Audiogram Production

The hearing thresholds of any organism possessing the appropriate receptor mechanism are illustrated in an audiogram (Myerberg, 1981), which presents the lowest level of sound that a species can hear as a function of frequency. Audiograms for marine animals are predominantly expressed in units of sound pressure, or dB (re. 1 µPa) and is the rationale for using them in this study. The techniques used to obtain fish audiograms may require a varying degree of time, surgical and technical expertise, or the use of behavioural paradigms to gain statistically sound data (see for instance, Yan, 1995). Behavioural methods require that fish are trained to react in a specified and measurable way (e.g. a reward based method by seeking food) when a tone at a given frequency is presented; however, in practice, the behavioural method is very time consuming and only effective with species that are easy to train. Conditioning can take up to 3 weeks (feeding 3-4 times per day) to get a stable association between stimulus, response and food reward (Fujiya 1974; Hughes 2001; Lovell 1999 and Russon 2002). The advantages of the operant (reward based) conditioning methodology is that invasive procedures are not required, and the stimulus equipment can be relatively simple, however, the feeding behaviour of the species under investigation needs to be suited to this type of experiment (Yan, 1995).

The measurement of microphonics from auditory end organs during acoustic stimulation is a technique favoured by a number of authors (e.g. Enger and Anderson, 1967; Fay and Popper, 1975; Fine, 1981). Although results can be obtained more rapidly than from behavioural paradigms, preparation can often be complex and require invasive surgery to implant the electrodes directly into the nerve (c.f. Enger and Anderson, 1967). The electrode is thus restricted to a specific end organ or region of macula, and the evoked potential does not necessarily represent the whole auditory pathway (Kenyon et al., 1998).

The Auditory Brainstem Response (ABR) technique of measuring hearing thresholds has been successfully applied to both mammalian and non-mammalian vertebrates (Corwin, Bullock and Schweitzer, 1982), Elasmobranchs (Casper et al., 2003), and marine invertebrates (Lovell et al., 2005 a). The ABR is a non-invasive far-field recording of synchronous neural activity in the eighth nerve and brainstem auditory nuclei elicited by acoustic stimuli (Jewett, 1970; Jewett and Williston, 1971; Jacobson, 1985; Kenyon et al., 1998), and waveforms clearly present with similarities between fish and higher vertebrates (Corwin, 1981) and between vertebrates and invertebrates (Lovell et al., 2005a). Electrophysiological studies of the ABR response is used routinely in the clinical evaluation of human hearing (Jacobson, 1985), allowing for the acquisition of thresholds from uncooperative or inattentive subjects and in situations where behavioural methods cannot be readily applied (Kenyon et al., 1998).

The literature review of current audiogram production methodologies has been divided up into three sections. The first section looks at the use of microphonics, or recordings taken directly from the saccular and VIII nerves. The second looks at behavioural methodologies, and includes classical shock conditioning and heart rate suppression to ascertain hearing thresholds. The final section reviews works that use the Auditory Brainstem Response (ABR) technique to measure Auditory Evoked Potentials (AEP’s). Audiograms produced using the Auditory Brainstem Response (ABR) technique show the range of sonic frequencies audible to both mammalian and non-mammalian vertebrates (Corwin, Bullock and Schweitzer 1982), and are regarded as being the least time consuming methodology for acquiring audiological data. Additionally, ABR recordings require no invasive procedural work as measurements are taken in the electro-physiological far field using cutaneous electrodes, resulting in significant stress reduction during the hearing assessment (Kenyon et al 1998).

2 Microphonics

Enger and Anderson (1967) conducted a field study of fish audiometry by measuring microphonic potentials from the cod (Gadus morhua) and the sculpin (Cottus scorpious) in the open sea. Electrode implantation involved a highly complex surgical procedure, and involved drilling small holes in the cranium close to the saccular nerve. A 0.5 mm diameter silver wire was inserted in the hole, and sealed using dental cement. Using this method, the authors recorded microphonics of 70 µv from both cod and sculpin, when stimulating with tone bursts presented from a J9 underwater sound projector driven by a Philips RC oscillator and a Quad II amplifier.

