KY16 8LB, UK. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,359-369doi:10.1242/jeb.091983 Received 7June2013; Accepted30September2013 1 al., 2000). from equalloudnesscontours(e.g.A-andC-weightings)(Kinsler et thresholds areobtainedusingauditoryweightingfunctionsderived levels (e.g.DeJongandAinslie,2008).Forhumans,weighted 2007), butthesetwomethodsoftenproduceverydifferent weighted bandwidthofhearing(M-weighting)(Southallet al., et al.,2006;Verboom andKastelein,2005)ortheapproximate auditory weightingfunctionsbasedontheaudiogram(e.g.Nedwell such weightedthresholdsformarinemammalswereobtained using many soundsirrespectiveoftheirfrequencyspectra.Untilrecently, perception ofthetarget species, sothatsinglethresholdsapplyto some formoffrequency-selectiveweightingaccordingto the underwater noise(Southalletal.,2007).Noisecriteriaoftencontain mammals hasledtoattemptsestablishacousticsafetycriteriafor Concern abouttheeffects ofanthropogenicnoiseonmarine Odontocete, Responselatency KEY WORDS:,Effectsofnoise,Frequencyweighting, reference valuesof150 latency contours,whichconnectequalRTs acrossfrequencies,for behavioural methodandanRT sensorbasedoninfraredlight.Equal narrowband frequency-modulatedsignalsweremeasuredusinga reaction times(RTs). SimpleRTs ofaharbourporpoiseto under theassumptionthatsoundsofequalloudnesselicit perceptionbyhumaninfantsandanimalscanbestudied Paul J.Wensveen harbour porpoise( Equal latencycontoursandauditoryweightingfunctionsforthe RESEARCH ARTICLE *Author ([email protected]) for correspondence 3843 CCHarderwijk,The Netherlands. 59–168 median RTs to1 INTRODUCTION ABSTRACT equal loudnesscontoursbecomeavailable. correlated noiseeffects intheharbourporpoise,atleastuntilactual smoothed functionsthatmaybeusedtopredictperceivedlevelsand contours. Auditoryweightingfunctionswerederivedfromthese smoothed functionsassumedtoberepresentativeofequalloudness latency contourlevelswiththehearingthresholdwereusedtocreate occurred forfrequenciesbelow63 pronounced athigherlevels(lowerRTs); aflatteningofthecontours thresholdandtheequallatencycontourswasmore sensation levels(higherRTs). Thedifference inshapebetweenthe contours roughlyparalleledthehearingthresholdatrelativelylow responded tothestimulus(medianRT 98− hearing thresholdoftheharbourporpoise,quickeranimal 63, 80and125 Sea Mammal Research Unit, University of StAndrews, StAndrews, Fife Sea MammalResearch Unit,University of

dB re.1 μPaandcentrefrequenciesof0.5,1,2,4,16,31.5, 2 Sea MammalResearch Company (SEAMARCO), Julianalaan 46, kHz. Thehigherthesignallevelwasabove s signalswithsoundpressurelevels(SPLs)of 1, − *, LéonieA.E.Huijser 200

ms (10

kHz. Relationshipsoftheequal ms intervals)werederivedfrom Phocoena phocoena 522 ms). Equallatency 2 , LeanHoek 2 and RonaldA.Kastelein been obtainedfortheharbourseal( ( finch (Carpodacusmexicanus)(Doolingetal.,1978)anddomestic ( squirrel monkey( crab-eating macaque( in humans(MarshallandBrandt,1980;Pfingstetal.,1975a). between SPL andRT, aresimilarinshapetoequalloudnesscontours contours, whichdescribethefrequency-dependentrelationships summation ofloudness(Wagner etal.,2004).Equallatency softness imperfection(Florentineetal.,2004)andspectral effects, suchasloudnessrecalibration(AriehandMarks,2003), Ahlstrom, 1984),andbyexploitingtemporalspectralloudness et al.,1982;Kohfeld1981)and1/3-octavebands(Humes demonstrated byloudnesscomparisontestswithpuretones(Buus correlation betweenRT andperceivedloudnesshas been when onlyonetypeofresponseispossible.Inhumans,astrong between theonsetofastimulusandinitiationresponse, Florentine, 2011). SimpleRT isdefinedasthetimethatelapses correlates withloudness(forreviews,seeLuce,1986;Marksand simple reactiontime(RT; orresponselatency)toasound,which difficult toquantifyinanimals.Itismorepracticalmeasure (Finneran andSchlundt,2013). threshold shift(TTS)onsetthresholdsoftwobottlenosedolphins from oneofthecontourscloselyagreedwithtemporary equal loudnesscontours.Anauditoryweightingfunctionderived somewhat shallowerasloudnessincreased,expectedfromhuman comparable inshapetotheanimal’s audiogram,andbecame contours wereobtained.Thethreeequalloudness thousands oftrialshadtobecompletedbeforetheequalloudness tone. Itwasdifficult toconveythecomplextaskdolphin,and indicate whetherthetesttonewaslouderorsofterthanreference with atesttoneandreferenceineachtrial,wastrainedto (Tursiops truncatus Schlundt, 2011) directlymeasuredtheequalloudnesscontoursofa Takeshima, 2004).Recently, FinneranandSchlundt(Finneran across frequency(FletcherandMunson,1933;Suzuki received soundpressurelevel(SPL)andtheperceivedloudness responding behaviourally tonarrowbandfrequency-modulated (FM) in aharbourporpoise,Phocoenaphocoena weighting basedondirectloudness estimates. method maybearelativelytime-efficient alternativetoauditory improves predictionsofnoise effects inmarinemammals, this loudness inotheranimals.If auditoryweightingbasedonRT dolphin, whichsuggeststhatRTs arealsorelatedtoperceived similar totheequalloudnesscontoursofhumansandbottlenose 2011). Theequallatencycontoursoftheanimalstestedtodateare Macaca mulatta In animals,equallatencycontourshavebeenobtainedfor the Perceived loudnessisasubjectivedescriptorofsoundthat Equal loudnesscontourspresenttherelationshipbetween In thisstudy, underwaterequallatencycontoursweremeasured Felis catus ) ) (Mayetal.,2009);near-threshold contourshave ) (Pfingstetal.,1975a;Pfingst1975b),house Saimiri sciureus) (Green,1975),rhesusmacaque Macaca irus)(Stebbins,1966),common 2 Phoca vitulina ). Thedolphinwaspresented (Linnaeus 1758), ) (Kasteleinetal., 359

