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Behavioral and auditory evoked potential (AEP) hearing measurements in odontocete cetaceans

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Title:
Behavioral and auditory evoked potential (AEP) hearing measurements in odontocete cetaceans
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Book
Language:
English
Creator:
Cook, Mandy Lee Hill
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects / Keywords:
Bottlenose dolphin
Beaked whale
Audiogram
Envelope following response (EFR)
modulation rate transfer function (MRTF)
Tursiops truncatus
Mesoplodon europaeus
Dissertations, Academic -- Marine Science -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Bottlenose dolphins (Tursiops truncatus) and other odontocete cetaceans rely on sound for communication, navigation, and foraging. Therefore, hearing is one of their primary sensory modalities. Both natural and anthropogenic noise in the marine environment could mask the ability of free-ranging dolphins to detect sounds, and chronic noise exposure could cause permanent hearing losses. In addition, several mass strandings of odontocete cetaceans, especially beaked whales, have been correlated with military exercises involving mid-frequency sonar, highlighting unknowns regarding hearing sensitivity in these animals.Auditory evoked potential (AEP) methods are attractive over traditional behavioral methods for measuring the hearing of marine mammals because they allow rapid assessments of hearing sensitivity and can be used on untrained animals. The goals of this study were to 1.) investigate the differences among underwater AEP, in-air AEP, and underwater behavioral heari ng measurements using two captive bottlenose dolphins, 2.) investigate the hearing abilities of a population of free-ranging bottlenose dolphins in Sarasota Bay, Florida, using AEP techniques, and 3.) report the hearing abilities of a stranded juvenile beaked whale (Mesoplodon europaeus) measured using AEP techniques.For the two captive dolphins, there was generally good agreement among the hearing thresholds determined by the three test methods at frequencies above 20 kHz. At 10 and 20 kHz, in-air AEP audiograms were substantially higher (about 15 dB) than underwater behavioral and underwater AEP audiograms.For the free-ranging dolphins of Sarasota Bay, Florida, there was considerable individual variation, up to 80 dB between individuals, in hearing abilities. There was no relationship between age, gender, or PCB load and hearing sensitivities. Hearing measured in a 52-year-old captive-born bottlenose dolphin showed similar hearing thresholds to the Sarasota dolphins up to 80 kHz,^ but exhibited a 50 dB drop in sensitivity at 120 kHz.Finally, the beaked whale was most sensitive to high frequency signals between 40 and 80 kHz, but produced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beaked whale hearing range and sensitivity were similar to other odontocetes that have been measured.
Thesis:
Dissertation (Ph.D. )--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Mandy Lee Hill Cook.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 123 pages.
General Note:
Includes vita.

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aleph - 001914875
oclc - 179658798
usfldc doi - E14-SFE0001769
usfldc handle - e14.1769
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Behavioral and Auditory Evoked Potential (AEP) Hearing Mea surements in Odontocete Cetaceans by Mandy Lee Hill Cook A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Major Professor: David A. Mann, Ph.D. Joseph J. Torres, Ph.D. Gordon B. Bauer, Ph.D. Sentiel A. Rommel, Ph.D. Randall S. Wells, Ph.D. Date of Approval: August 9, 2006 Keywords: bottlenose dolphin, beaked whale, audiogram, enve lope following response (EFR), modulation rate transfer function (MRTF), Tursiops truncatus Mesoplodon europaeus Copyright 2006 Mandy Lee Hill Cook

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Dedication This dissertation is dedicated to my family for their continued love, support, and guidance. Thank you for encouraging me to ask questions and for teaching me to love the ocean.

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Acknowledgments I would like to thank the numerous institutions, organizatio ns, and volunteers who helped make this project a reality. I would especially like to t hank my advisor, David Mann, for his never-ending enthusiasm and interest in my project; I am exceptionally grateful for your guidance along this journey. His wealth of knowledge a nd ideas made all of this possible. I would also like to thank my committee members Jose Torres, Gordon Bauer, Butch Rommel, and Randy Wells, for their helpful advice support, and encouragement, and for the valuable time they each invested in my resear ch to improve it by leaps and bounds. Finally, I would like to thank the past and present m embers of the Mann Lab who taught me a lot, enriched my graduate experience, and m ade life a little more interesting along the way. This work was funded in part by two Harbor Branch Oceanogra phic Institution Protect Wild Dolphins research grants to David A. Mann and Ra ndall S. Wells. I was funded in part by the University of South Florida’s College of Marine Science graduate assistantships, and the Lake, Getting, and Von Rosenstiel endowed fellowships. Support was also provided by a P.E.O. Scholar Award.

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i Table of Contents List of Tables...................................... ................................................... ........................iii List of Figures...................................... ................................................... .......................iv Abstract............................................ ................................................... ...........................vi Chapter One: Hearing Thresholds in Captive and Free-Ran ging Cetaceans: An Introduction......................................... ................................................... ...................1 References Cited................................. ................................................... ..............9 Chapter Two: Ground-Truthing In-Air Auditory Evoked Potentia l (AEP) Hearing Measurements with Traditional Behavioral Audiograms in Bottlenose Dolphins ( Tursiops truncatus ).................................................. ................................25 Abstract.......................................... ................................................... ................25 Introduction....................................... ................................................... .............26 Materials and Methods.............................. ................................................... ......29 Subjects.......................................... ................................................... ...........29 AEP Methods...................................... ................................................... ......30 Experiment 1: In-Air AEPs with a Jawphone....... ..................................33 Underwater Experimental Setup...................... .............................................34 Experiment 2: Underwater AEPs with a Free-Field Spe aker..................34 Experiment 3: Underwater Behavioral Audiogram with a FreeField Speaker......................................... ...........................................35 Jawphone and Free-Field Speaker Calibrations......... ....................................37 Results.......................................... ................................................... ..................38 Calvin........................................... ................................................... ............38 Ranier........................................... ................................................... ............38 Discussion......................................... ................................................... ..............39 References Cited................................. ................................................... ............45 Chapter Three: Auditory Evoked Potential (AEP) Hearing Th resholds of FreeRanging Bottlenose Dolphins ( Tursiops truncatus ).................................................. 61 Abstract.......................................... ................................................... ................61 Introduction....................................... ................................................... .............63 Materials and Methods.............................. ................................................... ......66 Subjects.......................................... ................................................... ...........66

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ii Sarasota Bay, FL, Bottlenose Dolphin Community..... ............................66 Dolphin Conservation Center at Marineland....... ....................................67 AEP Methods...................................... ................................................... ......68 Results.......................................... ................................................... ..................72 MRTF............................................ ................................................... ...........72 I-O Functions and AEP Audiograms.................... ........................................73 F195.............................................. ................................................... ............73 PCBs and Hearing Thresholds....................... ...............................................74 Nellie at Marineland............................. ................................................... .....74 Discussion......................................... ................................................... ..............75 References Cited................................. ................................................... ............80 Chapter Four: Beaked Whale Auditory Evoked Potential Heari ng Measurements.........98 Abstract.......................................... ................................................... ................98 Introduction....................................... ................................................... .............99 Materials and Methods.............................. ................................................... ....101 Subject........................................... ................................................... .........101 AEP Methods...................................... ................................................... ....101 Stimulus Control and Data Collection.............. ...........................................102 Jawphone and AEP Electrodes....................... ............................................103 Sounds............................................ ................................................... ........105 AEP Audiogram Calibration.......................... .............................................105 Results.......................................... ................................................... ................106 MRTF............................................ ................................................... .........106 EFR............................................. ................................................... ............106 Discussion......................................... ................................................... ............107 References Cited................................. ................................................... ..........110 Chapter Five: Hearing Thresholds in Captive and Free-Rangi ng Cetaceans: Concluding Remarks..................................... ................................................... ......118 References Cited................................. ................................................... ..........122 About the Author..................................... ................................................... ........End Page

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iii List of Tables Table 1-1 Behavioral hearing measurements for odontocete ce taceans..........................21 Table 1-2 Auditory evoked potential (AEP) hearing measurements for odontocete cetaceans.......................................... ................................................... ....................23 Table 2-1 Auditory evoked potential (AEP) and behavioral hearin g studies for which the same test subjects were used................. ................................................... 51 Table 2-2 Average number of hits, misses, false alarms, and correct rejections for the 25% and 50% catch trial sessions.................... ................................................... 52 Table 2-3 Calvin’s underwater AEP and in-air AEP hearing t hresholds........................53 Table 2-4 Ranier’s underwater behavioral, underwater AEP, a nd in-air AEP hearing thresholds................................... ................................................... ..............54 Table 3-1 Freeze-brand (FB) number, gender, age at AEP tes ting, and health assessment (H.A.) session for each animal tested.... .................................................86

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iv List of Figures Figure 2-1 Amplitude-modulated (AM) tone that was presented to the dolphins via a jawphone or a free-field speaker.................. ................................................... .55 Figure 2-2 An example of evoked potential data collected from Calvin in response to an 80 kHz AM tone at ten sound levels........ ..........................................56 Figure 2-3 In-air AEP hearing tests on Ranier............ ................................................... 57 Figure 2-4 Underwater AEP hearing tests on Calvin........ .............................................58 Figure 2-5 In-air and underwater AEP hearing thresholds fo r Calvin............................59 Figure 2-6 In-air AEP, underwater AEP, and behavioral hear ing thresholds for Ranier.............................................. ................................................... .....................60 Figure 3-1 Mean MRTF measured for seven free-ranging bottle nose dolphins ( Tursiops truncatus ).................................................. ...............................................88 Figure 3-2 Input-output function for FB75 for seven test fre quencies............................89 Figure 3-3 Mean ( SD) AEP audiograms measured for 32 male an d 29 female free-ranging bottlenose dolphins ( Tursiops truncatus )..............................................90 Figure 3-4 Plots of dolphin age, in years, versus SPL at h earing threshold, in dB re 1 Pa, for each frequency, separated by gender.......... ..........................................91 Figure 3-5 F195 showed no EP response to the 40 kHz tone burst a t 120 dB re 1 Pa even after 1100 sweeps (top), while FB75 showed a strong E P to the same tone burst at the same SPL after only 86 sweeps (bo ttom)...............................94 Figure 3-6 Nellie’s AEP audiogram compared to the mean ( S D) AEP audiograms measured for 32 male and 29 female free-ranging bot tlenose dolphins ( Tursiops truncatus ).................................................. ................................95

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v Figure 3-7 Nellie’s AEP audiogram compared to the predicted un derwater mean ( SD) AEP audiograms measured for 32 male and 29 female fre e-ranging bottlenose dolphins ( Tursiops truncatus ).................................................. ................96 Figure 3-8 Predicted behavioral audiograms based on AEP-beh avioral audiogram transfer functions................................. ................................................... .................97 Figure 4-1 Beaked whale ( Mesoplodon europaeus ) modulation rate transfer function measured with a 40 kHz carrier tone at 130 dB re 1 Pa at various amplitude modulation rates............................ ................................................... .....114 Figure 4-2 Beaked whale ( Mesoplodon europaeus ) input-output functions of evoked potential level as a function of stimulus sound pressur e level (SPL)...........115 Figure 4-3 Lowest sound pressure levels (SPLs) for which an evoked potential could be detected at each test frequency................. ................................................116 Figure 4-4 Comparison between auditory evoked potential (AEP ) and behavioral hearing thresholds determined for three bottlenose dolphins ( Tursiops truncatus ): a) WEN, b) BLU, and c) BEN...................... .......................................117

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vi Behavioral and Auditory Evoked Potential (AEP) Hearing Measuremen ts in Odontocete Cetaceans Mandy Lee Hill Cook ABSTRACT Bottlenose dolphins ( Tursiops truncatus ) and other odontocete cetaceans rely on sound for communication, navigation, and foraging. Therefore, hearing is one of their primary sensory modalities. Both natural and anthropogenic noise in the marine environment could mask the ability of free-ranging dolphins to detect sounds and chronic noise exposure could cause permanent hearing losses. In addition several mass strandings of odontocete cetaceans, especially beaked whales, have be en correlated with military exercises involving mid-frequency sonar, highlighting unknowns r egarding hearing sensitivity in these animals. Auditory evoked potential (AEP) methods are attractive ov er traditional behavioral methods for measuring the hearing of marine mam mals because they allow rapid assessments of hearing sensitivity and can be used o n untrained animals. The goals of this study were to 1.) investigate the differences a mong underwater AEP, in-air AEP, and underwater behavioral hearing measurements using two capt ive bottlenose dolphins,

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vii 2.) investigate the hearing abilities of a population of fr ee-ranging bottlenose dolphins in Sarasota Bay, Florida, using AEP techniques, and 3.) report t he hearing abilities of a stranded juvenile beaked whale ( Mesoplodon europaeus ) measured using AEP techniques. For the two captive dolphins, there was generally good agr eement among the hearing thresholds determined by the three test methods at frequencies above 20 kHz. At 10 and 20 kHz, in-air AEP audiograms were substantially higher (about 15 dB) than underwater behavioral and underwater AEP audiograms. For the free-ranging dolphins of Sarasota Bay, Florida, t here was considerable individual variation, up to 80 dB between individuals, in h earing abilities. There was no relationship between age, gender, or PCB load and hearing sensitivities. Hearing measured in a 52-year-old captive-born bottlenose dolphin s howed similar hearing thresholds to the Sarasota dolphins up to 80 kHz, but exhibited a 50 dB drop in sensitivity at 120 kHz. Finally, the beaked whale was most sensitive to high frequen cy signals between 40 and 80 kHz, but produced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beaked whale hearing range and sensitivity we re similar to other odontocetes that have been measured.

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1 Chapter One: Hearing Thresholds in Captive and Free-Ranging Cetaceans: An Introduction Bottlenose dolphins ( Tursiops truncatus ) have an impressive ability to both produce and perceive a wide variety of sounds. These sounds include echolocation clicks used for feeding and other functions, and whistles and burst-pulse so unds used for communication (Caldwell et al. 1990; Au 1993; Thomson and Richardson 1995). Pres umably, dolphins are capable of hearing all of the sounds they are capable o f producing; therefore, their hearing should be sensitive over a wide range of frequencie s. Additionally, because dolphins rely on sound for communication, navigation, and fo raging, their sense of hearing is one of their most important senses (Au 1993; Jani k and Slater 1998). This chapter presents a chronological review of the sound produc tion and hearing abilities of odontocetes to provide a framework for the hearing studie s presented in the following chapters. In particular, behavioral and auditory evoked pot ential techniques were used to evaluate the hearing capabilities of cetaceans. The sound production and reception abilities of bottlenose dolphins have been studied by several prominent researchers. In 1947 the first curator of Marineland, Florida, Arthur McBride, presented evidence that Atlantic bottlenose dolphins may detect objects underwater by means of echolocation. During the dol phin capture operations that took place at night in the turbid waters of Florida’s inla nd waterways, he noted that

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2 dolphins could avoid fine mesh nets and detect openings in the nets beyond visual range (McBride 1956). Kellogg and Kohler (1952) were the first to hypoth esize the production of echolocation by dolphins. They performed a crude sound a voidance experiment and found that dolphins could hear frequencies up to 50 kHz. A yea r later Kellogg and colleagues reported that dolphins could hear up to 80 kHz (Kel logg 1953; Kellogg et al. 1953). In 1953, Forrest Wood described spotted ( Stenella plagiodon ) and bottlenose dolphins producing rasping and grating sounds to “echo-investigate” a transducer (Wood 1953). Schevill and Lawrence (1953) reported Tursiops hearing frequencies as high as 120 kHz. In 1956, Schevill and Lawrence first described captive dolphins producing echolocation to find small, silent bits of food that we re placed into the water. These dolphins were producing sounds inaudible to a person listening fro m the bank of the pond, but they could be detected using sensitive underwater lis tening equipment. To eliminate vision as a possible cue for fish detection, Sch evill and Lawrence frequently worked on dark nights, and the pond that contained the dolphi n was extremely turbid. They extended a net from a boat perpendicular to the ba nk, and sat at opposite ends of the boat, each holding a fish at arm’s length. They would randomly take turns placing the fish below the water surface as the dolphin swam by and, in about 75% of the tests, the dolphin chose the correct side of the net from which to obtain a fish (Schevill and Lawrence 1956). In 1958 Kellogg published the results from a series of experime nts that provided strong evidence of echolocation in dolphins. He trained t he animals to swim through an obstacle course and to perform fish food discrimination t asks. These tasks required the

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3 dolphins to select the preferred fish or to select the fi sh not behind a clear glass pane (Kellogg 1958, 1961). Echolocation was unequivocally demonstrated by Kenneth Norr is and colleagues in the early 1960s; they used rubber suction cups to cover th e eyes of dolphins trained to perform echolocation tasks (Norris et al. 1961). The dolphi n was able to successfully retrieve fish tossed into the water as they drifted down ward. Many odontocetes have since been shown to produce echolocation. Most experim ents testing this ability have been conducted with animals wearing eye cups and trained in o bject retrieval, object discrimination, or obstacle course tasks. Au and many oth ers have studied various aspects of dolphin echolocation production since the mid-1970s (see Au 1993 for a general review). Kellogg and colleagues first described the whistles of bott lenose dolphins (Kellogg et al. 1953), although they were mentioned by both K ullenberg in 1947 and Essapian in 1953. In the 1960s several researchers, including Dreher (1961), Lilly (1962), Dreher and Evans (1964), Schevill (1964), and Evans (1967), desc ribed the shapes and early repetitive elements of dolphin whistles. L illy and Miller first hypothesized that dolphin whistles had specific functions ( Lilly and Miller 1961a, 1961b). They assigned discrete whistle contours to specific behavioral situations, and went on to propose the dolphin “distress call” (Lilly and Miller 1961a; Lilly 1963). David and Melba Caldwell first reported the presence of in dividualized whistles in captive bottlenose dolphins (Caldwell and Caldwell 1965). Over a three week period, they recorded the vocalizations of five newly-captured ani mals. These recordings showed that each animal from this group tended to produce an in dividually distinctive

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4 whistle that remained relatively unchanged regardless of context. The Caldwells called these individualized whistles “signature” whistles (Caldw ell and Caldwell 1968), and hypothesized that these whistles functioned in individual re cognition. They went on to show that dolphins could correctly classify different s ignature whistles in as little as a 0.5 second exposure to them (Caldwell et al. 1969). Their semi nal research on signature whistles during the 1960s and 1970s was summarized by Caldwell et a l. (1990). Although signature whistles were disputed by McCowan and Rei ss (1995, 2001), research by others produced an overwhelming amount of evidenc e supporting the signature whistle hypothesis, and demonstrated that free-ran ging bottlenose dolphins produce signature whistles in a variety of activity conte xts (e.g., Sayigh 1992; Sayigh et al. 1990, 1995, 1999; Watwood 2003; Watwood et al. 2004, 2005; Cook et al. 2004; Janik et al. 2006). For example, free-ranging bottlenose dolp hins respond significantly more often to signature whistles produced by related or fami liar animals than by unrelated or unfamiliar animals (Sayigh 1992; Sayigh et al. 1999; Janik et al. 2006). Burst-pulse sounds produced by bottlenose dolphins have recentl y been categorized as social and foraging sounds. Conner and Sm olker (1996) reported the use of ‘pop’ calls by male dolphins during consortship. Janik (2000) repor ted the production of food-related bray calls, and Nowacek (2005) reported the production of pop calls and suggested that perhaps they are used to startle fish. The hypothesis that dolphins use their lower jaws in the reception of sound, especially high frequency sounds, is generally accepted. Norr is (1964, 1968) originally proposed that the mandibular foramen and the fats assoc iated with it function as acoustic wave guides; electrophysiological (Bullock et al. 1968; McCo rmick et al. 1970, 1980)

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5 and behavioral (Brill et al. 1988, 2001) studies with bottleno se dolphins support this theory. Jawphones (contact hydrophones attached by suctio n cups) take advantage of this sound conduction pathway and have been used by several resea rchers to deliver acoustic stimuli to the mandibles of bottlenose dolphins (e.g., Moore and Pawloski 1993; Brill et al. 2001; Cook et al. 2006; Finneran and Houser 2006; Houser and Finne ran 2006). Behavioral hearing measurements of bottlenose dolphins began with Kellogg and Kohler’s study in 1952, which was quickly followed by reports from both Schevill and Lawrence (1953) and Kellogg (1953; see above). C. Scott Johnson performed the most detailed behavioral hearing measurement experiments in a bottlenose dolphin published to date (Johnson 1966, 1967). He trained an 8-9-year-old male bot tlenose dolphin to respond to 3-second pure-tone acoustic stimuli between 75 Hz and 150 kHz. The test procedure used a go/no-go response paradigm, and false ala rms were followed by 90second time-outs. This methodology probably caused the ani mal to respond very conservatively to the sound presentations and thus could have potentially elevated the results of the audiogram (Nachtigall et al. 2000). The lo west hearing thresholds occurred near 50 kHz at a level around 45 dB re 1 Pa, but sounds were detected by the dolphin throughout the range of 75 Hz to 150 kHz. Since Johnson’s s eminal work on bottlenose dolphin audiograms, Thompson and Herman (1975), Ljungblad et al (1982), Ridgway and Carder (1993, 1997), Au et al. (2002), Finneran et al. (2002a, 2002b, 2002c), Houser et al. (2004), Finneran and Houser (2006), Houser and Finneran (2006), and Cook et al. (in prep.) have reported additional behavioral audiograms f or this cetacean species. In the last 40 years, behavioral hearing thresholds have been reported for a wide variety of cetaceans, representing 13 different species (Table 1-1).

