OPENQ ACCESS Freely available online ~ PLOS one Distinguishing the Impacts of Inadequate Prey and Vessel Traffic on an Endangered Killer Orcinus orca! Population

KatherineL. Ayres"", RebeccaK. Booth",Jennifer A. Hempelmann, Kari L. Koski, CandiceK. Emmons, RobinW. Baird, Kelley Balcomb-Bartok,M. BradleyHanson, MichaelJ. Ford, SamuelK. Wasser" 1 Despartmentof Biology,Center for ConservationBiology, University of Washington,Seattle, Washington, United States of America,2 NationalMarine Service, NorthwestFisheries Science Center, Seattle, Washington, United States of America,3soundwatch Boater Education Program, The WhaleMuseum, Friday Harbor, Washington,United States of America,4Cascadia Research Collective, Olympia, Washington, United States of America,5 RentonCity Hall, Renton, Washington, United Statesof America

Abstract Managing endangered species often involves evaluating the relative impacts of multiple anthropogenic and ecological pressures. This challenge is particularly formidable for cetaceans, which spend the majority of their time underwater. Noninvasive physiological approaches can be especially informative in this regard. We used a combination of fecal thyroid 93! and glucocorticoid GC! measures to assess two threats influencing the endangered southern resident killer SRKW; Orcinus orca! that frequent the inland waters of , Canada and Washington, U.S.A. Glucocorticoids increase in response to nutritional and psychological stress, whereas thyroid hormone declines in response to nutritional stress but is unaffected by psychological stress. The inadequate prey hypothesis argues that the killer whales have become prey limited due to reductions of their dominant prey, Chinook tshawytscha!. The vessel impact hypothesis argues that high numbers of vessels in close proximity to the whales cause disturbance via psychological stress and/or impaired foraging ability. The GC and T3 measures supported the inadequate prey hypothesis. In particular, GC concentrations were negatively correlated with short-term changes in prey availability. Whereas, T3 concentrations varied by date and year in a manner that corresponded with more long-term prey availability. Physiological correlations with prey overshadowed any impacts of vessels since GCs were lowest during the peak in vessel abundance, which also coincided with the peak in salmon availability. Our results suggest that identification and recovery of strategic salmon populations in the SRKW diet are important to effectively promote SRKW recovery.

Citation:Ayres KL, Booth RK, Hempelmann JA, KoskiKL, Emmons CK, et al. 012! Distinguishingthe Impactsof InadequatePrey and VesselTraffic on an EndangeredKiller Whale Orcinusorca! Population. PLos ONE 7!: e36842.doi:10.1371/journal. pone.0036842 Editor:Brock Fenton, University of WesternOntario, Canada ReceivedOctober 20, 2011; Accepted April 13,2012; Published June 6, 2012 Thisis anopen-access article, free of all copyright,and may be freely reproduced, distributed, transmitted, modified, built upon,or otherwiseused by anyonefor any lawfulpurpose. The work is madeavailable under the CreativeCommons CCO public domain dedication. Funding:This work hasbeen funded by the NationalOceanic and AtmosphericAdministration-Northwest Center, the Joint Institutefor the Studyof the Atmosphereand , Washington Sea Grant No. NA10OAR417005, the Canadian Consulate General, Northwest Science Association, University of WashingtonDepartment of Biology,Lynn Riddifordand Jim Truman,Elizabeth Welch and the Centerfor ConservationBiology. MJ. Ford,M.B. Hanson, J. Hempelmann,and C. Emmons are employees of NorthwestFisheries Science Center. All authorswere involved in the preparationof the manuscript.M.B. Hanson, J. Hempelmann,and C. Emmons were involved in datacollection and analysis.K.L. Ayres, R.K. Booth are employees with the Centerfor ConservationBiology, a nonprofitdirected by S.K.Wasser. K.L. Ayres and S.K.Wasser designed the study,applied for funding,collected data and did mostof the analysis,decision to publishand preparation of the manuscript.R.K. Booth was involved in studydesign/methods and data analysis. The other funding agencies had no rolein study design,data collection and analysis,decision to publish,or preparationof the manuscript. CompetingInterests: Authors Michael J. Fordand M. BradleyHanson were partiallyresponsible for funding decisionsfrom NorthwestFisheries Science Center.Washington SeaGrant is alsoadministered by NOAA,however the NOAAemployees that areauthors on this paperwere not directlyinvolved with SeaGrantfunding decisions.Funding from this agencywas a majorpart of funding this research.Authors Katherine Ayres, Rebecca Booth and Samuel Wasserwork for the Centerfor ConservationBiology, which also partially funded this work.Author KariKoski is an employeefor the SoundwatchBoater Educationprogram that providedthe vesseltraffic data for this research.Soundwatch is a boater educationprogram that often consultson vessel regulationsdecisions based on their monitoringresearch. This does not alter the authors'adherence to all the PLosONE policies on sharingdata and materials. ' E-mail:kayresliharveyecology.corn u Currentaddress: H.T. Harvey 5 Associates,Fresno, California, United States of America

Introduction The endangered population of southern resident killer whales Orcinus orca; SRKWj that frequent the inland marine waters of Conservation management decisions often involve weighing the southern British Columbia, Canada and Washington, U.S.A. relative impacts of multiple, co-occurring anthropogenic and termed the ! provide a casein point. The three southern ecological pressures on wildlife health. Physiological measures resident "pods" each form long-term stable groups that frequent provide valuable tools for evaluating the relative importance of the Salish Sea for varying amounts of time from May through such impacts [1], an essential first step to guide mitigation and October [2 ]. From November through May, all three pods evaluate its success. spend the majority of their time along the outer coast P]. SRKWs are almost exclusively piscivorous, which distinguishes them from