The Journal of Experimental Biology values weresatisfactory:thecoefficient ofdetermination( towards themiddlefrequencies(Table , andincreasinglyshowedshallowlog–logslopes log–log slopes(α functions fittedtothemedianRTs generallyexhibitedthesteepest decreased withincreasingSPL ateveryfrequency. TheauditoryRT 360 after signalonsetwithmedianRTs ofbetween95and522 (SnL) (sensu 1asfunctionsofbothsensationlevel tonal signalsareshowninFig. was stillgettingusedtothetestprocedure). of which17occurredduringthefirstfivesessions(whenanimal occurred throughoutthestudy(0.5%oftotalnumbertrials), resulting in3822RT measurements.Only28pre-stimulusresponses A totalof5144trialswereconducted in167experimentalsessions, and correlatedeffects ofnoise. for theharbourporpoisethatcanbeusedtopredictperceivedlevels were thenusedtoderiveafamilyofauditoryweightingfunctions the equalloudnesscontoursofanimal.Thesmoothedfunctions create smoothedfunctionsthatareassumedtoberepresentativeof contours andtheaudiogramofporpoiseweredeterminedto Based ontheresults,relationshipsbetweenequallatency signalswithawiderangeofcentrefrequenciesandSPLs. RESEARCH ARTICLE The –6 between –72 from 2.7to6.4 from 0.90to0.99andtherootmeansquareerror(RMSE)ranged RESULTS al 2.Theweightinglevelat thelowestfrequency(250 Table 4);theparameter estimatesforEqns (Fig. 3)andauditoryweighting functions equal loudnesscontours(Fig. functions (Table that directlyrelatestothelog–logslopesofauditory RT and highrangeoftestfrequencies(63–125 (16–31.5 adjacent equallatencycontourswasgreaterinthemid-range 31 63 the hearingthreshold,includingnotchinaudiogram at (corresponding to190and200 auditory RT functions.EquallatencycontoursV andVI The medianobservedRTs oftheharbourporpoisetonineFM The sixequallatencycontours wereconvertedintohypothetical Six equallatencycontours(I T temporarythresholdshift soundpressurelevel sensationlevel W reactiontime TTS SPL mid-frequency rootmeansquareerror SnL lowfrequency RT light-emittingdiode RMSE MF LF LED L L L Fhighfrequency frequencymodulation I dataacquisition I HF FM DAQ List of symbolsand abbreviations List of dB apart(range20–41 2).Onaverage,theaudiogramandcontourVIwere kHz (Fig. 0 loud lat ht dB pointand–3 kHz, 11–13 dB and–41 Ellison etal.,2012)andSPL.Theporpoiseresponded SPL oftheequalloudnesscontour SPL ofthesmoothedequallatencycontour SPL ofthehearingthreshold sound intensityofthehearingthreshold weighting level sound intensity ms (Table

1, parameter closertounity)atthelowerandhigher dB) thaninthelowrange(0.5–4 dB pointmatched afrequencybetween4.6 dB, dependingontheweighting function. 1).

dB). Theaveragespacingbetween α ).

ms) roughlyfollowedtheshapeof − VI) wereconstructedfromthe

1, Fig.

3 and4areprovidedin

kHz, 5–8 1). Thegoodnessoffit dB), aneffect

kHz, 6–9 r 2

Hz) was ) ranged

ms. RT dB) threshold ofthe porpoisediffered significantly fromthoseforother humans (Chocholle,1940;Suzuki andTakeshima, 2004). expected fromtheequalloudness andequallatencycontoursof closely followtheshapeof hearingthresholdatlowSnLs,as similar. Thissuggeststhattheequallatencycontoursofporpoise differences inminimumlevel,the0.5to80 all frequenciesinasimilargraphasFig. 2004). ForSnLsbetween0and40 parameters presentedbyWagner andcolleagues(Wagner etal., SnLs –insufficient toinform theauditoryRT modelwithfour re-evaluated duringthestudyandrelativelyfewtestsignalshad low therefore, thehearingthresholdsofharbourporpoisewere not sources available. low frequencyprojectorwasoneofthemostpowerfulnon-military the hearingthresholdatthesefrequencies,despitefactthat the showed thatharmonicsoccurredatlevelsjudgedtobetooclose to 500 wide hearingrangeoftheanimal.Thelowesttestfrequencywas RT withincreasingSPL,atsufficient frequencies(nine)tocoverthe designed toprovidealarge enoughsampletocapturethedeclinein (Luce, 1986;Stebbins,1966).Thedatacollectionprotocolwas common inhumansandotherspeciesformediumhighSnLs contours (IandVI)wasashigh67 to getusedthenewprocedure. during thefirstfivesessionswasprobablybecauseanimalhad tasks (Blackwood,2003).Thehigherpre-stimulusresponserate because theanimalwashighlyexperiencedinstimulusdetection porpoise’s RTs wereprobablylowerthanitsspecies’ average found heremightrepresentconservativeestimates.However, the animal wasnottrainedtorespondasquicklypossible,sotheRTs the levelswerewellaboveanimal’s hearingthreshold.The porpoise mainlyrefrainedfromguessing,probablybecausemostof frequency independent. 2006; HumesandAhlstrom,1984),particularlyifthisvariationis variation inRT thatcommonlyoccurs(e.g.EpsteinandFlorentine, (Pfingst etal.,1975a).Thisapproachreducesthebetween-subject contours aredeterminedthatmatchpredefinedsensationlevels frequency atwhich,foreachindividual,thereferenceRTs ofthe averaged acrosssubjects,itismoreaccuratetouseonereference interpretation oftheresults.Whenequallatencycontoursare members ofthespecies. therefore, thesefunctionsmayalsoberepresentativeforother the equallatencycontoursandhearingthresholdsofanimal; al., 2010).Theauditoryweightingfunctions(Fig. porpoises (Kasteleinetal.,2002;Kastelein2009; 1.5–5 thresholds underunmaskedandmaskedconditionsmeasured representative forporpoisesofhisageandyounger, ashishearing The hearingabilitiesofthestudyanimalwereprobably weighting levelwas0 and 5.9 to 16 low frequencyroll-off rateoftheweightingfunctionrangedfrom10 Evaluation of theRT data Evaluation of DISCUSSION based upon. For testsinthe125 This studywasfocuseduponmid-rangeandhigh-rangeSnLs; The difference inSPL between the twoouterequallatency Very fewpre-stimulusresponsesoccurred,whichshowsthatthe The sixreferenceRTs of150–200 Hz. Acousticcalibrationsof250and400 dB peroctave,dependingontheequallatencycontouritwas years earlierweresimilartothoseoftwoothermaleharbour kHz andbetween7.88.2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983

dB forfrequenciesof

kHz band,themedianRTs near thehearing dB, plottingthemedianRTs for

ms werechosentosimplifythe dB. Similardifferences are

1 showedthat,despitethe

kHz functionswerevery kHz, respectively;the ≥ 17.1 to≥

Hz soundsignals

4) werebasedon 25.2 kHz. The

The Journal of Experimental Biology RESEARCH ARTICLE presentation ofthe125 the signaloperatorthroughmonitoringsystembeforeandduring porpoise (Blackwood,2003).Clickratesweresometimesheard by threshold level,especiallyforasmallodontoceteliketheharbour 522 frequencies. Atthelowesttestlevel(SnL=18 and Shown inthetablearetotalnumber oftrials,thetotalnum fo parameters Table 1.Best-fit sensation level(SnL) inFig.1. the testfrequenciesinthisstudy(900 mssignals)(Kastelein 125 0.0560.009 657 0.0450.004 500 80 9 0.0300.005 527 8 63 8 31.5 0.0220.006 578 0.0190.007 537 16 756 9 327.9 42.5 0.0320.004 8 4 265.3 12.6 0.0350.006 498 10 2 234.9 13.2 0.0280.004 598 8 1 8 234.7 19.5 0.0420.006 0.5 493 217.7 18.0 0.98 7 (kHz) 270.7 13.0 0.99 5.9 260.5 19.4 Frequency 0.98 2.7 243.9 11.8 4.4 0.93 43 0.90 278.1 15.0 46 6.4 0.98 6.0 55 0.98 5.1 0.98 4.1 47 49 3.3 0.99 60 71 3.9 85 102 RT (ms) 200 400 800 100 100 200 400 800 100 200 400 800  ms. Thisvaluewasexpectedtobemuchcloserthehearing (Eqn1),andthegoodness-of-fitparameters 102 55 60 0 0 0 40 47 49 50 122 50 75 80 50 20 20 20 trials No. of 50 52 50 51 142 100 50 95 50 40 40 40 51 54 55 50 162 115 120 51 56 60 60 60 r r r 51 2 55 2