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6 Auditory evoked potential (AEP) techniques, described in detail in the following chapters, can be used as an alternative to traditional behavioral techniques to measure hearing in cetaceans. Research projects using these proce dures were first attempted in the 1960s. Although Bullock et al. (1968) and Bullock and Ridgway (1972) reported cetacean evoked potentials recorded in response to auditory stimuli, both of these studies were done invasively (electrodes were placed near or withi n the inferior colliculus or the lateral lemniscus), and many of the animals were sacrif iced or succumbed to the experimental procedures soon after the completion of test ing (Bullock et al. 1968; Bullock and Ridgway 1972). Popov et al. (1986) reported the evo ked potentials of a harbor porpoise ( Phocoena phocoena ), but these techniques were also invasive. Ridgway (1980) reported less-invasive auditory evoked potentials rec orded in bottlenose dolphins, and Popov and Supin (1990 a, b) also repo rted similar experimental results (see Supin et al. 2001 for a general review). The AEP hearing abilities of 18 different species of cetaceans have been measured to date (Table 1-2). Most of these studies used subdermal or surface electrodes to record the auditory evoked potentials generated in response to acoustic stimuli. Dolphin and colleagues have also conducted several studie s that examine how the use of different test signals changes the evoked potential response (e.g., Dolphin and Mountain 1992, 1993; Dolphin 1995, 1997, 2000). For example, the magnitude o f the evoked potential response increases with both increased st imulus intensity and modulation depth (Dolphin and Mountain 1992). More recently, a uditory evoked potential measurements and behavioral hearing measurements have been collected on the same animals to accurately compare the threshold differ ences generated by each

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7 technique (e.g., Szymanski et al 1999; Houser et al. 2004; Yuen et al. 2005; Finneran and Houser 2006; Houser and Finneran 2006; Cook et al. 2006, in prep.). The major weaknesses of using behavioral techniques to study the hearing abilities of cetaceans include the large amounts of time required to train and test the animals (months to years) and the limited availability of animal subjects to test (e.g., generally smaller odontocetes maintained in captivity). In contrast, auditory evoked potential (AEP) techniques allow for the rapid measurement (minutes) of an individual’s hearing abilities with little or no training necessary. Th us, AEP techniques save large amounts of time, which potentially allow for larger samp le sizes. Furthermore, AEP techniques allow animals to be tested in the field, in air or in the water, and with nonmobile animals, which means stranded and larger cetaceans can be examined (e.g., Popov and Klishin 1998; Ridgway and Carder 2001; Andr et al. 2003; Nachti gall et al. 2005; Cook et al. 2006). Several research questions were addressed during the course of this dissertation. Chapter Two discusses the relationship among in-air AEP a udiograms, underwater AEP audiograms, and underwater behavioral audiograms. In this s tudy, two captive male bottlenose dolphins at The Living Seas, Epcot Walt Disney World Resort, Calvin and Ranier, participated in the in-air and underwater AEP meas urements, and Ranier participated in the underwater behavioral measurements. In addition, the acoustic stimuli used in each of the three experiments were the same. Therefore, the confounding issues of both subject and stimulus variability were removed fr om this study, and the three different methodologies could be directly compared. This chapter also addresses how

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8 well in-air AEP audiograms model or predict traditional underwater behavioral audiograms. Chapter Three investigates the hearing abilities of freeranging bottlenose dolphins in Sarasota Bay, Florida, using in-air AEP techniqu es. This is the first study to examine the hearing abilities of a population of wild odont ocetes. The effects of age and gender on an individual’s hearing abilities are discussed in this chapter. In addition, predicted underwater AEP and behavioral audiograms are calc ulated using the AEPbehavioral audiogram transfer function presented in Chapt er Two. Finally, this chapter emphasizes the need for larger sample sizes when making population-level assessments or management decisions. Chapter Four explores the hearing abilities of a live-st randed juvenile beaked whale ( Mesoplodon europaeus ). This study highlights the importance of stranded cetaceans, especially those that cannot be maintained in captivity, for addressing key scientific questions. In addition, these are the first hearing data collected for any member of the family Ziphiidae. Because several strandings of beaked whales have also been linked to the use of Naval sonar, the results of this st udy are discussed in terms of hearing sensitivity to sonar-like sounds. Chapter Five provides a brief summary of each chapter and th e concluding remarks to this dissertation. Each chapter has been formatted for the Journal of C omparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiolog y. Chapter Four was published there earlier this year.

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9 References Cited Andersen S (1970) Auditory sensitivity of the harbour porpo ise ( Phocoena phocoena ). In: Pilleri G (ed) Investigations on Cetacea, Vol. 2. AG Berne Bmpliz, Berne, Switzerland, pp 255-259 Andr M, Supin A, Delory E, Kamminga C, Degollada E, Alons o JM (2003) Evidence of deafness in a striped dolphin, Stenella coeruleoalba Aquat Mamm 29:3-8 Au WWL (1993) The Sonar of Dolphins. Springer-Verlag, New York Au WWL, Lemonds DW, Vlachos S, Nachtigall PE, Roitblat HL (2002) Atlantic bottlenose dolphin ( Tursiops truncatus ) hearing threshold for brief broadband signals. J Comp Psychol 116:151-157 Awbrey FT, Thomas JA, Kastelein RA (1988) Low frequency un derwater hearing sensitivity in belugas, Dephinapterus leucas J Acoust Soc Am 84:2273-2275 Beedholm K, Miller LA (2005) Stimulus-response characteris tics of a harbor porpoise during active echolocation and passive hearing studies with auditory brainstem recordings (ABR). J Acoust Soc Am 117:2441 Belkovich VM, Solntseva GN (1970) Anatomy and function of t he ear in dolphins. J Comp Zool 2:275-282 Brill RL, Sevenich ML, Sullivan TJ, Sustman JD, Witt RE (1988) Behavioral evidence for hearing through the lower jaw by an echolocating dolphi n ( Tursiops truncatus ). Mar Mamm Sci 4:223-230

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10 Brill RL, Moore PWB, Dankiewicz LA (2001) Assessment of dolphin ( Tursiops truncatus ) auditory sensitivity and hearing loss using jawphones. J Acoust Soc Am 109:1717-1722 Bullock TH, Ridgway SH (1972) Evoked potentials in the centra l auditory system of alert porpoises to their own and artificial sounds. J Neurobio l 3:79-99 Bullock TH, Grinnell AD, Ikezono E, Kameda K, Katsuki Y, Nomoto M, Sato O, Suga N, Yanagisawa K (1968) Electrophysiological studies of cent ral auditory mechanisms in cetaceans. Z vergl Physiol 59:117-156 Caldwell MC, Caldwell DK (1965) Individualized whistle contour s in bottlenosed dolphins ( Tursiops truncatus ). Nature 207:434-435 Caldwell MC, Caldwell DK (1968) Vocalization of nave capti ve dolphins in small groups. Science 159:1121-1123 Caldwell MC, Caldwell DK, Hall NR (1969) An experimental demonstration of the ability of an Atlantic bottlenosed dolphin to discriminate b etween whistles of other individuals of the same species. LA County Museum o f Natural History Foundation TR 6, 37 pp. Caldwell MC, Caldwell DK, Tyack PL (1990) Review of the si gnature-whistle hypothesis for the Atlantic bottlenose dolphin. In: Lea therwood S, Reeves RR (eds) The Bottlenose Dolphin. Academic Press, New York, pp 199-234 Carder DA, Ridgway SH (1990) Auditory brainstem response in a neonatal sperm whale, Physeter spp J Acoust Soc Am 88:S4 Connor RC, Smolker RA (1996) ‘Pop’ goes the dolphin: a vocali zation male bottlenose dolphins produce during consortships. Behaviour 133:643-662

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11 Cook MLH, Sayigh LS, Blum JE, Wells RS (2004) Signature-whi stle production in undisturbed free-ranging bottlenose dolphins ( Tursiops truncatus ). Proc R Soc Lond B 271:1043-1049 Cook MLH, Bauer GB, Fellner W, Mann DA (in prep.) Ground-tru thing in-air auditory evoked potential (AEP) hearing measurements with tradition al behavioral audiograms in bottlenose dolphins ( Tursiops truncatus ). Cook MLH, Varela RA, Goldstein JD, McCulloch SD, Boss art GD, Finneran JJ, Houser D, Mann DA (2006) Beaked whale auditory evoked potential heari ng measurements. J Comp Physiol A 192:489-495 Dolphin WF (1995) Steady-state auditory-evoked potentials in three cetacean species elicited using amplitude-modulated stimuli. In: Kastelein R A, Thomas JA, Nachtigall PE (eds) Sensory Systems of Aquatic Mammals. De Spil Publishers, The Netherlands, pp 25-47 Dolphin WF (1997) The envelope following response to multiple tone pair stimuli. Hear Res 110:1-14 Dolphin WF (2000) Electrophysiological measures of auditory processing in odontocetes. In: Au WWL, Popper AN, Fay RR (eds) Hearing by Whales and D olphins. Springer-Verlag, New York, pp 294-329 Dolphin WF, Mountain DC (1992) The envelope following response : scalp potentials elicited in the Mongolian gerbil using sinusoidally AM acousti c signals. Hear Res 58:70-78

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12 Dolphin WF, Mountain DC (1993) The envelope following response (EFR) in the Mongolian gerbil to sinusoidally amplitude-modulated signals in the presence of simultaneously gated pure tones. J Acoust Soc Am 94:3215-3226 Dreher JJ (1961) Linguistic considerations of porpoise soun ds. J Acoust Soc Am 33:1799-1800 Dreher JJ, Evans WE (1964) Cetacean communication. In: T avolga WN (ed) Marine BioAcoustics. Pergamon Press, New York, pp 373-393 Essapian FS (1953) The birth and growth of a porpoise. Nat H ist 62:392-399 Evans WE (1967) Vocalization among marine mammals. In: Ta volga WN (ed) Marine Bio-Acoustics, Vol. 2. Pergamon Press, New York, pp 159-186 Finneran JJ, Houser DS (2006) Comparison of in-air evoked poten tial and underwater behavioral hearing thresholds in four bottlenose dolphins ( Tursiops truncatus ). J Acoust Soc Am 119:3181-3192 Finneran JJ, Carder DA, Ridgway SH (2002a) Low-frequency acoustic pressure, velocity, and intensity thresholds in a bottlenose dolphin ( Tursiops truncatus ) and white whale ( Delphinapterus leucas ). J Acoust Soc Am 111:447-456 Finneran JJ, Schlundt CE, Carder DA, Ridgway SH (2002b) Auditor y filter shapes for the bottlenose dolphin ( Tursiops truncatus ) and the white whale ( Delphinapterus leucas ) derived with notched noise. J Acoust Soc Am 112:322-328 Finneran JJ, Schlundt CE, Dear R, Carder DA, Ridgway SH ( 2002c) Temporary shift in masked hearing thresholds in odontocetes after exposure to s ingle underwater impulses from a seismic watergun. J Acoust Soc Am 111:2929-2940

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13 Finneran JJ, Carder DA, Dear R, Belting T, McBain J, Da lton L, Ridgway SH (2005) Pure tone audiograms and possible aminoglycoside-induced hea ring loss in belugas ( Delphinapterus leucas ). J Acoust Soc Am 117:3936-3943 Hall JD, Johnson CS (1972) Auditory thresholds of a killer whale Orcinus orca Linnaeus. J Acoust Soc Am 51:515-517 Houser DS, Finneran JJ (2006) A comparison of underwater hea ring sensitivity in bottlenose dolphins ( Tursiops truncatus ) determined by electrophysiological and behavioral methods. J Acoust Soc Am 120:1713-1722 Houser DS, Finneran JJ, Carder DA, Ridgway SH, Moore PW (2004) Relationship between auditory evoked potential (AEP) and behavioral audio grams in odontocete cetaceans. J Acoust Soc Am 116:2503 Jacobs DW, Hall JD (1972) Auditory thresholds of a fresh water dolphin, Inia geoffrensis Blainville. J Acoust Soc Am 51:530-533 Janik VM (2000) Food-related bray calls in wild bottlenose dolphi ns ( Tursiops truncatus ). Proc R Soc Lond B 267:923-927 Janik VM, Slater PJB (1998) Context-specific use suggests that bottlenose dolphin signature whistles are cohesion calls. Anim Behav 56:829-838 Janik VM, Sayigh LS, Wells RS (2006) Signature whistle shape co nveys identity information to bottlenose dolphins. Proc Natl Acad Sci USA 103:8293-8297 Johnson CS (1966) Auditory thresholds of the bottlenosed porpo ise ( Tursiops truncatus Montagu). U.S. Naval Ordnance Test Station (NOTS) TP 4178, 37 pp. Johnson CS (1967) Sound detection thresholds in marine mammal s. In: Tavolga WN (ed) Marine Bio-Acoustics, Vol. 2. Pergamon Press, New York, pp 247-260

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14 Kastelein RA, Hagedoorn M, Au WWL, de Haan D (2003) Audiogram of a striped dolphin ( Stenella coeruleoalba ). J Acoust Soc Am 113:1130-1137 Kastelein RA, Bunskoek P, Hagedoorn M, Au WWL, de Haan D (2002 ) Audiogram of a harbor porpoise ( Phocoena phocoena ) measured with narrow-band frequencymodulated signals. J Acoust Soc Am 112:334-344 Kellogg WN (1953) Ultrasonic hearing in the porpoise, Tursiops truncatus J Comp Psychol 46:446-450 Kellogg WN (1958) Echo ranging in the porpoise. Science 128:982-988 Kellogg WN (1961) Porpoises and Sonar. The University of Chi cago Press, Chicago Kellogg WN, Kohler R (1952) Reactions of the porpoise to ultras onic frequencies. Science 116:250-252 Kellogg WN, Kohler R, Morris HN (1953) Porpoise sounds as so nar signals. Science 117:239-243 Kullenberg B (1947) Sounds emitted by dolphins. Nature 160:648 Lilly JC (1962) Vocal behavior of the bottlenose dolphin. Proc Amer Phil Soc 106:520529 Lilly JC (1963) Distress call of the bottlenose dolphin: s timuli and evoked behavioral responses. Science 139:116-118 Lilly JC, Miller AM (1961a) Sounds emitted by the bottlenose dolphin. Science 133:1689-1693 Lilly JC, Miller AM (1961b) Vocal exchanges between dolphins Science 134:1873-1876

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15 Ljungblad DK, Scoggins PD, Gilmartin WG (1982) Auditory thres holds of a captive Eastern Pacific bottle-nosed dolphin, Tursiops spp. J Acoust Soc Am 72:17261729 McBride AF (1956) Evidence for echolocation by cetaceans. D eep-Sea Res 3:153-154 McCormick JG, Wever EG, Palin J (1970) Sound conduction in t he dolphin ear. J Acoust Soc Am 48:1418-1428 McCormick JG, Wever EG, Ridgway SH, Palin J (1980) Sound rec eption in the porpoise as it relates to echolocation. In: Busnel RG, Fish J F (eds) Animal Sonar Systems. Plenum Press, New York, pp 449-467 McCowan B, Reiss D (1995) Quantitative comparison of whist le repertoires from captive adult bottlenose dolphins (Delphinidae, Tursiops truncatus ): a re-evaluation of the signature whistle hypothesis. Ethol 100:194-209 McCowan B, Reiss D (2001) The fallacy of ‘signature whist les’ in bottlenose dolphins: a comparative perspective of ‘signature information’ in anim al vocalizations. Anim Behav 62:1151-1162 Moore PWB, Pawloski DA (1993) Interaural time discriminati on in the bottlenose dolphin. J Acoust Soc Am 94:1829-1830 Nachtigall PE, Yuen MML, Mooney TA, Taylor KA (2005) Hear ing measurements from a stranded infant Risso’s dolphin, Grampus griseus J Exp Biol 208:4181-4188 Nachtigall PE, Au WWL, Pawloski JL, Moore PWB (1995) Risso ’s dolphin ( Grampus griseus ) hearing thresholds in Kaneohe Bay, Hawaii. In: Kast elein RA, Thomas JA, Nachtigall PE (eds) Sensory Systems of Aquatic Mamm als. DeSpil Publishers, The Netherlands, pp 49-53

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16 Nachtigall PE, Lemonds DW, Roitblat HL (2000) Psychoacousti c studies of dolphin and whale hearing. In: Au WWL, Popper AN, Fay RR (eds) Hearin g by Whales and Dolphins. Springer-Verlag, New York, pp 330-363 Norris KS (1964) Some problems of echolocation in cetacea ns. In: Tavolga WN (ed) Marine Bio-Acoustics. Pergamon Press, New York, pp 317-336 Norris KS (1968) The evolution of acoustic mechanisms in odo ntocete cetaceans. In: Drake ET (ed) Evolution and Environment. Yale University Pre ss, London, pp 297-324 Norris KS, Prescott JH, Asa-Dorian PV, Perkins P (1961) An experimental demonstration of echolocation behavior in the porpoise, Tursiops truncatus (Montagu). Biol Bull 120:163-176 Nowacek DP (2005) Acoustic ecology of foraging bottlenose dolphins ( Tursiops truncatus ), habitat-specific use of three sound types. Mar Mamm S ci 21:587-602 Popov VV, Klishin VO (1998) EEG study of hearing in the com mon dolphin, Delphinus delphis Aquat Mamm 24:13-20 Popov VV, Supin AY (1987) Characteristics of hearing in th e beluga Delphinapterus leucas Dokl Biol Sci 294:1255-1258 Popov VV, Supin AY (1990a) Auditory brain stem responses in characterization of dolphin hearing. J Comp Physiol A 166:385-393 Popov V, Supin A (1990b) Electrophysiological studies of hea ring in some cetaceans and a manatee. In: Thomas JA, Kastelein RA (eds) Sensory A bilities of Cetaceans: Laboratory and Field Evidence. Plenum Press, New York, pp 405 -415

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17 Popov VV, Supin AY (1990c) Electrophysiological investigation of hearing in the freshwater dolphin Inia geoffrensis Dokl Biol Sci 313:238-241 Popov VV, Ladygina TF, Supin AY (1986) Evoked potentials of the auditory cortex of the porpoise, Phocoena phocoena J Comp Physiol A 158:705-711 Popov VV, Supin AY, Wang D, Wang K, Xiao J, Li S (2005) Evo ked-potential audiogram of the Yangtze finless porpoise Neophocaena phocaenoides asiaeorientalis (L). J Acoust Soc Am 117:2728-2731 Ridgway SH (1980) Electrophysiological experiments on hearin g in odontocetes. In: Busnel RG, Fish JF (eds) Animal Sonar Systems. Plenum Press, New York, pp 483-493 Ridgway SH, Carder DA (1993) High-frequency hearing loss in ol d (25+ years old) male dolphins. J Acoust Soc Am 94:1830 Ridgway SH, Carder DA (1997) Hearing deficits measured in some Tursiops truncatus and discovery of a deaf/mute dolphin. J Acoust Soc Am 101:590-594 Ridgway SH, Carder DA (2001) Assessing hearing and sound production in cetaceans not available for behavioral audiograms: experiences wi th sperm, pygmy sperm, and gray whales. Aquat Mamm 27:267-276 Ridgway SH, Bullock TH, Carder DA, Seeley RL, Woods D, G alambos R (1981) Auditory brainstem response in dolphins. Proc Natl Acad Sc i USA 78:1943-1947 Sauerland M, Dehnhardt G (1998) Underwater audiogram of a tucuxi ( Sotalia fluviatilis guianensis ). J Acoust Soc Am 103:1199-1204 Sayigh LS (1992) Development and functions of signature whis tles of free-ranging bottlenose dolphins, Tursiops truncatus Ph.D. dissertation, MIT/WHOI, 348 pp.

