Temporary hearing threshold shift in harbor seals (Phoca vitulina) due to a one-sixth-octave noise band centered at 32 kHz

Marine mammals that are exposed to loud sounds may suffer hearing loss that can be temporary (TTS; temporary threshold shift) or permanent (PTS; permanent threshold shift; Melnick, 1991; Yost, 2007). TTS and PTS in marine mammals may be induced by anthropogenic offshore activities that generate high levels of underwater sound, such as percussion pile driving, seismic surveys, sonar, and detonations. For the regulation and management of such activities, it is important to know at what sound exposure level [SEL; a combination of received sound pressure level (SPL) and exposure duration] hearing loss may occur in marine mammals.

Studies of TTS can be used to estimate PTS onset SEL (Southall et al., 2019) and give insight into the effects of less harmful exposures by providing information on TTS onset SEL, rates of TTS increase, and critical levels (levels above which the TTS increases dramatically). TTSs of various magnitudes and durations may compromise foraging, orientation, communication, and predator detection, and may have negative effects on health and survival. Ecological effects of reduced hearing depend not only on the magnitude of the threshold shift and its duration (which in turn depends on exposure duration and recovery time), but also on the hearing frequency range affected and the ecological importance of that range. In some odontocete (toothed whale) species, susceptibility to TTS has been shown to depend on the frequency of the fatiguing sound (the sound causing TTS; Houser et al., 2017). Frequency dependence of TTS may also occur in other marine mammals, such as phocids (true seals).

The harbor seal (Phoca vitulina) occurs in temperate and Arctic coastal areas of the northern hemisphere (Burns, 2002). A large part of its geographic range has high levels of anthropogenic activity, where anthropogenic underwater sounds may cause TTS or PTS if the SEL is high enough. Susceptibility to TTS in harbor seals due to underwater sounds in a limited frequency range (0.1–16 kHz) has been tested in six studies (Table I).

Combined with the studies by Kastelein et al. (2019a, 2019b), the present study is part of a larger project on the susceptibility of harbor seals to TTS over their entire hearing range. Where possible, octave frequencies were chosen as fatiguing sound frequencies. The goal of the present study was to expose harbor seals to a continuous one-sixth-octave noise band centered at 32 kHz and to quantify the hearing frequencies affected, the recovery of hearing after the exposure stopped, and TTS in relation to the frequency and SEL of the fatiguing sound. The ultimate goal of the larger project is to use information on TTS induced by exposure to sound at various frequencies, first to establish equal-TTS curves for harbor seals and then to develop a research-based weighting curve for harbor seals (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 (assuming that all phocids are equally susceptible to TTS). In marine mammals, criteria that will facilitate defining the numbers of individuals that can be affected by human activities are important for conservation and mitigation.

The study animals were two adult female harbor seals, identified as F01 and F02. During the study they were both 11 years old and the body mass of each fluctuated around 44 kg in the summer and 55 kg in the early winter. Their girths at axilla fluctuated around 83 cm in the summer and 93 cm in the winter. Both body mass and girth were measured weekly. The seals were fed sprat (Sprattus sprattus), herring (Clupea harengus), and mackerel (Scomber scombrus), and they received most of their daily fish ration during hearing test sessions. The seals were healthy throughout the study period and had participated in nine previous hearing studies, which had shown that they had sensitive and very similar hearing (Kastelein et al., 2009a, 2009b, 2010, 2012, 2015, 2018a, 2018b, 2019a, 2019b). Their hearing was not damaged by these studies; in fact their measured hearing threshold became slightly lower over time, probably due to longer (4–6 months) testing of the same frequency in TTS studies (allowing them to focus on a particular frequency), and their increasing experience with hearing tests. Variations in motivation to participate in hearing tests were minimized by making weekly adjustments (usually in the order of 100 g) to their daily food ration, based on their weight and motivation during the previous week and the expected change in water and air temperatures in the following week.

The study was conducted at the SEAMARCO Research Institute (in a remote and quiet location in the Netherlands) in an outdoor pool [8 × 7 m and 2 m deep; see Kastelein et al. (2019b) for details] with haul-out areas. During the sound exposure sessions, these haul-out areas were barred with boards or plastic boxes, so that the seals could not leave the water.

