Blubber Biopsies Research Articles

Interannual variability in the lipid and fatty acid profiles of east Australia-migrating humpback whales (Megaptera novaeangliae) across a 10-year timeline

Southern hemisphere humpback whales are classified as high-fidelity Antarctic krill consumers and as such are vulnerable to variability and long-term changes in krill biomass. Evidence of heterogeneous feeding patterns of east coast of Australia migrating humpback whales has been observed, warranting a comprehensive assessment of interannual variability in their diet. We examined the lipid and fatty acid profiles of individuals of the east coast of Australia migrating stock sampled between 2008 and 2018. The use of live-sampled blubber biopsies showed that fatty acid profiles varied significantly among all years. The two trophic indicator fatty acids for Antarctic krill, 20:5ω3 and 22:6ω3 remained largely unchanged across the 10-year period, suggesting that Antarctic krill is the principal prey item. A distance-based linear model showed that 33% of the total variation in fatty acid profiles was explained by environmental variables and climate indices. Most of the variation was explained by the Southern Annular Mode (23.7%). The high degree of variability observed in this study was unexpected for a species that is thought to feed primarily on one prey item. We propose that the observed variability likely arises from changes in the diet of Antarctic krill rather than changes in the whale’s diet.

Corticosterone in central North Pacific male humpback whales (Megaptera novaeangliae): Pairing sighting histories with endocrine markers to assess stress

Developing a better understanding of the stress response is critical to ensuring the health and sustainability of marine mammal populations. However, accurately measuring and interpreting a stress response in free-ranging, large cetaceans is a nascent field. Here, an enzyme immunoassay for corticosterone was validated for use in biopsy samples from male humpback whales (Megaptera novaeangliae). Analyses were conducted on 247 male North Pacific humpback whale blubber samples, including 238 non-calves and 9 calves that were collected on the Hawaiian breeding and Southeast Alaskan feeding grounds from 2004 to 2006. Significant relationships were found when corticosterone concentrations were examined by year, age class and distribution between locations. When examined by year, corticosterone concentrations for male humpback whales were higher in Hawaii in 2004 than in 2005 and 2006 (p < 0.05). Corticosterone concentration also varied by age class with initially high concentrations at birth which subsequently tapered off and remained relatively low until sexual maturity was reached around age 8–10 years. Corticosterone concentrations appeared to peak in male humpback whales around 15–25 years of age. Blubber biopsies from Alaska and Hawaii had similar mean corticosterone concentrations, yet the variability in these samples was much greater for whales located in Hawaii. It is clear that much work remains to be done in order to accurately define or monitor a stress response in male humpback whales and that specific attention is required when looking at age, sex, and yearly trends. Our results suggest that a stress response may be most impacted by age and yearly oceanographic conditions and needs to be initially examined at the individual level.

First assessment of pollutant exposure in two balaenopterid whale populations sampled in the Svalbard Archipelago, Norway

Pollutant concentrations are poorly known for the largest animals on Earth, blue whales Balaenoptera musculus and fin whales Balaenoptera physalus. In this study, concentrations of persistent organic pollutants (POPs) were determined in blubber biopsies and stable isotope values for nitrogen (δ15N) and carbon (δ13C) were measured using skin biopsies for 18 blue whales and 12 fin whales sampled in waters surrounding the Svalbard Archipelago, Norway. The samples were collected in summer during the period 2014–2018. POPs were dominated by DDTs, PCBs and toxaphenes, with median concentrations in blue/fin whales being 208/341, 127/275 and 133/233 ng/g lipid weight, respectively. Linear models indicated that pollutant concentrations were 1.6–3 times higher in fin whales than in blue whales, which is likely related to the higher trophic positions of fin whales, as indicated by their higher δ15N. Lower δ13C in fin whales suggests that they feed at higher latitudes than blue whales; these values were not correlated with pollutant concentrations. Pollutant levels were approximately twice as high in males compared to females (intraspecifically), which indicates that females of these species offload pollutants to their offspring during gestation and lactation, similar to many other mammalian species. Pollutant concentrations in balaenopterid whales from Svalbard waters were generally much lower than in conspecific whales from the Mediterranean Sea or the Gulf of California, but higher than those in conspecifics from the Antarctic Peninsula.