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18 Sayigh LS, Tyack PL, Wells RS, Scott MD (1990) Signature whistles of free-ranging bottlenose dolphins Tursiops truncatus : stability and mother-offspring comparisons. Behav Ecol Sociobiol 26:247-260 Sayigh LS, Tyack PL, Wells RS, Scott MD, Irvine AB (1995) Sex differences in signature whistle production of free-ranging bottlenose dolp hins, Tursiops truncatus Behav Ecol Sociobiol 36:171-177 Sayigh LS, Tyack PL, Wells RS, Solow AR, Scott MD, Ir vine AB (1999) Individual recognition in wild bottlenose dolphins: a field test using playback experiments. Anim Behav 57:41-50 Schevill WE (1964) Underwater sounds of cetaceans. In: Tavo lga WN (ed) Marine BioAcoustics. Pergamon Press, New York, pp 307-316 Schevill WE, Lawrence B (1953) Auditory response of a bottl enosed porpoise, Tursiops truncatus to frequencies above 100 kc. J Exp Zool 124:147-165 Schevill WE, Lawrence B (1956) Food-finding by a captive porpoi se ( Tursiops truncatus ). Breviora, Mus Comp Zool, Harvard 53:1-15 Supin A, Popov V (1990) Frequency-selectivity of the audito ry system in the bottlenose dolphin, Tursiops truncatus In: Thomas JA, Kastelein RA (eds) Sensory Abilities of Cetaceans: Laboratory and Field Evidence. Plenum Press, New York, pp 385393 Supin AY, Popov VV (1995) Frequency tuning and temporal resolutio n in dolphins. In: Kastelein RA, Thomas JA, Nachtigall PE (eds) Sensory Systems of Aquatic Mammals. De Spil Publishers, The Netherlands, pp 95-110

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19 Supin AY, Popov VV, Klishin VO (1993) ABR frequency tuning curves in dolphins. J Comp Physiol A 173:649-656 Supin AY, Popov VV, Mass AM (2001) The Sensory Physiology of Aquatic Mammals. Kluwer Academic Publishers, Boston Supin AY, Nachtigall PE, Pawloski J, Au WWL (2003) Evoked p otential recording during echolocation in a false killer whale Pseudorca crassidens (L). J Acoust Soc Am 113:2408-2411 Szymanski MD, Bain DE, Kiehl K, Pennington S, Wong S, Henr y KR (1999) Killer whale ( Orcinus orca ) hearing: auditory brainstem response and behavioral audiograms. J Acoust Soc Am 106:1134-1141 Thomas J, Chun N, Au W, Pugh K (1988) Underwater audiogram of a false killer whale ( Psuedorca crassidens ). J Acoust Soc Am 84:936-940 Thompson RKR, Herman LM (1975) Underwater frequency discr imination in the bottlenosed dolphin (1-140 kHz) and the human (1-8 kHz). J Acous t Soc Am 57:943-948 Thomson DH, Richardson WJ (1995) Marine mammal sounds. In: Richardson WJ, Greene Jr CR, Malme CI, Thomson DH (eds) Marine Mamm als and Noise. Academic Press, San Diego, pp 159-204 Tremmel DP, Thomas JA, Ramirez KT, Dye GS, Bachman W A, Orban AN, Grim KK (1998) Underwater hearing sensitivity of a Pacific white-side d dolphin, Lagenorhynchus obliquidens Aquat Mamm 24:63-69

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20 Wang D, Wang K, Ziao Y, Sheng G (1992) Auditory sensitivity of a Chinese river dolphin ( Lipotes vexillifer ). In: Thomas JA, Kastelein RA, Supin AY (eds) Marine Mammal Sensory Systems. Plenum, New York, pp 213-221 Watwood SL (2003) Whistle use and whistle sharing by allied male bottlenose dolphins, Tursiops truncatus Ph.D. dissertation, MIT/WHOI, 227 pp. Watwood SL, Tyack PL, Wells RS (2004) Whistle sharing in paire d male bottlenose dolphins, Tursiops truncatus Behav Ecol Sociobiol 55:531-543 Watwood SL, Owen ECG, Tyack PL, Wells RS (2005) Signature w histle use by temporarily restrained and free-swimming bottlenose dolphi ns, Tursiops truncatus Anim Behav 69:1373-1386 White Jr MJ, Norris J, Ljungblad D, Barron K, di Sciara G (1978) Auditory thresholds of two beluga whales ( Delphinapterus leucas ). HSWRI TR 78-109, Hubbs/Sea World Research Institute, San Diego, CA, 37 pp. Wood Jr FG (1953) Underwater sound production and concurrent beh avior of captive porpoises, Tursiops truncatus and Stenella plagiodon Bull Mar Sci Gulf and Caribbean 3:120-133 Yuen MML, Nachtigall PE, Breese M, Supin AY (2005) Behavior al and auditory evoked potential audiograms of a false killer whale ( Pseudorca crassidens ). J Acoust Soc Am 118:2688-2695

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21 Table 1-1 Behavioral hearing measurements for odontocete cetacea ns. N indicates the sample size for the study. AUTHOR YEAR SPECIES n FREQUENCIES Kellogg & Kohler 1952 Tursiops truncatus Stenella plagiodon 10, 2 0.02-200 kHz Schevill & Lawrence 1953 Tursiops truncatus 1 0.15-153 kHz Kellogg 1953 Tursiops truncatus 13 0.1-200 kHz Johnson 1966, 1967 Tursiops truncatus 1 0.075-150 kHz Anderson 1970 Phocoena phocoena 1 1-150 kHz Belkovich & Solntseva 1970 Delphinus delphis 1 0.018-280 kHz Hall & Johnson 1972 Orcinus orca 1 0.5-32 kHz Jacobs & Hall 1972 Inia geoffrensis 1 1.0-105 kHz Thompson & Herman 1975 Tursiops truncatus 1 1-140 kHz White et al. 1978 Delphinapterus leucas 2 1-123 kHz Ljungblad et al. 1982 Tursiops spp. 1 2-160 kHz Awbrey et al. 1988 Delphinapterus leucas 3 0.125-8 kHz Thomas et al. 1988 Pseudorca crassidens 1 2-115 kHz Wang et al. 1992 Lipotes vexillifer 1 1-200 kHz Nachtigall et al. 1995 Grampus griseus 1 4-110 kHz Ridgway & Carder 1993, 1997 Tursiops truncatus 8 5-120 kHz Sauerland & Dehnhardt 1998 Sotalia fluviatilis guianensis 1 4-135 kHz Tremel et al. 1998 Lagenorhynchus obliquidens 1 0.075-150 kHz Szymanski et al. 1999 Orcinus orca 2 1-120 kHz Kastelein et al. 2002 Phocoena phocoena 1 0.250-180 kHz Au et al. 2002 Tursiops truncatus 1 40-140 kHz Finneran et al. 2002a Tursiops truncatus Delphinapterus leucas 1, 1 0.1 & 0.3 kHz Finneran et al. 2002b Tursiops truncatus Delphinapterus leucas 2, 1 20 & 30 kHz Finneran et al. 2002c Tursiops truncatus Delphinapterus leucas 1, 1 0.4, 4, & 30 kHz Kastelein et al. 2003 Stenella coeruleoalba 1 0.5-160 kHz Houser et al. 2004 Tursiops truncatus n/a n/a Finneran et al. 2005 Delphinapterus leucas 2 2-130 kHz

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22 Table 1-1 (Continued) AUTHOR YEAR SPECIES n FREQUENCIES Finneran & Houser 2006 Tursiops truncatus 4 5-150 kHz Houser & Finneran 2006 Tursiops truncatus 3 5-150 kHz Cook et al. in prep. Tursiops truncatus 2 5-80 kHz

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23 Table 1-2 Auditory evoked potential (AEP) hearing measurements fo r odontocete cetaceans. N indicates the sample size for the stud y. AUTHOR YEAR SPECIES n FREQUENCIES Bullock et al. 1968 Stenella coeruleoalba S. attenuata Steno bredanensis Tursiops gilli 29 total 5-150 kHz Bullock & Ridgway 1972 Tursiops truncatus 7 20-30 kHz Ridgway 1980 Tursiops truncatus 7 n/a Ridgway et al. 1981 Tursiops truncatus Delphinus delphis 2, 2 6, 66, & 124 kHz Popov et al. 1986 Phocoena phocoena 4 10-150 kHz Popov & Supin 1987 Delphinapterus leucas 2 15-120 kHz Carder & Ridgway 1990 Physeter spp. 1 2.5-60 kHz Supin & Popov 1990 Tursiops truncatus 4 25-100 kHz Popov & Supin 1990a Tursiops truncatus 4 5-150 kHz Popov & Supin 1990b Tursiops truncatus Inia geoffrensis Sotalia fluviatilis Delphinapterus leucas 4, 4, 2, 2 5-160 kHz Popov & Supin 1990c Inia geoffrensis 4 7-150 kHz Supin et al. 1993 Tursiops truncatus 2 16-128 kHz Dolphin 1995 Tursiops truncatus Delphinapterus leucas Pseudorca crassidens 2, 2, 1 0.5-10 kHz Supin & Popov 1995 Tursiops truncatus 4 16-128 kHz Popov & Klishin 1998 Delphinus delphis 1 5-150 kHz Szymanski et al. 1999 Orcinus orca 2 1-100 kHz Ridgway & Carder 2001 Eschrichtius robustus Kogia breviceps Physeter macrocephalus 1, 1, 1 0.02-200 kHz Andr et al. 2003 Stenella coeruleoalba 1 16-128 kHz Supin et al. 2003 Pseudorca crassidens 1 35 kHz Houser et al. 2004 Tursiops truncatus n/a n/a Beedholm & Miller 2005 Phocoena phocoena 1 80, 100, 125, & 160 kHz Nachtigall et al. 2005 Grampus griseus 1 4-150 kHz Popov et al. 2005 Neophocaena phocaenoides asiaeorientalis 2 8-152 kHz Yuen et al. 2005 Pseudorca crassidens 1 4-45 kHz

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24 Table 1-2 (Continued) AUTHOR YEAR SPECIES n FREQUENCIES Cook et al. 2006 Mesoplodon europaeus 1 5-80 kHz Finneran & Houser 2006 Tursiops truncatus 4 10-150 kHz Houser & Finneran 2006 Tursiops truncatus 3 5-150 kHz Cook et al. in prep. Tursiops truncatus 2 5-80 kHz

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25 Chapter Two: Ground-Truthing In-Air Auditory Evoked Potential (AEP) Heari ng Measurements with Traditional Behavioral Audiograms in Bottlenose Dolphins ( Tursiops truncatus ) Abstract Auditory evoked potential (AEP) methods are more attracti ve than traditional behavioral methods for measuring the hearing of marine mammals becau se they allow for rapid assessments of hearing sensitivity and can be used on untr ained animals. However, few studies have compared these two measurement types using the same individual. This study investigated the differences between underwater AEP an d in-air AEP measurements using two captive bottlenose dolphins ( Tursiops truncatus ). Underwater behavioral hearing measurements were also made with one of the dolphins using the same stimuli used for the AEP measurements. Frequencies tested ranged from 5 to 80 kHz. There was generally good agreement among the heari ng thresholds determined by these three methods at frequencies above 20 kHz. At 10 and 20 kHz, in-air AEP audiograms were substantially higher (about 15 dB) than un derwater behavioral and underwater AEP audiograms, suggesting multiple sound pathways t o the dolphins’ ears at lower frequencies.

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26 Introduction Bottlenose dolphins ( Tursiops truncatus ) have an impressive ability to both produce and perceive a wide variety of sounds over a large frequency range. Because dolphins rely on sound for communication, navigation, and foraging, their se nse of hearing is one of their most important senses (Au 1993; Janik and Slater 1998). The vas t majority of the information known about the hearing capabilities of dolp hins and other odontocetes (toothed whales) has been obtained using traditional behavior al and psychometric techniques. Behavioral audiograms have been reported for twelve odontocete species: bottlenose dolphin Tursiops spp. (Johnson 1966, 1967; Ljungblad et al. 1982), common dolphin Delphinus delphis (Belkovich and Solntseva 1970), Pacific white-sided dolphin Lagenorhynchus obliquidens (Tremmel et al. 1998), striped dolphin Stenella coeruleoalba (Kastelein et al. 2003), Risso’s dolphin Grampus griseus (Nachtigall et al. 1995), Amazon river dolphin Inia geoffrensis (Jacobs and Hall 1972), Chinese river dolphin Lipotes vexillifer (Wang et al. 1992), beluga whale Delphinapterus leucas (White et al. 1978; Awbrey et al. 1988; Finneran et al. 2005), false kil ler whale Pseudorca crassidens (Thomas et al. 1988; Yuen et al. 2005), tucuxi Sotalia fluviatilis guianensis (Sauerland and Dehnhardt 1998), harbor porpoise Phocoena phocoena (Andersen 1970; Kastelein et al. 2002), and killer whale Orcinus orca (Hall and Johnson 1972; Szymanski et al. 1999). Because most behavioral studies require repeat ed measurements using highly-trained subjects, sample sizes are generally smal l (one to two animals) and data can take up to several years to collect.

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27 As an alternative to traditional behavioral techniques, electrophysiological techniques can also be used to measure hearing abilities. Th e auditory evoked potential (AEP) response is a non-invasive electrophysiological tec hnique commonly used to measure hearing thresholds and other aspects of hearing (e.g. masking and sound localization) in humans, birds, fish, and other animals including cetaceans (e.g., Corwin et al. 1982; Supin and Popov 1995; Kenyon et al. 1998; Szymanski et al. 1999; Mann et al. 2001; Lucas et al. 2002). In general, when the auditory pat hway is presented with an acoustic stimulus that is above threshold levels, lar ge numbers of neurons within the acoustic pathway are excited. If the neuronal discharge s are time-locked to the acoustic stimulus, the electrical signals produced by the simultan eous firings of multiple neurons produce a synchronous discharge that can be detected by an e lectrode placed on the head. AEP hearing measurement techniques are advantageous over beha vioral hearing measurement techniques for several reasons: they are r elatively non-invasive, they require little to no animal training, and they can be done in short time periods. Therefore very rapid estimations of an individual’s hearing thresh old can be obtained. Finally, AEP techniques can be used to measure the hearing sensitivities of animals, particularly cetaceans, for which behavioral audiograms cannot be dete rmined (e.g., Ridgway and Carder 2001; Cook et al. 2006). One notable problem with AEP hearing measurements is that they are measures of neural activity rather than sensation and perception. Thus they need to be validated and calibrated against a direct measure of hearing, i.e., the behavioral audiogram. Although evoked potential and behavioral techniques have been used to asse ss hearing in many odontocetes (Dolphin 2000; Nachtigall et al. 2000; Supin et al 2001), they have only

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28 rarely been measured using the same animal (Szymanski et a l. 1999; Yuen et al. 2005; Cook et al. 2006; Finneran and Houser 2006; Houser and Finneran 2006). T herefore, any comparison between the two techniques is confounded by potent ial subject differences. Another potential source of variability comes from the type of sound stimulus used in each study condition. In general, pure tones (gen erally > 1 s) are used for behavioral hearing measurements (Nachtigall et al. 2000), whi le short clicks, tone pips, or tone bursts (all generally < 1 s) are used for AEP measur ements (Dolphin 2000). For example, Szymanski et al. (1999) collected behavioral hear ing data from killer whales using 2 s tones, and AEP data using 0.5-1 ms cosine-gated tone b ursts. Yuen et al. (2005) used 3 s pure tones to measure the behavioral audiogram o f a false killer whale and 20 ms sinusoidally amplitude-modulated (SAM) tone bursts to measure AEP thresholds. Cook et al. (2006), Finneran and Houser (2006), and H ouser and Finneran (2006) used 500 ms tones to measure behavioral hearing sensitivit ies in bottlenose dolphins. However, Cook et al. (2006) used 14 ms SAM tone bursts to measure AEP hearing thresholds, Finneran and Houser (2006) used 12-15 ms SAM to ne bursts to measure AEP thresholds, and Houser and Finneran (2006) used 23 ms SAM tone bursts to measure the majority of their AEP thresholds (Table 2-1). As a result, any comparisons between the two techniques are also complicat ed by the use of different acoustic stimuli and their potential to affect hearing sens itivity. This study addresses these differences by conducting both A EP and behavioral hearing tests using the same individual and the same acoust ic stimuli: 1.) AEP hearing measurements in air with sounds presented through a jawph one; 2.) AEP hearing measurements underwater using a free-field speaker; and 3.) Underwater behavioral

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29 hearing measurements using a free-field speaker. By compari ng differences among all three experiments, it then becomes possible to derive an appropriate calibration for the inair and underwater AEP audiograms. Thus, behavioral audiogr am estimates can be calculated for animals whose hearing can only be measured using AEP techniques, including live-stranded cetaceans, free-ranging cetaceans, a nd other untrained animals. Materials and Methods Subjects In-air and underwater AEP measurements were collected fr om Ranier and Calvin, two male bottlenose dolphins ( Tursiops truncatus ). They are currently housed at The Living Seas, Epcot Walt Disney World Resort in a 5.7-million-gallon circular exhibit housing many marine species, in Lake Buena Vista, Florida. Beh avioral data were collected from only Ranier due to time and research limitations. Ranier is an approximately 25-year-old male, 2.6 m in length and 190 kg in weight. Calvin is an 11-ye ar-old male (b. 1994), 2.5 m in length and 185 kg in weight. All research was approved by the IACUC of the Walt Disney World Animal Programs.

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30 AEP Methods The AEP technique involves repeatedly playing a test sound sti mulus while simultaneously recording the neural evoked potential from surface electrodes. The evoked potentials from each sound presentation are continuo usly added together to reduce background electrical noise in the recordings and revea l the underlying auditory response (Glasscock et al. 1987; Ferraro and Durrant 1994). A Tucker-Davis Technologies (TDT) AEP Workstation with Si gGen and BioSig software and laptop computer were used to control all stimu lus presentations and data acquisition. This Workstation has been previously used in f ield situations, and to record AEPs from cetaceans, including bottlenose dolphins and a be aked whale (Cook et al. 2006). The TDT Workstation was capable of sampling at 192 kHz, which meant it could test frequencies up to 80 kHz. Each sound trial lasted approximately one minute and consist ed of playing amplitude modulated (AM) tones at specific frequencies an d levels that were programmed using BioSig software. These AM tones consiste d of 14 ms tone bursts presented 21 times per second and 100% modulated at 600 Hz (Figure 21), a modulation rate which has been found to yield strong AEP responses i n bottlenose dolphins (Supin and Popov 1995). Using AM tones in AEP procedures results in an Envelope Fo llowing Response (EFR) in which the auditory system of the subject produce s neural responses that are phase-locked with the envelope of the stimulus (Dolphin 1996, 1997; Supin and Popov 1995). The advantages of using EFR are that 1.) it results in an AEP at the frequency of

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31 AM (Dolphin and Mountain 1992), making it easily distinguished from background electrical noise in the signal, and 2.) it has a narrow frequency spectrum, allowing for good frequency resolution in the audiogram (Dolphin 2000). Bottlenose dolphins use their lower jaws to receive sounds (Norris 1964, 1968). Jawphones (contact hydrophones attached to the dolphin u sing suction cups) take advantage of this pathway, and have been used by several re searchers to present acoustic stimuli to bottlenose dolphins via their lower jaws (Bul lock et al. 1968; McCormick et al. 1970, 1980; Brill et al. 2001; Cook et al. 2006; Finneran and Houser 2006; Houser and Finneran 2006). A jawphone composed of an ITC-1042 transducer em bedded in a suction cup (constructed from VI-SIL V-1062, Rhodia, Inc.) and powered by a Hafler P1000 amplifier was used to deliver the acoustic stimuli for the in-air AEP measurements. The jawphone suction cup is composed of a silicone-based RTV material which has an acoustic impedance similar to water (Brill et al. 2001). The jawphone was placed on the lower left jaw of each animal, correspon ding to position #38 in Mhl et al. (1999), which showed the greatest AEP response in their stud y. AEP signals were collected using suction cup electrodes ma de from standard 8 mm silver-silver chloride electrodes (Med-Associates, Inc .) embedded in either vinyl (VF65, Anver, Inc.) or RTV silicone (VI-SIL V-1062, Rhodia, Inc.) suction cups. Each dolphin’s skin was prepared by wiping the areas of suction cup attachment with a dry gauze pad in order to remove debris. Redux electrolyte paste (Parker Laboratories, Inc.), commonly used in human and veterinary applicatio ns, was used on the electrodes to establish a good electrical connection between the electr ode and the dolphin’s skin. A recording electrode was placed dorsally at the vertex of the skull, approximately six

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32 centimeters behind the blowhole, and a reference electro de was placed just anterior to the dorsal fin. A ground electrode was placed between the refe rence and recording electrodes with approximately 20 centimeters separating adj acent electrodes. All suction cups were removed as soon as tests were complete. The protected electrode leads were attached to a different ial amplifier (TDT DB4HS4) housed in a water-resistant case. The amplifier output was connected via a fiber optic cable to the TDT Workstation for data acquisition with the BioSig software. BioSig controlled both stimulus presentation and data acquisiti on. Electrical artifacts induced by dolphin breathing and locomotory muscle (or skeletal muscl e) movement of the electrodes were removed by artifact rejection in BioSig (excluding all sweeps with evoked potentials greater than a set threshold). Sounds in these experiments were played at levels less than or equal to 160 dB re 1 Pa, which is approximately the same sound pressure level ( SPL) as whistles produced by bottlenose dolphins (mean source level: 158 dB re 1Pa; Janik 2000). Furthermore, it is much lower than sound levels that have been found to cause temporary threshold shifts in dolphins (180-200 dB re 1 Pa; Schlundt et al. 2000). These sounds were attenuated in 6 or 10 dB steps and controlled by the computer using a progr ammable attenuator (TDT PA5). The following frequencies were measured: 5, 10 20, 30, 40, 60, and 80 kHz. Higher frequencies could not be measured due to the sa mpling rate limitations of the equipment. Up to 5000 averages were run for each test trial, although a few underwater AEP measurements contained up to 16,000 averages. Once an AEP re sponse was observed, averaging at that test level was ended, and the next lev el was tested, thus minimizing the

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33 amount of time required to collect data. An AEP response was determined to be present if the evoked signal, measured from 5 to 18 ms or from 20 to 33 ms (Calvin’s in-air AEP measurements only), was greater than the background noise, estimated from 0 to 4 ms in the same sweep (Figure 2-2). This is the same threshold de termination technique used by Cook et al. (2006). Experiment 1: In-Air AEPs with a Jawphone Ranier’s in-air AEP data were collected on July 20, 2004 f rom 0906 hrs to 0931 hrs. Calvin’s in-air AEP data were collected on April 19, 2005 fr om 0905 hrs to 0938 hrs and on May 30, 2006 from 0910 hrs to 0944 hrs. Each dolphin was isolate d in a medical pool and the water level was dropped or the false-bottom floo r was raised. The dolphin was then placed onto a closed-cell foam mat and kept wet usin g wet towels and water sprayers. Animal trainers were stationed laterally ar ound the dolphin to help support it. Once the animal was correctly stationed and not moving, the suction cup electrodes and jawphone were attached and AEP testing began (Figure 2-3). As soon as testing was complete, all suction cups were removed, the water leve l was raised or the false-bottom floor was lowered, and the dolphin was fed. It should be n oted that both dolphins were trained to voluntarily participate in this experiment. Also, because of changes in AEP procedures as part of other experiments, Calvin’s in-air AE P data were collected in response to 15 ms tones on April 19 and May 30, but only 13 ms o f the signals were analyzed to maintain consistency.

