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The measurement of glucocorticoid hormones, such as cortisol, is a technique being increasingly used as a welfare or stress indicator (Mormède et al., 2007; Palme, 2019). In response to perceived stressors, these hormones are released into the bloodstream through activation of the hypothalamic–pituitary–adrenal (HPA) axis. The release of cortisol, a principal glucocorticoid in many mammals, enables the body to cope with threatening or demanding situations. Although this adaptive response is usually beneficial for the animal, chronic activation of the HPA axis and long-term cortisol elevations can reduce growth and reproduction, compromise the immune system, and impair the individual's ability to respond to subsequent stressors (Moberg & Mench, 2000). info:eu-repo/semantics/publishedVersion

Little is known of the movement or presence of unregulated, emerging contaminants in top predators. The aim of the present study was to conduct the first screening of legacy and emerging contaminants in multiple tissues of killer whales (Orcinus orca) from Norway and investigate tissue partitioning and maternal transfer. Blubber was collected from 8 killer whales in 2015 to 2017, in addition to muscle from 5 of the individuals, and kidney, liver, heart, and spleen from a neonate. We screened for 4 unregulated brominated flame retardants and found pentabromotoluene (PBT) and hexabromobenzene (HBB) at low levels in the blubber of all individuals (median PBT 0.091 ng/g lipid wt, median HBB 1.4 ng/g lipid wt). Levels of PBT and HBB (wet wt) were twice as high in the blubber than the muscle for each individual, confirming preferential accumulation in lipid-rich tissues. Perfluoroalkyl substances and total mercury levels were lower in the neonate than adults, suggesting less efficient maternal transfer of these substances. Polychlorinated biphenyl levels in blubber exceeded the threshold for onset of physiological effects (9 µg/g lipid wt) in 7 of the 8 whales, including the neonate. The presence of PBT and HBB in the neonate is the first evidence of maternal transfer of these unregulated contaminants in marine mammals. Our results are relevant for the continued environmental monitoring of contaminants in the Arctic. Environ Toxicol Chem 2021;40:1850–1860. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Arctic climate change poses serious threats to polar bears (Ursus maritimus) as reduced sea ice makes seal prey inaccessible and marine ecosystems undergo bottom-up reorganization. Polar bears' elongated skulls and reduced molar dentition, as compared to their sister species the grizzly bear (Ursus arctos), are adaptations associated with hunting seals on sea ice and a soft, lipid-rich diet of blubber and meat. With significant declines in sea ice, it is unclear if and how polar bears may be altering their diets. Clarifying polar bear dietary responses to changing climates, both today and in the past, is critical to proper conservation and management of this apex predator. This is particularly important when a dietary strategy may be maladaptive. Here, we test the hypothesis that hard-food consumption (i.e., less preferred foods including bone), inferred from dental microwear texture analysis, increased with Arctic warming. We find that polar bears demonstrate a conserved absence of hard-object feeding in Alaska through time (including approximately 1000 years ago), until the 21st century, consistent with a highly conserved and specialized diet of soft blubber and flesh. Notably, our results also suggest that some 21st-century polar bears may be consuming harder foods (e.g., increased carcass utilization, terrestrial foods including garbage), despite having skulls and metabolisms poorly suited for such a diet. Prior to the 21st century, only polar bears with larger mandibles demonstrated increased hard-object feeding, though to a much lower degree than closely related grizzly bears which regularly consume mechanically challenging foods. Polar bears, being morphologically specialized, have biomechanical constraints which may limit their ability to consume mechanically challenging diets, with dietary shifts occurring only under the most extreme scenarios. Collectively, the highly specialized diets and cranial morphology of polar bears may severely limit their ability to adapt to a warming Arctic.

We would like to thank the funders: the Department for Environment Food and Rural Affairs and Marine Scotland, for a number of long‐term monitoring contracts awarded to SMASS, the Sea Mammal Research Unit National Capability Funding from the Natural Environment Research Council (grant no. SMRU 10001), and the Strategic Environmental Research and Development Program (award RC‐2337). Measuring the energy stores, or body condition, of cetaceans is vital for monitoring population health. Cetaceans are exposed to a range of anthropogenic stressors, including, for example, noise, contaminants, habitat degradation, reduced prey availability, ship strike, and entanglement. Tools to assess body condition in these inaccessible and vulnerable animals are required to better understand the energetic consequences of anthropogenic stressors that can impact population health and, ultimately, conservation status.Remotely obtained, dart biopsy samples are becoming an increasingly standard method of tissue collection from live, large cetaceans. Here, the potential applicability of using total lipid content in such biopsy samples to estimate overall body condition was assessed using full‐depth dorsal blubber samples from stranded ziphiids (n = 8) and balaenopterids (n = 9). First, variation in total lipid content through the blubber depth was investigated to assess the representativeness of shallow‐depth dart biopsies taken from live animals. Second, how lipid content varies by species, cause of death, sex, age class, and morphometric indices of body condition was assessed to evaluate what information about individual energy stores can be gained from such analyses. Total lipid content in dorsal, shallow‐depth blubber biopsy samples from both cetacean families provides little information on overall body condition. Stratification of lipid content through the blubber layer in the balaenopterids means that superficial biopsy samples are not representative of the lipid stores available for mobilization through the rest of the tissue. A lack of variation in blubber lipid content, both within and between the ziphiid individuals, resulted in no ability to correlate these measures with morphometrics or other health, sex, or age class covariates.Other potential markers in the blubber from remote biopsy sampling should be explored in order to further develop robust tools for estimating the body condition of free‐ranging cetaceans. Postprint

