Narrowband noise induces frequency-specific underwater temporary threshold shifts in freshwater turtles

Freshwater turtles can experience a temporary impairment of hearing sensitivity when exposed to intense underwater noise.¹ This recent finding added reptiles to the list of aquatic taxa, which includes fishes, cetaceans, and pinnipeds,^2,3 that are susceptible to noise-induced hearing loss. These results also suggest the hearing of aquatic turtles across diverse freshwater, brackish, and marine habitats may be affected by the rapidly increasing anthropogenic noise within underwater soundscapes.^4,5 Understanding the potential impacts of this globally recognized pollutant and environmental stressor is required to effectively manage anthropogenic noise and noise-exposed species. While temporary threshold shifts (TTS) had been predicted to occur in aquatic turtles,^2,3 these effects were estimated to begin at higher sound exposure levels (SELs) than what was observed when these predictions were tested.¹ These initial TTS results indicate that additional research is needed to understand the response of the turtle ear to the varied types of noise pollution these animals may encounter.

Even a temporary decline in hearing sensitivity may have consequences for a turtle's survival and fitness. Acoustic communication is common and widespread across the Testudine phylogenetic tree.⁶ This includes underwater vocalizations in both freshwater and marine species,^6–8 which are used for key functions, including courtship and parental care.^7,9 The large air-filled middle ear of Testudines is considered to have evolved to enhance underwater sound detection, enabling sound to be used for underwater communication and to provide information in often turbid and low-lit waters.^7,10–12 These functions of underwater sound and audition may be compromised by anthropogenic noise, which is varied in both frequency and temporal characteristics.^2,4

Understanding turtles' auditory responses to different types of noise pollution would enhance our ability to predict if and how a turtle's hearing may be affected by a given noise source. In the first studies on hearing loss in turtles, Salas et al.^1,13 explored TTS in red-eared sliders (Trachemys scripta elegans) and Eastern painted turtles (Chrysmys picta picta)—two species with acoustic communication^6,14—in response to continuous broadband (50–1000 Hz) white noise as a means to initially test if TTS can be induced in turtles. This broadband sound may also approximate noise in these species' natural environments produced by small boats and vessel traffic. Here, we tested if TTS occurs in these same turtles upon exposure to $16$-octave narrowband noise centered at 400 Hz; this frequency band both is within the range of their highest hearing sensitivity^1,11 and represents a noise source analogous to low-frequency sonar signals. Our first goal was to test if freshwater turtles are susceptible to TTS upon exposure to narrowband noise. Second, we considered if this TTS occurred predominantly at the noise center frequency (400 Hz) or $12$ octave above this frequency (570 Hz), as observed for mammals.^15,16 Taken together, these results provide key data to support criteria for evaluating and managing the impacts of sound exposures on aquatic turtles. The similarity of the turtle ear across Testudines^11,12 allows both tested species to represent freshwater turtles more broadly.

Full methodological details on collection of auditory evoked potentials (AEPs) and experimental protocols are provided in Salas et al.;^1,13 here, we give an abbreviated description. We tested three T. s. elegans and three C. p. picta, all adult females. These six animals also participated in prior underwater TTS studies using broadband continuous noise.^1,13 All thresholds returned to baseline hearing sensitivity at the conclusion of those studies. There is the potential that repeated exposures from the first broadband study affected TTS susceptibility in the current study in a way not captured in our experimental design.

The experimental procedure consisted of sedation, followed by a 5- or 20-min noise exposure (or ambient sound control) and subsequent AEP measurements to assess auditory thresholds following the noise exposure or control. A combination of mild mechanical restraint and sedation kept the turtles in one location for the hearing tests;^17,18 sedation was intramuscular midazolam (5 mg/ml) and dexmedetomidine (0.5 mg/ml). One turtle (RES05) required the addition of ketamine (100 mg/ml) to achieve the minimum required level of sedation. Doses were adjusted as needed to achieve sufficient relaxation while not compromising AEP measurements or airway control. First effects of sedation were typically observed approximately 5 min post injection, and the desired sedation level was reached after approximately 40 min, at which time we began AEP measurements. Thus, 5-min exposures (or controls) began 35 min post injection and 20-min exposures or controls began 20 min post injection.

Exposure and control sessions took place in a fiberglass circular tank (0.6-m depth, 1-m diameter) with the speaker (UW-30; Lubell Labs, Inc., Columbus, OH) facing upwards on the tank bottom placed on rubber padding. Turtles rested on a custom plastic mesh platform underwater 0.2 m above the speaker (see Salas et al.¹ for diagram) and were maintained in a defined location to ensure consistent received levels. The animals were raised when they showed signs of needing to surface (e.g., raising the head) and/or every 5 min to breathe during the 20-min durations. The animals were raised so that only the nares broke the surface, and surfacing time was sufficiently short (<5 s), relative to exposure durations, for the noise exposure to be considered continuous.

