Thresholds for noise induced hearing loss in harbor porpoises and phocid seals.

Intense sound sources, such as pile driving, airguns, and military sonars, have the potential to inflict hearing loss in marine mammals and are, therefore, regulated in many countries. The most recent criteria for noise induced hearing loss are based on empirical data collected until 2015 and recommend frequency-weighted and species group-specific thresholds to predict the onset of temporary threshold shift (TTS). Here, evidence made available after 2015 in light of the current criteria for two functional hearing groups is reviewed. For impulsive sounds (from pile driving and air guns), there is strong support for the current threshold for very high frequency cetaceans, including harbor porpoises (Phocoena phocoena). Less strong support also exists for the threshold for phocid seals in water, including harbor seals (Phoca vitulina). For non-impulsive sounds, there is good correspondence between exposure functions and empirical thresholds below 10 kHz for porpoises (applicable to assessment and regulation of military sonars) and between 3 and 16 kHz for seals. Above 10 kHz for porpoises and outside of the range 3-16 kHz for seals, there are substantial differences (up to 35 dB) between the predicted thresholds for TTS and empirical results. These discrepancies call for further studies.

Preliminary study of oxidative stress biomarkers and trace elements in North Sea Harbour Seals.

This preliminary study investigated the potential correlations between trace elements (mercury, zinc, cadmium, copper, selenium, lead, nickel, chromium, lithium and vanadium) concentrations, measured in red blood cells, and oxidative stress biomarkers (total thiols, total glutathione, total and selenium-dependent glutathione peroxidases, triglycerides, malondialdehyde) assessed in the respective serum, in males and females P. vitulina, sampled in the Wadden Sea in spring and autumn 2015. Only concentrations of total mercury and zinc showed significant differences by sex, and only lipid peroxidation was different by season. Moreover, significant positive and negative correlations were observed between biomarkers (triglycerides, thiols, malondialdehyde, glutathione) and trace element concentrations (copper, lead, mercury, nickel, zinc). These findings suggest that the studied biomarkers could be useful for the assessment of oxidative stress in harbour seals exposed to trace elements, but further research with larger sample sizes is needed to better understand their specific associations.

Long-term evidence of noise-induced permanent threshold shift in a harbor seal (Phoca vitulina).

In psychophysical studies of noise-induced hearing loss with marine mammals, exposure conditions are often titrated from levels of no effect to those that induce significant but recoverable loss of auditory sensitivity [temporary threshold shift (TTS)]. To examine TTS from mid-frequency noise, a harbor seal was exposed to a 4.1-kHz underwater tone that was incrementally increased in sound pressure level (SPL) and duration. The seal's hearing was evaluated at the exposure frequency and one-half octave higher (5.8 kHz) to identify the noise parameters associated with TTS onset. No reliable TTS was measured with increasing sound exposure level until the second exposure to a 60-s fatiguing tone of 181 dB re 1 µPa SPL (sound exposure level 199 dB re 1 µPa2s), after which an unexpectedly large threshold shift (>47 dB) was observed. While hearing at 4.1 kHz recovered within 48 h, there was a permanent threshold shift of at least 8 dB at 5.8 kHz. This hearing loss was evident for more than ten years. Furthermore, a residual threshold shift of 11 dB was detected one octave above the tonal exposure, at 8.2 kHz. This hearing loss persisted for more than two years prior to full recovery.

Aerial low-frequency hearing in captive and free-ranging harbour seals (Phoca vitulina) measured using auditory brainstem responses.

The hearing sensitivity of 18 free-ranging and 10 captive harbour seals (Phoca vitulina) to aerial sounds was measured in the presence of typical environmental noise through auditory brainstem response measurements. A focus was put on the comparative hearing sensitivity at low frequencies. Low- and mid-frequency thresholds appeared to be elevated in both captive and free-ranging seals, but this is likely due to masking effects and limitations of the methodology used. The data also showed individual variability in hearing sensitivity with probable age-related hearing loss found in two old harbour seals. These results suggest that the acoustic sensitivity of free-ranging animals was not negatively affected by the soundscape they experienced in the wild.

Comparative analysis of the fecal bacterial community of five harbor seals (Phoca vitulina).

