concluded that differences of the vaterite presence in otoliths had no relation with sound exposure, but was probably explained by a deficiency in nutrition associated to captivity as has been shown in previous studies [14]. In terms of behaviour, despite minor and non-permanent behavioural responses were reported (small level of startle response with habituation at subsequent exposures) to very high sound levels produced by air-guns (196 dB re 1 µPa at 1 m from the source) [16,17] we did not find any change in the behaviour of the salmon. In addition, the lateral line superficial neuromasts did not show any alteration in the sensory epithelia. Acoustic trauma was, however, observed in larval zebrafish lateral line hair cells using underwater cavitation to stimulate a response [25]. Gross pathology and histopathological analysis on salmon did not show any lesion that could be associated to sound exposure. Some works have described different pathologies associated to sound exposure. Various levels of barotrauma after sound exposure—mild injuries (eye haemorrhage, fin haematoma), moderate injuries (liver haemorrhage, bruised swim bladder), and mortal injuries (intestinal haemorrhage and kidney haemorrhage)— were observed after exposing Chinook salmon (Oncorhynchus tshawytscha) to impulsive pile-driving sounds using a high-intensity controlled impedance fluid filled wave tube [26]. However, the sound exposure level received during this experiment greatly exceeded the levels we reached in our study. None of these consequences were observed during our experiments. Parasitic sea lice (L. salmonis) infestations are one of the most important concerns in salmonid farming, reducing productivity and causing economic losses to the aquaculture industry (around 0.39 € per kg of salmon produced) [27,28]. Currently the most common treatment against salmon lice are chemicals. The pharmaceuticals currently used for the control of sea lice (cypermethrin, deltamethrin, azamethiphos, hydrogen peroxide) are applied through in situ immersion treatments [28]. Although these treatments have been effective in managing sea lice outbreaks, they have negative effects on fish welfare, reducing appetite and growth. Furthermore, over time salmon lice have built up a resistance to the three major classes of chemicals being used [29]. In addition, these pharmaceuticals are released into the surrounding environment, exposing non-target species to lethal and sub-lethal doses, and are harmful to the human health [28]. Other methods (e.g., in-feed treatments and use of skirts) are very expensive. Skirts have low impact on salmon welfare and the environment but reduce oxygen flow, affecting the respiratory functions of fish. From this perspective, this exhaustive assessment of the effects of acoustic treatment against J. Mar. Sci. Eng. 2021, 9, 1114 19 of 20 lice infestation on salmon confirmed that the chosen sound dose did not affect the exposed fish and, therefore, confirmed it as a potentially safe and sustainable protocol to address the problem. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jmse9101114/s1, Figure S1: Salmon tanks and refrigeration system; Figure S2: Incidence of vaterite on the salmon otoliths. (A,B) Incidence of vaterite versus time. (C) Incidence of vaterite on total sampled fishes. (D) Incidence of vaterite in right and left otolith on total sampled fishes. (E) Incidence of vaterite on right and left otolith versus time. (F) Incidence of vaterite on total sampled fishes versus control/exposed cages; Figure S3: Swimbladder with normal aspect of salmons from sea trial experiment. (A) Control. (B) 3 weeks after exposure. (C) 6 weeks after exposure. (D) 9 weeks after exposure. (E) 12 weeks after exposure. Scale bar: 100µm. Author Contributions: M.S. and M.A. planned the research and designed the study. M.S., M.A. and M.v.d.S. conducted experimental/lab work. M.S. and M.A. conducted sea trial work. A.L. and M.S. performed the otolith organ dissection. M.S. and J.-M.F. performed SEM analysis. M.C. and F.P. performed the histopathological examination. M.S. and M.v.d.S. analysed the data. M.S. and M.v.d.S. prepared the figures. M.S. wrote the article and all authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: Funding for this project was provided by SEASEL SOLUTIONS AS. 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