Exposure and control sessions were identical in timing and procedure except for the presence of the speaker broadcasting $16$-octave narrowband white noise (center frequency = 400 Hz) (Fig. 1) in the exposure sessions. The 570-Hz auditory test frequency was outside this noise band, and fatiguing sound received levels at this frequency were substantially diminished (by ≈ 40 dB) (Fig. 1). Towards our goal of exploring TTS responses to two SELs, we tested two noise treatments: a 5-min duration at a target root mean square sound pressure level (RMS SPL) of 131 dB re 1 μPa and a 20-min duration at a target RMS SPL of 141 dB. Immediately before placing each turtle in the exposure tank, we collected recordings of the noise at the position of the head to calculate the RMS SPL and SEL of the fatiguing noise for that turtle and trial. Recordings were made using an HTI-96-MIN hydrophone (sensitivity, –165.2 dB re 1 V/uPa) (High-Tech, Inc., Long Beach, MS) connected to an autonomous recorder (Ocean Instruments SoundTrap 4300, –4 dB gain) (Ocean Instruments, Auckland, New Zealand). SPLs were within ±1 dB of target RMS SPLs; RMS SPL and SEL calculations using these recordings are described in Salas et al.¹ The exposure conditions provided SELs of approximately 155 dB or 172 dB re 1 μPa² s. The four noise exposures (2 SELs × 2 auditory test frequencies) per turtle took place from October 2022 to February 2023 for C. p. picta as part of a larger narrowband noise TTS study. The T. s. elegans testing took place from June to July 2023. Here, we present the exposure data that allow direct comparison between the two species. We worked with all three individuals of one species each test day, and noise exposure sessions generally took place on Mondays, followed by control sessions two days later. We tested TTS in response to lower SELs before moving to higher SELs and to both 400 and 570 Hz under the same exposure conditions in approximately consecutive weeks.

Recordings of AEPs were used to measure auditory thresholds. Following removal from the exposure tank, turtles were gently wrapped with an elastic bandage, securing them to a plastic platform. This platform was raised and lowered on a custom frame to allow the turtles to breathe during the AEPs, which took place in a rectangular PVC tank (0.84 m long × 0.38 m high × 0.53 m wide) (for a diagram, see Salas et al.¹). Turtle sedation and condition were monitored by a veterinarian during AEP sessions, and turtles were raised approximately every 2 min to breathe, or when they showed signs of needing to surface.¹⁷ The time elapsed following the end of the noise exposure session to the beginning of AEP collection ranged from 2.5 to 6 min [mean = 3.2; standard deviation (s.d.) = 0.52]. A recording 27-gauge, 6-mm stainless steel subdermal electrode (Rochester Electromedical Inc., Coral Springs, FL) was inserted 2–3 mm dorsal to the dorsal margin of the right tympanum and <1 mm under the epidermal skin layer in a rostral-to-caudal direction. The reference electrode was inserted in the subcutaneous tissue left or right of the tail, and the ground electrode hung in the water. The water temperature for both AEP and exposure tanks ranged from 24 °C to 26 °C, similar to the daytime temperature of the turtles' home tanks. After a noise exposure, we collected five consecutive auditory thresholds, taking approximately 40 to 60 min, to monitor for the occurrence of TTS and track recovery. Following the highest observed shift (RES01), seven thresholds were collected. Development of sedation drug resistance in RES05 led to the collection of only one post-exposure threshold following the 172 dB SEL exposure trial for 400 Hz and no final 400-Hz control session following that exposure.

Hearing test tones and evoked responses were generated and measured, respectively, via custom Labview software (National Instruments, Austin, TX). Tones were 30 ms duration and either 400 Hz (noise center frequency) or 570 Hz (⁠ $12$ octave above center frequency) at a rate of 1/10.1 s (alternating polarities) from a UW-30 speaker 0.45 m from the turtle's head.¹ For the auditory response waveform, we used the average of 250–500 responses; more responses were collected, as necessary, to achieve a stabilized average response frequency spectrum. Tone pips were initially presented at starting amplitudes of 107–114 dB re 1 μPa SPL. This stimulus was decreased in 5 dB steps until no response could be seen in the averaged frequency spectrum, after which two additional 5 dB steps and measurements were taken. Refer to Salas et al.¹ for test tone calibration methods.