'... PLoSONE l vvvvvv.plosone.org June 2012 l Volume 7 l Issue 6 l e36842 Prey and VesselImpacts on Killer Whale Physiology sympatric "transient" killer whales that forage on other marine distinguish the relative contributions of psychological and nutri- mammals [5,6]. A near 20'/0 decline from 1995 2001 precipitated tional stress to a population's physiological health P3,34]. GC the SRKW being listed as an endangered population under the concentrations rise in response to nutritional stress as well as a Canadian Species at Risk Act in 2001 [7] and the United States wide variety of psychological stressors, including circumstances Endangered SpeciesAct in 2005 [4,0]. Both the US and Canadian triggering fight or flight or an animal's perceived lack of control SRKW Recovery Plans outline three main threats that may have over its environment P4 37]. By contrast, T3 concentrations contributed to the past decline and may currently slow recovery: decrease in response to nutritional stress Pfl 40], but are largely vessel disturbance " vessel impact hypothesis"!, nutritional stress unaffected by psychological stress [41 44]. from inadequate prey availability " inadequate prey hypothesis"!, Glucocorticoids are steroid released from the adrenal and exposure to persistent organic pollutants " toxin hypothesis"! cortex that help regulate a suite of physiological and behavioral [9,10]. Here we use noninvasive endocrine measures in SRKW coping mechanisms in response to nutritional as well as scat to evaluate the inadequate prey and vessel impact hypotheses psychologically stressful situations P7,45]. T3 is a modified amino in an effort to help guide mitigation priorities. acid that helps regulate metabolism [46]. In vertebrates, both The vessel impact hypothesis argues that exposure to a high hormones are excreted as metabolites in , at concentrations abundance of vessel traflic is associated with behavioral changes, that reflect biological activity P1,32]. However, the GC response increased energy expenditure and/or foraging interference [11 16], to nutritional and other emergencies tends to be more rapid than resulting in psychological and/or nutritional stress.The SRKW are the T3 response. Short-term nutritional emergencies cause a rise the focus of the whale watching industry in the inland waters of in GC concentrations that promote quick glucose mobilization Washington and southern British Columbia, which includes a followed by rapid metabolism and clearance of GCs from combination of private and commercial whale watching vessels. circulation once the stressor has passed. By contrast, sustained The whales are also exposed to private and commercial fishing boats, food deprivation causes a decrease in T3 concentrations, slowing recreational powerboats, sailboats, kayaks, research vessels,military metabolism to conserve energy stores [46]. vesselsand freight carrying ships.Reducing potential vesselimpacts is While in the Salish Sea from May through September, the complicated by the collective contribution of thesevessels to U.S. and SRKW primarily eat heading to the Fraser River Canadian economies, along with treaty and international trade system [20]. Fraser River Chinook salmon counts are relatively agreements. In 2011, NOAA Fisheries implemented federal regula- low when the whales first arrive sometime in the late spring and tions restricting the approach of vesselswithin 200 yards of killer early summer, as are the number of vesselsin the area Figure la whales in U.S. coastalwaters www.nwr.noaa.gov/Publications/FR- and lb respectively!. Both Fraser River Chinook salmon counts Notices/2011/upload/76FR20070.pdfI as well as prohibiting park- and vessel abundance peak around August-September, progres- ing a vesselin the path of traveling killer whales. sively declining thereafter. These coincident peaks allow us to use The inadequate prey hypothesis arguesthat the SRKW population GC and T3 measures to distinguish between the inadequate prey experiences times of prey limitation due to marked declines and and vessel impact hypotheses. Under the inadequate prey fluctuations in the availability of their primary food source, adult hypothesis, GC concentrations should be relatively high upon Chinook salmon Onrorbpnrbrrstsbazeptsrba! on the west coast of the SRKW arrival when Fraser River Chinook salmon counts arelow. United Statesand Canada [9,10,17,10]. During the summer months, GC concentrations should reach their nadir around August- SRKWs eat a diet estimated to be 00 90'/0 Chinook salmon [19,20]. September the peak of Fraser River Chinook salmon counts and Adult Chinook salmon are the largest of the salmonids and have the then increase as Fraser River Chinook salmon decline thereafter. highest caloric and fat content, which may explain the whales' strong The vessel impact hypothesis makes the opposite prediction. GC preference for them [19]. However, most Chinook salmon stocks in concentrations should be relatively low due to low vessel traffic the eastern North Pacific are at a fraction of their historic levels due to when SRKW arrive in late Spring, peak around August- a combination of historical , loss and dams and September with the peak in vessel abundance, and decline with other blockages to migration and large-scale climate variation declining vessel traflic thereafter. If prey availability and vessel [21,22]. Long-term demographic studies show that SRKW survival impacts act cumulatively, we predict an interaction between Fraser [17], fecundity [lfl] and social cohesion [23,24] are positively River Chinook salmon counts and vessel abundance on GC correlated with annual indices of Chinook salmon abundance. concentrations. Specifically, GC concentrations should show a Salmon conservation and restoration is economically and politically steeper positive correlation with vessel abundance during years of complicated by a large number of factors that impact salmon low Fraser River Chinook salmon returns. throughout their complex life-cycle. The inadequate prey hypothesis also predicts a relation between The toxin hypothesis stems from biopsy studies, revealing T3 and Fraser River Chinook salmon; T3 should be positively persistent organic pollutants in SRKW that exceed an correlated with Fraser River Chinook salmon. However, if the T3 established health-effects threshold, presumably due to biomagni- response to nutrition is more protracted, we expect the T3 fication in these long-lived, top-level predators [25 20]. Although concentration of arriving whales to initially reflect the abundance the present study focuses on the inadequate prey and vesselimpact and nutritional quality of the food source the whales were eating just hypotheses, impacts of these lipophilic toxicants on SRKW are prior to their arrival in the Salish Sea e.g., during the previous 1 2 likely tied to periods of food deprivation due to associated months!. If the prior food source was relatively more nutritious, increases in fat metabolism [29,30]. Eliminating legacy toxins in SRKW T3 concentrations at first arrival should still be high despite the international Salish Sea ecosystem is yet another economic, Fraser River Chinook salmon being relatively low at that time. logistic and politically complicated task. Under those circumstances, we expect SRKW T3 concentrations to To test the inadequate prey and vessel impact hypotheses, we progressively decline from their time of first arrival, increase again measured fecal glucocorticoid GC! PI] and thyroid triiodothy- around the peak in Fraser River Chinook salmon, and then decline ronine or "T3"! P2] hormone concentrations in relation to continuously until the late fall departure. Since T3 is uncorrelated temporal changes in Chinook salmon availability and vesseltraffic with psychological stress,T3 should only be correlated with vessel over a three-year period. The combination of GC and T3 abundance if an increase in vesselabundance persistently interferes hormone measures from the same sample are well suited to with killer whale foraging efliciency.

'... PLoSONE ~ www.plosone.org June 2012 ~ Volume 7 ~ Issue 6 ~ e36842 Prey and VesselImpacts on Killer Whale Physiology

C 0 E IAlQ 0 G! 0 Z c3 o0 o IAI/I LL

30 IA Gl 25 E o0 20 0 E Ct Q>IC0 IA C !< c 10 Ilt Ill IC0

5000

4000 o0 0 3000

2000 Ql lt 1000

400

4I c 300 C 0 0 ot 0 c 200

olll 100

0 IA 0 IA Ct IA 0 IA Ct Itt C 0 Itt 0 IA 0 Itt 0 Itt 0 IA 0 0 IA 0 IA 0 IA Ct IA 0 Itt 0 0 N IA I 0 N NN IA N NI 0 tt tt IA 0 N IA I 0 N NN IA N N IA0 N IA IA 0 N IA > 0 N N N IA N NN 0 tt N IA IA

2007 2008 2009

Day of the year

Figure 1. Temporal trends in variables used to test the inadequate prey and vessel impacts hypotheses. Temporal variation in Eraser RiverChinook salmon at the Albion test a!; vesseltraffic in proximity to Southern resident killer whales b!; physiological stress indexed by fecal glucocorticoid concentrations! c!; nutrition indexed by fecal triiodothyronine concentrations! d!. Trend lines determined using general linear model selection with predictor variables year, Julian date linear, quadratic, cubic, etc.; see Table Sl! and the interactions between year and Julian date parameters.Hashed lines indicate 95% confidence intervals. Dotted vertical lines indicate Julian day 230 August 18!, the time of maximum vessel traffic and approximately ten days before the maximum Chinook salmon catch each year. Horizontal dotted lines indicate dependent variable marginal means for each year on day 230 within the individual model. doi:10.1371/journal.pone.0036842.g001

'... PLoSONE www.plosone.org June 2012 J Volume 7 J Issue 6 J e36842 Prey and VesselImpacts on Killer Whale Physiology