Acoustical terminology for sound pressure level (SPL) and sound exposure level (SEL) follows ISO 18405:2017 (2017). The ambient noise was measured, and the fatiguing sound and hearing test signals were calibrated, once every 2 months during the study period by an acoustic consulting agency (TNO, the Hague, Netherlands; see Kastelein et al., 2019a, 2019b). Great care was taken to make the harbor seals' listening environment as quiet as possible (see Kastelein et al., 2019a, 2019b). Under test conditions (i.e., only researchers involved in the study within 15 m of the pool, water circulation system off, no rain, and wind force Beaufort 4 or below), the ambient noise in the pool was very low and fairly constant in amplitude (Fig. 1).

A continuous (100% duty cycle for 60 min), one-sixth-octave noise band centered at 32 kHz, without harmonics, was selected as the fatiguing sound. The sound was played by an omnidirectional transducer (ITC, model 6084, International Transducer Corporation, Santa Barbara, California, USA) in the center of the pool at 1 m depth (see Kastelein et al., 2019a, 2019b). Before each sound exposure test, the voltage output of the emitting system to the transducer and the voltage output of the sound-receiving system were checked (see Kastelein et al., 2019a, 2019b).

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 × 1.3 m, at three depths per location on the grid (0.5, 1.0, and 1.5 m below the surface). Only small differences existed in SPL per depth and per location, and no gradient existed in the SPL in relation to the distance to the transducer, resulting in a fairly homogeneous sound field (Fig. 2). The fatiguing sound was projected at various source levels, resulting in average received SPLs ranging from 92 to 152 dB re 1 μPa. For 60-min exposures, the resulting SELs were between 128 and 188 dB re 1 μPa²s. To avoid potential startle responses, the SPL of the fatiguing sound started 20 dB below the desired SPL and was increased in 2 dB steps over a period of 2 min to the SPL required for that session.

During one session, one of the open plastic boxes (60 × 40 × 40 cm) that were used to prevent the seals from hauling out was blown into the water by the wind and filled with water. It floated against the transducer, where seal F02 played with it, mainly from below the water surface, so that she remained ≤1 m from the transducer for most of the session. Thus, during that one session, she inadvertently experienced an SPL of around 155 dB re 1 μPa, resulting in an estimated SEL of ∼191 dB re 1 μPa²s, instead of the mean SEL of 185 dB re 1 μPa²s that she experienced in sessions with the same source level while swimming throughout the pool.

The harbor seals were trained to detect signals presented during hearing tests before and after exposure to the fatiguing sound. The hearing test signals were generated digitally (Adobe Audition, version 3.0, Adobe: Sunrise, Florida, USA). The hearing thresholds were tested at the center frequency of the fatiguing sound (32 kHz), half an octave above the frequency of the fatiguing sound (45 kHz), and one octave above the center frequency of the fatiguing sound (63 kHz).

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, as described by Kastelein et al. (2019b), but using a different transducer (EDO, model 337, EDO Corporation Salt Lake City, Utah). Two hydrophones, at the two locations of the seals' ears when they would be positioned at the listening station, were used to measure the SPL received by the seals during the hearing tests. The SPL at the two hydrophones differed by 0 to 2 dB, depending on the test frequency. The mean SPL of the two hydrophones was used to calculate the stimulus level during hearing threshold tests.

The audiometric method used is described by Kastelein et al. (2019a, 2019b). Each hearing test session consisted of ∼25 trials and lasted for up to 12 min per seal. Both the first and second session after the fatiguing sound stopped were divided into three 4-min periods: 1–4, 4–8, and 8–12 min, and 12–16, 16–20, and 20–24 min, respectively. During the hearing tests, the seal not being tested was kept in the water next to the main haul-out area, while a trainer used hand signals to ask her to perform quiet husbandry behaviors to keep her occupied. Sessions consisted of two-thirds signal-present and one-third signal-absent trials, offered in quasi-random order (see Kastelein et al., 2019a, 2019b).

One total sound exposure test, consisting of (1) pre-exposure hearing test session, (2) fatiguing sound exposure, and (3) post-sound exposure (PSE) hearing test session(s), was conducted per day. The PSE hearing test (using the same test signals as in the pre-exposure hearing test) commenced within 1 min after the fatiguing sound had stopped for seal F02 and 12 min after the sound had stopped for seal F01. The seals were always tested in the same order to ensure a quick and efficient hearing test start after sound exposure stopped.

