Temporary hearing threshold shift in harbor seals (Phoca vitulina) due to a one-sixth-octave noise band centered at 16 kHz

When marine mammals such as pinnipeds are exposed to high-amplitude sounds, they may suffer hearing loss. That is, their hearing threshold may shift temporarily (i.e., temporary threshold shift or TTS) or permanently (i.e., permanent threshold shift or PTS; Melnick, 1991; Yost, 2007). For the regulation and management of anthropogenic offshore activities that generate high levels of underwater sound (e.g., percussion pile driving, seismic surveys, sonars, detonations), it is important to know at what sound exposure level (SEL) hearing may be reduced in various marine mammal species. SEL is a combination of the sound pressure level (SPL) generated by the sound source and the duration of the exposure. Studies of TTS can be used to estimate PTS-onset SELs (Southall et al., 2019), but also give insight into the effects of less harmful exposures, by providing information on TTS-onset SELs, rates of increase of TTS, and critical levels (i.e., levels above which TTS increases strongly; Chen et al., 2014). TTSs of various magnitudes and durations have the potential to compromise foraging, orientation, communication, and predator detection in marine mammals. This may affect individual health and survival, which in turn may have negative impacts at the population level. The ecological effect of reduced hearing depends not only on the magnitude of TTS and its duration (which in turn depends on the exposure duration plus the recovery time after exposure stops), but also on the hearing frequency range affected and the ecological importance of this frequency range for the animal.

The harbor seal (Phoca vitulina) is a widely distributed pinniped species in the coastal waters of the northern hemisphere (Burns, 2002) that is present in areas with high levels of anthropogenic activity. Anthropogenic underwater sounds may cause TTS or PTS in harbor seals if the received SPL is high enough and/or if the exposure duration is long enough. So far, only five studies have been conducted in which TTS due to underwater sounds was studied in harbor seals (Kastak et al., 2005; Kastelein et al., 2012; Kastelein et al., 2013a; Kastelein et al., 2018a; Kastelein et al., 2019a). After exposing a harbor seal to octave-band noise centered at 2.5 kHz, Kastak et al. (2005) tested its hearing at two frequencies (2.50 and 3.53 kHz) and found that the highest TTS had occurred at the center frequency. Kastelein et al. (2012) tested the hearing of two harbor seals at 4 kHz after exposing them to octave-band noise centered at the same frequency. After exposing two harbor seals to playbacks of broadband pile-driving sound (the one-third-octave band centered at 630 Hz contained the most energy), Kastelein et al. (2018a) tested their hearing at 0.25, 0.5, 1, 2, 4, 8, 16, and 32 kHz, and found TTS only at 4 and 8 kHz (the highest TTS occurred at 4 kHz). Kastelein et al. (2019a) exposed harbor seals to a continuous 6.5 kHz sinusoidal wave and tested their hearing at 6.5, 9.2, and 13 kHz; the hearing frequency most affected depended on the SEL the animals were exposed to. The highest TTSs occurred at 6.5 kHz for the lower SELs and at 9.2 kHz for higher SELs.

Susceptibility to TTS depends on the frequency of the fatiguing sound (i.e., the sound intended to cause TTS) in odontocete cetaceans (Houser et al., 2017), but frequency-dependence of TTS has not been studied extensively in pinnipeds. So far, the susceptibility of harbor seals to TTS has only been tested in a small frequency range (2.5–6.5 kHz); more frequencies need to be tested in order to assess frequency-dependence. As part of a larger project on the susceptibility of harbor seals to TTS over their entire hearing range, in the present study, susceptibility to TTS after exposure to a sound of 16 kHz was tested in this species. The goal of the present study was to expose harbor seals to different SELs of a continuous one-sixth-octave noise band centered at 16 kHz and to quantify TTS-onset SEL, changes in TTS with increasing SEL of the fatiguing sound, the affected hearing frequencies, and the recovery of hearing after exposure. The ultimate goal of the larger project is to establish equal-TTS curves for harbor seals, upon which a research-based weighting curve for phocid seals can be based (see Houser et al., 2017). These data can then be used by government regulators to set safe sound level criteria for harbor seals and other phocids or they can be used to determine the area around a sound source in which TTS or PTS can be elicited in a particular species. In marine mammals, criteria defining the numbers of individuals that can be affected by human activities are vital for conservation and mitigation.

