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An animal-borne active acoustic tag for minimally invasive behavioral response studies on marine mammals

An animal-borne active acoustic tag for minimally invasive behavioral response studies on marine... Background: There is a variety of evidence that increased anthropogenic noise (e.g., shipping, explosions, sonar) has a measureable effect on marine mammal species. Observed impacts range in severity from brief interruptions of basic life functions to physiological changes, acute injury, and even death. New research tools are needed to better meas- ure and understand the potential effects of anthropogenic noise on marine mammals. Current behavioral response studies typically utilize ship-based sound sources to study potential acute behavioral responses in tagged animals experimentally exposed to noise. Integrating the sound source within animal-mounted passive acoustic and motion- sensing tags provides a novel tool for conducting additional highly controlled response studies. Results: We developed and conducted pilot field trials of a prototype tag on five juvenile northern elephant seals, Mirounga angustirostris, using experimental exposures to both natural and anthropogenic noise stimuli. Results indicate behavioral responses were elicited in tagged individuals. However, no pattern was found in the occurrence and types of response compared to stimulus type. Responses during the ascending dive phase consisted of a dive inversion, or sustained reversal from ascending to descending (8 of 9 exposures). Dive inversions following exposure were 4–11 times larger than non-exposure inversions. Exposures received during the descending dive phase resulted in increased descent rates in 9 of 10 exposures. All 8 exposures during dives in which maximum dive depth was lim- ited by bathymetry were characterized by increased flow noise in the audio recordings following exposure, indicating increased swim speed. Conclusions: Results of this study demonstrate the ability of an animal-mounted sound source to elicit behavioral responses in free-ranging individuals. Behavioral responses varied by seal, dive state at time of exposure, and bathym- etry, but followed an overall trend of diving deeper and steeper and swimming faster. Responses did not consistently differ based on stimulus type, which may be attributable to the unique exposure context of the very close proximity of the sound source. Further technological development and focused field efforts are needed to advance and apply these tools and methods in subsequent behavioral response studies to address specific questions. Keywords: Acoustic, Tag, Behavioral response, Northern elephant seal, Controlled exposure experiment and predator avoidance [1]. Anthropogenic activities Background have contributed to increased ocean ambient noise lev- Marine mammals rely on acoustic cues for many life els in certain areas [2, 3]. The potential adverse effects functions including navigation, foraging, communication, of both acute and chronic human-generated noise on *Correspondence: selene.fregosi@oregonstate.edu marine mammals are a major conservation concern [e.g., Cooperative Institute for Marine Resources Studies, Hatfield Marine 4–14]. Naval sonar activities have been linked to cetacean Science Center, Oregon State University and NOAA Pacific Marine stranding events, where necropsies have shown physical Environmental Laboratory, 2030 SE Marine Science Drive, Newport, OR 97365, USA damage to vital organs [5, 6]. Noise has also been shown Full list of author information is available at the end of the article © 2016 Fregosi et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 3 of 15 to increase stress hormone levels [9], reduce foraging that measure changes in pressure, acceleration, strength activity [8, 15], alter migration routes [16], mask com- of magnetic field, and turn rate [13, 37]. munication sounds [10], and displace marine mammals Here we describe a pilot study testing an animal- from primary feeding and breeding grounds [11, 12, 15]. mounted active acoustic and motion-sensing tag on free- Understanding the differential responses of marine mam - ranging juvenile northern elephant seals. The main goals mals to noise stimuli is necessary for managing human of this study were (1) to test whether an animal-borne impacts in the ocean [17, 18]. acoustic tag elicits any behavioral response and if so (2) Behavioral response studies (BRSs) using a controlled to determine whether responses are related to stimulus exposure experiment (CEE) paradigm have emerged as content. Development and application of an active acous- an effective tool to study discrete behavioral responses tic tag in a behavioral response paradigm could answer of individual, free-ranging marine mammals to particu- targeted questions regarding the nature and magnitude lar sounds [19, 20]. These studies typically use animal- of responses of marine mammals to anthropogenic noise mounted archival motion-sensing (via pressure sensors and their potential physiological consequences, lead- and triaxial accelerometers and magnetometers) and ing to better informed evaluation of human-generated acoustic recording tags as well as visual observations to sound. measure individual focal animal response to a controlled exposure from a sound source deployed nearby. Meas- Methods urements of swim speed, dive depth, dive duration, head- Tag development ing, vocal behavior, group spacing, ascent and descent The prototype tag contained three subsystems: an active rate, and other metrics are made before, during, and after acoustic playback system, a passive acoustic record- the CEE in order to assess response [e.g., 21–25]. ing system, and a motion monitoring system (Fig.  1). Combining the sound source with behavioral sensors An OpenTag single board controller (SBC, Logger- and an acoustic recorder into a single animal-mounted head Instruments, Inc, Sarasota, FL, USA) controlled tag could enable an alternative BRS methodological playbacks and contained a three-axis accelerometer, approach. Such an instrument could offer a more cost- magnetometer, and gyroscope. It was programmed (Log- effective tool that would allow for better control of expo - gerhead Instruments, Arduino programming language) sure sound levels. While the exposure context is unique to execute a playback of a single stimulus at a predeter- in being physically attached to the animal, this con- mined time interval, using sound files stored on a SOMO text is consistent for all exposures whereas other BRS playback module (4D Systems, Minchinbury, NSW, Aus- approaches often have variability in the context of differ - tralia), through an amplifier board (LM48580 Evaluation ent exposures. Finally, it could enable investigation of the Board, Texas Instruments, Dallas, TX, USA) and two or effects of varied sound levels, multiple or sustained expo - three piezo electric ceramic cylinder transducers (Steiner sures, and behavioral habituation. & Martin, Inc, Doral, FL, USA; 26  mm external diame- The northern elephant seal, Mirounga angustirostris ter  ×  22 internal diameter  ×  13  mm high, resonant fre- [26], presents an ideal study species to test the effective - quency 43 ± 1.5 kHz). The playback system was powered ness of such a tag. Northern elephant seals are accessible by a 3.7  V, 3000  mAh lithium-ion polymer rechargeable as they haul out twice a year to breed and molt [27, 28]. battery (Tenergy, Fremont, CA, USA). Elephant seal diving behavior is well studied and found to Six playback stimuli were used: sperm whale (Physeter be highly stereotypic, with almost continuous, repetitive macrocephalus) clicks, common dolphin (Delphinus sp.) deep diving [28–31]. They regularly reach depths simi - whistles, killer whale (Orcinus orca) whistles and clicks, lar to many of the cetacean species thought to be most simulated mid-frequency active sonar, and white noise. affected by anthropogenic noise (i.e., beaked whales; [28, White noise and the simulated mid-frequency sonar were 32]) and dive as deep as or deeper than the deep sound created using Adobe Audition 3 (Adobe System Incorpo- channel (~1000  m). Previous studies have shown that rated, San Jose, CA, USA). Sperm whale clicks and com- carrying relatively large instruments does not inhibit the mon dolphin whistles were obtained from the Discovery ability of juvenile seals to swim or forage, and instrument of Sound in the Sea sound library. Both killer whale recovery rate is above 90  % [28, 33, 34]. Additionally, vocalizations were obtained from the Vancouver Aquar- northern elephant seals have acute underwater hearing ium. An inverse of the transducer’s transmitting sensi- sensitivity with relatively low hearing thresholds occur- tivity function was generated and used to normalize each ring over a very broad frequency range [35, 36]. High-res- playback’s output over the entire frequency bandwidth. olution behavioral responses to sound stimuli from our device can be collected for elephant seals using sensors http://www.dosits.org/audio/marinemammals/toothedwhales/. http://killerwhale.vanaqua.org/page.aspx?pid=1331. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 4 of 15 Table 1 Calibrated source levels for exposure stimuli Stimulus Source level White noise 125.56 (0.94) @ 1 m (dB re 1 µPa) RMS Common dolphin whistles 130.66 (1.48) @ 1 m (dB re 1 µPa) RMS Sperm whale clicks 131.77 (1.89) @ 1 m (dB re 1 µPa) 0-peak Killer whale whistles 121.06 (4.60) @ 1 m (dB re 1 µPa) RMS Killer whale clicks Estimated 126 @ 1 m (dB re 1 µPa) 0-peak Sonar 124.27 (0.58) @ 1 m (dB re 1 µPa) RMS Mean and standard deviation of two calibrated recordings are given. All sources levels are given in dB re 1 µPa @ 1 m over the stimulus duration, except for RMS the sperm whale click track , which was measured in dB re 1 µPa @ 1 m and SOMO 0-peak the killer whale click track which was measured by relative comparison to killer playback whale whistles module hydrophone (High Tech, Inc, Long Beach, MS, USA; OpenTag pre- power −1 controller sensitivity −190.5  dB re 1  V  µPa ; frequency response amplifier supply board 2 Hz–30 kHz) recorded for 30 min starting 15 min prior loudspeaker to each scheduled playback. This provided a recorded confirmation that a single playback occurred and allowed DSG passive power acoustic hydrophone detection of other environmental noise events occurring supply recorder at the time of the playback. Recordings were sampled at 32  kHz (16  bit resolution) with 20  dB of gain. The pas - sive acoustic system was powered by a second 3.7  V, 3000  mAh lithium-ion polymer battery (Tenergy, Fre- mont, CA, USA). All tag components were placed in an oil-filled Delrin housing, rated to 1500 m depth. Clear, food-grade min- eral oil was used as a non-compressible and non-conduc- tive filler so no air remained in the housing and to limit acoustic impedance between the tag and seawater. The tag package weighed approximately 1.5  kg (<1.2  % total Fig. 1 Prototype tag was built, calibrated, and field-tested on north- body mass of smallest seal) and was slightly negatively ern elephant seals (center black and orange tag). Tag components buoyant. The original tag design was modified follow - included a DSG passive acoustic recorder and hydrophone, an active ing the first three deployments. Changes in pressure and acoustic preamplifier, playback module, transducers, and OpenTag temperature between the surface and at depth caused single board controller with a nine-axis motion sensor (three axes each for accelerometer, magnetometer, and gyroscope). A Wildlife changes in mineral oil density. The slight flexibility of Computers Mk10-AF TDR was mounted on the head. An additional Delrin caused a pumping action to develop that moved TDR was attached to the shoulder but was not used in this study seawater past the o-ring seals into the oil-filled tag body. Larger o-rings were used, and flexible tubing with small air pockets was attached to the outside of the housing to allow minor changes in mineral oil density and limit sea- Playbacks of stimuli were limited to 30  s to reduce the water intrusion. possibility of seals associating the stimulus with the device mounted on their back. Playback track source lev- Field efforts els were measured using a calibrated hydrophone system We conducted field experiments in March and April (G.R.A.S. 42AC High Pressure Pistonphone, G.R.A.S. 2012 on juvenile northern elephant seals at Año Nuevo Sound & Vibration A/S, Holte, Denmark) and ranged State Park, San Mateo County, CA, USA (Fig. 2; Table 2). from 121 to 132 dB re 1 µPa at 1 m, depending on expo- Seals were instrumented with the active acoustic tag sure type (Table 1). Estimated received levels were calcu- placed on the animal’s back (30–60  cm from the ears) lated assuming spherical spreading, based on the distance and an ARGOS-Fastloc GPS time-depth recorder (TDR) of tag from the animals’ ears. on the head (Mk10-AF, Wildlife Computers, Redmond, A DSG acoustic recorder (Loggerhead Instruments, WA, USA). Tags were attached by gluing a flexible mesh Inc, Sarasota, FL, USA) connected to an HTI-96-MIN Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 5 of 15 Fig. 2 Tracks of all successful seal deployments. The track line is the straight-line connection between subsequent surface locations. Numbered black bars are approximate locations where exposures occurred, between the surface locations before and after the time of the exposure. Bathym- etry data are from NOAA’s National Geophysical Data Center (NGDC), Southern California Coastal Relief Map, one-arc second resolution base to the animal’s fur using quick-cure adhesive (Dev- CA, USA, and released. Seals were recaptured upon con 14270—5 Minute Epoxy, ITW Polymers Adhesives return to Año Nuevo, and tags (including the metal ties) North America, Danvers, MA, USA), weaving stainless were manually removed from the flexible mesh base. steel locking ties through the mesh, and securing the tag Seals molted off the base shortly after tag recovery. Depth with the locking ties (similar to [30, 38]). Animals were was sampled every 1 s for seal G5449 and every 4 s for all other seals. Resolution of TDRs were 0.5 m and accuracy transported approximately 100  km south to Monterey, Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 6 of 15 Table 2 Summary dive statistics for all study individuals Seal Age Sex Body Deployment Number Number Mean dive depth (m) Mean dive duration (s) Median dive duration (s) mass (kg) duration (h) of dives of dives All dives Dives deeper All dives Dives deeper All dives Dives deeper recorded deeper than 100 m than 100 m than 100 m than 100 m (% of total) G5449 1 M 134 207.22 972 509 (52.4 %) 136.618 (±106.0357) 217.114 (±84.7149) 646.6337 (±267.9752) 829.34 (±191.822) 637 815 G5520 1 F 167 63.11 402 20 (5.0 %) 36.6157 (±53.6568) 223.425 (±116.878) 365.34.33 (±251.5771) 958.0 (±398.2795) 340 868 G6009 1 F 182 121.26 518 124 (23.9 %) 94.9913 (±128.4333) 290.395 (±129.899) 608.7259 (±436.105) 1185.8 (±324.3298) 556 1236 G6110 2 F 262 30.24 148 28 (18.9 %) 75.0811 (±108.5156) 258.375 (±165.32) 585.4324 (±379.2734) 1146.3 (±279.5604) 526 1146 G6651 1 F 206 73.01 321 115 (35.8 %) 145.829 (±167.2949) 339.274 (±136.802) 748.2991 (±580.3701) 1419.1 (±364.688) 560 1488 Age was either known from initial tagging as a pup or estimated in the field based on standard length and weight. Sex was assessed and body mass was measured before instrumentation. Deployment duration was dependent on the time it took the seal to return to the beach. Number of dives recorded includes all instances where the animal dove deeper than 4 m. Mean (and SD) dive duration, in min, are given for all dives and the subset of dives deeper than 100 m Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 7 of 15 ±1 m. The accelerometer, magnetometer, and gyroscope ambient noise levels, and thus a decreased signal-to- were all