Materials and Methods for detection dog sampling Grady White!. The second team utilized 6 m and 7 m rigid-hulled inflatable boats with a bow Ethics Statement platform Avon and Zodiac respectively!. Fecal samples were collected in United States waters under Samples were located using two different sampling methods: National Marine Fisheries Service permits 532-1022-00, 532-1022 focal animal follows and detection dog assistedsampling of one or and 10045 and in Canadian waters under more clustered individuals. The first research team conducted License numbers 2000 16 and 2009 00 as well as Species at Risk focal animal follows in 2007 and for two months in 2000. Act permits numbered 91 and 102. Sample collection methods Detection dog techniques were then implemented for one month were approved by the University of Washington's Institutional in 2000 and all of 2009. The second research team conducted Animal Care and Use Committee IACUCl although no permit focal animal follows exclusively. Focal animal follows were was required, because the research was non-invasive. conducted by following closely behind the whale, searching for scat floating in the fluke prints a series of calm circles of displaced Study area and Population water left after a whale surfaces and then submerges [19,20]. We The Salish Sea is an estuarine fjord habitat that supports a great confirmed the target whale's identity whenever possible using diversity of species,including many salmon populations. The May published photo-identification catalogs [61,62]. through October SRKW occurrence in the Salish Sea coincides Detection dog sampling was conducted using a modification of with annual Fraser River Chinook salmon migrations [47 49], previously published methods [63] for fecal sample collection in which comprise 80 90'/0 of the prey consumed by SRKWs during marine environments [64]. Use of a detection dog enabled us to the summer [20]. The Fraser River system includes multiple rivers sample at an average distance of 400 meters from the target and tributaries throughout British Columbia that eventually whale sl, minimizing any potential disturbance from the research converge and empty into the Strait of Georgia. Occasionally, vessel.The detection dog was selected for its obsessivedrive to play SRKWs are also observed in coastal waters near the mouths of the with a ball. Sample localization was paired with a brief <2 mini Sacramento River and the Columbia River, two other U.S.A. play reward with a ball. Once the dog associated sample west-coast river systems that currently support large Chinook localization with receipt of the reward, it would change its salmon populations [0,10]. However, these sightings most often behavior to an alert searching mode as soon as the target scent was occur during winter and early spring when sighting effort and diet detected. Training the dog on samples from a variety of data are both very limited. individuals taught the dog to generalize its alert response to scent The Center for Whale Research has maintained an annual common to all individuals of the target species [63]. photo-identification census of all the whales in the population, During sampling, the dog rode on the bow of the vesselwith the tracking age and life history stage for all individuals since the dog handler. The driver maneuvered the vessel in transects 1970s. The SRKW population is made up of three familial groups perpendicular to the wind and downwind from a group of whales or pods: J, K and L. Each individual is identified by a unique or the area that they previously swam through. When the vessel combination of saddle patch and dorsal fin morphology and is was in the cone of the scent emanating downwind of the floating designated alphanumerically with the letter representing its pod g, scat, the dog indicated sample detection by changing his behavior K, or Ll and the number its order of initial identification within from a relaxed sit or stand to leaning over the bow of the vessel the pod e.g., Jl; www.whaleresearch.coml. with tensed muscles, anticipating a reward. The dog maintained The three pods interact and interbreed with each other, but not this position as long as the scent concentration increased from low with other killer whale populations P,50 52]. Each pod is made to high. The dog alerted the handler as soon as the scent up of multiple matrilines a highly stable group of individuals concentration began to change from high to low concentration by linked by maternal descent P,53,54]. Neither males nor females standing erect and turning in the direction of the more disperse from their natal group P,53,55]. Maternal pedigrees are concentrated scent. The handler communicated this to the driver, well described through the annual census and many confirmed who made an en course correction confirmed by the dog's return through a combination of mitochondrial and microsatellite DNA to a tensed muscle position on the bow. As we got close to the scat, analyses [50,52,56]. Of the three pods, J pod spends the greatest the dog often stood up and began to whimper, presumably amount of time in the Salish Sea. because the scent was surrounding the vessel and he could no In 2011, the SRKW population consisted of about 88 longer follow a concentration gradient. Throughout this whole individuals www.whaleresearch.coml. In the 1960s and 1970s, process, crewmembers visually scanned for the sample floating on approximately 50 SRKW individuals were live-captured from the the water's surface. population for marine aquaria [10,57 59]. The population had Fecal samples were identified via appearance and odor. Killer recovered to pre-capture numbers by the early 1990s, but then whale feces are observed as clumped patches, having a mucousy experienced a near 20'/0 decline from 1995 2001 that could not and/or semi-cohesive texture. Killer whale feces are usually brown be explained by demographic effects from the live captures [60]. or green, but can also appear grey, yellow or orange. Samples The decline resulted from increased mortality across all sex/age often have a characteristic fishy odor that can be recognized with classesand several periods of low reproduction [4,0]. experience. We have observed SRKW fecal samples floating on the water's surface for up to 45 minutes. If scat is defecated below Sample Collections the surface or the surface tension is disturbed than the fecal pieces Collection of floating fecal samples occurred from May through sink. Once a sample was identified, it was collected with a scoop or October in the Salish Sea: Haro, Rosario, Juan de Fuca, and fine mesh net mounted on a telescoping pole. The scoop proved Georgia Straits as well as Swanson Channel and Boundary optimal for samples floating on the surface, because it minimized Passage. Additional samples were opportunistically collected in sample disturbance and provided better sample recovery com- November and December when some whales were in Puget pared to the net Ayres unpublished data!. Nets were more Sound, Washington, U.S.A. Two research teams were involved in effective for collecting samples below the surface. When samples fecal collections. The first utilized a 6 m fiberglass motorboat were collected with scoops, excesswater was carefully poured off. Boston ! and a 6 m fiberglass motorboat with an open bow The sample was then transferred to a 50 ml polypropylene screw-

'... PkoSONE l www.plosone.org June 2012 l Volume 7 l Issue 6 l e36842 Prey and VesselImpacts on Killer Whale Physiology top vial, promptly centrifuged at 1,000 rpm for approximately 5 emaciated, physiologically stressed adult male killer whale that minutes and the excesswater decanted from the fecal pellet. When stranded on the coast of Kauai, Hawai'i, expecting its GC collected with a net, the water was drained off and the sample concentration to be markedly elevated compared to that of adult transferred to the 50 ml polypropylene screw-top vial. Approxi- males in the SRKW population. A similar opportunistic challenge mately 2 5 sub-samples for separate DNA analyses were taken was not possible for thyroid hormone because we could not whenever possible with sterile cotton swabs or small pieces of ascertain the degree to which disease contributed to the whale's sterile gauze. All samples were stored on ice for up to 12 hours emaciation. while in the field, and then at 20'C upon return to the field station. Sub-samples were shipped on dry ice at the end of each DNA Analyses field season to the Center for for hormone DNA analyses were conducted on all fecal samples to confirm analyses and to NOAA, Northwest Fisheries Science Center for species,sex and individual identification at the Northwest Fisheries DNA analyses. All samples were stored until extraction at 20'C. Science Center, NOAA, in Seattle, Washington, USA. DNA extraction and analyses were performed according to previously Hormone Extraction and Radioimmunoassay published methods [52]. Species was confirmed by fragment In the lab, each sample was thawed once and centrifuged at length of 16 s ribosomal DNA. Sex was confirmed by amplifica- 2,200 rpm for 20 minutes. Excess salt-water was decanted from tion of the SRY and ZFX genes [69]. Individual identification was the fecal pellet, taking care not to lose the fecal pellet. The samples made by amplification of 26 polymorphic microsatellite loci, were then lyophilized for 48 hours in a Labconco FreeZone Freeze subsequently matched to other fecal and biopsy samples acquired Dry System. Samples were lyophilized prior to extraction and from known individual killer whales [52]. If a genotype could not hormone concentrations expressed per gram dry weight to control be matched to a known individual, the genotype was recorded and for inter-sample variation due to diet and variable moisture [65]. given a unique identification number, therefore, that unknown Freeze-dried fecal material was thoroughly mixed and up to 0.1 g individual could still be included in the analyses to control for weighed and transferred to a new 50 ml polypropylene screw-top pseudoreplication. Occasionally, unique genotypes could also be tube for extraction. Samples smaller than 0.02 g dried weight were assigned to pod, if there was only one pod in the area at the time of excluded from analysis to avoid inflation effects of low sample mass sampling. Thus, using genotypes we were able to track samples on hormone concentrations [66,67]. Fecal material was extracted that were from the same individual and sometimes identify the in 15 ml of 70'/0 ethanol according to previously published genotype to pod even if the identity of the individual could not yet methods P2], with one modification. The fecal pellet was only be determined. extracted once since previous validation showed very low hormone concentrations in the second extract for GCs and T3 in killer Prey and Vessel Traffic Measures whale samples Ayres unpublished data!. The extract was then Approximately 80 90'/0 of the SRKWs diet from May through stored at 20'C until hormone analysis. September is made up of Fraser River Chinook salmon [20]. Radioimmunoassay was performed to measure fecal hormone metabolitesusing ' I corticosteroneRIA kits +07-120103;MP Therefore, we compared changes in hormone concentrations over time with changes in the Department of Fisheries and ' Biomedicals, Costa Mesa, CAl and MP Biomedicals' Total T3 Fraser River Albion test fishery, which is the most consistent data coated tube assaykits +06-B254216l for GC metabolites and T3, set available to index relative availability of Fraser River Chinook respectively. The T3 assay was previously validated for killer salmon [70]. Data are reported as catch per unit effort CPUEl. whales P2]. The GC assay P I] was validated for killer whales in Chinook salmon CPUEs on days when the test fishery did not the present study see below!. Commercial controls from each operate were estimated by averaging the CPUE from the day prior assay kit were used to assessinter-assay coeflicients of variation. and the day after. Commercial T3 controls were prepared as previously described Vessel abundance was quantified using data collected by The P2]. Whale Museum's Soundwatch Boater Education Program. Observers count the total number of vessels observed within a Hormone Assay Validations half mile ca. 000 ml of any whale in view, at 30-minute intervals Standard parallelism and accuracy tests [68] were performed on during day light hours, with the aid of laser range finders to a pooled extract from 5 different killer whale fecal samples. measure distances [71]. Vessel data were gathered from May Parallelism tests compare the slope of a curve generated from through September in 2007 and 2000 and through October in serially diluted fecal extracts to that of the standard curve; parallel 2009. There is approximately a 24-hour lag time between slopes indicate that hormone metabolites are being reliably hormone secretion in blood and its excretion in feces in large measured across their range of concentration. Accuracy tests plot mammals P1,32,72], making the previous days' vesselcounts most concentrations of standards spiked with fecal extract against those relevant to hormone concentrations in a given sample. Therefore, of unspiked standards. A slope of 1.0, after adjusting for the added vessel traflic was averaged throughout a given day and compared hormone concentration in the added extract, indicates that to hormone concentrations from the following day. products in the extract are not interfering with antibody binding in the radioimmunoassay. Challenge experiments are also used in validation studies to Distinguishing between Inadequate Prey and Vessel assesswhether excreted hormone metabolites reflect biological Impacts activity. A tropic hormone e.g., adrenocorticotropic hormone for To test the inadequate prey and the vessel impact hypotheses as GC or thyroid stimulating hormone for T3l is injected to induce well as their potential interaction, we used general linear mixed secretion of the respective target hormone, which should then be effects models to test the effects of year, sex, pod, Fraser River measured as a significant increase in excretion of its metabolites in Chinook salmon counts and vessel abundance as main effects, and feces. We were unable to obtain permission to conduct such all two-way interactions of main effects on natural log transformed challenge studies on captive killer whales. So, we used the fecal GC and T3 concentrations. Individual differences were alternative of obtaining an opportunistic sample from a severely controlled in these analyses by including individual identity as a