Variation in outer blubber lipid concentration does not reflect morphological body condition in humpback whales.

An animal's body condition provides valuable information for ecophysiological studies, and is an important measure of fitness in population monitoring and conservation. While both the external body shape of an animal and its internal tissues (i.e. fat content) can be used as a measure of body condition, the relationship between the two is not always linear. We compared the morphological body condition (external metric obtained through aerial photogrammetry) of migrating humpback whales (Megaptera novaeangliae) with their outer blubber lipid concentration (internal metric obtained through blubber biopsy sampling) off the coast of south-west Australia early and late in the breeding season (spanning ∼4.5 months). The external body condition index of juvenile and adult humpback whales decreased by 26.9 (from 18.8% to -8.1%) and 12.0 percentage points (from 8.6% to -3.4%), respectively, between the early and late phase. In contrast, we found no intra-seasonal change in blubber lipid concentration, and no difference between reproductive classes (juveniles, adults and lactating females); however, the small sample size prevented us from effectively testing these effects. Importantly, however, in the 33 animals for which paired metrics were obtained, we found no correlation between the morphometric body condition index and the blubber lipid concentration of individual whales. The lack of a linear relationship suggests that changes in outer blubber lipid concentration do not reflect external changes in body shape, thus limiting the utility of outer blubber lipid reserves for individual body condition evaluation. The wider spectrum of change in body morphometry captured with aerial photogrammetry supports the use of body morphometry as a reliable and well-tested method.

Blubber proteome response to repeated ACTH administration in a wild marine mammal

While the response to acute stress is adaptive in nature, repeated or chronic stress can impact an animal's fitness by depleting its energy stores and suppressing immune function and reproduction. This can be especially deleterious for species that rely on energy reserves to fuel key life history stages (e.g. reproduction), already experience physiological extremes (e.g. fasting), and/or have undergone significant population declines, such as many marine mammals. However, identifying chronically stressed individuals is difficult due to the practical challenges to sample collection from large aquatic animals and a paucity of information on downstream consequences of the stress response. We previously simulated repeated stress by ACTH administration in a model marine mammal, the northern elephant seal, and showed that changes in blubber gene expression, but not circulating cortisol levels, could distinguish between single and repeated responses to ACTH. Here, we profiled changes in the proteome of the same blubber cell population and identified a set of differentially expressed proteins that included extracellular matrix components, heat shock and mitochondrial proteins, metabolic enzymes, and metabolite transporters. Differentially expressed proteins and genes shared similar functions that suggest that repeated corticosteroid elevation may affect blubber tissue proteostasis, mitochondrial activity, adipogenesis, and metabolism in marine mammals. For marine mammal species from which blubber biopsies, but not blood can be obtained by remote sampling, measurement of abundance of such proteins may serve as a novel method for identifying chronically stressed animals.

The influence of physiological status on the reproductive behaviour of humpback whales (Megaptera novaeangliae)

For most cetacean species, there is little known about how an individual's physiology influences its behaviour. Humpback whales (Megaptera novaeangliae) are a good candidate to examine such links as they have a well-described distribution and behaviour, can be consistently sampled using remote biopsy systems, and have been the subject of several previous endocrine studies. The objective here was to examine whether a female humpback whale's social state (i.e. escorted by a male or not) is related to her endocrine condition, and whether male dominance ranking is related to testosterone levels. Skin and blubber biopsies were collected from the east and west Australian humpback whale populations in 2010–2016 (n = 252) at multiple times throughout the winter-spring breeding season. Steroid hormones were extracted from blubber and concentrations of progesterone (a marker for pregnancy), testosterone (a marker of male testicular activity) and oestradiol (a potential marker of ovarian activity) measured using enzyme-immunoassays. Principal escorts—the dominant males in mixed sex groups—had significantly higher blubber testosterone levels (mean ± SE; 1.43 ± 0.20 ng/g wet weight) than subordinate, secondary escorts (0.69 ± 0.06 ng/g wet weight). Females that were escorted by males typically possessed elevated blubber oestradiol levels (1.96 ± 0.25 ng/g wet weight; p = 0.014); few were considered to be pregnant (p = 0.083). ‘Unescorted’ females displayed characteristically lower blubber oestradiol levels (0.56 ± 0.06 ng/g wet weight). Together, these results are consistent with ‘challenge hypothesis’ theory and suggest the existence of associated reproductive patterns in humpback whales.