The auditory responses occurred at twice the test frequency.^1,18 We used the regression method to estimate hearing thresholds^19,20 (following Salas et al.^1,21) Briefly, we first calculated fast-Fourier transforms of the AEP response waveforms in matlab (vR2019a; MathWorks, Natick, MA). The amplitude (in μV) of the frequency representing the maximum response peak (approximately twice the test tone) was plotted relative to the SPLs (in dB re 1 μPa) that generated those responses; a regression was then fitted to those points. Suprathreshold responses or points comparable to the ambient electrophysiological background noise (<0.02 μV; beyond the one required by the analysis) were excluded. In general, the r² of these regression lines was >0.9. Auditory thresholds were defined as the SPL at which the regression crossed 0 μV, indicating the theoretical no response.¹⁹ 

We defined TTS to have occurred following a noise exposure if at least one of the five post-exposure thresholds was >6 dB above the control threshold.^22,23 We also tested that these post-exposure thresholds were significantly elevated above the control means, relative to control thresholds, using a Wilcoxon rank sum test (p < 0.05; matlab). The shift for a given noise exposure was calculated as the maximum post-exposure threshold minus the mean control threshold for each test frequency and individual. The mean T. s. elegans 400-Hz control thresholds were calculated as the mean of the thresholds collected in this narrowband study combined with those collected for the same individuals in Salas et al.¹ The control 570-Hz thresholds for T. s. elegans and the control 400- and 570-Hz thresholds for C. p. picta were calculated as the mean of all thresholds collected for this narrowband study. EPT04 did not participate in the 20-min noise exposure trials for either 400 or 570 Hz. For sample sizes, refer to Table 1. Refer to the supplementary material for additional details about EPT04 and combining control samples.

We defined recovery to have occurred within a noise exposure session when a threshold and all subsequent thresholds were within 6 dB of the control threshold.¹ Time to recovery was calculated as the time elapsed between removal from the noise exposure and the temporal middle of the collection of the first recovered threshold. To consider the relationship between noise received level and shift magnitude at 400 and 570 Hz, we extracted the pressure spectral density at 398 and 574 Hz from the noise frequency spectra from each noise exposure for each turtle.

All turtles exhibited TTS as a result of narrowband sound exposure (Table 1; Fig. 2). TTS ranged from 10.7 to >38.3 dB and were dependent on exposure SEL and auditory test frequency (Fig. 2). At 400 Hz, there were no significant shifts at the lower SEL of 155 dB re 1 μPa² s for either species (mean threshold differences = 0.9 dB for C. p. picta and 1.9 dB for T. s. elegans) (Fig. 2). At 172 dB SEL, all individuals showed TTS at 400 Hz (mean shift for C. p. picta = 18.5 dB; mean shift for T. s. elegans = 15.4 dB) for a combined species mean shift of 16.7 dB (Fig. 2). There was also a high degree of species overlap in the amount of TTS across the frequencies measured and exposure conditions. Post-exposure thresholds representing TTS were significantly elevated above the control means, compared to control thresholds, for both test frequencies (see Fig. S1 in the supplementary material). The 400-Hz control thresholds (the mean of all available auditory data for these individuals collected over two studies) used to calculate TTS are shown in Table 1, including the control threshold means (s.d.) for both frequencies and species.

Both species showed greater TTS at 570 Hz (relative to 400 Hz) despite the considerably lower fatiguing sound received level at 570 Hz (Fig. 3). This was true in comparing TTS at 400 and 570 Hz for 155 dB SEL (p = 0.026) and 172 dB SEL (p = 0.0079) (Wilcoxon rank sum test) (Fig. 2). At 155 dB SEL, the mean shift at 570 Hz, combining both species, was 8.5 dB (mean for C. p. picta = 7.6 dB; mean for T. s. elegans = 9.3 dB). This combined species mean shift increased to 29.2 dB at 172 dB SEL (mean for C. p. picta = 26.2 dB; mean for T. s. elegans = 31.2 dB) (Fig. 2). For RES01 following the 172 dB SEL exposure, the 570-Hz threshold exceeded the maximum amplitude of the hearing test tone, so this stimulus amplitude was used as the minimum threshold. Thus, the mean TTS at 172 dB was likely underestimated.

Across all individuals, TTS was observed in 13 of the 22 noise exposure trials. In 10 of the 13 cases of TTS, the first post-exposure threshold was the highest and represented the greatest shift. In the three instances of TTS following the 155 dB SEL exposure, recovery occurred by 25.5–38 min post-sound exposure (Table 1). For the ten 172 dB SEL exposures inducing TTS, recovery was observed twice in the post-exposure AEP testing (within 18.5 or 39.5 min). In the remaining eight cases, recovery required longer than the apportioned sedation time to acquire the post-exposure thresholds (38.5–50 min). For those sessions, recovery occurred by the following control session, or in one case, it could not be evaluated because sufficient sedation for AEP measurements could not be reached (RES05).