'... PkoSONE l www.plosone.org June 2012 l Volume 7 l Issue 6 l e36842 Prey and VesselImpacts on Killer Whale Physiology random effect in the models. We also tested GC concentrations as not interfere with hormone binding. Inter-assay coefficients of a predictor variable for T3 and vice versa to test for inter-hormone variation for T3 and GCs were 14.6'/0 and 10'/0, respectively. effects. Intra-assay coefficients of variation for T3 and GCs were 1.9'/0 All statistical analyses were conducted using the model fit and 3 /0, respectively. application in the JMP 9 statistical package. Candidate models The opportunistic hormone challenge study showed the were compared using the R adjusted of the model [73], where the stranded male killer whale in Hawai'i had a fecal GC concentra- best-fit model was indicated by the highest R adjusted value. tion that was 27 times higher than the average male SRKW The Albion test fishery is approximately 140 km travel distance Figure Sll. This result suggeststhat fecal GC concentration is a from the west side of San Juan Island, the whales' primary feeding reliable index of biological activity. area where the majority of our samples were collected. We used a best-fit model to estimate the time lag from the date of SRKW Distinguishing between Inadequate Prey and Vessel fecal collection until the date the Chinook salmon were caught at Impacts the test fishery. As a cross-check, the best fit time lag was Figure 1 summarizes the annual and seasonalpatterns of Fraser compared to travel time for a fish to swim from prime whale River Chinook salmon CPUE, vesseltraffic, fecal GC and fecal T3 foraging grounds off the west side of San Juan Island [20] to the patterns from 2007 to 2009. Each variable was examined Albion test fishery based on documented Chinook salmon swim separately to assesshow it changed within and between years speeds multiplied by the distance traveled [74]. Both analyses during the study period. Raw data are presented in Figure 1 with indicated a 10-day time lag, and this was the lag we subsequently trend lines determined using general linear model selection based used in our analyses predicting hormone levels. on maximum likelihood model comparisons. Each variable was analyzed as a response to year, Julian date and higher orders of Addressing Pseudoreplication Julian date quadratic, cubic, etc.l along with their interactions On twelve occasions, multiple samples were collected from the Table Sll. FraserRiver Chinook CPUE wasbest fit by a 9' order same individual on the same day. For these twelve cases,hormone polynomial of Julian date across years Figure Ial. A 9th order concentrations were averaged between the samples or the largest, polynomial was necessary to capture the timings of multiple runs more representative sample was used for that individual on that day. of different Chinook subpopulations returning to the Fraser River through the Albion test fishery [70]. Fraser River Chinook CPUE Results varied markedly between years. Early season brunel Chinook CPUE was lowest in 2007, intermediate in 2000 and highest in Sampling 2009 Figure lal. Peaks in Chinook runs ca. Julian date 240 or We collected 154 fecal samples that were large enough August 2gthl were intermediate in 2007, highest in 2000, and >0.02 gl to be confidently assayed for hormone concentrations lowest in 2009. However, the width of the August peak was also see Methods!. Of these, 130 samples were successfully genotyped narrowest in 2000, followed by 2007, and broadest in 2009. for sex determination. Twice as many males as females were Mean vessel abundance in proximity to whales did not differ sampled in 2007, while males and females were sampled in significantly between years. The best-fit model for explaining roughly equal proportions in 2000 and 2009 Table Il. Of the 154 vessel patterns was a 3' order polynomial of Julian date samples, 113 were identified to pod. J pod, which is the most Figure Ibl. On any given day from June through September, frequently occurring pod in the Salish Sea, was sampled most often average vesseltraffic was consistently between 10 10 boats around Table Il, followed by K and then L pod. Pods were sampled in groups of whales Figure Ibl. Vessel traffic progressively increased similar proportions in 2000 and 2009. to its peakaround Julian day 230 August10'"l andthen steadily declined into the fall. Validations To explore temporal patterns in physiological stress, the entire Both corticosterone and T3 assaysexhibited excellent parallel- GC data set was used to test linear models of GCs as a response to ism; slopes of serially diluted extracts were not significantly year and Julian date Table Sll. Fecal GCs which index the different from the slopes of the standard curves GC: combination of acute psychological and nutritional stress! over F, i =0.41 p=0.54; T3: F, s 2.09, p = 0.12l. Fifty percent time was best predicted by a 2" order quadratic of Julian date binding of the radioactively labeled hormone occurred at target Figure I cl. GC concentrations were always intermediate when the dilutions of 1:240 for GC and 1:30 for T3 concentrations. Both T3 whales arrived in the spring, and Fraser River Chinook were and corticosterone assaysexhibited good accuracy at their target relatively low. GC concentrations progressively declined thereafter dilutions Linear regression; GC: slope = 1.2, R = 0.90; T3: slope untilJulian date 230 August18'"l approximately10 days before = 1.09 and R = 1.0l, indicating that substancesin fecal extract do the annual peak in Fraser River Chinook CPUE. GC concentra- tions increased from that point as salmon declined into the fall and Table 1. Distribution and percent of fecal samples winter. The highest observed GC concentrations occurred in successfully identified to sex and pod. November and December Figure Icl. Average annual GC concentrations were comparable across years after controlling for Julian date. Sex percent We tested for effects of prey and vessel traffic on GC identified! Pod percentidentified! concentrations by fitting fecal GC concentrations to Fraser River Chinook CPUE, vessel abundance, Julian date, sex, pod and fecal T3 concentrations, including individual identity as a random 2007 18 7/0! 9 3/0! 13 6/0! 3 0/0! 12 2/0! effect. The best-fit models are presented in Table 2, however more 269S.::.:f7.:: std%'!::.::::::1:7::t5P/ti'!:::::::::::::::::.::.::.::.::1.::7::.:6%'i!.::.::.::.::.::.::.::.:::7::.:5%!:.::.::.::.::.::3.:0:.: detailed model selection data'29%! can be found in Tables S2 and S3. 2009 37 8/0! 40 2/0! 25 9/0! 11 2/0! 15 9/0! Fecal GC concentrations were best modeled as a response to year, Fraser River Chinook CPUE with a 10-day time Iagl, vessel doi:10.1371/journal.pone.0036842.t001 abundance in proximity to whales and the interaction of prey and