Both the magnitude of TTS after exposure and the subsequent hearing recovery was recorded. Recovery was defined as a return of hearing threshold to within 2 dB of the pre-exposure hearing threshold, based on the fluctuations in control sessions of the present study and previous TTS studies with these harbor seals. The hearing sensitivity of both seals was tested during several PSE periods: 1–4 (PSE_1–4), 4–8 (PSE_4–8), 8–12 (PSE_8–12), 60 (PSE₆₀), 120 (PSE₁₂₀), 240 (PSE₂₄₀) min, and 24 (PSE_24h), 48 (PSE_48h), 72 (PSE_72h), and 96 (PSE_96h) h after the fatiguing sound exposure ended for seal F02, and 12–16 (PSE_12–16), 16–20 (PSE_16–20), 20–24 (PSE_20–24), 72 (PSE₇₂), 132 (PSE₁₃₂), 252 (PSE₂₅₂) min, and 24 (PSE_24h), 48 (PSE_48h), and 72 (PSE_72h) h after the fatiguing sound exposure ended for seal F01. For both seals, hearing was always tested during the first three PSE periods; hearing was only tested in the following PSE periods if it was not shown to have recovered in the previous PSE period.

Sample sizes were chosen to maximize the study time available for testing SELs which seemed to cause TTS, while minimizing the risk of hearing damage from repeated exposure to the highest SELs, and avoiding repeated testing of SELs for which TTS obviously did not occur. In order to protect their hearing, the seals were exposed to fatiguing sound only once per day for 60 min up to 7 days per week. Consequently, randomizing the order in which the seals were tested while maintaining the sample sizes would have doubled the study period. As a precaution, sound exposure tests were not conducted on days following exposure to the highest SELs.

Control tests were conducted in the same way as sound exposure tests, but without fatiguing sound exposure, under low ambient noise conditions (Fig. 1). The post-ambient exposure (PAE) hearing test sessions were divided into three 4-min periods; 1–4 (PAE_1–4), 4–8 (PAE_4–8), and 8–12 (PAE_8–12) min after ambient noise exposure for seal F02, and 12–16 (PAE_12–16), 16–20 (PAE_16–20), and 20–24 (PAE_20–24) min after ambient noise exposure for seal F01. Control tests were randomly dispersed among the fatiguing sound exposure tests during the study period. Data were collected between July and December 2017.

The mean incidence of pre-stimulus responses (“pre-stimuli”) by the seals for both signal-present and signal-absent 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.

A video camera on a pole above the pool recorded the behavior of the seals during the sessions. To determine the SEL the seals were exposed to, their locations in the pool and the time they spent (1) completely submerged, (2) at the water surface with their nose in the air and mandible and lower part of the skull under water, or (3) at the water surface with their head completely out of the water was determined. By lifting their heads out of the water, they may have been able to reduce their exposure to the fatiguing sounds. Via a grid on the monitor screen, the pool was divided into 36 approximately 1.2 × 1.3 m rectangles (two of which coincided with the floating platform). The approximate overall percentage of time that the seals spent completely submerged, with their heads at the water surface, and with their heads completely out of the water was estimated from the video recordings of a subset of the total number of sessions by using stopwatches. For each average received SPL, one to twelve 60-min fatiguing sound exposures were randomly selected for this analysis, depending on the varying number of times each average received SPL was tested. Seal F02 was marked with a patch of zinc ointment on her head, so that she could be distinguished from seal F01 on the video recordings.

In the hearing tests, a staircase method using 2 dB steps was used (see Kastelein et al., 2019a, 2019b). A switch from a test signal level that the harbor seal responded to (a hit), to a level that she did not respond to (a miss), and vice versa, was called a reversal. The pre-exposure mean 50% hearing threshold (PE_50%) for each hearing test session was determined by calculating the mean SPL of all reversals in the pre-exposure hearing session. Only pre-exposure sessions with at least ten reversals were included in the analysis. The TTS quantified in seal F02 during PSE_1–4 (TTS_1–4) for each hearing test frequency was calculated by subtracting the PE_50% from the mean 50% hearing threshold during PSE_1–4. The same method was used to calculate TTS_12–16 for seal F01.