sampled at 50 Hz. noise ratio of the low-level stimulus, near the water’s Playbacks occurred every 6 h for the first two seals and surface. every 3  h for the remaining seals. Each playback con- To determine potential responses during ascent or sisted of one exposure to a single stimulus. The order of bottom phases of deep (>100  m) dives, dive inversions stimuli presentation varied between seals, but remained (Fig.  4) were measured and compared. Dive inversions the same within an individual seal, unless the tag reset were defined as a change from ascending or horizontal itself, at which point it started back at the beginning of swimming to descending, with a change in depth >3  m the sequence. over 4  s. Change in depth and change in time from the inversion point to the next maximum depth were meas- Behavioral analysis ured and normalized by maximum depth and total dive Passive recordings were screened for successful expo- duration, respectively. In order to statistically compare sures (Table  3), and timing of exposures across multi- dive inversions following exposures to dive inversions ple tag systems was synced. Depth data from the TDRs outside of exposure events, an Anderson–Darling k-sam- were examined for possible seal responses to exposures ple (ADK; [40]) test was run on seal G6651, the only seal (Fig.  3). To account for zero offset drift, dive data were to receive multiple exposures during the ascending phase. analyzed using the IKNOS toolbox (Y. Tremblay, unpub- The ADK test was selected because it is distribution free lished) in MATLAB (MathWorks, Inc, Natick, MA, and does not rely on equal sample sizes; however, it USA). Dives were defined as any instance where the indi - requires that samples are taken from independent distri- vidual dove deeper than 4 m. This threshold was chosen butions, in this case, different seals. G5449 was chosen to be greater than the sum of the accuracy of the TDR for comparison as it had the largest deployment duration (±1 m) and the body length of an individual. and therefore the most non-exposure dive inversions. To We hypothesized that responses would differ accord - first test whether different seals could be directly com - ing to dive type and state, so different dives and expo - pared, an ADK test was used to compare non-exposure sures were analyzed separately. Dive shapes were used dive inversions for seals G6651 and G5449. The magni - to identify different dive types (e.g., transit vs. foraging tude of the dive inversion following the single playback [39]). Dives were grouped into deep (>100  m) and shal- during the bottom phase of a deep dive was measured low (≤100  m) dives (Table  3). Exposures were catego- and reported. rized by dive phase (descent, ascent, bottom phase, and Descent rates for descending phases of deep dives were surface; Table  3). Descents were defined as the segment measured and compared to identify possible responses from 0  m to the point at which the seal descended at a during descents. Average descent rate over 60  s before −1 rate <0.1 m s for 20 s. The beginning of an ascent was and 60  s after exposure onset were calculated. A time defined as the point at which an individual ascended for window of 60  s was selected in order to differenti - −1 more than 4  m over 20  s (an ascent rate of 0.2  m  s ). ate sustained changes in descent rate from brief startle Bottom phases were defined as the segment between responses during the playback only. Because an increase the descending and ascending segments. Exposures that in descent rate is considered a subtle response that likely occurred at depths shallower than 20  m were labeled as happens regularly in non-exposure dives, statistical anal- “surface” and excluded from all analyses due to increased ysis of changes in descent rate was not performed. Table 3 Deployment duration and number of exposures for the successful deployments of the prototype tag Seal Exposure Number of  Exposures by dive stage interval (h) exposures received Surface Descent Bottom phase Ascent Deep Shallow Deep Shallow Deep Shallow G5449 3 6 2 – – 1 1 – 2 G5520 3 2 – – – 1 – 1 – G6009 6 2 – – – – – – 2 G6110 6 4 1 – – – 1 – 2 G6651 3 24 7 1 1 3 7 1 4 The number of exposures was dependent on the success of the tag and the length of the deployment. Exposures during descent, bottom phase, and ascent are the subset of exposures used for analysis in each respective phase, split by shallow (<100 m) and deep (>100 m) exposures. Surface exposures were excluded from analysis Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 8 of 15 G5449 -100 -200 -300 -400 -500 0 20 40 60 80 100120 140160 180200 G5520 -100 -200 -300 -400 -500 05 10 15 20 25 30 35 40 45 G6009 -100 -200 -300 -400 -500 -600 G6110 -100 -200 -300 -400 -500 -600 -700 05 10 15 20 25 G6651 -200 -400 -600 -800 -1000 time (hours) Fig. 3 Depth profiles and seafloor bathymetry for complete deployments for all seals. Seal dive profile is in blue, seafloor bathymetry is in gray, and exposures are indicated by black dots. Bathymetry data are from NOAA’s NGDC, Southern California Coastal Relief Map, one-arc second resolution. Instances where a dive profile overlaps the seafloor are an artifact of data resolution mismatch; bathymetry data were extracted only for known GPS surface positions and linearly connected in the gray bathymetry plot, leading to areas with reduced detail depth (meters) depth (meters) depth (meters) depth (meters) depth (meters) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 9 of 15 G5449 Exposure 2 G6110 Exposure 3 G6651 Exposure 3 0 0 -100 -100 -100 -300 -300 -300 -500 -500 -500 470 490 510 1085 1105 1125 440 460 480 G6651 Exposure G6651 Exposure 5 G6651 Exposure 9 -100 -100 -100 -300 -300 -300 -500 -500 -500 610 630 650 802 822 842 1520 1540 1560 G6651 Exposure 12 G6651 Exposure 18 G6651 Exposure 21 0 0 -100 -100 -100 -300 -300 -300 -500 -500 -500 2060 2080 2100 3135 3155 3175 3685 3705 3725 time (minutes) sperm whale clicks white noise common dolphin whistles killer whale whistles killer whale clicks simulated sonar Fig. 4 Dive profiles for all exposures received during ascending phases of dives from seals G5449, G6110, and G6651. Shapes indicate time of exposure and type Shallow dives occurred along the shelf edge and were different stimuli, each playback was categorized as either limited by the seafloor so diving deeper or reversing an “response” or “no response,” using a minimum level of ascent was not possible. To identify possible responses response based on percent change, which varied for each for these shallow dives, changes in flow noise on the dive state (ascending, bottom, descending, or shallow/ passive acoustic recorder (a proxy for changes in swim bottom-limited). Ascending or bottom-phase playbacks speed) were measured and compared. Previous studies were categorized as “response” if there was a dive inver- have found an 18- to 20-dB increase at very low frequen- sion magnitude >2 SDs from non-playback inversions. cies (8–18 Hz) corresponds to a doubling of current flow, Descending playbacks were considered a response if regardless of tag or hydrophone design [34, 41]. Average descent rate increased by >50 % following exposure, and RMS in the 8–18  Hz frequency band was measured for for shallow dives, an increase in flow noise of >10 % was 30–60 and 0–30 s before exposure and 0–30 and 30–60 s considered a response. Percent of overall response was after the exposure ended (Adobe Audition CC, Adobe calculated. Systems Incorporated, San Jose, CA, USA). The before and after periods were divided to detect shorter-term Results changes in swim speed, but the entire 60-s period was Tag development taken into account when assessing a response. Seven deployments of the prototype tag resulted in suc- The normalized change in depth for exposure and cessful CEEs for five individuals (Table  2). Received levels non-exposure dive inversions for seal G6651 was plot - at the individuals’ ears, estimated from the source level ted against time to investigate possible habituation to and assuming spherical spreading, ranged from 128 to multiple playbacks over the duration of the deploy- 138  dB re 1  µPa, depending on seal and stimulus type. ment. To investigate differential response of all seals to Mechanical (flooding) or electrical (tag control board depth (m) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 10 of 15 reset) tag failure rendered the remaining two unsuccess- during the bottom phase of a deep dive (351 m). She dove ful. The playback control of the tag would occasionally to 614 m immediately following exposure, a 75 % increase experience a reset for undetermined reasons, resulting in maximum depth of dive (killer whale whistle exposure, in a stimulus that was played out of sequence. In some Table 3). cases, this reset caused the tag to stop working entirely. Three seals received exposures during the descending phase of a deep dive, and two of three (G5549 and G6651) Deployments and basic dive behavior exhibited an increased descent rate (35.9–271.9 %; mean Five successful deployments ranged from 30 to 207  h in 125.2  %; ±95.8  %) following exposure (Fig.  6; 9 of 10 duration. Animals received 2–24 playbacks, each a sin- total deep descending exposures). Seal G6110 had a gle 30-s exposure, at an interval of 3 or 6  h, and non- lower descent rate following exposure to sonar (72.7  % exposure dives served as control dives (Table  3; Fig.  2). decrease). Four of five animals dove along the seafloor (depths up Flow noise (swim speed) increased by 10.0 (±8.0)  dB to ~100  m) until reaching the continental shelf edge, re 1 µPa from 0 to 30 s before exposure to 30–60 s after after which they exhibited deeper, pelagic transit dives exposure (30.3  ±  14.7  % increase) in all 8 exposures (to with mean depths of 217–340  m over the deep water of three seals) that occurred in shallow water (<100 m). For Monterey Canyon. They returned to the shallow dive pat - two of the exposures, flow noise initially decreased in tern when returning to shallow coastal waters near Año the 0–30  s immediately after exposure but by 60  s after, Nuevo State Park (Fig.  3). We found no evidence of for- reached above pre-exposure levels. aging or drift dives, and the observed dive patterns were Dive inversion responses seal G6651 were as pro- similar to those of other translocated juveniles [13, 42]. nounced as or more pronounced than those later in the Dive depths <100  m were likely limited by the seafloor, deployment compared to earlier (Fig. 7). Seals responded while deeper dives were not bottom-limited, which may to 23 of 28 total exposures (82 %), regardless of stimulus have an effect on responses measured (Fig.  3) [43]. One type, indicating no differential response. individual (G5520) rarely dove deeper than 100 m (5.0 % of all dives), even over deep water (Fig.  3). We found no Discussion significant difference in magnitude of non-exposure dive We identify behavioral responses in juvenile northern inversions for seals G5449 and G6651 (ADK, p  =  0.14, elephant seals elicited by an animal-mounted sound n = 283 non-exposure inversions, seal G5449 and n = 30 source. This is the first dedicated BRS conducted on any non-exposure inversions, seal G6651). Both exposures to marine mammal using an animal-borne sound source seal G6009 were received at <10  m depth, so they were (see [44] for incidental BRS with an active sonar tag). excluded from further analyses. After this exclusion, This approach has the potential to expand and comple - there were 28 usable exposures on four seals, made up of ment current CEE methodologies. Such data will pro- 9 ascending, 10 descending, 1 bottom phase, and 8 shal- vide enhanced understanding of longer-term effects of low water exposures. anthropogenic noise. Controlled exposure experiment Tag performance We observed dive inversions following all playbacks that Deployments that resulted in unsuccessful or reduced occurred during the ascending portion of a deep dive. duration CEEs were due to issues with either the SBC or Individuals descended an additional 116 m (SD ±51.6 m) the housing. We were unable to resolve the cause of the on average (Fig.  4). 8 of 9 exposures resulted in changes SBC reset, but believe it was caused by the formation of in depth >2 SDs from the mean of all non-exposure inver- a ground loop due to differential ground potentials of the sions (Figs.  4, 5; Table  4). Seal G6651 was the only seal independent hardware components comprising the sin- which received multiple exposures during ascent (n  =  7 gle tag system. Although the housing was rated to 1500 m exposures), and the magnitudes of dive inversions follow- depth, either changes in temperature and pressure with ing these exposures were statistically different than non- depth or incorrectly sized o-rings likely caused failure exposure inversions (Fig. 5; Table 4; ADK test, p < 0.001, of the slightly flexible Delrin housing, allowing seawa- n = 283 non-exposure inversions, seal G5449 and n =  7 ter to enter the housing and short circuit the electron- exposure inversions, seal G6651). We observed this dive ics. Future tags would benefit greatly from being potted inversion response for white noise, sperm whale clicks, in solid resin; however, the non-permanent setup used in killer whale whistles, and simulated mid-frequency sonar this trial allowed us to monitor and modify physical tag exposure, but not following an exposure to common dol- components and programming as needed during the field phins, received by seal G6651 (Fig. 4, G6651 Exposure 4). effort. Additionally, we found no evidence of instrumen - Seal G6651 was the only seal that received an exposure tation or drag affecting normal swimming behavior, as Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 11 of 15 G5449 0.4 n=283 and 1 0.3 0.2 0.1 G6110 0.4 n=8 and 1 0.3 0.2 0.1 non-exposures white noise common dolphin whistles G6651 0.4 sperm whale clicks n=30 and 7 killer whale whistles 0.3 + killer whale clicks simulated sonar 0.2 0.1 0 0.1 0.2 0.3 0.40.5 0.60.7 normalized change in depth Fig. 5 Plot of normalized change in depth versus normalized change in time for all dive inversions during the ascending phase of all deep dives (>100 m) for seals G5449, G6110, and G6651. Black dots symbolize non-exposure dive inversions, and open symbols indicate dive inversions following exposures Table 4 Mean change in depth for non-exposure and exposure dive inversions Seal Mean change in depth for  Mean change in depth Anderson–Darling k-sample test non-exposure inversions (m) for exposure inversions (m) G5449 14.875 (±13.997) n = 283 70.5 n = 1 n/a G6110 16.749 (±16.293) n = 8 186 n = 1 n/a G6651 24.867 (±39.981) n = 30 96.36 (±59.23) n = 7 p = <0.001 Dive inversions are defined by a change from ascending to descending >3 m over 4 s, for all dives deeper than 100 m. Standard deviation is given in parentheses, and n is given for each. Results from an Anderson–Darling k-sample test comparing the change in depth of an inversion from ascending to descending, normalized by maximum depth of dive, for exposure inversions of seal G6651 to non-exposure inversions from seal G5449 (n = 283) are given dive profiles were similar to those of other translocated to smaller-scale dive variations seen naturally in these seals carrying smaller devices [13, 42, 45]. and other translocated seals carrying a depth sensor [13, 46]. The exposure context here is unique in terms of the Animal response to disturbance extreme proximity of the sound source to the animal, and Behavioral responses measured during CEEs varied the extrapolation of results to longer-range exposures with dive state at the time of exposure. Most responses should thus be considered cautiously. However, this con- involved seals diving deeper and/or longer after expo- text is more constant than in previous studies providing a sure. The clearest responses (Fig.  4; Table 4) involved dive stronger basis for the comparison of responses within an inversions (reversal of an ascent) followed by the seal div- individual across exposure conditions. ing deeper than the initial maximum dive depth. These Responses observed are similar to hypothesized ele- extended dive inversions served as strong indicators phant seal behavior to escape predators by diving deeper of response. Quantitatively, dive inversions resulted in [37], and responses to anthropogenic sound sources are greater depths and longer dive durations, when compared not unexpected [13, 44]. Responses elicited during CEEs normalized change in time Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 12 of 15 for observed responses. In future studies, initiating Descent Rates Before and After Exposure exposure during one dive phase would control experi- 2 before exposure mental variability and allow robust statistical analyses of after exposure responses. 