RATIONALE The coupled analysis of δ13 C and δ15 N stable isotope values of blubber and skin biopsy samples is widely used to study the diet of free-ranging cetaceans. Differences in the lipid content of these tissues can affect isotopic variability because lipids are depleted in 13 C, reducing the bulk tissue 13 C/12 C. This variability in carbon isotope values can be accounted for either by chemically extracting lipids from the tissue or by using mathematical lipid normalisation models. METHODS This study examines (a) the effects of chemical lipid extraction on δ13 C and δ15 N values in blubber and skin of southern hemisphere humpback whales, (b) whether chemical lipid extraction is more favourable than mathematical lipid correction and (c) which of the two tissues is more appropriate for dietary studies. Strategic comparisons were made between chemical lipid extraction and mathematical lipid correction and between blubber and skin tissue δ13 C and δ15 N values, as well as C:N ratios. Six existing mathematical normalisation models were tested for their efficacy in estimating lipid-free δ13 C for skin. RESULTS Both δ13 C and δ15 N values of lipid-extracted skin (δ13 C: -25.57‰, δ15 N: 6.83‰) were significantly higher than those of bulk skin (δ13 C: -26.97‰, δ15 N: 6.15‰). Five of the six tested lipid normalisation models had small error terms for predicting lipid-free δ13 C values. The average C:N ratio of lipid-extracted skin was within the lipid-free range reported in other studies, whereas the average C:N ratio of blubber was higher than previously reported. CONCLUSIONS These results highlight the need to account for lipids when analysing δ13 C and δ15 N values from the same sample. For optimised dietary assessments using parallel isotope analysis from a single sample, we recommend the use of unextracted skin tissue. δ15 N values should be obtained from unextracted skin, whereas δ13 C values may be adequately lipid corrected by a mathematical correction.

In cetaceans, blubber is the primary and largest lipid body reservoir. Our current understanding about lipid stores and uses in cetaceans is still limited, and most studies only focused on a single narrow snapshot of the lipidome. We documented an extended lipidomic fingerprint in two cetacean species present in northern Norway during wintertime. We were able to detect 817 molecular lipid species in blubber of killer whales (Orcinus orca) and humpback whales (Megaptera novaeangliae). The profiles were largely dominated by triradylglycerols in both species and, to a lesser extent, by other constituents including glycerophosphocholines, phosphosphingolipids, glycerophosphoethanolamines, and diradylglycerols. Through a unique combination of traditional statistical approaches, together with a novel bioinformatic tool (LION/web), we showed contrasting fingerprint composition between species. The higher content of triradylglycerols in humpback whales is necessary to fuel their upcoming half a year fasting and energy‐demanding migration between feeding and breeding grounds. In adipocytes, we assume that the intense feeding rate of humpback whales prior to migration translates into an important accumulation of triacylglycerol content in lipid droplets. Upstream, the endoplasmic reticulum is operating at full capacity to supply acute lipid storage, consistent with the reported enrichment of glycerophosphocholines in humpback whales, major components of the endoplasmic reticulum. There was also an enrichment of membrane components, which translates into higher sphingolipid content in the lipidome of killer whales, potentially as a structural adaptation for their higher hydrodynamic performance. Finally, the presence of both lipid‐enriched and lipid‐depleted individuals within the killer whale population in Norway suggests dietary specialization, consistent with significant differences in δ15N and δ13C isotopic ratios in skin between the two groups, with higher values and a wider niche for the lipid‐enriched individuals. Results suggest the lipid‐depleted killer whales were herring specialists, while the lipid‐enriched individuals might feed on both herrings and seals., We documented an extended lipidomic fingerprint in two cetacean species. We were able to detect 817 molecular lipid species in blubber of killer whales and humpback whales. The profiles are described in terms of lipid classes. Through a unique combination of traditional statistical approaches and a novel bioinformatic tool, we showed contrasting fingerprint composition between species. The higher content of triradylglycerols in humpback whales is necessary to fuel their migration. The intense feeding rate of humpback whales prior to migration translates into an important accumulation of triacylglycerol content in lipid droplets. Upstream, the endoplasmic reticulum is operating at full capacity to supply acute lipid storage. There was also an enrichment of membrane components, which translates into higher sphingolipid content in the lipidome of killer whales, potentially as a structural adaptation for their higher hydrodynamic performance. The presence of both lipid‐enriched and lipid‐depleted individuals within the killer whale population in Norway suggests dietary specialization, consistent with significant differences in δ15N and δ13C isotopic ratios in skin tissue. This suggests the lipid‐depleted killer whales were herring specialists, while the lipid‐enriched individuals might feed on both herrings and seals.

Odontocetes have specialized mandibular fats, the extramandibular (EMFB) and intramandibular fat bodies (IMFB), which function as acoustic organs, receiving and channeling sound to the ear during hearing and echolocation. Recent strandings of beaked whales suggest that these fat bodies are susceptible to nitrogen (N2 ) gas embolism and empirical evidence has shown that the N2 solubility of these fat bodies is higher than that of blubber. Since N2 gas will diffuse from blood into tissue at any blood/tissue interface and potentially form gas bubbles upon decompression, it is imperative to understand the extent of microvascularity in these specialized acoustic fats so that risk of embolism formation when diving can be estimated. Microvascular density was determined in the EMFB, IMFB, and blubber from 11 species representing three odontocete families. In all cases, the acoustic tissues had less (typically 1/3 to 1/2) microvasculature than did blubber, suggesting that capillary density in the acoustic tissues may be more constrained than in the blubber. However, even within these constraints there were clear phylogenetic differences. Ziphiid (Mesoplodon and Ziphius, 0.9 ± 0.4% and 0.7 ± 0.3% for EMFB and IMFB, respectively) and Kogiid families (1.2 ± 0.2% and 1.0 ± 0.01% for EMFB and IMFB, respectively) had significantly lower mean microvascular densities in the acoustic fats compared to the Delphinid species (Tursiops, Grampus, Stenella, and Globicephala, 1.3 ± 0.3% and 1.3 ± 0.3% for EMFB and IMFB, respectively). Overall, deep-diving beaked whales had less microvascularity in both mandibular fats and blubber compared to the shallow-diving Delphinids, which might suggest that there are differences in the N2 dynamics associated with diving regime, phylogeny, and tissue type. These novel data should be incorporated into diving physiology models to further understand potential functional disruption of the acoustic tissues due to changes in normal diving behavior.