2 0.5 kHz 56 63 kHz kHz signals,anditisthereforepossiblethat =0.977 test levels No. of =0.983 =0.990 50 182 135 140 4 kHz 55 80 80 80 56 202 155 160 100 100 100 55 r theauditoryRTfunctions(Eqn 100 200 400 800 800 100 200 400 800 100 200 400 Best-fit value 95% CI 95% Best-fit value log–log slope 49 46 85 0 0 0 34 56 SPL (dBre.1µPa) 105 69 66 40 20 20 20 54 457 54 r 50 2 454 54

androotmeansquareerror(RMSE). Alsoshownare dB) themedianwas SnL (dB) 125 89 86 50 40 40 40 58 50 54 55 Intercept 109 106 145 50 60 60 60 54 etal.,2010),whichwereusedforthe conversionbetween sound 55 r r r 2 2 2 50 ber oftestlevels,thebest-fit (CIs)ofparameters estimates and 95%confidenceintervals 54 16 kHz 80 kHz =0.984 =0.900 =0.994 54 129 165 126 1 kHz 50 80 80 80 54 53 50 149 185 146 100 100 100

 1) Best-fit value 95% CI 95% Best-fit value 100 200 400 800 100 200 400 800 100 200 400 800 47 71 43 0 0 0 thresholds wasrecentlyperformedwhichshowednosubstantial below 125 relative to80 also explaintheslightincreaseinequallatencycontourvalues echolocation clicktrainsmaskeddetectionofthesignals.Thismay some testsignalswerenotaudibletotheporpoisebecausehis 36 34  67 91 63 20 20 20 50 (ms) 50 53 51 052 50 111 59 87 83 40 40 40 50 57 kHz weretested.A re-evaluationofthesubject’s hearing 50 50 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983 107 131 103 51 60 60 60 50 50 31.5 kHz

r r r 125 kHz kHz. Clicktrainswerenotheardwhenfrequencies 50 2 2 2 =0.983 =0.981 =0.931 50 51 127 151 123 2 kHz 50 80 80 80 50 50 50

Goodnessoffit 50 50 147 171 143 100 100 100 50 the hearingthresholds ofthesameporpoisefor r 2 RMSE (ms) fitting apowerlaw(Eqn functions (blacklines;Eqn 0.5–125 tonal signalswithcentrefrequenciesof narrowband frequency-modulated(FM) time (RT)oftheharbourporpoise.Nine level (SPL;bottomaxes)andreaction level (SnL;topaxes),soundpressure 1.Relationshipbetweensensation Fig. scale ontheordinate. interquartile ranges.Notethelogarithmic above eachmedian,anderrorbarsindicate The numberofRT measurementsisshown threshold medians(crosses)wereomitted. RTs (circles),afteroneortwonear-

kHz weretested.TheauditoryRT pressure level(SPL) and

1) tothemedian Hearing threshold (dB re.1

1) resultfrom μ Pa)  361

The Journal of Experimental Biology Brandt, 1980;Pfingst etal.,1975a).Ingeneral, RTs donotvarywith contours aresimilarinshape (Chocholle,1940;Marshalland moderate tohighSnLs,and equal latencyandloudness of loudnessandauditoryRT functionsarefrequencydependentat most sensitivehearing.Whenmore testfrequenciesareused,slopes loudness haveusedonlyoneor twotestfrequenciesintherangeof Takashima etal.,2003). power law(Chocholle,1940;HellmanandZwislocki,1961; function andtheauditoryRT functiondiverge fromthissimple Reason, 1972).ForSnLslowerthan~30 functions fortheindividuallistener(HumesandAhlstrom,1984; functions arenegativelycorrelatedwiththeslopesofauditory RT At thesemoderatetohighlevels,theslopesofsuchloudness simple powerlawthatisalmostidenticaltoEqn estimation) andsensationlevelabove~30 and Greenbaum,1966). body size(Blackwood,2003)andmaskingnoiselevels(Chocholle alone butalsoby, forexample,age(BirrenandBotwinick,1955), RT isnotdeterminedbypropertiesofthereceivedsoundstimulus motor processesneededtogeneratetheresponse(Sanders,1998). the RT reflectsthecombineddurationofsensory, cognitiveand et al.,1975a;Stebbins,1966;Ridgway2001).Functionally, 1975; Kasteleinetal.,2011; Mayetal.,2009;Moody, 1973;Pfingst Werner, 2002)andnon-humananimals(Doolingetal.,1978;Green, very difficult orimpossible,suchashumaninfants(Leiboldand subjects forwhichloudnessassessmentwithstandardmethodsis al., 2004;Wagner etal.,2004).RT hasthereforebeenusedin a proxymeasureofloudness(AriehandMarks,2003;Florentineet (Luce, 1986;MarksandFlorentine,2011), andRT isoftenusedas In humans,simpleRTs correlatewithdirectestimatesofloudness echolocate duringresearchtrials. 2013). Inthismorerecentstudy, theporpoisewasnotallowedto changes intheaudiogramovera3–4 362 900 with dottedmarkersatthebottomisporpoise’s hearingthresholdfor Materials andmethods,‘Reactiontimemeasurements’ forrationale).Theline RTs were includedandexcludedduringthefittingprocess,respectively(see frequencies. Circlesandcrossesindicatethetestlevelsforwhichmedian the auditoryRT functionsinFig. corresponding referenceRTvalues. 2.EquallatencycontoursI− Fig. RESEARCH ARTICLE Relationship between RT andloudness Most researchersinvestigating therelationshipbetweenRT and The relationshipbetweenloudness(derivedfrommagnitude

ms signals(Kasteleinetal.,2010). SPL (dB re. 1 µPa) 100 120 140 160 180 40 60 80 VI (200 IV (180 III (170 V (190 II (160 I (150

ms) ms) ms) ms) ms) ms) 1

1 bymatchingthesixreferenceRTs across Fre VI oftheharbourporpoisewith q uenc The contourSPLswerederivedfrom year period(Kasteleinetal., y

( 10 kHz

dB isbestdescribedbya

dB, boththeloudness )

1 (Stevens,1955). 100 causes thecontours toflattentowardshigherloudness levels(Suzuki the rangeofmostsensitivehearing thanatlowerfrequencies,which increases inloudnesswithSnL areobserved atfrequencieswithin between thecontoursissmaller andviceversa).Inhumans,smaller (i.e. loudnessincreasesmoresteeply withSnL wherethespacing loudness withSnL andthespacingbetween equalloudnesscontours loudness-matching procedurethatwasused(Buusetal.,1982). lower levels(20and40 ),butthiswaslaterattributedtothe between equallatencycontoursandloudness at and colleagues(Kohfeldetal.,1981)alsoreportedadiscrepancy reported forsomelisteners(EpsteinandFlorentine,2006).Kohfeld shape ofthehearingthreshold,althoughdeviationshave been frequency atlowSnLs,sothatequallatencycontoursfollow the audiogram ofthesubjectisshownhere(solidlinewithdots). was fitted(solidlines)totheseextendedsmoothedcontours.Onlythe loudness contours(dashedlineswithsquares).A closed-formmodel(Eqn functions’ forparametervalues),whichresultedinsixhypotheticalequal (Eqn thresholds atthesefrequenciesandthethreshold–contourrelationships to thesmoothedequallatencycontoursusinganimal’s ownhearing the bottomofgraph.(B)Values for0.25,8and50 line (Andersen,1970)anddotted(Kasteleinetal.,2010)]areshownat 2010)] andtheaudiogramsoftwootherharbourporpoises[dashed–dotted Fig. lines withsquares)fromtheoriginalcontours(dashedlines;sameasin a templatetocreatesmoothedversionsoftheequallatencycontours(solid contours. 3. Conversion fromequallatencycontourstoloudness Fig. There isanegativerelationship betweentheincreaseinperceived

2). Theaudiogramofthesubject[solidlinewithdots(Kasteleinetal., SPL (dB re. 1 µPa) 2; seeMaterialsandmethods,‘Derivationoftheauditoryweighting 100 120 140 160 180 120 140 160 180 100 40 60 80 40 60 80 (A) Theshapeoftheharbourporpoise’s audiogramwasusedas A B The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983 VI IV III V II I 1 1 Frequency (kHz) 10 10 − 150

kHz wereadded 100 100

3)