Fay and Popper (1974) recorded microphonic potentials from the ear of the goldfish (Carrassius auratus) in a situation where sound pressure and particle displacement could be independently varied. When two transducers positioned facing each other are operated in phase, the water between them is compressed, creating a sound field dominated by pressure and minimal particle displacement. When the transducers are operated out of phase, one compresses the water whilst the other pulls it, creating a field dominated by particle displacement with minimal sound pressure (both modes of sound presentation are discussed further in Chapter 5). The authors also tested the fish with the swim bladder present and after its removal. The fish were tested in a 330 mm diameter PVC cylinder 1500 mm high, located in a soundproof acoustic chamber. A water bag containing the fish was suspended in the middle of the cylinder; air speakers were positioned above and below the bag containing the fish and the stimulus sounds presented using a Dyna 120 amplifier and a 7056 function generator.

In a second series of experiments measuring microphonics, Fay and Popper (1975) recorded potentials from the saccule of the African mouthbreeders (Tilapia macrocephala) and the catfish (I. nebulosus) using submerged glass insulated tungsten electrodes. The fish were tested in a soundproof acoustic chamber to both acoustic and vibrational stimulation, and for sound reception with the swim bladder filled with water. The test tank was a 250 mm diameter PVC cylinder 200 mm high, filled to a height of 160 mm. The floor of the cylinder was made from 5 mm thick “Rho C” rubber supported by a plastic grating. A loudspeaker with a diameter of 200 mm was suspended facing upwards 250 mm below the test tank in an airtight extension of the cylinder. The sound pressure level required to evoke a 1 μv RMS Auditory Evoked Potential (AEP), was determined using a Clevite Model CH-17T hydrophone positioned adjacent to the fish’s ear.

Fine (1981) investigated the mismatch between sound production and hearing in the oyster toadfish (Opsanus tau). Anaesthetised fish were and clamped in a tank with the top of the head above the water surface. Single nerve fibres were then isolated from the saccular afferents, and the response to 300 ms tone burst from a speaker in air was measured. The tone bursts were phase-locked, had rise-fall times of 5 ms, and presented to the subject at a rate of 1 burst per second. The stimulus sound and background noise were measured using a Celesco LC34 hydrophone connected to A B&K 2508 amplifier, and wave analyser with a 3Hz filter.

3 Behavioural approaches

Popper (1972) used an avoidance conditioning procedure to define auditory thresholds for the carp (Cyprinus carpio). In this experiment, the fish were trained to cross a barrier in the middle of tank whenever a pure tone was presented through a loudspeaker mounted in the air, about 100mm from the test tank. The experimental tank was placed in an acoustic chamber to reduce ambient noise, and the experiment tested the hearing of 6 animals ranging in standard length from 50 to 60 mm. When the fish failed to cross the barrier during presentation of the stimulus, it was concluded that that the fish had not heard the sound, and thresholds were determined at the 50 % level using the up-down staircase method.

Offutt (1974) used classical conditioning of heart rate to determine hearing thresholds in the Atlantic cod (Gadus morhua). Fish were held in a nylon mesh net, in a tubular tank 530 mm long, and a diameter of 305 mm, positioned lengthwise in a wooden framework. The test tank and all test equipment was housed in an underground concrete room, and the pure tone stimulus sounds were generated by a 410 mm speaker built into the wall of the chamber. ECG’s were obtained using an electrode inserted in the pericardial cavity, and a reduction in the heart rate indicated fish had heard signal. Thresholds were determined by the staircase method, with the stimulus attenuated in 2dB steps and a minimum of 10 reversals.

