Blubber stratification in white whales and killer whales: variability in ...

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Blubber stratification in white whales and killer whales: variability in contaminant concentrations, fatty acid profiles, lipid percent and lipid class profiles that has implications for biopsy sampling M ARGARET M. KRAHN*, D AVID P. HERMAN*, G INA M. YLITALO*, C ATHERINE A. SLOAN*, D OUGLAS G. B URROWS*, RODERICK C. H OBBS+, B ARBARA A. M AHONEY# , GLADYS K. YANAGIDA * AND JOHN C ALAMBOKIDIS ¥ Contact e-mail: [email protected] . ABSTRACT The biopsy—either via dart or surgery—is becoming the preferred protocol for sampling many cetacean species, so it is essential to compare the results obtained from biopsy samples to those found by analyzing a ull-thickness f blubber sample. This manuscript takes a broad look at blubber stratification in two important odontocete species—white whales (Delphinapterus leucas) and killer whales (Orcinus orca). The results of this study—in which five parameters (i.e., lipid percent and classes, contaminant concentrations and profiles, fatty acid profiles) were measured by blubber depth—demonstrate that blubber biopsy techniques seldom result in samples that give information completely representative of that obtained from full-thickness blubber samples obtained via necropsy. Thus, biopsy results are best interpreted with caution and in conjunction with results from blubber depth profiling on the same cetacean species. Often the biopsy is the only means of obtaining samples (e.g., for threatened or endangered species), so results obtained from analyzing the sample can provide useful information when carefully interpreted. This study showed that biopsy samples adequately measure the lipid classes present in the sampled blubber layer, but cannot provide information on depth-related changes in the lipid class profile. In addition, contaminant concentrations of biopsy samples from these species are generally within a factor of 2 of those obtained via necropsy and that may be sufficient information to determine whether an animal is highly contaminated and therefore at risk for contaminant -related health effects. In contrast, fatty acid profiles from outer blubber layers collected via biopsy are unlikely to be useful in determining the probable prey species consumed by these odontocetes due to the high level of fatty acid stratification observed between the outer blubber layer that would be collected by biopsy and the metabolically active inner layer.

INTRODUCTION Fat (lipid) comprises a large proportion of the body mass of many cetaceans and is consolidated as a blubber layer. Analysis of blubber can provide a great deal of information about the body condition and health of marine mammals. For example, blubber thickness and lipid content can be indicative of the nutritive condition of cetaceans (Aguilar and Borrell, 1990). In addition, profiles of fatty acids in blubber can be used to estimate the diet of marine mammals (Adams et al., 1997; Iverson et al., 1997; Iverson et al., 2002; Walton et al., 2000). Furthermore, measuring concentrations of lipophilic organochlorine contaminants (O Cs) in the blubber of top predators (e.g., odontocetes) provides information on potential adverse health effects resulting from exposure to these contaminants, because high OC concentrations have been associated with immunosuppression, reproductive impairment, alteration in bone development and growth and in increased susceptibility to disease (Beckmen et al., 1999; Beckmen et al., 2003; De Guise et al., 1996; De Guise et al. , 1997; De Swart et al., 1996; Kamrin and Ringer, 1996; Olsson et al., 1994; Reijnders, 1986; Ross et al., 1996a; Zakharov et al., 1997), Finally, changes in lipid class profiles (i.e., proportions of triglycerides, free fatty acids, phospholipids, wax esters and cholesterol) may influence the concentrations of contaminants in a blubber layer (Koopman et al., 1996). Collecting biopsy samples (i.e., blubber and epidermis) from free-ranging cetaceans—through surgical or punch biopsies on captured-released small cetaceans and through remote biopsy darting of larger cetaceans—is becoming more frequent as part of an effort to develop non-destructive techniques for monitoring population status (genetics), contaminant concentrations and trophic levels (stable isotope ratios) (Barrett-Lennard et al., 1996; Fossi et al., 1997a; Fossi et al., 1997b; Fossi et al., 1999; Fossi et al., 2000). In addition, biop sy samples * Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service,

National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, WA 98112, USA. + National Marine Mammal Laboratory, Alaska Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 7600 Sand Point Way NE, Seattle, WA 98115, USA. # Alaska Regional Office, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 222 West 7th Avenue, Anchorage, AK 95513, ¥USA Cascadia Research, 218 1/2 W 4th Avenue, Olympia, WA 98501, USA. 1 07/05/03

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are often preferred over samples from necrop sies of stranded cetaceans, because stranded animals often include animals with chronic diseases that have altered feeding habits or are emaciated and, thus, are not representative of the overall population (Aguilar et al., 1999; Borrell and Aguilar, 1990). However, only a few studies have tested whether the blubber collected in these small samples is representative of the entire blubber layer in cetaceans (Aguilar and Borrell, 1991; Gauthier et al., 1997) and these studies have looked primarily at the distribution of OCs within the blubber of mysticetes . Studies that have evaluated differences in OC concentration by blubber depth have found that results appear to be species -specific, i.e., some species show pronounced stratification and others have blubber that is more homogenous . For example, two studies of OC stratification in blubber of balaenopterid whales have led to different conclusions. Aguilar and Borrell (1991) studied the stratification of OCs in full-depth blubber of fin (B. physalus) and sei whales (B. borealis ) and found significantly higher OC concentrations in the outer layer compared to the inner layer of blubber in both species. They concluded that blubber samples collected from cetaceans for pollutant analyses should include all layers in order to be representative of an individual animal’s pollutant load. In contrast, Gauthier et al. (1997) found no statistically significant differences in lipidnormalized OCs among the outer, middle or inner blubber layers (subsampled from full-thickness blubber samples) in minke and blue whales (B. musculus). Recently, as interest in using blubber fatty acids to provide information about cetacean diets has increased, researchers have questioned whether biopsy samples can provide adequate samples for fatty acid determinations. Koopman (1996; 2001; 2002) found that vertical stratification of fatty acids was evident between the inner and outer blubber layers in odontocetes, suggesting that the inner blubber layer is more active metabolically than the outer layer in terms of lipid deposition and mobilization. Similarly, Olsen and Grahl-Nielsen (2003) found vertical stratification of fatty acids in minke whale (Balaenoptera acutorostrata) blubber and concluded that studies to trace dietary influence of fatty acids should be made using the inner blubber layer. Furthermore, Hooker et al. (2001) reported that fatty-acid stratification was present throughout the depth of the blubber in northern bottlenose whales (Hyperoodon ampullatus), but was much less pronounced than that found in smaller cetaceans (Koopman, 2001; Koopman et al., 2002) . This manuscript takes a broad look at blubber stratification in two odontocete species—white whales (Delphinapterus leucas) and killer whales (Orcinus orca)—by measuring five parameters (i.e., percent lipid, lipid classes, OC concentrations, OC patterns and fatty acid profiles) by blubber depth. Furthermore, because the dart biopsy is becoming a standard protocol for obtaining tissue samples from many cetaceans, it is essential to compare the results obtained from biopsy samples to those found by analyzing a full-thickness blubber sample. Two questions must be asked: (1) does the outer blubber layer of a particular cetacean species (presumably the portion that would be sampled via dart biopsy) have measured parameters (e.g., percent lipid or contaminant concentrations) that are representative of the blubber layer as a whole; and (2) does a sample obtained by dart or other biopsy procedures have measured parameters that are very similar to those in a sample of the outer blubber al yer collected via necropsy? The results of this study demonstrate that certain of the measured parameters show stratification by blubber depth, so biopsy results must be interpreted with caution and in conjunction with results from blubber depth profiling on the same cetacean species. METHODS Cetaceans sampled Samples from five white whales were available: four from Cook Inlet (near Anchorage, AK) and one from Bristol Bay (on the Bering Sea just north of the Alaska Peninsula). White whales from Cook Inlet and Bristol Bay are genetically distinct populations (O'Corry-Crowe et al. , 1997) . Cook Inlet white whales necropsied were 692-BLKA-073 and 692-BLKA-076 (referenced as CI-73 and CI-76 in this paper) from a subsistence harvest in Cook Inlet. White whale CI-73 (sampled in 2001) was a lactating female, 345cm in length and CI-76 (captured in 2002) was an adult male, 457cm in length. In addition, a juvenile male white whale from Bristol Bay that was entangled and drowned in a net during an attempted tagging capture in 2002 was necropsied and used by local hunters for subsistence (692-BLKA-075; referenced as BB-75; 287cm in length). Finally, two adult female white whales, captured for satellite tagging, were biopsied by trocar and then released in Cook Inlet in 2001— one (CI-01-05) was 362cm in length and the second (CI-01-06) was 401cm in length. Tissue samples were available from two killer whales that stranded in 2002. The first (CA189) stranded in January in inland Washington State waters and the second (L60) stranded in April on Washington’s outer coast. CA189 was a “fresh-dead” adult female transient-type whale—length of 671cm and weight of 4000kg—with a “good” body condition and no emaciation. According to necropsy results, CA189 may have given birth to a stillborn calf or aborted a late-term fetus. CA189 had been observed most often in California waters (Black et 2 07/05/03