Nerves that supply the floor of the oral cavity in rorqual whales are extensible to accommodate the dramatic changes in tissue dimensions that occur during "lunge feeding" in this group. We report here that the large nerves innervating the muscle component of the ventral grooved blubber (VGB) in fin whales are branches of cranial nerve VII (facial nerve). Therefore, the muscles of the VGB are homologous to second branchial arch derived muscles, which in humans include the muscles of "facial expression." We speculate, based on the presence of numerous foramina on the dorsolateral surface of the mandibular bones, that general sensation from the VGB likely is carried by branches of the mandibular division (V3) of cranial nerve V (trigeminal nerve), and that these small branches travel in the lipid-rich layer directly underlying the skin. We show that intercostal and phrenic nerves, which are not extensible, have a different wall and nerve core morphology than the large VGB nerves that are branches of VII. Although these VGB nerves are known to have two levels of waviness, the intercostal and phrenic nerves have only one in which the nerve fascicles in the nerve core are moderately wavy. In addition, the VGB nerves have inner and outer parts to their walls with numerous large elastin fibers in the outer part, whereas intercostal and phrenic nerves have single walls formed predominantly of collagen. Our results illustrate that overall nerve morphology depends greatly on location and the forces to which the structures are exposed. Anat Rec, 300:1963-1972, 2017. © 2017 Wiley Periodicals, Inc.

Rationale Analysis of steroids from precious blubber biopsies obtained from marine mammals, especially endangered species, can provide valuable information on their endocrine status. Challenges with currently used ELISA methodology include lack of absolute quantitation and incompatibility with multiple steroids analysis due to limited biopsy mass. Development of a sensitive, accurate analytical method for this purpose is critical. Methods A nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) method was validated for sensitive, specific and quantitative analysis of three steroid hormones, without derivatization, extracted from 50 mg blubber samples. Data was acquired with an LTQ XL ion trap mass spectrometer in positive ion mode, using single reaction monitoring. All three steroids were analyzed in a single run. Cholic acid was used as a surrogate internal standard for quantitation due to its steroidal structure and lack of measurable endogenous levels in blubber. Results The lowest limits of quantitation for progesterone, testosterone, and hydrocortisone were significantly improved compared to previous studies using conventional LC/MS/MS. The lowest limit of detection was 7 fg/μL using a 1 μL injection volume. Calibration curves for steroid quantification showed good linearity (r2 >0.99) between 14 and 3620 fg/μL, and accuracy was

Stress hormones, released into circulation as a consequence of disturbance, are classically assayed from blood samples but may also be detected in a variety of matrices. Blubber and fecal samples can be remotely collected from free-ranging cetaceans without the confounding hormone elevations associated with chase, capture, and handling required to collect blood samples. The relationship between cortisol concentrations in circulation with that of blubber and feces, however, is unknown. To assess these associations, we elevated cortisol by orally administering hydrocortisone for five days in five bottlenose dolphins. Voluntary blood and fecal samples were collected daily; blubber biopsies were collected on day one, just prior to hydrocortisone administration, and days three and five of hydrocortisone administration. We evaluated subsequent changes in several circulating stress hormones as well as cortisol and glucocorticoid metabolites in blubber and feces, respectively. There was a significant association between cortisol levels in serum and in blubber (F1,12.7 = 14.3, P < 0.01, mR2 = 0.57) despite substantial variability in blubber cortisol levels. Counterintuitively, fecal cortisol metabolite levels were inversely related to serum cortisol. The relationship between serum and blubber cortisol levels suggests blubber samples from remote sampling may be useful to detect stress loads in this species.

Polychlorinated biphenyls (PCBs) from the commercial mixture Aroclor 1268 were historically released into the Turtle-Brunswick River estuary (southeastern Georgia, USA) from industrial operations. Sum PCBs (ΣPCBs) in blubber samples from Turtle-Brunswick River estuary bottlenose dolphins (Tursiops truncatus) have been reported at concentrations more than 10-fold higher than those observed in dolphins from adjacent regional estuaries. Given that toxicity data specific to Aroclor 1268 and applicable to marine mammals are limited, predicting the toxic effects of Aroclor 1268 in dolphins is uncertain, particularly because of its unique congener profile and associated physiochemical characteristics compared with other PCB mixtures. American mink (Neovison vison) were chosen as a surrogate model for cetaceans to develop marine mammalian PCB toxicity benchmarks. Mink are a suitable surrogate species for cetaceans in toxicity studies because of similarities in diet and taxonomic class, and a characteristic sensitivity to PCBs provides a potential safety factor when using mink toxicology data for cross-species extrapolations. Effects of dietary exposure to Aroclor 1268 on reproduction, growth, and mortality in mink were compared with both a negative control and a positive control (3,3',4,4',5-pentachlorobiphenyl, PCB 126). Aroclor 1268 dietary ΣPCB concentrations ranged from 1.8 µg/g feed wet weight to 29 µg/g feed wet weight. Whelp success was unaffected by Aroclor 1268 exposure at any level. Treatment mean litter size, kit growth, and kit survival were adversely affected relative to the negative control at dietary ΣPCB concentrations of 10.6 µg/g feed wet weight and greater.

Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River Estuary (TBRE, Georgia, USA) suggests the potential for adverse effects. However, PCBs in TBRE dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain due to the mixture's unique composition and associated physiochemical characteristics. These differences suggest toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126) positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 to 29 µg/g ww. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation (SEP) jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of SEP jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor (AhR)-mediated pathway are associated with Aroclor 1268 exposure. This article is protected by copyright. All rights reserved

Monitoring reproductive rates in wild cetaceans is a crucial step towards adequate management of their populations. Their reproductive hormone profiles can be of importance, especially when combined with behavioural or life history monitoring. Sampling biological matrices of wild, free-ranging cetaceans can be a difficult task. The purposes of this review are: to describe briefly the different methods available for monitoring the reproductive endocrine parameters of wild and captive cetaceans, and to evaluate the feasibility of using various matrices – blood, urine, faeces, saliva, bubbler, muscle, ocular secretions, blow (respiratory vapour), milk, baleen, and earplug materials – to measure sex hormones in free-ranging cetaceans. We performed a retrospective analysis of the measurement and sample collection methods and the most commonly used matrices for constructing reproductive hormone profiles. The PubMed and Google Scholar data bases were used, and scientific articles and book chapters published from 1980 to the present were considered. In the past, the most commonly used matrix for measuring hormones of free-ranging cetaceans was blood. This has been changing since studying live whales became more common: researchers have dispensed with using blood samples from dead animals. Matrices such as blow, which can be obtained non-invasively, and other matrices that can be obtained with minimal invasion, such as blubber, appear to be feasible options for monitoring the hormones of live, free-ranging cetaceans.