A Field study of hearing in two species of flatfish Pleuronectes Platessa (L.) and Limanda limanda (L.) was conducted by Chapman and Sand (1974) in Upper Loch Torridon, Scotland, using a PVC frame located 15m below the water surface and 6m from the seabed. A pair of stainless steel electrodes was built into the cage, to administer an electric shock to the subject’s tail, and the potentials generated by the cardiac muscles were recorded using a subcutaneous electrode. The conditioning stimulus was a pure tone pulse presented to the fish for 10 seconds, paired with a 6 to 12 V dc electric pulse administered to the fish from the loch shore. The cardiac potentials from the fish were amplified and recorded using a storage oscilloscope, and a hydrophone positioned 10 mm below the head of the fish recorded the sound pressure of the stimulus tone. The sound was presented to the fish through two sound projectors located 0.7 m, and 3 m from the holding cage. In order to condition the fish, the electric shock was administered after presentation of the stimulus sounds. Conditioning using this methodology was repeated until the fish showed an alteration in heart rate after onset of sound but before the shock. Full conditioning was considered to have occurred when 5 consecutive trials had yielded positive responses. In some experiments a small 34 mm diameter spherical air-filled rubber balloon was placed behind the cranium to simulate the presence of a swim bladder.

Coombs and Popper (1979) conditioned Squirrelfish (Myripristis kuntee) and (Adioryx xantherythrus) to respond to sound in a 410 x 240 x 170mm Plexiglas tank situated in an anechoic chamber. The stimulus sounds were presented to the fish through two air mounted loudspeakers, which produced a series of 600ms tone bursts with a 5ms rise and fall, followed by 400ms of silence. A shock avoidance technique was used to measure auditory sensitivity, and the fish trained to swim across a barrier on hearing a sound or risk an electric shock. The staircase method was used to determine threshold, and the sound level increased or decreased in 5dB steps depending on the response of the fish during the test.

A behavioural study of hearing in damselfish (Eupomacentrus dorsopunicans, E. mellis, E. variabilis, E. diencaeus, and E. planifrons) by Myrberg and Spires (1980) looked at hearing in these closely related species. The audiological tests were conducted in a 5m long, 150 mm internal diameter glass tube, divided into two sections, and filled with seawater. The farthest end of the section in which fish was placed had a type J-9 underwater transducer mounted on anti-vibration pads. The second section was filled with sponges to act as sound absorbers, and the entire assemblage was suspended from the floor by rubber bungees attached to a beam above the tube. For some tests, in order to increase ratio between sound pressure and velocity, a 150 mm ø hollow rubber ball was placed at the end of the first tube opposite the speaker. The fish was restrained in a transparent Plexiglas cylinder positioned so the fish was equidistant from the surrounding wall of the glass tube. Little sideways movement was possible, but the fish could easily be moved vertically. Stainless steel rods were located on each side of the restrainer as electrodes for applying a shock to the fish, and the sound pressure was measured by an Aquadyne AQ-12 hydrophone placed in the restrainer below the head of the fish. The fish was stationed either 400 mm or 1.45 m from the speaker face. The fish was conditioned to respond to sound by moving downwards if it detected a tone, and the staircase method was used to determine the threshold (the sound level was varied in 2dB steps). Threshold was determined from the average sound level attained after 50 sound presentations beyond the point where the levels accompanying response and no-response varied by no more than 8dB.

Coombs and Popper (1982) studied the structure and function of the auditory system in 3 specimens of the clown knifefish (Notopterus chitala). The association between the ear and anterior projections of the swim bladder were subjected to an anatomical investigation. Auditory sensitivity was determined using an operant conditioning technique, where the fish was trained to cross a barrier in the centre of a tank on hearing the audio cue, in order to avoid being given an electric shock. The sound pressure level was decreased in 5 dB steps following each successful avoidance response, and increased by 5 dB if the fish did not avoid the shock. Two tanks were placed in an anechoic chamber, and the stimulus sound source was a single 203 mm diameter speaker positioned in air, above the test tanks. Vertical particle velocity was also measured with a velocity hydrophone at four positions in the tank.

Hawkins and Myrberg (1983) used cardiac suppression to define the hearing abilities of 43 immature cod (Gadus morhua) ranging in length between 210 mm and 470 mm. The fish were anaesthetized in a 1:15000 solution of MS-222 whilst silver electrodes were inserted subcutaneously into the body cavity, in order to detect the electric potentials from the heart. Experiments were performed in a framework immersed in the sea 100 m from the shore, and the top of the framework was located 15 m below the sea surface, and 6 m above the seabed. The test cages contained stainless steel electrodes, which were used to administer a shock on presentation of a sound during conditioning. Two sound projectors were placed on a line from the shore, at right angles to the axis of the cage. The intensity of the stimulus sounds were recorded using a hydrophone and filtered to bandwidth of between 10 Hz to 1000 Hz. For some experiments, a high level of random noise was continuously transmitted from the sound projector and the pure tone stimulus superimposed.