1.5 We suspect the tag reset described above, and the resulting stimulus playback, is the most likely explana- tion for three non-exposure dive inversions of similar magnitude to inversions following exposures observed in 0.5 seal G6651 (Fig.  5). The inversions could have occurred naturally (e.g., exposure to an external alarming sound) or from an accidental exposure by the prototype tag fol- G5449 G6110 G6651 lowing a reset; however, the independent passive acous- exposure number and type white noise common dolphin whistles sperm whale clicks tic system was not recording so we cannot know for killer whale whistles + killer whale clicks simulated sonar sure. Continuous recording of ambient noise on future −1 Fig. 6 Mean descent rates, in m s , for seals G5449, G6110, and tag iterations, and longer-duration deployments, would G6651 during the 60 s before (white bars) and 60 s after (black bars) allow for potential opportunistic measurement of behav- exposure. Open symbols correspond to exposure type ioral responses to naturally occurring threatening sounds such as predators, boats, or other man-made or tag-made sounds. G6651 0.5 Differential response by stimulus type 0.45 We found no indication of differential responses based on 0.4 stimulus type or order (Table 5). Notably, seals responded 0.35 to most (82  %) but not all exposures, indicating a possi- non-exposures ble differential response that was not detected because of 0.3 white noise common dolphin whistles limited sample size. 0.25 sperm whale clicks The observed results suggest that the response may be killer whale whistles 0.2 killer whale clicks a result of acoustic limitations of the playback apparatus simulated sonar 0.15 rather than the stimuli (see [48]). Limitations included 0.1 the nonlinear nature of the small transducers and 0.05 extreme proximity of the source to the animal. This prox - imity likely created an unusual perceptual context com- 12 24 36 48 60 pared to a sound produced naturally at realistic ranges time (hours) due to the effects of sound propagation (e.g., rever - Fig. 7 Plot of normalized change in depth versus elapsed time (in beration, directional cues, and the relative presence or h) for all dive inversions during the ascending phase of all deep absence of harmonics). Many details of pinniped under- dives (>100 m) for seal G6651. Black dots symbolize non-exposure dive inversions, and open symbols indicate dive inversions following water hearing, including directionality, are still poorly exposures understood [36, 49, 50]. Sudden sounds may trigger a response, regardless of the sound type or the distance it were similar to those found by other BRSs on deep-div- Table 5 Differences in  response rate by  exposure type ing marine mammals. For instance, dive inversions and and for all exposures to all seals remaining at depth longer were demonstrated in two spe- cies of beaked whale exposed to simulated mid-frequency Exposure Number of exposures Response measured sonar from an external, ship-mounted sound source [22, White noise 6 5 47]. A third species of beaked whale responded to expo- Common dolphin 4 2 sures of both sonar and killer whale sounds by inter- whistles rupting foraging and performing an unusually long and Sperm whale clicks 5 5 shallow ascent [12]. Killer whale whistles 6 5 Responses to exposures that occurred during descents Killer whale click 1 1 or during bottom-limited dives were more difficult to Sonar 6 5 measure. Natural variation in swim speed and descent Total 28 23 angle limited our ability to define statistical significance -1 normalized change in depth descent rate (ms ) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 13 of 15 is perceived to be coming from. Future testing using play- beginning to better understand how animals behaviorally back stimuli that are filtered to simulate different source respond to sound, but we still do not fully understand locations could examine the effects of reverberation and the underlying physiological changes that are linked to localization in detail. Comparable parallel CEEs would these behavioral changes (e.g., oxidative stress). Physi- need to be conducted on pinnipeds, using an external ological parameters such as oxygen utilization, heart rate, sound source that closely approximate actual signals, to and post-dive recovery times can be monitored in free- fully evaluate the potential of this tag for BRS. ranging animals [53, 54], and with this tag, dives can be While individual differences could also contribute experimentally extended, allowing quantitative investiga- to different responses, no single seal received multiple tion of physiological responses. To support the potential exposures of all exposure types because of tag failures, use of this tag to study physiological effects of extended which precludes further examination of this possibility. dives possibly related to responses to anthropogenic BRS exposing animals to multiple exposures of all stimuli noise, it is promising that the responses were similar to are needed to investigate individual differences to stimuli. other deep-diving species of interest. For species that are A differential response to dolphin whistles was recorded specialized for oxygen efficiency during prolonged, deep for seal G6651, the only seal exposed to two playbacks of dives, physiological responses to changes in planned dive dolphin whistles while ascending from a dive. No extreme duration could help physiologists better understand these dive inversions were recorded for dolphin whistles, while adaptations [55]. other stimuli resulted in extreme evasive behavior. This anecdotal example provides evidence for the possibility Conclusions of differential response to different stimuli. This pilot study was the first of its kind to investigate Repeated exposures to various stimuli may cause habit- the potential use of an animal-borne sound source to uation over time [51]. Although there was no evidence of conduct BRS on marine mammals to further investigate habituation over short-term deployments (<3  days), we the effects of anthropogenic noise. Five juvenile north - cannot rule out the possibility of a sensitization response. ern elephant seals were instrumented and translocated The consistent response to most exposures independ - south of their colony at Año Nuevo State Park. They ent of stimulus type and the apparent lack of reduction received playbacks of multiple stimulus types (both in response within individuals suggest either that seals man-made and natural) from the tag while diving con- are generally sensitive to a relatively wide range of audi- tinuously on their return to the colony. Dive behavior ble exposures or possibly that seals became sensitized to before and after exposures were compared to assess subsequent exposure. Preliminary evidence from captive whether animal-borne tags holding sound sources were elephant seals suggests some sensitization, as opposed to capable of eliciting a response from a free-ranging ani- habituation, upon repeated exposure to certain acous- mal and whether potential responses differed with stim - tic stimuli [52]. However, the sound source placement, ulus content. context, and limited number of exposures of each stimu- Projecting sound from a tag mounted to the back lus type to each individual limited our ability to test for of a free-ranging juvenile northern elephant seal did potential sensitization. elicit behavioral responses; however, the responses The tag and method may be useful in physiological were not consistently different with different stimulus studies of dive limits and studies of hearing ranges. The types. Responses varied by seal, by dive state, and by the tag could be used as a mobile, programmable sound bathymetry where the exposure occurred, but in general, source to study frequency-dependent hearing in marine seals dove deeper following playback. Animals responded mammal species that cannot be studied using captive to 82 % of exposures overall, with no clear evidence of a psychophysical methods. Little is known about hearing reduction in response to repeated exposures of various in many marine mammals because laboratory studies of stimuli. The unique and biologically unrealistic context hearing are not feasible due to constraints with keeping of a sudden sound exposure coming from directly behind very large, migratory, deep-diving, and social species in the animal is likely the greatest limiting factor preventing captivity, but using controlled, incrementally increas- use of this tag in examining differential responses to par - ing exposure levels of tonal sounds that trigger drastic ticular sounds and relating them to responses that may changes in behavior could investigate differences in hear - occur over more realistic source-animal ranges. Mak- ing sensitivities in previously inaccessible species. ing conclusions about the effects of particular sounds on Additionally, this tag could be used to study the physi- juvenile northern elephant seals was beyond the scope of ological effects of unanticipated extensions of dives, this pilot study, but additional tests using this technology another important research need for understanding may help develop additional questions and hypotheses in the effects of noise on marine mammals [18]. We are the future. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 14 of 15 Received: 30 October 2015 Accepted: 11 March 2016 Tag improvements and additional field testing could strengthen results found here, but the concept of an animal-borne sound source for triggering behavio- ral responses has been validated. The use of this novel animal-borne sound source in future studies could help References 1. 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An animal-borne active acoustic tag for minimally invasive behavioral response studies on marine mammals

An animal-borne active acoustic tag for minimally invasive behavioral response studies on marine mammals

Background: There is a variety of evidence that increased anthropogenic noise (e.g., shipping, explosions, sonar) has a measureable effect on marine mammal species. Observed impacts range in severity from brief interruptions of basic life functions to physiological changes, acute injury, and even death. New research tools are needed to better meas- ure and understand the potential effects of anthropogenic noise on marine mammals. Current behavioral response studies typically utilize ship-based sound sources to study potential acute behavioral responses in tagged animals experimentally exposed to noise. Integrating the sound source within animal-mounted passive acoustic and motion- sensing tags provides a novel tool for conducting additional highly controlled response studies. Results: We developed and conducted pilot field trials of a prototype tag on five juvenile northern elephant seals, Mirounga angustirostris, using experimental exposures to both natural and anthropogenic noise stimuli. Results indicate behavioral responses were elicited in tagged individuals. However, no pattern was found in the occurrence and types of response compared to stimulus type. Responses during the ascending dive phase consisted of a dive inversion, or sustained reversal from ascending to descending (8 of 9 exposures). Dive inversions following exposure were 4–11 times larger than non-exposure inversions. Exposures received during the descending dive phase resulted in increased descent rates in 9 of 10 exposures. All 8 exposures during dives in which maximum dive depth was lim- ited by bathymetry were characterized by increased flow noise in the audio recordings following exposure, indicating increased swim speed. Conclusions: Results of this study demonstrate the ability of an animal-mounted sound source to elicit behavioral responses in free-ranging individuals. Behavioral responses varied by seal, dive state at time of exposure, and bathym- etry, but followed an overall trend of diving deeper and steeper and swimming faster. Responses did not consistently differ based on stimulus type, which may be attributable to the unique exposure context of the very close proximity of the sound source. Further technological development and focused field efforts are needed to advance and apply these tools and methods in subsequent behavioral response studies to address specific questions. Keywords: Acoustic, Tag, Behavioral response, Northern elephant seal, Controlled exposure experiment and predator avoidance [1]. Anthropogenic activities Background have contributed to increased ocean ambient noise lev- Marine mammals rely on acoustic cues for many life els in certain areas [2, 3]. The potential adverse effects functions including navigation, foraging, communication, of both acute and chronic human-generated noise on *Correspondence: selene.fregosi@oregonstate.edu marine mammals are a major conservation concern [e.g., Cooperative Institute for Marine Resources Studies, Hatfield Marine 4–14]. Naval sonar activities have been linked to cetacean Science Center, Oregon State University and NOAA Pacific Marine stranding events, where necropsies have shown physical Environmental Laboratory, 2030 SE Marine Science Drive, Newport, OR 97365, USA damage to vital organs [5, 6]. Noise has also been shown Full list of author information is available at the end of the article © 2016 Fregosi et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 3 of 15 to increase stress hormone levels [9], reduce foraging that measure changes in pressure, acceleration, strength activity [8, 15], alter migration routes [16], mask com- of magnetic field, and turn rate [13, 37]. munication sounds [10], and displace marine mammals Here we describe a pilot study testing an animal- from primary feeding and breeding grounds [11, 12, 15]. mounted active acoustic and motion-sensing tag on free- Understanding the differential responses of marine mam - ranging juvenile northern elephant seals. The main goals mals to noise stimuli is necessary for managing human of this study were (1) to test whether an animal-borne impacts in the ocean [17, 18]. acoustic tag elicits any behavioral response and if so (2) Behavioral response studies (BRSs) using a controlled to determine whether responses are related to stimulus exposure experiment (CEE) paradigm have emerged as content. Development and application of an active acous- an effective tool to study discrete behavioral responses tic tag in a behavioral response paradigm could answer of individual, free-ranging marine mammals to particu- targeted questions regarding the nature and magnitude lar sounds [19, 20]. These studies typically use animal- of responses of marine mammals to anthropogenic noise mounted archival motion-sensing (via pressure sensors and their potential physiological consequences, lead- and triaxial accelerometers and magnetometers) and ing to better informed evaluation of human-generated acoustic recording tags as well as visual observations to sound. measure individual focal animal response to a controlled exposure from a