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34 Underwater Experimental Setup The experimental conditions were similar between the underwater AEP measurements and the underwater behavioral audiogram measurements. Both experiments were conducted from a 0.4 m 0.5 m floating dock within the 5.7-mill ion-gallon circular environment. An underwater PVC stationing apparatus was secur ely attached to the floating dock. Each dolphin was trained to station in the apparatus one meter below the water surface by placing its rostrum into a plastic chinstra p. Animal trainers helped the dolphin maintain its position within the water column by ge ntly holding its dorsal fin during testing. This also helped to prevent the animal from moving its flukes to maintain position, which reduced data contamination for the underwate r AEP measurements. An ITC-1042 transducer, identical to the one used as the jawpho ne, was placed one meter underwater and attached to the PVC apparatus approximately 15 c m in front of the dolphin’s rostrum (Figure 2-4). Experiment 2: Underwater AEPs with a Free-Field Speaker The AEP methods, stimulus control, data collection, suc tion cup electrodes, and sound frequencies and levels used in the underwater AEP measure ments were all identical to those used for the in-air AEP measurements with a few notable exceptions. Each dolphin was trained to wear the recording and reference suction cup electrodes while the ground electrode was placed freely in the water instead of on t he animal. In addition the sounds

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35 were presented from a free-field speaker instead of an att ached jawphone. Finally the animals were trained to station one meter underwater for data collection. The dolphin was called over to the floating platform, an d the area between its blowhole and dorsal fin was wiped dry with a gauze pad. The r ecording and reference suction cup electrodes were subsequently attached to the ani mal in the same locations used for the in-air AEP measurements. A hand signal wa s then used to send the dolphin to the underwater PVC stationing apparatus. Once the dolphin was stationed correctly, underwater AEP measurements began (Figure 2-4). If the dolp hin vocalized or moved excessively during testing, the trial was ended and the dolphi n was signaled to return to the surface. Otherwise, the dolphin remained stationed f or up to two minutes of data collection, after which time he was recalled to the sur face and rewarded. Two-minute trials were conducted in 15-20 minute sessions, up to four times a day. Behavioral training for both Ranier and Calvin began on October 23, 2003. Ranier’s underwater AEP measurements were collected on May 11, 13, 19, and 28, 2004. Calvin’s underwater AEP measurements were collected on September 1, 2, 8, and 9, 2004. Experiment 3: Underwater Behavioral Audiogram with a Free-Field Speaker Ranier’s behavioral audiogram was measured using a modified go /no-go procedure (Schusterman 1980), in which he was trained to vocalize in the presence of a tone and remain silent in the absence of a tone. A hand signal was used to send Ranier to the underwater PVC stationing apparatus, which was the same appar atus used for the

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36 underwater AEP measurements. Once Ranier was stationed correctly, an underwater light illuminated, indicating the start of a trial. T he time between the light illumination and sound presentation (excluding catch trials) varied from two to three seconds. Ranier was trained to whistle within six seconds of detecting a sound and to remain silent for 10 seconds if no sound was detected. He was recalled to th e surface at the end of each trial. A research assistant used a Sonatech (Model 8234-1) hydrophone and headset to monitor for the dolphin’s response (whistle or no whistle) during each trial. This information was electronically relayed to the TDT Works tation, which recorded whether Ranier’s response was correct or incorrect and then autom atically determined the next trial. The TDT Workstation flashed a green LED for ea ch of Ranier’s correct responses, and a red LED for each of Ranier’s incorrect responses. This alerted the trainer whether or not to reward Ranier’s response. Each correct respon se was rewarded, and each incorrect response was neither rewarded nor punished. The trainer and the assistant were both nave as to whether a tone was present or absent during each trial, except at 5 and 10 kHz, which the assistant could hear through the headphones at only the loudest sound presentations. Continuous acoustic recordings were also collected during each session using Avisoft SASLab Pro v 4.38 (Avisoft Bioacoustics, Berlin ). The sound stimuli used for the behavioral audiogram measure ments were the same as those used for the AEP measurements. Thus, eac h trial lasted approximately one minute and consisted of playing 600 Hz AM tone bursts 14 ms in dur ation and repeated 21 times per second. A modified staircase method was used. For each tone frequency, testing was started at a sound intensity level that was easily detectable, based on previously published reports for bottlenose dolphins (Johnson 1966, 1967) and on

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37 preliminary analyses of Ranier’s in-air AEP data. Initi al step sizes were 6 dB until the first error, after which 3 dB step sizes were used. Catch trials (no sound presented) varied between 25 and 50% of the total trials per session. Session with 25% catch trials allowed more sound trials to be run, but these sessions were alternated with 50% catch trial sessions in order to avoid biasing Ranier’s response pa ttern toward whistling. Table 2-2 shows that Ranier’s responses were not biased between the 25% and 50% catch trial sessions because similar response patterns were seen bet ween the two types of sessions. Run lengths for sound or catch trials varied pseudo-randomly (Gellermann 1933) with a maximum of three of one trial type in a row. A session consisted of 30 trials; this was designed to elicit approximately eight sound intensity reversals per session. A threshold was defined as two consecutive sessions with m ean amplitude levels of reversals differing by no more than 3 dB. Because of log istical constraints, only Ranier participated in this experiment. Training for the underwater behavioral audiogram began on September 23, 2004. Underwater behavioral audiogram data were collected over the course of 69 sessions between December 29, 2004 and June 6, 2005. Jawphone and Free-Field Speaker Calibrations The jawphone was calibrated for the in-air AEP measurem ents by placing a Reson calibrated hydrophone (Reson TC4013; -212 dBV re 1 Pa) 10 cm from the end of the suction cup, and calibrating it in the test tank at approxima tely one meter water depth. For the underwater AEP and behavioral hearing measurements the free-field speaker was

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38 calibrated by placing a calibrated hydrophone (Reson TC4013 or HT I 96-min; -164 dBV re 1 Pa) in the center of the chinstrap either before or after each test session without the dolphins present. Background tank noise was also measured wit h the HTI hydrophone, which provided better sensitivity than the Reson hydrophone for measuring the low background noise levels. Results Calvin Table 2-3 and Figure 2-5 present the in-air and underwater AEP hearing thresholds for Calvin. In-air AEP thresholds closely matched underwater AEP thresholds, except at 10 and 20 kHz, where the underwater thresholds were much lowe r than the thresholds measured in air. Ranier Table 2-4 and Figure 2-6 present the in-air AEP, underwater A EP, and behavioral hearing thresholds for Ranier. Underwater AEP thresholds could only be determined for four of the seven frequencies tested, and in-air AEP thresholds could only be determined for six of the seven frequencies tested. Underwater AEP thresho lds were lower than in-air AEP

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39 thresholds at 10 and 20 kHz and more closely resembled the behavioral audiogram at these frequencies. Nonetheless, there was generally go od agreement among the different measurements, especially at 40, 60, and 80 kHz. Discussion While behavioral psychoacoustic methods provide the most direct measures of hearing (Nachtigall et al. 2000), the time and training required with these techniques limit their broad application. Alternatively, AEP methods can be use d to rapidly assess hearing abilities and can be used on minimally or untrained animals. Studies comparing behavioral and AEP hearing measurements on the same anima l have only recently been conducted (Szymanski et al. 1999; Yuen et al. 2005; Cook et al. 2006; Finneran and Houser 2006; Houser and Finneran 2006), and only two of these studi es measured AEPs in air (Cook et al. 2006; Finneran and Houser 2006). Szymanski et al. (1999) found that AEP thresholds were, on average, 12 dB less sensitive tha n behavioral thresholds across the entire frequency range tested. Yuen et al. (2005) obtai ned similar results, with behavioral thresholds always lower than AEP thresholds Behavioral hearing thresholds were also lower than AEP thresholds for two of the t hree animals measured by Cook et al. (2006). Behavioral and AEP hearing thresholds were in close agreement for the animals evaluated by Finneran and Houser (2006) and Houser and Finne ran (2006), and measured differences between the two methods were gener ally the result of more sensitive behavioral measurements.

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40 The current study measured in-air AEP, underwater AEP, and behavioral audiograms in the same individual using the same acoustic stimuli; this combination allowed for differences among methodologies to be direct ly compared. These results show that behavioral, underwater AEP, and in-air AEP h earing measurements produce similar thresholds, especially at higher frequencies. At 10, 20, and 60 kHz both Calvin and Ranier had higher in-air AEP thresholds than underwate r AEP thresholds, and at 40 kHz they both had higher underwater AEP thresholds than in-air AEP thresholds (Tables 2-2 and 2-3). At both 30 and 80 kHz, Calvin’s underwater AEP thre shold measurements were higher than his in-air AEP measurements. Ranier’s underwater AEP hearing thresholds were not determined at either of these freque ncies because there were no AEP signals larger than the AEP noise floor at 80 kHz and bec ause the 30 kHz data were contaminated by low-frequency electrical noise near the rate of amplitude modulation. Additionally, Ranier’s in-air and underwater AEP hearing t hresholds were not measured at 5 kHz due to time limitations. Ranier’s underwater behavioral hearing thresholds were lo wer than both his underwater AEP and in-air AEP hearing thresholds at 10, 20 30 (in-air AEP only), and 40 kHz, while at 60 kHz his behavioral hearing threshold was higher than either AEP threshold. At 80 kHz, Ranier’s underwater behavioral and i n-air AEP thresholds were very similar, differing by only 0.6 dB re 1 Pa. These res ults show that in-air AEP measurements collected using a jawphone accurately repres ent underwater behavioral measurements at higher frequencies, and exhibit both the gene ral shape and highfrequency cutoff of behavioral audiograms. In addition, th ese results are consistent with previous studies, which also found similar thresholds betwe en behavioral and in-air AEP

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41 measurements at higher frequencies (Cook et al. 2006; Finneran and Houser 2006). Somewhat surprisingly, these measurements also demonstra te Ranier’s substantial hearing losses at 60 and 80 kHz which had previously gone undetec ted. At lower frequencies, behavioral measurements resulted in the lowest measured hearing thresholds, followed by underwater AEP measuremen ts and finally in-air AEP measurements, with the highest measured hearing threshold s. These results support the idea that bottlenose dolphins transmit lower frequency soun ds to their ears using multiple sound pathways, not solely via the acoustic window area o f their lower jaws (Popov et al. 2006). However, it is also possible that this is an acous tic phenomenon related to the size of the jawphone suction cup, which itself can act as an acoustic waveguide. At 20 kHz, the acoustic wavelength is approximately 7.5 cm, while the jawphone diameter is 5.0 cm. At higher frequencies, the acoustic wavelengths are shorter than the jawphone diameter. The results of this study allow behavioral audiogram thre sholds to be estimated for dolphins whose hearing can only be measured using in-air A EP techniques, including live-stranded, free-ranging, and other untrained cetaceans. The transfer function of the in-air AEP audiogram to the underwater behavioral audiogram ( the numerical difference between the two hearing threshold measurements at each frequency) accounts for all differences between the two test procedures, includin g differences in calibration procedures. One of the challenges of in-air AEP audiogram s is measuring the sound level at the dolphin ear. In this study, a free-field calibration of the jawphone measured at 10 cm was used to estimate the jawphone sound levels. However, the jawphone is not used in a free-field situation when it is attached to a do lphin in air. The calibration performed underwater is relatively straightforward, si nce the sound level can be

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42 measured at the same location as the dolphin in the sta tioning apparatus. The transfer function thus accounts for errors in the estimation of the delivered sound level in air, as well as for differences between the AEP and behavioral methods. When comparing among studies that measure both behavioral a nd AEP hearing thresholds in the same individuals, other factors must also be considered. Behavioral test paradigms and step sizes, number of AEP sweeps averaged per trial, signal lengths, background noise levels, and methods of threshold determina tion all factor into the final threshold value assigned to each test frequency. For exa mple, Szymanski et al. (1999), Yuen et al. (2005), and the current study all used variations of the go/no-go test paradigm, while Cook et al. (2006), Finneran and Houser (2006), and Houser and Finneran (2006) used both go/no-go and Method of Free Response t est paradigms. Behavioral step sizes in Szymanski et al. (1999) were 6-8 dB, and in both Yuen et al. (2005) and Finneran and Houser (2006) they were 2 dB. In the curr ent study they were 3 dB. The number of AEP sweeps averaged per trial also vari ed considerably among studies. Szymanski et al. (1999) averaged 350 sweeps, Yuen et al (2005) averaged 1000 sweeps, Cook et al. (2006) averaged up to 2000 sweeps, Finneran and H ouser (2006) averaged between 500 and 1000 sweeps, and Houser and Finneran (2006) av eraged 500 sweeps. The current study averaged up to 16,000 sweeps, due in par t to the difficulty of obtaining robust AEP signals underwater. Background noise can also affect the final hearing thresho ld calculations. Finneran and Houser (2006) measured behavioral hearing threshold s for one of their subjects, BLU, in an above ground pool and in San Diego Bay. Because of higher background noise levels, BLU’s behavioral hearing threshol ds in San Diego Bay were

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43 substantially elevated compared to her hearing thresholds i n the pool, especially below 40 kHz (Finneran and Houser 2006). Perhaps the most important factor in comparing among hea ring thresholds in cetaceans is the method used to determine the threshold. For example, Szymanski et al. (1999) defined the behavioral threshold as “two detections at one intensity level, and two failures to detect the tone level below”; thresholds r eported were the average of three determinations. Yuen et al. (2005) defined the behavioral thre shold as a minimum of five reversals, and where the threshold values of two consecut ive sessions varied by no more than 3 dB. Finneran and Houser (2006) defined their behavioral th resholds as 6-10 consecutive reversals averaged between 2-3 independent sess ions. The current study defined a threshold as two consecutive sessions where the t hreshold value varied by no more than 3 dB, and was the result of at least eight r eversals. AEP threshold determination is equally variable. Szymansk i et al. (1999) defined AEP thresholds as a 350 nV PIII-NIV level (peak-to-peak). Yuen et al. (2005) and Houser and Finneran (2006) calculated a linear regression of the AEP data and extrapolated to 0 V; this was defined as the AEP hearing th reshold. Cook et al. (2006) defined thresholds as the quietest SPL for which an AEP was detected above the noise floor, and Finneran and Houser (2006) used magnitude-squared coheren ce to determine the AEP hearing thresholds in their study. Thus, diffe rences in the way thresholds are determined could affect the final value reported at each tes t frequency. Until systematic calculations are used to determine these values, it will r emain difficult to compare results from different studies.

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44 Nonetheless, the results of this study and previous studies (Szymanski et al. 1999; Yuen et al. 2005; Cook et al. 2006; Finneran and Houser 2006; Houser a nd Finneran 2006) show that each of the three methods used to measure ce tacean hearing (in-air AEPs, underwater AEPs, and underwater behavioral measuremen ts) can reliably determine both the general shape and high-frequency cutoff of an individual’s audiogram. Furthermore, these results demonstrate that AEP hearin g measurements are acceptable alternatives to traditional behavioral measurements. In situations where behavioral hearing measurements cannot be made, i.e., temporarily-c aptured and stranded cetaceans, AEP hearing measurements will provide valuable information regarding the auditory capabilities of these animals.

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45 References Cited Andersen S (1970) Auditory sensitivity of the harbour porpo ise ( Phocoena phocoena ). In: Pilleri G (ed) Investigations on Cetacea, Vol. 2. AG Berne Bmpliz, Berne, Switzerland, pp 255-259 Au WWL (1993) The Sonar of Dolphins. Springer-Verlag, New York Awbrey FT, Thomas JA, Kastelein RA (1988) Low frequency un derwater hearing sensitivity in belugas, Dephinapterus leucas J Acoust Soc Am 84:2273-2275 Belkovich VM, Solntseva GN (1970) Anatomy and function of t he ear in dolphins. J Comp Zool 2:275-282 Brill RL, Moore PWB, Dankiewicz LA (2001) Assessment of dolphin ( Tursiops truncatus ) auditory sensitivity and hearing loss using jawphones. J Acoust Soc Am 109:1717-1722 Bullock TH, Grinnell AD, Ikezono E, Kameda, K, Katsuki Y, Nomoto M, Sato O, Suga N, Yanagisawa K (1968) Electrophysiological studies of cent ral auditory mechanisms in cetaceans. Z vergl Physiol 59:117-156 Cook MLH, Varela RA, Goldstein JD, McCulloch SD, Boss art GD, Finneran JJ, Houser D, Mann DA (2006) Beaked whale auditory evoked potential heari ng measurements. J Comp Physiol A 192:489-495 Corwin JT, Bullock TH, Schweitzer J (1982) The auditory b rain stem response in five vertebrate classes. Electroencephalogr Clin Neurophysiol 54:629-641 Dolphin WF (1996) Auditory evoked responses to amplitude modula ted stimuli consisting of multiple envelope components. J Comp Physiol A 179:113-121

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46 Dolphin WF (1997) The envelope following response to multiple tone pair stimuli. Hear Res 110:1-14 Dolphin WF (2000) Electrophysiological measures of auditory processing in odontocetes. In: Au WWL, Popper AN, Fay RR (eds) Hearing by Whales and D olphins. Springer-Verlag, New York, pp 294-329 Dolphin WF, Mountain DC (1992) The envelope following response : scalp potentials elicited in the Mongolian gerbil using sinusoidally AM acousti c signals. Hear Res 58:70-78 Ferraro JA, Durrant JD (1994) Auditory evoked potentials: ove rview and basic principles. In: Katz J (ed) Handbook of Clinical Audiolo gy. Williams & Wilkins, London, pp 317-338 Finneran JJ, Houser DS (2006) Comparison of in-air evoked poten tial and underwater behavioral hearing thresholds in four bottlenose dolphins ( Tursiops truncatus ). J Acoust Soc Am 119:3181-3192 Finneran JJ, Carder DA, Dear R, Belting T, McBain J, Da lton L, Ridgway SH (2005) Pure tone audiograms and possible aminoglycoside-induced hea ring loss in belugas ( Delphinapterus leucas ). J Acoust Soc Am 117:3936-3943 Gellermann LW (1933) Chance orders of alternating stimuli in visual discrimination experiments. J Genet Psychol 42: 206-208 Glasscock III ME, Jackson CG, Josey AF (1987) The ABR Han dbook: Auditory Brainstem Response. Thieme Medical Publishers, Inc., Ne w York Hall JD, Johnson CS (1972) Auditory thresholds of a killer whale Orcinus orca Linnaeus. J Acoust Soc Am 51:515-517

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47 Houser DS, Finneran JJ (2006) A comparison of underwater hea ring sensitivity in bottlenose dolphins ( Tursiops truncatus ) determined by electrophysiological and behavioral methods. J Acoust Soc Am 120:1713-1722 Jacobs DW, Hall JD (1972) Auditory thresholds of a fresh water dolphin, Inia geoffrensis Blainville. J Acoust Soc Am 51:530-533 Janik VM (2000) Source levels and the estimated active space of bottlenose dolphin ( Tursiops truncatus ) whistles in the Moray Firth, Scotland. J Comp Physiol A 186:673-680 Janik VM, Slater PJB (1998) Context-specific use suggests that bottlenose dolphin signature whistles are cohesion calls. Anim Behav 56:829-838 Johnson CS (1966) Auditory thresholds of the bottlenosed porpo ise ( Tursiops truncatus Montagu). U.S. Naval Ordnance Test Station (NOTS) TP 4178, 37 pp. Johnson CS (1967) Sound detection thresholds in marine mammal s. In: Tavolga WN (ed) Marine Bio-Acoustics, Vol. 2. Pergamon Press, New York, pp 247-260 Kastelein RA, Hagedoorn M, Au WWL, de Haan D (2003) Audiogram of a striped dolphin ( Stenella coeruleoalba ). J Acoust Soc Am 113:1130-1137 Kastelein RA, Bunskoek P, Hagedoorn M, Au WWL, de Haan D (2002) Audiogram of a harbor porpoise ( Phocoena phocoena ) measured with narrow-band frequencymodulated signals. J Acoust Soc Am 112:334-344 Kenyon TN, Ladich F, Yan HY (1998) A comparative study of hearing ability in fishes: the auditory brainstem response approach. J Comp Physiol A 182:307-318