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al., 1997), L60 (born 1972; died 2002; 606cm long), was a Southern Resident killer whale from a pod that inhabits the inland waters of Puget Sound in the summer months (Black et al., 1997; Ford et al., 2000). She had given birth to at least two calves, the first has died (1990 -1998), but the second calf is still alive (1995-) (Ford et al., 2000 ). Subsampling of blubber by depth and position on animal Full-thickness blubber samples (epidermis to muscle) were collected from CI-73, CI-76 and BB-75. These animals had sufficient blubber to allow subdividing the samples (epidermis to muscle), but because of differences in blubber thickness and total mass, blubber from CI-73 and CI-76 was divided into fourths and blubber from BB-75 was divided into thirds. Blubber samples from CI-01-05 and CI-01-06 were collected by trocar (a 6mm core from epidermis to the muscle sheath—a deep biopsy) and were of sufficient length/mass to allow division into halves —near-epidermis and near-muscle subsamples —that were analyzed separately. Full-thickness samples of blubber from the stranded transient killer whale (CA189) were collected from the dorsal and mid-lateral regions. Subsequently, blubber from thes e two regions was subdivided by depth into three layers from 0-2cm (from the epidermis), 2-4cm and > 4cm. Full-thickness blubber samples were collected from the dorsal and lateral region of the resident killer whale L60. In addition, a large portion of the saddle patch region of the killer whale was removed from the whale carcass and transported on ice and frozen. A fullthickness blubber sample from the anterior, central and posterior (to the dorsal fin) regions of the saddle patch were subsampled. Blubb er from each region (n = 5; dorsal, lateral, anterior, central and posterior) was subdivided by depth into three layers from 0-2cm (from the epidermis), 2-4cm and > 4cm (not all blubber regions were thick enough to include the third layer; see Table 3). At the same time, simulated ‘biopsy’ samples were taken using a dart (5 x 20mm) that was thrust fully into the anterior, central and posterior regions of the saddle patch. The ‘biopsy’ sample was estimated to be ~2cm in depth. To simplify references, the near-epidermis blubber layer will be called the ‘outer’ layer and that nearest to the muscle will be the ‘inner’ layer—the other layer(s) will be termed ‘middle’ layers. TLC/FID lipid percent and lipid class determinations Blubber samples were analyzed for total lipids by thin layer chromatography coupled with flame ionization detection (TLC/FID) using an Iatroscan Mark 5 (Iatron Laboratories, Tokyo, Japan) (Shantha, 1992) . The lipid sample extracts were spotted on Chromarods (Type SIII) and developed in a solvent system containing 60:10:0.02 hexane:diethyl ether:formic acid (v/v/v). The various classes of lipids (i.e., wax esters, triglycerides, free fatty acids, cholesterol and phospholipids) were separated based on polarity, with the nonpolar compounds (e.g., wax esters) eluting first, followed by the more polar lipids (e.g., phospholipids). The Iatroscan was operated with a hydrogen flow rate of 160mL/min and air flow of 2000mL/min. A four-point linear external calibration was used for quantitation. Duplicate TLC/FID analyses were performed for each sample extract and the mean value reported. Total lipid concentrations were calculated by adding the concentrations of the five lipid classes for each sample and were reported as percent total lipid. Fatty acid concentrations and profiles The analytical method used to measure fatty acid concentrations in these tissues was recently developed in our laboratory and represents a compilation of several different methodologies reported in the literature. Briefly, the method involves: (1) extraction of approximately 1g of tissue by Accelerated Solvent Extraction (ASE) using 50 ml methylene chloride at 100°C and 2000psi; (2) partition of the extract into three fractions (approximately 46% for OC analysis, 46% for total lipid by our standard gravimetric method and 8% for fatty acid and lipid class (Iatroscan) analysis ; (3) derivatization of the fatty acid fraction to fatty acid methyl esters (FAMEs) using 3% sulfuric acid in methanol; (4) extraction of the FAMEs into iso-octane; (5) drying the extract over a bed of sodium sulfate; and finally (6) separation and analysis of the FAME extracts on a DB-23 capillary column using a quadrupole gas chromatography/mass spectrometry (GC/MS) operated in the selected ion monitoring (SIM) mode. In most cases, the molecular ion was chosen for quantitation and a confirmation ion was also monitored. Eighty-three different fatty acids were determined as their methyl esters (Table 1). A standard nomenclature system was used for naming these fatty acids, where ‘n’ follow ed by a number refers to the position of the first double bond relative to the alkyl end of the molecule (Table 1). OC contaminant concentrations and profiles Blubber samples (1.0 to 3.0g ) were extracted, following add ition of internal standards, using the ASE procedure (above). The methylene chloride extract was then filtered through a column of silica gel and alumina and 3

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concentrated for further cleanup by high -performance liquid chromatography (HPLC) with a size-exclusion column that separated lipids and other biogenic material from the OC fraction (Krahn et al. , 1988; Krahn et al., 1994; Sloan et al. , 1993). Finally, the fraction containing OCs was analyzed by GC/MS. Although 40 PCBs and 24 chlorinated pesticides were determined in thes e samples, in the interest of brevity, this paper reports only ∑PCB and ∑ DDTs as representative of the entire list of contaminants. T he killer whale blubber samples were also analyzed for selected OCs, including dioxin-like and other PCB congeners, as well as for selected pesticides (e.g., DDTs), using HPLC coupled with photodiode array detection (PDA) (Krahn et al., 1994). Briefly, the analytes were extracted from the blubber by maceration with pentane/hexane (50/50, v/v) and were separated from interfering compounds on a gravity-flow cleanup column (packed with neutral, basic and acidic silica gels) that was eluted with methylene chloride/hexane (50/50, v/v). The dioxin-like PCB congeners and pesticides were determined using HPLC coupled with an ultraviolet/visible photodiodearray ( PDA) detector and a Cosmosil PYE column. Again, although a number of PCB congeners and pesticides were measured, only ∑ PCB and ∑DDTs are reported. Results from samples analyzed by HPLC/PDA have been shown to be comparable to those of the same samples analyzed by GC/MS (Krahn et al., 1994). Moreover, many of the samples reported in this paper were analyzed by both GC/MS (reported) and HPLC/PDA with very good comparability between the results. Statistical analyses Principal Component Analysis (PCA) analysis of the blubber OC contaminant data was used as the statistical ® method (JMP Statistical Discovery Software version 9.0) by which the similarity of contaminant and fatty acid patterns was revealed. Analyte concentrations were normalized by dividing concentrations of each analyte by total OCs (sum of ?PCBs, ?DDTs, and other analytes measured). PCA was then computed on the correlation matrix of these normalized data. When PCA is used, the number of samples should exceed the number of variables, preferably by a factor of two (McGarigal et al., 2000) . First, all analytes that had values below the limits of detection were excluded from the data set because values below this limit distort the pattern and strongly affect the PCA analysis. Because of the need to further reduce the number of variables (64 variables and 15 samples for white whales; 58 variables and 20 samples for killer whales), only those OC analytes exhibiting the largest positive and largest negative eigen vector projections along the first four principal component axes were used for the final PCA analysis. These eight analytes were— for white whales: PCB congeners 28, 52, 70, 118, 151, dieldrin, p,p’-DDD, o,p’-DDT; and for killer whales: congeners 87, 153, 191, 199, beta-HCH, o,p’DDT, o,p’-DDD, p,p’-DDE. PCA fatty acid concentrations were normalized by dividing concentrations of each analyte by total FAMEs (sum of all FAME analytes measured). All analytes that had values below detection limits were excluded and PCA was computed on the correlation matrix. Again, only those eight analytes that were the major contributors to the first four principal components were used for the final PCA analysis. These analytes were —for white whales: C16:1n7, iso-C18:0, C18:1n9, C18:1n13, C18:2n7, C18:3n3, C19:0, C20:1n7; and for killer whales: C14:0, C14:1n5, 261014-M e-C15:0, iso-C16:0, C18:1n5, C18:1n13, C20:2n11, C20:4n6. Quality assurance Quality assurance procedures for determining OC and fatty acid concentrations and percent lipid included analyses of National Institute of Standards and Technology (NIST) standard reference materials (SRMs)— specifically SRM 1945 for OC contaminants and percent lipids and SRM 1946 for fatty acids. The QA procedures also included the use of certified calibration standards, method blanks, solvent blanks and replicate samples. Acceptance criteria for analyses of NIST SRM 1945 were those that NIST uses for its Intercomparison Exercises . In addition, our laboratory has successfully participated in NIST and other Quality Assurance Intercomparison Exercises each year. RESULTS Percent lipid in blubber Percent lipid did not vary greatly by depths in the samples from the necropsied white whales (RSDs = 14%, 1.6% and 9.6% for BB-75, CI-73 and CI-76 respectively; Table 2), The middle layer(s) tended to have higher percent lipid compared to the outer (epidermis ) or inner (muscle) layer. For each white whale sampled, the outer layer had percent lipid that was very close to the mean of the 3 or 4 layers analyzed (Table 2). In contrast, blubber samples collected by trocar (Table 2) showed a larger variation in percent lipid between the halves (RSDs = 79% and 54% for CI-01-05 and CI-01-06 respectively). Furthermore, both the inner and outer layers 4