We define the onset of TTS as occurring at the lowest SEL at which a 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 the sample size was ≤3, we compared the hearing threshold to the control level without statistical analysis in order to judge whether TTS occurred or not—if the hearing shift was ≥2 dB, it was considered to be TTS (this was decided based on variation in the control sessions of the present study and data from several previous TTS studies with these seals using similar methods). For larger sample sizes, statistical significance was established by conducting a one-way analysis of variance (ANOVA) on the TTS, for each seal and for each hearing test frequency separately, with the factor SEL (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 analyses were conducted in Minitab 18 (Coventry, United Kindom), and data conformed to the assumptions of the tests used (Zar, 1999).

During the 60-min control sessions, the seals spent ∼17% of their time with their heads at the water surface and ∼83% of their time with their heads completely submerged. During 60-min exposures to the continuous one-sixth-octave noise band centered at 32 kHz, the seals spent 15%–39% of their time with their heads at the water surface (Table II). There was no correlation between SEL and the percentage of time spent at the water surface (Spearman's rho [ρ] = 0.485, P = 0.157 for seal F02; ρ = 0.437, P = 0.207 for seal F01). Both seals only raised their head completely out of the water a few times for up to 2 s in an attempt to look over the edge of the pool; therefore, it can be assumed that they were fully exposed to the fatiguing sound during the exposure periods. During control and exposure sessions, the seals swam throughout the pool, at all depths, so their average received SPL was calculated as the energetic average of the SPL at all 105 individual measurement locations (Fig. 2).

After the 60-min sound exposure periods, the seals were always willing to participate in the hearing tests. The pre-stimulus response rates for all trials in the pre-exposure, post-exposure, and control hearing tests were of the same order of magnitude (Table III).

The ANOVAs showed that both TTS_1–4 (seal F02) and TTS_12–16 (seal F01) were significantly affected by the fatiguing sound's SEL at hearing frequency 45 kHz. TTS_1–4 (seal F02) was also significantly affected by the fatiguing sound's SEL at hearing frequency 32 kHz. Comparisons with the control revealed that the statistically significant onset of TTS varied depending on the seal and the hearing test frequency (Table IV).

For a hearing test signal of 32 kHz, TTS_1–4 occurred in seal F02 after exposure to fatiguing sounds with SELs of ≥134 dB re 1 μPa²s [Table IV, Fig. 3(a)], but the TTS_1–4 increased very little when the SELs increased from 134 to 185 dB re 1 μPa²s; in all cases, hearing recovered within 60 min [Fig. 4(a)]. For a hearing test signal of 45 kHz, TTS_1–4 occurred with SELs of ≥176 dB re 1 μPa²s [Table IV, Fig. 3(a)]. The rate of increase in TTS with increasing SEL for this hearing frequency was ∼0.9 dB/dB fatiguing sound between 170 and 182 dB SEL, and ∼3.7 dB/dB between 182 and 185 dB SEL (Table IV). Thus, the critical level for 45 kHz was between 182 and 185 dB SEL. Recovery of hearing occurred within 60 min for exposures up to an SEL of 182 dB re 1 μPa²s, within 72 h for an SEL of 185 dB re 1 μPa²s, and within 96 h for SELs of 188 and 191 dB re 1 μPa²s [Fig. 4(b)]. For a hearing test signal of 63 kHz, no TTS_1–4 occurred even after exposure to an SEL of 185 dB re 1 μPa²s [Fig. 4(c)]. No change in susceptibility to TTS was observed over the duration of the study. The control sessions showed that the hearing thresholds for all three hearing test signals before and after 60 min exposure to low ambient noise were very similar (Table IV, Fig. 4).

During the one session with the plastic box in the water, seal F02 experienced a mean SEL ∼191 dB re 1 μPa²s because she swam within 1 m of the fatiguing sound transducer, instead of the mean SEL of 185 dB re 1 μPa²s that she experienced under normal conditions when she swam throughout the pool. Seal F02 spent 27% of her time at the water surface during this session, which was typical. Her TTS_1–4 and TTS₆₀ at 45 kHz were >45 dB; exact thresholds for PSE_1–4 to PSE₆₀ could not be measured, as they were above the maximum level at which the hearing test signals could be produced [Fig. 5(a)]. Her TTS₁₂₀ at 45 kHz was 45 dB and her hearing was tested at all three hearing test frequencies until hearing at each frequency recovered. Four hours after the sound stopped, she had 45 dB TTS at 45 kHz, 7 dB TTS at 32 kHz, and 4 dB TTS at 63 kHz. Forty-eight hours after the sound stopped, her hearing had almost completely recovered at 32 and 63 kHz, but it took 4 days for her hearing at 45 kHz to recover completely. Her recovery pattern differed from the recovery of TTS after exposures to 185 and 188 dB SEL [Fig. 5(b)].