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48 Ljungblad DK, Scoggins PD, Gilmartin WG (1982) Auditory thres holds of a captive Eastern Pacific bottle-nosed dolphin, Tursiops spp. J Acoust Soc Am 72:17261729 Lucas JR, Freeberg TM, Krishnan A, Long GR (2002) A comparat ive study of avian auditory brainstem responses: correlations with phylogeny a nd vocal complexity, and seasonal effects. J Comp Physiol A 188:981-992 Mann DA, Higgs DM, Tavolga WN, Souza MJ, Popper AN (2001) Ul trasound detection by clupeiform fishes. J Acoust Soc Am 109:3048-3054 McCormick JG, Wever EG, Palin J (1970) Sound conduction in t he dolphin ear. J Acoust Soc Am 48:1418-1428 McCormick JG, Wever EG, Ridgway SH, Palin J (1980) Sound rec eption in the porpoise as it relates to echolocation. In: Busnel RG, Fish J F (eds) Animal Sonar Systems. Plenum Press, New York, pp 449-467 Mhl B, Au WWL, Pawloski J, Nachtigall PE (1999) Dolphin hear ing: relative sensitivity as a function of point of application of a contact sound source in the jaw and head region. J Acoust Soc Am 105:3421-3424 Nachtigall PE, Au WWL, Pawloski JL, Moore PW (1995) Risso’ s dolphin ( Grampus griseus ) hearing thresholds in Kaneohe Bay, Hawaii. In: Kast elein RA, Thomas JA, Nachtigall PE (eds) Sensory Systems of Aquatic Mamm als. DeSpil Publishers, The Netherlands, pp 49-53 Nachtigall PE, Lemonds DW, Roitblat HL (2000) Psychoacousti c studies of dolphin and whale hearing. In: Au WWL, Popper AN, Fay RR (eds) Hearin g by Whales and Dolphins. Springer-Verlag, New York, pp 330-363

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49 Norris KS (1964) Some problems of echolocation in cetacea ns. In: Tavolga WN (ed) Marine Bio-Acoustics. Pergamon Press, New York, pp 317-336 Norris KS (1968) The evolution of acoustic mechanisms in odo ntocete cetaceans. In: Drake ET (ed) Evolution and Environment. Yale University Pre ss, London, pp 297-324 Popov VV, Supin AY, Klishin VO, Bulgakova TN (2006) Monaural a nd binaural hearing directivity in the bottlenose dolphin: evoked-potential stud y. J Acoust Soc Am 119:636-644 Ridgway SH, Carder DA (2001) Assessing hearing and sound production in cetaceans not available for behavioral audiograms: experiences wi th sperm, pygmy sperm, and gray whales. Aquat Mamm 27:267-276 Sauerland M, Dehnhardt G (1998) Underwater audiogram of a tucuxi ( Sotalia fluviatilis guianensis ). J Acoust Soc Am 103:1199-1204 Schlundt CE, Finneran JJ, Carder DA, Ridgway SH (2000) Tempora ry shift in masked hearing thresholds of bottlenose dolphins, Tursiops truncatus and white whales, Delphinapterus leucas after exposure to intense tones. J Acoust Soc Am 107:3496-3508 Schusterman RJ (1980) Behavioral methodology in echolocatio n by marine mammals. In: Busnel RG, Fish JF (eds) Animal Sonar Systems. Plenum, New York, pp 11-41 Supin AY, Popov VV (1995) Frequency tuning and temporal resolutio n in dolphins. In: Kastelein RA, Thomas JA, Nachtigall PE (eds) Sensory Systems of Aquatic Mammals. De Spil Publishers, The Netherlands, pp 95-110

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50 Supin AY, Popov VV, Mass AM (2001) The Sensory Physiology of Aquatic Mammals. Kluwer Academic Publishers, Boston Szymanski MD, Bain DE, Kiehl K, Pennington S, Wong S, Henr y KR (1999) Killer whale ( Orcinus orca ) hearing: auditory brainstem response and behavioral audiograms. J Acoust Soc Am 106:1134-1141 Thomas J, Chun N, Au W, Pugh K (1988) Underwater audiogram of a false killer whale ( Pseudorca crassidens ). J Acoust Soc Am 84:936-940 Tremmel DP, Thomas JA, Ramirez KT, Dye GS, Bachman W A, Orban AN, Grim KK (1998) Underwater hearing sensitivity of a Pacific white-side d dolphin, Lagenorhynchus obliquidens Aquat Mamm 24:63-69 Wang D, Wang K, Ziao Y, Sheng G (1992) Auditory sensitivity of a Chinese river dolphin ( Lipotes vexillifer ). In: Thomas JA, Kastelein RA, Supin AY (eds) Marine Mammal Sensory Systems. Plenum, New York, pp 213-221 White Jr MJ, Norris J, Ljungblad D, Barron K, di Sciara G (1978) Auditory thresholds of two beluga whales ( Delphinapterus leucas ). HSWRI TR 78-109, Hubbs/Sea World Research Institute, San Diego, CA, 37 pp. Yuen MML, Nachtigall PE, Breese M, Supin AY (2005) Behavior al and auditory evoked potential audiograms of a false killer whale ( Pseudorca crassidens ). J Acoust Soc Am 118:2688-2695

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51 Table 2-1 Auditory evoked potential (AEP) and behavioral hearing stud ies for which the same test subjects were used. AUTHOR YEAR SPECIES (n) TEST SIGNAL TEST TYPE Szymanski et al. 1999 Orcinus orca (2) 0.5 ms or 1 ms cosinegated tone bursts AEP 2 s tones Behavioral Yuen et al. 2005 Pseudorca crassidens (1) 20 ms SAM tone bursts AEP 3 s pure tones Behavioral Cook et al. 2006 Tursiops truncatus (3) 14 ms SAM tone bursts AEP 500 ms tones Behavioral Finneran & Houser 2006 Tursiops truncatus (4) 12-15 ms SAM tone bursts and/or continuous SAM tones AEP 500 ms tones Behavioral Houser & Finneran 2006 Tursiops truncatus (3) 23, 32, or 62 ms SAM tone bursts AEP 500 ms tones Behavioral Present Study 2006 Tursiops truncatus (2) 14 ms SAM tone bursts AEP 14 ms SAM tone bursts Behavioral

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52 Table 2-2 Average number of hits, misses, false alarms, and corr ect rejections for the 25% and 50% catch trial sessions. Hits and misses are for sound trials, and false alarms and correct rejections are for catch trials. These numbers show that Ranier’s responses were not biased during the 25% catch trial sessions because similar patterns were seen during the 50% catch trial sessions. 25 % CATCH TRIALS 50 % CATCH TRIALS Frequency (kHz) Avg. # Hits Avg. # Misses Avg. # False Alarms Avg. # Correct Rejections Avg. # Hits Avg. # Misses Avg. # False Alarms Avg. # Correct Rejections 10 13.3 3.7 0.3 6.7 8.5 3.5 0.5 11.5 30 13.0 4.0 0.0 7.0 9.3 2.7 0.8 11.2 40 10.0 2.5 1.0 5.5 11.0 1.0 4.0 8.0

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53 Table 2-3 Calvin’s underwater AEP and in-air AEP hearing threshol ds. FREQUENCY (kHz) UNDERWATER AEP THRESHOLD (dB re 1 Pa) IN-AIR AEP THRESHOLD (dB re 1 Pa) 5 112.4 107.9 10 90.0 108.9 20 85.2 116.7 30 90.2 85.3 40 72.7 68.0 60 76.0 81.3 80 74.0 71.3

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54 Table 2-4 Ranier’s underwater behavioral, underwater AEP, and inair AEP hearing thresholds. FREQUENCY (kHz) UNDERWATER BEHAVIORAL THRESHOLD (dB re 1 Pa) UNDERWATER AEP THRESHOLD (dB re 1 Pa) IN-AIR AEP THRESHOLD (dB re 1 Pa) 5 103.8 not tested not tested 10 95.5 107.2 122.2 20 81.2 86.8 101.1 30 79.2 not determined 93.4 40 84.4 95.0 86.3 60 132.9 119.3 122.1 80 136.2 not determined 136.8

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55 Figure 2-1 Amplitude-modulated (AM) tone that was presented to the dolphins via a jawphone or a free-field speaker. This example is of a 40 kHz tone modulated at 600 Hz generated using TDT SigGen software.

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56 0 5 10 15 20 25 30 35 40 45 Time (ms) 92 98 104 110 116 122 128 134 140 146 1 uVSPL (dB re 1 uPa) Figure 2-2 An example of evoked potential data collected from Calv in in response to an 80 kHz AM tone at ten sound levels. Time, in millisecon ds, is on the x-axis, and sound pressure level (SPL), in dB re 1 Pa, is on the y-axis.

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57 Figure 2-3 In-air AEP hearing tests on Ranier.

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58 Figure 2-4 Underwater AEP hearing tests on Calvin.

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59 30 40 50 60 70 80 90 100 110 120 130 0102030405060708090100 Frequency (kHz)SPL (dB re 1 Pa) In-Air AEP UW AEP BackgroundNoise Figure 2-5 In-air and underwater AEP hearing thresholds for Calvi n. Spectrum level (dB re 1 Pa 2 /Hz) background noise from the underwater tests is also pl otted.

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60 30 50 70 90 110 130 0102030405060708090100 Frequency (kHz)SPL (dB re 1 Pa) In-Air AEP UW AEP Behavioral BackgroundNoise Figure 2-6 In-air AEP, underwater AEP, and behavioral hearing thre sholds for Ranier. Spectrum level (dB re 1 Pa 2 /Hz) background noise from the underwater tests is also plotted.

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61 Chapter Three: Auditory Evoked Potential (AEP) Hearing Thresholds of FreeRanging Bottlenose Dolphins ( Tursiops truncatus ) Abstract Bottlenose dolphins ( Tursiops truncatus ) rely on sound for communication, navigation, and foraging. Therefore hearing is one of their primar y sensory modalities. Both natural and anthropogenic noise in the marine environment could mask t he ability of free-ranging dolphins to detect sounds, and chronic noise exposure could cause permanent hearing losses. The goal of this study was to investigate the h earing abilities of a population of free-ranging bottlenose dolphins in Sarasota Bay, Florida. The hearing abilities of 62 bottlenose dolphins (32 males and 30 females), ranging in age from 2 to 36 years, were measured in the field using non-invasive auditory evoked potenti al (AEP) techniques during brief capture-release sessions for health assessme nt. Evoked potentials in response to amplitude-modulated (AM) tones ranging from 5-120 kHz elicited a robust envelope following response (EFR), and allowed an entire aud iogram to be obtained in approximately 40 minutes. There was considerable individual variation, up to 80 dB between individuals, in hearing abilities. With the poss ible exception of dolphin F195, which did not produce a detectable evoked potential in response to a 120 dB signal at 40

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62 kHz, none of the Sarasota dolphins demonstrated substantial hearing losses. There was no relationship between age, gender, or PCB load and hearing sensitivities. It is possible that the oldest animals in this population (> 36 years old) do exhibit hearing losses, but they were not tested in this study. Hearing measured in a 52-year-old captive-born bottlenose dolphin showed similar hearing thresholds to th e Sarasota dolphins up to 80 kHz, but exhibited a 50 dB drop in sensitivity at 120 kHz. It i s also possible that individuals experiencing hearing losses do not survive long in th e wild as a result of compromised echolocation abilities.

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63 Introduction Bottlenose dolphins ( Tursiops truncatus ) have an impressive ability to both produce and perceive a wide variety of sounds including echolocation c licks, whistles, and burst-pulse sounds (Au 1993; Caldwell et al. 1990; Thomson and Richardson 1995) Because dolphins rely on these sounds for communication, navigatio n, and foraging, their sense of hearing is one of their most important senses (Au 1993; Jani k and Slater 1998). Anthropogenic noises, including boat engine noise, ultrasoni c noise from depth sounders and fish finders, marine construction, and industrial noise could be impacting dolphins and other cetaceans by impairing their hearing or otherwise interfering with their detection of biologically relevant sounds. The effect of noise on marine mammals is currently a hotly-debated topic in scientific and environme ntal communities. Much of this debate is contentious due to a lack of data on the ac tual impacts of noise on these animals, especially on their hearing abilities. Behavioral audiograms have been reported for several of t he at least 70 odontocete (toothed whale) species (Reeves et al. 2002) inc luding the bottlenose dolphin (Johnson 1966, 1967; Jacobs 1972; Thompson and Herman 1975). However most behavioral paradigms require repeated measurements using hi ghly trained animals; therefore, sample size is generally limited to one or two individuals. In addition, the subjects must be maintained in captivity for long periods, which limits the number of species available for study. As an attractive alternative to traditional behavioral techniques, auditory evoked potential (AEP) techniques can also be used to measure heari ng abilities in odontocetes

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64 (e.g., Ridgway et al. 1981; Supin et al. 1993; Supin and Popov 1995; Szymanski et al. 1999; Yuen et al. 2005; Finneran and Houser 2006; Houser and Finneran 2006; Cook et al. in prep.). In general, when the auditory pathway is presented with an acoustic stimulus that is above threshold levels, large numbers of neurons within the acoustic pathway are excited. The simultaneous firing of multipl e neurons produces an electrical signal that can be detected by an electrode placed on the head. AEP hearing measurements are advantageous over behavioral measurements because they are non-invasive, require little to no trai ning on the part of the animal, and can be completed in short time segments. Thus, they allow researchers to perform very rapid estimates of an individual’s audiogram. Finally, t hey can be used to test hearing thresholds of animals for which behavioral audiograms ca nnot be determined (Ridgway and Carder 2001), including live-stranded (Popov and Klishin 1998; A ndr et al. 2003; Nachtigall et al. 2005; Cook et al. 2006) and free-ranging odonto cetes. Data from both behavioral and AEP techniques have resulte d in fewer than a dozen published audiograms for bottlenose dolphins. Thus, lit tle is known about intraspecific variability in their hearing capacities. Only f our studies published to date have even begun to examine this variability (Ridgway and Carder 1993, 1997; Finneran and Houser 2006; Houser and Finneran 2006). Ridgway and Carder (1993, 1997) foun d that high-frequency hearing loss is common in elderly captive bottlenose dolphins. Out of the eight individuals they tested, three males over the age of 25 (25, 29, and 35) and one female, age 33, showed hearing losses at higher frequencies The remaining four animals, two older females, age 32 and 36, one younger male, age 9, and one younger female, age 13, showed no noticeable hearing losses. These studies suggest that hearing

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65 loss in bottlenose dolphins may not be just a factor of increasing age, but may also depend to some extent on the gender of the individual. The National Research Council (NRC) noted that “[audio metric] measurements from a single animal should be viewed as only a temporary substitute for average hearing capabilities across members of wild populations” (NRC 2000), yet no study to date has investigated the hearing thresholds of free-ranging dolphins. In Sarasota Bay, Florida, bottlenose dolphin capture-release projects have been ca rried out since 1970, with health assessment of the wild community being the primary goal o f the research since the late 1980s (Wells and Scott 1990; Wells et al. 2004). These studies ha ve collected detailed information about these animals including age, gender, and gene tic relatedness to other animals in the community. In addition, individuals are sampled for levels of environmental contaminants including several PCB congeners (Wells et al. 2005). Thus, this community provides a rare opportunity to assess hearin g in a natural population for which age, gender, contaminant loads, and relatedness of ind ividuals could be correlated to differences in hearing sensitivity. This study reports the auditory temporal resolution and evoke d potential hearing measurements for 62 free-ranging bottlenose dolphins ( Tursiops truncatus ) determined using AEP techniques. Two different hearing tests were perfo rmed. The modulation rate transfer function (MRTF), which measures the strength of the AEP using different modulation rates, was determined for a subset of seven do lphins. The second test, the envelope following response (EFR) procedure, was used to estima te AEP hearing thresholds for all 62 animals. Because PCBs have been l inked to hearing losses in mammals including humans (Murata et al. 1999; Grandjean et al 2001), hearing

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66 thresholds were also compared to PCB concentrations f or several male dolphins. PCBs tend to bio-accumulate in male dolphins, but in female dol phins concentrations tend to decline with reproductive activity (Wells et al. 2005). This suggests that PCBs and other lipid-soluble contaminants are transferred to the fetus a nd/or calf, either through direct transfer across the placenta or through lactation; thus the congener concentrations found in many of the female Sarasota dolphins may not accurat ely represent exposure levels (Wells et al. 2005). Finally, the hearing of a 52-year-old captive-born dolphin, the oldest bottlenose dolphin in captivity, was measured to compare wit h the measurements of the free-ranging dolphins. Materials and Methods Subjects Sarasota Bay, FL, Bottlenose Dolphin Community AEP hearing measurements were collected on individual bott lenose dolphins within the Sarasota Bay bottlenose dolphin community (Wells 2003). Bot tlenose dolphins were encircled in a net (500 m 5 m, 15-20 cm stretch mesh) in shallow water (< 2 m). Most individuals were then brought onboard a veterinary examina tion boat to be evaluated. A full assessment typically required up to one hour, after which the individual was returned to the water and released. In-air AEP data were simult aneously collected during this

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67 onboard examination. The AEP procedures did not significan tly increase the amount of time that dolphins were on the examination boat nor did they adversely impact other ongoing projects. AEP tests were conducted during six health assessment sess ions conducted between June 2003 and June 2006. Each dolphin brought onboard the e xamination boat for a complete veterinary work-up had its hearing tested ( n=32 males and n=30 females). These animals ranged in age from 2 to 36 years (Table 3-1). D uring the study period, five animals were sampled twice and one animal was sampl ed three times, for a total of 69 AEP tests. During the same health assessment sessions, blubber samp les were taken from individuals to determine the concentrations of 63 different P CB congeners. Dolphin Conservation Center at Marineland AEP hearing measurements were collected on Nellie, a 52-y ear-old female bottlenose dolphin, born and raised at Marineland of Florida, on August 17, 2005 from 0931 hrs to 0955 hrs and from 1612 hrs to 1627 hrs. Because of her advanced age, she remained in the water during testing, with her lower jaw below the water and her melon and dorsal surface above the water. The jawphone was attached t o her lower left jaw during AEP testing, similar to the procedure used by Cook et al. (2006) t o measure the hearing of a stranded beaked whale ( Mesoplodon europaeu s). Animal trainers gently restrained

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68 Nellie at the surface of the water during testing to keep her from making large movements. AEP Methods The AEP technique involves repeatedly playing a test sound sti mulus while simultaneously recording the synchronized neural evoked pote ntial from surface electrodes. Because the evoked potential from a single s ound stimulus is small and is less than the electrical noise in the recordings, the neur al potentials in response to each tone presentation are summed to increase the signal above the noise and reveal the underlying evoked potential (Glasscock et al. 1987; Ferraro and Durrant 1994) ; this process is called “signal averaging”. All stimulus presentation and data acquisition were con trolled from a TuckerDavis Technologies (TDT) AEP Workstation with SigGen an d BioSig software. The TDT Workstation was controlled with a laptop computer, an d was powered using a marine battery and inverter on the veterinary examinati on boat. This Workstation has been used previously to record AEPs from other odontocetes in field situations (Cook et al. 2004, 2005, 2006, in prep.). This Workstation used programmed test frequencies and test levels that were controlled using BioSig software. Two different AEP hearing tests were performed. The firs t test was a measurement of the MRTF, which determines how well the auditory system is able to follow the temporal envelope of an acoustic stimulus (Do lphin et al. 1995). For this test,

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69 a 40 kHz stimulus carrier (132 dB re 1 Pa) was 100% amplitude m odulated with amplitude modulation (AM) rates ranging from 200 Hz to 2000 Hz, in 100 Hz steps. The second AEP hearing test employed the EFR technique (Supin a nd Popov 1995; Dolphin 1996, 1997, 2000) and was used to determine the hearing thresholds of each animal. For this test, a 600 Hz AM rate was chosen 1.) because it yi elds a robust EFR response in bottlenose dolphins (Supin and Popov 1995; Cook et al. in prep.) and 2.) because signals modulated at 600 Hz have a relatively narrow frequency sp ectrum, which allows for good frequency resolution in the audiogram, especially at low er carrier frequencies. Each trial lasted approximately one minute and consisted of playing AM tones at specific frequencies and levels. These AM tones consi sted of 14 ms tone bursts modulated at 600 Hz; beginning in February 2005 the signal length w as increased to 15 ms to allow for nine complete cycles of the 600 Hz modulat ion rate. This sound was presented 21 times per second, with simultaneous averaging o f the evoked potential sweeps. Sounds in these experiments were presented at l evels less than or equal to 160 dB re 1 Pa. These sound stimuli are quieter than sounds the animals are normally exposed to on a daily basis, and are much lower than soun d levels that have been found to cause temporary threshold shifts in dolphins (180-200 dB re 1 Pa ; Schlundt et al. 2000). The frequencies tested were divided into two groups that spann ed the dolphin hearing range from 5 kHz-120 kHz. Thus, if an animal’s time o n the examination boat was less than expected, there were still data that s panned the hearing range. The following frequencies were initially measured: 10, 20, 40, an d 80 kHz. Once these frequencies had been tested and if time was still availa ble, the following frequencies were

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70 then tested: 5, 30, and 60 kHz. In February 2005 the TDT AEP W orkstation was upgraded from the RP2.1 to the RX6, so that 120 kHz could also be tested. A jawphone composed of an ITC-1042 transducer embedded in a suc tion cup (constructed from VI-SIL V-1062, Rhodia, Inc.) and powered by a Hafler P1000 amplifier was used to deliver the acoustic stimulus. The jawphone suction cup is composed of an RTV silicone-based material which has an acoustic impedance similar to water (Brill et al. 2001). The jawphone was placed on the lower left jaw of each animal corresponding to position #38 in Mhl et al. (1999), which sh owed the greatest AEP response in their study. The jawphone was calibrated by placing a Reson calibrated hydrophone (Reson TC4013; -212 dBV re 1 Pa) 10 cm from the end of the suction cup, and calibrating it in the field at approximately one mete r water depth. Sound levels were controlled by the computer with a programmable attenuator ( TDT PA5). AEP signals were collected with vinyl (V-F65, Anver, Inc. ) or RTV silicone (VISIL V-1062, Rhodia, Inc.) suction cups that incorporated sta ndard 8 mm Ag-AgCl electrodes (Med-Associates, Inc.). The skin of each individual was prepared by wiping the areas of suction cup attachment with a dry gauze pad in order to remove debris. Redux electrolyte paste (Parker Laboratories, Inc.) was use d on the electrodes to establish a good electrical connection between each elect rode and the dolphin’s skin. All suction cups were removed as soon as tests were complet e. Recordings were made with three suction cup electrodes at tached to a differential amplifier (TDT DB4-HS4). A recording electrode was place d dorsally at the vertex of the skull, approximately six centimeters behind the blowhole The reference electrode was located just anterior to the dorsal fin. A ground elec trode was placed between the

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71 reference and recording electrodes, with approximately 20 ce ntimeters separating adjacent electrodes. The output signal of the amplifier was connected via a fiber optic cable to the TDT Workstation for data acquisition with the BioSig sof tware, which was located in the bow of the examination boat. BioSig controlled both sti mulus presentation and data acquisition. Electrical artifacts induced by dolphin brea thing and movement of the electrodes were removed by artifact rejection in BioSig (excluding all sweeps with evoked potentials greater than a set threshold). Informat ion about the test subject, placement of the jawphone, date, time, amplifier gai n, number of sweeps, and any additional information was stored with the AEP data in BioSig and was also recorded separately in a field notebook. The number of sweeps analyzed ranged from 200 to 7176, with an average of 1795 ( 1424) sweeps analyzed for each trial. Once an AEP re sponse was observed, averaging at that test level ended, and the next level w as tested. Evoked potential levels in response to the AM tones were measured by performing a 1220 -point Fast Fourier Transform (FFT) on the portion of the evoked potential waveform containing the evoked potential in response to the sound. Evoked potentials were included in the analysis if there was a peak in the spectrum that was greater in amp litude than an estimate of the noise level from the same sweep. Because input-output functions, plots of evoked potential str ength against sound pressure level (SPL), are non-linear, they were not use d to extrapolate hearing thresholds. Rather, the lowest SPL for which an evoked potential wa s detected with a signal strength less than or equal to -150 dBV (31.62 nV) was determined to be the threshold SPL for

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72 each individual at each frequency. Thus, if an evoked pote ntial signal greater than -150 dBV was still detected at the lowest SPL presented for a n individual at a given frequency it was excluded from further analyses. The -150 dBV cutoff level indicated that sufficient averaging had been performed and that extrane ous electrical noise did not produce artificially high thresholds. A multiple linear regression model (STATISTICA v. 6, St atSoft, Inc.) was calculated for each frequency to determine if hearing thr esholds were affected by the age and/or gender of the individual. Correlations between he aring thresholds and PCB concentrations were also calculated for the male dolp hins at each frequency. Results MRTF The MRTFs of seven bottlenose dolphins were measured to de termine the effect of AM rate on the evoked potential amplitude (Figure 3-1). Respons es were detected at all modulation rates tested from 200 to 2000 Hz. Although there was a large amount of variability among the individuals tested, a 600 Hz modulatio n rate consistently gave a robust response to the 40 kHz stimulus carrier with high si gnal-to-noise ratios. Moderate peaks occurred at modulation rates of 1000-1200 Hz, while a trough occurred at 800 Hz.