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sampled by trocar had much lower percent lipid values than were found in the corresponding layers of any of the blubber samples collected via necropsy. Percent lipid varied more by depth in the necropsied resident killer whale L60, than in the necropsied white whales (Table 3). For the body positions (i.e., central, posterior, dorsal) having blubber that was thick enough to be split into three layers, RSDs for percent lipid ranged from 12-50%. The middle layer of each had the highest percent lipid; the inner layer had the same or lower percent lipid than was found in the outer layer. Interestingly, for each body position sampled, the outer layer had percent lipid that was very close to the mean of the 2 or 3 layers analyzed (Table 3). In contrast, the samples collected by simulated dart biopsy showed a substantially lower range of percent lipid values (8.3-10%) in comparison to the samples collected via necropsy (28-40%) from the same body positions and depths. Percent lipid varied less by depth in the transient killer whale CA189 than in the resident whale, but more than in the white whales (Table 4). T he middle layer from each of the two positions sampled had the highest percent lipid, but the inner layer had lower percent lipid than the outer layer in the dorsal sample, but the reverse was true for the mid-lateral sample. Again, the outer layer provided an adequate representation of the mean lipid percent for the three depths analyzed. Lipid classes Triglycerides were the predominant lipid class found in blubber samples from all the white whales, irrespective of depth in the blubber layer (Fig. 1). In addition, some of the samples show a small proportion (~5%) of free fatty acids (necropsy samples CI-73 and CI-76; trocar sample CI-01-05). In contrast, the lipid classes in killer whale blubber vary substantial ly by depth (Fig. 2). Both the resident and transient animals have a high proportion (> 50%) of wax esters in the outer layer (0-2cm) and that proportion decreases with depth. As the wax ester proportion decreases, the triglyceride proportion increases to > 80% in the layer nearest the muscle. The lipid in blubber sampled by dart biopsy from the resident killer whale contained about 40% wax esters and most resembled the outer layer in the sample taken by necropsy from the same animal. Fatty acid profiles Fatty acid profiles for blubber from white whale CI-73 (divided into four layers) and from the ‘central’ position (on the saddle patch) of resident killer whale L60 (divided into three layers) are shown in Fig. 3 (identities of the 83 fatty acids are given in Table 1). The lines at 25%, 50% and 75% (white whale sample) and at 33.33% and 66.66% (killer whale sample) indicate the proportion of each fatty acid that would be expected if thes e acids were homogeneously distributed among the layers. Only a few fatty acids were evenly distributed (e.g., 29, 46, 47, 62 and 66 in the white whale; 6 and 21 in the killer whale) and the others were more heterogeneously distributed among layers. Furthermore, the lower molecular weight fatty acids (those having lower identification numbers) were found in higher proportions in the outer (epidermis) layer than in the inner layer—particularly in the white whales, but also to a lesser extent in the killer whales. PCA was used to assess the homogeneity of the fatty acids profiles in the white whale blubber samples. The fatty acid profiles for each white whale generally did not group closely by depth in the blubber layers (evenly dashed ovals—Fig. 4A), indicating a high degree of depth stratification for these animals. However, the four animals from the Cook Inlet stock could be distinguished from the single animal from the Bristol Bay stock by their fatty acid profiles (unevenly dashed lines—Fig 4A) and this resolution is predominantly due to a larger relative abundance of branched-chain fatty acids in the latter. When PCA was used to depict patterns for the killer whale fatty acids, all lateral body positions sampled on each killer whale were highly clustered when grouped by depth (solid ovals —Fig. 4B). For example, the fatty acid profiles at the anterior and mid-lateral positions at 0-2cm for the transient killer whale (CA189) were similar to one another, but distinctly different from those at the two other depths (Fig. 4B). Similarly, the fatty acid profiles of the five lateral positions sampled for the resident killer whale (L60) were highly similar and thus clustered when grouped by depth (solid ovals). In contrast, the fatty acid profiles for the two killer whales were not well-correlated by depth, with the outer layer well-separated from the inner layer in both animals (Fig. 4B). The two killer whales could, however, be readily distinguished from one another by means of their fatty acid profiles, regardless of either the blubber depth or lateral position at which the blubber sample was acquired (dashed ovals in Fig. 4B).

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Contaminant concentrations Concentrations of selected contaminants (∑PCBs and ∑DDTs) for all depths and positions sampled are given in Table 2 (white whales), Table 3 (resident killer whale L60) and Table 4 (transient killer whale CA189). The highest wet weight contaminant concentrations for the white whales were found in CI-76 (an adult male), next was BB-75 (a juvenile male) and then CI-73 (a lactating female). The lowest concentrations were foun d in the two female white whale s sampled by trocar—CI-01-06 had higher concentrations of OCs than were found in CI01-05. Although reproductive status of both adult female white whales is not known, no calves were observed near these whales. Concentrations of ∑PCBs and ∑ DDTs in the outer layer of white whales differed from concentrations in the inner layer, but generally by no more than a factor of 2. In some samples the outer layer had higher concentrations and in others the inner layer was higher (T able 2). ‘Normalization ’ of the wet weight concentrations to a lipid basis (i.e., the concentrations reported in ng/g lipid in Table 2) did not equalize the distribution of the contaminants, but instead the outer layer now had higher concentrations (ng/g lipid) of ∑PCBs and ∑DDTs than those found in the inner layer in every Cook Inlet white whale, whereas the reverse was true for the Bristol Bay white whale. The resident killer whale L60 had mean concentrations of ∑PCBs and ∑DDTs that were about 4-fold higher than those found in the necropsied white whale (CI-76) with the highest concentrations. Furthermore, the samples taken by simulated dart biopsy had mean wet weight concentrations of ∑ PCBs and ∑DDTs that were about onethird of the mean concentrations found in the necropsy samples. The mean lipid adjusted concentrations of ∑PCBs and ∑DDTs for the two sampling methods showed reasonable agreement (biopsy samples were about 20% higher, due to substantially lower lipid levels in the biopsy samples ). Differences between wet weight concentrations of ∑PCBs and ∑DDTs in the inner (> 4cm) and outer (0 -2cm) layers of the resident killer whale were somewhat smaller (less than a factor of 1.7) than those found for the white whales, but the differences increased when concentrations were lipid adjusted (factors of 1.6 to 3; Table 3). The transient killer whale CA 189 had mean concentrations of ∑PCBs and ∑DDTs (wet weight) in blubber that were much higher than those of the necropsied resident killer whale—by a factor of about 100 for ∑ PCBs and about 300 for DDTs (Table 4). F or the two lateral body positions, differences between wet weight concentrations of ∑PCBs and ∑DDTs in the inner (> 4cm) and outer (0-2cm) layers of this killer whale were quite low (factors of 1.0 of 1.4). These differences among the layers increased only for the mid-lateral position (to a factor ~2) when the concentrations were expressed as lipid weight. The layer with higher lipid normalized concentrations of ∑PCBs and ∑DDTs was not the same for the two lateral sampling positions —the outer layer had higher lipid normalized concentrations than found in the inner layer for the mid-lateral sampling position, but the reverse was true for the dorsal position. Contaminant profiles PCA was used to depict patterns in the OC data for both the white whales and killer whales. For white whales, the OC profiles of samples from different blubber depths of each individual animal were clustered (evenly dashed ovals and lines in Fig. 5A) and were well-separated from the clusters observed for the other animals. The two stocks, Cook Inlet and Bristol Bay, were also separated from each other (unevenly dashed ovals). The OC profiles of the five lateral positions sampled for the resident killer whale L60 were highly similar at each depth (solid ovals in Fig. 5B) and the groups for each depth were well separated from each other. Contaminant patterns in samples from the dorsal and mid-lateral body positions for the transient killer whale CA189 were also grouped, but the p atterns for the 0-2cm depth samples were less similar than those found for the other depths. Similar to results for the white whales, OC profiles from different blubber depths of the transient killer whale CA189 were well-separated from those of the resident L60 (evenly dashed ovals in Fig. 5B). DISCUSSION Additional samples of each species would have strengthened this study, but it is difficult to obtain good quality, full thickness blubber samples. A limited number of white whales are still being harvested for subsistence purposes in Alaska, so full-thickness blubber samples from subsistence animals from Cook Inlet and Bristol Bay were obtained. Although our laboratory has analyzed a number of white whale blubber samples previously (Krahn et al., 1999), these were not full-thickness blubber samples. In an effort to obtain full-thickness blubber samples from live animals, trocar biopsy sampling was attempted with Cook Inlet white whales captured for satellite tagging, but this effort was not fully successful (detailed below). Most of the recent killer whale samples in the Northeast Pacific have been collected via biopsy darts, because killer whale strandings are unusual and this species is not harvested for subsistence. However, due to two rare killer whales strandings in 2002, good quality samples were available for use in this study. Although non-stranded animals are preferred for 6