The study animals were two healthy adult female harbor seals (F01 and F02). During the study, they were both 12 years old and weighed ∼67 kg. Nine previous hearing studies showed that they had sensitive and very similar hearing, likely representative of similarly aged conspecifics, and were not affected by previous TTS studies (Kastelein et al., 2009b; Kastelein et al., 2009a; Kastelein et al., 2010; Kastelein et al., 2012; Kastelein et al., 2013b; Kastelein et al., 2015b; Kastelein et al., 2018a; Kastelein et al., 2018b; Kastelein et al., 2019a). The feeding regime was as described by Kastelein et al. (2019a).

The study was conducted at the SEAMARCO Research Institute, the Netherlands, in a remote and quiet location. The seals were kept in an outdoor, seawater-filled pool (8 × 7 m, 2 m deep, with haul-out areas; see Fig. 1 in Kastelein et al., 2012), which also served as the test setting. During sound exposure sessions, barriers were placed to prevent the seals from using the haul-out areas. During hearing tests, one seal was tested at a time. The seal not being tested remained in the water next to the main haul-out area and was kept occupied with quiet husbandry behaviors. During the sound exposure periods, the total surface area of the pool was filmed with a wide-angle video camera on a pole, in order to record the behavior of the seals, their distance from the transducer, and the percentage of time they spent fully submerged and at the water surface.

Acoustical terminology follows ISO (2017). All SELs reported here are unweighted.

The ambient noise was measured, and the fatiguing sound and hearing test signals were calibrated once every two months by an acoustic consulting agency (TNO, Den Haag, the Netherlands), as described by Kastelein et al. (2019a). Before each TTS test, the hearing test signals and fatiguing sound in the pool were measured with a spectrum analyzer and results were compared to the levels measured during calibration. The maximum deviation from the calibration value was 2 dB. Care was taken to keep the harbor seals' listening environment during hearing tests as quiet as possible, by allowing only researchers involved in the study within 15 m of the pool, having the water circulation system turned off, and only performing tests under favorable weather conditions (i.e., no rain and wind force Beaufort 4 or below; see also Kastelein et al., 2019a). Under test conditions, the ambient noise in the pool was therefore very stable and low (Fig. 1), and did not mask the hearing test signals.

A continuous (100% duty cycle) one-sixth-octave noise band centered at 16 kHz, without harmonics, was used as the fatiguing sound. This digitally generated sound was played by a laptop computer to a data acquisition card (National Instruments, Model No. USB 6229) the output of which went via a ground loop isolator and a custom-built buffer to a custom-built passive low-pass filter set to 20 kHz, after which it went to a high-frequency power amplifier (HLLY 2012–01, Model No. LS5002), which drove the transducer (ITC, Model No. 6084; see Kastelein et al., 2019a for more information). The transducer was placed in the center of the pool at 1 m depth. The linearity of the transmitter system was consistent to 1 dB within a 42 dB range (including the entire SPL range used in the study).

To determine the fatiguing sound's distribution in the pool, the SPL was measured at 35 locations (on a horizontal grid of 1.2 m × 1.2 m), at three depths per location on the grid (0.5, 1.0, and 1.5 m below the surface; Fig. 2). To investigate the received SPL, the seals' location in the pool and their head position (fully submerged, or at the water surface) was monitored during the fatiguing sound exposure periods (see Sec. II D). Since they swam throughout the pool, and at all depths, the average received SPL experienced by the harbor seals was calculated as the energetic average of the SPL at all 105 individual measurement locations. Differences in SPL per depth and per location were small, and, probably as a result of reflections from the pool walls and water surface, no gradient existed in the SPL in relation to the distance to the transducer, resulting in a fairly homogeneous sound field {Fig. 2, example mean [± standard deviation (SD)] SPL 140 ± 2 dB re 1μPa, range 134–152 dB re 1 μPa}. The one-sixth-octave noise band fatiguing sound was projected at various source levels, resulting in average received SPLs ranging from 128 to 149 dB re 1 μPa. For 60 min exposures, the resulting SELs were between 164 and 185 dB re 1 μPa²s.