sound source deployed nearby. Meas- Methods urements of swim speed, dive depth, dive duration, head- Tag development ing, vocal behavior, group spacing, ascent and descent The prototype tag contained three subsystems: an active rate, and other metrics are made before, during, and after acoustic playback system, a passive acoustic record- the CEE in order to assess response [e.g., 21–25]. ing system, and a motion monitoring system (Fig.  1). Combining the sound source with behavioral sensors An OpenTag single board controller (SBC, Logger- and an acoustic recorder into a single animal-mounted head Instruments, Inc, Sarasota, FL, USA) controlled tag could enable an alternative BRS methodological playbacks and contained a three-axis accelerometer, approach. Such an instrument could offer a more cost- magnetometer, and gyroscope. It was programmed (Log- effective tool that would allow for better control of expo - gerhead Instruments, Arduino programming language) sure sound levels. While the exposure context is unique to execute a playback of a single stimulus at a predeter- in being physically attached to the animal, this con- mined time interval, using sound files stored on a SOMO text is consistent for all exposures whereas other BRS playback module (4D Systems, Minchinbury, NSW, Aus- approaches often have variability in the context of differ - tralia), through an amplifier board (LM48580 Evaluation ent exposures. Finally, it could enable investigation of the Board, Texas Instruments, Dallas, TX, USA) and two or effects of varied sound levels, multiple or sustained expo - three piezo electric ceramic cylinder transducers (Steiner sures, and behavioral habituation. & Martin, Inc, Doral, FL, USA; 26  mm external diame- The northern elephant seal, Mirounga angustirostris ter  ×  22 internal diameter  ×  13  mm high, resonant fre- [26], presents an ideal study species to test the effective - quency 43 ± 1.5 kHz). The playback system was powered ness of such a tag. Northern elephant seals are accessible by a 3.7  V, 3000  mAh lithium-ion polymer rechargeable as they haul out twice a year to breed and molt [27, 28]. battery (Tenergy, Fremont, CA, USA). Elephant seal diving behavior is well studied and found to Six playback stimuli were used: sperm whale (Physeter be highly stereotypic, with almost continuous, repetitive macrocephalus) clicks, common dolphin (Delphinus sp.) deep diving [28–31]. They regularly reach depths simi - whistles, killer whale (Orcinus orca) whistles and clicks, lar to many of the cetacean species thought to be most simulated mid-frequency active sonar, and white noise. affected by anthropogenic noise (i.e., beaked whales; [28, White noise and the simulated mid-frequency sonar were 32]) and dive as deep as or deeper than the deep sound created using Adobe Audition 3 (Adobe System Incorpo- channel (~1000  m). Previous studies have shown that rated, San Jose, CA, USA). Sperm whale clicks and com- carrying relatively large instruments does not inhibit the mon dolphin whistles were obtained from the Discovery ability of juvenile seals to swim or forage, and instrument of Sound in the Sea sound library. Both killer whale recovery rate is above 90  % [28, 33, 34]. Additionally, vocalizations were obtained from the Vancouver Aquar- northern elephant seals have acute underwater hearing ium. An inverse of the transducer’s transmitting sensi- sensitivity with relatively low hearing thresholds occur- tivity function was generated and used to normalize each ring over a very broad frequency range [35, 36]. High-res- playback’s output over the entire frequency bandwidth. olution behavioral responses to sound stimuli from our device can be collected for elephant seals using sensors http://www.dosits.org/audio/marinemammals/toothedwhales/. http://killerwhale.vanaqua.org/page.aspx?pid=1331. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 4 of 15 Table 1 Calibrated source levels for exposure stimuli Stimulus Source level White noise 125.56 (0.94) @ 1 m (dB re 1 µPa) RMS Common dolphin whistles 130.66 (1.48) @ 1 m (dB re 1 µPa) RMS Sperm whale clicks 131.77 (1.89) @ 1 m (dB re 1 µPa) 0-peak Killer whale whistles 121.06 (4.60) @ 1 m (dB re 1 µPa) RMS Killer whale clicks Estimated 126 @ 1 m (dB re 1 µPa) 0-peak Sonar 124.27 (0.58) @ 1 m (dB re 1 µPa) RMS Mean and standard deviation of two calibrated recordings are given. All sources levels are given in dB re 1 µPa @ 1 m over the stimulus duration, except for RMS the sperm whale click track , which was measured in dB re 1 µPa @ 1 m and SOMO 0-peak the killer whale click track which was measured by relative comparison to killer playback whale whistles module hydrophone (High Tech, Inc, Long Beach, MS, USA; OpenTag pre- power −1 controller sensitivity −190.5  dB re 1  V  µPa ; frequency response amplifier supply board 2 Hz–30 kHz) recorded for 30 min starting 15 min prior loudspeaker to each scheduled playback. This provided a recorded confirmation that a single playback occurred and allowed DSG passive power acoustic hydrophone detection of other environmental noise events occurring supply recorder at the time of the playback. Recordings were sampled at 32  kHz (16  bit resolution) with 20  dB of gain. The pas - sive acoustic system was powered by a second 3.7  V, 3000  mAh lithium-ion polymer battery (Tenergy, Fre- mont, CA, USA). All tag components were placed in an oil-filled Delrin housing, rated to 1500 m depth. Clear, food-grade min- eral oil was used as a non-compressible and non-conduc- tive filler so no air remained in the housing and to limit acoustic impedance between the tag and seawater. The tag package weighed approximately 1.5  kg (<1.2  % total Fig. 1 Prototype tag was built, calibrated, and field-tested on north- body mass of smallest seal) and was slightly negatively ern elephant seals (center black and orange tag). Tag components buoyant. The original tag design was modified follow - included a DSG passive acoustic recorder and hydrophone, an active ing the first three deployments. Changes in pressure and acoustic preamplifier, playback module, transducers, and OpenTag temperature between the surface and at depth caused single board controller with a nine-axis motion sensor (three axes each for accelerometer, magnetometer, and gyroscope). A Wildlife changes in mineral oil density. The slight flexibility of Computers Mk10-AF TDR was mounted on the head. An additional Delrin caused a pumping action to develop that moved TDR was attached to the shoulder but was not used in this study seawater past the o-ring seals into the oil-filled tag body. Larger o-rings were used, and flexible tubing with small air pockets was attached to the outside of the housing to allow minor changes in mineral oil density and limit sea- Playbacks of stimuli were limited to 30  s to reduce the water intrusion. possibility of seals associating the stimulus with the device mounted on their back. Playback track source lev- Field efforts els were measured using a calibrated hydrophone system We conducted field experiments in March and April (G.R.A.S. 42AC High Pressure Pistonphone, G.R.A.S. 2012 on juvenile northern elephant seals at Año Nuevo Sound & Vibration A/S, Holte, Denmark) and ranged State Park, San Mateo County, CA, USA (Fig. 2; Table 2). from 121 to 132 dB re 1 µPa at 1 m, depending on expo- Seals were instrumented with the active acoustic tag sure type (Table 1). Estimated received levels were calcu- placed on the animal’s back (30–60  cm from the ears) lated assuming spherical spreading, based on the distance and an ARGOS-Fastloc GPS time-depth recorder (TDR) of tag from the animals’ ears. on the head (Mk10-AF, Wildlife Computers, Redmond, A DSG acoustic recorder (Loggerhead Instruments, WA, USA). Tags were attached by gluing a flexible mesh Inc, Sarasota, FL, USA) connected to an HTI-96-MIN Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 5 of 15 Fig. 2 Tracks of all successful seal deployments. The track line is the straight-line connection between subsequent surface locations. Numbered black bars are approximate locations where exposures occurred, between the surface locations before and after the time of the exposure. Bathym- etry data are from NOAA’s National Geophysical Data Center (NGDC), Southern California Coastal Relief Map, one-arc second resolution base to the animal’s fur using quick-cure adhesive (Dev- CA, USA, and released. Seals were recaptured upon con 14270—5 Minute Epoxy, ITW Polymers Adhesives return to Año Nuevo, and tags (including the metal ties) North America, Danvers, MA, USA), weaving stainless were manually removed from the flexible mesh base. steel locking ties through the mesh, and securing the tag Seals molted off the base shortly after tag recovery. Depth with the locking ties (similar to [30, 38]). Animals were was sampled every 1 s for seal G5449 and every 4 s for all other seals. Resolution of TDRs were 0.5 m and accuracy transported approximately 100  km south to Monterey, Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 6 of 15 Table 2 Summary dive statistics for all study individuals Seal Age Sex Body Deployment Number Number Mean dive depth (m) Mean dive duration (s) Median dive duration (s) mass (kg) duration (h) of dives of dives All dives Dives deeper All dives Dives deeper All dives Dives deeper recorded deeper than 100 m than 100 m than 100 m than 100 m (% of total) G5449 1 M 134 207.22 972 509 (52.4 %) 136.618 (±106.0357) 217.114 (±84.7149) 646.6337 (±267.9752) 829.34 (±191.822) 637 815 G5520 1 F 167 63.11 402 20 (5.0 %) 36.6157 (±53.6568) 223.425 (±116.878) 365.34.33 (±251.5771) 958.0 (±398.2795) 340 868 G6009 1 F 182 121.26 518 124 (23.9 %) 94.9913 (±128.4333) 290.395 (±129.899) 608.7259 (±436.105) 1185.8 (±324.3298) 556 1236 G6110 2 F 262 30.24 148 28 (18.9 %) 75.0811 (±108.5156) 258.375 (±165.32) 585.4324 (±379.2734) 1146.3 (±279.5604) 526 1146 G6651 1 F 206 73.01 321 115 (35.8 %) 145.829 (±167.2949) 339.274 (±136.802) 748.2991 (±580.3701) 1419.1 (±364.688) 560 1488 Age was either known from initial tagging as a pup or estimated in the field based on standard length and weight. Sex was assessed and body mass was measured before instrumentation. Deployment duration was dependent on the time it took the seal to return to the beach. Number of dives recorded includes all instances where the animal dove deeper than 4 m. Mean (and SD) dive duration, in min, are given for all dives and the subset of dives deeper than 100 m Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 7 of 15 ±1 m. The accelerometer, magnetometer, and gyroscope ambient noise levels, and thus a decreased signal-to- were all sampled at 50 Hz. noise ratio of the low-level stimulus, near the water’s Playbacks occurred every 6 h for the first two seals and surface. every 3  h for the remaining seals. Each playback con- To determine potential responses during ascent or sisted of one exposure to a single stimulus. The order of bottom phases of deep (>100  m) dives, dive inversions stimuli presentation varied between seals, but remained (Fig.  4) were measured and compared. Dive inversions the same within an individual seal, unless the tag reset were defined as a change from ascending or horizontal itself, at which point it started back at the beginning of swimming to descending, with a change in depth >3  m the sequence. over 4  s. Change in depth and change in time from the inversion point to the next maximum depth were meas- Behavioral analysis ured and normalized by maximum depth and total dive Passive recordings were screened for successful expo- duration, respectively. In order to statistically compare sures (Table  3), and timing of exposures across multi- dive inversions following exposures to dive inversions ple tag systems was synced. Depth data from the TDRs outside of exposure events, an Anderson–Darling k-sam- were examined for possible seal responses to exposures ple (ADK; [40]) test was run on seal G6651, the only seal (Fig.  3). To account for zero offset drift, dive data were to receive multiple exposures during the ascending phase. analyzed using the IKNOS toolbox (Y. Tremblay, unpub- The ADK test was selected because it is distribution free lished) in MATLAB (MathWorks, Inc, Natick, MA, and does not rely on equal sample sizes; however, it USA). Dives were defined as any instance where the indi - requires that samples are taken from independent distri- vidual dove deeper than 4 m. This threshold was chosen butions, in this case, different seals. G5449 was chosen to be greater than the sum of the accuracy of the TDR for comparison as it had the largest deployment duration (±1 m) and the body length of an individual. and therefore the most non-exposure dive inversions. To We hypothesized that responses would differ accord - first test whether different seals could be directly com - ing to dive type and state, so different dives and expo - pared, an ADK test was used to compare non-exposure sures were analyzed separately. Dive shapes were used dive inversions for seals G6651 and G5449. The magni - to identify different dive types (e.g., transit vs. foraging tude of the dive inversion following the single playback [39]). Dives were grouped into deep (>100  m) and shal- during the bottom phase of a deep dive was measured low (≤100  m) dives (Table  3). Exposures were catego- and reported. rized by dive phase (descent, ascent, bottom phase, and Descent rates for descending phases of deep dives were surface; Table  3). Descents were defined as the segment measured and compared to identify possible responses from 0  m to the point at which the seal descended at a during descents. Average descent rate over 60  s before −1 rate <0.1 m s for 20 s. The beginning of an ascent was and 60  s after exposure onset were calculated. A time defined as the point at which an individual ascended for window of 60  s was selected in order to differenti - −1 more than 4  m over 20  s (an ascent rate of 0.2  m  s ). ate sustained changes in descent rate from brief startle Bottom phases were defined as the segment between responses during the playback only. Because an increase the descending and ascending segments. Exposures that in descent rate is considered a subtle response that likely occurred at depths shallower than 20  m were labeled as happens regularly in non-exposure dives, statistical anal- “surface” and excluded from all analyses due to increased ysis of changes in descent rate was not performed. Table 3 Deployment duration and number of exposures for the successful deployments of the prototype tag Seal Exposure Number of  Exposures by dive stage interval (h) exposures received Surface Descent Bottom phase Ascent Deep Shallow Deep Shallow Deep Shallow G5449 3 6 2 – – 1 1 – 2 G5520 3 2 – – – 1 – 1 – G6009 6 2 – – – – – – 2 G6110 6 4 1 – – – 1 – 2 G6651 3 24 7 1 1 3 7 1 4 The number of exposures was dependent on the success of the tag and the length of the deployment. Exposures during descent, bottom phase, and ascent are the subset of exposures used for analysis in each respective phase, split by shallow (<100 m) and deep (>100 m) exposures. Surface exposures were excluded from analysis Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 8 of 15 G5449 -100 -200 -300 -400 -500 0 20 40 60 80 100120 140160 180200 G5520 -100 -200 -300 -400 -500 05 10 15 20 25 30 35 40 45 G6009 -100 -200 -300 -400 -500 -600 G6110 -100 -200 -300 -400 -500 -600 -700 05 10 15 20 25 G6651 -200 -400 -600 -800 -1000 time (hours) Fig. 3 Depth profiles and seafloor bathymetry for complete deployments for all seals. Seal dive profile is in blue, seafloor bathymetry is in gray, and exposures are indicated by black dots. Bathymetry data are from NOAA’s NGDC, Southern California Coastal Relief Map, one-arc second resolution. Instances where a dive profile overlaps the seafloor are an artifact of data resolution mismatch; bathymetry data were extracted only for known GPS surface positions and linearly connected in the gray bathymetry