The gut microbiota has many beneficial effects on host metabolism and health, and its composition is determined by numerous factors. It is also assumed that there was a co-evolution of mammals and the bacteria inhabiting their gut. Current knowledge of the mammalian gut microbiota mainly derives from studies on humans and terrestrial animals, whereas those on marine mammals are sparse. However, they could provide additional information on influencing factors, such as the role of diet and co-evolution with the host. In this study, we investigated and compared the bacterial diversity in the feces of five male harbor seals (Phoca vitulina). Because this small population included two half-brother pairs, each sharing a common father, it allowed an evaluation of the impact of host relatedness or genetic similarity on the gut microbial community. Fresh feces obtained from the seals by an enema were analyzed by fluorescence in situ hybridization and amplicon sequencing of 16S rRNA genes. The results showed that the bacterial communities in the seals' feces mainly consisted of the phyla Firmicutes (19-43%), Bacteroidetes (22-36%), Fusobacteria (18-32%), and Proteobacteria (5-17%) . Twenty-one bacterial members present in the fecal samples of the five seals contributed an average relative abundance of 93.7 + 8.7% of the total fecal microbial community. Contrary to all expectations based on previous studies a comparison of the fecal community between individual seals showed a higher similarity between unrelated than related individuals.

Absence of selenium protection against methylmercury toxicity in harbour seal leucocytes in vitro.

Previous studies described high concentrations of mercury (Hg) and selenium (Se) in the blood of harbour seals, Phoca vitulina from the North Sea. In the present study, we evaluated the in vitro potential protective effects of sodium selenite (Na2SeO3) and selenomethionine (SeMet) on cell proliferation of harbour seal lymphocytes exposed to MeHgCl 0.75µM. In vitro exposure of ConA-stimulated T lymphocytes resulted in severe inhibition of DNA synthesis, likely linked to severe loss of mitochondrial membrane potential at 0.75µM. Neither selenite nor SeMet showed a protective effect against MeHg toxicity expressed at the T lymphocyte proliferation level for harbour seals. Selenite and SeMet did not show negative effects regarding lymphocyte proliferation and mitochondrial membrane potential. To conclude, our results clearly demonstrated that MeHg affected in vitro immune cells exposure with no protective effects of selenium at a molar ratio Hg:Se of 1:10 in harbour seals from the North Sea.

Auditory sensitivity of seals and sea lions in complex listening scenarios.

Standard audiometric data, such as audiograms and critical ratios, are often used to inform marine mammal noise-exposure criteria. However, these measurements are obtained using simple, artificial stimuli-i.e., pure tones and flat-spectrum noise-while natural sounds typically have more complex structure. In this study, detection thresholds for complex signals were measured in (I) quiet and (II) masked conditions for one California sea lion (Zalophus californianus) and one harbor seal (Phoca vitulina). In Experiment I, detection thresholds in quiet conditions were obtained for complex signals designed to isolate three common features of natural sounds: Frequency modulation, amplitude modulation, and harmonic structure. In Experiment II, detection thresholds were obtained for the same complex signals embedded in two types of masking noise: Synthetic flat-spectrum noise and recorded shipping noise. To evaluate how accurately standard hearing data predict detection of complex sounds, the results of Experiments I and II were compared to predictions based on subject audiograms and critical ratios combined with a basic hearing model. Both subjects exhibited greater-than-predicted sensitivity to harmonic signals in quiet and masked conditions, as well as to frequency-modulated signals in masked conditions. These differences indicate that the complex features of naturally occurring sounds enhance detectability relative to simple stimuli.

Auditory detection of ultrasonic coded transmitters by seals and sea lions.

Ultrasonic coded transmitters (UCTs) are high-frequency acoustic tags that are often used to conduct survivorship studies of vulnerable fish species. Recent observations of differential mortality in tag control studies suggest that fish instrumented with UCTs may be selectively targeted by marine mammal predators, thereby skewing valuable survivorship data. In order to better understand the ability of pinnipeds to detect UCT outputs, behavioral high-frequency hearing thresholds were obtained from a trained harbor seal (Phoca vitulina) and a trained California sea lion (Zalophus californianus). Thresholds were measured for extended (500 ms) and brief (10 ms) 69 kHz narrowband stimuli, as well as for a stimulus recorded directly from a Vemco V16-3H UCT, which consisted of eight 10 ms, 69 kHz pure-tone pulses. Detection thresholds for the harbor seal were as expected based on existing audiometric data for this species, while the California sea lion was much more sensitive than predicted. Given measured detection thresholds of 113 dB re 1 µPa and 124 dB re 1 µPa, respectively, both species are likely able to detect acoustic outputs of the Vemco V16-3H under water from distances exceeding 200 m in typical natural conditions, suggesting that these species are capable of using UCTs to detect free-ranging fish.