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blubber stratification studies, neither stranded killer whale was believed to have died from a chronic illness resulting in emaciation, so the results should not be biased from this cause. The prospects for obtaining additional full-thickness blubber samples of this species are limited. Percent lipid in blubber Percent lipid was fairly uniform in the quartered depths of blubber from the two adult Cook Inlet subsistence white whales (CI-73 and -76) and the juvenile from Bristol Bay (BB-75). Furthermore, the lipid percent in outer layer was a good representation of the mean for the entire blubber layer for each white whale. These results are similar to those found by Gauthier et al. (1997) for minke and blue whales and by Aguilar and Borrell (1991) for fin whales , where no significant difference in lipid content was found among blubber layers. In contrast, Aguilar and Borrell (1991) found a significant difference in lipid content between the inner and outer layers of sei whales (outer layer had higher percent lipid). T he trocar samples collected from live white whales exhibited an uneven distribution of lipid between the blubber halves. Percent lipid in the outer half was lower than the mean for the entire blubber thickness and also was much lower than the percent lipid in outer blubber layers of white whales sampled by necropsy. Individuals collecting the trocar samples noted that white whale blubber at body temperature is quite fluid, so the lipid near the trocar tip seeped from the trocar as it was removed from each animal. Initial attempts at removing the blubber from the trocars resulted in a large loss of lipid from the samples, so these earlier attempts at trocar sampling have not been included in this paper. Although the two samples (CI-01-05 and -06) reported in this paper were frozen in the trocar soon after collection, lipid was likely lost from the samples as the trocar was removed from the animal. As a result, lipid was present in lower proportions in thes e trocar samples (mean = 924%; Table 2) compared to those found for the two Cook Inlet necropsy samples (C I-73 and CI-76; mean = 7275%). These large differences in measured lipid content between trocar and necropsy blubber samples likely represent a true sampling bias. It is expected that the blubber thickness and lipid content for these four Cook Inlet animals should, in fact, be similar, because they were all sampled during the summer season, were of the same approximate age and were feeding within the same geographic location. Thus, although the outer layer of white whale blubber from necropsy can provide a good estimate of percent lipid for the entire blubber thickness, the trocar biopsy samples provided a less accurate representation of the actual percent lipid present in the outer blubber layer. In contrast, Gauthier et al. (1997) reported that their ‘deep’ (19-26mm) biopsy samples were within the ranges measured in the blubber of necropsied whales. Percent lipid varied somewhat among the blubber depths for the resident and transient killer whales, but the outer layer still provided a good estimate of mean percent lipid for the entire blubber thickness, similar to the results found for white whales. In contrast, the simulated dart biopsy samples taken from the resident whale had much lower percent lipid (8-10%) in each body position sampled (anterior, central, posterior) than found for the 0-2cm layer from necropsy samples of the same body position (28-40%). Thus, as observed for the white whales, the biopsy samples from the killer whales provided a less accurate value for percent lipid. These results are consistent with previous results for dart biopsies of free-ranging cetaceans that showed lower levels of percent lipid than would be expected from blubber sampled via necropsy in that species. For example, Krahn et al. (2001) reported that lipid levels in gray whale blubber samples taken by biopsy were lower than those found for samples of subsistence animals taken by necropsy. Furthermore, percent lipid was lower in biopsied blubber samples from Alaskan killer whales (residents = 28 ± 9.8%; transients = 24 ± 9.5%) (Ylitalo et al., 2001) than in L60 and CA189 sampled by necropsy (resident = 35 ± 11%, Table 3; transient = 50 ± 13%, Table 4), even though stranded animals are more likely to be emaciated (Krahn et al., 2001). A number of theories have been advanced to explain the lower percent lipid in biopsy samples : (1) lipid may seep from the blubber structural matrix as the biopsy dart is removed from the animal; (2) some of the lipid could be washed away when the dart falls into the water before being retrieved; (3) and the dart often hits the animal at an oblique angle so that more epidermis and connective tissue are collected than actual blubber. Because the simulated biopsy dart entered the blubber as vertically as possible, did not fall into water and the carcass was sampled at room temperature so the lipid would be less likely to seep from the matrix, these explanations are not likely relevant. However, the dart was pushed into the carcass by hand, so perhaps it did not penetrate as deeply as the same dart would have if fired from a gun or cross-bow and the blubber may not have been sampled to the depth desired. Lipid class profiles For the white whales, triglycerides comprised the greatest proportion of the lipid (> 90% ) regardless of depth, with much smaller proportions of free fatty acids present (Fig. 1). These results agree with previous studies showing that blubber of healthy cetaceans contained primarily neutral lipids, e.g., triglycerides and nonesterified 7

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free fatty acids (Kawai et al., 1988; Krahn et al., 2001; Tilbury et al., 1997). Furthermore, the trocar samples showed the same profile of lipid classes as that found for the necropsy samples. As a result, a trocar biopsy would accurately represent the lipid class profile for the white whales. Unlike the blubber of white whales (F ig. 1), all blubber layers of killer whales contained wax esters, with the greatest proportion found in the outer layer (Fig. 2). These results were unexpected because previous studies suggested that cetacean blubber is composed primarily of triglycerides and contains little or no wax esters (Koopman et al., 1996; Krahn et al., 2001; Lockyer et al., 1984). Litchfield et al. (1975) examined the lipid composition of fatty tissues of 20 different species of odontocetes and found that the blubber of all nine species of Dephinidae examined in the study contained mostly triglycerides, with the exception of false killer whales which contained 96% triglycerides and 4% wax esters. In fact, the only odontocetes that have been reported to contain appreciable proportions of wax esters (> 40%) in their blubber were whales from the Ziphiidae (beaked whale) and Physeteridae (sperm whale) families (Hooker et al. , 2001; Litchfield et al., 1975) . T he simulated dart biopsy provided a reasonably accurate lipid class profile of the outer layer of the resident killer whale, showing only slightly smaller proportions of wax esters than found in the outer layers from necropsy (Fig. 2). Therefore, the biopsy dart sample could provide lipid class information that adequately portrays the profile of the same-depth sample obtained via necropsy, but would not provide information about the manner in which lipid class profiles of killer whales change by blubber depth. Fatty acid profiles Fatty acid profiles of a predator have been statistically linked to fatty acid profiles from potential prey species to provide an estimate of relative proportions of specific prey species consumed by the predator (Adams et al., 1997; Iverson et al., 1997; Iverson et al., 2002; Walton et al., 2000). Because the inner blubber layer is more metabolically active than the outer layer, the inner layer is thought to correlate best with the fatty acid profiles of the prey consumed (Hooker et al., 2001). Thus, it is important to ascertain whether biopsy sampling techniques can provide blubber samples that are representative of the fatty acid profiles of the metabolically active inner blubber layer. In both white whales and killer whales, fatty acids were disproportionately distributed among the blubber layers (Fig. 3), similar to the results reported by other researchers (Hooker et al., 2001; Olsen and Grahl-Nielsen, 2003). When PCA was used to determine how the differ ent blubber layers were grouped based on the fatty acids present, profiles from the white whales showed that the inner and outer layers were not highly correlated (Fig. 4A). Similarly, fatty acid profiles of the inner blubber layer were very different from the outer layer for both the resident killer whale L60 and the transient CA189 (Fig. 4B). Thus, a biopsy sample comprising only the outer blubber layer of white whales or killer whales would not be sufficiently representative of the metabolically active inner layer and thus likely would fail to correlate well with the fatty acid signatures of their primary prey species when applying fatty acid signature analysis to determine the relative diets of these two odotocetes. Olsen and Grahl-Nielsen (2003) have also indicated that blubber fatty acid profiles may be a method suitable for population (stock) identification in minke whales that is convenient, rapid and cost-effective. F atty acid profiles were able to markedly separate the Bristol Bay white whale from the Cook Inlet animals (Fig. 4A), so stock identification may be possible. However, additional samples from each stock would help to verify these results Similarly , the two ecotypes of killer whales were cleanly separated using fatty acid profiles (Fig. 4B), so ecotype identification may be possible for these animals, but more samples from killer whales with identified ecotypes are needed to confirm this observation. Contaminant concentrations Age and sex were known for white whales CI-73, CI-76 and BB-73, so contaminant levels in these animals could be interpreted relative to these demographic parameters. For example, the Bristol Bay white whale BB-73 was a juvenile male with contaminant concentrations (wet weight and lipid weight) that were lower than those of CI-76, the Cook Inlet adult male (Table 2). Juveniles generally have contaminant concentrations that are lower than those found in adult males (Aguilar et al., 1999). In addition, the foraging areas used by the Bristol Bay population (to which BB-73 belongs) are probably very different from those of the Cook Inlet population. However, this is the only animal from Bristol Bay analyzed for contaminants, and relative levels of contamination in the Bristol Bay white whale population, compared to those in animals from Cook, Inlet, cannot be established from a single animal. Among the Cook Inlet animals, CI-76, an adult male, had higher concentrations of ∑PCBs and ∑DDTs than found for CI-73, a lactating female. These results were in agreement with those reported in a number of studies where reproductive females of many marine mammal species were found to have lower contaminant 8 07/05/03

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concentrations than were found in adult males (Aguilar and Borrell, 1994; Aguilar et al., 1999; Krahn et al., 1997; Krahn et al., 1999; Muir et al. , 1992; Norstrom and Muir, 1994), as the result of maternal transfer of contaminant burdens to offspring during gestation and lactation (Aguilar and Borrell, 1994; Aguilar et al., 1999; Ridgway and Reddy, 1995). Furthermore, the lactating female CI-73 from Cook Inlet had concentrations of ∑PCBs and ∑DDTs (Table 2) that were very similar those reported previously (790 ± 560 and 530 ± 450 ng/g wet weight, respectively) for Cook Inlet females (n = 10) (Krahn et al., 1999). In addition, the adult male CI-76 had concentrations in the same range as those of previously-reported Cook Inlet males (∑PCBs = 1490 ± 700 and ∑DDTs = 1350 ± 730ng/g wet weight; n = 10) (Krahn et al., 1999). Concentrations of ∑ PCBs and ∑DDTs by depth on a wet or lipid weight basis did not vary by much more than a factor of 2 in the white whales sampled by necropsy. However, the variations were inconsistent —sometimes the outer layer had higher concentrations, similar to results reported by Aguilar and Borrell (1991) and other times (e.g., the Bristol Bay animal), the inner layer had higher concentrations. T rocar biopsies from the adult female white whales contained much lower concentrations of ∑ PCBs and ∑DDTs on a wet weight basis than were found for the Cook Inlet samples collected by necropsy (Table 2) and for fem ale white whales reported previously (Krahn et al., 1999). Consequently, the trocar samples did not seem to provide accurate wet weight OC concentrations. Because these trocar samples also had very low percent lipid compared to the necropsied samples , contaminant concentrations based on lipid weight increased up to 20-fold compared to the wet weight, resulting in ∑ PCB and ∑ DDT concentrations that were in the same ranges as those reported for necropsied females (Krahn et al., 1999). If better trocar sampling techniques can be developed, the samples obtained should be more suitable for determining both wet weight and lipid weight contaminant concentrations. The ∑PCBs and ∑ DDTs found in the female transient killer whale CA189 (Table 4) were higher than those reported previously for either male or female transient killer whales (Ross et al. , 2000; Ylitalo et al., 2001). Females generally contain lower contaminant concentrations than are found in males due to contaminant transfer to offspring during gestation and lactation. Although CA189 apparently had given birth to a calf that did not survive or had aborted a late-term fetus (see Methods), she still had a very high contaminant load. T he high contam inant concentrations found in the transient killer whale (Table 4) compared to those found in the resident animal (Table 3) can be explained by the different diets of the two killer whale ecotypes, i.e., transient killer whales feed primarily on marine mammals and resident animals eat fish (Baird, 1994). Thus, the transients feed at a higher trophic level on prey that contain higher contaminant levels as a result of bioaccumulation of persistent contaminants (e.g., ∑PCBs and ∑DDTs) (Fisk et al., 2001; Kucklick et al., 1994; Muir et al., 1988). These results agree with those reported for resident and transient killer whales in Alaska by Ylitalo et al. (2001) and in Canadian waters by Ross et al (2000). Both ecotypes of killer whales showed inconsistent values for ∑ PCBs and ∑DDTs by depth, with more variation when contaminant concentrations were expressed on a lipid weight basis. On a wet weight basis, the inner blubber layer in the resident usually had higher levels of OC contaminants than found for the outer layer (Table 3), but the reverse was true for the transient (Table 4). When contaminant concentrations in the resident whale L60 were expressed as lipid weight, the inner blubber layer still contained higher levels of ∑ PCBs and ∑DDTs than found in the outer layer and the differences were more pronounced. Results for the transient CA189 were inconsistent on a lipid weight basis; the dorsal sample had higher contaminant levels in the inner layer, but the mid-lateral sample had higher concentrations in the outer layer. These variations in contaminant concentrations within the blubber layer point out the need to have a full-thickness blubber sample to accurately represent contaminant concentrations as suggested by Aguilar and Borrell (1991). None of the simulated dart biopsy samples from the three body positions on the resident killer whale accurately portrayed wet weight concentrations of ∑PCBs and ∑ DDTs when results were compared to those in the 0-2cm necropsy samples from the same body positions. Contaminant concentrations in biopsy samples were about half as great as those from necropsy samples. In contrast, when OC contaminant concent rations were expressed as lipid weight, the dart samples were about 20% higher than the necropsy samples. The percent lipid in the dart samples was very low (about 25% of that found in the necropsy samples), so this was another indication that dart biopsies produced samples with measured parameters that are different from the samples taken via necropsy. Thus, additional work on dart sampling techniques is warranted to obtain more information about their suitability for obtaining accurate OC contaminant data. For example, other researchers report using darts that are larger in diameter (6.4mm) (Barrett-Lennard, 2000) or longer (30mm) (Matkin, 2003) than the one used in this study. The larger darts may provide biopsy samples more similar to those taken via necropsy.