The Journal of Experimental Biology RESEARCH ARTICLE the fovealregion.Inaddition,RTs ofothermammalianspecies may havebeentheresultofincreasedneuralactivitygenerated in (Ketten, 1997);therelativelyshortRTs thatwerefoundinthisstudy the regionwheretheseecholocationfrequenciesareprocessed fovea onthebasilarmembranewithhighganglioncelldensities in other porpoises.Theharbourporpoiseinnerearhasanacoustic 125 not onlyforlowfrequenciesbutalsoof63,80and these frequencies. which suggestsastrongcorrelationbetweenRT andloudness at the porpoiseshowedasimilartrendforfrequenciesupto31.5 and Takeshima, 2004).Inthisstudy, theequallatencycontoursof at 160 is toemphasizethattheeffective hearingrangeoftheporpoiseendsabruptly (I beinghighandVIlow).Theextensionofthefunctions(dashedline) porpoise’s hearingsystem,areassociatedwiththeSnL ofthereceivedsignal weighting functions,whichshouldreflectthefrequencyresponseof 4.Sixauditoryweightingfunctionsfortheharbourporpoise. Fig. levels: 178 echolocation clicksandhighintensity(peak-to-peaksource their dailylife;theyexperienceveryfaintechoesofown animals encounterlarge differences inSPL atthesefrequenciesin dynamic hearingrangeatthesefrequenciesseemsunlikely. The 110 echolocation clickscontainsoundenergy mainlyatfrequenciesof very narrowatthesehighfrequencies.Harbourporpoise results indicatethatthedynamichearingrangeofporpoiseis equal loudnesstrulyelicitRTs inharbourporpoises,thenthe or ofabottlenosedolphin(FinneranandSchlundt,2011). Iftonesof equal loudnesscontoursofhumans(SuzukiandTakeshima, 2004) I 86 56 .0 .0 55 45 .4 .0 15.9 14.7 0.90 0.84 13.6 0.78 12.3 2.64 11.1 4758 0.71 2.45 9.7 4667 0.65 2.26 0.58 4550 5856 2.05 5722 5.60 4393 1.84 5.17 4180 1.62 5550 3322 4.73 5318 0.00 5004 5.60 4.27 0.00 4563 5.17 3.80 0.01 3.32 78.62 4.73 88.41 0.01 4.27 Data areshownfor parameters 0.02 98.71 3.80 0.03 VI 109.62 3.32 CI 95% V 121.17 Best-fitvalue 133.48 IV III CI 95% II Best-fitvalue I Contour functions (Eqn4;Fig.4) Table 2.Parameterestimateswith95% CIs fo same forparameters Less spacingbetweentheequallatencycontourswasobserved

− Weighting level (dB) kHz (Fig. 150 –80 –70 –60 –50 –40 –30 –20 –10 kHz. 10 0 kHz (MøhlandAndersen,1973);therefore,anarrow 0.1 − 205 K VI IV III 1 V II 2), aneffect thatcannotbeexpectedbasedonthe (dBre.1μ I dB re.1 μPa K 1 and Pa) K 1 2 because K K 1 Frequency (kHz) , K m) (Villadsgaard etal.,2007)from 2 , a and K 2 wasderivedfrom x (seeMaterialsand methods,‘Derivationoftheauditory weighting functions’). Notethatthe95% CIsarethe 2 (dB) 10 r thehypotheticalequalloudnesscontours a K 1 . Thelowfrequency roll-off rate,whichdependson parameter 100 Best-fit value 95% Best-fit CI value 95% CI The

kHz, (Hz) behavioural responsethresholdsindependentofthefrequency of the frequency signals,orviceversa. too conservativeforlowfrequencysignals,andliberal high regulations areappropriate.Thesemay, forinstance,be may alsohelptodeterminewhetherthecurrentnoisesafety Southall etal.,2007).Frequencyweightingbasedonequallatency sound thanwiththeunweightedSPL (FinneranandSchlundt,2013; mammals tonoisecorrelatebetterwiththeperceivedloudnessofa because behaviouralandphysiologicalresponsesofmarine weighing functionssuchasthoseobtainedinthepresentstudy, anthropogenic noisecanbemademoreaccuratewithauditory these effects arehighlycorrelatedwithloudness. appropriate forpredictingspecificbehaviouraleffects, incaseswhere be usedtodeterminewhichoftheweightingfunctionsismost in thisspecies.Similarly, behaviouralresponsethresholdSPLsmay weighting functionsisthemostappropriateforpredictingTTSonset Schlundt, 2013)maybeusedtodeterminewhichofthesixauditory frequencies, themethodofFinneranandSchlundt(Finneran TTS onsetinharbourporpoisesbecomeavailableformultiple always perceivedasequallyloudacrossfrequencies.Whendataon Schlundt, 2013).ThismaysuggestthatTTSonsetlevelsarenot onset levelsmostaccuratelyinbottlenosedolphins(Finneranand loudness contour, thuswithrelativelymorecurvature,predictedTTS curve. Incontrast,aweightingfunctionbasedonlowerequal expected tobesimilarinshapetheinverseofflattestweighting therefore, afunctionrelatingTTSonsetlevelstofrequencyis the loudestsounds.TTSisgenerallyinducedbyloudsounds; relationship atveryhighfrequencies. and RT atlowandmiddlefrequencies,(2)divergence fromthis from theassumptionsof(1)astrongrelationshipbetweenloudness subject wouldminimizetheuncertaintyinoutcomethatresults loudness contoursoverawiderangeoffrequenciesinthesame equal latency. A directcomparisonbetweenequallatencyand only indirectevidenceinfavouroftheweightingmethodbasedon perform psychophysicalgo/no-gotasks.However, thereiscurrently estimation, andcouldbeappliedtoanyspeciesthatcantrained used inthisstudyisrelativelyfastcomparedwithdirectloudness relative loudnessperceptionintheporpoise.Theexperimentalmethod The sixauditoryweightingfunctions(Fig. consistently weakerattheseveryhighfrequencies. suggesting thatthecorrelationbetweenRT andloudnessis expected fromtheequalloudnesscontoursmeasuredtodate, 1975; Mayetal.,2009;Kastelein2011) werealsolowerthan for frequencieshigherthan~16 Ecological significanceandrecommendations The weightingfunctionsinthisstudymaybeusedtopredict Acoustic safetycriteriafortheexposureofmarinemammalsto The flattestweightingfunction(curveIinFig. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983 (Eqn 3;Fig.3B)andtheauditoryweight (Eqn

x

kHz reportedelsewhere(Green, x 4) areassumedtorepresent , isalsoshown.