Before each sound exposure test, the voltage output of the emitting system to the transducer and of the sound-receiving system were checked by producing a 16 kHz continuous tone from the laptop, recording it underwater and visualizing it on a spectrum analyzer (see Kastelein et al., 2019a for details). If the values corresponded to those obtained during SPL calibrations by the acoustic consulting agency, the SPLs were assumed to be correct and a sound exposure test could be performed.

The harbor seals were trained to detect signals presented during hearing tests before and after exposure to the fatiguing sound. The hearing test signals (tonal sweeps ranging ±2.5% of the center frequency, 1000 ms duration including 50 ms amplitude on- and off ramps) were generated digitally, exactly as described by Kastelein et al. (2019a); the test signal SPL at the location of a harbor seal's head positioned at the listening station was varied in 2 dB increments. Hearing thresholds were tested at the center frequency of the fatiguing sound (sweep centered at 16 kHz) and at half an octave above that frequency (sweep centered at 22.4 kHz).

Calibration measurements were conducted with two hydrophones, one at the location of each auditory meatus of the harbor seal when it was positioned at the listening station (see Kastelein et al., 2019a). At the listening station, the distance between each seal's head and the transducer was stable to within 1 cm, and both seals were trained not to move their body more than ±2° in the horizontal and vertical planes. The linearity of the transmitter system was consistent to 1 dB within a 30 dB range (from 10 dB above the hearing threshold). The mean SPL of the two hydrophones was used to calculate the acoustic stimulus level during hearing tests. The difference in SPL between the two locations was <3 dB.

The audiometric method used is described in detail by Kastelein et al. (2019a). Each hearing test session consisted of ∼25 trials and lasted for up to 12 min per animal (the first session after the fatiguing sound stopped was divided into three periods: 1–4, 4–8, and 8–12 min). The test tone was reduced by 2 dB following each correct detection and increased by 2 dB following each non-detection. One-third of the trials were catch trials, in which no hearing test tone was produced (i.e., signal-absent trials).

One total sound exposure test, consisting of (1) pre-exposure hearing test, (2) fatiguing sound exposure, and (3) post-sound exposure (PSE) hearing test or tests, was conducted per day. The SPL of the fatiguing sound during the exposure period was increased slowly to the intended SPL during the first 60 s. This was to avoid startle responses which may otherwise have led to large changes in the seals' swimming pattern. PSE hearing tests (using the same acoustic stimuli as used in the pre-exposure hearing tests) commenced within 1 min after the fatiguing sound had stopped for F02 and 12 min after the sound had stopped for F01. The two seals were always tested in this order to ensure a consistently quick and efficient start after the sound exposure stopped.

Besides the magnitude of TTS immediately after sound exposure, the subsequent hearing recovery was recorded. Recovery was defined as a return to <2 dB TTS, based on the fluctuations in control sessions of the present study and previous TTS studies with these animals. The hearing sensitivity of seal F02 was tested during up to five PSE periods: 1–4 (PSE_1-4), 4–8 (PSE_4-8), 8–12 (PSE_8-12), 60 (PSE₆₀), and 120 (PSE₁₂₀) min after the fatiguing sound exposure ended. The hearing of seal F01 was tested 12–16 (PSE_12-16), 16–20 (PSE_16-20), and 20–24 (PSE_20-24) min after the fatiguing sound exposure ended. PSE_1-4 (measured in F02) was considered to be the most informative period, as the TTS_1-4 is closest to the maximum TTS induced by the fatiguing sound. PSE_12-16, the earliest the hearing of F01 was measured, was considered to be the second most informative.

Sample sizes were chosen to maximize the study time available for testing SPLs in which TTS seemed to occur, while minimizing the risk of hearing damage (from repeated exposure to the loudest sounds) and avoiding repeated testing of SPLs for which TTS obviously did not occur (see Sec. III). In order to protect their hearing, the study animals were exposed to fatiguing sounds only once per day. Consequently, randomizing the order in which they were tested, while maintaining the sample sizes would have doubled the length of the study period. As a precaution, sound exposure tests were not conducted on days after the seals had been exposed to the highest fatiguing sound SPL.

Control tests were conducted in the same way as sound exposure tests, but without fatiguing sound exposure. Each control test started with a pre-exposure hearing test session, but was followed by exposure to the normal, very low ambient noise in the pool for 60 min (Fig. 1). Just like the PSE hearing test sessions, post-ambient exposure (PAE) sessions were divided into three periods: 1–4 (PAE_1-4), 4–8 (PAE_4-8), and 8–12 (PAE_8-12) min for seal F02, and 12–16 (PAE_12-16), 16–20 (PAE_16-20), and 20–24 (PAE_20-24) min for seal F01. Control tests were randomly dispersed during the study period among the fatiguing sound exposure tests. Data were collected between January and March 2018.