'... PkoSONE I www.plosone.org June 2012 I Volume 7 I Issue 6 I e36842 Prey and VesselImpacts on Killer Whale Physiology vessel abundance Table 2; GC Top model A!. Chinook CPUE modeled as an additive response to sex, year and Julian date was the only significant main effect in this model, however less Table 2!. Females had significantly higher average T3 concen- variance was explained by the model if any of the other trations in the model, as St. Aubin et al. [75] also reported for parameters were removed. There was a highly significant negative bottlenose dolphins. The best-fit model showed a linear response relationship between GC concentrations and Fraser River of T3 to Julian date, after controlling for sex. T3 concentrations Chinook CPUE each year; GC concentrations consistently were highest when the whales arrived in the spring with a steady decreased as Chinook counts increased Figure 2a!. Both year decline into fall Figure 3!. Overall, T3 marginal means were and Chinook CPUE were significant if vessel abundance and its highest in 2007, intermediate in 2009 and lowest in 200B for any interactions were removed, with GCs being significantly lower in given day of the year horizontal lines in Figure 3!. Fraser River 2007 compared to 2009 Table 2; GC Top model B!. Sex, pod and Chinook CPUE, vessel abundance, pod and fecal GC concentra- fecal T3 concentrations did not improve any of the tested models. tions did not improve any of the tested models. Similar to GCs, temporal patterns in the entire T3 data set were examined in response to year and various orders of Julian date Discussion Table Sl!. Fecal T3 presumed to index long-term nutritional The temporal pattern in GC concentrations closely corresponds status! was best fit by a third order quadratic of Julian date across to relative Fraser River Chinook salmon counts from the time years Figure ld!. T3 concentrations were consistently highest SRKW first arrive in the Salish Sea. This pattern appeared to indicating relatively good nutrition! in the spring when the whales result from a rapid GC responsiveness to prey availability. GC arrived in the Salish Sea Figure 1 d!. T3 concentrations concentrations reached their nadir when Fraser River Chinook progressively declined from time of arrival until Julian date 230 salmon lagged by 10 days! peaked at the test fishery even though August 1B' ! followedby a slightbut sustainedupturn that began vessel abundance was also peaking around this time. GC coincident with the Fraser River Chinook salmon peak Figure la!, concentrations then progressively rose to their highest levels of but never rose to the spring arrival levels within any given year. T3 the year as Fraser River Chinook salmon declined, even though concentrations then progressively declined into the late fall/early vessel numbers in proximity to the whales also markedly declined. winter. SRKW arrived with the highest T3 concentrations in When prey and vesselabundance indices were tested directly, GCs 2007, but also showed the greatest percent decline over the entire were significantly correlated with Fraser River Chinook salmon. study season in that year. Mean T3 was lowest in 200B compared Vessel abundance and its interaction with prey improved model to 2007 and 2009. Although 200B had the highest peak in Chinook performance as indicated by the amount of variance explained, CPUE, the 200B peak was also the narrowest Figure la!. however neither vessel abundance or the interaction were To test for effects of prey and vessel traffic on T3 concentra- significant as parameters within the model. When vessel abun- tions, the T3 data set was restricted to the tested predictor dance and its interaction was removed from the model, year variables: Fraser River Chinook CPUE, vessel abundance, Julian became significant, with 2007 the year with the lowest average date, sex, pod and fecal GC concentrations, with individual ID salmon counts being lower than 2009. This suggests that some included as a random effect. Fecal T3 concentrations were best combination of year, salmon availability and vessel abundance

Table 2. ]3est-fit general linear mixed effects models explaining southern resident killer whale fecal glucocorticoid GC! and triiodothyronine ff3! concentrations.

Model Response Parameter Estimate SE P R Adj 1'nfl'sl 8 1.::.::.::.:..:..::.:::::..:Year..:f2667I:.::.:::.::::.::.::.:..::..::.:::.::.::.::.::.::.::.::.::.::.::..::.::.::.:..::.::.::::.::.:..::.9:,:3 1.::.::.::.::.::.:.::::.:::.::..:::.::.::.::.:..::.::9'A3:.::.::.::.::.::.::.::.::.::.::.::.::.::.:6]39.::.::.::.::.::.::.:..:::::::::::::::6:75 top modelA Year008] 0.11 0.35 0.65 Cbiimok:.::sal'mo'n: i:9-:Chy'..:.ta'6].::.::.::.::.::.::.::.:::.::.::.::.::::::9:,42:.::.::.::.::.:::.:..::.:::.::.::.::.::.:..::.::.::9::.1.:2:.::.::.::.::.:..:..:..:..:..:..:..:..:..:.<6 091.:as Vesselabundance 0.02 0.02 0.44 Carinyoyoyk:.:k:Vers'sets:.:::::::::.::::::::::::::::::::::::::::::.::.::.9:.62::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::.::9:.62:.::.::.::.::.::.::.::.::.::.::.::.::.::.:6]29 Individual Random!

GC In GCs! 81 Year007] 0.63 0.29 0.04~ 0.71

Chinooksalmon 0.37 0.11 <0.01~ 0-day lag! I'n'div'I'd'u'at:.:]]t'a'odom!

In T3! 79 sex Female] 25.62 11.58 <0.03~ 0.51 top:.::rIIodeI::A Year008] 59.40 20.80 <0.01~ J6]fan.:.:date Individual Random!

doi:10.1371/journal.pone.0036842.t002

'... PLoSONE ~ www.plosone.org June 2012 ~ Volume 7 ~ Issue 6 ~ e36842 Prey and VesselImpacts on Killer Whale Physiology

5 CD 4 V! C 3

0 O 7 0 O 8 0 O 5 U O D U C 8

0 1 2 3 4 5 10 15 20 25 30 35 40 45

Chinook Salmon CPUE! Vessel Abundance ¹ of boats!

Figure 2. Physiological stress correlates with year, Chinook availability, vessel abundance and an interaction between Chinook and vessel abundance. According to the best-fit mixed effects model, glucocorticoid concentrations decreasedwith increasedChinook salmon CPUE, after taking into account a 10-day lag time for fish to swim from the study site to the test fishery column A!. The best-fit model also includes an interaction between Chinook counts and vesselabundance on glucocorticoids,whereby fecal glucocortiods are always high at times of low Chinook counts. However,an increasein glucocorticoids with increasingvessel abundance is observed only during times of relatively high Chinook counts column 8 set to the Chinook value indicated by the vertical line in the corresponding panel of column A!. The y-axis represents glucocorticoid concentration marginal means predicted from the best-fit model. The hashed blue lines indicate 95% confidence intervals.Vertical red dotted lines indicate Julian day 230 August 18!,the time of maximum vesseltraffic and approximately ten days before the maximum Chinook salmon catch each year. Horizontal red dotted lines indicate dependent variable marginal means for each year on day 230 within the model. doi:10.1371/journal.pone.0036842.g002 may be required to fully explain variance in GCs, however our there was a slight increase in T3 starting in August, roughly sample size may have had insuAicient statistical power to coincident with the peak in Fraser River Chinook salmon, but T3 demonstrate that. concentrations never reached those observed when the whales first In contrast to GCs, T3 concentrations were not directly arrived in the Salish Sea. The estimated T3 decline with Julian correlated with Fraser River Chinook counts, but were instead date was linear when the data were restricted by sex, potentially associated with Julian date. Temporal patterns in T3 concentra- due to the restricted sample size that limited the statistical power to tions across years indicate that SRKW nutrition is consistently show a higher order relationship. highest when the whales first arrive in the Salish Sea during The temporal trend in T3 concentrations within and between spring/early summer. Since Fraser River salmon counts were years suggest that the sampled SRKWs might be feeding on a relatively low at that time, this suggests that the SRKW may nutritious early spring food source acquired prior to their arrival in consistently be foraging on an early spring, nutrient-rich food the Salish Sea. The trend further suggeststhat the whales become source just prior to their late spring arrival in the Salish Sea. T3 somewhat food limited during the course of the summer. This concentrations progressively declined from the time of SRKW result is somewhat unexpected, because the more confined arrival until the whales' late fall/early winter departure. When the waterways of the Salish Sea, combined with large runs of salmon entire data set was used and did not include sex as a parameter, returning through the area would seem to provide easier foraging

'... PLo5ONE l www.plosone.org June 2012 l Volume 7 l Issue 6 l e36842 Prey and VesselImpacts on Killer Whale Physiology

2007 2008 2009 6.5

C 6

5.5

cr cr 5

V 4.5

i: 4 120 140 160 180 200 220 240 260 120 140 160 180 20D 220 240 260 120 140 160 180 20D 220 240 260 280 Day of the Year