Selenium and mercury concentrations in harbor seals (Phoca vitulina) from central California: health implications in an urbanized estuary.

We measured total selenium and total mercury concentrations ([TSe] and [THg]) in hair (n=138) and blood (n=73) of harbor seals (Phoca vitulina) from California to assess variation by geography and sex, and inferred feeding relationships based on carbon, nitrogen, and sulfur stable isotopes. Harbor seals from Hg-contaminated sites had significantly greater [THg], and lesser [TSe] and TSe:THg molar ratios than seals from a relatively uncontaminated site. Males had significantly greater [THg] than females at all locations. Sulfur stable isotope values explained approximately 25% of the variability in [THg], indicating increased Hg exposure for seals with a greater use of estuarine prey species. Decreased [TSe] in harbor seals from Hg-contaminated regions may indicate a relative Se deficiency to mitigate the toxic effects of Hg. Further investigation into the Se status and the potential negative impact of Hg on harbor seals from Hg-contaminated sites is warranted.

Temporal processing of low-frequency sounds by seals (L).

In a recent study, Kastelein et al. [(2010) J. Acoust. Soc. Am. 127, 1135-1145] reported auditory integration times for harbor seals (Phoca vitulina) exceeding 3000 ms for 200 Hz tonal signals. This finding is unexpected and potentially significant given that time constants measured in mammals for tones above 1 kHz are typically less than 500 ms. To further explore this result, the hearing of another harbor seal was measured in air and water for 200 Hz tones with durations of 500 and 2500 ms. Threshold comparisons, as well as reaction time measures, revealed no gain in audibility as signal duration increased above 500 ms.

Hearing threshold shifts and recovery in harbor seals (Phoca vitulina) after octave-band noise exposure at 4 kHz.

Safety criteria for underwater sounds from offshore pile driving are needed to protect marine mammals. As a first step toward understanding effects of impulsive sounds, two harbor seals were exposed to octave-band white noise centered at 4 kHz at three mean received sound pressure levels (SPLs; 124, 136, and 148 dB re 1 µPa) at up to six durations (7.5, 15, 30, 60, 120, and 240 min); mean received sound exposure level (SEL) range was 166-190 dB re 1 µPa(2) s. Hearing thresholds were determined before and after exposure. Temporary hearing threshold shifts (TTS) and subsequent recovery were quantified as changes in hearing thresholds at 1-4, 4-8, 8-12, 48, and 96 min after noise exposure in seal 01, and at 12-16, 16-20, 20-24, 60, and 108 min after exposure in seal 02. Maximum TTS (1-4 min after 120 min exposure to 148 dB re 1 µPa; 187 dB SEL) was 10 dB. Recovery occurred within ~60 min. Statistically significant TTSs (>2.5 dB) began to occur at SELs of ~170 (136 SPL, 60 min) and 178 dB re 1 µPa(2) s (148 SPL, 15 min). However, SEL is not an optimal predictor of TTS for long duration, low SPL continuous noise, as duration and SPL play unequal roles in determining induced TTS.

Hair sampling location in harbor seals (Phoca vitulina) affects selenium and mercury concentrations: implications for study design of trace element determination in pinnipeds.

Hair is used to determine trace elements exposure and status of pinnipeds because it is an excretory route for many elements and can be collected non-lethally. Despite increased use, there have been few studies on how sampling designs and procedures (e.g., hair type, collection site) affect results. The objective of this study was to determine whether concentrations of an essential (selenium; Se) and non-essential element (mercury; Hg) differed between hair samples collected from two body locations on harbor seals (Phoca vitulina). Concentrations of Se and total Hg (THg) differed between mid-dorsal midline and neck samples, and although the absolute differences were relatively small (Δ(absolute) Se = 0.69 µg g(-1), Δ(absolute) THg = 2.86 µg g(-1)), the relative differences were large (Δ(relative) Se = 49%, Δ(relative) THg = 17%). These differences highlight the need to standardize the collection site for trace element determination in pinnipeds.

Near-threshold equal-loudness contours for harbor seals (Phoca vitulina) derived from reaction times during underwater audiometry: a preliminary study.