Elevated levels of persistent organic pollutants (POPs) have been reported in tropical Pacific Island cetaceans and their environment. In addition, recent health concerns in cetacean populations have warranted investigation into potential physiological effects from POP exposure for this region. Cytochrome P450 1A1 (CYP1A1) is a candidate for examining such effects. This well-studied biomarker of exposure and effect was examined in stranded cetacean liver using immunoblot (n = 39, 16 species) and blubber using immunohistochemistry (n = 23, 10 species). Paired tissue samples allowed for CYP1A1 comparisons not only between species but also within each individual animal to examine differences between tissue types. Liver CYP1A1 expression correlated positively and significantly with blubber concentrations of all POP categories (n = 39, p 0.100). Among Stenella species, liver CYP1A1 tissue expression was correlated negatively with the sum of all blubber layer endothelial cell CYP1A1 expression (n = 14, p = 0.049). Overall, elevated expression of liver CYP1A1 confirms its use as a biomarker of POP exposure to cetaceans stranded in the tropical Pacific basin. Environ Toxicol Chem 2015;34:1989–1995. © 2015 SETAC

Cetacean evolution was shaped by an extraordinary land-to-sea transition in which the ancestors of whales became fully aquatic. As part of this transition, these mammals evolved unusually thick blubber which acts as a metabolic reservoir as well as an insulator and provides buoyancy and streamlining. This study describes blubber stratification and correlates it to seasonal variation, feeding patterns, and ontogeny in an arctic-adapted mysticete, the bowhead whale (Balaena mysticetus). Bowheads are unique among mammals for possessing the largest known blubber stores. We found that adipocyte numbers in bowheads, like other mammals, do not vary with season or feeding pattern but that adipocyte size and structural fiber densities do vary with blubber depth. Anat Rec, 298:1416–1423, 2015. © 2015 Wiley Periodicals, Inc.

Summary 1. Current studies of fine-scale baleen whale diving and foraging behaviour rely on archival suction cup tags that remain attached over time scales of hours. However, skin irregularities can make suction cup attachment unreliable, and traditional pole deployment of suction cup tags is challenging in moderate sea conditions or when whales are evasive. 2. We developed a new tag attachment to overcome these limitations. The attachment features a short (6� 5– 7� 5 cm) needle that anchors in the whale’s dermis (epidermis and blubber) to which a free-floating tag is attached via a severable tethered link. The needle, tag and a detachable ‘carrier rocket’ with fletching are fitted together to form a projectile that can be deployed at distances of up to 20 m using a compressed-air launcher. A corrosive release mechanism allows the tag to separate from the needle after a specified period of time so that the tag can be recovered. 3. The dermal attachment was evaluated during a study of humpback whales (Megaptera novaeangliae )i n the Gulf of Maine and then subsequently deployed on bowhead whales (Balaena mysticetus) near Barrow, Alaska. Monitoring of tagged humpback whales indicated that the needle was shed several days after deployment, the attachment site healed shortly thereafter, and there were no discernible behavioural or health effects over time scales of days to months after tagging. Bowhead whales showed little immediate reaction to tagging; the most common response was a prolonged dive right after tag deployment. On average, respiration rates of tagged bowhead whales were elevated after tag attachment, but returned to the same rate as undisturbed bowheads within 1–1� 5h . 4. When compared to suction cups, the dermal anchor provided a more reliable attachment and it can be applied from greater distances and in rougher sea conditions; it is therefore a useful alternative in circumstances where suction cup tags cannot be easily deployed.

ELIZABETH T. VU, Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive La Jolla, California 92093, U.S.A.; CASEY CLARK, Moss Landing Marine Laboratories, 8272 Moss Landing Road Moss Landing, California 95039, U.S.A.; KRISTA CATELANI, and NICHOLAS M. KELLAR, Southwest Fisheries Science Center, NOAA, 8901 La Jolla Shores Drive, La Jolla, California 92037, U.S.A.; JOHN CALAMBOKIDIS, Cascadia Research Collective, 2181⁄2 4th Avenue West, Olympia, Washington 98501, U.S.A.

Pacific walruses may be unable to meet caloric requirements in the changing Arctic ecosystem, which could affect body condition and have population-level consequences. Body condition has historically been monitored by measuring blubber thickness over the xiphoid process (sternum). This may be an unreliable condition index because blubber at other sites along the body may be preferentially targeted to balance energetic demands. Animals in aquaria provided an opportunity for controlled study of how blubber topography is altered by caloric intake. Morphology, body mass, blubber thickness (21 sites), and caloric intake of five mature, nonpregnant, nonlactating female walruses were measured monthly (12 month minimum). Body condition (mass × standard length−1) was described by a model that included caloric intake and a seasonal effect, and scaled positively with estimates of total blubber mass. Blubber thicknesses (1.91–10.69 cm) varied topographically and were similar to values reported for free-ranging female walruses. Body condition was most closely related to blubber thickness measured dorsomedially in the region of the anterior insertion of the pectoral flippers (shoulders); sternum blubber thickness was a relatively poor indicator of condition. This study demonstrates the importance of validating condition metrics before using them to monitor free-ranging populations.