McCormick and Popper (1984) studied auditory sensitivity and psychophysical tuning curves in the elephant nose fish, Gnathonemus petersi using a behavioural method. The auditory tests were carried out in tanks located in a chamber which had 150mm thick sand-filled walls. The test fish had to cross a barrier dividing the tank within 10sec of the sound being presented, to avoid being given an electric shock. The sound projector was a 203 mm diameter speaker positioned in the air above the test tank, and the stimulus tones were generated with a 5 ms rise and decay time. The staircase method was used for threshold determination, and the sound level was varied in 5dB steps. The threshold was calculated from the final 8 trials over a 24 hour period. The sound level in the tank was measured with a Clevite hydrophone, at 10 locations, and the median values of the levels was used as the calibrated value. Particle velocity was also measured at 4 locations using a velocity hydrophone. The ambient sound pressure was found to be well below threshold levels at all frequencies, and tests were also conducted to ascertain if the fish might be influenced by electric fields; it was concluded that this was highly unlikely.

Yan and Popper (1992) defined the auditory sensitivity of the cichlid (Astronotus ocellatus) using a non invasive reward based methodology, and present a behavioural audiogram for the goldfish from Yan and Popper (1991). The experiment involved using an automatic feeding device to train 3 A. ocellatus to respond to an acoustic cue. A clear plastic tube delivered the food pellets to the fish, feeding tube was clear to allow the fish to receive visual as well as acoustic clues to a feeding event. 2 paddles were suspended from a platform and sent response signals to a PC which controlled food delivery if the correct sequence of paddles were pressed during acoustic stimulation. The experiments were conducted in a soundproof chamber, and the stimulus tones were presented to the fish using an underwater speaker (University Sound UW-30). The fish were trained to peck the O-paddle and then to peck the R-paddle if they detected the stimulus sound; a correct response resulted in the fish obtaining food. Once trained, thresholds were determined from the sound level at which 50% of the trials resulted in a correct responses.

Mann, Lu & Popper (1997) also used a cardiac suppression methodology to determine ultrasonic hearing by the American shad (Alosa spp). The experiment involved training 5 fish to reduce their heart rates on presentation of an audible sound; however the experiment was conducted with an active pump system, which may have masked responses to low frequencies.

Casper, Lobel and Yan (2003) studied the hearing sensitivity of the little skate (Raja erinacea) using both behavioural and ABR methods (see next section for ABR description). 3 test subjects were conditioned in a tank 1.5 m x 1.08 m x 0.65 m using a 60-s pulsed recording of brown noise (low-passed noise that has a 4 dB drop per octave), played through an underwater speaker 1 m from the skate’s head. The fish were trained to associate noise with food provision, and feeding/conditioning events were conducted 3 to 4 times per day for 6 weeks. Conditioning was considered a success if the skate showed response 10 times without the introduction of food. A positive response was acknowledged if skate began swimming on presentation of the stimulus sound, or an increase in the rate of respiration was observed. Following training, audiological tests were conducted using 500 ms pulsed tones emitted from a Lubell Corp. LL-98A projector positioned 200 mm above bottom of tank, and 1m from the skate. An Interocean Systems Model 902 hydrophone was used to record the sound pressure at a distance of 150 mm above the skate’s head. If the skate responded 5 times consecutively, it was deemed to be responding to the sound stimulus at that intensity, so the pulsed tone was attenuated in 5 dB steps until the fish did not respond to the sound (threshold was determined from the lowest sound pressure where a 100 % response could be observed).