Equal-loudness functions describe relationships between the frequencies of sounds and their perceived loudness. This pilot study investigated the possibility of deriving equal-loudness contours based on the assumption that sounds of equal perceived loudness elicit equal reaction times (RTs). During a psychoacoustic underwater hearing study, the responses of two young female harbor seals to tonal signals between 0.125 and 100 kHz were filmed. Frame-by-frame analysis was used to quantify RT (the time between the onset of the sound stimulus and the onset of movement of the seal away from the listening station). Near-threshold equal-latency contours, as surrogates for equal-loudness contours, were estimated from RT-level functions fitted to mean RT data. The closer the received sound pressure level was to the 50% detection hearing threshold, the more slowly the animals reacted to the signal (RT range: 188-982 ms). Equal-latency contours were calculated relative to the RTs shown by each seal at sound levels of 0, 10, and 20 dB above the detection threshold at 1 kHz. Fifty percent detection thresholds are obtained with well-trained subjects actively listening for faint familiar sounds. When calculating audibility ranges of sounds for harbor seals in nature, it may be appropriate to consider levels 20 dB above this threshold.

Metallomics approach for the identification of the iron transport protein transferrin in the blood of harbour seals (Phoca vitulina).

The health status of marine mammals such as harbour seals (Phoca vitulina) represents an indirect but powerful way for the assessment of environmental changes. The present work illustrates the first investigation and characterisation of Tf isolated from blood samples of North Sea harbour seals with a view to using changes in Tf isoform patterns as an additional parameter in extended studies of their health status. Therefore, an HPLC-ICP-MS approach has been developed which allows the highly resolved separation and fractionation of up to eight different Tf isoforms, as well as their sensitive and specific detection on the basis of their characteristic iron content. Molecule-specific detection techniques such as nanoLC-ESI-QTRAP-MS or MALDI-TOF-MS were used as complementary techniques to unambiguously identify the isolated proteins as Tf via cross species protein identification and to further characterise the molecular weight as well as the sialic acid content, which is responsible for the elution behaviour of the different isoforms during their ion exchange separation. A molecular mass above 80 kDa has been measured for the different seal Tf isoforms, which is in good agreement with the known molecular mass in other mammalian species, while the estimated pI of the different isoforms indicates some differences in comparison to other species. A number of homologies to known Tf sequences have been observed, which finally allows the cross species protein identification. The combined metallomics orientated analytical approach, which includes the complementary application of element and molecule-specific detection techniques, opens up interesting possibilities for the fast and targeted isolation and identification of a diagnostically relevant metal containing protein from an un-sequenced mammalian species prior to its utilisation in extended studies.

The effect of signal duration on the underwater hearing thresholds of two harbor seals (Phoca vitulina) for single tonal signals between 0.2 and 40 kHz.

The underwater hearing sensitivities of two 2-year-old female harbor seals were quantified in a pool built for acoustic research by using a behavioral psycho-acoustic technique. The animals were trained only to respond when they detected an acoustic signal ("go/no-go" response). Detection thresholds were obtained for pure tone signals (frequencies: 0.2-40 kHz; durations: 0.5-5000 ms, depending on the frequency; 59 frequency-duration combinations). Detection thresholds were quantified by varying the signal amplitude by the 1-up, 1-down staircase method, and were defined as the stimulus levels, resulting in a 50% detection rate. The hearing thresholds of the two seals were similar for all frequencies except for 40 kHz, for which the thresholds differed by, on average, 3.7 dB. There was an inverse relationship between the time constant (tau), derived from an exponential model of temporal integration, and the frequency [log(tau)=2.86-0.94 log(f);tau in ms and f in kHz]. Similarly, the thresholds increased when the pulse was shorter than approximately 780 cycles (independent of the frequency). For pulses shorter than the integration time, the thresholds increased by 9-16 dB per decade reduction in the duration or number of cycles in the pulse. The results of this study suggest that most published hearing thresholds

Underwater hearing sensitivity of harbor seals (Phoca vitulina) for narrow noise bands between 0.2 and 80 kHz.

The underwater hearing sensitivities of two 1.5-year-old female harbor seals were quantified in a quiet pool built specifically for acoustic research, by using a behavioral psychoacoustic technique. The animals were trained to respond when they detected an acoustic signal and not to respond when they did not ("go/no-go" response). Fourteen narrowband noise signals (1/3-octave bands but with some energy in adjacent bands), at 1/3-octave center frequencies of 0.2-80 kHz, and of 900 ms duration, were tested. Thresholds at each frequency were measured using the up-down staircase method and defined as the stimulus level resulting in a 50% detection rate. Between 0.5 and 40 kHz, the thresholds corresponded to a 1/3-octave band noise level of approximately 60 dB re 1 microPa (SD+/-3.0 dB). At lower frequencies, the thresholds increased to 66 dB re 1 microPa and at 80 kHz the thresholds rose to 114 dB re 1 microPa. The 1/3-octave noise band thresholds of the two seals did not differ from each other, or from the narrowband frequency-modulated tone thresholds at the same frequencies obtained a few months before for the same animals. These hearing threshold values can be used to calculate detection ranges of underwater calls and anthropogenic noises by harbor seals.