For a hearing test signal of 32 kHz, no TTS_12–16 occurred in seal F01, even after exposure to a fatiguing sound with an SEL of 185 dB re 1 μPa²s [Table IV, Figs. 3(b) and 6(a)]. For a hearing test signal of 45 kHz, TTS_12–16 occurred after exposure to fatiguing sounds with SELs of ≥176 dB re 1 μPa²s [Table IV, Fig. 3(b)]. After exposure to ≤182 dB SEL, recovery of hearing occurred within 132 mins [Fig. 6(b)]. After exposure to >185 dB SEL, recovery of hearing occurred within 24 h, and after exposure to 188 dB SEL, recovery occurred within 72 h [Fig. 6(b)]. For a hearing test signal of 63 kHz, no TTS_12–16 occurred, even after exposure to a SEL of 188 dB re 1 μPa²s [Fig. 6(c)]. No change in susceptibility to TTS was observed over the duration of the study. The control sessions showed that the hearing thresholds for both hearing test signals before and after 60-min exposure to low ambient noise were very similar (Fig. 6, Table IV).

The seals in the present study had normal hearing for adult female harbor seals (Kastelein et al., 2018a). The pre-exposure hearing thresholds found in the present study for the 32 and 63 kHz hearing test signals were within 2 dB of the hearing thresholds measured in these harbor seals for tonal signals approximately 10 years before the present study (Kastelein et al., 2009a, 2010).

Although the hearing of seal F02 was always tested before the hearing of seal F01, their TTSs can be compared. The closest possible comparison is of TTS_8–12 in seal F02 and TTS_12–16 in seal F01. Comparing the recovery of both seals for hearing test signals of 45 kHz after exposure to the highest SEL [Figs. 4(b), 6(b), and Table IV] shows that TTS_12–16 in seal F01 was ∼12 dB lower than TTS_8–12 in seal F02. Seal F02 was also more susceptible (∼6 dB) to fatiguing sound around 16 kHz (Kastelein et al., 2019a). Studies on humans and other terrestrial mammals show large 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). Therefore, further replication with more harbor seals is needed to assess the generality of the results.

The mean pre-stimulus response rates in hearing tests before and after the sound exposures and during the control sessions were similar in both seals (Table III). This suggests that the seals' decision-making process in the post-exposure hearing tests was not influenced by the sound exposure.

No change in the seals' susceptibility to TTS was observed over time during the present study. The TTSs were less severe than those elicited in studies on terrestrial mammals, in which susceptibility to TTS did change due to conditioning of the ear after repetitive sound exposures (Kujawa and Liberman, 1997; Mannström et al., 2015).

No aerial sound was projected during exposure sessions, based on results from a noise band study in which a similar TTS_1–4 was found in harbor seals exposed to the same underwater fatiguing sound with and without aerial fatiguing sound (Kastelein et al., 2012). Harbor seal hearing thresholds for underwater sounds are similar when they are swimming with their heads at the water surface (nose in the air, mandible and lower part of skull under water) and when they are completely submerged (Kastelein et al., 2018a). The seals in the present study spent approximately the same percentage of their time swimming at the water surface during exposure to the fatiguing sound and during control periods (Table II). In two previous TTS studies, these seals increased the percentage of time they spent at the water surface during exposure to high SPLs (pile driving sounds: Kastelein et al., 2018b; 6.5 kHz continuous sinusoidal wave: Kastelein et al., 2019b). However, in another part of the project on the susceptibility of harbor seals to TTS over their entire hearing range, in which they were exposed to a one-sixth-octave noise band centered at around 16 kHz, the seals spent a similar percentage of their time at the water surface in control and exposure periods, possibly because the received levels for the noise band at 16 kHz were lower than those in the two previous TTS studies (Kastelein et al., 2019a), or because the signal types were different (impulsive sound and continuous wave compared to noise bands).