The mean rate of pre-stimulus responses (“pre-stimuli”) by the seals for both signal-present and signal-absent (catch) trials (in the latter, a whistle indicating the end of the test period was the stimulus) was calculated as the number of pre-stimuli as a percentage of all trials in each hearing test period.

Underwater sound reaches the harbor seal's ears directly and also via tissue conduction through the lower part of the head (Kastelein et al., 2018a), so the received level is probably similar at, or just below, the water surface and in air. However, it is unknown whether hearing sensitivity corresponds directly with susceptibility to TTS, so the percentage of time that each seal spent with her head at the water surface (i.e., nose in the air, mandible and lower part of skull under water) during ambient and fatiguing sound exposure was quantified from the video recordings by using stopwatches. For each average received SPL, one to nine 60-min fatiguing sound exposures were randomly selected for this analysis (the number varied with the numbers of SPLs that were tested) to obtain an average percentage per SPL. Via a grid on the monitor screen in the research cabin next to the pool, the pool was divided into 36 approximately 1.2 × 1.3 m rectangles (two of which coincided with the floating haul-out area). The grid location of the seals was recorded every 5 s for the selected sessions.

The pre-exposure mean 50% hearing threshold for each hearing test session (PE_50%) was determined by calculating the mean SPL of all reversal pairs (see Kastelein et al., 2019a) in the pre-exposure hearing sessions consisting of at least ten reversals. The TTS_1-4 in seal F02 was calculated by subtracting the mean 50% hearing threshold obtained during the PE_50% from the mean 50% hearing thresholds during PSE_1-4. The same method was used to calculate TTS in the other hearing test periods after the sound stopped, and after the control periods. Only pre-exposure hearing sessions consisting of 10 or more reversals were used for analysis and only PSE periods with four or more reversals were used.

We define the onset of TTS as occurring at the lowest SEL at which a statistically significant difference could be detected between the hearing threshold shift due to the fatiguing sound exposures and the hearing threshold shift as measured after the control exposures (this shift was close to zero). When statistical analysis of significance was not possible due to a sample size of 1 or 2, we compared the hearing threshold to the control level without statistical analysis in order to judge whether TTS occurred or not (a hearing shift of ≥2 dB was considered to be TTS, a shift of <2 dB was not, as such small shifts were normal variations in hearing threshold measurements in these seals). For sufficiently large sample sizes (i.e., n ≥ 4), the level of significance was established by conducting a one-way analysis of variance (ANOVA) on the TTS, separately for each seal and for each hearing test frequency, with the factor SPL (including zero as the control). When the ANOVA produced a significant value overall, the levels were compared to the control by means of Dunnett multiple comparisons. All analysis was conducted in Minitab 18, and data conformed to the assumptions of the tests used (Zar, 1999).

During the 60 min control sessions (only ambient noise), the seals spent ∼15% of their time with their heads at the water surface, and thus ∼85% of their time with their heads completely submerged. During 60 min exposures to the continuous one-sixth-octave noise band centered at 16 kHz, the mean percentage of time the seals spent with their heads at the water surface varied between 16% and 24% in seal F02, and between 13% and 22% in seal F01 (Table I). No clear relationship between the SEL and the percentage of time spent with heads at the water surface was observed (seal F02, r = 0.35, n = 6, t = 0.74, p = 0.50; seal F01, r = 0.16, n = 6, t = 0.32, p = 0.77). During the control and exposure sessions, the seals used most of the pool, and did not specifically avoid the location of the transducer. On a few occasions per session (both in control and test sessions), they raised their heads fully above the water surface for up to 2 s.

After the 60 min sound exposure periods, the seals were always willing to participate in the hearing tests. The pre-stimulus response rates in the pre-exposure, post-exposure, and control hearing tests were similarly low for both signal-present and signal-absent trials (in the latter, the whistle was the stimulus, indicating a correct signal-absence response; Table II).