4. Measurements of the Auditory Brainstem Response

Kenyon, Ladich and Yan (1998) used the ABR audiometric method on goldfish (Carassius auratus) and the cichlid (Astronotus ocellatus) to generate audiograms. The experiments were conducted in a soundproof booth (2 m x 3 m x 2 m), into which anaesthetised fish were clamped in place using a net mesh and positioned so the top of the head was 1 mm above the water surface. Two electrodes were pressed against the exposed cranium above the medulla, with the reference electrode positioned 5 mm anterior of the recording electrode. Frequencies below 3 kHz were generated using a 300 mm loudspeaker suspended 1 m above the water surface in the holding tank, and for frequencies above 3 kHz, a 120 mm loudspeaker was used. The Sound Pressure Level (SPL) was recorded using a hydrophone placed near the ear of fish, and the tones and clicks were presented over a range of intensities, in order to obtain evoked potential thresholds defined by visual inspection of two overlayed traces from a repeat test at a particular frequency and intensity. Clicks were 0.1 ms in duration, and presented at a rate of 38.2 clicks per second. The number of cycles in a tone burst was set to get best compromise between stimulus rapidity and peak frequency bandwidth, with bursts gated using a Blackman window function applied to reduce spectral leakage from the signal (e.g. if a 3 Hz sine wave is sampled for .9 seconds, a discontinuity results). In total, eight fish were given Flaxedil (gallamine triethiodode) to immobilise them, whilst three fish remained untreated. The authors reported that thresholds were significantly lower for the Flaxedil treated fish, demonstrating that the restraining methodology allows untreated fish enough gross movement to contaminate the ABR trace.

Ladich and Yan (1998) used the ABR method to study hearing in the paradise fish (Macropodus opercularis). The experiments were conducted on an air table located in a soundproof booth (see Kenyon, Laditich and Yan, 1998 for dimensions). During the investigation, Flaxedil immobilised fish were held in place using a net mesh, with just 1 mm of top of head above the water surface. 2 electrodes were pressed against the head, with the reference electrode positioned 10 mm anterior of the recording electrode. Sound was generated by a loudspeaker suspended 1 m above the surface of the water, with a 300 mm speaker used to generate frequencies below 3 kHz, and a 120 mm speaker was used for frequencies above 3 kHz. The SPL was obtained using a hydrophone (Celesco LC-10) placed in proximity to the ear of the fish. Tones and clicks were presented at various pressure levels to obtain thresholds, which were identified by visual inspection of the averaged ABR traces when superimposed over the first run. Clicks were 0.1 ms in duration, and presented at 38.2 clicks per second, and the number of cycles in each of the tone bursts was programmed to optimise stimulus rapidity and peak frequency bandwidth and gated using a Blackman window.

Yan, (2001) tested a number of hearing specialists including the goldfish (Carassius auratus), blue gourami (Trichogaster trichopterus), kissing gourami (Helostoma temminckii), dwarf gourami (Colisa lalia), and a mormyrid (Brienomyrus brachyistius) using ABR audiometry. In addition, Yan studied auditory thresholds from the oyster toadfish (Opsanus tau), a hearing generalist. The experiments took place in a soundproof booth (see Kenyon, Laditich and Yan, 1998 for dimensions). The fish were sedated with Flaxedil (gallamine triethiodode) and clamped in a mesh net suspended in a tank (see Scholik and Yan, 2002 for dimensions) standing on an air table. The top of the head was positioned 1 mm above water level, and tissue placed on head to prevent it from drying out. The electrodes were pressed against the head, and the reference electrode positioned 5 mm anterior to the recording electrode. Sound was presented to the fish through a speaker suspended 1 m above subject, with a 300 mm speaker used for frequencies below 3 kHz and a 120 mm speaker for frequencies above 3 kHz. Clicks with a duration of 0.1 ms, were presented at a rate of 38.2 clicks per second. The number of cycles in each of the tone bursts was set to get best compromise between stimulus rapidity and peak frequency bandwidth, and gated using a Blackman window. The Sound Pressure Level of the stimulus sounds was obtained using a hydrophone placed near the fish ear, and once the baseline audiogram had been taken, the gas inside the swim bladder was removed using a syringe and needle. The audiogram procedure was repeated with the swim bladder deflated to show that the organ enhanced hearing sensitivity.