In December 2009 seven male sperm whales (Physeter macrocephalus) stranded along the Adriatic coast of Southern Italy. Genetic and photo-identification data indicated that these specimens belonged to the Mediterranean Sea population. Complete necropsies were performed on the three best preserved animals. Blubber and muscle samples were collected from all seven specimens for contaminants analysis (DDTs, PCBs and PAHs) and biomarker responses (CYP1A1 and CYP2B) were evaluated from the three better preserved animals using western blot analysis. Fibroblast cell cultures from two specimens were set up by culturing cell lines from integument tissue, to explore the susceptibility of these marine mammals to different xenobiotic compounds. The levels of CYP1A1 and CYP2B, proteins that are induced by some POPs (DDTs and PCBs) and PAHs were detected using the indirect immunofluorescence assay. The results showed relatively high levels of contaminants, in particular from organochlorine xenobiotics, and strong responses of CYP1A1 and CYP2B reflecting toxicological stress of these sperm whales. This is not considered to be their cause of death, but may have contributed to lowering the defences of their immune system. Copyright © 2014 John Wiley & Sons, Ltd.

Humpback whales are iconic mammals at the top of the Antarctic food chain. Their large reserves of lipid-rich tissues such as blubber predispose them to accumulation of lipophilic contaminants throughout their lifetime. Changes in the volume and distribution of lipids in humpback whales, particularly during migration, could play an important role in the pharmacokinetics of lipophilic contaminants such as the organochlorine pesticide hexachlorobenzene (HCB). Previous models have examined constant feeding and nonmigratory scenarios. In the present study, the authors develop a novel heuristic model to investigate HCB dynamics in a humpback whale and its environment by coupling an ecosystem nutrient-phytoplankton-zooplankton-detritus (NPZD) model, a dynamic energy budget (DEB) model, and a physiologically based pharmacokinetic (PBPK) model. The model takes into account the seasonal feeding pattern of whales, their energy requirements, and fluctuating contaminant burdens in the supporting plankton food chain. It is applied to a male whale from weaning to maturity, spanning 20 migration and feeding cycles. The model is initialized with environmental HCB burdens similar to those measured in the Southern Ocean and predicts blubber HCB concentrations consistent with empirical concentrations observed in a southern hemisphere population of male, migrating humpback whales. Results show for the first time some important details of the relationship between energy budgets and organochlorine pharmacokinetics.

Narwhals (Monodon monoceros) are sentinel species in the Arctic and to investigate marine food web changes from 1982–2011 we examined diet using fatty acids, δ15N, and δ13C, in narwhals from Baffin Bay (BB) and northern Hudson Bay (NHB). We predicted temporal changes would be greater in NHB due to a significant reduction in summer ice cover. In NHB, δ15N significantly increased, δ13C displayed a parabolic trend, and fatty acids gradually shifted, albeit not significantly, over time. δ15N was stable, δ13C decreased, and fatty acids significantly changed over time in BB. Stable isotope mixing models indicated a dietary reduction in capelin and increase in Greenland halibut from 1994–2000 to 2006–2011 in BB, while capelin was an important dietary component for narwhals in NHB in recent years (2006–2011). These dietary changes may be attributed to changes in sea ice and narwhal migration. Seasonal dietary changes, as evidenced by changes in blubber fatty acids and skin and muscle stable isotopes, were not as apparent in the NHB population, which may be indicative of a reduced migratory distance. Long-term monitoring of narwhal diet and migratory patterns associated with reduced sea ice provides invaluable information about how the marine ecosystem will redistribute with global warming.

Summary: Understanding how female body condition (FBC) influences foetal development, and hence offspring production, is fundamental for our understanding of species reproductive physiology and life history. We investigated the effects of FBC on foetus growth in common minke whales. Pregnant minke whales were sampled around Iceland during the summer feeding seasons between 2003 and 2007 and the length and weight of their foetuses were measured. FBC was modelled as the relative difference between measured blubber volume and the average expected blubber volume of individual whales. Generalized linear models were used to test the effect of FBC on foetus length, while accounting for the daily growth in foetus size through gestation, as well as other covariates. Foetus length increased curvilinearly through the study period at an average rate of 0·964 cm day-1 (SE = 0·138). The effect of FBC on foetal length was nonlinear, showing an almost linear positive relationship for females in poorer body condition (FBC 0). The curvilinear relationship between FBC and foetus growth was confirmed by fitting a generalized additive model and by running separate analyses on two subsets of data separating females in poorer and better condition. Our findings suggest that females that are in poorer body condition reduce their energetic investment in their foetus proportionately to their condition, most likely to help maintain a high survival probability. That foetus length did not increase for females in better body condition suggests that females have an upper limit on the amount of energy they will or can invest in their foetus. Reducing the size at birth by reducing the gestation period is also unlikely, because the reproductive cycle of balaenopterids is strongly linked to their seasonal migration between feeding grounds and breeding grounds. This study is the first to demonstrate that FBC can affect foetus growth in a capital breeding mysticete.

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ15N and δ13C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ15N value within 3 d at 20°C. Storage at −20°C was as effective as −80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze-dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO-salt solution, frozen or at room temperature, did not significantly change the δ15N and δ13C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.

Remote biopsy sampling has been used for almost 40 yr (Winn et al. 1973) to obtain tissue samples for various analyses, and is particularly important because most cetaceans are difficult or impossible to capture safely at sea (Noren and Mocklin 2011). It is generally assumed that there are few, if any, detrimental effects on the fitness of sampled animals. In support of this assumption, there has only been one documented case of death of a cetacean from a biopsy dart (Bearzi 2000), although it is possible deaths have occurred that have not been described in the published literature. Failure of the dart stopper and the poor body condition of the common dolphin (Delphinus delphis) were believed responsible for the death of this animal, as the dart penetrated through its thin blubber layer. Many studies have reported qualitative and semiquantitative descriptions of the reactions of individual whales and dolphins to biopsy attempts. In most cases, descriptions of the response to biopsy sampling used qualitative and sometimes subjective observations, all limited to the behavior of sampled animals at the surface. Furthermore, the criteria used to assess response varied across studies, hindering comparisons across species and sites. Recently Noren and Mocklin (2011) performed an extensive and very useful review of the behavioral and physical reactions of mysticetes and odontocetes to biopsy attempts and standardized categories of behavioral response. They concluded that most odontocetes exhibit a low level of response (e.g., short-term startle response, immediate dive, increase of speed), while low and moderate levels of responses were equally prevalent for mysticetes. Their review also revealed that wounds from biopsy darts typically heal quickly, with no signs of infection. It is apparent from Noren and Mocklin’s (2011) review that there has been little quantitative analysis of the behavioral reactions of cetaceans to biopsy sampling due