In one session, seal F02 was inadvertently exposed to an SPL of around 155 dB re 1 μPa while playing with the plastic box. Maybe she accepted the sound as it was a continuous band noise (rather than an intermittent tonal or impulsive sound); probably her gradually acquired TTS reduced her sensation level over time during exposure. Playing with the box was unexpected, as in general, the seals at SEAMARCO do not play with toys, although toys have been provided. The two study animals have similar audiograms (Kastelein et al., 2009a, 2009b), but seal F02 often approaches sound sources, whereas seal F01 avoids them. This shows that the effect of a sound is not simply due to its sensation level (the number of dB above the basic hearing threshold), but depends on how an individual animal perceives the sound.

Seals with very high TTS, such as occurred in the present study, may not show abnormal behavior during recovery. The study animals continued to perform trained husbandry behaviors and participated well in the post-exposure hearing tests. Kastelein et al. (2012) also observed no behavioral changes in these seals during exposure to an octave noise band centered at 4 kHz, although they did acquire TTS.

In harbor seals, similar TTSs caused by different fatiguing sounds require similar recovery times, as evidenced by the present study and previous TTS studies with harbor seals (in which the fatiguing sounds were: a continuous one-octave noise band centered at 4 kHz, an intermittent impulsive sound, a continuous sinusoidal wave of 6.5 kHz, and a continuous one-sixth-octave noise band centered at 16 kHz; Kastelein et al., 2012, 2018b, 2019a, 2019b).

The results of the present study show that, after exposure to a one-sixth-octave noise band centered at 32 kHz, the harbor seals had TTSs at 32 and/or 45 kHz, and the hearing frequency at which the greatest TTS occurred depended on the SEL (for <176 dB SEL, the highest TTS occurred at 32 kHz; for >176 dB SEL, the highest TTS occurred at 45 kHz). The hearing frequency that was most affected at higher SELs was 45 kHz, half an octave higher than the center frequency of the fatiguing sound (32 kHz). TTS research in terrestrial mammals also suggests that the maximum TTS is induced half an octave above the fatiguing sound's frequency, and that the magnitude of TTS induced at frequencies higher than the fatiguing sound's center frequency is related to the SPL of the fatiguing sound (Cody and Johnstone, 1981; McFadden and Plattsmier, 1983; McFadden, 1986). Cody and Johnstone (1981) found that in guinea pigs (Cavia porcellus), the TTS increased more in response to hearing frequencies ≥0.5 octave above than for frequencies <0.5 octave above the frequency of the fatiguing sound.

In marine mammals, the relationship between SEL and affected hearing frequency has so far only been studied in one odontocete species, the harbor porpoise (Phocoena phocoena; Kastelein et al., 2014) and one pinniped species, the harbor seal (Kastelein et al., 2019a, 2019b). For both species, the affected hearing frequency increased with increasing SEL within the SEL range that was tested. Therefore, the SEL received by marine mammals should be incorporated when determining the affected hearing frequency, in order to evaluate the potential effect and thus the ecological impact of anthropogenic sounds on marine mammal hearing.

An SEL that causes a TTS of 40 dB is considered to be the PTS onset SEL (Southall et al., 2019), but after a TTS_1–4 of >45 dB, the hearing of seal F02 recovered within 4 days. In a previous study, after unintentional exposure to an octave band noise centered at 4 kHz for 60 min at a mean received SPL of 163 dB re 1 μPa (SEL: 199 dB re 1 μPa²s), seal F02 also recovered within 4 days from a TTS_12–16 of 44 dB (Kastelein et al., 2013). This suggests that a hearing shift of 40 dB is not as close to a PTS as has been suggested by Southall et al. (2019). However, recovery may vary over the untested hearing range of the harbor seal, and severe hearing damage may occur if harbor seals are exposed to fatiguing sounds on multiple occasions over short time periods, as reported by Reichmuth et al. (2019). In their study, a harbor seal was exposed twice to a 4.1 kHz continuous wave for 60 s at an SPL of 181 dB re 1 μPa SPL (SEL: 199 dB re 1 μPa²s). After the second exposure, a large threshold shift (>47 dB) was observed. While hearing at 4.1 kHz recovered within 48 h, there was a PTS of at least 8 dB at 5.8 kHz. This hearing loss was evident for more than 10 years. Furthermore, a threshold shift of 11 dB was detected at 8.2 kHz, one octave above the tonal exposure, which persisted for more than 2 years prior to full recovery (Reichmuth et al., 2019). Thus, until more information is available, it is safest to continue to assume that 40 dB TTS is close to a PTS threshold, as proposed by Southall et al. (2019).