The four ANOVAs showed that both TTS_1-4 (harbor seal F02) and TTS_12-16 (harbor seal F01) were significantly affected by the fatiguing sound's SEL at hearing frequency 16 kHz. At hearing frequency 22.4 kHz, TTS was affected by the SEL only in seal F02 1–4 mins after exposure ended. Comparisons with the control revealed that the statistically significant onset of TTS varied depending on the animal and the hearing test frequency (Table III).

For hearing test signals of 16 kHz (i.e., the center frequency of the fatiguing sound), statistically significant TTS_1-4 occurred in seal F02 at and above an SEL of 182 dB re 1μPa²s [Table III, Fig. 3(a)]. Hearing recovered within 12 min [Fig. 4(a)]. For hearing test signals of 22.4 kHz (i.e., half an octave above the center frequency of the fatiguing sound), TTS_1-4 was assumed to differ from the control value after exposure to SELs of 176 and 179 dB re 1 μPa²s, and TTS_1-4 was significantly different from the control value after exposure to SELs of 182 and 185 dB re 1 μPa²s [Table III, Fig. 3(a)]. The rate of increase in TTS with increasing SEL (hearing test signal of 22.4 kHz) was 0.6 dB/dB fatiguing sound between 170 and 182 dB SEL, and 3.3 dB/dB between 182 and 185 dB SEL (Table III). Thus, the critical level was close to 182 dB SEL. The highest measured TTS_1-4 at 22.4 kHz was 17 dB. When TTS had occurred, hearing recovered within 60 min for exposures up to an SEL of 182 dB re 1 μPa²s, and within 120 min for an SEL of 185 dB re 1 μPa²s [Fig. 4(b)]. The control sessions showed that the hearing thresholds for both hearing test signal frequencies before and after 60 min exposure to low ambient noise were very similar (Fig. 4, Table III).

For hearing test signals of 16 kHz (i.e., the center frequency of the fatiguing sound), no statistically significant TTS_12-16 occurred in seal F01, even after exposure to an SEL of 185 dB re 1 μPa²s [Table III, Fig. 3(b)]. For hearing test signals of 22.4 kHz (i.e., half an octave above the center frequency of the fatiguing sound), statistically significant TTS_12-16 occurred only after exposure to an SEL of 185 dB re 1 μPa²s [Table III, Fig. 3(b)]. Recovery of hearing occurred within 20 min after the sound exposure stopped. The control sessions showed that the hearing thresholds for both hearing test signal frequencies before and after 60 min exposure to low ambient noise were very similar (Fig. 5, Table III).

As the hearing of seal F02 was always tested before the hearing of seal F01, their TTSs cannot be compared directly. However, the closest possible comparison is between TTS_8-12 in seal F02 who was tested first, and TTS_12-16 in seal F01 who was tested second. Comparing the recovery of both animals for hearing test signals of 22.4 kHz after exposure to the highest SEL [Figs. 4(b) and 5(b)] shows that TTS_12-16 was ∼6 dB lower in seal F01 than TTS_8-12 in seal F02.

The animals in the present study had normal hearing for young adult female harbor seals (Kastelein et al., 2018a). The pre-exposure hearing thresholds found in the present study for the two hearing test signal frequencies were within a few dB of the hearing thresholds measured in these harbor seals for tonal signals during two studies conducted approximately ten years before the present study (Kastelein et al., 2009a; Kastelein et al., 2010).

TTS_12-16 in seal F01 was just ∼6 dB lower than TTS_8-12 in seal F02. This is probably partly due to the recovery of hearing in the minute that the animals changed positions between the hearing tests, as well as to the recovery which occurred during the 4 min that seal F01's hearing was tested (12–16 min). Thus, the seals' susceptibility to TTS caused by the 16 kHz fatiguing sound appeared to be similar. Studies on humans and other terrestrial mammals show individual, genetic, and population-level differences in susceptibility to TTS (Kylin, 1960; Kryter et al., 1962; Henderson et al., 1993; Davis et al., 2003; Spankovich et al., 2014). Further replication with more harbor seals is needed to assess the generality of the results obtained in the present study.

The mean pre-stimulus response rates in hearing tests before and after the sound exposures and during the control sessions were similar in both animals (Table II). This indicates that the seals used the same decision criteria in all conditions, and that their decision-making process in the post-exposure hearing tests was not influenced by the sound exposure.