Many Arctic animals carry high body burdens of organochlorine contaminants (OCs) as a result of long-range transport of persistent pollutants. It has been shown that seasonal mobilization of body fat in these species results in increased blood concentration of OCs. The authors investigated OC assimilation, tissue distribution, and biotransformation in farmed Arctic fox (Vulpes lagopus) continuously fed a diet containing contaminated minke whale blubber or lard (control) from 8 wk of age in August 2003, until sampling when they were at their fattest (in November 2004) and leanest (in June 2005). Markedly higher tissue (liver, adrenals, brain, and blood) OC levels were found in June than in November despite low exposure to OCs during emaciation, suggesting that OCs had been redistributed from adipose tissues to vital organs. There were no differences in the activities of hepatic biotransforming enzymes between exposed fat and control fat foxes, except for 16α-hydroxylation, which was higher in exposed fat foxes. In emaciated foxes, ethoxyresorufin activity was higher in exposed than in control foxes, indicating an enhanced potential for toxicity of OCs with emaciation. Lower activities of 6β- and 2β-hydroxylation were found in lean than in fat foxes, irrespective of OC treatment. The results show that emaciation increase the toxic potential of accumulated OCs and emphasize that body adiposity must be considered when time-trend analyses, risk assessments, and effect studies are designed. Environ Toxicol Chem 2013;32:1784–1792. © 2013 SETAC

RATIONALE: Stable isotope values (?(13)C and ?(15)N) of darted skin and blubber biopsies can shed light on habitat use and diet of cetaceans, which are otherwise difficult to study. Non-dietary factors affect isotopic variability, chiefly the depletion of (13)C due to the presence of (12)C-rich lipids. The efficacy of post hoc lipid-correction models (normalization) must be tested. METHODS: For tissues with high natural lipid content (e.g., whale skin and blubber), chemical lipid extraction or normalization is necessary. C:N ratios, ?(13)C values and ?(15)N values were determined for duplicate control and lipid-extracted skin and blubber of fin (Balaenoptera physalus), humpback (Megaptera novaeangliae) and minke whales (B. acutorostrata) by continuous-flow elemental analysis isotope ratio mass spectrometry (CF-EA-IRMS). Six different normalization models were tested to correct ?(13)C values for the presence of lipids. RESULTS: Following lipid extraction, significant increases in ?(13)C values were observed for both tissues in the three species. Significant increases were also found for ?(15)N values in minke whale skin and fin whale blubber. In fin whale skin, the ?(15)N values decreased, with no change observed in humpback whale skin. Non-linear models generally out-performed linear models and the suitability of models varied by species and tissue, indicating the need for high model specificity, even among these closely related taxa. CONCLUSIONS: Given the poor predictive power of the models to estimate lipid-free ?(13)C values, and the unpredictable changes in ?(15)N values due to lipid-extraction, we recommend against arithmetical normalization in accounting for lipid effects on ?(13)C values for balaenopterid skin or blubber samples. Rather, we recommend that duplicate analysis of lipid-extracted (?(13)C values) and non-treated tissues (?(15)N values) be used.

Blubber, a specialized form of subdermal adipose tissue, surrounds marine mammal bodies. Typically, adipose tissue is perfused by capillaries but information on blubber vascularization is lacking. This study's goals were to: 1) describe and compare the microvasculature (capillaries, microarterioles, and microvenules) of blubber across odontocete species; 2) compare microvasculature of blubber to adipose tissue; and 3) examine relationships between blubber's lipid composition and its microvasculature. Percent microvascularity, distribution, branching pattern, and diameter of microvessels were determined from images of histochemically stained blubber sections from shallow-diving bottlenose dolphins (Tursiops truncatus), deeper-diving pygmy sperm whales (Kogia breviceps), deep-diving beaked whales (Mesoplodon densirostris; Ziphius cavirostris), and the subdermal adipose tissue of domestic pigs (Sus scrofa). Tursiops blubber showed significant stratification in percent microvascularity among the superficial, middle, and deep layers and had a significantly higher percent microvascularity than all other animals analyzed, in which the microvasculature was more uniformly distributed. The percent microvasculature of Kogia blubber was lower than that of Tursiops but higher than that of beaked whales and the subdermal adipose tissue of domestic pigs. Tursiops had the most microvascular branching. Microvessel diameter was relatively uniform in all species. There were no clear patterns associating microvascular and lipid characteristics. The microvascular characteristics of the superficial layer of blubber resembled the adipose tissue of terrestrial mammals, suggesting some conservation of microvascular patterns in mammalian adipose tissue. The middle and deep layers of blubber, particularly in Tursiops, showed the greatest departure from typical mammalian microvascular arrangement. Factors such as metabolics or thermoregulation may be influencing the microvasculature in these layers. © J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.

Stable carbon and nitrogen isotope ratios (δ13C and δ15N) of serum, red blood cells (RBC), muscle, and blubber were measured in captive and wild northeast Pacific harbor seals (Phoca vitulina richardii) at three coastal California sites (San Francisco Bay, Tomales Bay, and Channel Islands). Trophic discrimination factors (ΔTissue-Diet) were calculated for captive seals and then applied in wild counterparts in each habitat to estimate trophic position and feeding behavior. Trophic discrimination factors for δ15N of serum (+3.8‰), lipid-extracted muscle (+1.6‰), and lipid-blubber (+6.5‰) are proposed to determine trophic position. An offset between RBC and serum of +0.3‰ for δ13C and −0.6‰ for δ15N was observed, which is consistent with previous research. Specifically, weaner seals (

Three adult captive Steller sea lions (SSL; two females, one male) housed in Alaska were longitudinally sampled for organochlorine contaminant (OC) analysis in both blubber (n= 19) and blood (n= 69) over a 2 yr period (March 2001 to March 2003). Blood OC concentrations were similar between individuals, and exhibited significant increases in summer months (July through September) relative to winter (January through March). Additionally, paired blood and blubber sample (n= 18) OC were significantly related for all animals. The relationship of blubber OC concentrations to lipid content was significant in all animals. Although limited to a small number of animals, our study results indicate that in SSLs, blood OC were both consistent among all animals and likely changed in association with physiologically driven metabolism of blubber.