These results show that freshwater turtles can experience TTS after exposure to narrowband sound. This finding supports the apparent sensitivity of turtles to sound, as we observed significant shifts at a relatively low SEL (see Ref. 3) (155 dB re 1 μPa² s). Further, following the 172 dB re 1 μPa² s noise exposure, the mean 570-Hz shift was nearly 30 dB. In one individual (RES01), TTS was >38 dB and AEP responses were not detected for up to 15 min. These TTS results—combined with recent evidence of acoustic communication in the study species and across the Testudine phylogenetic tree^6,14—suggest that anthropogenic noise pollution and subsequent hearing loss are a valid concern for noise-exposed populations. Inducing shifts with narrowband noise suggests that similar sounds could induce TTS in turtles.

A key finding was that more TTS was observed at a half-octave (570 Hz) above the center frequency of the noise (400 Hz). Shifts occurred at 570 Hz in 50% of the exposure trials at 155 dB SEL (Table 1 and Fig. 2), whereas no TTS was observed at 400 Hz at this SEL. For the 172 dB SEL exposure trials, the mean TTS at 570 Hz was nearly twice that observed at 400 Hz (Fig. 2). While recovery rates were not fully explored here, these shifts led to extended recovery durations in the 570-Hz sessions.¹ Notably, the fatiguing noise received levels were considerably lower at 570 Hz (Fig. 1); Fig. 3 in fact shows a negative relationship between TTS magnitude and sound received level. This half-octave shift has been observed in both marine and terrestrial mammals,^15,16,23 but this is the first time such an observation has been made in reptiles.

The lack of 400-Hz TTS in response to the 155 dB SEL narrowband sound coincides with expectations from earlier studies using broadband sound to induce TTS.^1,13 Those studies found 400-Hz TTS onsets (the minimum SEL predicted to induce TTS) at 158 (Ref. 13) and 160 (see Ref. 1) dB re 1 μPa² s for C. p. picta and T. s. elegans, respectively. The lower exposure SEL (155 dB re 1 μPa² s) used in this study fell just below those empirically predicted TTS onset values. The result of no observed TTS suggests that those models may reasonably predict TTS occurrence for these sound types (e.g., non-impulsive, continuous) and frequencies.

However, the TTS growth models for 400 Hz^1,13 underpredicted the amount of shift we observed for the 172 dB SEL narrowband exposures; those models estimated shifts of 11.1 dB (T. s. elegans) and 13.7 dB (C. p. picta). We observed 4.3 dB (T. s. elegans) and 4.8 dB (C. p. picta) greater TTS than these predictions. This could be because the received level at 400 Hz was higher in this narrowband study compared to the energy at 400 Hz in the broadband noise (see Fig. 1 in this study compared to Fig. 1 in Salas et al.¹) or simply due to variance in the models. More TTS data following narrowband exposures across additional SELs and frequencies are needed to compare how the turtle ear responds to the two types of noise, but these initial results suggest a general similarity considering the narrowband center frequency.

Because turtles are more closely related phylogenetically to birds than to mammals,²⁴ we might have predicted that turtles, like birds,²⁵ would not show the half-octave shift, but our results were counter to this prediction. While there was a clear upward spread of TTS,^16,23 we did not determine the extent of this spread (i.e., maximum TTS may have occurred at an auditory frequency we did not test). Future research is needed to explore how TTS changes with respect to hearing frequency. The results herein align with our observation of greater TTS at 600 Hz, compared to 400 Hz, in C. p. picta following broadband continuous noise, despite relatively equal received levels at those two frequencies.^1,13 Differences in nonlinear processes along the sensory epithelium were given as a potential explanation for why birds did not show the half-octave shift compared to mammals.^25,26 Thus, basilar membrane nonlinearities in turtles may be more similar to mammals compared to birds, at least in how they contribute to the frequency spread of TTS. Also potentially contributing to the frequency spread of TTS in turtles is their sole reliance on electrical tuning, which has the trait of broadening the tuning at higher sound levels.²⁷ Additional testing of narrowband noise and the half-octave shift in turtles is important to generalize these initial findings. Identifying how TTS manifests across frequencies—and particularly the frequencies of maximum TTS—is critical to understand and predict noise-induced hearing loss in turtles, optimizing noise management across freshwater and marine habitats.

See the supplementary material for additional details regarding sample sizes and Fig. S1.

We thank the Woods Hole Oceanographic Institution Facilities team for turtle husbandry support. Research was funded by the U.S. Navy's Living Marine Resources Program (Project No. N39430-19-C-2173) and the National Oceanographic and Atmospheric Administration's National Marine Fisheries Service.

The authors have no conflicts to disclose.

The Massachusetts Division of Fisheries and Wildlife (Permit No. 075.20LP) and the Institutional Animal Care and Use Committee (WHOI ID Nos. 25252.01 and 25999) approved animal acquisition and protocols.

The data that support the findings of this study are available within the article and from the corresponding author upon reasonable request.