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Contaminant patterns Contaminant patterns (profiles) from PCA of blubber samples collected by necropsy from white whales have been used to distinguish among stocks, both from Alaska (Krahn et al., 1999) and Canada (Muir et al., 1996). Thus, it would be informative to ascertain whether biopsy samples are sufficiently accurate in portraying contaminant patterns in both white whales and killer whales , thus allowing the various stocks or ecotypes to be identified. A PCA analysis of OC contaminant profiles in the white whale indicated that the intra-animal depth variation in OC patterns was small relative to the inter-animal differences, such that each individual animal can be readily distinguished from the others, regardless of the blubber depth sampled. More importantly, in the context of stock identification, OC pattern s at all blubber depths for the single animal from the Bristol Bay stock (BB-73) were readily discernable from those of the Cook Inlet stock (stocks identified by unevenly dashed lines in Fig. 5A), but additional Bristol Bay animals are needed to determine whether thes e two stocks can be distinguished by contaminant patterns alone. PCA of contaminant patterns showed separation by depth for each white whale, with the inner and outer layers well separated from each other (Fig. 5A). It may be possible for trocar samples to provide adequate contaminant profiles with which to make comparisons. Among the Cook Inlet samples, the two trocar samples (CI-01-05 and CI-01-06; Fig. 5A) were closely associated with each other and with CI-73 (necropsy), but CI-76 (necropsy ) exhibited OC patterns that were substantially different. However, the three similar samples were all from females white whales, whereas the sample that was most different was from a male. This species has previously demonstrated resolution of OC profiles by sex (Krahn et al., 1999), so it is not surprising to find these samples grouping by sex. Again, additional samples could help determine the range of values for the species and confirm whether the trocar samples are typical of those found by necropsy. T he transient killer whale CA189 had contaminant patterns that were well correlated by blubber depth and body position sampled (Fig. 5B). In contrast, although the contaminant profiles of the resident killer whale L60 at all lateral positions and blubber depths were somewhat more varied than those of the transient, the profiles of all resident L60 blubber samples were substantially different from those of the transient, as would be expected due to their different trophic positions and diets (Baird, 1994). When the lateral blubber samples for the resident were grouped by blubber depth, it was found that the outer blubber layers were distinctly different from the middle and inner layers. Because only two animals (one animal of each ecotype) were sampled, it is difficult to predict whether resident and transient killer whales could routinely be distinguished using PCA contaminant profiles. Conclusions The conclusions from this study (summarized in Table 5) demonstrate that blubber biopsy techniques seldom result in samples that give information completely representative of that obtained from full-thickness or even outer layer blubber samples obtained via necropsy. However, there are many instances in which a biopsy is the only means of obtaining samples (e.g., for threatened or endangered species). However, if carefully interpreted, biopsy samples can provide useful information for several types of analyses. For example, biopsy samples can provide adequate information on lipid classes present in the sampled blubber layer, but cannot provide information on depth-related changes in the lipid class profile. In addition, because measured contaminant concentrations of biopsy samples are generally within a factor of 2 of those obtained via necropsy for these species, results based on analyses of biopsy samples may be of sufficient accuracy to determine whether the animals are highly contaminated and thus at risk for contaminant-related health effects. In contrast, fatty acid profiles from outer blubber layers collected via biopsy are substantially different from the met abolically active inner layer and are therefore unlikely to be useful in attempting to make correlations with the fatty acid profiles of potential prey. In the future, we will continue to profile cetacean blubber by depth as samples become available. In the meantime, this preliminary study has provided information that demonstrates the need for caution when in interpreting results when analyzing tissue collected via biopsy. ACKNOWLEDGEMENTS We appreciate the assistance provided by a number of individuals and organizations in collecting the samples for this manuscript. White whale samples were provided by hunters from Bristol Bay and Cook Inlet. In addition, the 2001 Cook Inlet white whale tagging crew collected the trocar necropsy samples. Brent Norberg, Robyn Angliss and other members of the Northwest Regional Stranding Network, Marine Mammal Health and Stranding Response Program, coordinated the response and sampling efforts for the transient killer whale (CA189) that stranded in January 2002 and the resident killer whale (L60) that stranded on Long Beach, WA in April 2002.. Marilyn Dahlheim, Stephanie Norman and Brad Hanson (National Marine Mammal Laboratory, Alaska Fisheries Science Center), Deb bie Duffield (Northwest Regional Stranding Network, Port land State University Vertebrate Biology Museum) John Calambokidis (Cascadia Research Collective), Pete Schroeder 10 07/05/03

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(Marine Mammal Research Associates), Dave Huff (Vancouver Aquarium) and Stephen Raverty (Ministry of Agriculture, Food & Fisheries ), as well as personnel from the Olympic Coast National Marine Sanctuary, the Whale Museum and the Washington Department of Fish and Wildlife, were involved in the necropsy of the killer whales. We value the technical assistance of Don Brown, Daryle Boyd, Richard Boyer, Ron Pierce, Jennie Bolton and Karen Tilbury in sample and data analysis. Many thanks to Sue Moore for suggesting that a paper addressing these issues should be written and also for reviewing the manuscript. We are grateful for the support of Teri Rowles and for funding, in part, from the Marine Mammal Health and Stranding Response Program of NOAA Fisheries. REFERENCES Adams, T.C., Davis, R.W. and Iverson, S.J. 1997. The use of fatty acid profiles in determining the diet of Steller sea lions (Eumetopias jubatus ). FASEB J. 11(3):168-. Aguilar, A. and Borrell, A. 1990. Patterns of lipid content and stratification in the blubber of fin whales (Balaenoptera physalus). J. Mammal. 71(4):544-54. Aguilar, A. and Borrell, A. 1991. Heterogeneous distribution of organochlorine contaminants in the blubber of baleen whales: Implications for sampling procedures. Mar. Environ. Res. 31:275-86. Aguilar, A. and Borrell, A. 1994. Reproductive transfer and variation of body load of organochlorine pollutants with age in fin whales (Balaenoptera physalus). Arch. Environ. Contam. Toxicol. 27:546-54. Aguilar, A., Borrell, A. and Pastor, T. 1999. Biological factors affecting variability of persistent pollutant levels in cetaceans. J. Cetacean Res. Manage. (special issue 1):83-116. Baird, R. 1994. Foraging behaviour and ecology of transient killer whales., Ph.D. thesis, Simon Fraser University, Burnaby, B.C., Canada. Barrett-Lennard, L.G., Smith, T.G. and Ellis, G.M. 1996. A cetacean biopsy system using lightweight pneumatic darts, and its effect on the behavior of killer whales. Mar. Mamm. Sci. 12(1):14-27. Barrett-Lennard, L.G. 2000. Population Structure and Mating Patterns of Killer Whales as Revealed by DNA Analysis. Ph.D. thesis, University ofBritish Columbia, Vancouver, B.C. Beckmen, K.B., Ylitalo, G.M., Towell, R.G., Krahn, M.M., O'Hara, T.M. and Blake, J.E. 1999. Factors affecting organochlorine contaminant concentrations in milk and blood of northern fur seal (Callorhinus ursinus) dams and pups from St. George Island, Alaska. Sci. Total Environ. 231:183-200. Beckmen, K.B., Blake, J.E., Ylitalo, G.M., Stott, J.L. and O'Hara, T.M. 2003. Organochlorine contaminant exposure and associations with hematological and humoral immune functional assays with dam age as a factor in free-ranging northern fur seal pups (Callorhinus ursinus). Mar. Pollut. Bull. 46:594-606. Black, N.A., Schulman -Janiger, A., Ternullo, R.L. and Guerrero-Ruiz, M. 1997. Killer whales of California and western Mexico: a catalog of photo-identified individuals. U.S. Dep. Commer., NOAA Tech. Memo. NMFS-SWFSC, La Jolla, CA. 247pp. Borrell, A. and Aguilar, A. 1990. Loss of organochlorine compounds in the tissues of a decomposing stranded dolphin. Bull. Environ. Contam. Toxicol. 45:46-53. De Guise, S., Bernier, J., Dufresne, M.M., Martineau, D., Beland, P. and Fournier, M. 1996. Immune function in beluga whales (Delphinapterus leucas): Evaluation of mitogen-induced blastic transformation of lymphocytes from peripheral blood, spleen and thymus. Vet. Immunol. Immunopathol. 50:117 -26. De Guise, S., Ross, P.S., Osterhaus, A.D.M.E., Martineau, D., Beland, P. and Fournier, M. 1997. Immune functions in beluga whales (Delphinapterus leucas): Evaluation of natural killer cell activity. Vet. Immunol. Immunopathol. 58:345-54. De Swart, R.L., Ross, P.S., Vos, J.G. and Osterhaus, A.D.M.E. 1996. Impaired immunity in harbour seals (Phoca vitulina) exposed to bioaccumulated environmental contaminants: Review of a long-term feeding study. Environ. Health Persp. 103:62-72. Fisk, A.T., Hobson, K.A. and Norstrom, R.J. 2001. Influence of chemical and biological factors on trophic transfer of persistent organic pollutants in the northwater polynya marine food web. Environ Sci Technol 35(4):732-8. Ford, J.K.B., Ellis, G.M. and Balcomb, K.C. 2000. Killer Whales: The Natural History and Genealogy of Orcinus orca in British Columbia and Washington, Second Edition. University of British Columbia Press and University of Washington Press, Vancouver, BC, Canada and Seattle, WA, USA. 104pp. Fossi, M.C., Marsili, L., Junin, M., Castello, H., Lorenzani, J.A., Casini, S., Savelli, C. and Leonzio, C. 1997 a. Use of nondestructive biomarkers and residue analysis to assess the health status of endangered species of pinnipeds in the Southwest Atlantic. Mar. Pollut. Bull. 34(3):157-62.