No change in the seals' susceptibility to TTS over time, regardless of fatiguing sound level or hearing test signal frequency, was observed during the present study. In terrestrial mammals, susceptibility to TTS sometimes changed, due to conditioning of the ear, after repetitive sound exposures (Kujawa and Liberman, 1997; Mannström et al., 2015). Maybe this conditioning of the ears did not occur in the present study because the TTSs in the present study were smaller than those elicited in terrestrial mammals.

No aerial sound was projected during exposure sessions because, in a noise-band study, similar TTS_1-4 was found in harbor seals with and without aerial fatiguing sound in addition to the underwater fatiguing sound (Kastelein et al., 2012). When harbor seals are swimming with their heads at the water surface (nose in the air, mandible, and lower part of skull under water), their hearing thresholds for underwater sounds are the same as when they are fully submerged (Kastelein et al., 2018a). The seals in the present study spent from 0% to 9% more time swimming at the water surface during exposure to the fatiguing sound than during control periods (Table I). In two previous TTS studies, the same two harbor seals spent more time than normal at the water surface during high SPL exposures (pile driving sounds: Kastelein et al., 2018b; 6.5 kHz sinusoidal wave: Kastelein et al., 2019a). The seals in the present study did not spend more time with their heads at the surface at higher SELs, however. On the rare occasions during sound exposure sessions when the seals raised their heads fully above the water for one or a few seconds, their hearing may have recovered slightly, but the effect on the SEL they experienced would have been minimal.

Statistically significant TTS, as reported in the present study, is not necessarily the same as ecologically significant TTS, and it is unclear what the specific ecological effects of TTS are. These effects depend on the magnitude of the TTS, the duration of the exposure (because TTS begins to occur during sound exposure), the time needed for recovery of hearing, and the hearing frequency that is affected. As pinnipeds rely on hearing as one of their main sensory modalities (alongside vision and mechano-reception with their whiskers), reduced hearing in seals may impede ecologically important activities such as orientation, communication, foraging, and predator detection. Reduction in hearing may reduce their fitness, reproductive output, and longevity, which in turn may negatively impact population sizes.

The present study shows that, after exposure to a one-sixth-octave noise band centered at 16 kHz, harbor seal F02 suffered TTS_1-4 at 16 and 22.4 kHz, and harbor seal F01 suffered TTS_12-16 only at 22.4 kHz. If F01 had been tested 1–4 min after the sound stopped, she may have shown TTS also at 16 kHz, although this is speculative. When TTS_1-4 occurred in F02, the hearing frequency at which it was greatest depended on the SPL, and above 174 dB SEL the hearing frequency that was most affected was 22.4 kHz, half an octave above the center frequency of the fatiguing sound (16 kHz). TTS research in terrestrial mammals also shows that the maximum TTS is induced at half an octave above the fatiguing sound's center frequency (Cody and Johnstone, 1981; McFadden and Plattsmier, 1983; McFadden, 1986), and that the magnitude of TTS induced at frequencies above the fatiguing sound's center frequency is related to the SPL of the fatiguing sound. In guinea pigs (Cavia porcellus), the rate of increase in TTS is much higher for frequencies ≥0.5 octave above the fatiguing sound's frequency than for frequencies <0.5 octave above it (Cody and Johnstone, 1981).

The results of the present study and those of other studies with the same harbor seals suggest that similar TTSs caused by different fatiguing sounds require similar recovery times (Kastelein et al., 2012; Kastelein et al., 2018b; Kastelein et al., 2019a, using a one-octave continuous noise band, intermittent impulsive sounds, and a continuous sinusoidal wave, respectively).