Persistent organic pollutants in killer whales (Orcinus orca) of the Russian Far East.

Exposure to persistent organic pollutants (POPs) is a key factor in predicting the collapse of global killer whale (Orcinus orca) populations due to reproductive and immune impacts. Blubber biopsies from killer whales (n = 25) were collected in the Russian Far East in 2002-2004. Biopsies were analyzed for ΣDDT, ΣPCB, and HCB concentrations. A subset of biopsies was further examined for additional contaminants, ΣPBDE, ΣHCH, ΣCHLD, mirex, and dieldrin. Mean concentrations were compared across resident (fish-eating) and transient (mammal-eating) ecotypes and between sexes. ΣPCB analytes (resident males 18,000, resident females 1200, and transient males 420,000 ng g-1 lw) and HCB (resident males 750, resident females 81, and transient males 6200 ng g-1 lw) differed significantly (p < 0.001). No significant difference was observed between sexes. Notable disparities in contaminant levels between ecotypes support the major toxicological theories of contaminant bioaccumulation and dietary impacts on individual contaminant load.

Examining the relationships between blubber steroid hormones and persistent organic pollutants in common bottlenose dolphins

Odontocete cetaceans bioaccumulate high concentrations of endocrine disrupting persistent organic pollutants (POPs), including dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyltrichloroethylene (DDE), and dichlorodiphenyldichloroethane (DDD) – collectively DDTs – but few studies have explored DDTs-mediated endocrine disruption in cetaceans. Herein, we use remotely collected blubber biopsies from common bottlenose dolphins (Tursiops truncatus) inhabiting a site with high localized DDTs contamination to study the relationships between DDTs exposure and steroid hormone homeostasis in cetaceans. We quantified blubber steroid hormone concentrations by liquid chromatography-tandem mass spectrometry and blubber POP concentrations by gas chromatography-mass spectrometry. We detected six steroid hormones in blubber, including progesterone (P4), 17-hydroxyprogesterone (17OHP4), androstenedione (AE), testosterone (T), cortisol (F), and cortisone (E). Sampled dolphins (n = 62) exhibited exposure to DDT, DDE, DDD, chlordanes (CHLDs), mirex, dieldrin, hexachlorobenzene, polychlorinated biphenyls (PCBs), and brominated diphenyl ethers (BDEs). Using principal components analysis (PCA), we determined that blubber DDTs primarily loaded to the first principal component (PC1) explaining 81.6% of the total variance in POP exposure, while the remaining POPs primarily loaded to the PC2 (10.4% of variance). PC1 scores were negatively correlated with blubber T in males and blubber F in females, suggesting that exposure to DDTs impacted androgen and corticosteroid homeostasis. These conclusions were further supported by observed negative correlations between T and o,p’-DDE, o,p’-DDD, and p,p’-DDD in males sampled in the fall, and between F and the six individual DDTs and ∑6DDTs in females. Overall, these results suggest that POP-mediated endocrine disruption may have occurred in this stock of dolphins, which could negatively impact their health and fitness. However, this study relied on uncontrolled incidental exposures, making it impossible to establish a causal relationship between DDTs exposure and endocrine effects. Importantly, this study demonstrates that remotely collected blubber biopsies are a useful matrix for studying endocrine disruption in marine mammals.

Sex steroid hormones and behavior reveal seasonal reproduction in a resident fin whale population.