1. Using measurements of naturally occurring stable isotopes in animal tissues is useful for monitoring diets of wide-ranging species that would otherwise be logistically difficult to evaluate. However, differential metabolic routing of macromolecules within a consumer can be problematic when using stable isotope analysis of bulk tissues to trace dietary input. 2. We used stable isotope (δ13C, δ15N) analysis to examine polar bear Ursus maritimus diet, which includes both lipid-rich blubber and the proteinaceous tissues of their marine mammal prey. Because the proportion of proteins and lipids consumed may depend on prey type and size, it was necessary to consider metabolic routing of these macromolecules separately in isotope mixing models. 3. Bayesian mixing models (MixSIR, version 1.04) were used to separately estimate protein (δ13C, δ15N) and lipid (δ13C) dietary inputs. We used existing knowledge of the relative lipid and protein intake for polar bears and isotopic information from both macromolecules to estimate overall diet composition. 4. The results for both male and female polar bears indicated that smaller prey (e.g. ringed seal Pusa hispida) contributed the largest proportion to the protein-metabolic pathway. In contrast, the largest proportion of the lipid-metabolic pathway for both sexes tended to consist of larger prey (e.g. bearded seal Erignathus barbatus). 5. The diet composition of male polar bears consisted of more large than small prey. Diet estimates for females overlapped to some degree with males but tended to consist of less large prey. 6. Synthesis and applications. Monitoring polar bear diet may help determine the effects of climate-induced environmental changes in Arctic marine ecosystems including shifts in prey composition. Additionally, tracing origins of anthropogenic pollutants is currently a priority for wildlife managers concerned with the health of marine mammals. However, our results indicate using stable isotopes to infer dietary inputs when proportions of macromolecules fluctuate amongst food sources requires the sampling and analysis of multiple tissues representing distinct macromolecular metabolic pathways. In such cases, utilizing only proteinaceous tissues for analysis will result in erroneous dietary source estimates and inaccuracies when examining trophic-level transfer of contaminants, especially those that are lipophylic.

Persistent organic pollutants (POPs) have been associated with adverse health effects in marine mammals. However, the complex mixtures to which free-ranging populations are exposed constrain the elucidation of cause-and-effect relationships between specific POPs and the observed health risks. In this study, we 1) assembled data from studies showing polychlorinated biphenyl (PCB)-associated effects on the health of free-ranging harbor seals in the northeastern Pacific Ocean, 2) carried out additional POP analyses on seal samples to broaden the available data on contaminant residues, and 3) derived estimates of individual POPs and their toxic risks. Taken together, these components were used to generate a new toxicity reference value (TRV) for the protection of marine mammal health. In this case study of seals in British Columbia, Canada, and Washington State, USA, PCBs were the single most abundant POP and were correlated with several adverse health effects. PCB exposures consistently exceeded regulatory toxicity thresholds for fish-eating wildlife. Nursing seal pups were at particular risk, reflecting their greatly increased dietary intake of PCBs and their sensitivity to developmental toxicity. Based on the collective evidence obtained, we propose TRVs (consisting of 5% tissue residue concentration and dose) of 1.3 mg/kg lipid weight tissue residue in blubber and 0.05 mg/kg lipid weight tolerable daily intake in prey. Insofar as the TRVs are lower than previously established TRVs and regulatory guidelines, our study highlights the current underestimation of risks associated with PCBs in high-trophic-level wildlife. Integr Environ Assess Manag 2010;6:631–640. © 2010 SETAC

Few birds parasitize living tissue of other birds or mammals. At Golfo San Jose, Argentina, kelp gulls, Larus domincanus, fed on live southern right whale, Eubalaena australis, mothers, landing on their backs and pecking at peeling skin or gouging into the energy-rich hypodermal blubber layer. Most attacks occurred while mothers were resting at the surface in the central nursery area. The typical immediate reaction of mothers to gull attacks was to lower the mid-back and rapidly raise the head and tail stock. Less frequently mothers submerged quickly. After their initial response mothers traveled rapidly, usually underwater, or adopted resting postures that submerged the vulnerable midback area. Gulls attacked singly or in small groups and alternated between following a single pair of whales and attacking several pairs sequentially. The intensity of maternal responses to these attacks affects the energy budget of mothers and calves during a time when energy conservation is important. Subtle postural shifts to submerge the back require less energy than rapid, frantic fleeing. Since gulls feed on whales without causing fatal injury this relationship is termed parasitic.

A novel molecular technique was used to measure blubber testosterone (BT) in 114 male short-beaked common dolphins, Delphinus delphis, collected from incidental fishery bycatch and strandings. When these concentrations were compared across maturity states, the mean (± SEM) BT levels of mature D. delphis (14.3 ± 3.0 ng/g) were significantly higher than those of pubertal (2.5 ± 0.5 ng/g, P = 0.006) and immature animals (2.2 ± 0.3 ng/g, P < 0.0001). BT concentrations in mature males were significantly higher in summer months (53.9 ± 2.0 ng/g) than during the rest of the year (7.9 ± 0.69 ng/g, P < 0.0001), indicating reproductive seasonality. An analysis of BT in different anatomical locations showed that hormone concentrations were not homogenous throughout the body; the levels in the dorsal fin were significantly lower than in most other areas (F = 5.39, P = 0.043). Conversely, we found no significant differences in BT concentration with respect to subepidermal depth (F = 2.09, P = 0.146). Finally, testosterone levels in biopsies from 138 free-swimming male D. delphis, of unknown maturity state, sampled off California were found to be of concentrations similar to those from the fishery bycatch and stranding samples and revealed an analogous trend with respect to ordinal date. Little is known about androgen levels in cetaceans, and yet they are important components of male endocrinology that can provide pertinent data on reproduction, development, and seasonal reproductive trends. The increase of androgens during postnatal development is a key factor defining puberty in male mammals (Preslock 1980). Consequently, concentrations of androgens, particularly testosterone, are