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Fossi, M.C., Savelli, C., Marsili, L., Casini, S., Jimenez, B., Junin, M., Castello, H. and Lorenzani, J.A. 1997b. Skin biopsy as a nondestructive tool for the toxicological assessment of endangered populations of pinnipeds: Preliminary results on mixed function oxidase in Otaria flavescens. Chemosphere 35(8):1623-36. Fossi, M.C., Casini, S. and Marsili, L. 1999. Nondestructive biomarkers of exposure to disrupting chemicals in endangered species endocrine of wildlife. Chemosphere39(8):1273-85. Fossi, M.C., Marsili, L., Neri, G., Casini, S., Bearzi, G., Politi, E., Zanardelli, M. and Panigada, S. 2000. Skin biopsy of Mediterranean cetaceans for the investigation of interspecies susceptibility to xenobiotic contaminants. Mar. Environ. Res. 50(1 -5):517 -21. Gauthier, J.M., Metcalfe, C.D. and Sears, R. 1997. Validation of the blubber biopsy technique for monitoring of organochlorine contaminants in balaenopterid whales. Mar. Environ. Res. 43(3):157-79. Hooker, S.K., Iverson, S.J., Ostrom, P. and Smith, S.C. 2001. Diet of northern bottlenose whales inferred from fatty-acid and stable-isotope analyses of biopsy samples. Can. J. Zool. 79:1442-54. Iverson, S.J., Frost, K.J. and Lowry, L.F. 1997. Fatty acid signatures reveal fine scale structure of foraging distribution of harbor seals and their prey in Prince William Sound, Alaska. Mar. Ecol.-Prog. Ser. 151(1-3):255-71. Iverson, S.J., Frost, K.J. and Lang, S.L.C. 2002. Fat content and fatty acid composition of forage fish and invertebrates in Prince William Sound, Alaska: factors contributing to among and within species variability. Mar. Ecol.-Prog. Ser. 241:161-81. Kamrin, M.A. and Ringer, R.K. 1996. Toxicological implications of PCB residues in mammals. pp. 153-64. In: W. Beyer, G. Heinz and A. Redmon -Norwood (eds.) Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations. SETAC Special Publication Series, Lewis Publishers, Boca Raton, FL, USA. 494pp. Kawai, S., Fukushima, M., Miyazaki, N. and Tatsukawa, R. 1988. Relationship between lipid composition and organochlorine levels in the tissue of striped dolphin. Mar. Pollut. Bull. 19:129-33. Koopman, H.N., Iverson, S.J. and Gaskin, D.E. 1996. Stratification and age-related differences in blubber fatty acids of the male harbour porpoise (Phocoena phocoena). J. Comp. Physiol. B 165(8):628-39. Koopman, H.N. 2001. The structure and function of the blubber of odontocetes. Ph.D. dissertation, Duke University, Durham, NC. Koopman, H.N., Pabst, D.A., McLellan, W.A., Dillaman, R.M. and Read, A.J. 2002. Changes in bubber distribution and morphology associated with starvation in the harbour porpoise (Phocoena phocoena ): Evidence for regional differences in bubber structure and function. Physiol. Biochem. Zool. 75(5):498 -512. Krahn, M.M., Moore, L.K., Bogar, R.G., Wigren, C.A., Chan, S.-L. and Brown, D.W. 1988. High -performance liquid chromatographic method for isolating organic contaminants from tissue and sediment extracts. J. Chrom atogr. 437:161-75. Krahn, M.M., Ylitalo, G.M., Buzitis, J., Sloan, C.A., Boyd, D.T., Chan, S.-L. and Varanasi, U. 1994. Screening for planar chlorobiphenyls in tissues of marine biota by high -performance liquid chromatography with photodiode array detection. Chemosphere29:117-39. Krahn, M.M., Becker, P.R., Tilbury, K.L. and Stein, J.E. 1997. Organochlorine contaminants in blubber of four seal species: Integrating biomonitoring and specimen banking. Chemosphere 34:2109-21. Krahn, M.M., Burrows, D.G., Stein, J.E., Becker, P.R., Schantz, M.M., Muir, D.C. and O'Hara, T.M. 1999. White whales (Delphinapterus leucas) from three Alaskan stocks: concentrations and patterns of persistent organochlorine contaminants in blubber. J. Cetacean Res. Manage. 1(3):239-49. Krahn, M.M., Ylitalo, G.M., Burrows, D.G., Calambokidis, J., Moore, S.E., Gosho, M., Gearin, P., Plesha, P.D., Robert L. Brownell, J., Tilbury, K.L., Rowles, T. and Stein, J.E. 2001. Organochlorine contaminant concentrations and lipid profiles in eastern North Pacific gray whales (Eschrichtius robustus). J. Cetacean Res. Manage. 3(1):19-29. Kucklick, J.R., Bidleman, T.F., McConnell, L.L., Michael D, W. and Ivanov, G.P. 1994. Organochlorines in the water and biota of Lake Baikal, Siberia. Environ. Sci. Technol. 28:31-7. Litchfield, C., Greenberg, A.J., Caldwell, D.K., Caldwell, M.C., Sipos, J.C. and Ackman, R.G. 1975. Comparative lipid patterns in acoustical and nonacoustical fatty tissues of dolphins. Comp. Biochem. Physiol., B: Comp. Biochem. 50:591-7. Lockyer, C.H., McConnell, L.C. and Waters, T.D. 1984. The biochemical composition of fin whale blubber. Can. J. Zool. 62(12):2553 -62. Matkin, C.O. 2003. pers. commun. McGarigal, K., Cushman, S. and Stafford, S. 2000. Multivariate statistics for wildlife and ecology research. Springer, New York, NY. Muir, D.C.G., Norstrom, R.J. and Simon, M. 1988. Organochlorine contaminants in Arctic marine food chains: accumulation of specific polychlorinated biphenyls and chlordane-related compounds. Environ. Sci. Technol. 22:1071-79. Muir, D.C.G., Wagemann, R., Hargrave, B.T., Thomas, D.J., Peakall, D.B. and Norstrom, R.J. 1992. Arctic marine ecosystem contamination. Sci. Total Environ. 122:75 -134.