Fin whales in the Gulf of California constitute a resident population genetically isolated from the rest of the North Pacific Ocean. Its small population size and the scarce information available about its dynamics in a semi-enclosed sea underline the importance of conducting studies about its reproduction. Given the monsoonal regime that dominates the oceanographic habitat of this region, we hypothesized seasonality in the population’s reproductive activity. To test this, we validated and assayed testosterone and progesterone from blubber biopsies of free-ranging individuals. Lactating females exhibited low progesterone concentrations, whereas a group of females of unknown reproductive stage, but with extremely high progesterone concentrations, showed strong evidence of separation and were considered to be likely ovulating or pregnant. A seasonal model of testosterone concentrations showed a high peak during the late summer. This trend was supported by the first documentation of courtship events and by the recording of a female with high progesterone concentration during summer and re-sighted with a calf 1 year later. Therefore, the breeding in this resident population would be seasonal, as it is in migratory baleen whales, but occurring during the summer/autumn, which is the least productive season in the Gulf of California. Our study represents an important input to assist in future management policies of this protected population.

Comprehensive endocrine response to acute stress in the bottlenose dolphin from serum, blubber, and feces

Several hormones are potential indicators of stress in free-ranging animals and provide information on animal health in managed-care settings. In response to stress, glucocorticoids (GC, e.g. cortisol) first appear in circulation but are later incorporated into other tissues (e.g. adipose) or excreted in feces or urine. These alternative matrices can be sampled remotely, or by less invasive means, than required for blood collection and are especially valuable in highly mobile species, like marine mammals. We characterized the timing and magnitude of several hormones in response to a stressor in bottlenose dolphins (Tursiops truncatus) and the subsequent incorporation of cortisol into blubber, and its metabolites excreted in feces.We evaluated the endocrine response to an acute stressor in bottlenose dolphins under managed care. We used a standardized stress protocol where dolphins voluntarily beached onto a padded platform and remained out of water for two hours; during the stress test blood samples were collected every 15 min and blubber biopsies were collected every hour (0, 60, and 120 min). Each subject was studied over five days: voluntary blood samples were collected on each of two days prior to the stress test; 1 and 2 h after the conclusion of the out-of-water stress test; and on the following two days after the stress test. Fecal samples were collected daily, each afternoon.The acute stressor resulted in increases in circulating ACTH, cortisol, and aldosterone during the stress test, and each returned to baseline levels within 2 h of the dolphin’s return to water. Both cortisol and aldosterone concentrations were correlated with ACTH, suggesting both corticosteroids are at least partly regulated by ACTH. Thyroid hormone concentrations were generally unaffected by the acute stressor. Blubber cortisol increased during the stress test, and fecal GC excretion was elevated on the day of the stress test. We found that GCs in bottlenose dolphins can recover within hours of acute stress, and that cortisol release can be detected in alternate matrices within a few hours—within 2 h in blubber, and 3.5–5 h in fecal samples.

Not just fat: investigating the proteome of cetacean blubber tissue.

Mammalian adipose tissue is increasingly being recognized as an endocrine organ involved in the regulation of a number of metabolic processes and pathways. It responds to signals from different hormone systems and the central nervous system, and expresses a variety of protein factors with important paracrine and endocrine functions. This study presents a first step towards the systematic analysis of the protein content of cetacean adipose tissue, the blubber, in order to investigate the kinds of proteins present and their relative abundance. Full depth blubber subsamples were collected from dead-stranded harbour porpoises (Phocoena phocoena) (n = 21). Three total protein extraction methods were trialled, and the highest total protein yields with the lowest extraction variability were achieved using a RIPA cell lysis and extraction buffer based protocol. Extracted proteins were separated using 1D Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry (nLC-ESI-MS/MS). A range of proteins were identified (n = 295) and classed into eight functional groups, the most abundant of which were involved in cell function and metabolism (45%), immune response and inflammation (15%) and lipid metabolism (11%). These proteins likely originate both from the various cell types within the blubber tissue itself, and from the circulation. They therefore have the potential to capture information on the cellular and physiological stresses experienced by individuals at the time of sampling. The importance of this proteomic approach is two-fold: Firstly, it could help to assign novel functions to marine mammal blubber in keeping with current understanding of the multi-functional role of adipose tissue in other mammals. Secondly, it could lead to the development of a suite of biomarkers to better monitor the physiological state and health of live individuals though remote blubber biopsy sampling.