4) isassociatedwith (dB octave Roll-off rate ing 363  1 )

The Journal of Experimental Biology during researchsessions. about 2 length was145 cmandhisgirthataxilla73 364 during whichtheanimalagedfrom 6to7 and athistarget bodymass.Thisstudywasconductedin2011 and2012, blubber thickness)wascheckedonce aweektoensurethathewashealthy medication. Theporpoise’s bodycondition (bodymass,length,girthand 2012). Veterinary recordsoftheanimal showednoexposuretoototoxic psychoacoustic studies,includingarecentstudyonTTS(Kastelein et al., porpoise waswelltrainedandhadparticipatedinanumber of rehabilitated afterbeingstrandedattheageofabout21 The subjectwasamaleharbourporpoise(Jerry;ID02)thathad been avoidance ofthesoundsource,occurinmarinemammals. such astheonsetofTTS,andbehaviouralresponses, as from avarietyofsoundsourcesatwhichphysiologicalresponses, functions mayalsoenablemoreaccurateestimationsofthedistances sources (e.g.Milleretal.,2012;Tyack etal.,2011). Theweighting that weremeasuredduringexposuretorelativelynarrowband behavioural responsethresholdsformarinemammalsinthewild extrapolations wouldgreatlyincreasetheapplicabilityof thresholds tosignalsofknownfrequencyspectra.Such signal thatcausedtheresponse,frommeasuredbehaviouralresponse 3 RESEARCH ARTICLE 2 3 Study animal MATERIALS ANDMETHODS B A

m m m Sideview Top view cabin Research

cabin Research kg ofthawedfish perdayandwasfedfourtimesa dayingeneral To outdoorpool

Channel

Videomonitor

Operator

Operator LF projector LF

Net gate

Trainer Box

Trainer

projectors

MF + HF + MF Box

0.9

projectors MF + HF + MF

Start/response buoy Start/response

m 0.9

LF projector LF m

years, weighed39 1

Baffleboard 1

m m Baffleboard cm. Theanimalreceived Platform reaction timesensor Listening stationand

Underwater videocamera

Underwater video camera video Underwater

Platform

reaction time sensor time reaction Listening station and station Listening

Bio-filter Swimming path Swimming

months. The kg, hisbody 8 8

m m Indoor pool chloride tube,1 listening stationwasattheendof a 32 listening station,inaresearchcabin nexttotheindoorpool(Fig. used toproducethesoundsignalswereoutofsightanimal at the of theresearchpoolduringsessions.Thesignaloperatorandequipment with Endangered Species PermitFF/75A/2005/048. Economy, AgricultureandInnovation,DepartmentofNatureManagement, were conductedunderauthorization ofTheNetherlandsMinistry School ofBiologyEthicsCommittee. Animaltraininganddatacollection systems canbefoundelsewhere(Kasteleinetal.,2009). the backgroundnoiselevel.Informationonwatercirculationandaeration the skimmersandthroughpipes,reducinginfluenceofflownoise on commenced. Bythetimeasessionhadstarted,littletonowaterflowed over pool andneighbouringpoolswereshutoff 15 the salinitywasaround34‰.Thewaterpumpsandairforresearch thick layerofslopingsandonwhichaquaticvegetationgrew. 2 quiet area.Thetestsessionswereperformedinanindoorpool(8×7 Netherlands; afacilityforpsychophysicalresearchlocatedinremoteand effective at>25 thick coconutmatswiththeirfibresembeddedin4 absorb soundenergy fromreflections,thewallswerecoveredwith3

m deep;Fig. The researchprotocolwasapproved bytheUniversityofStAndrews’ To avoiddistractingtheanimal,nobodywasallowedtomove within 15 The watertemperatureduringthestudyvariedbetween14and18°C, and Test sessionswereconductedattheSEAMARCOResearchInstitute,The 2

m The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983

5) thatwaspartoftheporpoise’s ownpoolcomplex.To

kHz), andthebottomofpoolwascoveredwitha20 m belowthewatersurface(i.e.mid-water). 1 7

m

m 80− (MF; 16− low frequency(LF;0.5 a researchtrial.Signalsweretransmittedwith are shownintheirpositionsatthebeginningof harbour porpoise,trainerandsignaloperator (A) fromaboveand(B)theside.The 5.Theindoortestpool. Fig. 125

kHz) projector. 63

mm diameterwater-filled polyvinyl kHz) orhighfrequency(HF;

min beforetestsessions − 4

mm thickrubber(most

kHz), mid-frequency The poolisshown

5). The

cm

cm m,

m

The Journal of Experimental Biology Corporation 6084, SantaBarbara,CA,USA).The80–125 transmitted byacylindrical piezoelectricprojector(International Transducer driven byanisolationtransformer(LL AC202).The16–63 unamplified andwithabalancedtonpilz piezoelectricprojector(LL 916) 30 a highlevel),and(2)theSPL ofanygivenharmonichadtobeatleast responses (e.g.hesitationtoapproachthelisteningstationafteratrialwith hearing thresholdshiftintheanimalorcauseadversebehavioural determined al., 2010)inthisharbourporpoise.Themaximumtestlevelswere other sessions,only4 for 900 as thenumberofdBabovesubject’s 50%detectionhearingthreshold across frequenciesfrom3to22 and testlevelswerespaced10 station rangedfrom59to168 mammals (Fleshler, 1965;GötzandJanik,2011)]. reflexes [risetimeispositivelyrelatedtostartlereflexthresholdsin prevent onsetandoffset clicksandreducetheprobabilityofelicitingstartle ramp oneithersideofthewaveform(10%Tukey window),inorderto also functionedasalow-passreconstructionfilter. Harderwijk, TheNetherlands)beforegoingtotheprojector. Theattenuator custom-built digitallycontrolledattenuator(AS2009-01,Smink, transmission system,theelectricoutputofDAQcardwentthrough a connected toalaptopcomputer. To increasethedynamicrangeof the (DAQ) device(NationalInstrumentsUSB-6251BNC,Austin,TX, USA) RESEARCH ARTICLE projector wasalsousedtotransmit 4 OH, USA]drivenbyanisolation transformer(LL AC1424HP).This high-power piezoelectricprojector [LubellLabs(LL)1424HP, Columbus, (Vellerman HQVPA2450MB, Gent,Belgium)andthentransmittedbya The 0.5–2 rate 1 underwater soundfieldisshowninFig. measurements’, below),andmonitortheanimal’s behaviourandthe electrical signalsfromthereactiontimesensor(see‘Reaction The equipmentusedtogenerateandtransmitthesounds,record signal wasalways1 than puretones(FinneranandSchlundt,2007).Thedurationofthetest such signalsproduceamoreuniformsoundfieldwithfewerstandingwaves and 1.01 actual frequencyofthesignalfluctuated100timespersecondbetween0.99 100 deviation was2%ofthecentrefrequency, andthemodulationfrequencywas USA) usingtheFMsynthesisequation(Chowning,1973).Thefrequency created digitallyinMATLAB (version7.5;TheMathWorks, Natick,MA, frequencies of0.5,1,2,4,16,31.5,63,80and125 The soundstimuliwerenarrowbandsinusoidalFMsignalswithcentre Sound production andmonitoring Sound production Test stimuli II 160 I 150 Contour Reference RT functions ofthetestfrequenciesincluded Table 3.TheRMSEbetweentheSPLsoft (ms) The highestsimilaritybetweenthetwocu each RMSEcolumn.Thesmoothedcontourwa Test frequenciesincludedinthedatasetsforsimilarityan III 170 IV 180 VI 200 V 190 The SPL ofthestimulireceivedbyporpoisewhileatlistening Four projectorswereusedtotransmitthesignalsintowater(Fig. dB belowtheSPL ofthefundamentalfrequency. Hz. Therefore,forexample,whenthecentrefrequencywas1 MHz) wereconvertedtoanalogsignalsusinga16 ms tonalsignals,whichwasmeasured2–3 kHz. FMstimuliwereusedbecauseinsmall,reverberantpools kHz signalswerefirstfedintoanaudiopoweramplifier a priori

s. Eachsignalwascosine-taperedtocreatea50 based ontwocriteria:(1)signalscouldnotinduce kHz signalsofSnLs≤

dB re.1 μPa(dependingonthefrequency), dB apart.Theminimumtestlevelvaried

dB intermsofSnL.SnL isdefinedhere

kHz signalsofSnLs≥

6. Thedigitalsoundsignals(sample rves, indicatedbythesmallestRMSE(in 4.6 RMSE 3.4 3.8 5.0 6 4 . 5.9 5.9 6.4 3.1 he unsmoothed equal latency contourandtheSP he unsmoothedequal s thetransformedhearingthreshold(Eqn 58