Figure 3. Nutrition correlates with sex, year and Julian date. Nutritional status, indexed by fecal triiodothyronine concentrations,is highest when the southern resident killer whales return to the SalishSea in the spring and declines throughout the summer into the fall and winter. The y- axis representstriiodothyronine concentration marginal means predicted from the best-fit mixed effects model after controlling for individual and sex.The hashed blue lines indicate 95'/o confidence intervals. Vertical red dotted lines indicate Julian day 230 August 18!, the time of maximum vessel traffic and ten days before maximal FraserRiver Chinook salmon catch each year. Horizontal red dotted lines indicate dependent variable marginal means for each year on day 230 within the model. doi:10.1371/journal.pone.0036842.g003 opportunities for the whales than the outer coast. Nonetheless, the and lowest number of births and surviving calves observed during declining trend in T3 levels at least suggeststhe possibility that the our three-year study. Eight whales went missing from December of early spring period when the whales are typically in coastal waters 2007 through October 2008, two of which were reproductive age might be a more important foraging time than was previously females Center for Whale Research unpublished data! and included believed. a visually emaciated pregnant female L67; Ayres et al. in The spring range of SRKW is not well defined but available preparation!. Loss of multiple reproductive age females is uncom- information indicates that their range includes the coastal waters mon in long-lived mammals and is particularly detrimental to of California, Oregon, Washington and British Columbia [8, population recovery in a population of this size. It is also noteworthy NMFS unpublished data]. Several stocks of Chinook occur in that while the Fraser River Chinook salmon peak in 2008 had the these coastal waters in the spring [76]. Some of the most abundant highest amplitude of the three study years, the peak was relatively Chinook stocks available to the whales in the spring are the brief Figure la!. Perhaps this brief pulse in relative fish availability Columbia River spring Chinook [77], and if the whales are during 2008 overwhelmed the predator, actually making a relatively foraging on these stocks, that may contribute to the elevated spring small proportion of the total fish returns accessibleto the whales that T3 concentrations prior to the whales' arrival in the Salish Sea. year. Consistent Chinook availability throughout the season, as These early spring Chinook are "interior race" salmon known to occurred in 2009 Fig. la!, may be much more important to SRKW have particularly high fat content to sustain their long spawning sustained nutrition compared to high numbers of fish that are only migrations upstream to interior river systems [78,79]. In contrast available for a short period of time. to the summer period, direct observation of the coastal feeding Oritz et al. [8 I] found that captive bottlenose dolphins events are very limited. However, the available information does responded to a 38 hour fast by elevating lipid metabolism to suggest that the whales may be feeding on Columbia River spare lean tissue. They observed an initial decline in serum T3 salmon. In particular, the only scale samples collected from followed by recovery, although the trend was not significant. foraging killer whales off the Washington coast in March were There was, however, an increase in biologically inactive reverseT3 interior Columbia River Chinook salmon n = 2; Hanson unpub- rT3! by 38 hours, suggesting that such conversion to rT3 may lished data!. In addition, some SRKWs have been observed protect dolphins from excess cellular metabolism during caloric foraging near the mouth of the Columbia in late March, when the restriction. Our results suggest that more sustained periods of spring run Chinook salmon stocks return to the Columbia River reduced food availability in SRKW likely results in a lowering of [80]. Our results therefore reinforce the importance of gaining a basal T3, which is probably a critical strategy for conserving better understanding of the whale's diet during this potentially energy and slowing the need for lipid metabolism. Such a strategy important time period, and suggest the possibility that these may be crucial during sustained periods of food decline, given the spring-run stocks might be of particular importance for the importance oflipids as a long-term energy store in addition to their nutrition of this population. importance in buoyancy and thermoregulation. The end of 2007 through 2008 appeared to represent the poorest Despite previous reported pod differences in movement patterns overall nutritional state of the SRKW population during our three- and the locations of prey consumed in the winter and early spring year study. The whales left the Salish Sea in 2007 following the most [28], including pod as a predictor variable did not improve any of precipitous T3 decline and GC elevation over the three years the models we tested. While these preliminary analyses do not Figure lc and Id!. Their T3 concentrations upon arrival in late indicate a significant difference in physiological trends between spring 2008 were the lowest observed during that time of year over pods, J pod was represented more often in our data than K and L the three-year study period and remained low throughout 2008. pods, suggesting that more data may be needed to address pod This period also corresponded with the highest number of deaths differences in physiology.

'... PkoSONE ~ www.plosone.org June 2012 ~ Volume 7 ~ Issue 6 ~ e36842 Prey and VesselImpacts on Killer Whale Physiology

Our findings that glucocorticoids are correlated with an index of average!, indicating stress-induced adrenal activation. Similar Chinook salmon availability are consistent with studies indicating results from a tangled in a fishing net were observed a high percentage of Chinook salmon in the SRKW diet by Hunt et al 006!. [19,20,02] as well as correlations of SRKW demographic trends TIFF! with coast-wide indices of Chinook abundance [17,18]. Our Table Sl Best-fit general linear models testing annual results suggest that prey availability has a greater physiological and seasonal patterns in Fraser River Ghinook salmon, impact on SRKWs than does vessel traffic. However, we cannot vessel traffic, fecal glucocorticoid and triiodothyronine yet rule out a cumulative effect of vessel traffic on the overall concentrations. SRKW stress response, particularly during years of relatively low Fraser River Chinook abundance. Exposure to toxicants may also DOC! add to these cumulative effects if food deprivation promotes Table S2 Model comparisons for the final set of mixed metabolism of lipid stores, releasing sequestered toxicants into effects models tested to explain fecal glucocorticoid GG! circulation. Combined, these results suggest that promoting concentrations. salmon recovery is vital to the long-term persistence of SRKW. DOC! Conservation of early spring salmon runs consumed by SRKW prior to arrival in the Salish Sea may be especially important to Table S3 Model comparisons for the final set of mixed these recovery efforts. Future studies should aim to better identify effects models tested to explain fecal triiodothyronine these early spring food sources to better target recovery efforts. T3! concentrations. It is a modern reality that anthropogenic impacts and DOC! are forever intertwined. As anthropogenic disturbances continue to affect wildlife, it is important for conservation biologists and Acknowledgments managers to prioritize mitigation efforts. To this end, conservation Our thanks go to Gregory Schorr, David Ellifrit, Heath Smith, D. Giles, biologists need tools that better clarify anthropogenic and Erin Heydenreich,Lynn Minor, JessicaMorten, Nicole Brandt, Robert ecological impacts on the health of endangered populations before Ottis, Adam U, Kylie Nulty, MacKenzie Consoer, Noah Wasser,Jeff devastating demographic incidents occur. This study shows that Hogan, Kathleen Gobush, Pema Kitaetf, Bud Marks, Ron Bates, the combining GC and T3 hormone measures enables investigators to Centerfor Whale Research,Friday Harbor Labs,the Whale Museumand partition the relative impacts of psychological and nutritional the SoundwatchBoater Educanon Program, Straitwatch Boater Education stressors, along with their short versus long-term metabolic Program,the whale watch operatorsand multiple volunteersthat assisted consequences. As such, these combined tools offer more timely with field work and provided field support. Thank you to Kenneth evaluation of anthropogenic disturbances, their ecological signif- Balcomb III for the generoususe of his permit and boat. Thank you to Kim Parsonsfor help with DNA analyses.Thank you to Krisn West and icance and provide means to monitor the success of mitigation BrendaJensen of Hawai'i Pacific University'sMarine Mammal Response efforts in free-ranging vertebrates. Network for providing the samplefrom the strandedkiller whale. Thank you to James Ha, Fred Felleman,Laurie Weitcamp, Dawn Noren, Lisa Disclaimer Hayward, Jennifer White, JessicaLundin, Kathleen Gobush, Jennifer The findings and conclusions in this article are those of the Ruesink,Walton Dickotf, andJim Kenagyfor providing valuablefeedback author s! and do not necessarily represent the views of the National on the study and this manuscript. Special thanks go to Tucker, the Oceanic and Atmospheric Administration. ConservationCanine, for his keen noseand assistancewith non-invasive fecal sampling. Supporting Information Author Contributions Figure Sl Biological relevance of fecal glucocorticoids Conceived and designedthe experiments:KLA SKW. Performed the in a stranded killer whale. All genetically confirmed male experiments:KLA RKB JAH. Analyzedthe danu KLA SKW RKB JAH Southern resident killer whale fecal glucocorticoid concentrations MJF. Contributed reagents/materials/analysistools: KLA SKW CKE KK n = 36! were compared to a killer whale that stranded in Hawai'i. MBH RWB MJFJAH KBB. Wrote the paper: KLA SKW MJF. Provided The killer whale was severely emaciated and later euthanized. The significantefforts in field work and data collection:MBH CKE RWB KBB stranded killer whale had exceptionally high fecal glucocorticoid KK. Providedcomments and feedbackon the manuscript:RWB KK CKE concentrations ca. 28 times higher than the male SRKW MBH. Providedsignificant field support and assistance:KBB KK.