Scholik and Yan (2001) studied the effects of underwater noise on auditory sensitivity of the fathead minnow (Pimephales promelas) exposed for selected durations. A mesh screen prevented the fish from jumping out of the tub (see Kenyon, Laditich and Yan, 1998 for dimensions); though the fish were free to swim around during noise exposure. The bandwidth of the noise was limited to between 300 Hz to 4 kHz, and presented at a Sound Pressure Level (SPL) of 142 dB (re 1µPa). The fish were mildly sedated with Flaxedil and the ABR technique used to obtain the threshold values. The experiment was designed to establish hearing thresholds immediately after 24 hours of continuous exposure to the noise, then at 1, 2, 4, 8 and 24 hours after exposure.

In a similar experiment, Scholik and Yan (2002) produced several ABR generated audiograms to ascertain the effects of noise on the auditory sensitivity of the bluegill sunfish (Lepomis). Specimens of L macrochirus were exposed to white noise presented at 142 dB re 1µPa, and a bandwidth of between 300 Hz to 2000 Hz. The fish were sedated with Flaxedil, and the ABR technique was used to obtain the threshold values after exposure to the noise. The stimulus sounds used to test for threshold shifts were generated using an air mounted transducer, and the evoked response recorded using two cutaneous electrodes held in place using micromanipulators, with the fish placed in a plastic tub (380 mm x 24.5 mm x 145 mm).

Casper, Lobel and Yan (2003) studied the hearing sensitivity on 4 specimens of the little skate (Raja erinacea), using ABR audiometry. The fish were immobilised by an injection of d-tubocurarine chloride and suspended in a 380 mm x 245 mm x 145 mm plastic tray, suspended at an angle so the entire body of the skate was immersed. A small portion of the head (near the medulla region), posterior to the eyes, was exposed to the air, and chosen for the primary site for the placement of the electrodes. The plastic tub was placed on a vibration-isolating table, in a sound proof booth (2 m × 3m × 2 m). Tone bursts with a duration of 20 ms, were presented through a Pioneer 300 mm speaker, positioned 1 m above the subject’s head. 3000 iterations of the stimulus sound were averaged at each Sound Pressure Level (SPL), which was reduced in 5 dB steps until the threshold was reached. The threshold SPL value was measured with a Celesco LC-10 hydrophone placed where the subject’s head was during the audiometric examination.

Akamatsu, Nanami and Yan (2003) defined the hearing abilities of the spotlined sardine (Sardinops melanostictus) using the ABR technique. Audiograms were generated from fish stationed in a seawater-filled plastic tub, 280 mm x 200 mm x 35 mm deep, and placed on a vibration isolating table in a soundproof chamber. The stimulus sound was presented through a ceiling-mounted loudspeaker positioned 450 mm above the head of the fish. The stimulus sounds were digitally generated 5-cycle tone bursts, multiplied with a Gaussian function. The sound in the water was monitored with a B&K Type 8103 hydrophone located adjacent to the subject’s head, and the fish restrained using a neoprene rubber sling with stainless steel plates attached to sides. The fish were held horizontally, with the inner ear and anterior end of gas bladder kept at the same depth to ensure equal levels of incident sound pressure on both organs. A small area of skin on the top of the head was exposed above the water line to facilitate in the placement of the electrodes. The potentials were amplified and filtered to a bandwidth of between 50 Hz to 10 kHz. Only 300 stimulus exposures at each frequency were used, thus cutting back on the time it takes to produce the audiogram as it was found to be difficult to sustain life support for the test sardine. The sound level at each frequency tested was varied initially in 6 dB steps, and then in 3 dB steps as the threshold was approached. Water was continually supplied to the mouth of the subject, with the flow maintained by gravity to avoid the noise generated by an electric pump. The electrodes, through which the evoked potential was conducted, were placed along the midline of the skull over the medulla region, with the cables twisted in an effort to reduce the electromagnetic noise generated outside the chamber.

Lugli, Yan and Fine (2003) studied the relationship between ambient noise, hearing thresholds and sound spectra in acoustic communication between two freshwater gobies Padogobius martensii and Gobius nigricans. A total of 5 fish (2 females, 3 males) were tested to generate the ABR audiograms; in each case the fish was held with the nape of the head just above the water surface, in a 380 mm x 245 mm x 145 mm plastic tub. The stimulus sounds were presented to the fish through a 300 mm Pioneer speaker located 1 m above the subject. The sound used was a tone burst 20 ms in duration, and used for each frequency tested; the sound level in the water was monitored with a Celesco LC-10 hydrophone located adjacent to the head of the fish. During the experiment, the sound level was reduced in 5 dB steps until threshold was reached. Part of the experiment was to study the sound produced by the fish, and how their hearing might be related to the ambient noise in their normal environment (shallow stony streams); a relationship was found between the sound spectrum of the ambient noise and hearing sensitivity.