A years ago(Kasteleinet

kHz. Thesignalswere t  dB weretransmitted 16 kHz

bit dataacquisition

kHz signalswere kHz signalswere (dB) alyses (see ‘Derivation oftheauditoryweighting functions’ fo 48

dB, butin

kHz, the

ms 6). 4.3 A 3.2 3.0 3.7 4.7 t  31.5 kHz the measurements. sensor wascleaneddailytoprevent algalgrowththatwouldhaveinfluenced component oftheresponse)was minimal, withoutfalsedetections.The response andthemomentthatsensor indicated‘absence’ (i.e.themotor into fine-tuningthedimensionsso theintervalbetweenstartof porpoise’s rostrumwasoutsidetheoptical beam.Significanteffort was put porpoise’s rostrum(whenthebeamwas broken),and‘absence’ when the The sensorindicated‘presence’ whentheinfraredlightwas blockedbythe effective opticalbeam,which wasabout8 facing eachother. Thetipofthelisteningstationreachedjustinside (Batbox III,Steyning,UK). Denmark] andamplifiedloudspeaker, oramodifiedultrasounddetector fed intoeitheracharge amplifier[BruelandKjær(B&K)2635,Nærum, to theholeinbaffle board.Theoutputofthemonitoringhydrophonewas hydrophone (Labforce190.02.01,Gouda,TheNetherlands)positionednext further verifiedbylisteningtotheunderwatersoundviaamonitoring oscilloscope andvoltmeter. Beforeandduringsessions,thesystemwas signals andbackgroundnoiseinthewaterweremonitoredusingsame signal withaknownrootmeansquarevoltagefromthecomputer. Thetest voltmeter (HewlettPackard3478A,PaloAlto,CA,USA),byplayinga digital storageoscilloscope(Voltcraft 632FG,Hirschau,Germany)anda rubber matonthesidefacingprojector. high, 1.2 water surfacereachingthelisteningstation.Theboardwasmadeof2.4 and theanimaltoreducereflectionsfrombottomofpool board witha30 acoustic beamaxispointedatthecentreofporpoise’s head.A baffle (Fig. stainless steelcage,at1.2 box, sothisprojectorwashunginfrontoftheboxbyropesattachedtoits listening station.ThehighpowerLL 1424HP didnotfitintheprotective from theporpoise’s externalauditorymeatuswhiletheanimalwasat was linedwithrubberanirregularsurface.Theseprojectorswere2 placed inacornerofthepoolprotectivewoodenbox(Fig. caused bymulti-patharrivals,allprojectorsexcepttheLL 1424HP were Honolulu, HI,USA)(seeKasteleinetal.,2009). transmitted byacustom-builtdiscoidpiezoelectricprojector(WAU q7b, (LED). Theintensityoftheinfraredlightwasmodulated(38 Japan) connectedtoa319 consisted ofaninfrareddetectorintegratedcircuit(SharpIS471FE,Osaka, and builtforthisstudy. Thereactiontimesensor’s electroniccircuit An opticalsensorsystemtomeasuretheanimal’s responseswasdesigned below thetipoflisteningstation,respectively, spaced13 Fig. polyurethane epoxy, insidetwobracket-shapedpolyvinylchloridepipes(see external light.Theelectroniccomponentswereembeddedintransparent by theintegratedcircuit,makingdetectorimpervioustodisturbing Reaction timemeasurements italics), wasgenerallyobtainedwiththe0.5 The outputofthesoundsystemwascheckedbeforeeverysessionwitha To minimizetemporalandspatialvariationsintheunderwatersoundfield 5B). Theinfraredemitteranddetectorwereplaceddirectlyabove 5). ThedirectionalWAU q7bprojectorwaspositionedsothatthe  2) that was the most similar to the unsmoothed contour. 2) thatwasthemostsimilartounsmoothedcontour. m wide,4 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983 cm diameterholewasplacedhalfwaybetweentheprojector 7.2 10.4 10.7 A 6.0 8.3 8.2 5.2 6.6 6.3 5.0 5.5 5.2 5.3 5.1 5.3 cm thickplywood,coveredwitha2 t  63 kHz Ls ofthesmoothedequallatencycontour, m fromtheporpoise’s externalauditorymeatus

THz narrow-beaminfraredlight-emittingdiode A 5.5 r moredetails) aregivenabove t 

mm indiameteratthatlocation. 80 kHz  31.5 kHzdataset. 31.5

cm thickclosed-cell A 6.2 t 

kHz frequency) 125 kHz

cm apart,and

5), which 365 as

m m

The Journal of Experimental Biology 3.5 measurements the backgroundnoiseinthepool was verylow;above visible tothetraineronamonitornearstart/responsebuoy. screen tothesignaloperatorduringresearchsessions.Theimageswere also converter (GeniatechEZGrabber, Shenzhen,China)andshownonalaptop the researchcabin,weredigitizedbyusingavideoanalog-to-digital images fromthecamera,togetherwithsoundamicrophone inside underwater cameramadetheinfraredlightvisibleonmonitorimage. The Puerto Montt,Chile)filmingthelisteningstationfromabove(Fig. present, andwascapturedbyanunderwatercamera(MariscopeMicro, correctly. TheLEDwasswitchedonautomaticallywhentheanimal signal operatortocheckwhetherthereactiontimesensorwasworking measure theporpoise’s RTs. 6.Equipmentset-up. Fig. 366 in the100 background noiseweredetermined byaveragingthesquaredsoundpressure and windforceBeaufort4or below. 1/3-OctavebandSPLsofthe research sessionconditions:water andaircirculationsystemoff, norain, by thecorresponding90%timeduration (Madsen,2005). of eachtestsignalwasderivedfromthe90%energy fluxdensity, divided system wascalibratedwithapistonphone(B&K4223).Thereceived SPL laptop computerwithB&KPULSEsoftware(Labshopversion12.1). The with amultichannelhighfrequencyanalyser(B&KPULSE3560D), anda The soundcalibrationequipmentconsistedoftwohydrophones(B&K 8106) RESEARCH ARTICLE simulation ofatesttrial. achieve stablesampling,whichwasverifiedbeforeeachresearchsessionby 125 optical beam].Theoutputofthesensorswassampledreal-timeatarate memory beforetriggering)andthemomentanimalmovedoutof between thetriggeroftestsignal(whichwasloadedintocomputer the stimuluslevelandmeasureanimal’s RT [definedhere astheinterval program. Theprogramallowedtheoperatorduringresearchsessionstoset the DAQdevice,whichwascontrolledbyacustom-writtenMATLAB Acoustic calibration The backgroundnoiselevelswere measuredmultipletimes,under A second 319 The reactiontimesensorcommunicatedviabinaryelectricalsignalswith kHz itwasjustabove theself-noiseofrecording equipment. Hz (8

Hz to160 ms resolution).Thisratewasthemaximumpossibleto Underwater for trainer THz infraredLEDinthetopsensorbracketallowed Microphone Monitor camera kHz bandsoveraperiodof10 Block diagramoftheequipmentusedtogeneratesoundstimuli,monitorsoundsandharbourporpoiseunderwater, and Laptop Amplifier digital converter acquisition Analog to DAQ Data card A D

s. Duringcalibration Ground loop Video image distorter isolator Reaction time sensor Attenuator