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73 I-O Functions and AEP Audiograms Input-output functions were plotted for each animal at e ach frequency to compare evoked potential strength to the SPL of the stimulus. Although t he input-output functions were non-linear, in general, higher SPLs resulted in larger evoked potentials and lower SPLs resulted in smaller evoked potentials (Figure 3-2). Threshold AEP values were used to calculate the mean mal e and mean female Sarasota Bay bottlenose dolphin AEP audiograms (Figure 3-3). A multiple linear regression performed on each frequency determined that hear ing thresholds were not significantly influenced by the age and/or gender of the indi vidual being tested (p > 0.05 for gender and p > 0.05 for age at each frequency). For purpose s of illustration and clarity, simple linear regressions for male and female data are plotted separately for each frequency in Figure 3-4. Note that the coefficients of det ermination (r 2 ) are generally low, except for when only a few data points are availa ble, such as at 5 kHz. In the case of 5 kHz and some of the other frequencies tested, the slo pe of the regressions are opposite of what one would expect for age-related hearing lo ss. F195 AEPs measured on one individual, F195, indicated that this f emale may have had substantial mid-frequency hearing losses. She showed no evo ked potential response to the 40 kHz tone burst at 120 dB re 1 Pa after 1100 sweeps, while FB75, a 31-year-old

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74 female, showed a strong response to the same stimulus af ter only 86 sweeps (Figure 3-5). In addition, F195 showed no evoked potential response to th e 20 kHz tone burst at 153 dB re 1 Pa after 1000 sweeps, while FB75 showed a strong respon se to the same stimulus after only 348 sweeps. Although the exact age of F 195 remains unknown, she was likely old when her hearing was tested based on the wo rn condition of her few remaining teeth. To definitively determine the extent of F195’s possible hearing losses, additional AEP hearing data would need to be collected. PCBs and Hearing Thresholds There were no strong relationships among hearing threshol ds and the concentrations levels of total PCBs or of the 69 PCB congeners. The l argest positive correlation in the correlation matrix over all frequencies (5, 10, 20, 30, 40, 60 80, and 120 kHz) was 0.55 for PCB 174 at 20 kHz. This correlation did not hold for t his PCB at other frequencies. Nellie at Marineland Animals under the age of two or over the age of 40-45 years are not generally sampled during health assessments in Sarasota Bay. As a result it was not possible to measure the hearing of the oldest individuals in this population. Howe ver, during this study period, the hearing of the oldest known bottlenose dolphin in ca ptivity, Nellie, was measured.

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75 Nellie’s AEP audiogram was very similar to the mean Sa rasota male and female dolphin audiograms, except at 120 kHz, where she exhibited a substanti al hearing loss (Figure 36). At the four lower frequencies tested (5, 10, 20, and 40 kHz), Nellie’s audiogram was slightly lower than the mean Sarasota audiograms, and a t 80 kHz, Nellie’s hearing threshold was slightly higher than the mean Sarasota audiograms. Discussion The MRTF data collected from seven dolphins in this study a re very similar to MRTF data collected previously on captive dolphins (Supin et al. 2001) with large peaks at 600 Hz and 1000 Hz. These data demonstrate the high temporal res olution of free-ranging bottlenose dolphins. The robust evoked potential values me asured at 600 Hz justify the use of the 600 Hz AM rate for EFR data collection on bott lenose dolphins. The results of the PCB concentrations-hearing thresho lds correlation matrices suggest that the hearing thresholds of bottlenose dolphins are not negatively affected by PCB levels, at least at levels of exposure occurring i n Sarasota Bay. It is thought that PCBs cause hearing loss by blocking thyroid hormones during fet al development, resulting in inner ear defects (Goldey et al. 1995). Theref ore, the PCB concentrations of a young calf’s mother or its own PCB concentrations be fore the age of three may be more relevant to the calf’s hearing thresholds than its ow n PCB concentrations later in life. However, because animals under the age of two are rarely sampled during health assessments, it is difficult to measure PCB concentr ations in new mothers and very young

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76 calves. Additionally, because PCBs are lipid-soluble a nd are excreted in milk (Wells et al. 2005), measuring their concentrations in mothers of ol der calves may not accurately represent the load received by that calf as a developing fetus and newborn. Concurrent AEP and PCB data are presently available for very few c alves; therefore, these analyses were not conducted. However, these are important analy ses worthy of future consideration. The two most interesting findings from the AEP audiogram data are as follows: first, there is a large amount of variability among the hearing thresholds of free-ranging bottlenose dolphins that occurs independently of the age or gender of the individual; second, none of the individuals tested had a substantial hearing loss, with the possible exception of F195. There are two obvious explanations for the results of t his study. First, it is possible that the free-ranging bottlenose dolphins of Sar asota Bay, Florida, experience no significant hearing losses during the majority of their lifetime. Alternatively, it is possible that individuals that experience significant hear ing losses do not survive long in the wild because hearing is so vital for both navigation and foraging. Most published AEP studies have been conducted on captive (e.g., Ridgway and Carder 1993, 1997; Szymanski et al. 1999; Yuen et al. 2005; Finneran an d Houser 2006; Houser and Finneran 2006; Cook et al. in prep.) or stranded (Po pov and Klishin 1998; Andr et al. 2003; Nachtigall et al. 2005; Cook et al. 2006) odon tocetes, where the pressures of food-finding, predator avoidance, and navigation have largely been removed. Because of this, hearing losses reported in these animals (Ridgway and Carder 1993, 1997; Finneran and Houser 2006; Houser and Finneran 2006; Cook et al. i n prep.), while

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77 most likely detrimental to the individual, are not as life-threatening as they might be for free-ranging animals. The results of Nellie’s AEP testing indicate that she has a significant highfrequency hearing loss. Although data from one individual mu st be interpreted cautiously, they do support the idea that bottlenose dolphi ns could experience presbycusis, increasing hearing loss with increasing age ( Ridgway and Carder 1993, 1997). Nellie’s hearing at lower frequencies further supports t he idea that bottlenose dolphins transmit these frequencies to their inner ears usi ng more than just the acoustic window of their lower jaws (Popov et al. 2006; Cook et a l. in prep.). Because the Sarasota animals’ hearing was measured in air using a jawph one, alternate sound pathways to the inner ear were unavailable. Therefore, the hearing thresholds determined for these animals at lower frequencies (5, 10, and 20 kHz) ar e likely elevated compared to analogous underwater measurements. Cook et al. (in prep.) found that AEP hearing measurements in air using a jawphone were up to 32 dB (20.0 8.0 dB) higher than AEP hear ing measurements made underwater for two captive bottlenose dolphins at 10 and 20 kH z. In addition, in-air AEP measurements were approximately 20 dB (20.3 6.2 dB) higher than underwater behavioral measurements for one captive dolphin at 10, 20, an d 30 kHz (Cook et al. in prep.). At 40, 60, and 80 kHz, however, there was good agreem ent between the in-air and underwater AEP measurements (Cook et al. in prep.). Us ing the results of Cook et al. (in prep.), the mean in-air AEP measurements for t he Sarasota animals were adjusted at 10 and 20 kHz to more accurately represent their likely A EP hearing thresholds in water. These adjusted values were determined by subtracti ng the mean difference

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78 between in-air AEP and underwater AEP measurements for the two captive dolphins at each frequency (Cook et al. in prep.) from the mean in-a ir AEP measurements for the Sarasota animals at each corresponding frequency. Figure 3 -7 shows these adjusted AEP audiograms. The mean in-air AEP measurements for the Sarasota anima ls were also modified at 10, 20, and 30 kHz to model their theoretical behavioral hearing thresholds. These values were calculated by subtracting the difference be tween in-air AEP and underwater behavioral hearing measurements for one captive dolphin a t each frequency (Cook et al. in prep.) from the mean in-air AEP measurements for th e Sarasota animals at each corresponding frequency. Nellie’s underwater AEP hearin g thresholds were also adjusted to model her theoretical behavioral hearing thre sholds at these frequencies using the surface AEP-behavioral audiogram transfer function fr om Cook et al. (2006). The data point representing her hearing threshold at 120 kHz was removed because there was no correction value at this frequency. These audiograms ar e shown in Figure 3-8. With the possible exception of F195, the free-ranging bott lenose dolphins of Sarasota Bay do not exhibit substantial hearing losses. The animals exhibiting hearing losses in the two studies by Ridgway and Carder (1993, 1997) were all at least 25 years old; however, none of the six 25-year-old or older anim als tested in this study showed any hearing deficits. Because they were not tested, it is not possible to say whether or not the very oldest animals in the Sarasota Bay population have higher hearing thresholds. The considerable variability in hearing thresholds among th ese individuals further substantiates the idea that data from individual animals do not accurately represent entire populations (NRC 2000). For example, at 80 kHz there was as much as a 47 dB hearing

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79 threshold difference between individuals within the Sarasot a dolphin population. This hearing variability can perhaps be best appreciated in term s of echolocation: assuming a spherical spreading loss model of 1/r 2 a 47 dB hearing difference could result in minimum signal detection differences of up to 15-fold. So a target detectable by a dolphin with good hearing at 150 m would only be detectable at 10 m by a dolphin with a 47 dB hearing deficit. The substantial differences in he aring thresholds in these dolphins could be the result of several factors working independent ly or in concert with each other, including genetic differences and differences in levels of instantaneous or chronic environmental noise exposure. For perspective on noise ex posure, more than 41,000 boats are registered within the home range of the resi dent Sarasota dolphin community (Florida Fish and Wildlife Conservation Commission 2002, un published data), and there are occasional marine construction/demolition projects t hat introduce exceptionally loud noise into the environment from time to time (R. Wells, personal communication). With the increasing portability and decreasing cost of AEP equipment, hearing threshold data from larger sample sizes of a wider vari ety of odontocetes should continue to become more easily obtained. In addition, AEP measur ements on temporarilycaptured and stranded animals will continue to provide powerful insights into the auditory capabilities of these animals.

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80 References Cited Andr M, Supin A, Delory E, Kamminga C, Degollada E, Alons o JM (2003) Evidence of deafness in a striped dolphin, Stenella coeruleoalba Aquat Mamm 29:3-8 Au WWL (1993) The Sonar of Dolphins. Springer-Verlag, New York Brill RL, Moore PWB, Dankiewicz LA (2001) Assessment of dolphin ( Tursiops truncatus ) auditory sensitivity and hearing loss using jawphones. J Acoust Soc Am 109:1717-1722 Caldwell MC, Caldwell DK, Tyack PL (1990) Review of the si gnature-whistle hypotheses for the Atlantic bottlenose dolphin. In: Leath erwood S, Reeves RR (eds) The Bottlenose Dolphin. Academic Press, New York, pp 199-234 Cook MLH, Wells RS, Mann DA (2004) Auditory brainstem response hearing measurements in free-ranging bottlenose dolphins ( Tursiops truncatus ). J Acoust Soc Am 116:2504 Cook, MLH, Manire CA, Mann DA (2005) Auditory evoked potential (A EP) measurements in stranded rough-toothed dolphins ( Steno bredanensis ). J Acoust Soc Am 117:2441 Cook MLH, Bauer GB, Fellner W, Mann DA (in prep.) Ground-tru thing in-air auditory evoked potential (AEP) hearing measurements with tradition al behavioral audiograms in bottlenose dolphins ( Tursiops truncatus ). Cook MLH, Varela RA, Goldstein JD, McCulloch SD, Boss art GD, Finneran JJ, Houser D, Mann DA (2006) Beaked whale auditory evoked potential heari ng measurements. J Comp Physiol A 192:489-495

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81 Dolphin WF (1996) Auditory evoked responses to amplitude modula ted stimuli consisting of multiple envelope components. J Comp Physiol A 179:113-121 Dolphin WF (1997) The envelope following response to multiple tone pair stimuli. Hear Res 110:1-14 Dolphin WF (2000) Electrophysiological measures of auditory processing in odontocetes. In: Au WWL, Popper AN, Fay RR (eds) Hearing by Whales and Dolphins. Springer-Verlag, New York, pp 294-329 Dolphin WF, Au WWL, Nachtigall PE, Pawloski J (1995) Modulat ion rate transfer functions to low-frequency carriers in three species o f cetaceans. J Comp Physiol A 177:235-245 Ferraro JA, Durrant JD (1994) Auditory evoked potentials: ove rview and basic principles. In: Katz J (ed) Handbook of Clinical Audiolo gy. Williams & Wilkins, London, pp 317-338 Finneran JJ, Houser DS (2006) Comparison of in-air evoked poten tial and underwater behavioral hearing thresholds in four bottlenose dolphins ( Tursiops truncatus ). J Acoust Soc Am 119:3181-3192 Glasscock III ME, Jackson CG, Josey AF (1987) The ABR Han dbook: Auditory Brainstem Response. Thieme Medical Publishers, Inc., Ne w York Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM (1995) Dev elopmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating t hyroid hormone concentrations and causes hearing deficits in rats. Toxi col Appl Pharmacol 135:77-88

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82 Grandjean P, Weihe P, Burse VW, Needham LL, Storr-Hanse n E, Heinzow B, Debes F, Murata K, Simonsen H, Ellefsen P, Budtz-Jrgensen E, Ke iding N, White RF (2001) Neurobehavioral deficits associated with PCB in 7-yea r-old children prenatally exposed to seafood neurotoxicants. Neurotoxicol T eratol 23:305-317 Houser DS, Finneran JJ (2006) A comparison of underwater hea ring sensitivity in bottlenose dolphins ( Tursiops truncatus ) determined by electrophysiological and behavioral methods. J Acoust Soc Am 120:1713-1722 Jacobs DW (1972) Auditory frequency discrimination in the A tlantic bottlenose dolphin, Tursiops truncatus Montague: a preliminary report. J Acoust Soc Am 52:696-698 Janik VM, Slater PJB (1998) Context-specific use suggests that bottlenose dolphin signature whistles are cohesion calls. Anim Behav 56:829-838 Johnson CS (1966) Auditory thresholds of the bottlenosed porpo ise ( Tursiops truncatus Montagu). U.S. Naval Ordnance Test Station (NOTS) TP 4178, 37 pp. Johnson CS (1967) Sound detection thresholds in marine mammal s. In: Tavolga WN (ed) Marine Bio-Acoustics, Vol. 2. Pergamon Press, New York, pp 247-260 Mhl B, Au WWL, Pawloski J, Nachtigall PE (1999) Dolphin hear ing: relative sensitivity as a function of point of application of a contact sound source in the jaw and head region. J Acoust Soc Am 105:3421-3424 Murata R, Weihe P, Araki S, Budtz-Jrgensen E, Grandjean P (1999) Evoked potentials in Faroese children prenatally exposed to methylmercury. Neur otoxicol Teratol 21:471-472 Nachtigall PE, Yuen MML, Mooney TA, Taylor KA (2005) Hear ing measurements from a stranded infant Risso’s dolphin, Grampus griseus J Exp Biol 208:4181-4188

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83 National Research Council (NRC) (2000) Marine Mammals an d Low-Frequency Sound: Progress Since 1994. National Academy Press, Washington, D.C Popov VV, Klishin VO (1998) EEG study of hearing in the com mon dolphin, Delphinus delphis Aquat Mamm 24:13-20 Popov VV, Supin AY, Klishin VO, Bulgakova TN (2006) Monaural a nd binaural hearing directivity in the bottlenose dolphin: evoked-potential stud y. J Acoust Soc Am 119:636-644 Reeves RR, Stewart BS, Clapham PJ, Powell JA (2002) Guide to Marine Mammals of the World. Knopf, New York Ridgway SH, Carder DA (1993) High-frequency hearing loss in ol d (25+ years old) male dolphins. J Acoust Soc Am 94:1830 Ridgway SH, Carder DA (1997) Hearing deficits measured in some Tursiops truncatus and discovery of a deaf/mute dolphin. J Acoust Soc Am 101:590-594 Ridgway SH, Carder DA (2001) Assessing hearing and sound production in cetaceans not available for behavioral audiograms: experiences wi th sperm, pygmy sperm, and gray whales. Aquat Mamm 27:267-276 Ridgway SH, Bullock TH, Carder DA, Seeley RL, Woods D, G alambos R (1981) Auditory brainstem response in dolphins. Proc Natl Acad Sc i USA 78:1943-1947 Schlundt CE, Finneran JJ, Carder DA, Ridgway SH (2000) Tempora ry shift in masked hearing thresholds of bottlenose dolphins, Tursiops truncatus and white whales, Delphinapterus leucas after exposure to intense tones. J Acoust Soc Am 107:3496-3508

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84 Supin AY, Popov VV (1995) Frequency tuning and temporal resolutio n in dolphins. In: Kastelein RA, Thomas JA, Nachtigall PE (eds) Sensory Systems of Aquatic Mammals. De Spil Publishers, The Netherlands, pp 95-110 Supin AY, Popov VV, Klishin VO (1993) ABR frequency tuning curves in dolphins. J Comp Physiol A 173:649-656 Supin AY, Popov VV, Mass AM (2001) The Sensory Physiology of Aquatic Mammals. Klewer Academic Publishers, Boston, MA Szymanski MD, Bain DE, Kiehl K, Pennington S, Wong S, Henr y KR (1999) Killer whale ( Orcinus orca ) hearing: auditory brainstem response and behavioral audiograms. J Acoust Soc Am 106:1134-1141 Thompson RKR, Herman LM (1975) Underwater frequency discr imination in the bottlenosed dolphin (1-140 kHz) and the human (1-8 kHz). J Acous t Soc Am 57:943-948 Thomson DH, Richardson WJ (1995) Marine mammal sounds. In: Richardson WJ, Greene Jr CR, Malme CI, Thomson DH (eds) Marine Mamm als and Noise. Academic Press, San Diego, pp 159-204 Wells RS (2003) Dolphin social complexity: lessons from l ong-term study and life history. In: de Waal FBM, Tyack PL (eds) Animal Social C omplexity: Intelligence, Culture, and Individualized Societies. Harv ard University Press, Cambridge, pp 32-56 Wells RS, Scott MD (1990) Estimating bottlenose dolphin popul ation parameters from individual identification and capture-release techniques. Rep I nt Whal Commn, Special Issue 12, 407-415