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Muir, D.C.G., Ford, C.A., Rosenberg, B., Norstrom, R.J., Simon, M. and Beland, P. 1996. Persistent organochlorines in beluga whales (Delphinapterus leucas) from the St. Lawrence River Estuary—I. Concentrations and patterns of specific PCBs, chlorinated pesticides and polychlorinated dibenzo-p-dioxins and dibenzofurans. Environ. Pollut. 93:219-34. Norstrom, R.J. and Muir, D.C.G. 1994. Chlorinated hydrocarbon contaminants in arctic marine mammals. Sci. Total Environ. 154:107-28. O'Corry-Crowe, G.M., Suydam, R.S., Rosenberg, A., Frost, K.J. and Dizon, A.E. 1997. Phylogeography, population structure and dispersal patterns of the beluga whale Delphinapterus leucas in the western Nearctic by mitochondrial DNA. Mol. Ecol. 6:955 -70. Olsen, E. and Grahl-Nielsen, O. 2003. Blubber fatty acids of minke whales: stratification, population identification and relation to diet. Mar. Biol. 142:13-24. Olsson, M., Karlsson, B. and Ahnland, E. 1994. Diseases and environmental contaminants in seals from the Baltic and the Swedish west coast. Sci Total Environ 154(2 -3):217-27. Reijnders, P.J.H. 1986. Reproductive failure in common seals feeding on fish from polluted coastal waters. Nature 324:456-7. Ridgway, S. and Reddy, M. 1995. Residue levels of several organochlorines in Tursiops truncatus milk collected at various stages of lactation. Mar. Pollut. Bull. 30(9):609 -14. Ross, P.S., De Swart, R.L., Timmerman, H.H., Reijnders, P.J.H., Vos, J., Van Lov eren, H. and Osterhaus, A. 1996. Suppression of natural killer cell activity in harbor seals (Phoca vitulina) fed Baltic Sea herring. Aquatic. Toxicol. 34:71-84. Ross, P.S., Ellis, G.M., Ikonomou, M.G., Barrett -Lennard, L.G. and Addison, R.F. 2000. High PCB concentrations in free-ranging Pacific killer whales, Orcinus orca: effects of age, sex and dietary preference. Mar. Pollut. Bull. 40(6):504-15. Shantha, N.C. 1992. Thin-layer chromatography–flame ionization detection Iatroscan® system. J. Chromatogr. 624:21–35. Sloan, C.A., Adams, N.G., Pearce, R.W., Brown, D.W. and Chan, S.-L. 1993. Northwest Fisheries Science Center Organic Analytical Procedures. pp. 53-96. In: G.G. Lauenstein and A.Y. Cantillo (eds.) Sampling and analytical methods of the National Status and Trends Program: National Benthic Surveillance and Mussel Watch Projects 1984-1992. NOAA Coastal Monitoring and Bioeffect s Assessment Division, Office of Ocean Resources Conservation and Assessment, National Ocean Service, Silver Spring, Maryland. Tilbury, K.L., Stein, J.E., Meador, J.P., Krone, C.A. and Chan, S.-L. 1997. Chemical contaminants in harbor porpoise (Phocoena phocoena ) from the north Atlantic coast: tissue concentrations and intra- and inter - organ distribution. Chemosphere 34:2159-81. Walton, M.J., Henderson, R.J. and Pomeroy, P.P. 2000. Use of blubber fatty acid profiles to distinguish dietary differences between grey seals (Halichoerus grypus ) from two UK breeding colonies. Mar. Ecol. Prog. Ser. 193:201-8. Ylitalo, G.M., Matkin, C.O., Buzitis, J., Krahn, M.M., Jones, L.L., Rowles, T. and Stein, J.E. 2001. Influence of life -history parameters on organochlorine concentrations in free-ranging killer whales (Orcinus orca) from Prince William Sound, AK. Sci. Total Environ. 281:183203. Zakharov, V.M., Valetsky, A.V. and Yablokov, A.V. 1997. Dynamics of developmental stability of seals and pollution in the Baltic Sea. Acta-Theriologica Supplement 4:9-16.

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Table 1. Fatty acids analyzed (as methyl esters) in the blubber of marine mammals. The fatty acid number (as shown in figure 3), abbreviation, systematic name and trivial (common name) are provided. # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Abbreviation C10.0 C11.0 C11.1 C12.0 C12.1 Me4812C13.0 C14.0 isoC14.0 Me11C14.0 C14.1n5 C14.1n7 C14.1n9 C15.0 isoC15.0 anteisoC15.0 tMeC15.0 C15.1n5 C16.0 isoC16.0 anteisoC16.0 Me7C16.1 Me78C16.1na C16.1n11 C16.1n5 C16.1n7 C16.1n9 C16.2n4 C16.2n6 C16.3n4 C16.3n6 C16.4n1 C16.4n3 C17.0 anteisoC17.0 isoC17.0 C17.1n7 C17.1ne C18.0 isoC18.0 anteisoC18.0 C18.1n11 C18.1n13

Systematic Name n-Decanoic acid n-Undecanoic acid 10-Undecenoic acid n-Dodecanoic acid 11-Dodecenoic acid 4,8,12-Trimethyltridecanoic acid n-Tetradecanoic acid 12-Methyltridecanoic acid 11-Methyltetradecanoic acid 9-Tetradecenoic acid 7-Tetradecenoic acid 5-Tetradecenoic acid n-Pentadecanoic acid 13-Methyltetradecanoic acid 12-Methyltetradecanoic acid 2,6,10,14-Tetramethylpentadecanoic acid 10-Pentadecenoic acid n-Hexadecanoic acid 14-Methylpentadecanoic acid 13-Methylpentadecanoic acid 7-Methylhexadecenoic acid 7,8-Dimethylhexadecenoic acid 5-Hexadecenoic acid 11-Hexadecenoic acid 9-Hexadecenoic acid 7-Hexadecenoic acid 9,12-Hexadecadienoic acid 7,10-Hexadecadienoic acid 6,9,12-Hexadecatrienoic acid 4,7,10-Hexadecatrienoic acid 6,9,12,15-Hexadecatetraenoic acid 4,7,10,13-Hexadecatetraenoic acid n-Heptadecanoic acid 14-Methylhexadecanoic acid 15-Methylhexadecanoic acid 10-Heptadecenoic acid Heptadecenoic acid n-Octadecanoic acid 16-Methylheptadecanoic acid 15-Methylheptadecanoic acid 7-Octadecenoic acid 5-Octadecenoic acid

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Trivial Name Capric Hendecanoic Hendecenoic Lauric

Myristic

Myristoleic

Palmitic

Palmitoleic

Margaric

Stearic

# 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

Abbreviation C18.1n5 C18.1n7 C18.1n9 C18.2n4 C18.2n6 C18.2n7 C18.3n1 C18.3n3 C18.3n4 C18.3n6 C18.4n1 C18.4n3 C19.0 C20.0 C20.1n11 C20.1n15 C20.1n5 C20.1n7 C20.1n9 C20.2n11 C20.2n6 C20.2n9 C20.3n3 C20.3n6 C20.4n3 C20.4n6 C20.5n3 C21.5n3 C22.0 C22.1n11 C22.1n5 C22.1n7 C22.1n9 C22.2n6 C22.3n3 C22.4n3 C22.4n6 C22.5n3 C22.6n3 C24.0 C24.1n9

Systematic Name 13-Octadecenoic acid 11-Octadecenoic acid 9-Octadecenoic acid 11,14-Octadecadienoic acid 9,12-Octadecadienoic acid 8,11-Octadecadienoic acid 11,14,17-Octadecatrienoic acid 9,12,15-Octadecatrienoic acid 8,11,14-Octadecatrienoic acid 6,9,12-Octadecatrienoic acid 8,11,14,17-Octadecatetraenoic acid 6,9,12,15-Octadecatetraenoic acid n-Nonadecanoic acid n-Eicosanoic 9-Eicosenoic acid 5-Eicosenoic acid 15-Eicosenoic acid 13-Eicosenoic acid 11-Eicosenoic acid 6,9-Eicosadienoic acid 11,14-Eicosadienoic acid 8,11-Eicosadienoic acid 11,14,17-Eicosatrienoic acid 8,11,14-Eicosatrienoic acid 8,11,14,17-Eicosatetraenoic acid 5,8,11,14-Eicosatetraenoic acid 5,8,11,14,17-Eicosapentaenoic acid 6,9,12,15,18-Heneicosapentaenoic acid n-Docosanoic acid 11-Docosenoic acid 17-Docosenoic acid 15-Docosenoic acid 13-Docosenoic acid 13,16-Docosadienoic acid 13,16,19-Docosatrienoic acid 10,13,16,19-Docosatetraenoic acid 7,10,13,16-Docosatetraenoic acid 7,10,13,16,19-Docosapentaenoic acid 4,7,10,13,16,19-Docosahexaenoic acid n-Tetracosanoic acid 15-Tetracosenoic acid

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Trivial Name Vaccenic Oleic Linoleic

alpha-Linolenic gamma-Linolenic

Arachidic Gadoleic

Gondoic

hono-gamma-Linolenic Arachidonic EPA

Erucic

DPA DHA Nervonic

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Table 2. Concentrations of ? PCBs and ? DDTs determined by GC/MS in white whale blubber sampled at various depths. ng/g, wet weight ? PCBs# ? DDTs

ng/g, lipid ? PCBs

Blubber depth†

%lipid‡

? DDTs

BB-75 necropsy

1st third (outer-nearest epidermis) 2nd third 3rd third (inner-nearest muscle) BB-75 necropsy (mean) SD RSD (%)

66 85 69 73 10 14

1,100 1,500 2400 1700 670 39

1,000 1,300 2000 1400 510 36

1,700 1,800 3500 2300 1000 43

1,500 1,500 2,900 2,000 810 41

CI-73 necropsy

1st quarter (outer-nearest epidermis) 2nd quarter 3rd quarter 4th quarter (inner-nearest muscle) CI-73 necropsy (mean) SD RSD (%)

71 73 73 71 72 1.2 1.6

860 820 650 590 730 130 18

920 790 610 560 720 170 24

1200 1100 890 830 1,000 170 17

1,300 1,100 840 790 1,000 240 24

CI-76 necropsy

1st quarter (outer-nearest epidermis) 2nd quarter 3rd quarter 4th quarter (inner-nearest muscle) CI-76 necropsy (mean) SD RSD (%)

68 85 73 74 75 7.2 9.6

2,200 2,600 1,300 1,200 1,800 690 38

3,000 3,200 1,400 1,400 2,300 990 43

3,200 3,100 1,800 1,600 2,400 840 35

4,400 3,800 1,900 1,900 3,000 1,300 43

CI-01-05¥ trocar biopsy

1st half (outer-nearest epidermis) 2nd half (inner-nearest muscle) CI-01-05 trocar biopsy (mean) SD RSD (%)

10 37 24 19 79

66 120 93 38 41

70 120 95 35 37

660 320 490 240 49

700 320 510 270 53

CI-01-06¥ trocar biopsy

1st half (outer-nearest epidermis) 2nd half (inner-nearest muscle) CI-01-06 trocar biopsy (mean) SD RSD (%)

5.8 13 9.4 5.1 54

110 230 170 85 50

150 280 220 92 42

1,900 1,800 1,800 70 3.9

2,600 2,200 2,400 280 12

† A full-thickness portion of blubber was subdivided into quarters for the necrosy samples; CI-73's blubber layer was 5 cm thick, BB75's was 2.2 cm and CI-76's was 6 cm. The trocar biopsy sample was divided in half; CI-01-05's sample measured 6.8 cm and CI-0106's was 3.3 cm (see Methods). ‡ TLD-FID lipid analyses (see Methods). # Sum of 40 PCBs (see Methods). ¥ Samples collected with a trocar (see Methods)

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Table 3. Concentrations of ? PCBs and ? DDTs determined by HPLC/PDA in blubber sampled at various depths and positions in a resident killer whale (L60) that stranded in 2002.