5). The operator signal Monitor for amplifier Power was consistentto1 The linearityofthetransmittersystemwascheckedat0.5,1and4 hydrophone locationsandmeasurementdays. frequency). Thefinalcalibrationvaluewastakenasthegrandmeanover acoustic beamaxisoftheprojector(Fig. rostrum againstit,sothathisanterior–posterioraxiswasalignedwith the 7A)andpositionedhis the porpoiseswamtolisteningstation(Fig. rostrum. Whenthetrainergaveavocalcommandandpointeddownwards, A trial beganwhentheporpoisetouchedstart/responsebuoywithhis indicated tothetrainer thattheresponsewascorrectusing ahandgesture,after test sound.Ifthe animal respondedwithin2 the operatortodeterminewhether or nottheporpoisehadrespondedto distorter producedhorizontallinesin thevideoimage(Fig. after therandomwaitingtime.When thesoundwasbeingtransmitted,avideo operator playedthetestsignalfrom thecustom-writtenMATLAB program for arandomperiodbetween4and 10 absent (or‘catch’)trials.Inalltrials the porpoisewaitedatlisteningstation station untilheheardasignal. returning tothestart/responsebuoy(Fig. (Fig. positioned atthelisteningstation,porpoisewastrainedtorespond and thetrainersenthimstraightbacktolisteningstation. Once trainer knockedonthestart/responsebuoy, theporpoisereturnedtobuoy, positioned correctly. Ifhewas,thetrialwouldcontinue;ifwasnot, underwater camera,thetrainerjudgedwhetherornotanimal was average SPL betweenmeasurementdayswas1 averaging oftheSPL overthetwohydrophonelocations,difference in locations differed by0 porpoise whenhewaspositionedatthelisteningstation.TheSPL atthetwo the twohydrophones,oneateachlocationofauditorymeatus (depending onthefrequency).Thesemeasurementswereconductedusing Experimental procedure Received SPLswerecalibratedusingrelativeoutputlevelsof60 Research sessionsconsistedof75%signal-presenttrialsand25%signal- The receivedSPL ofeachtestsignalwasmeasuredonceortwice 7C) upondetectingeithertheteststimulusortrainer’s whistleby transformer transformer Isolation Isolation Oscilloscope The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983 Voltmeter V dB withinthe40 (16–63 kHz) (>63 kHz) (<16 kHz) P Projector Projector Projector (4 kHz) rojector − 7 Loudspeaker

dB (meanabsolutedifference 3

dB range. hydrophone Monitoring

s. Insignal-presenttrials,thesignal

7B). Usingtheimagesfrom 7D), andtostayatthelistening

s ofsignalonset, the operator detector Variable amplifier resistor Charge − 3

dB (dependingonthe

7C), whichhelped

dB). After − 100

kHz; it dB.

The Journal of Experimental Biology available tocalculatetheequallatencycontours. until atleast50RT measurementsperlevel/frequencycombinationwere measured, andtheexistingdatasetsforotherfrequencieswereincreased high frequencies).In2012,RTs forfrequenciesof0.5,1and2 usually testedonsuccessivedays(goingfromhightolowand to frequency waschangedfromdaytoandadjacentfrequencies were measurements werecollectedperlevel/frequencycombination.The test test frequenciesrangedfrom4to125 one extrasessionwasperformeddailyin2012(startingat16:00 RESEARCH ARTICLE asymptotic parameter, whichissuggested toreflectaminimumprocessing reasonable approximations tothedata.However, theestimateof model, knownasPiéron’s law, tothemedianRTs oftheporpoiseprovided intensities (Luce,1986;Piéron,1920). Initialfitsofthisthree-parameter described byadecayingpowerlaw approachinganasymptoteathigh relationship betweenmeanormedian RT andstimulusintensityhasbeen especially forlevelsnearthehearing threshold,wereoftenskewed.The Medians werecalculatedratherthan means,becausethedistributionsofRTs, 5 September 2012.Threeexperimentalsessionsperdaywereconducted ( the leveldifference betweensuccessivetrialswasnotmorethan30 per signallevel.Thelevelswererandomized,withtherestrictionthat sheet hadarandomseriesofwaitingtimesandbalancednumbertrials 20 continued assoonthesoundhadstopped. were ignoredwhentheyclearlyinitiatedbyexternalsounds;sessions the animalforabout10 response), thesignaloperatorindicatedthistotrainer, whothenignored was asignal-presentorsignal-absenttrial. received afishreward.Thetrainerdidnotknowbeforehandwhethertrial returning tothestart/responsebuoydirectlyafterawhistle,animalalso three timesonthesideofpool(inrelativeproportions1:1).For the operatorgesturedtotrainereitherblowonawhistleorsoftlytap signal-absent trials,theporpoisestationed,andafterrandomwaitingtime tapping threetimesonthesideofpool,andnofishrewardwasgiven.In trainer thencalledtheporpoisebacktostart/responsebuoybysoftly within 2 which thetrainergaveporpoiseafishreward.Ifanimaldidnotrespond Analysis of the reaction times thereaction of Analysis sensu

days aweekin2011 (sessionsstartedat09:00 Research sessionswereconductedinMaytoJuly2011 andinAugust An experimentalsessionconsistedof30 If theanimalrespondedbeforeasignalwasproduced(pre-stimulus min. Foreachsession,oneoffourdatacollectionsheetswasused; Wagner etal.,2004). s, theoperatorsignalledtotrainerthattrialhadended.The

s beforestartinganewtrial.Pre-stimulusresponses

kHz, andonaverage39RT − 35 trialsandlastedforabout

h, 11:00 h and14:00

kHz werealso h). In2011, h), and

dB six contoursalwaysfellwithintherangeoftestedlevels. were selectedbecause,exceptforonedatapointat16 equal latencycontours(labelledI 180, 190and200 fitting auditoryRT modelswereevaluatedatreferenceRTs of150,160,170, improved themodelfits(omitteddataareshowninFig. of astimulusatthreshold( median RTs (inms)foreachtestfrequency: became negative.Therefore,atwo-parameterpowerlawwasfittedtothe time andmotorcomponent(LuceGreen,1972),wasunstableoften to havesimilarshapes,exceptpossibly atveryhighfrequencies. rates; and(4)theequallatencyloudnesscontourswereexpected expected tohavesimilarshapesbutdifferent lowfrequencyroll-off Kastelein etal.,2010);(3)theequalloudnesscontoursandaudiogram were of twootherharbourporpoises(Andersen,1970;Kasteleinetal., 2002; of thesubjecthadbeendeterminedveryaccurately, andwassimilartothat and weightingfunctionsaregenerallyidealizedcurves;(2)theaudiogram follows: (1)smoothingwasjustifiedbecausetherangeofRTs wassmall, own audiogramasatemplate.Therationalebehindthisapproachwas as the datasetswereadaptedandsmoothedusingshapeofanimal’s To derivesixauditoryweightingfunctionsfromtheequallatency contours, threshold (L where low intensitysignals(SnL <30 2004). Therefore,oneortwomedianRTs (dependingonthefrequency)to behaviour (PinsandBonnet,2000;StebbinsMiller, 1964;Wagner etal., long RTs oftenoccurthatresultindeviationfromsimplepowerlaw affected bytheanimal’s responsecriterion(Heiletal.,2006)andrelatively linear method(Trust Regionalgorithm)inMATLAB. Piéron’s law. AllfitsoftheauditoryRT functionsweremadeusinganon- the medianRT andgavesimilarresultsintermsofgoodnessfitas SnL=0 the slopeonalog–logscale,and where Derivation of the auditory weighting functions theauditory Derivation of contour andtheSPLs ofthetransformedhearingthreshold wascalculatedfor parameter, respectively. TheRMSEbetweentheSPLsof equallatency Each smoothedcontour( For levelsnearthehearingthreshold,statisticalmeasuresofRT are f I fish rewardfromthetrainer. (D) Theporpoiseswimsbacktothestart/responsebuoyreceivea video distorterarevisibleintheimagebecausesoundison). while thesoundisstillbeingemitted(horizontallinesproducedby arrow). (C)Theporpoisehasrespondedandstartsmovingaway top sensorbracketabovethelisteningstation(indicatedby light beaminthereactiontimesensor, andilluminatingtheLEDin its rostrumagainstthelisteningstation,therebybreakinginfrared porpoise approachesthelisteningstation.(B)Thepositions 7.Stillframesequenceofaresearchtrial. Fig. dB). Thistwo-parameterfunctionwaslesssensitivetovariationin / is avectoroftestfrequencies,and I 0 is theratioofintensityteststimulus( ht The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091983 ): ms todeterminetheSnLs(and,hence,SPLs)of I L 0 ) (calculatedusing lat RT =β L ( f lat ) =γ