plot, leading to areas with reduced detail depth (meters) depth (meters) depth (meters) depth (meters) depth (meters) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 9 of 15 G5449 Exposure 2 G6110 Exposure 3 G6651 Exposure 3 0 0 -100 -100 -100 -300 -300 -300 -500 -500 -500 470 490 510 1085 1105 1125 440 460 480 G6651 Exposure G6651 Exposure 5 G6651 Exposure 9 -100 -100 -100 -300 -300 -300 -500 -500 -500 610 630 650 802 822 842 1520 1540 1560 G6651 Exposure 12 G6651 Exposure 18 G6651 Exposure 21 0 0 -100 -100 -100 -300 -300 -300 -500 -500 -500 2060 2080 2100 3135 3155 3175 3685 3705 3725 time (minutes) sperm whale clicks white noise common dolphin whistles killer whale whistles killer whale clicks simulated sonar Fig. 4 Dive profiles for all exposures received during ascending phases of dives from seals G5449, G6110, and G6651. Shapes indicate time of exposure and type Shallow dives occurred along the shelf edge and were different stimuli, each playback was categorized as either limited by the seafloor so diving deeper or reversing an “response” or “no response,” using a minimum level of ascent was not possible. To identify possible responses response based on percent change, which varied for each for these shallow dives, changes in flow noise on the dive state (ascending, bottom, descending, or shallow/ passive acoustic recorder (a proxy for changes in swim bottom-limited). Ascending or bottom-phase playbacks speed) were measured and compared. Previous studies were categorized as “response” if there was a dive inver- have found an 18- to 20-dB increase at very low frequen- sion magnitude >2 SDs from non-playback inversions. cies (8–18 Hz) corresponds to a doubling of current flow, Descending playbacks were considered a response if regardless of tag or hydrophone design [34, 41]. Average descent rate increased by >50 % following exposure, and RMS in the 8–18  Hz frequency band was measured for for shallow dives, an increase in flow noise of >10 % was 30–60 and 0–30 s before exposure and 0–30 and 30–60 s considered a response. Percent of overall response was after the exposure ended (Adobe Audition CC, Adobe calculated. Systems Incorporated, San Jose, CA, USA). The before and after periods were divided to detect shorter-term Results changes in swim speed, but the entire 60-s period was Tag development taken into account when assessing a response. Seven deployments of the prototype tag resulted in suc- The normalized change in depth for exposure and cessful CEEs for five individuals (Table  2). Received levels non-exposure dive inversions for seal G6651 was plot - at the individuals’ ears, estimated from the source level ted against time to investigate possible habituation to and assuming spherical spreading, ranged from 128 to multiple playbacks over the duration of the deploy- 138  dB re 1  µPa, depending on seal and stimulus type. ment. To investigate differential response of all seals to Mechanical (flooding) or electrical (tag control board depth (m) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 10 of 15 reset) tag failure rendered the remaining two unsuccess- during the bottom phase of a deep dive (351 m). She dove ful. The playback control of the tag would occasionally to 614 m immediately following exposure, a 75 % increase experience a reset for undetermined reasons, resulting in maximum depth of dive (killer whale whistle exposure, in a stimulus that was played out of sequence. In some Table 3). cases, this reset caused the tag to stop working entirely. Three seals received exposures during the descending phase of a deep dive, and two of three (G5549 and G6651) Deployments and basic dive behavior exhibited an increased descent rate (35.9–271.9 %; mean Five successful deployments ranged from 30 to 207  h in 125.2  %; ±95.8  %) following exposure (Fig.  6; 9 of 10 duration. Animals received 2–24 playbacks, each a sin- total deep descending exposures). Seal G6110 had a gle 30-s exposure, at an interval of 3 or 6  h, and non- lower descent rate following exposure to sonar (72.7  % exposure dives served as control dives (Table  3; Fig.  2). decrease). Four of five animals dove along the seafloor (depths up Flow noise (swim speed) increased by 10.0 (±8.0)  dB to ~100  m) until reaching the continental shelf edge, re 1 µPa from 0 to 30 s before exposure to 30–60 s after after which they exhibited deeper, pelagic transit dives exposure (30.3  ±  14.7  % increase) in all 8 exposures (to with mean depths of 217–340  m over the deep water of three seals) that occurred in shallow water (<100 m). For Monterey Canyon. They returned to the shallow dive pat - two of the exposures, flow noise initially decreased in tern when returning to shallow coastal waters near Año the 0–30  s immediately after exposure but by 60  s after, Nuevo State Park (Fig.  3). We found no evidence of for- reached above pre-exposure levels. aging or drift dives, and the observed dive patterns were Dive inversion responses seal G6651 were as pro- similar to those of other translocated juveniles [13, 42]. nounced as or more pronounced than those later in the Dive depths <100  m were likely limited by the seafloor, deployment compared to earlier (Fig. 7). Seals responded while deeper dives were not bottom-limited, which may to 23 of 28 total exposures (82 %), regardless of stimulus have an effect on responses measured (Fig.  3) [43]. One type, indicating no differential response. individual (G5520) rarely dove deeper than 100 m (5.0 % of all dives), even over deep water (Fig.  3). We found no Discussion significant difference in magnitude of non-exposure dive We identify behavioral responses in juvenile northern inversions for seals G5449 and G6651 (ADK, p  =  0.14, elephant seals elicited by an animal-mounted sound n = 283 non-exposure inversions, seal G5449 and n = 30 source. This is the first dedicated BRS conducted on any non-exposure inversions, seal G6651). Both exposures to marine mammal using an animal-borne sound source seal G6009 were received at <10  m depth, so they were (see [44] for incidental BRS with an active sonar tag). excluded from further analyses. After this exclusion, This approach has the potential to expand and comple - there were 28 usable exposures on four seals, made up of ment current CEE methodologies. Such data will pro- 9 ascending, 10 descending, 1 bottom phase, and 8 shal- vide enhanced understanding of longer-term effects of low water exposures. anthropogenic noise. Controlled exposure experiment Tag performance We observed dive inversions following all playbacks that Deployments that resulted in unsuccessful or reduced occurred during the ascending portion of a deep dive. duration CEEs were due to issues with either the SBC or Individuals descended an additional 116 m (SD ±51.6 m) the housing. We were unable to resolve the cause of the on average (Fig.  4). 8 of 9 exposures resulted in changes SBC reset, but believe it was caused by the formation of in depth >2 SDs from the mean of all non-exposure inver- a ground loop due to differential ground potentials of the sions (Figs.  4, 5; Table  4). Seal G6651 was the only seal independent hardware components comprising the sin- which received multiple exposures during ascent (n  =  7 gle tag system. Although the housing was rated to 1500 m exposures), and the magnitudes of dive inversions follow- depth, either changes in temperature and pressure with ing these exposures were statistically different than non- depth or incorrectly sized o-rings likely caused failure exposure inversions (Fig. 5; Table 4; ADK test, p < 0.001, of the slightly flexible Delrin housing, allowing seawa- n = 283 non-exposure inversions, seal G5449 and n =  7 ter to enter the housing and short circuit the electron- exposure inversions, seal G6651). We observed this dive ics. Future tags would benefit greatly from being potted inversion response for white noise, sperm whale clicks, in solid resin; however, the non-permanent setup used in killer whale whistles, and simulated mid-frequency sonar this trial allowed us to monitor and modify physical tag exposure, but not following an exposure to common dol- components and programming as needed during the field phins, received by seal G6651 (Fig. 4, G6651 Exposure 4). effort. Additionally, we found no evidence of instrumen - Seal G6651 was the only seal that received an exposure tation or drag affecting normal swimming behavior, as Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 11 of 15 G5449 0.4 n=283 and 1 0.3 0.2 0.1 G6110 0.4 n=8 and 1 0.3 0.2 0.1 non-exposures white noise common dolphin whistles G6651 0.4 sperm whale clicks n=30 and 7 killer whale whistles 0.3 + killer whale clicks simulated sonar 0.2 0.1 0 0.1 0.2 0.3 0.40.5 0.60.7 normalized change in depth Fig. 5 Plot of normalized change in depth versus normalized change in time for all dive inversions during the ascending phase of all deep dives (>100 m) for seals G5449, G6110, and G6651. Black dots symbolize non-exposure dive inversions, and open symbols indicate dive inversions following exposures Table 4 Mean change in depth for non-exposure and exposure dive inversions Seal Mean change in depth for  Mean change in depth Anderson–Darling k-sample test non-exposure inversions (m) for exposure inversions (m) G5449 14.875 (±13.997) n = 283 70.5 n = 1 n/a G6110 16.749 (±16.293) n = 8 186 n = 1 n/a G6651 24.867 (±39.981) n = 30 96.36 (±59.23) n = 7 p = <0.001 Dive inversions are defined by a change from ascending to descending >3 m over 4 s, for all dives deeper than 100 m. Standard deviation is given in parentheses, and n is given for each. Results from an Anderson–Darling k-sample test comparing the change in depth of an inversion from ascending to descending, normalized by maximum depth of dive, for exposure inversions of seal G6651 to non-exposure inversions from seal G5449 (n = 283) are given dive profiles were similar to those of other translocated to smaller-scale dive variations seen naturally in these seals carrying smaller devices [13, 42, 45]. and other translocated seals carrying a depth sensor [13, 46]. The exposure context here is unique in terms of the Animal response to disturbance extreme proximity of the sound source to the animal, and Behavioral responses measured during CEEs varied the extrapolation of results to longer-range exposures with dive state at the time of exposure. Most responses should thus be considered cautiously. However, this con- involved seals diving deeper and/or longer after expo- text is more constant than in previous studies providing a sure. The clearest responses (Fig.  4; Table 4) involved dive stronger basis for the comparison of responses within an inversions (reversal of an ascent) followed by the seal div- individual across exposure conditions. ing deeper than the initial maximum dive depth. These Responses observed are similar to hypothesized ele- extended dive inversions served as strong indicators phant seal behavior to escape predators by diving deeper of response. Quantitatively, dive inversions resulted in [37], and responses to anthropogenic sound sources are greater depths and longer dive durations, when compared not unexpected [13, 44]. Responses elicited during CEEs normalized change in time Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 12 of 15 for observed responses. In future studies, initiating Descent Rates Before and After Exposure exposure during one dive phase would control experi- 2 before exposure mental variability and allow robust statistical analyses of after exposure responses. 1.5 We suspect the tag reset described above, and the resulting stimulus playback, is the most likely explana- tion for three non-exposure dive inversions of similar magnitude to inversions following exposures observed in 0.5 seal G6651 (Fig.  5). The inversions could have occurred naturally (e.g., exposure to an external alarming sound) or from an accidental exposure by the prototype tag fol- G5449 G6110 G6651 lowing a reset; however, the independent passive acous- exposure number and type white noise common dolphin whistles sperm whale clicks tic system was not recording so we cannot know for killer whale whistles + killer whale clicks simulated sonar sure. Continuous recording of ambient noise on future −1 Fig. 6 Mean descent rates, in m s , for seals G5449, G6110, and tag iterations, and longer-duration deployments, would G6651 during the 60 s before (white bars) and 60 s after (black bars) allow for potential opportunistic measurement of behav- exposure. Open symbols correspond to exposure type ioral responses to naturally occurring threatening sounds such as predators, boats, or other man-made or tag-made sounds. G6651 0.5 Differential response by stimulus type 0.45 We found no indication of differential responses based on 0.4 stimulus type or order (Table 5). Notably, seals responded 0.35 to most (82  %) but not all exposures, indicating a possi- non-exposures ble differential response that was not detected because of 0.3 white noise common dolphin whistles limited sample size. 0.25 sperm whale clicks The observed results suggest that the response may be killer whale whistles 0.2 killer whale clicks a result of acoustic limitations of the playback apparatus simulated sonar 0.15 rather than the stimuli (see [48]). Limitations included 0.1 the nonlinear nature of the small transducers and 0.05 extreme proximity of the source to the animal. This prox - imity likely created an unusual perceptual context com- 12 24 36 48 60 pared to a sound produced naturally at realistic ranges time (hours) due to the effects of sound propagation (e.g., rever - Fig. 7 Plot of normalized change in depth versus elapsed time (in beration, directional cues, and the relative presence or h) for all dive inversions during the ascending phase of all deep absence of harmonics). Many details of pinniped under- dives (>100 m) for seal G6651. Black dots symbolize non-exposure dive inversions, and open symbols indicate dive inversions following water hearing, including directionality, are still poorly exposures understood [36, 49, 50]. Sudden sounds may trigger a response, regardless of the sound type or the distance it were similar to those found by other BRSs on deep-div- Table 5 Differences in  response rate by  exposure type ing marine mammals. For instance, dive inversions and and for all exposures to all seals remaining at depth longer were demonstrated in two spe- cies of beaked whale exposed to simulated mid-frequency Exposure Number of exposures Response measured sonar from an external, ship-mounted sound source [22, White noise 6 5 47]. A third species of beaked whale responded to expo- Common dolphin 4 2 sures of both sonar and killer whale sounds by inter- whistles rupting foraging and performing an unusually long and Sperm whale clicks 5 5 shallow ascent [12]. Killer whale whistles 6 5 Responses to exposures that occurred during descents Killer whale click 1 1 or during bottom-limited dives were more difficult to Sonar 6 5 measure. Natural variation in swim speed and descent Total 28 23 angle limited our ability to define statistical significance -1 normalized change in depth descent rate (ms ) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 13 of 15 is perceived to be coming from. Future testing using play- beginning to better understand how animals behaviorally back stimuli that are filtered to simulate different source respond to sound, but we still do not fully understand locations could examine the effects of reverberation and the underlying physiological changes that are linked to localization in detail. Comparable parallel CEEs would these behavioral changes (e.g., oxidative stress). Physi- need to be conducted on pinnipeds, using an external ological parameters such as oxygen utilization, heart rate, sound source that closely approximate actual signals, to and post-dive recovery times can be monitored in free- fully evaluate the potential of this tag for BRS. ranging