Validation of a blubber-based endocrine pregnancy test for humpback whales.

Baleen whales have few identifiable external indicators of pregnancy state, making it challenging to study essential aspects of their biology and population dynamics. Pregnancy status in other marine mammals has been determined by measuring progesterone concentrations from a variety of sample matrices, but logistical constraints have limited such studies in free-swimming baleen whales. We use an extensive blubber sample archive and associated calving history data to retrospectively identify samples that correspond to pregnant females and develop a progesterone-based pregnancy test for humpback whales. The lowest pregnant blubber progesterone concentration was 54.97 ng g-1, and the mean for the known-pregnant group was 198.74 ± 180.65 ng g-1. Conversely, females known to be below the minimum age of sexual maturity (juvenile females) had an overall low mean progesterone concentration (0.59 ± 0.25 ng g-1), well below the known-pregnant range. Of the mature females that did not return with a calf (n = 11), three fell within the known-pregnant range (320.79 ± 209.34 ng g-1), while the levels for the remaining eight were two orders of magnitude below the lowest known-pregnant level (1.63 ± 1.15 ng g-1). The proportion of females that did not return with a calf but had values similar to known-pregnant females are consistent with rates of calf mortality, but other potential explanations were considered. Our findings support a validated blubber endocrine assignment of pregnancy corroborated with field life history information, a first for any baleen whale species. The progesterone values we measured were similar to those found in different pregnancy states of other cetaceans and support using blubber biopsy samples for assigning pregnancy in humpback whales. This method can be applied to existing archives or new samples to better study life history and population demography broadly across species and populations.

Nanospray liquid chromatography/tandem mass spectrometry analysis of steroids from gray whale blubber

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. 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. 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 <20% for interday and <10% for intraday. After validation, the method was successfully applied to quantification of steroids in gray whale blubber samples. The nanoLC/MS/MS method is more sensitive than traditional LC/MS/MS for steroid analysis. It is also compatible with other important biopsy analyses due to its small blubber mass requirement. This will benefit the reproductive and stress assessments for all marine mammals, particularly endangered populations. Copyright © 2017 John Wiley & Sons, Ltd.

Blubber cortisol qualitatively reflects circulating cortisol concentrations in bottlenose dolphins

AbstractStress 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 &lt; 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.

Seasonal and developmental differences in blubber stores of beluga whales in Bristol Bay, Alaska using high-resolution ultrasound.

Diving mammals use blubber for a variety of structural and physiological functions, including buoyancy, streamlining, thermoregulation, and energy storage. Estimating blubber stores provides proxies for body condition, nutritional status, and health. Blubber stores may vary topographically within individuals, across seasons, and with age, sex, and reproductive status; therefore, a single full-depth blubber biopsy does not provide an accurate measure of blubber depth, and additional biopsies are limited because they result in open wounds. We examined high-resolution ultrasound as a noninvasive method for assessing blubber stores by sampling blubber depth at 11 locations on beluga whales in Alaska. Blubber mass was estimated as a proportion of body mass (40% from the literature) and compared to a function of volume calculated using ultrasound blubber depth measurements in a truncated cone. Blubber volume was converted to total and mass-specific blubber mass estimates based on the density of beluga blubber. There was no significant difference in mean total blubber mass between the 2 estimates (R2 = 0.88); however, body mass alone predicted only 68% of the variation in mass-specific blubber stores in juveniles, 7% for adults in the fall, and 33% for adults in the spring. Mass-specific blubber stores calculated from ultrasound measurements were highly variable. Adults had significantly greater blubber stores in the fall (0.48±0.02kg/kgMB) than in the spring (0.33±0.02kg/kgMB). There was no seasonal effect in juveniles. High-resolution ultrasound is a more powerful, noninvasive method for assessing blubber stores in wild belugas, allowing for precise measurements at multiple locations.