The present study reports unique data on concentrations of several classes of organochlorine pesticides and polychlorinated biphenyls in blubber biopsies from healthy living fin whales (Balaenoptera physalus) from the Gulf of California, Mexico, one of the most isolated and unstudied population in the world. OC levels in this population were generally lower than levels reported in fin whales from other regions. The rank order of OCs were ∑DDTs (range from 300 to 2400 ng g−1 lw) > ∑PCBs (range from 40 to 290 ng g−1 lw) > ∑HCHs (range from

In his letter of criticism of our two recent papers on foraging habits of Arctic marine mammals (Thiemann et al. 2007, 2008), Grahl-Nielsen (2008) questions our interpretations and methods in using blubber fatty acids (FAs) as indicators of diet. The objections raised by Grahl-Nielsen are not new: they are the same arguments he has made for nearly a decade (Grahl-Nielsen 1999) and which have been refuted or addressed elsewhere in the literature (references below). Although FA analysis as an ecological tool has seen dramatic advancements in recent years, Grahl-Nielsen continues to ignore the substantial and growing body of scientific evidence that supports its use. It is unfortunate that we must repeat what has been thoroughly addressed and reviewed by a number of authors, including Budge et al. (2006) in this journal. However, because Grahl-Nielsen’s comments are so flawed, and because we have been given the opportunity to respond, we endeavor to address his specific objections as succinctly as possible. Grahl-Nielsen’s letter has two main contentions: the first is that because other factors besides diet (e.g., population, age, sex, condition, etc.) may influence the FA composition of marine mammal blubber, FA profiles cannot be used as an indicator of diet; concurrent with this argument, he states that only two captive experimental studies have ever been conducted and that the effect of dietary FAs on blubber FAs has never been established. The other main contention of the letter is that when FA profiles are used as an indicator of diet, only the inner portion of blubber should

The composition of predator adipose stores can provide important insights into foraging patterns and the ecological relationships among species. We determined the fatty acid (FA) composition of 843 blubber samples from 80 bearded seals (Erignathus barhatus), 33 harbor seals (Phoca vitulina), 239 harp seals (Pagophilus groenlandicus), 32 hooded seals (Cystophora cristata), 281 ringed seals (Phoca hispida), 53 walruses (Odobenus rosmarus rosmarus), 105 beluga whales (Delphinapterus leucas), and 20 narwhals (Monodon monoceros) across the Canadian Arctic to examine patterns of variability among and within species. FA signatures accurately distinguished phocid seals, walruses, and whales. Belugas and narwhals had the most similar FA signatures of any two species, suggesting substantial overlap in their diets, especially in the narwhal-wintering area off eastern Baffin Island. Among phocid seals, harp and hooded seals had the most similar FA signatures. Bearded seals were most similar to walruses, which was consistent with the benthic feeding habits of both species. Within species, geographic differences in FA signatures were found over both large (>4,000 km) and small (< 100 km) spatial scales. Overall, within-species differences were smaller than among-species differences. In general, FA signature patterns were consistent with previous studies of the ecology and diets of arctic marine mammals.

Determining body composition in gray seals (Halichoerus grypus) is important when studying their physiology and life history. In this study we investigated the predictability of total body fat (TBF) and protein (TBP) in postweaned gray seal pups from morphometric measurements, blubber thickness using ultrasonographs and bioelectrical impedance analysis (BIA). In postweaned pups, TBF (kg) could be estimated from girth measurements and sex (n = 45, r 2 = 0.878) using hydrogen isotope dilution methods as the reference. However, TBP could not be reliably estimated from morphometric data. TBF (kg) in yearlings was best predicted from mass (n = 6, r 2 = 0.776) and TBP (kg) from mass/length (r 2 = 0.949). Dorsal blubber thickness using B-mode ultrasound was also a significant predictor of TBF (kg) in postweaned pups (r2 = 0.725) but BIA was not. Marked pups were recaptured during their first few months of life (n = 48) and body composition changes investigated. Animals lost mass and TBF after leaving the breeding beach, largely during the first 5-6 mo of life. Postweaned pups were ∼40% TBF and ∼13% TBP whereas yearlings were ∼12% TBF and ∼20% TBP. Pups that survived beyond 6 mo of age then regained mass as protein. Morphometric measurements are a useful field indication of body condition when isotope dilution is impractical.

We determined the blubber fatty acid (FA) composition of 281 ringed seals (Phoca hispida) across the Canadian Arctic to make inferences about spatial, temporal, and demographic patterns of foraging. Seals were sampled in nine locations between 1992 and 2004. Regional differences in FA signatures were related to the distance between groups, with the greatest similarity occurring among seals sampled in three locations within the Beaufort Sea-Amundsen Gulf. Seals in the western and southeastern portions of Hudson Bay also had similar FA signatures. Discriminant analysis on seventeen FAs classified ringed seals to their correct geographic region with 95% accuracy. Although location accounted for most of the variability in FA signatures, adult and juvenile ringed seals in Frobisher Bay-Labrador Sea and Jones Sound showed significant FA differences, as did male and female ringed seals in Jones Sound and Qaanaaq. Demographic differences were not detected among ringed seals in the Beaufort Sea-Amundsen Gulf or in western Hudson Bay. Seals off the coast of Labrador showed significant seasonal variability in FA signatures. Overall, seasonal, regional, and demographic patterns in FA signatures were consistent with differences in ringed seal diets, as inferred from stomach content and stable isotope analyses.