animals [53, 54], and with this tag, dives can be While individual differences could also contribute experimentally extended, allowing quantitative investiga- to different responses, no single seal received multiple tion of physiological responses. To support the potential exposures of all exposure types because of tag failures, use of this tag to study physiological effects of extended which precludes further examination of this possibility. dives possibly related to responses to anthropogenic BRS exposing animals to multiple exposures of all stimuli noise, it is promising that the responses were similar to are needed to investigate individual differences to stimuli. other deep-diving species of interest. For species that are A differential response to dolphin whistles was recorded specialized for oxygen efficiency during prolonged, deep for seal G6651, the only seal exposed to two playbacks of dives, physiological responses to changes in planned dive dolphin whistles while ascending from a dive. No extreme duration could help physiologists better understand these dive inversions were recorded for dolphin whistles, while adaptations [55]. other stimuli resulted in extreme evasive behavior. This anecdotal example provides evidence for the possibility Conclusions of differential response to different stimuli. This pilot study was the first of its kind to investigate Repeated exposures to various stimuli may cause habit- the potential use of an animal-borne sound source to uation over time [51]. Although there was no evidence of conduct BRS on marine mammals to further investigate habituation over short-term deployments (<3  days), we the effects of anthropogenic noise. Five juvenile north - cannot rule out the possibility of a sensitization response. ern elephant seals were instrumented and translocated The consistent response to most exposures independ - south of their colony at Año Nuevo State Park. They ent of stimulus type and the apparent lack of reduction received playbacks of multiple stimulus types (both in response within individuals suggest either that seals man-made and natural) from the tag while diving con- are generally sensitive to a relatively wide range of audi- tinuously on their return to the colony. Dive behavior ble exposures or possibly that seals became sensitized to before and after exposures were compared to assess subsequent exposure. Preliminary evidence from captive whether animal-borne tags holding sound sources were elephant seals suggests some sensitization, as opposed to capable of eliciting a response from a free-ranging ani- habituation, upon repeated exposure to certain acous- mal and whether potential responses differed with stim - tic stimuli [52]. However, the sound source placement, ulus content. context, and limited number of exposures of each stimu- Projecting sound from a tag mounted to the back lus type to each individual limited our ability to test for of a free-ranging juvenile northern elephant seal did potential sensitization. elicit behavioral responses; however, the responses The tag and method may be useful in physiological were not consistently different with different stimulus studies of dive limits and studies of hearing ranges. The types. Responses varied by seal, by dive state, and by the tag could be used as a mobile, programmable sound bathymetry where the exposure occurred, but in general, source to study frequency-dependent hearing in marine seals dove deeper following playback. Animals responded mammal species that cannot be studied using captive to 82 % of exposures overall, with no clear evidence of a psychophysical methods. Little is known about hearing reduction in response to repeated exposures of various in many marine mammals because laboratory studies of stimuli. The unique and biologically unrealistic context hearing are not feasible due to constraints with keeping of a sudden sound exposure coming from directly behind very large, migratory, deep-diving, and social species in the animal is likely the greatest limiting factor preventing captivity, but using controlled, incrementally increas- use of this tag in examining differential responses to par - ing exposure levels of tonal sounds that trigger drastic ticular sounds and relating them to responses that may changes in behavior could investigate differences in hear - occur over more realistic source-animal ranges. Mak- ing sensitivities in previously inaccessible species. ing conclusions about the effects of particular sounds on Additionally, this tag could be used to study the physi- juvenile northern elephant seals was beyond the scope of ological effects of unanticipated extensions of dives, this pilot study, but additional tests using this technology another important research need for understanding may help develop additional questions and hypotheses in the effects of noise on marine mammals [18]. We are the future. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 14 of 15 Received: 30 October 2015 Accepted: 11 March 2016 Tag improvements and additional field testing could strengthen results found here, but the concept of an animal-borne sound source for triggering behavio- ral responses has been validated. The use of this novel animal-borne sound source in future studies could help References 1. 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Copyright © 2016 by Fregosi et al.
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Abstract

Background: There is a variety of evidence that increased anthropogenic noise (e.g., shipping, explosions, sonar) has a measureable effect on marine mammal species. Observed impacts range in severity from brief interruptions of basic life functions to physiological changes, acute injury, and even death. New research tools are needed to better meas- ure and understand the potential effects of anthropogenic noise on marine mammals. Current behavioral response studies typically utilize ship-based sound sources to study potential acute behavioral responses in tagged animals experimentally exposed to noise. Integrating the sound source within animal-mounted passive acoustic and motion- sensing tags provides a novel tool for conducting additional highly controlled response studies. Results: We developed and conducted pilot field trials of a prototype tag on five juvenile northern elephant seals, Mirounga angustirostris, using experimental exposures to both natural and anthropogenic noise stimuli. Results indicate behavioral responses were elicited in tagged individuals. However, no pattern was found in the occurrence and types of response compared to stimulus type. Responses during the ascending dive phase consisted of a dive inversion, or sustained reversal from ascending to descending (8 of 9 exposures). Dive inversions following exposure were 4–11 times larger than non-exposure inversions. Exposures received during the descending dive phase resulted in increased descent rates in 9 of 10 exposures. All 8 exposures during dives in which maximum dive depth was lim- ited by bathymetry were characterized by increased flow noise in the audio recordings following exposure, indicating increased swim speed. Conclusions: Results of this study demonstrate the ability of an animal-mounted sound source to elicit behavioral responses in free-ranging individuals. Behavioral responses varied by seal, dive state at time of exposure, and bathym- etry, but followed an overall trend of diving deeper and steeper and swimming faster. Responses did not consistently differ based on stimulus type, which may be attributable to the unique exposure context of the very close proximity of the sound source. Further technological development and focused field efforts are needed to advance and apply these tools and methods in subsequent behavioral response studies to address specific questions. Keywords: Acoustic, Tag, Behavioral response, Northern elephant seal, Controlled exposure experiment and predator avoidance [1]. Anthropogenic activities Background have contributed to increased ocean ambient noise lev- Marine mammals rely on acoustic cues for many life els in certain areas [2, 3]. The potential adverse effects functions including navigation, foraging, communication, of both acute and chronic human-generated noise on *Correspondence: selene.fregosi@oregonstate.edu marine mammals are a major conservation concern [e.g., Cooperative Institute for Marine Resources Studies, Hatfield Marine 4–14]. Naval sonar activities have been linked to cetacean Science Center, Oregon State University and NOAA Pacific Marine stranding events, where necropsies have shown physical Environmental Laboratory, 2030 SE Marine Science Drive, Newport, OR 97365, USA damage to vital organs [5, 6]. Noise has also been shown Full list of author information is available at the end of the article © 2016 Fregosi et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 3 of 15 to increase stress hormone levels [9], reduce foraging that measure changes in pressure, acceleration, strength activity [8, 15], alter migration routes [16], mask com- of magnetic field, and turn rate [13, 37]. munication sounds [10], and displace marine mammals Here we describe a pilot study testing an animal- from primary feeding and breeding grounds [11, 12, 15]. mounted active acoustic and motion-sensing tag on free- Understanding the differential responses of marine mam - ranging juvenile northern elephant seals. The main goals mals to noise stimuli is necessary for managing human of this study were (1) to test whether an animal-borne impacts in the ocean [17, 18]. acoustic tag elicits any behavioral response and if so (2) Behavioral response studies (BRSs) using a controlled to determine whether responses are related to stimulus exposure experiment (CEE) paradigm have emerged as content. Development and application of an active acous- an effective tool to study discrete behavioral responses tic tag in a behavioral response paradigm could answer of individual, free-ranging marine mammals to particu- targeted questions regarding the nature and magnitude lar sounds [19, 20]. These studies typically use animal- of responses of marine mammals to anthropogenic noise mounted archival motion-sensing (via pressure sensors and their potential physiological consequences, lead- and triaxial accelerometers and magnetometers) and ing to better informed evaluation of human-generated acoustic recording tags as well as visual observations to sound. measure individual focal animal response to a controlled exposure from a sound source deployed nearby. Meas- Methods urements of swim speed, dive depth, dive duration, head- Tag development ing, vocal behavior, group spacing, ascent and descent The prototype tag contained three subsystems: an active rate, and other metrics are made before, during, and after acoustic playback system, a passive acoustic record- the CEE in order to assess response [e.g., 21–25]. ing system, and a motion monitoring system (Fig.  1). Combining the sound source with behavioral sensors An OpenTag single board controller (SBC, Logger- and an acoustic recorder into a single animal-mounted head Instruments, Inc, Sarasota, FL, USA) controlled tag could enable an alternative BRS methodological playbacks and contained a three-axis accelerometer, approach. Such an instrument could offer a more cost- magnetometer, and gyroscope. It was programmed (Log- effective tool that would allow for better control of expo - gerhead Instruments, Arduino programming language) sure sound levels. While the exposure context is unique to execute a playback of a single stimulus at a predeter- in being physically attached to the animal, this con- mined time interval, using sound files stored on a SOMO text is consistent for all exposures whereas other BRS playback module (4D Systems, Minchinbury, NSW, Aus- approaches often have variability in the context of differ - tralia), through an amplifier board (LM48580 Evaluation ent exposures. Finally, it could enable investigation of the Board, Texas Instruments, Dallas, TX, USA) and two or effects of varied sound levels, multiple or sustained expo - three piezo electric ceramic cylinder transducers (Steiner sures, and behavioral habituation. & Martin, Inc, Doral, FL, USA; 26  mm external diame- The northern elephant seal, Mirounga angustirostris ter  ×  22 internal diameter  ×  13  mm high, resonant fre- [26], presents an ideal study species to test the effective - quency 43 ± 1.5 kHz). The playback system was powered ness of such a tag. Northern elephant seals are accessible by a 3.7  V, 3000  mAh lithium-ion polymer rechargeable as they haul out twice a year to breed and molt [27, 28]. battery (Tenergy, Fremont, CA, USA). Elephant seal diving behavior is well studied and found to Six playback stimuli were used: sperm whale (Physeter be highly stereotypic, with almost continuous, repetitive macrocephalus) clicks, common dolphin (Delphinus sp.) deep diving [28–31]. They regularly reach depths simi - whistles, killer whale (Orcinus orca) whistles and clicks, lar to many of the cetacean species thought to be most simulated mid-frequency active sonar, and white noise. affected by anthropogenic noise (i.e., beaked whales; [28, White noise and the simulated mid-frequency sonar were 32]) and dive as deep as or deeper than the deep sound created using Adobe Audition 3 (Adobe System Incorpo- channel (~1000  m). Previous studies have shown that rated, San Jose, CA, USA). Sperm whale clicks and com- carrying relatively large instruments does not inhibit the mon dolphin whistles were obtained from the Discovery ability of juvenile seals to swim or forage, and instrument of Sound in the Sea sound library. Both killer whale recovery rate is above 90  % [28, 33, 34]. Additionally, vocalizations were obtained from the Vancouver Aquar- northern elephant seals have acute underwater hearing ium. An inverse of the transducer’s transmitting sensi- sensitivity with relatively low hearing thresholds occur- tivity function was generated and used to normalize each ring over a very broad frequency range [35, 36]. High-res- playback’s output over the entire frequency bandwidth. olution behavioral responses to sound stimuli from our device can be collected for elephant seals using sensors http://www.dosits.org/audio/marinemammals/toothedwhales/. http://killerwhale.vanaqua.org/page.aspx?pid=1331. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 4 of 15 Table 1 Calibrated source levels for exposure stimuli Stimulus Source level White noise 125.56 (0.94) @ 1 m (dB re 1 µPa) RMS Common dolphin whistles 130.66 (1.48) @ 1 m (dB re 1 µPa) RMS Sperm whale clicks 131.77 (1.89) @ 1 m (dB re 1 µPa) 0-peak Killer whale whistles 121.06 (4.60) @ 1 m (dB re 1 µPa) RMS Killer whale clicks Estimated 126 @ 1 m (dB re 1 µPa) 0-peak Sonar 124.27 (0.58) @ 1 m (dB re 1 µPa) RMS Mean and standard deviation of two calibrated recordings are given. All sources levels are given in dB re 1 µPa @ 1 m over the stimulus duration, except for RMS the sperm whale click track , which was measured in dB re 1 µPa @ 1 m and SOMO 0-peak the killer whale click track which was measured by relative comparison to killer playback whale whistles module hydrophone (High Tech, Inc, Long Beach, MS, USA; OpenTag pre- power −1 controller sensitivity −190.5  dB re 1  V  µPa ; frequency response amplifier supply board 2 Hz–30 kHz) recorded for 30 min starting 15 min prior loudspeaker to each scheduled playback. This provided a recorded confirmation that a single playback occurred and allowed DSG passive power acoustic hydrophone detection of other environmental noise events occurring supply recorder at the time of the playback. Recordings were sampled at 32  kHz (16  bit resolution) with 20  dB of gain. The pas - sive acoustic system was powered by a second 3.7  V, 3000  mAh lithium-ion polymer battery (Tenergy, Fre- mont, CA, USA). All tag components were placed in an oil-filled Delrin housing, rated to 1500 m depth. Clear, food-grade min- eral oil was used as a non-compressible and non-conduc- tive filler so no air remained in the housing and to limit acoustic impedance between the tag and seawater. The tag package weighed approximately 1.5  kg (<1.2  % total Fig. 1 Prototype tag was built, calibrated, and field-tested on north- body mass of smallest seal) and was slightly negatively ern elephant seals (center black and orange tag). Tag components buoyant. The original tag design was modified follow - included a DSG passive acoustic recorder and hydrophone, an active ing the first three deployments. Changes in pressure and acoustic preamplifier, playback module, transducers, and OpenTag temperature between the surface and at depth caused single board controller with a nine-axis motion sensor (three axes each for accelerometer, magnetometer, and gyroscope). A Wildlife changes in mineral oil density. The slight flexibility of Computers Mk10-AF TDR was mounted on the head. An additional Delrin caused a pumping action to develop that moved TDR was attached to the shoulder but was not used in this study seawater past the o-ring seals into the oil-filled tag body. Larger o-rings were used, and flexible tubing with small air pockets was attached to the outside of the housing to allow minor changes in mineral oil density and limit sea- Playbacks of stimuli were limited to 30  s to reduce the water intrusion. possibility of seals associating the stimulus with the device mounted on their back. Playback track source lev- Field efforts els were measured using a calibrated hydrophone system We conducted field experiments in March and April (G.R.A.S. 42AC High Pressure Pistonphone, G.R.A.S. 2012 on juvenile northern elephant seals at Año Nuevo Sound & Vibration A/S, Holte, Denmark) and ranged State Park, San Mateo County, CA, USA (Fig. 2; Table 2). from 121 to 132 dB re 1 µPa at 1 m, depending on expo- Seals were instrumented with the active acoustic tag sure type (Table 1). Estimated received levels were calcu- placed on the animal’s back (30–60  cm from the ears) lated assuming spherical spreading, based on the distance and an ARGOS-Fastloc GPS time-depth recorder (TDR) of tag from the animals’ ears. on the head (Mk10-AF, Wildlife Computers, Redmond, A DSG acoustic recorder (Loggerhead Instruments, WA, USA). Tags were attached by gluing a flexible mesh Inc, Sarasota, FL, USA) connected to an HTI-96-MIN Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 5 of 15 Fig. 2 Tracks of all successful seal deployments. The track line is the straight-line connection between subsequent surface locations. Numbered black bars are approximate locations where exposures occurred, between the surface locations before and after the time of the exposure. Bathym- etry data are from NOAA’s National Geophysical Data Center (NGDC), Southern California Coastal Relief Map, one-arc second resolution base to the animal’s fur using quick-cure adhesive (Dev- CA, USA, and released. Seals were recaptured upon con 14270—5 Minute Epoxy, ITW Polymers Adhesives return to Año Nuevo, and tags (including the metal ties) North America, Danvers, MA, USA), weaving stainless were manually removed from the flexible mesh base. steel locking ties through the mesh, and securing the tag Seals molted off the base shortly after tag recovery. Depth with the locking ties (similar to [30, 38]). Animals were was sampled every 1 s for seal G5449 and every 4 s for all other seals. Resolution of TDRs were 0.5 m and accuracy transported approximately 100  km south to Monterey, Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 6 of 15 Table 2 Summary dive statistics for all study individuals Seal Age Sex Body Deployment Number Number Mean dive depth (m) Mean dive duration (s) Median dive duration (s) mass (kg) duration (h) of dives of dives All dives Dives deeper All dives Dives deeper All dives Dives deeper recorded deeper than 100 m than 100 m than 100 m than 100 m (% of total) G5449 1 M 134 207.22 972 509 (52.4 %) 136.618 (±106.0357) 217.114 (±84.7149) 646.6337 (±267.9752) 829.34 (±191.822) 637 815 G5520 1 F 167 63.11 402 20 (5.0 %) 36.6157 (±53.6568) 223.425 (±116.878) 365.34.33 (±251.5771) 958.0 (±398.2795) 340 868 G6009 1 F 182 121.26 518 124 (23.9 %) 94.9913 (±128.4333) 290.395 (±129.899) 608.7259 (±436.105) 1185.8 (±324.3298) 556 1236 G6110 2 F 262 30.24 148 28 (18.9 %) 75.0811 (±108.5156) 258.375 (±165.32) 585.4324 (±379.2734) 1146.3 (±279.5604) 526 1146 G6651 1 F 206 73.01 321 115 (35.8 %) 145.829 (±167.2949) 339.274 (±136.802) 748.2991 (±580.3701) 1419.1 (±364.688) 560 1488 Age was either known from initial tagging as a pup or estimated in the field based on standard length and weight. Sex was assessed and body mass was measured before instrumentation. Deployment duration was dependent on the time it took the seal to return to the beach. Number of dives recorded includes all instances where the animal dove deeper than 4 m. Mean (and SD) dive duration, in min, are given for all dives and the subset of dives deeper than 100 m Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 7 of 15 ±1 m. The accelerometer, magnetometer, and gyroscope ambient noise levels, and thus a decreased signal-to- were all sampled at 50 Hz. noise ratio of the low-level stimulus, near the water’s Playbacks occurred every 6 h for the first two seals and surface. every 3  h for the remaining seals. Each playback con- To determine potential responses during ascent or sisted of one exposure to a single stimulus. The order of bottom phases of deep (>100  m) dives, dive inversions stimuli presentation varied between seals, but remained (Fig.  4) were measured and compared. Dive inversions the same within an individual seal, unless the tag reset were defined as a change from ascending or horizontal itself, at which point it started back at the beginning of swimming to descending, with a change in depth >3  m the sequence. over 4  s. Change in depth and change in time from the inversion point to the next maximum depth were meas- Behavioral analysis ured and normalized by maximum depth and total dive Passive recordings were screened for successful expo- duration, respectively. In order to statistically compare sures (Table  3), and timing of exposures across multi- dive inversions following exposures to dive inversions ple tag systems was synced. Depth data from the TDRs outside of exposure events, an Anderson–Darling k-sam- were examined for possible seal responses to exposures ple (ADK; [40]) test was run on seal G6651, the only seal (Fig.  3). To account for zero offset drift, dive data were to receive multiple exposures during the ascending phase. analyzed using the IKNOS toolbox (Y. Tremblay, unpub- The ADK test was selected because it is distribution free lished) in MATLAB (MathWorks, Inc, Natick, MA, and does not rely on equal sample sizes; however, it USA). Dives were defined as any instance where the indi - requires that samples are taken from independent distri- vidual dove deeper than 4 m. This threshold was chosen butions, in this case, different seals. G5449 was chosen to be greater than the sum of the accuracy of the TDR for comparison as it had the largest deployment duration (±1 m) and the body length of an individual. and therefore the most non-exposure dive inversions. To We hypothesized that responses would differ accord - first test whether different seals could be directly com - ing to dive type and state, so different dives and expo - pared, an ADK test was used to compare non-exposure sures were analyzed separately. Dive shapes were used dive inversions for seals G6651 and G5449. The magni - to identify different dive types (e.g., transit vs. foraging tude of the dive inversion following the single playback [39]). Dives were grouped into deep (>100  m) and shal- during the bottom phase of a deep dive was measured low (≤100  m) dives (Table  3). Exposures were catego- and reported. rized by dive phase (descent, ascent, bottom phase, and Descent rates for descending phases of deep dives were surface; Table  3). Descents were defined as the segment measured and compared to identify possible responses from 0  m to the point at which the seal descended at a during descents. Average descent rate over 60  s before −1 rate <0.1 m s for 20 s. The beginning of an ascent was and 60  s after exposure onset were calculated. A time defined as the point at which an individual ascended for window of 60  s was selected in order to differenti - −1 more than 4  m over 20  s (an ascent rate of 0.2  m  s ). ate sustained changes in descent rate from brief startle Bottom phases were defined as the segment between responses during the playback only. Because an increase the descending and ascending segments. Exposures that in descent rate is considered a subtle response that likely occurred at depths shallower than 20  m were labeled as happens regularly in non-exposure dives, statistical anal- “surface” and excluded from all analyses due to increased ysis of changes in descent rate was not performed. Table 3 Deployment duration and number of exposures for the successful deployments of the prototype tag Seal Exposure Number of  Exposures by dive stage interval (h) exposures received Surface Descent Bottom phase Ascent Deep Shallow Deep Shallow Deep Shallow G5449 3 6 2 – – 1 1 – 2 G5520 3 2 – – – 1 – 1 – G6009 6 2 – – – – – – 2 G6110 6 4 1 – – – 1 – 2 G6651 3 24 7 1 1 3 7 1 4 The number of exposures was dependent on the success of the tag and the length of the deployment. Exposures during descent, bottom phase, and ascent are the subset of exposures used for analysis in each respective phase, split by shallow (<100 m) and deep (>100 m) exposures. Surface exposures were excluded from analysis Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 8 of 15 G5449 -100 -200 -300 -400 -500 0 20 40 60 80 100120 140160 180200 G5520 -100 -200 -300 -400 -500 05 10 15 20 25 30 35 40 45 G6009 -100 -200 -300 -400 -500 -600 G6110 -100 -200 -300 -400 -500 -600 -700 05 10 15 20 25 G6651 -200 -400 -600 -800 -1000 time (hours) Fig. 3 Depth profiles and seafloor bathymetry for complete deployments for all seals. Seal dive profile is in blue, seafloor bathymetry is in gray, and exposures are indicated by black dots. Bathymetry data are from NOAA’s NGDC, Southern California Coastal Relief Map, one-arc second resolution. Instances where a dive profile overlaps the seafloor are an artifact of data resolution mismatch; bathymetry data were extracted only for known GPS surface positions and linearly connected in the gray bathymetry plot, leading to areas with reduced detail depth (meters) depth (meters) depth (meters) depth (meters) depth (meters) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 9 of 15 G5449 Exposure 2 G6110 Exposure 3 G6651 Exposure 3 0 0 -100 -100 -100 -300 -300 -300 -500 -500 -500 470 490 510 1085 1105 1125 440 460 480 G6651 Exposure G6651 Exposure 5 G6651 Exposure 9 -100 -100 -100 -300 -300 -300 -500 -500 -500 610 630 650 802 822 842 1520 1540 1560 G6651 Exposure 12 G6651 Exposure 18 G6651 Exposure 21 0 0 -100 -100 -100 -300 -300 -300 -500 -500 -500 2060 2080 2100 3135 3155 3175 3685 3705 3725 time (minutes) sperm whale clicks white noise common dolphin whistles killer whale whistles killer whale clicks simulated sonar Fig. 4 Dive profiles for all exposures received during ascending phases of dives from seals G5449, G6110, and G6651. Shapes indicate time of exposure and type Shallow dives occurred along the shelf edge and were different stimuli, each playback was categorized as either limited by the seafloor so diving deeper or reversing an “response” or “no response,” using a minimum level of ascent was not possible. To identify possible responses response based on percent change, which varied for each for these shallow dives, changes in flow noise on the dive state (ascending, bottom, descending, or shallow/ passive acoustic recorder (a proxy for changes in swim bottom-limited). Ascending or bottom-phase playbacks speed) were measured and compared. Previous studies were categorized as “response” if there was a dive inver- have found an 18- to 20-dB increase at very low frequen- sion magnitude >2 SDs from non-playback inversions. cies (8–18 Hz) corresponds to a doubling of current flow, Descending playbacks were considered a response if regardless of tag or hydrophone design [34, 41]. Average descent rate increased by >50 % following exposure, and RMS in the 8–18  Hz frequency band was measured for for shallow dives, an increase in flow noise of >10 % was 30–60 and 0–30 s before exposure and 0–30 and 30–60 s considered a response. Percent of overall response was after the exposure ended (Adobe Audition CC, Adobe calculated. Systems Incorporated, San Jose, CA, USA). The before and after periods were divided to detect shorter-term Results changes in swim speed, but the entire 60-s period was Tag development taken into account when assessing a response. Seven deployments of the prototype tag resulted in suc- The normalized change in depth for exposure and cessful CEEs for five individuals (Table  2). Received levels non-exposure dive inversions for seal G6651 was plot - at the individuals’ ears, estimated from the source level ted against time to investigate possible habituation to and assuming spherical spreading, ranged from 128 to multiple playbacks over the duration of the deploy- 138  dB re 1  µPa, depending on seal and stimulus type. ment. To investigate differential response of all seals to Mechanical (flooding) or electrical (tag control board depth (m) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 10 of 15 reset) tag failure rendered the remaining two unsuccess- during the bottom phase of a deep dive (351 m). She dove ful. The playback control of the tag would occasionally to 614 m immediately following exposure, a 75 % increase experience a reset for undetermined reasons, resulting in maximum depth of dive (killer whale whistle exposure, in a stimulus that was played out of sequence. In some Table 3). cases, this reset caused the tag to stop working entirely. Three seals received exposures during the descending phase of a deep dive, and two of three (G5549 and G6651) Deployments and basic dive behavior exhibited an increased descent rate (35.9–271.9 %; mean Five successful deployments ranged from 30 to 207  h in 125.2  %; ±95.8  %) following exposure (Fig.  6; 9 of 10 duration. Animals received 2–24 playbacks, each a sin- total deep descending exposures). Seal G6110 had a gle 30-s exposure, at an interval of 3 or 6  h, and non- lower descent rate following exposure to sonar (72.7  % exposure dives served as control dives (Table  3; Fig.  2). decrease). Four of five animals dove along the seafloor (depths up Flow noise (swim speed) increased by 10.0 (±8.0)  dB to ~100  m) until reaching the continental shelf edge, re 1 µPa from 0 to 30 s before exposure to 30–60 s after after which they exhibited deeper, pelagic transit dives exposure (30.3  ±  14.7  % increase) in all 8 exposures (to with mean depths of 217–340  m over the deep water of three seals) that occurred in shallow water (<100 m). For Monterey Canyon. They returned to the shallow dive pat - two of the exposures, flow noise initially decreased in tern when returning to shallow coastal waters near Año the 0–30  s immediately after exposure but by 60  s after, Nuevo State Park (Fig.  3). We found no evidence of for- reached above pre-exposure levels. aging or drift dives, and the observed dive patterns were Dive inversion responses seal G6651 were as pro- similar to those of other translocated juveniles [13, 42]. nounced as or more pronounced than those later in the Dive depths <100  m were likely limited by the seafloor, deployment compared to earlier (Fig. 7). Seals responded while deeper dives were not bottom-limited, which may to 23 of 28 total exposures (82 %), regardless of stimulus have an effect on responses measured (Fig.  3) [43]. One type, indicating no differential response. individual (G5520) rarely dove deeper than 100 m (5.0 % of all dives), even over deep water (Fig.  3). We found no Discussion significant difference in magnitude of non-exposure dive We identify behavioral responses in juvenile northern inversions for seals G5449 and G6651 (ADK, p  =  0.14, elephant seals elicited by an animal-mounted sound n = 283 non-exposure inversions, seal G5449 and n = 30 source. This is the first dedicated BRS conducted on any non-exposure inversions, seal G6651). Both exposures to marine mammal using an animal-borne sound source seal G6009 were received at <10  m depth, so they were (see [44] for incidental BRS with an active sonar tag). excluded from further analyses. After this exclusion, This approach has the potential to expand and comple - there were 28 usable exposures on four seals, made up of ment current CEE methodologies. Such data will pro- 9 ascending, 10 descending, 1 bottom phase, and 8 shal- vide enhanced understanding of longer-term effects of low water exposures. anthropogenic noise. Controlled exposure experiment Tag performance We observed dive inversions following all playbacks that Deployments that resulted in unsuccessful or reduced occurred during the ascending portion of a deep dive. duration CEEs were due to issues with either the SBC or Individuals descended an additional 116 m (SD ±51.6 m) the housing. We were unable to resolve the cause of the on average (Fig.  4). 