dB) wereomittedwhenthissubstantially − ) wasatransformationofthehearing VI, respectively).Thesereferencevalues β L ( isthey I ht / I ( 0 f ) ) +δ γ – and α , -intercept (equaltotheRT at ,(2) , δ I areascalingandtranslation / I 0 =10

kHz, theSPLsof SnL/10

(A) Theharbour 1). Finally, thebest- I ) totheintensity ), exponentα 367 (1) is

The Journal of Experimental Biology the levelis6 was settoaveryhighnumber(10 Parameters was effectively non-existentwithinthefrequencyrangeofhearing. where each ofthesecontours: hypothetical equalloudnesscontours.A closed-formmodelwasfittedto contour/threshold relationships(Eqn frequencies (Table 8 and50− 368 L.A.E.H. andL.H.collected andprocessedthereactiontime data.P.J.W. analysed P.J.W. andR.A.K.designedtheexperimental studyandgainedfunding.P.J.W., The authorsdeclarenocompetingfinancial interests. manuscript. anonymous reviewersareacknowledged fortheirhelpfulcommentsonthe Environment), WimVerboom (JunoBioacoustics,TheNetherlands)andtwo Iceland), RenéDekeling(TheNetherlandsMinistryofInfrastructureandthe Miller (UniversityofStAndrews),FilipaSamarra(MarineResearchInstitute, Figs group] forconductingtheacousticcalibrations,andRobTriesscheijn formaking Organisation forAppliedScientificResearch(TNO);AcousticsandSonarresearch designing andbuildingthereactiontimesensor, ErwinJansen[TheNetherlands Krijger fortheirhelpwiththedatacollection.We alsothankArieSminkfor We thankstudentMartijnRambagsandtrainersTess vanderDriftandKrista ( is averticaloffset thatresults fromthenormalizationofcontours and 24.37 and 1.104 best-fit estimatesforparameter and VI;thesmallestRMSEwas~5 similarity betweentheunsmoothedandsmoothedversionsofcontoursV with partofthedataset(<31.5 on thesimilarity:oncewithfulldataset(allfrequencies)andfourtimes each ofthesixcontourstoinvestigateinfluencehighfrequencydata function (the‘smoothed’ contour).Thisprocesswasrepeatedfivetimesfor determined thecombinationof indicated thesimilaritybetweentwocurves;minimumRMSE combinations of RESEARCH ARTICLE parameter (Finneran andSchlundt,2011) isaspecialcaseofEqn et al.,2000;Southall2007)alsousedbyFinneranandSchlundt of thefunction.ThemathematicalformC-andM-weightings(Kinsler frequency inHz,andK Author contributions Competing interests Acknowledgements where which thentaketheform: sensitive frequencyandinvertedtoobtaintheauditoryweightingfunctions, 0.5− to aweakRT–loudness correlation(seeDiscussion),soonlythesmoothed and 125 (Table I the similaritybetweenunsmoothedandsmoothedversionsofcontours VI, independentoftherangefrequenciesincluded(Table K − IV increasedsignificantlyaftertheexclusionofhighfrequencydata The best-fittingfunctionsofEqn The smoothedcontourdatasetswereextendedwithfrequenciesof0.25, Exclusions ofhighfrequencydatadidnothavecleareffects onthe 2 =min{L 5 and6.NancyJennings(Dotmoth.co.uk),MichaelAinslie(TNO),Patrick 31.5 L 3), andthebestresults(smallestRMSEs)wereobtainedwhen63,80 W loud kHz wereomitted.Thedecreasedsimilaritywassuspectedtobedue ( kHz datasetswereusedinfurtheranalyses.Forthesesets,the f 150 dB, forcontoursI− ) istheweightinglevelindB, dB/dB andforparameter x loud a is theSPL oftheequalloudnesscontourindBre.1 μPa, was fittedasafreeparametertoallowtheroll-off ratetovary.

dB abovetheminimumofcurve,respectively. Parameter and }–K kHz byusingtheporpoise’s ownhearingthresholdsforthese LfK fK Wf od110 1 loud γ b () 1 andδ ). in Eqn ()

1) (Kasteleinetal.,2010)inthecalculated =+ 1 = , a usingasimpleiterativealgorithm.TheRMSE 210 , 3 representthelowerandupperfrequencywhere b − 20log VI, respectively. and

kHz, <63 20log γ andδ γ x were:0.610,0.721,0.825,0.924,1.016 9

dB forcontourV and~6 are parametersthatdeterminetheshape

3 werenormalizedto0 Hz) sothatthehighfrequencyroll-off δ

⎣ ⎢ ⎢ ⎡ : 103.77,85.94,69.22,53.39,38.52 2). Thisresultedinafamilyofsix thatcorrespondedtothebest-fitting ()() ⎣ ⎢ ⎢ ⎡ afbf kHz, <80 ()() a afbf xxxx , xxxx ++ b ++ and bf bf xx xx kHz and<125 x are asinEqn

3 wherex ⎦ ⎥ ⎥ ⎤

⎦ ⎥ ⎥ ⎤ dB atthemost (4) , dB forcontour

(3) , 3). However, kHz). is 2.Here, 3, andK f is the b 2 Fletcher M. Fleshler, C.E. Schlundt, and J. Finneran, A.S. Frankel, and C.W. Clark, B.L., Southall, W. T., Ellison, oln,R . ooh .R n als J.R. Baylis, and S.R. Zoloth, R.J., Dooling, M.A. Ainslie, and C.A.F. De Jong, J.M. Chowning, inrn .J n clnt C.E. Schlundt, and J. Finneran, C.E. Schlundt, and J. Finneran, M. Florentine, and M. Epstein, el . ebur . ifnu .advnSeh,H. vonSpecht, and A. Tiefenau, H., Neubauer, P., Heil, S. Green, Environment ResearchCouncil[toP.J.W.]. Netherlands MinistryofDefence(administeredbyTNO)andtheUKNatural Environment [grantnumber4500182046],andbymatchedfundingfromThe This workwassupportedbyTheNetherlandsMinistryofInfrastructureandthe from allauthors. the dataandpreparedmanuscriptwithadviceonresultsinterpretation hcol,R. Chocholle, J. Botwinick, and J.E. Birren, L.E. Marks, and Y. Arieh, S. Andersen, hcol,R n rebu,H. Greenbaum, and R. Chocholle, öz .adJnk V. M. Janik, and T. Götz, M. Rosenberg, and S. Buus, M., Florentine, atli,R . usok . aeor,M,A,W .L n eHa,D. deHaan, and W. W. L. Au, M., Hagedoorn, P., Bunskoek, R.A., Kastelein, J. Zwislocki, and R.P. Hellman, ue,L .adAlto,J.B. Ahlstrom, and L.E. Humes, References Funding us . rebu,H n caf B. Scharf, and H. Greenbaum, S., Buus, D.J. Blackwood, atli,R . esen .J,He,L,A,W .L,True .M n de and J.M. Terhune, W. W. L., Au, L., Hoek, P. J., Wensveen, R.A., Kastelein, atli,R . ok . eJn,C .F n esen P. J. Wensveen, and C.A.F. deJong, L., Hoek, R.A., Kastelein, Physiol. Psychol. Am. temporary thresholdshiftinbottlenosedolphins( Soc. Am. Soc. 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