Validating the use of biopsy sampling in contamination assessment studies of small cetaceans

Remote biopsy sampling is the most common technique for acquiring samples from free-ranging marine mammals. However, such techniques may result in variable sampling being sometimes superficial skin and blubber biopsies. For decades, blubber has been used to monitor the exposure of marine mammals to persistent organic pollutants (POPs), but little is known regarding the variability of POPs as a function of blubber depth in small cetaceans and the available literature offers variable results. Thus, the aim of the present study was to validate biopsy sampling for monitoring contaminant concentrations in small, free-ranging cetaceans. Samples from the dorsal blubber of 10 incidentally captured Atlantic spotted dolphins (Stenella frontalis) were separated into two different layers (outer and inner) to investigate the influence of sampling depth on POP concentrations. POP concentrations were compared to those of the full blubber layer. The results revealed no significant differences in lipid content between males and females or among the inner, outer and full blubber layers (p>0.05). Moreover, the wet and lipid weight concentrations of all POP classes analysed [i.e. polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), polybrominated diphenyl ethers (PBDEs), hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), chlordanes (CHLs) and mirex] did not differ significantly with blubber depth (p>0.05). POP classes followed the same decreasing order of wet weight concentrations in blubber layers and full blubber: PCBs>DDTs>PBDEs>mirex>HCB>HCHs>CHLs. Moreover, there was a low degree of differentiation in the accumulation of POP congeners. The present findings indicated that the distribution of contaminants was homogenous with blubber depth, which validates the use of biopsy sampling for the assessment of contaminants in small cetaceans.

Organochlorine contaminant concentrations in multiple tissues of free-ranging Steller sea lions (Eumetopias jubatus) in Alaska

The relationships of selected organochlorine (OC) contaminants between blubber, blood, feces, and milk of young, free-ranging Steller sea lions (Eumetopias jubatus) were examined. Both between and within each tissue there was considerable individual variation. In spite of the variation, similar patterns were observed across the tissues for most of the selected PCB congeners. In all four tissues, the major PCB congeners were PCB101, PCB118, PCB138, and PCB153. The most prominent congener, both as a weight (ng/g lipid) and as a percentage of summed PCBs (∑PCBs), was PCB 153. Comparisons between paired tissues showed that ∑DDTs in blubber samples were related to concentrations in blood, feces, and milk. The ∑PCBs in blubber were related to concentrations in milk and fecal samples, though the relationship with feces was weak. Our findings show milk samples, in particular, are useful for assessing OCs in young sea lions. Blubber concentrations of PCB101, PCB118, and PCB138 were an order of magnitude higher than those in milk, supporting the biomagnification of these PCB congeners in SSL tissues. The findings indicate alternative tissues may be used as indicators of relative contaminant exposure in lieu of surgical blubber biopsy.

Persistent Organic Pollutant Determination in Killer Whale Scat Samples: Optimization of a Gas Chromatography/Mass Spectrometry Method and Application to Field Samples.

Biologic sample collection in wild cetacean populations is challenging. Most information on toxicant levels is obtained from blubber biopsy samples; however, sample collection is invasive and strictly regulated under permit, thus limiting sample numbers. Methods are needed to monitor toxicant levels that increase temporal and repeat sampling of individuals for population health and recovery models. The objective of this study was to optimize measuring trace levels (parts per billion) of persistent organic pollutants (POPs), namely polychlorinated-biphenyls (PCBs), polybrominated-diphenyl-ethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs), and hexachlorocyclobenzene, in killer whale scat (fecal) samples. Archival scat samples, initially collected, lyophilized, and extracted with 70 % ethanol for hormone analyses, were used to analyze POP concentrations. The residual pellet was extracted and analyzed using gas chromatography coupled with mass spectrometry. Method detection limits ranged from 11 to 125 ng/g dry weight. The described method is suitable for p,p'-DDE, PCBs-138, 153, 180, and 187, and PBDEs-47 and 100; other POPs were below the limit of detection. We applied this method to 126 scat samples collected from Southern Resident killer whales. Scat samples from 22 adult whales also had known POP concentrations in blubber and demonstrated significant correlations (p < 0.01) between matrices across target analytes. Overall, the scat toxicant measures matched previously reported patterns from blubber samples of decreased levels in reproductive-age females and a decreased p,p'-DDE/∑PCB ratio in J-pod. Measuring toxicants in scat samples provides an unprecedented opportunity to noninvasively evaluate contaminant levels in wild cetacean populations; these data have the prospect to provide meaningful information for vital management decisions.