8 of 9 exposures resulted in changes SBC reset, but believe it was caused by the formation of in depth >2 SDs from the mean of all non-exposure inver- a ground loop due to differential ground potentials of the sions (Figs.  4, 5; Table  4). Seal G6651 was the only seal independent hardware components comprising the sin- which received multiple exposures during ascent (n  =  7 gle tag system. Although the housing was rated to 1500 m exposures), and the magnitudes of dive inversions follow- depth, either changes in temperature and pressure with ing these exposures were statistically different than non- depth or incorrectly sized o-rings likely caused failure exposure inversions (Fig. 5; Table 4; ADK test, p < 0.001, of the slightly flexible Delrin housing, allowing seawa- n = 283 non-exposure inversions, seal G5449 and n =  7 ter to enter the housing and short circuit the electron- exposure inversions, seal G6651). We observed this dive ics. Future tags would benefit greatly from being potted inversion response for white noise, sperm whale clicks, in solid resin; however, the non-permanent setup used in killer whale whistles, and simulated mid-frequency sonar this trial allowed us to monitor and modify physical tag exposure, but not following an exposure to common dol- components and programming as needed during the field phins, received by seal G6651 (Fig. 4, G6651 Exposure 4). effort. Additionally, we found no evidence of instrumen - Seal G6651 was the only seal that received an exposure tation or drag affecting normal swimming behavior, as Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 11 of 15 G5449 0.4 n=283 and 1 0.3 0.2 0.1 G6110 0.4 n=8 and 1 0.3 0.2 0.1 non-exposures white noise common dolphin whistles G6651 0.4 sperm whale clicks n=30 and 7 killer whale whistles 0.3 + killer whale clicks simulated sonar 0.2 0.1 0 0.1 0.2 0.3 0.40.5 0.60.7 normalized change in depth Fig. 5 Plot of normalized change in depth versus normalized change in time for all dive inversions during the ascending phase of all deep dives (>100 m) for seals G5449, G6110, and G6651. Black dots symbolize non-exposure dive inversions, and open symbols indicate dive inversions following exposures Table 4 Mean change in depth for non-exposure and exposure dive inversions Seal Mean change in depth for  Mean change in depth Anderson–Darling k-sample test non-exposure inversions (m) for exposure inversions (m) G5449 14.875 (±13.997) n = 283 70.5 n = 1 n/a G6110 16.749 (±16.293) n = 8 186 n = 1 n/a G6651 24.867 (±39.981) n = 30 96.36 (±59.23) n = 7 p = <0.001 Dive inversions are defined by a change from ascending to descending >3 m over 4 s, for all dives deeper than 100 m. Standard deviation is given in parentheses, and n is given for each. Results from an Anderson–Darling k-sample test comparing the change in depth of an inversion from ascending to descending, normalized by maximum depth of dive, for exposure inversions of seal G6651 to non-exposure inversions from seal G5449 (n = 283) are given dive profiles were similar to those of other translocated to smaller-scale dive variations seen naturally in these seals carrying smaller devices [13, 42, 45]. and other translocated seals carrying a depth sensor [13, 46]. The exposure context here is unique in terms of the Animal response to disturbance extreme proximity of the sound source to the animal, and Behavioral responses measured during CEEs varied the extrapolation of results to longer-range exposures with dive state at the time of exposure. Most responses should thus be considered cautiously. However, this con- involved seals diving deeper and/or longer after expo- text is more constant than in previous studies providing a sure. The clearest responses (Fig.  4; Table 4) involved dive stronger basis for the comparison of responses within an inversions (reversal of an ascent) followed by the seal div- individual across exposure conditions. ing deeper than the initial maximum dive depth. These Responses observed are similar to hypothesized ele- extended dive inversions served as strong indicators phant seal behavior to escape predators by diving deeper of response. Quantitatively, dive inversions resulted in [37], and responses to anthropogenic sound sources are greater depths and longer dive durations, when compared not unexpected [13, 44]. Responses elicited during CEEs normalized change in time Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 12 of 15 for observed responses. In future studies, initiating Descent Rates Before and After Exposure exposure during one dive phase would control experi- 2 before exposure mental variability and allow robust statistical analyses of after exposure responses. 1.5 We suspect the tag reset described above, and the resulting stimulus playback, is the most likely explana- tion for three non-exposure dive inversions of similar magnitude to inversions following exposures observed in 0.5 seal G6651 (Fig.  5). The inversions could have occurred naturally (e.g., exposure to an external alarming sound) or from an accidental exposure by the prototype tag fol- G5449 G6110 G6651 lowing a reset; however, the independent passive acous- exposure number and type white noise common dolphin whistles sperm whale clicks tic system was not recording so we cannot know for killer whale whistles + killer whale clicks simulated sonar sure. Continuous recording of ambient noise on future −1 Fig. 6 Mean descent rates, in m s , for seals G5449, G6110, and tag iterations, and longer-duration deployments, would G6651 during the 60 s before (white bars) and 60 s after (black bars) allow for potential opportunistic measurement of behav- exposure. Open symbols correspond to exposure type ioral responses to naturally occurring threatening sounds such as predators, boats, or other man-made or tag-made sounds. G6651 0.5 Differential response by stimulus type 0.45 We found no indication of differential responses based on 0.4 stimulus type or order (Table 5). Notably, seals responded 0.35 to most (82  %) but not all exposures, indicating a possi- non-exposures ble differential response that was not detected because of 0.3 white noise common dolphin whistles limited sample size. 0.25 sperm whale clicks The observed results suggest that the response may be killer whale whistles 0.2 killer whale clicks a result of acoustic limitations of the playback apparatus simulated sonar 0.15 rather than the stimuli (see [48]). Limitations included 0.1 the nonlinear nature of the small transducers and 0.05 extreme proximity of the source to the animal. This prox - imity likely created an unusual perceptual context com- 12 24 36 48 60 pared to a sound produced naturally at realistic ranges time (hours) due to the effects of sound propagation (e.g., rever - Fig. 7 Plot of normalized change in depth versus elapsed time (in beration, directional cues, and the relative presence or h) for all dive inversions during the ascending phase of all deep absence of harmonics). Many details of pinniped under- dives (>100 m) for seal G6651. Black dots symbolize non-exposure dive inversions, and open symbols indicate dive inversions following water hearing, including directionality, are still poorly exposures understood [36, 49, 50]. Sudden sounds may trigger a response, regardless of the sound type or the distance it were similar to those found by other BRSs on deep-div- Table 5 Differences in  response rate by  exposure type ing marine mammals. For instance, dive inversions and and for all exposures to all seals remaining at depth longer were demonstrated in two spe- cies of beaked whale exposed to simulated mid-frequency Exposure Number of exposures Response measured sonar from an external, ship-mounted sound source [22, White noise 6 5 47]. A third species of beaked whale responded to expo- Common dolphin 4 2 sures of both sonar and killer whale sounds by inter- whistles rupting foraging and performing an unusually long and Sperm whale clicks 5 5 shallow ascent [12]. Killer whale whistles 6 5 Responses to exposures that occurred during descents Killer whale click 1 1 or during bottom-limited dives were more difficult to Sonar 6 5 measure. Natural variation in swim speed and descent Total 28 23 angle limited our ability to define statistical significance -1 normalized change in depth descent rate (ms ) Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 13 of 15 is perceived to be coming from. Future testing using play- beginning to better understand how animals behaviorally back stimuli that are filtered to simulate different source respond to sound, but we still do not fully understand locations could examine the effects of reverberation and the underlying physiological changes that are linked to localization in detail. Comparable parallel CEEs would these behavioral changes (e.g., oxidative stress). Physi- need to be conducted on pinnipeds, using an external ological parameters such as oxygen utilization, heart rate, sound source that closely approximate actual signals, to and post-dive recovery times can be monitored in free- fully evaluate the potential of this tag for BRS. ranging animals [53, 54], and with this tag, dives can be While individual differences could also contribute experimentally extended, allowing quantitative investiga- to different responses, no single seal received multiple tion of physiological responses. To support the potential exposures of all exposure types because of tag failures, use of this tag to study physiological effects of extended which precludes further examination of this possibility. dives possibly related to responses to anthropogenic BRS exposing animals to multiple exposures of all stimuli noise, it is promising that the responses were similar to are needed to investigate individual differences to stimuli. other deep-diving species of interest. For species that are A differential response to dolphin whistles was recorded specialized for oxygen efficiency during prolonged, deep for seal G6651, the only seal exposed to two playbacks of dives, physiological responses to changes in planned dive dolphin whistles while ascending from a dive. No extreme duration could help physiologists better understand these dive inversions were recorded for dolphin whistles, while adaptations [55]. other stimuli resulted in extreme evasive behavior. This anecdotal example provides evidence for the possibility Conclusions of differential response to different stimuli. This pilot study was the first of its kind to investigate Repeated exposures to various stimuli may cause habit- the potential use of an animal-borne sound source to uation over time [51]. Although there was no evidence of conduct BRS on marine mammals to further investigate habituation over short-term deployments (<3  days), we the effects of anthropogenic noise. Five juvenile north - cannot rule out the possibility of a sensitization response. ern elephant seals were instrumented and translocated The consistent response to most exposures independ - south of their colony at Año Nuevo State Park. They ent of stimulus type and the apparent lack of reduction received playbacks of multiple stimulus types (both in response within individuals suggest either that seals man-made and natural) from the tag while diving con- are generally sensitive to a relatively wide range of audi- tinuously on their return to the colony. Dive behavior ble exposures or possibly that seals became sensitized to before and after exposures were compared to assess subsequent exposure. Preliminary evidence from captive whether animal-borne tags holding sound sources were elephant seals suggests some sensitization, as opposed to capable of eliciting a response from a free-ranging ani- habituation, upon repeated exposure to certain acous- mal and whether potential responses differed with stim - tic stimuli [52]. However, the sound source placement, ulus content. context, and limited number of exposures of each stimu- Projecting sound from a tag mounted to the back lus type to each individual limited our ability to test for of a free-ranging juvenile northern elephant seal did potential sensitization. elicit behavioral responses; however, the responses The tag and method may be useful in physiological were not consistently different with different stimulus studies of dive limits and studies of hearing ranges. The types. Responses varied by seal, by dive state, and by the tag could be used as a mobile, programmable sound bathymetry where the exposure occurred, but in general, source to study frequency-dependent hearing in marine seals dove deeper following playback. Animals responded mammal species that cannot be studied using captive to 82 % of exposures overall, with no clear evidence of a psychophysical methods. Little is known about hearing reduction in response to repeated exposures of various in many marine mammals because laboratory studies of stimuli. The unique and biologically unrealistic context hearing are not feasible due to constraints with keeping of a sudden sound exposure coming from directly behind very large, migratory, deep-diving, and social species in the animal is likely the greatest limiting factor preventing captivity, but using controlled, incrementally increas- use of this tag in examining differential responses to par - ing exposure levels of tonal sounds that trigger drastic ticular sounds and relating them to responses that may changes in behavior could investigate differences in hear - occur over more realistic source-animal ranges. Mak- ing sensitivities in previously inaccessible species. ing conclusions about the effects of particular sounds on Additionally, this tag could be used to study the physi- juvenile northern elephant seals was beyond the scope of ological effects of unanticipated extensions of dives, this pilot study, but additional tests using this technology another important research need for understanding may help develop additional questions and hypotheses in the effects of noise on marine mammals [18]. We are the future. Fregosi et al. Anim Biotelemetry (2016) 4:9 Page 14 of 15 Received: 30 October 2015 Accepted: 11 March 2016 Tag improvements and additional field testing could strengthen results found here, but the concept of an animal-borne sound source for triggering behavio- ral responses has been validated. The use of this novel animal-borne sound source in future studies could help References 1. 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Animal BiotelemetrySpringer Journals

Published: Apr 1, 2016

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