Based on the published data on TTS caused by underwater sound in harbor seals (Kastak et al., 2005; Kastelein et al., 2012; Kastelein et al., 2019a; present study), their susceptibility to TTS in the fatiguing sound frequency range tested (2.5–16 kHz) varies little with hearing frequency (Fig. 6). Kastak et al. (2005) did not specify how soon after cessation of the fatiguing sound (octave-band noise centered at 2.5 kHz), or for how long exactly they tested the seal's hearing (at 2.5 kHz), so recovery before and during the post-exposure hearing tests cannot be evaluated precisely. Sessions lasted ca. 15 min, so hearing had more time to recover than in the PSE_1-4 hearing test in the present study, leading to a higher SEL being needed to cause 6 dB initial TTS (a marker of TTS onset used in marine mammal hearing tests; Houser et al., 2017) than in the present study. However, in the studies by Kastelein et al. (2012; 2019a) and in the present study (using the same seals), the timing of hearing tests was precisely specified and consistent. In the present study, the SEL required to elicit 6 dB TTS within 4 min after sound exposure stopped at 22.4 kHz was ∼181 dB re 1 μPa²s in seal F02 [Fig. 4(b)]. Six dB TTS was not reached at 22.4 kHz in seal F01 12–16 min after the sound stopped (or, if it was reached, hearing had recovered), but extrapolation of the TTS curve at 22.4 kHz [Fig. 5(b)] suggests that 6 dB TTS may have been demonstrable 12–16 min after the sound stopped if the SEL of the fatiguing sound had been higher (∼188 dB re 1 μPa²s).

The two harbor seals in the present study and the seal studied by Kastak et al. (2005) seem to have similar susceptibility to TTS in the frequency range within which their hearing was tested. The hearing thresholds of the seals for this frequency range are very similar as well, and this range represents the “flat” part of the audiogram (corresponding to the most sensitive part of the entire hearing range; Kastelein et al., 2009a; Kastelein et al., 2009b; Fig. 6). More fatiguing sound frequencies need to be tested in harbor seals in order to produce equal-TTS curves, on which weighting functions can be based that can be used to set safety criteria for seals for broadband sounds in the marine environment (Houser et al., 2017).

Harbor porpoises and harbor seals have overlapping geographic ranges and are therefore exposed to similar anthropogenic underwater sounds. In a study on TTS in a harbor porpoise, Kastelein et al. (2019b) used the same fatiguing sound (one-sixth-octave noise band centered at 16 kHz) and methodology as used in the present study, so these studies can be compared directly. At 22.4 kHz, the harbor porpoise showed TTS_1-4 onset (3.6 dB) at ∼165 dB SEL; harbor seal F02 experienced a similar 4 dB TTS_1-4 at ∼179 dB SEL. Thus, at least for fatiguing sounds centered at 16 kHz, harbor porpoises appear to be more susceptible to TTS than harbor seals, possibly because at 16 kHz, the tonal hearing threshold of the harbor porpoise is ∼7 dB lower than that of the harbor seal (Kastelein et al., 2009a; Kastelein et al., 2017). However, caution is needed, as the sample sizes this generalization is based on are very small. For a 6.5 kHz sinusoidal wave, the difference in susceptibility to TTS between the harbor seal (Kastelein et al., 2019a) and the harbor porpoise (Kastelein et al., 2014) was only 4 dB (the tonal hearing threshold difference at this frequency is also small; ∼ 4 dB). In a review of the marine mammal TTS and PTS literature, all of which is limited in terms of species and individuals tested, Finneran (2015) also concluded that odontocetes appear more susceptible to TTS than pinnipeds. Weighting functions based on equal-TTS curves are already in use for both species (NMFS, 2016) to set safety criteria for anthropogenic underwater noise in areas where harbor porpoises and harbor seals co-occur, but require refinement.

We thank research assistants Naomi Claeys, Kimberly Biemond, Rowanne Huisman, and Ruby van Kester; students Tessa Kreeft, Tom Beudel, Britt Spinhoven, and Jennifer Smit; and volunteers Stacey van der Linden, Simone de Winter, Susan Jansen, Thaana van Dessel, Jolien Hakvoort, and Brigitte Slingerland for their help in collecting the data. We thank Arie Smink for the design, construction, and maintenance of the electronic equipment. We thank Bert Meijering (Topsy Baits) for providing space for the SEAMARCO Research Institute. Erwin Jansen (TNO) conducted the acoustic calibration measurements. We also thank Nancy Jennings (Dotmoth.co.uk) for the statistical analysis and for her valuable constructive comments on this manuscript. Funding for this project was obtained from the U.S. Navy's Living Marine Resources program (LMR; Contract No. N39430-18-C-2041). We thank Mandy Shoemaker and Anu Kumar for their guidance on behalf of the LMR program. The harbor seals were made available for the research by Ecomare. The training and testing of the seals was conducted under authorization of the Netherlands Ministry of Economic Affairs, Department of Nature Management, with Endangered Species Permit no. FF/75A/2016/031.