The SEL at which TTS onset occurs is an indication of the susceptibility of an animal to TTS, but so is the rate at which TTS increases as SEL increases. In the present study, TTS at 45 kHz increased rapidly as SEL increased [Fig. 3], showing that small increases in SEL above the TTS onset SEL can be very dangerous to seal hearing.

Finneran (2015) and Southall et al. (2019) define TTS onset as a threshold shift of 6 dB. Based on the very few data available, the TTS onset for phocids in water was defined by Southall et al. (2019) as 181 dB SEL (weighted) at the most sensitive frequency. As more TTS data become available for fatiguing sounds of different frequencies, the safety threshold defined by Southall et al. (2019) can be adjusted to improve fit. In both seals in the present study, TTS onset (6 dB) occurred after exposure to a 32 kHz sound between 176 and 181 dB (unweighted), which is lower than the 187 dB SEL at 32 kHz predicted by Southall et al. (2019). Therefore, TTS onset for harbor seals seems to be 6–11 dB below the presently used weighted value.

The available data for harbor seals suggest that their susceptibility to TTS in the underwater fatiguing sound frequency range that has been tested (2.5–32 kHz) varies little with hearing frequency (Kastak et al., 2005; Kastelein et al., 2012, 2018b, 2019a, 2019b, and the present study; Table I, Fig. 7). Kastak et al. (2005) used octave-band noise centered at 2.5 kHz as the fatiguing sound, but did not specify precisely how soon after the sound stopped or for how long they tested the hearing (at 2.5 kHz) of the seal (although they mention that the seal's hearing test was completed 15 min after the fatiguing sound stopped), so recovery before and during the post-exposure hearing tests cannot be evaluated. In the studies by Kastelein et al. (2012, 2019a, 2019b) and in the present study (using the same seals), the timing of hearing tests was consistent and precisely specified. In the present study, the SEL required to elicit 6 dB TTS_1–4 (a marker of TTS onset used by Finneran, 2015) at 45 kHz was ∼177 dB re 1 μPa²s in seal F02 [Fig. 4(b)]; TTS_12–16 reached 6 dB in seal F01 after exposure to ∼181 dB re 1 μPa²s [Fig. 6(b)]. The two harbor seals in the present study have similar (±6 dB) susceptibility to TTS in the fatiguing sound frequency range of 4 to 32 kHz. The seal studied by Kastak et al. (2005) also appears to have similar susceptibility to TTS just below this frequency range. The frequency range in which TTS has been tested in harbor seals corresponds to the flat part of the audiogram and the range of most sensitive hearing; in this range, the hearing thresholds of the seals are very similar (Kastelein et al., 2009a, 2009b; Fig. 7).

More fatiguing sound frequencies need to be tested in harbor seals in order to produce equal TTS curves, on which weighting functions for phocids can be based. These in turn can be used to set safety criteria for broadband sounds emitted in the marine environment (Houser et al., 2017). Evidence from the present study and from the others making up the project on the susceptibility of harbor seals to TTS over their entire hearing range (Kastelein et al., 2019a, 2019b) suggests that, when attempting to evaluate the impacts of anthropogenic sounds on marine mammal hearing, it is important to take into account the frequency of the sound, the type of sound, the SPL received by the marine mammals, and the duration of exposure to the fatiguing sound, since all four parameters are likely to affect the outcome of exposure. Ecological effects of sounds depend on the magnitude of the TTS, the time needed for hearing to recover, and the hearing frequency that is affected. Damaged hearing in seals may reduce the efficiency of ecologically important activities such as orientation, communication, foraging, and predator detection, thus potentially reducing seals' fitness. In turn, these individual-level effects might negatively impact entire populations.

We thank research assistants Kimberly Biemond, Rowanne Huisman, and Ruby van Kester; students Tessa Kreeft, Daniek Kuipers, Tom Beudel, Britt Spinhoven, and Jennifer Smit; and volunteers Stacey van der Linden, Susan Jansen, Lotte Dalmeijer, Thaana van Dessel, 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 her valuable constructive comments on this manuscript and for the statistical analysis, and Christ de Jong (TNO) for his comments on part of the discussion. Funding for this project was obtained from the U.S. Navy's Living Marine Resources program (Contract No. N39430-18-C-2041). We thank Mandy Shoemaker and Anu Kumar for their guidance on behalf of the LMR program. The seals were made available for the research by Ecomare. The training and testing of the harbor 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.