Underwater detection of tonal signals between 0.125 and 100 kHz by harbor seals (Phoca vitulina).

The underwater hearing sensitivities of two 1-year-old female harbor seals were quantified in a pool built for acoustic research, using a behavioral psychoacoustic technique. The animals were trained to respond when they detected an acoustic signal and not to respond when they did not (go/no-go response). Pure tones (0.125-0.25 kHz) and narrowband frequency modulated (tonal) signals (center frequencies 0.5-100 kHz) of 900 ms duration were tested. Thresholds at each frequency were measured using the up-down staircase method and defined as the stimulus level resulting in a 50% detection rate. The audiograms of the two seals did not differ statistically: both plots showed the typical mammalian U-shape, but with a wide and flat bottom. Maximum sensitivity (54 dB re 1 microPa, rms) occurred at 1 kHz. The frequency range of best hearing (within 10 dB of maximum sensitivity) was from 0.5 to 40 kHz (6(1/3) octaves). Higher hearing thresholds (indicating poorer sensitivity) were observed below 1 and above 40 kHz. Thresholds below 4 kHz were lower than those previously described for harbor seals, which demonstrates the importance of using quiet facilities, built specifically for acoustic research, for hearing studies in marine mammals. The results suggest that under unmasked conditions many anthropogenic noise sources and sounds from conspecifics are audible to harbor seals at greater ranges than formerly believed.

Trace element concentrations in blood of harbor seals (Phoca vitulina) from the Wadden Sea.

Concentrations of 23 elements (Be, Al, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Pd, Cd, Sn, Pt, Pb) were evaluated in whole blood samples of live harbor seals (Phoca vitulina) from two different locations in the Wadden Sea, the Lorenzenplate in Germany, and the Danish island Rømø. Elemental blood levels were compared to data from literature of seals, other marine mammals and humans. While homeostatically controlled elements showed no differences, concentrations of As, Cr, Mn, Mo, Se, and V were higher than human levels. Furthermore, animals from both locations showed significant geographical differences in whole blood concentrations of Al, Mn, Cu, and Pt. These findings could be explained by differences in feeding areas. The element pattern was not affected by gender. In conclusion, these findings indicate an impact of the environment on biochemical blood parameters of the harbor seals. The significant differences of elements in blood samples of two groups of seals, which were associated with geographical variations of prey support the use of element pattern in blood as tool for investigation of environmental impact on seals.

Deterring effects of 8-45 kHz tone pulses on harbour seals (Phoca vitulina) in a large pool.

The marine aquaculture industry suffers losses due to pinniped attacks which damage net enclosures and fish stocks. Acoustic harassment devices (AHDs) emit loud sounds which are intended to deter pinnipeds from approaching aquaculture enclosures. At present, many AHDs emit sounds in the 8-20 kHz frequency range. It is not known whether sounds of higher frequencies have a deterrent effect on seals. Therefore five captive harbour seals (Phoca vitulina) were subjected to four series of tone pulses together spanning a broad frequency range (8, 16, 32 and 45 kHz). Pulse duration was 250 ms and pulse interval was 5s. Each of the four sounds was made deterrent by increasing the amplitude. The seals reacted by swimming away from the sounds. The displacement effect of each sound was judged by comparing the animals' surface positions, and number of surfacings, during ten 45 min baseline periods with ten 45 min test periods per frequency (one frequency per day in rotation, 40 sessions in total). The seals were displaced by all four frequencies throughout the 40 trial days. The seals came to the surface more often when the test tones were produced than in the baseline periods. The initial displacement distances did not change over the 40 test days. This suggests that operating AHDs for only short periods will be more effective and less likely to result in habituation by the seals than operating them continuously. The discomfort threshold sound pressure level (SPL) was established for each of the four pulse frequencies. The acoustic discomfort threshold SPL is defined as the boundary SPL between the area that the animals generally occupied during the transmission of the sounds and the area that they generally did not enter during sound transmission. The discomfort threshold SPL may depend on the context.