Blubber position

Blubber depth†

%lipid‡

ng/g, wet weight ? PCBs ? DDTs

ng/g, lipid ? PCBs ? DDTs

Female resident (L60)—dart biopsy Anterior 0 - ~2 cm Central 0 - ~2 cm Posterior 0 - ~2 cm L60—dart biopsy (0 - ~2 cm) (mean) SD RSD (%)

10 9.0 8.3 9 0.9 9.9

2,700 2,800 3,000 2,800 150 5.4

2,700 2,700 3,000 2,800 170 6.1

27,000 31,000 36,000 31,000 4,500 15

27,000 30,000 36,000 31,000 4,600 15

40 40 40 0

6,500 9,200 7,900 1,900 24

6,400 10,000 8,200 2,500 30

16,000 23,000 20,000 5,000 25

16,000 25,000 21,000 6,400 30

0 - 2 cm 2 - 4 cm > 4 cm Central necropsy (mean) SD RSD (%)

28 35 11 25 12 50

5,900 8,000 6,500 6,800 1,100 16

6,000 9,200 5,800 7,000 1,900 27

21,000 23,000 59,000 34,000 21,000 62

21,000 26,000 53,000 33,000 17,000 52

0 - 2 cm 2 - 4 cm > 4 cm Posterior necropsy (mean) SD RSD (%)

37 46 38 40 5 12

7,200 9,600 11,000 9,300 1,900 20

6,900 12,000 12,000 10,000 2,900 29

19,000 21,000 30,000 23,000 5,900 26

19,000 26,000 32,000 26,000 6,500 25

Dorsal

0 - 2 cm 2 - 4 cm > 4 cm Dorsal necropsy (mean) SD RSD (%)

42 51 17 37 18 48

7,400 11,000 6,300 8,200 2,500 30

7,000 11,000 6,100 8,000 2,600 33

18,000 22,000 37,000 26,000 10,000 38

17,000 22,000 36,000 25,000 9,800 39

Lateral

0 - 2 cm 2 - 4 cm Lateral necropsy (mean) SD RSD (%)

31 38 35 5 14

6,800 9,600 8,200 2,000 24

7,200 10,000 8,600 2,000 23

22,000 25,000 24,000 2,100 8.8

23,000 26,000 25,000 2,100 8.4

L60—necropsy (mean, n=13) SD RSD (%)

35 11 31

8,100 1,800 22

8,400 2,300 27

26,000 11,000 42

26,000 9,800 38

Female resident (L60)—necropsy Anterior

Central

Posterior

0 - 2 cm 2 - 4 cm Anterior necropsy (mean) SD RSD (%)

-

† Measured from bottom of epidermis ‡ Pentane/hexane extraction with TLD-FID lipid analyses # Dart was inserted into the skin/blubber by hand.

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Table 4. Concentrations of ? PCBs and ? DDTs determined by HPLC/PDA in blubber sampled at various depths and positions in a transient killer whale that stranded in 2002. Blubber position Blubber depth†

ng/g, wet weight ? PCBs ? DDTs

%lipid‡

ng/g, lipid ? PCBs

? DDTs

Female transient (CA 189)—necropsy Dorsal

0 - 2 cm 2 - 4 cm > 4 cm Dorsal necropsy—(mean) SD RSD (%)

48 64 42 51 11 22

820,000 900,000 800,000 840,000 53,000 6.3

2,700,000 2,500,000 2,600,000 2,600,000 100,000 3.8

1,700,000 1,400,000 1,900,000 1,700,000 250,000 15

5,600,000 3,900,000 6,200,000 5,200,000 1,200,000 23

0 - 2 cm 2 - 4 cm > 4 cm Mid-lateral necropsy—(mean) SD RSD (%)

42 64 58 55 11 21

1,000,000 1,000,000 830,000 940,000 98,000 10

3,400,000 2,900,000 2,400,000 2,900,000 500,000 17

2,400,000 1,600,000 1,400,000 1,800,000 530,000 29

8,100,000 4,500,000 4,100,000 5,600,000 2,200,000 39

CA189 (mean n=6) SD RSD (%)

50 13 26

890,000 90,000 10

2,750,000 360,000 13

1,730,000 380,000 22

5,400,000 1,600,000 30

Mid-lateral

† Measured from bottom of epidermis ‡ Pentane/hexane extraction with TLD-FID lipid analyses

Table 5. Summary of conclusions. Outer layer represents full-thickness sample or inner layer?

Biopsy sample represents layer(s) sampled via necropsy? simulated dart trocar (white whales) (resident killer whale)

white whales

killer whales

lipid percent

yes

yes

no

no

lipid classes

yes

no

yes

yes, but changes in profile with depth would not be seen

fatty acid profiles

no

no

no*

not applicable

inconsistent variations by layer by factor of ~2

inconsistent variations by layer by factor of < 2

wet weight—no lipid weight—OK

wet weight—no lipid weight—20% high bias

possibly

resident—no transient—yes

possibly—additional samples needed

not applicable

contaminant concentrations

contaminant patterns

#

#

# If the outer layer is unsuitable for determinations of fatty acid profiles, dart biopsies could not collect the needed samples. * If trocar sampling techniques improve so that full-thickness samples of blubber are obtained without loss of lipid, it might be possible to obtain fatty acid profiles from the metabolically active inner layer.

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1st third (nearest epidermis) % lipid = 66

BB-75 (necropsy)

2nd third % lipid = 85 3rd third (nearest muscle) % lipid = 69

CI-73 (necropsy) 1st quarter (nearest epidermis) % lipid = 71 2nd quarter % lipid = 73 3rd quarter % lipid = 73 4th quarter (nearest muscle) % lipid = 71

CI-76 (necropsy) 1st quarter (nearest epidermis) % lipid = 68 2nd quarter % lipid = 85 3rd quarter % lipid = 73 4th quarter (nearest muscle) % lipid = 74

1st half (nearest epidermis) % lipid = 10

CI-01-05 (trocar biopsy)

2nd half (nearest muscle) % lipid = 37

CI-01-06 (trocar biopsy) 1st half (nearest epidermis) % lipid = 5.8 2nd half (nearest muscle) % lipid = 13

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Percent of each lipid class by weight

wax esters triglycerides free fatty acids cholesterol phospholipids

Figure 1. Lipid classes (i.e., wax esters, triglycerides, free fatty acids, cholesterol and phospholipids) determined in each layer of white whale blubber by depth (i.e., each half, third or quarter).

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L 60 stranded resident (necropsy) 0-2 cm % lipid = 36 ± 5.9 n=5 2-4 cm % lipid = 42 ± 6.4 n=5 > 4 cm % lipid = 22 ± 14 n=3

L 60 stranded resident (dart biopsy) Biopsy (~0-2 cm) %lipid = 9.1 ± 0.9 n=3

CA 189 stranded transient (necropsy) 0-2 cm % lipid = 45 ± 4.2 n=2 2-4 cm % lipid = 64 ± 0 n=2 > 4 cm % lipid = 50 ± 11 n=2 0%

20%

40%

60%

80%

100%

Percent of each lipid class by weight

wax esters triglycerides free fatty acids cholesterol

Figure 2. Lipid classes (i.e., wax esters, triglycerides, free fatty acids, cholesterol and phospholipids) determined in each layer of killer whale blubber by depth (i.e., 0-2 cm, 2-4 cm, > 4 cm).

phospholipids

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Fatty acid profiles White whale C I-73

layer nearest epidermis

100%

1st quarter 2nd quarter 3rd quarter 4th quarter

75%

50%

25%

0% 1

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83

layer nearest muscle

Resident killer whale L60

layer nearest epidermis

99.99%

0-2 cm 2-4 cm >4 cm

66.66%

33.33%

0.00% 1

3

5

7

9

11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83

layer nearest muscle

Figure 3. Proportions of 83 fatty acids (see Table 1 for identification) found in the blubber layers of a white whale from Cook Inlet (CI-73) and the resident killer whale L60. Blubber from CI-73 was divided into four layers and that from L60 was divided into thirds.

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Figure 4. Plot of the first three principal components derived from the fatty acid composition of (A) white whale blubber and (B) killer whale blubber. The quartered white whale blubber samples are grouped by animal (evenly dashed ovals) and by stock (unevenly dashed ovals). Killer whale blubber samples from each body position (individual symbols) were divided into thirds and grouped by depth from epidermis (solid ovals) and by animal (dashed ovals). The percent of the total variation among samples explained by each principal component is given on the label for each axis. Depth 1 is the layer closest to the epidermis and depth 4 is closest to the muscle. 21

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Figure 5. Plot of the first three principal components derived from the OC composition of (A) white whale blubber and (B) killer whale blubber . The quartered white whale blubber samples are grouped by animal (dashed ovals) and by stock (unevenly dashed ovals). Killer whale blubber samples from each body position (individual symbols) were divided into thirds and grouped by depth from epidermis (solid ovals) and by animal (dashed ovals). The percent of the total variation among samples explained by each principal component is given on the label for each axis. Depth 1 is the layer closest to the epidermis and depth 4 is closest to the muscle. 22

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