References WikelskiM, CookeSJ 120061 Conservation physiology. Trends Ecol Evol 21: 7. BairdRW 120011Status of killerwhales, Ornnus orra, in Canada.Can Field Nat. 38 46. 115:676 701. BiggM 119821An assessmentof killer whale I,Oinnus oira! stocks off Vancouver 8. Krahn MM, FordMJ, PerrinWF, WadePR, Angliss RP, et al. 1200412004 Island,British Columbia. Report of theInternational Commission 32: Statusreview of SouthernResident killer whales Ornnusorra! under the 655&66. EndangeredSpecies Act. U.S. Departmentof Commerce,NOAA Technical FordJKB, EllisGM, BalcombKC 120001Killer whales: The natural history and Memo,National Marine Fisheries Service- Northwest Fisheries Science Center, genealogyof Ornnusorra in BritishColumbia and WashingtonState. Second Seattle,Washington. edition.Vancouver: University of BritishColumbia Press and Seattle:U. of 9. Fisheries,Oceans Canada 120081 Recovery Strategy for the Northernand WashingtonPress. 102 p. SouthernResident Killer Whales Ornnus orra! in Canada.DFO, Ottawa, KrahnMM, WadePR, Kalinowski ST, Dahlheim ME, TaylorBL, et al. 120021 Canada. StatusReview of SouthernResident killer whales Ornnus orra! under the 10. NationalMarine Fisheries Service 120081 Recovery Plan for SouthernResident EndangeredSpecies Act. U.S. Departmentof Commerce,NOAA Technical Killer WhalesI, Ornnus orra!. National Marine FisheriesService, Northwest Memo,National Marine Fisheries Service-Northwest Fisheries Science Center, Region,Seattle, Washington. Seattle,Washington. I l. WilliamsR, LusseauD, HammondPS 120061 Estimating relative energetic costs JeffersonTA, StaceyPJ, Baird RW 119911A reviewof killerwhale interactions of humandisturbance to killerwhales I,Ornnus orra!. Biol Conserv 133: 301 311. with othermarine mammals: predation to co-existence.Mammal Review 21: 12. WilliamsR, AsheE 120071Killer whaleevasive tactics vary with boatnumber. 151 180. J Zool 272:390 397. FordJKB, EllisGM 119991Transients: mammal- hunting killer whales of British Columbia,Washington, and southeastern Alaska. Vancouver: UBC Press. 96 p.

'... PLoSONE l www.plosone.org 10 June 2012 l Volume 7 l Issue 6 l e36842 Prey and VesselImpacts on Killer Whale Physiology

13. Holt MM, NorenDP, Veirs V, EmmonsCK, VeirsS 008lSpeaking up: Killer 42. GerisKL, BergmanLR, Kuhn ER, DarrasVM 999l The drop in plasma whalesI,Ornnui orza! increase their callamplitude in responseto vesselnoise. thyrotropinconcentrations in fasted chickens is causedby an actionat thelevel J AcoustSoc Amer 125:EL27 EL32. of thehypothalamus: role of corticosterone.Domest Anim Endo16: 231 237. 14. NorenDP, Johnson AH, RehderD, LarsonA 009l Closeapproaches by 43. KitayskyAS, RomanoMD, PiattJF, WingfieldJC, Kikuchi M 005l The vesselselicit surface active behaviors by southernresident killer whales. Endang adrenocorticalresponse of tuftedpuffin chicksto nutritionaldeficits. Horm SpRes 8: 179 192. Behav47: 606 619. 15. WilliamsR, NorenDP 009l Swimmingspeed, respiration rate, and estimated 44. WalpitaCN, GrommenSVH, DarrasVM, Van der GeytenS 007l The costof transportin adultkiller whales. Mar MammalSci 25: 327 350. influenceof stresson thyroidhormone production and peripheral deiodination 16. WilliamsR, BainDE, SmithJC, LusseauD 009l Effectsof vesselson behavior in theNile tilapia.Gen Comp Endocr 150: 18 25. patternsof individualsouthern resident killer whalesOrnnus orca. Endang Sp 45. RomeroLM 004l Physiologicalstress in ecology:lessons from biomedical Res6: 199 209. research.Trends Ecol Evol 19l: 249 255. 17. FordJKB, Ellis GM, OlesiukPF, BalcombKC 009l Linkingkiller whale 46. NelsonRJ 005l An introductionto behavioralendocrinology " edl. survivaland prey abundance:foodlimitation in the oceans'apex predatory Sunderland:Sinauer Associates. 822 p. BiologyLetters 6: 139 142. 47. BalcombKC III, BoranJR, OsborneRW, HaenelNJ 980l Observationsof 18. Ward EJ, HolmesEE, BalcombKC 009l Quantifyingthe effectsof prey killerwhales I,Ornnui orza! in greaterPuget Sound, State of Washington.Report abundanceon killerwhale reproduction. J Appl Ecol46: 632 640. MMI300731 7, U.S.Marine Mammal Commission, Washington, D. C p. 19. FordJKB, EllisGM 006l Selectiveforaging by fish-eatingkiller whales Ornnui 48. Heimlich-BoranJR 986l Fisherycorrelations with the occurrenceof killer orzain BritishColumbia. Marine Ecol Progress Series 316: 185 199. whalesin GreaterPuget Sound. In: KirkevoldBC, Lockard JS, eds. Behavioral 20. HansonMB, Baird RW, Ford JKB, Hempelmann-Halos J, Van Doornik DM, et biologyof killerwhales. New York: Alan R. Liss,New York. Pp. pp 113 131. al. 010l Speciesand stockidentification of prey consumedby endangered 49. FeflemanFL, Heimlich-BoranJR, OsborneRW 991l Thefeeding ecology of southernresident killer whalesin their summerrange. Endang Sp Res11: killerwhales I,Ornnui orza! in thePacific Northwest. In: PryorK, NorrisKS, eds. 69 82. Dolphinsocieties: discoveries and puzzles.Berkley: University of California 21. MyersJM, et al. 998l Statusreview of chinooksalmon from Washington, Press.pp 113 147. Idaho, Oregonand California.NOAA TechnicalMemorandum NMFS- 50. HoelzelAR, DahlheimM, SternSJ 998l Lowgenetic variation among killer NWFSC-35. whalesI,Ornnui orza! in the easternnorth Pacificand geneticdifferentiation 22. GustafsonRG, WaplesRS, Myers JM, WeitkampLA, BryantGJ, et al. 007l betweenforaging specialists. J Heredity 89: 121 128. Pacificsalmon extinctions: Quantifying lost and remainingdiversity. Conserv 51. Barrett-LennardLG, Ellis GM 001l Populationstructure and genetic Biol 21: 1009 1020. variabilityin northeasternPacific killer whales:towards an assessmentof 23. ParsonsKM, BalcombKC III, FordJKB, DurbanJW 009l The social populationviability. ResearchDocument 2001/065, Canadian Science dynamicsof the southernresident killer whalesand implicationsfor the AdvisorySecretariat, Fisheries and Oceans Canada, Ottowa, Ontario. conservationof thisendangered population. Anim Behav77: 963 971. 52. FordMJ, Hanson MB, HempelmannJA, AyresKL, EmmonsCK, et al.011l 24. FosterEA, FranksDW, MorreflLJ, BalcombKC, ParsonsKM, et al. 012l Inferredpaternity and male reproductive success in a kiflerwhale Orcinus orcal Socialnetwork correlates of food availabilityin an endangeredpopulation of population.J Hereditydoi:10.1093/jhered/esr067. killerwhales, Orcinus orca. Anim Beh83: 731 736. 53. BairdRW 000l Thekiller whale: foraging specializations and group hunting. 25. RossPS, Eflis GM, IkonomouMG, Barrett-LennardLG, AddisonRF 000l In: MannJ, ConnorRC, TyackPL, Whitehead H, eds.Cetacean societies: field High PCBconcentrations in free-rangingPacific killer whales,Orcinus orca: studiesof dolphinsand whales.Chicago: University of ChicagoPress. pp Effectsof age,sex and dietary preference. Mar PoflutBull 40:504 515. 127 153. 26. RossPS 006l Fireproofkifler whales Orcinus orcal: flame-retardant chemicals 54. Ford JKB 002l Killer whale Ornnuiorza. In: Perrin WF, Wursig B and the conservationimperative in the charismaticicon of BritishColumbia, ThewissenJGM, eds.Encyclopedia of marine mammals. San Diego: Academic Canada.Can.J. Fish. Aquat. Sci. 63: 224 234. Press.pp 669&76. 55. BiggMA, OlesiukPF, Ellis GM, FordJKB, BalcombKC III 990l Social 27. KrahnMM, HansonMB, BairdRW, Boyer RH, BurrowsDG 007l Persistent organizationand genealogy of residentkiller whales I,Ornnui orza! in the coastal organicpollutants and stableisotopes in biopsysamples 004/2006l from watersof BritishColumbia and Washington State. Report of the International SouthernResident killer whales. Mar PoflutBull 54: 1903 1911. WhalingCommission, Special Issue 12: 383 405. 28. KrahnMM, HansonMB, SchorrGS, Emmons CK, BurrowsDG, et al.009l 56. Pilot M, DahlheimME, HoelzelAR 010l Socialcohesion among kin, gene Effectsof age,sex and reproductivestatus on persistentorganic pollutant flow withoutdispersal and the evolutionof populationgenetic structure in the concentrationsin "SouthernResident" kifler whales.Mar Poflut Bull 58: killerwhale Orcinusorcal. J EvolBio 23:20 31. 1522 1529. 57. BiggMA, WolmanAA 975l Live-capturekiller whale Ornnuiorza! fishery, 29. AguilarA, BorreflA, PastorT 999l Biologicalfactors affecting variability of BritishColumbia and Washington,1962 73. J Fish ResBoard of Can 32: persistentpollutant levels in cetaceans.JCetacean Res Manage, Special Issue 1: 1213 1221. 83 116. 58. OlesiukPF, Bigg MA, EllisGM 990l Life historyand population dynamics of 30. O'Shea TJ 999l Environmentalcontaminants and marinemammals. In: residentkiller whales I,Ornnui orza! in thecoastal waters of BritishColumbia and ReynoldsIII JE, RommelSA, eds. Biology of marinemammals. Washington WashingtonState. Report of the InternationalWhaling Commission, Special D.Cu SmithsonianInstitution Press. pp 485 563. Issue12: 209 243. 31. WasserSK, Hunt KE, BrownJL, CooperK, CrockettCM, et al. 000l A 59. Wiles GJ 004l WashingtonState StatusReport for the Kifler Whale. generalizedfecal glucocorticoid assay for usein a diversearray of nondomestic WashingtonDepartment of Fishand Wildlife, Olympia, Washington. mammalianand avian species. Gen Comp Endocr 120: 260 275. 60. Taylor M 004l Southernresident orcas: population change, habitat 32. WasserSK, Azkarate JC, BoothRK, HaywardL, Hunt KE, et al.010l Non- degradationand habitat protection. Report number SC/56/E32, International invasivemeasurement of thyroid hormone in fecesof a diversearray of avian WhalingCommission, Cambridge, United Kingdom. andmammalian species. General and Comparative Endocrinology 168: 1 7. 61. Eflifrit D, BalcombKC IH, van GinnekenAM 006l Officialorca survey: 33. WasserSK, KeimJL, TaperML, SubhashRL 011l The influencesof wolf Photo-identificationguide to orcawhales of the Southernresident community predation,habitat loss, and human activity on caribouand moose in theAlberta Spring2006 edition. Friday Harbor: Center for WhaleResearch. oil sands.Front Ecol Environ doi:10.1890/100071. 62. Eflifrit D, BalcombKC III, Balcomb-BartokK 008l Officialorca survey: 34. HaywardLS, Bowles AE, Ha JC, WasserSK 011l Impactsof acuteand long- Naturalist'sfamily tree guide to orca whalesof the Southernresident termvehicle exposure on physiologyand reproductive success of the northern community,2008 First Edition. Friday Harbor: Center for WhaleResearch. spottedowl. Ecosphere 2l: 1 20. 63. WasserSK, DavenportB, RamageER, Hunt KE, ParkerM, et al. 004l Scat 35. WeissJM 968l Effectsof copingresponses on stress.J Comp Physiol Psychol detectiondogs in wildliferesearch and management: application to grizzlyand 65: 251 260. blackbears in the YeflowheadEcosystem, Alberta, Canada. Can J Zoo 82: 36. LevineS, Goldman L, CooverGD 972l Expectancyand the pituitary adrenal 475W92. system.Ciba Found. Symp. 8: 281 291. 64. RoflandRM, HamiltonPK, KrausSD, Davenport B, GiflettRM, et al. 006l 37. SapolskyRM, RomeroLM, Munck AU 000l How do glucocorticoids Faecalsampling using detection dogs to studyreproduction and health in North influencestress responsesg Integrating permissive, suppressive, stimulatory, and Atlanticright whales Eubalaena glacialisl.J Cetacean Res Manage 8: 121 125. preparativeactions. Endo Rev 21: 55 89. 65. WasserSK, ThomasR, Nair PP,Guidry C, SouthersS, et al. 993l Effectsof 38. FlierJ, HarrisM, HoflenbergAN 000l Leptin,nutrition, and the thyroid: the dietaryfiber on faecalsteroid measurements. J Reprod Fertil 97: 569 574. why,the wherefore, and the wiring. J Clin Invest105: 859 861. 66. Miflspaughg, WashburnBE 004l Use of fecalglucocorticoid metabolite 39. DouyonL, SchteingartDE 002l Effectof obesityand starvationon thyroid measuresin conservationbiology research: considerations for applicationand hormone,growth hormone, and cortisol secretion. Endo and Metab Clin N Amer interpretation.Gen Comp Endocr 138: 189 99. 31: 173 189. 67. HaywardLS, BoothRK, WasserSK 010l Eliminatingthe artificialeffect of 40. EalesJG 988l Theinfluence of nutritionalstate on thyroidfunction in various samplemass on avianfecal hormone metabolite concentration. Gen and Comp vertebrates.Amer Zoo 28:351 362. Endocr169: 117 122. 41. SchewWA, McNabb FMA, Scanes CG 996l Comparisonof theontonogenesis 68. DiamandisEP, Christopoulos, TK 996l Immunoassay.San Diego: Academic of thyroidhormones, growth hormone, and insulin-likegrowth factor-I in ad Press.579 p. libitumand food-restricted altriciall European starlings and precociallJapanese 69. RoselPE 003l PCR-basedsex determinationin Odontocetecetaceans. quail.Gen Comp Endo 101: 304 316. ConservGenet. 4: 647 649.