5 Previous uses of ABR in cetacean audiometry

Popov and Supin (1990) studied hearing in the beluga dolphin (Delphinapterus leucas), the bottlenose dolphin (Tursiops truncates), the Amazon River dolphin (Inia geoffrensis), tucuxi dolphin (Sotalia fluviatilis) and the Manatee (Trichechus inunquis) using the ABR technique. The hearing tests were conducted in either a 4 m x 0.6 m x 0.6 m bath, in a round pool, or in an enclosed sea bay. During the tests, the subject was supported on a stretcher positioned so only the top of the head with the blowhole and the back, as far as the dorsal fin was out of the water. The Auditory Evoked Potentials (AEP’s) were recorded using 0.4 mm to 0.6 mm diameter subcutaneous needle electrodes inserted into the skin at depths of between 3 mm to 5 mm (see also: Popov, Ladygina and Supin, 1986). The record electrode was placed on the dorsal head surface 60 mm to 90 mm caudal from the blowhole, and the reference electrode placed on the back near to the dorsal fin. The potential difference between the two electrodes was fed to a biological amplifier (gated between 5 Hz to 5000 Hz) and the signal averaged to reveal the AEP. The stimulus sounds used in the audiological tests were clicks, square enveloped noise or ramped tone bursts of frequencies of between 5 kHz to 160 kHz, generated using piezo-ceramic transducers with diameters of 20 mm, 30 mm and 50 mm. The array was stationed 300 mm below the water surface, at distances of between 1 m to 2 m anterior of the subject’s head.

In a second series of experiments using the ABR technique on odontocetiforms, Bibikov (1992) studied hearing in the harbour porpoise (Phocoena phocoena) using both cutaneous and implanted electrodes. The porpoise was loosely restrained in a bath with dimensions of 2.5 m x 0.6 m x 0.65 m, which had been lined with sound absorbing rubber and filled with seawater. The record electrode used in the first experiment was a 10 mm diameter silver disc placed on the surface of the skin above the muscles overlying the vertex of the head, whilst the second experiment used an implanted electrode. In both experiments, the reference electrode was a subcutaneous needle electrode inserted into the skin close to the dorsal fin, and the AEP’s gated between 50 Hz and 4 kHz for the subcutaneous electrode and 200 Hz to 5 kHz for the surface electrode.

André et al. (2002) found evidence of deafness in a young stranded female striped dolphin, Stenella coeruleoalba, which cancelled her possibility to process correctly any acoustic information. The experiments took place in a large seawater pool, with the dolphin held in a stretcher made from a sound transparent fabric, stationed at a depth of 40 to 50 cm in the centre of the pool. This allowed the body of the dolphin to remain under the water, while the dorsal part of the head and the blowhole stayed above the surface. The stimuli used during the study were sinusoidal amplitude-modulated tones, generated using a function generator and amplified using a B&K 2713 amplifier driving a piezoceramic transducer (B&K 8104 hydrophone). Tone bursts were presented for a duration of 20 ms, at a rate of 20 s -1. The stimulating transducer was placed in front of the dolphin, at a distance of 1 m from the head and a depth of 20 cm, with stimulus intensity specified in units of dB (re. 1 μPa) RMS. The evoked potentials were recorded using 1-cm disk electrodes secured at the body surface inside 6-cm suction cups. The active electrode was placed at the head vertex, just behind the blowhole, with the reference electrode placed on the back (both electrodes were above the water surface). The recorded potentials were digitised using an A/D converter and averaged over 1000 sweeps of 30 ms, using a standard personal computer. The analysis of the results from the experiment suggests that the dolphin had great difficulty processing acoustic stimulus, and most likely explains the cause of stranding.

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