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85 Wells RS, Rhinehart HL, Hansen LJ, Sweeney JC, Townse nd FI, Stone R, Casper DR, Scott MD, Hohn AA, Rowles TK (2004) Bottlenose dolphins as m arine ecosystem sentinels: developing a health monitoring system EcoHealth 1:246254 Wells RS, Tornero V, Borrell A, Aguilar A, Rowles TK, Rhinehart HL, Hofmann S, Jarman WM, Hohn AA, Sweeney JC (2005) Integrating life-hist ory and reproductive success data to examine potential relationships with organochlorine compounds for bottlenose dolphins ( Tursiops truncatus ) in Sarasota Bay, Florida. Sci Total Environ 349:106-119 Yuen MML, Nachtigall PE, Breese M, Supin AY (2005) Behavior al and auditory evoked potential audiograms of a false killer whale ( Pseudorca crassidens ). J Acoust Soc Am 118:2688-2695

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86 Table 3-1 Freeze-brand (FB) number, gender, age at AEP testing, and health assessment (H.A.) session for each animal tested. Animals tested during multiple sessions are listed separately for each session. F173 was tested, but no usable data were obtained; therefore, she was excluded from all subsequent analyses. # FB # GENDER AGE AT AEP TESTING H.A. SESSION 1 10 M 25 JUN 06 2 100 M 17 JUN 06 3 106 M 22 JUN 03 4 109 F 8 JUN 03 5 11 F 19.5 FEB 04 6 113 F 10 JUN 06 7 118 M 11 JUN 03 8 118 M 11.5 FEB 04 9 118 M 12.5 FEB 05 10 125 F 5.5 FEB 04 11 128 M 11 JUN 03 12 133 F 7 JUN 06 13 135 F 5 FEB 05 14 138 M 12 JUN 04 15 139 F 3 JUN 03 16 146 M 9 JUN 05 17 148 M 7 JUN 03 18 148 M 8 JUN 04 19 151 F 6 JUN 06 20 155 F 15 JUN 05 21 157 F ADULT JUN 06 22 159 F 9 JUN 04 23 164 M 17 JUN 06 24 167 F 14 JUN 03 25 171 F 5 JUN 03 26 173 F 1.5 FEB 04 27 175 F 12.5 FEB 04 28 178 M 8.5 FEB 04 29 179 F 1.5 FEB 04 30 179 F 4 JUN 06 31 181 F ADULT JUN 04 32 182 M 18 JUN 05 33 185 F UNKNOWN JUN 04 34 188 M 7.5 FEB 04 35 188 M 8 JUN 04 36 195 F ADULT JUN 05 37 196 M 6 JUN 04

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87 Table 3-1 (Continued) # FB # GENDER AGE AT AEP TESTING H.A. SESSION 38 198 M 7 JUN 03 39 199 F 3 JUN 05 40 2 M 12.5 FEB 04 41 20 M 15 JUN 04 42 20 M 17 JUN 06 43 218 M 6 FEB 05 44 220 M 6 FEB 05 45 220 M 6 FEB 05 46 222 M 5 JUN 03 47 224 M 1.5 FEB 04 48 226 M 1.5 FEB 04 49 228 M 4.5 FEB 04 50 230 M 2 JUN 04 51 232 M 2.5 FEB 05 52 234 M 2 JUN 05 53 236 M UNKNOWN JUN 05 54 240 M 2 JUN 06 55 242 M UNKNOWN JUN 06 56 244 M 2 JUN 06 57 27 F 25.5 FEB 04 58 33 F 21.5 FEB 04 59 36 M 34 JUN 06 60 54 F 35 JUN 06 61 6 M 19 JUN 03 62 65 F 20.5 FEB 04 63 7 F 19.5 FEB 04 64 75 F 31 FEB 05 65 79 F 24 JUN 03 66 9 F 19.5 FEB 04 67 90 F 36 JUN 06 68 92 M 16 JUN 04 69 99 F 17 JUN 04

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88 -100 0 100 200 300 400 500 600 700 800 02004006008001000120014001600180020002200 AM Rate (Hz)EP Level (nV) Figure 3-1 Mean MRTF measured for seven free-ranging bottlenose dolphins ( Tursiops truncatus ). Individual MRTFs were measured from 200 to 2000 Hz using a 40 kH z carrier frequency at ~130 dB re 1 Pa. Mean ( SD) evoked potential level (nV) is plotted against AM rate (Hz).

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89 -170 -165 -160 -155 -150 -145 -140 -135 -130 -125 -120 20406080100120140160 SPL (dB re 1 Pa)EP Level (dBV) 5 10 20 30 40 60 80 Figure 3-2 Input-output function for FB75 for seven test frequencies Sound pressure level (SPL), in dB re 1 Pa, is plotted on the x-axis a nd evoked potential (EP) level, in dBV, is plotted on the y-axis. The input-output function s are generally non-linear.

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90 40 50 60 70 80 90 100 110 120 130 140 0102030405060708090100110120130 Frequency (kHz)SPL (dB re 1 Pa) Male Female Figure 3-3 Mean ( SD) AEP audiograms measured for 32 male and 29 fema le freeranging bottlenose dolphins ( Tursiops truncatus ).

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91 105 110 115 120 125 130 Female dolphins at 5 kHz 121416182022 y = 157.32 2.487x R2= 0.99482 SPL (dB re 1 uPa)Age (years) n = 3 70 80 90 100 110 120 130 05101520 Male dolphins at 5 kHz y = 104.93 + 0.42498x R2= 0.024866 SPL (dB re 1 uPa)Age (years) n = 10 90 95 100 105 110 115 120 05101520253035 Female dolphins at 10 kHz y = 105.12 0.28765x R2= 0.12225 SPL (dB re 1 uPa)Age (years) n = 9 90 100 110 120 130 140 150 0510152025 Male dolphins at 10 kHz y = 101.67 + 0.731x R2= 0.12802 SPL (dB re 1 uPa)Age (years) n = 16 60 70 80 90 100 110 120 130 0510152025 Male dolphins at 20 kHz y = 86.45 + 0.75504x R2= 0.097664 SPL (dB re 1 uPa)Age (years) n = 9 80 90 100 110 120 130 140 150 160 05101520253035 Female dolphins at 20 kHz y = 116.62 0.64526x R2= 0.088258 SPL (dB re 1 uPa)Age (years) n = 12 Figure 3-4 Plots of dolphin age, in years, versus SPL at hearing t hreshold, in dB re 1 Pa, for each frequency, separated by gender. Regression equations, r 2 values, and sample sizes are reported on each plot for each freque ncy.

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92 75 80 85 90 95 100 105 110 0510152025 Male dolphins at 30 kHz y = 81.428 + 0.48994x R2= 0.075491 SPL (dB re 1 uPa)Age (years) n = 11 70 80 90 100 110 120 5101520253035 Female dolphins at 30 kHz y = 92.202 0.14181x R2= 0.014285 SPL (dB re 1 uPa)Age (years) n = 9 40 50 60 70 80 90 100 0510152025303540 Female dolphins at 40 kHz y = 66.335 + 0.26726x R2= 0.05006 SPL (dB re 1 uPa)Age (years) n = 16 40 50 60 70 80 90 100 110 05101520253035 Male dolphins at 40 kHz y = 71.393 0.28497x R2= 0.027249 SPL (dB re 1 uPa)Age (years) n = 20 60 65 70 75 80 85 90 5101520253035 Female dolphins at 60 kHz y = 90.721 0.70229x R2= 0.50477 SPL (dB re 1 uPa)Age (years) n = 5 60 70 80 90 100 110 0246810121416 Male dolphins at 60 kHz y = 87.839 0.86762x R2= 0.057038 SPL (dB re 1 uPa)Age (years) n = 9 Figure 3-4 (Continued)

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93 50 55 60 65 70 75 80 85 05101520253035 Male dolphins at 120 kHz y = 65.599 0.1055x R2= 0.017034 SPL (dB re 1 uPa)Age (years) n = 5 62 64 66 68 70 72 74 76 78 0510152025303540 Female dolphins at 120 kHz y = 72.104 0.17519x R2= 0.42338 SPL (dB re 1 uPa)Age (years) n = 5 40 50 60 70 80 90 100 110 0510152025303540 Female dolphins at 80 kHz y = 78.806 0.49724x R2= 0.10547 SPL (dB re 1 uPa)Age (years) n = 15 40 50 60 70 80 90 100 110 05101520253035 Male dolphins at 80 kHz y = 71.389 0.30851x R2= 0.03754 SPL (dB re 1 uPa)Age (years) n = 24 Figure 3-4 (Continued)

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94 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 010203040 Time (ms)AEP Level (V) -0.6 -0.4 -0.2 0 0.2 0.4 0.6 010203040 Time (ms)AEP Level (V) Figure 3-5 F195 showed no EP response to the 40 kHz tone burst at 120 dB re 1 Pa even after 1100 sweeps (top), while FB75 showed a strong EP t o the same tone burst at the same SPL after only 86 sweeps (bottom). It is like ly that F195 exhibited a midfrequency hearing loss.

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95 40 50 60 70 80 90 100 110 120 130 140 0102030405060708090100110120130 Frequency (kHz)SPL (dB re 1 Pa) Male Female Nellie Figure 3-6 Nellie’s AEP audiogram compared to the mean ( SD) AEP audiograms measured for 32 male and 29 female free-ranging bottlenose dolphins ( Tursiops truncatus ).

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96 40 50 60 70 80 90 100 110 120 130 140 0102030405060708090100110120130 Frequency (kHz)SPL (dB re 1 Pa) Male Female Nellie Figure 3-7 Nellie’s AEP audiogram compared to the predicted underwater mean ( SD) AEP audiograms measured for 32 male and 29 female free-rangi ng bottlenose dolphins ( Tursiops truncatus ). The audiograms of the free-ranging animals have been a djusted at 10 and 20 kHz to more accurately represent underwater AEP hea ring thresholds.

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97 40 50 60 70 80 90 100 110 120 130 140 0102030405060708090100110120130 Frequency (kHz)SPL (dB re 1 Pa) Sarasota Male Sarasota Female Nellie Figure 3-8 Predicted behavioral audiograms based on AEP-behavioral audiogram transfer functions. The mean ( SD) male and female AEP audiograms of the freeranging animals have been adjusted at 10, 20, and 30 kHz, and Ne llie’s AEP audiogram has been adjusted at each frequency (except 120 kHz) to mor e accurately represent theoretical underwater behavioral hearing thresholds.

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98 Chapter Four: Beaked Whale Auditory Evoked Potential Hearing Measurements Abstract Several mass strandings of beaked whales have recently been correlated with military exercises involving mid-frequency sonar, highlighting unknowns r egarding hearing sensitivity in these species. The hearing abilities of a stranded juvenile beaked whale ( Mesoplodon europaeus ) were measured with auditory evoked potentials (AEPs). T he beaked whale’s modulation rate transfer function (MRTF ), measured with a 40 kHz carrier, showed responses up to an 1800 Hz amplitude modulation (AM) rate. The MRTF was strongest at the 1000 Hz and 1200 Hz AM rates. The envelope following response (EFR) input-output functions were non-linear. T he beaked whale was most sensitive to high frequency signals between 40-80 kHz, but pr oduced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beake d whale hearing range and sensitivity are similar to other odontocetes that have been measured.

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99 Introduction Beaked whales (e.g., Ziphius cavirostris and Mesoplodon densirostris ) produce echolocation clicks with estimated source levels of 200-220 dB re 1 Pa peak-peak at 1 m (Johnson et al. 2004; Zimmer et al. 2005) and with the energ y of the click centered on 42 kHz and -10 dB bandwidths of 22 kHz (Zimmer et al. 2005). Given that other odontocetes demonstrate similar structure in their ech olocation clicks and have sensitive hearing within the range of echolocation frequencies, it se ems likely that the beaked whales would also have good high-frequency hearing sensitivity. How ever, no direct assessment of hearing sensitivity has ever been perfor med on a beaked whale to verify this assumption. This lack of information is an impedime nt to understanding the effects that anthropogenic sound can have on marine mammals, part icularly since several mass strandings of beaked whales have been linked both spatiall y and temporally to military exercises involving mid-frequency sonar (Balcomb and Claridge 2001; US Dept. of Commerce 2001; Frantzis 1998; Simmonds and Lopez-Jurado 1991). Auditory evoked potential (AEP) techniques are commonly used t o measure hearing thresholds and other aspects of hearing in humans, birds, fishes, and other animals, including cetaceans (e.g., Ridgway et al. 1981; Corwin et al. 1982; Szymanski et al. 1999; Lucas et al. 2002). In general, when the auditory pat hway is presented with an acoustic stimulus that is above threshold levels, lar ge numbers of neurons within the acoustic pathway are excited. If the neuronal discharge s are time-locked to the acoustic stimulus, the electrical signals produced by the simultan eous firings of multiple neurons produce an evoked potential (EP) that can be detected by an electrode placed on the head.

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100 AEP hearing measurements are advantageous over behavioral hearing measurements because they may be performed non-invasively, they require no training on the part of the animal, and they can be done in a short time frame. T herefore, they allow researchers to perform rapid estimations of an individual’s hearing thre sholds. This often becomes critical when working with stranded marine mammals becaus e of time limitations and the nature of the stranding event itself. This study reports the auditory temporal resolution and evoke d potential hearing measurements of a live-stranded juvenile male beaked whale ( Mesoplodon europaeus ) as determined using auditory evoked potential techniques. The modulat ion rate transfer function (MRTF), which measures the strength of the AE P using different modulation rates, was first measured for the animal. The results of MRTF testing determined the amplitude-modulation (AM) rate employed in the envelope f ollowing response (EFR) procedure used to estimate AEP hearing thresholds. To dete rmine the similarity between these AEP EFR hearing threshold estimates and tradition al behavioral hearing threshold estimates, AEP hearing measurements were conducted on capt ive bottlenose dolphins ( Tursiops truncatus ) for whom behavioral hearing abilities had previously been measured (Houser et al. 2004). This study is the first to report data on the auditory system of any whale in the family Ziphiidae, and provides insights regard ing the use of military sonar and coincidental mass strandings of several species of w hales from this family.

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101 Materials and Methods Subject A single, 181 kg juvenile male beaked whale ( Mesoplodon europaeus ; HBOI-Me-0402) live-stranded ocean-side near the south edge of St. Lucie Inlet, FL, on July 20, 2004. It presented in underweight nutritional condition with decr eased post-nuchal fat and a slight concavity to its epaxial muscles with visible peduncular vert ebral processes and ribs, as well as a prominent scapular ridge. The animal was trans ported to Harbor Branch Oceanographic Institution, where it was maintained in an aboveground pool (approximately 1.5 m depth) until its death on July 22, 2004 at 1821 h rs. AEP measurements were performed on the animal on July 22, 2004 from 1527 hrs to 1611 hrs, under the direct supervision of Dr. Greg Bossart, V.M.D., Ph.D. and in accordance with NMFS Permit No. 932-1489-06. During this time the animal was stationed at the surface of the water with passive rest raint, and remained relatively motionless. AEP Methods Evoked potentials were measured by repeatedly playing a sound stimulus while simultaneously recording the neural evoked potential from surface electrodes. Because the evoked potential from a single sound stimulus is smal l and is less than the electrical

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102 noise in the recordings, the neural potentials in respon se to each tone presentation are averaged together to reduce noise and reveal the underlying ev oked potential (Ferraro and Durrant 1994). Stimulus Control and Data Collection All stimulus presentation and data acquisition were con trolled from a Tucker-Davis Technologies (TDT) AEP Workstation. The TDT Workstati on was run from a laptop computer. This Workstation has pre-programmed test frequenc ies and test levels that were run with BioSig software. Two hearing tests were performed. The first test was a measurement of the MRTF, which determines how well the auditory system is a ble to follow the temporal envelope of an acoustic stimulus (Dolphin et al. 1995). Fo r this test, a 40 kHz stimulus carrier (130 dB re 1 Pa) was 100% amplitude modulated with AM rates ranging from 200 Hz to 1800 Hz, in 200 Hz steps. The MRTF results were use d to determine the AM rate that yielded the strongest AEP response. This AM rate was then used to conduct the second hearing test, hearing threshold determination, using the EFR technique. AM tones used in an AEP procedure result in an EFR in whi ch the auditory system of the subject produces neural responses that are phase-locked with the envelope of the stimulus (Dolphin 1996; Dolphin 1997). The advantages of such a stimulus are that it results in an AEP at the frequency of AM, whic h can be distinguished from background electrical noise in the electrode signal, and that it has a narrow frequency

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103 spectrum, which allows for good frequency resolution in the audiogram. Each trial lasted approximately one minute and consisted of playing AM tones a t specific frequencies and levels. These AM tones consisted of 14 ms tone bursts m odulated at 1200 Hz, the AM rate that yielded the strongest AEP response. This soun d was presented 21 times per second, with simultaneous averaging of the evoked potential Jawphone and AEP Electrodes The hypothesis that dolphins use their lower jaws in the reception of sound is generally accepted. Norris (1964, 1968) originally proposed that the mand ibular foramen and the fats associated with it function as acoustic wave guide s; electrophysiological (Bullock et al. 1968; McCormick et al. 1970, 1980) and behavioral (Brill et al 1988, 2001) studies with bottlenose dolphins support this theory. Taking advan tage of this sound reception pathway, jawphones (contact hydrophones attached by sucti on cups) have been used by several researchers to deliver acoustic stimuli to the lower jaw of bottlenose dolphins (e.g., Moore and Pawloski 1993; Brill et al. 2001; Houser and F inneran 2005). A jawphone composed of an ITC-1042 transducer embedded in a suctio n cup with an acoustic impedance similar to water (constructed from VI-S IL V-1062, Rhodia, Inc.) and powered by a Hafler P1000 amplifier was used to deliver the ac oustic stimulus in this study. The jawphone was placed on the lower left jaw of the animal corresponding to a position scaled to that of position #38 in Mhl et al. (1999), which showed the greatest AEP response in their study on bottlenose dolphins. The jawphone was located below

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104 the water surface during data collection. The jawphone was calibrated in reference to the sound level 10 cm from the suction cup using a calibrated hydropho ne (Reson TC4013; -212 dB re 1 V/Pa). Sound levels were controlled by the co mputer with a programmable attenuator (TDT PA5). The sound field in water is complicated by constructive a nd destructive interference from reflections off of the water surfac e and bottom. Thus, it is often more difficult to deliver a consistent sound stimulus in sha llow water than in air. More precise stimulus levels were presented to the animal via the jawph one than if a free-standing underwater speaker were used to deliver sounds because the dist ance between the ear and the jawphone did not change as it might with a free s peaker. Evoked potentials were collected with suction cup electrode s made from standard 8 mm silver-silver chloride electrodes (Med-Associates, In c.) embedded in a RTV silicone rubber compound (VI-SIL V-1062, Rhodia, Inc.). Re dux electrolyte paste (Parker Laboratories, Inc.) was used on the electrodes t o establish a good electrical connection between the electrodes and the whale’s ski n. All electrodes and suction cups were removed as soon as testing was complete. Recordings were made with two suction cup electrodes and a ground electrode attached to a differential amplifier (TDT DB4-HS4). Th e recording electrode was placed behind the nuchal crest approximately 2 cm lateral to the dorsal midline and approximately 15 cm behind the blowhole. The reference elec trode was placed approximately 20 cm caudal to the recording electrode, and a gr ound electrode was placed in the water. The output of the amplifier was connected via a fiber optic cable to the TDT Workstation for data acquisition with the BioS ig software. BioSig controlled

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105 both stimulus presentation and data acquisition. Elect rical artifacts induced by the whale breathing and movement of the electrodes were removed by a rtifact rejection in BioSig (excluding all sweeps with evoked potentials greater than 90 V). Sounds The carrier frequencies tested included 5, 10, 20, 40, 60, and 80 kHz. Sound pressure levels (SPLs) were attenuated in 10 dB steps. Up to 2000 sweeps were averaged for each test trial, although most trials consisted of about 500 sw eeps. Once an evoked potential was observed, averaging at that test level was ended, an d the next level was tested. Evoked potential levels in response to the AM tones were measured by performing a 1220-point Fast Fourier Transform (FFT) on the evoked potent ial waveform from 5-20 ms (the portion containing the EP in response to the sound) EPs were included in the analysis if there was a peak in the spectrum that was gr eater in amplitude than an estimate of the noise level from 0-5 ms in the same sweep. AEP Audiogram Calibration AEP measurements were also conducted using the same metho ds and equipment as above on three bottlenose dolphins ( Tursiops truncatus ) for which behavioral audiograms had already been measured (WEN: 21 yr. old male, BLU: 39 yr. old female, and BEN: 41 y r.