'... PLoSONE ~ www.plosone.org June 2012 ~ Volume 7 ~ Issue 6 ~ e36842 Prey and VesselImpacts on Killer Whale Physiology

70. ParkenCK, CandyJR, IrvineJR, BeachamTD 008l Geneticand coded wire 77. PacificFishery Management Council 011l Reviewof 2010Ocean Salmon tagresults combine to allowmore-precise management of a complexChinook Fisheries. Document prepared for theCouncil and its advisoryentities.! Pacific salmonaggregate. N AmJ FishManage 28: 828 840. FisheryManagement Council, 7700 NE AmbassadorPlace, Suite 101, Portland, 71. KoskiK 010l 2010Soundwatch Program Annual Contract Report. Sound- Oregon97220 1884. watchPublic Outreach/Boater Education Project, The Whale Museum, Friday 78. BrettJR 995l Energetics.In: C. Groot,L. Margolis,and W.C. Clarke, editors. Harbor, Washington.Contract report submittedto U.S. Departmentof PhysiologicalEcology of PacificSalmon. Vancouver: UBC Press,8&8. Commerce,NOAA, National Marine Fisheries Service, Seattle, Washington. 79. MesaMG, MagicCD 006l Evaluationof energyexpenditure in adultspring 72. WasserSK, Monfort SL, SouthersJ, Wildt DE 994l Excretionrates and Chinook salmonmigrating upstream in the ColumbiaRiver basin: an metabolitesof oestradioland progesterone in baboon Papto riinorephalus! faeces. assessmentbased on sequentialproximate analysis. River Res Appl 22: J ReproFertil 101:218 220. 1085 1095. 78. HurvichCM, Tsai CL 989l Regressionand time seriesmodel selection in 80. ZamonJE, Guy TJ, BalcombK, Ellifrit D 007l Winter observationsof smallsamples. Biometrika 76: 297 807. southernresident killer whales Orcinus orcal near the ColumbiaRiver plume 74. CandyJR, QuinnTP 999l Behaviorof adultchinook salmon Oncorhynchus duringthe 2005spring Chinook salmon Oncorhynchus tshawytschal spawning tshawytschalin BritishColumbia coastal waters determined from ultrasonic migration.Northwestern Naturalist 88: 198 198. telemetry.Can J Zoo 1169:1161 1169. 81. Ortiz RM, LongB, CasperD, Ortiz CL, WilliamsTM 010l Biochemicaland 75. St. Aubin DJ, RidgwaySH, WellsRS 996l Dolphinthyroid and adrenal hormonalchanges during acute fasting and re-feedingin bottlenosedolphins hormones:circulating levels in wild andsemidomesticated Tursiops truncatus, andinfluence of sex,age and season. Mar MammalSci 12:1 18. Tursiopstruncatusl. Mar MammalSci 26: 409WI9. 76. WeitcampLA 010l Marinedistributions of Chinooksalmon from the west 82. FordJKB, EllisGM, Barrett-LennardLG, MortonAB, PalmRS, et al. 998l coastof NorthAmerica determined by codedwire tag recoveries. T Am Fish Soc Dietaryspecialization in two sympatric populations of killerwhales I,Ornnui orra! 189:147 170. in coastalBritish Columbia and adjacent waters. Can J Zoo 76: 1456 1471.

'... PLoSONE ~ www.plosone.org 12 June 2012 ~ Volume 7 ~ Issue 6 ~ e36842