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106 old male; Houser et al. 2004). WEN and BEN were tested in San Diego Bay, while BLU was tested in a 6.1 m diameter, 1.5 m deep above-ground pool Results MRTF The beaked whale MRTF was measured to determine the effect of AM rate on the evoked potential amplitude (Figure 4-1). While responses were detecte d at all modulation rates tested, a 1200 Hz modulation rate gave a robust response to t he 40 kHz stimulus carrier with a high signal-to-noise ratio. Thus, this modulati on rate was chosen for subsequent EFR measurements. Strong peaks occurred at modulation rat es of 600 and 1000-1200 Hz, while a trough occurred at 800 Hz. Evoked potentials were detected in response to AM rates up to 1800 Hz, the highest AM rate tested. EFR An EFR was detected at each frequency tested, but was stro ngest at the highest frequencies tested (40, 60, and 80 kHz). Input-output function s were plotted for each frequency to compare evoked potential strength to the SPL of the stimulus (Figure 4-2). The input-output functions were non-linear. In general, higher SPLs resulted in larger

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107 evoked potentials, except at 80 kHz where mid-level sounds (110-128 dB re 1 Pa) evoked the strongest potentials. Because of the non-li nearity in these data, the inputoutput functions were not used to extrapolate hearing th resholds (Popov and Supin 1990). Rather, only the lowest SPLs for which an evoked potentia l was detected at each frequency are reported here (Figure 4-3). It is also import ant to note that the whale showed no reaction to the presentation of the acousti c stimuli. To establish the equivalence between these AEP EFR hear ing threshold estimates and traditional behavioral hearing threshold estimates, A EP hearing measurements were conducted on three bottlenose dolphins (WEN, BLU, BEN) fo r whom behavioral hearing abilities had previously been measured by the U.S. Navy Mar ine Mammal Program (Houser and Finneran 2005; Finneran et al. 2005). The AEP thres holds tended to be higher than the behavioral thresholds, especially at low er frequencies (Figure 4-4). Discussion The lowest detected AEPs of this beaked whale resemble h earing thresholds of other cetaceans reported in the literature (Johnson 1966; Nacht igall et al. 2000) with decreasing hearing sensitivity at lower frequencies and increasing se nsitivity at higher frequencies. These findings show that beaked whales are capable of dete cting sounds between 5 and 80 kHz, and are most likely capable of detecting frequencies much higher than 80 kHz; however, higher frequencies could not be tested due to the sampling rate limitations of the equipment. The results of the MRTF procedure suggest t hat beaked whales have a

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108 high temporal resolution, similar to that of other cetac eans (Supin et al. 2001). Beaked whale ( Ziphius cavirostris ) echolocation clicks have energy centered on 42 kHz, wi th energy up to about 80 kHz (Zimmer et al. 2005). This range appe ars to be lower than the high frequency limits of the beaked whale tested in this st udy, based on the data obtained at 80 kHz. It is important to note however, that the wh ale tested was a juvenile of a different genus. Although behavioral psychoacoustic methods provide the most direct measures of hearing abilities (Nachtigall et al. 2000), the training and t ime involved with these techniques can limit their broad application. Alternati vely, AEP techniques allow for rapid hearing assessment of untrained or minimally trai ned animals. However, the equivalence between hearing thresholds determined using thes e two testing paradigms has only recently been investigated (Szymanski et al. 1999; Houser et al. 2004; Yuen et al. 2005). Therefore, the hearing abilities of bottlenose dolphins measured behaviorally in a direct-field were compared with hearing estimates made with a jawphone in the same testing configuration that was used with the beaked whale (i .e., at the surface with the jawphone attached). The most similar situation was B LU who was tested in a pool similar to that of this beaked whale. WEN and BEN wer e tested in San Diego Bay, which has much higher ambient noise levels compared to the test pool (Finneran et al. 2005). The results with BLU showed that the AEP audiogram had consistently higher thresholds than the behavioral audiogram, with the great est differences at the lowest frequencies. The U.S. Navy’s mid-frequency tactical sonar AN/SQS-53 h as center frequencies of 2.6 and 3.3 kHz and nominal source levels of 235 dB re 1 Pa at 1 m; the AN/SQS-56

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109 has center frequencies of 6.8 to 8.2 kHz and nominal source l evels of 223 dB re 1 Pa at 1 m (U.S. Dept. of Commerce 2001). Several hypotheses hav e been put forth concerning the potential mechanism of sonar-induced stranding including a coustic or pressure trauma, in vivo bubble formation, and high auditory sensitivity of beaked wh ales to midrange sonar (Balcomb and Claridge 2001; U.S. Dept. of Commer ce 2001; Jepson et al. 2003; Fernndez et al. 2004). The lowest SPL to produce a detec table evoked potential in the beaked whale at 5 kHz was 132 dB re 1 Pa. Based o n the differences between AEP thresholds and behavioral thresholds observed in capt ive bottlenose dolphins (Figure 4-4), it is likely that the beaked whale behavioral threshold at 5 kHz would be lower than 132 dB re 1 Pa. However, until a beaked whale c an be kept alive in captivity, the behavioral data will be impossible to obtai n. The hearing sensitivity of the beaked whale at 5 kHz appear s to be similar to or less than that of bottlenose dolphins measured with evoked potentials. Thus, the beaked whale AEP measurements do not support the hypothesis that th ese species have a particularly high auditory sensitivity at the frequencies used in mid-range sonar. The data presented here, along with accurate sound propagation models, should be useful for estimating minimum distances at which beaked whales could a coustically detect midfrequency sonar.

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110 References Cited Balcomb III KC, Claridge DE (2001) A mass stranding of ceta ceans caused by naval sonar in the Bahamas. Bahamas J Sci 8:2-12. Brill RL, Sevenich ML, Sullivan TJ, Sustman JD, Witt RE (1988) Behavioral evidence for hearing through the lower jaw by an echolocating dolphi n ( Tursiops truncatus ). Mar Mamm Sci 4:223-230 Brill RL, Moore PWB, Dankiewicz LA (2001) Assessment of dolphin ( Tursiops truncatus ) auditory sensitivity and hearing loss using jawphones. J Acoust Soc Am 109:1717-1722 Bullock TH, Grinnell AD, Ikezono E, Kameda K, Katsuki Y, Nomoto M, Sato O, Suga N, Yanagisawa K (1968) Electrophysiological studies of cent ral auditory mechanisms in cetaceans. Z vergl Physiol 59:117-156 Corwin JT, Bullock TH, Schweitzer J (1982) The auditory b rain stem response in five vertebrate classes. Electroencephalogr Clin Neurophysiol 54:629-641 Dolphin WF (1996) Auditory evoked responses to amplitude modula ted stimuli consisting of multiple envelope components. J Comp Physiol A 179:113-121 Dolphin WF (1997) The envelope following response to multiple tone pair stimuli. Hear Res 110:1-14 Dolphin WF, Au WWL, Nachtigall PE, Pawloski J (1995) Modulat ion rate transfer functions to low-frequency carriers in three species o f cetaceans. J Comp Physiol A 177:235-245

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111 Fernndez A, Arbelo M, Deaville R, Patterson IAP, Cas tro P, Baker JR, Degollada E, Ross HM, Herrez P, Pocknell AM, Rodrguez E, Howie FE, Espinosa A, Reid RJ, Jaber JR, Martin V, Cunningham AA, Jepson PD (2004) Whales sonar and decompression sickness (reply). Nature 428:1-2 Ferraro JA, Durrant JD (1994) Auditory evoked potentials: ove rview and basic principles. In: Katz J (ed) Handbook of Clinical Audiolo gy. Williams & Wilkins, London, pp 317-338 Finneran JJ, Carder DA, Schlundt CE, Ridgway SH (2005) Temporar y threshold shift in bottlenose dolphins ( Tursiops truncatus ) exposed to mid-frequency tones. J Acoust Soc Am 118:2696-2705 Frantzis A (1998) Does acoustic testing strand whales? Nat ure 392:29 Houser DS, Finneran JJ (2005) Auditory evoked potentials (AEP) methods for population-level assessment of hearing sensitivity in bo ttlenose dolphins. J Acoust Soc Am 117:2408 Houser DS, Finneran JJ, Carder DA, Ridgway SH, Moore PW (2004) Relationship between auditory evoked potential (AEP) and behavioral audio grams in odontocete cetaceans. J Acoust Soc Am 116:2503 Jepson PD, Arbelo M, Deaville R, Patterson IAP, Castr o P, Baker JR, Degollada E, Ross HM, Herrez P, Pocknell AM, Rodrguez F, Howie FE, Espin osa A, Reid RJ, Jaber JR, Martin V, Cunningham AA, Fernndez A (2003) Gas-bu bble lesions in stranded cetaceans: was sonar responsible for a spate of whale deaths after an Atlantic military exercise? Nature 425:575-576

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112 Johnson CS (1966) Auditory thresholds of the bottlenosed porpo ise ( Tursiops truncatus Montagu). U.S. Naval Ordinance Test Station (NOTS) TP 4178, 37 pp. Johnson M, Madsen PT, Zimmer WMX, Aguilar de Soto N, Tya ck PL (2004) Beaked whales echolocate on prey. Proc R Soc Lond B (Suppl.) 271: S383-S386 Lucas JR, Freeberg TM, Krishnan A, Long GR (2002) A comparat ive study of avian auditory brainstem responses: correlations with phylogeny a nd vocal complexity, and seasonal effects. J Comp Physiol A 188:981-992 McCormick JG, Wever EG, Palin J (1970) Sound conduction in t he dolphin ear. J Acoust Soc Am 48:1418-1428 McCormick JG, Wever EG, Ridgway SH, Palin J (1980) Sound rec eption in the porpoise as it relates to echolocation. In: Busnel RG, Fish J F (eds) Animal Sonar Systems. Plenum Press, New York, pp 449-467 Mhl B, Au WWL, Pawloski J, Nachtigall PE (1999) Dolphin hear ing: relative sensitivity as a function of point of application of a contact sound source in the jaw and head region. J Acoust Soc Am 105:3421-3424 Moore PWB, Pawloski DA (1993) Interaural time discriminati on in the bottlenose dolphin. J Acoust Soc Am 94:1829-1830 Nachtigall PE, Lemonds DW, Roitblat HL (2000) Psychoacousti c studies of dolphin and whale hearing. In: Au WWL, Popper AN, Fay RR (eds) Hearing by Whales and Dolphins. Springer-Verlag, New York, pp 330-363 Norris KS (1964) Some problems of echolocation in cetacea ns. In: Tavolga WN (ed) Marine Bio-Acoustics. Pergamon Press, New York, pp 317-336

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113 Norris KS (1968) The evolution of acoustic mechanisms in odo ntocete cetaceans. In: Drake ET (ed) Evolution and Environment. Yale University Pre ss, London, pp 297-324 Popov VV, Supin AY (1990) Auditory brain stem responses in characterization of dolphin hearing. J Comp Physiol A 166:385-393 Ridgway SH, Bullock TH, Carder DA, Seeley RL, Woods D, G alambos R (1981) Auditory brainstem response in dolphins. Proc Natl Acad Sc i USA 78:1943-1947 Simmonds MP, Lopez-Jurado LF (1991) Whales and the militar y. Nature 351:448 Supin AY, Popov VV, Mass AM (2001) The Sensory Physiology of Aquatic Mammals. Kluwer Academic Publishers, Boston Szymanski MD, Bain DE, Kiehl K, Pennington S, Wong S, Henr y KR (1999) Killer whale ( Orcinus orca ) hearing: auditory brainstem response and behavioral audiograms. J Acoust Soc Am 106:1134-1141 US Department of Commerce and US Navy (2001) Joint Interim Report: Bahamas Marine Mammal Stranding Event of 15-16 March 2000. www.nmfs.noaa.gov/pr/pdfs/acoustics/bahamas_stranding.pdf Yuen MML, Nachtigall PE, Breese M, Supin AY (2005) Behavior al and auditory evoked potential audiograms of a false killer whale ( Pseucorca crassidens ). J Acoust Soc Am 118:2688-2695 Zimmer WMX, Johnson MP, Madsen PT, Tyack PL (2005) Echoloc ation clicks of freeranging Cuvier’s beaked whales ( Ziphius cavirostris ). J Acoust Soc Am 117:3919-3927

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114 0 0.05 0.1 0.15 0.2 0.25 0500100015002000 Amplitude Modulation Rate (Hz)EFR Amplitude (V) Figure 4-1 Beaked whale ( Mesoplodon europaeus ) modulation rate transfer function measured with a 40 kHz carrier tone at 130 dB re 1 Pa at various amplitude modulation rates.

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115 0 0.05 0.1 0.15 0.2 0.25 0.3 7090110130150170 Sound Pressure Level (dB re 1 Pa)EP Level (V) 5 kHz 10 kHz 20 kHz 40 kHz 60 kHz 80 kHz Figure 4-2 Beaked whale ( Mesoplodon europaeus ) input-output functions of evoked potential level as a function of stimulus sound pressure l evel (SPL). Carrier tones were amplitude modulated at 1200 Hz.

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116 80 90 100 110 120 130 140 020000400006000080000100000 Frequency (Hz)Sound Pressure Level (dB re 1 Pa) Figure 4-3 Lowest sound pressure levels (SPLs) for which an evoked p otential could be detected at each test frequency.

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117 60 80 100 120 140 160 020000400006000080000100000 Frequency (Hz) Sound Pressure Level (dB re 1 Pa) WEN_AEP WEN_behavioral 60 80 100 120 140 160 020000400006000080000100000 Frequency (Hz) Sound Pressure Level (dB re 1 Pa) BLU_AEP BLU_behavioral 60 80 100 120 140 160 020000400006000080000100000 Frequency (Hz) Sound Pressure Level (dB re 1 Pa) BEN_AEP BEN_behavioral Figure 4 4 Comparison between auditory evoked potential (AEP) and be havioral hearing thresholds determined for three bottlenose dolphins ( Tursiops truncatus ): a) WEN, b) BLU, and c) BEN. a). b). c).

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118 Chapter Five: Hearing Thresholds in Captive and Free-Ranging Cetaceans: Co ncluding Remarks In-air AEP, underwater AEP, and underwater behavioral audi ograms have been measured in several species of cetaceans by many prominent researc hers. Several of these studies have been discussed in detail throughout this dissertat ion. Chapter One presented a brief overview of the sound productio n and hearing abilities of odontocetes in order to provide a framework for the auditory evoked potential (AEP) and behavioral hearing studies that were presented i n the chapters that followed. Chapter Two investigated the differences between underwater AEP and in-air AEP measurements in two bottlenose dolphins ( Tursiops truncatus ). Underwater behavioral hearing measurements were also conducted with o ne of the dolphins using the same stimuli used for the AEP measurements. There was generally good agreement among the hearing thresholds determined by these three met hods at frequencies above 20 kHz. At 10 and 20 kHz, in-air AEP audiograms were considerabl y higher than underwater behavioral and underwater AEP audiograms. This s uggests multiple sound pathways to the dolphins’ ears at lower frequencies and/o r poor transmission of lower frequency stimuli through the jawphone. This chapter also provided an in-air AEP to underwater behavioral audiogram transfer function that co uld be applied to the in-air AEP data. Thus, it validated the used of in-air AEP hear ing measurements for animals

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119 whose hearing cannot be measured using traditional techniques, including live-stranded and free-ranging cetaceans. Chapter Three presented the first hearing measurements ev er collected on freeranging bottlenose dolphins. The hearing abilities of 62 bot tlenose dolphins (32 males and 30 females), ranging in age from 2 to 36 years, were mea sured in the field using AEP techniques during brief capture-release sessions for health assessment. Evoked potentials in response to AM tones ranging from 5-120 kHz elicited a rob ust envelope following response. There was considerable individual variation in hearing abilities, up to 80 dB, between individuals. With the possible exception of dolp hin F195, which did not produce a detectable evoked potential in response to a 120 dB re 1 Pa signal at 40 kHz, none of the Sarasota dolphins demonstrated substantial he aring losses. There was no relationship among age, gender, or PCB load and hearing sensit ivities. Because they were not tested, it is not possible to say whether or not the very oldest animals (> 36 years old) in the Sarasota Bay population have higher hear ing thresholds. Hearing measured in a 52-year-old captive-born bottlenose dolphin s howed similar hearing thresholds to the Sarasota dolphins up to 80 kHz, but exhibited a 50 dB drop in sensitivity at 120 kHz. It is possible that individuals experiencing he aring losses do not survive long in the wild as a result of compromised echolocation abi lities. Chapter Four provided the first hearing measurements made o n any member from the Ziphiidae family, a juvenile beaked whale, Mesoplodon europaeus measured with auditory evoked potentials. The beaked whale’s modulation rate transfer function measured with a 40 kHz carrier showed responses up to an 1800 Hz amplitude modulation rate. The MRTF was strongest at the 1000 Hz a nd 1200 Hz AM rates. The

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120 envelope following response input-output functions were non-l inear. The beaked whale was most sensitive to high frequency signals between 40-80 kHz, but produced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beaked whale hearing range and sensitivity were similar to other odontocetes that h ave been measured. These hearing data were discussed in terms of sonar-type sounds, as sev eral species from this family of cetaceans have been shown to strand in close spatial a nd temporal proximity to Naval sonar exercises (Balcomb and Claridge 2001; US Dept. of Com merce 2001; Frantzis 1998; Simmonds and Lopez-Jurado 1991). These studies show that for odontocete cetaceans, aud itory evoked potential hearing measurements capture both the shape and upper hearing c utoff of behaviorally determined audiograms. Furthermore, AEP hearing measurement s can be adjusted with a transfer function to estimate the behavioral threshol d. Thus, AEP audiograms are a good approximation of hearing abilities for animals whose hear ing cannot be measured behaviorally. The ease and rapidity of AEP data colle ction compared to behavioral methods dictates their expanded, though not exclusive, use i n marine mammal audiometry. The hearing abilities of a large population of animals ca n be highly variable from individual to individual, regardless of age or gender. This underscores the need for larger numbers of individuals to be sampled prior to management or policy decisions. Unlike previous studies on captive dolphins (Ridgway and Carder 1993, 1997; Finneran and Houser 2006; Houser and Finneran 2006), the wild dolphins in Saraso ta Bay, Florida, did not exhibit substantial hearing losses, with the possible e xception of F195. Also unlike previous studies (Ridgway and Carder 1993, 1997), hearing loss in t he Sarasota animals

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121 did not increase with increasing age, and males were no more likely than females to have higher hearing thresholds. Perhaps the most important use of auditory evoked potentia l hearing measurements is in the hearing assessment of stranded ce taceans. Many whales, dolphins, and porpoises cannot be maintained in captivity and a re difficult to find and study in the wild. Stranded animals, therefore, can provide valuable data that may otherwise never be obtained. AEP hearing data collected fr om stranded animals provide key information about their basic biology, and allow mor e informed decisions to be made regarding their management, conservation, and protection Finally, AEP hearing work with stranded cetaceans will al low for the effects of aminoglycosidic antibiotics commonly used in marine mamm al rehabilitation to be carefully monitored. For example, gentamicin sulfate, amikacin sulfate, and vancomycin hydrochloride capsules (vancocin HCL) are all currently u sed to treat stranded cetaceans in very poor health. However, it is unknown if these dr ugs cause hearing losses in cetaceans similar to the known hearing losses they caus e in both rodents (Rybak and Whitworth 2005) and humans (Garca et al. 2001; Black et al. 2004) Because the foremost goal of the rehabilitation process is to succes sfully return the animal to the wild, it is important to know whether or not these drugs do more h arm than good. AEP hearing measurements collected on stranded individuals shortly aft er the stranding event (prior to treatment with aminoglycosidic antibiotics), followe d with repeat measurements throughout the rehabilitation process will allow for do se-effect tables to be determined for these drugs and for the ethical consequences of their a dministration to be considered for odontocete cetaceans.

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122 References Cited Balcomb III KC, Claridge DE (2001) A mass stranding of ceta ceans caused by naval sonar in the Bahamas. Bahamas J Sci 8:2-12 Black FO, Pesznecker S, Stallings V (2004) Permanent gentami cin vestibulotoxicity. Otol Neurotol 25:559-569 Finneran JJ, Houser DS (2006) Comparison of in-air evoked poten tial and underwater behavioral hearing thresholds in four bottlenose dolphins ( Tursiops truncatus ). J Acoust Soc Am 119:3181-3192 Frantzis A (1998) Does acoustic testing strand whales? Nat ure 392:29 Garca VP, Martnez FA, Agust EB, Menca LA, Asenjo V P (2001) Drug-induced otoxicity: current status. Acta Otolaryngol 121:569-572 Houser DS, Finneran JJ (2006) A comparison of underwater hea ring sensitivity in bottlenose dolphins ( Tursiops truncatus ) determined by electrophysiological and behavioral methods. J Acoust Soc Am 120:1713-1722 Ridgway SH, Carder DA (1993) High-frequency hearing loss in ol d (25+ years old) male dolphins. J Acoust Soc Am 94:1830 Ridgway SH, Carder DA (1997) Hearing deficits measured in some Tursiops truncatus and discovery of a deaf/mute dolphin. J Acoust Soc Am 101:590-594 Rybak LP, Whitworth CA (2005) Ototoxicity: therapeutic opportunitie s. Drug Discov Today 10:1313-1321 Simmonds MP, Lopez-Jurado LF (1991) Whales and the militar y. Nature 351:448

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123 US Department of Commerce and US Navy (2001) Joint Interim Report: Bahamas Marine Mammal Stranding Event of 15-16 March 2000. www.nmfs.noaa.gov/pr/pdfs/acoustics/bahamas_stranding.pdf

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About the Author Mandy Lee Hill Cook graduated summa cum laude from the University of North Carolina at Wilmington (UNCW) in May 1999 with a Bachelor of Science degree in Marine Biology and a minor in Spanish. She completed a n undergraduate honors thesis entitled “Quantification of Signature Whistle Production by Free-ranging Bottlenose Dolphins ( Tursiops truncatus )” during her senior year. She graduated with a Master of Science Degree in Marine Biology from UNCW in May 2002. Her thesis was titled “Signature Whistle Production, Development, and Perceptio n in Free-ranging Bottlenose Dolphins”, and her major advisor was Dr. Laela Sayigh. W hile in graduate school at the University of South Florida, Mandy received numerous awar ds including the Von Rosenstiel, Getting, and Lake Endowed Fellowships through th e College of Marine Science, and a P.E.O. Scholar Award. Mandy currently lives with her husband in Hillsboro, Oregon.