POPs in free-ranging pilot whales, sperm whales and fin whales from the Mediterranean Sea: Influence of biological and ecological factors

The pilot whale Globicephala melas, the sperm whale Physeter macrocephalus, and the fin whale Balaenoptera physalus are large cetaceans permanently inhabiting the Mediterranean Sea. These species are subjected to numerous anthropogenic threats such as exposure to high levels of contaminants. Therefore, selected persistent organic pollutants POPs (29 PCBs, 15 organochlorine compounds, 9 PBDEs and 17 PCDD/Fs) were analysed in blubber biopsies of 49 long-finned pilot whales, 61 sperm whales and 70 fin whales sampled in the North Western Mediterranean Sea (NWMS) from 2006 to 2013. Contamination profile and species feeding ecology were then combined through the use of stable isotopes. δ13C, δ15N values and POPs levels were assessed through IR-MS and GC–MS respectively. To assess the toxic potency of the dioxin-like compounds, the TEQ approach was applied. δ15N values were 12.2±1.3‰ for sperm whales, 10.5±0.7‰ for pilot whales and 7.7±0.8‰ in fin whales, positioning sperm whales at higher trophic levels. δ13C of the two odontocetes was similar and amounted to −17.3±0.4‰ for sperm whales and −17.8±0.3‰ for pilot whales; whilst fin whales were more depleted (−18.7±0.4‰). This indicates a partial overlap in toothed-whales feeding habitats, while confirms the differences in feeding behaviour of the mysticete. Pilot whales presented higher concentrations than sperm whales for ΣPCBs (38,666±25,731ngg−1lw and 22,849±15,566ngg−1lw respectively), ΣPBDEs (712±412ngg−1lw and 347±173ngg−1lw respectively) and ΣDDTs (46,081±37,506ngg−1 lw and 37,647±38,518ngg−1lw respectively). Fin whales presented the lowest values, in accordance with its trophic position (ΣPCBs: 5721±5180ngg−1 lw, ΣPBDEs: 177±208ngg−1 lw and ΣDDTs: 6643±5549ngg−1 lw). Each species was characterized by large inter-individual variations that are more related to sex than trophic level, with males presenting higher contaminant burden than females. The discriminant analysis (DA) confirmed how DDTs and highly chlorinated PCBs were influential in differentiating the three species. Pollutant concentrations of our species were significantly higher than both their Southern Hemisphere and North Atlantic counterparts, possibly due to the particular Mediterranean geomorphology, which influences pollutants distribution and recycle. Dioxin-like PCBs accounted for over 80% of the total TEQ. This study demonstrated (1) an important exposure to pollutants of Mediterranean cetaceans, often surpassing the estimated threshold toxicity value of 17,000ngg−1 lw for blubber in marine mammals; and (2) how the final pollutant burden in these animals is strongly influenced not only by the trophic position but also by numerous other factors such as sex, age, body size and geographical distribution.

Age estimation of belugas, Delphinapterus leucas, using fatty acid composition: A promising method

AbstractData about age‐specific survival and mortality rate, as well as life history parameters are essential for studying population demography. However, noninvasive methods for ageing free‐ranging marine mammals are generally lacking. Recently, a few studies have highlighted the potential of using fatty acid (FA) composition in blubber biopsy samples to estimate age in some cetaceans. Here, we explore the opportunity of using this technique to estimate the age of free‐ranging belugas from three different populations. Belugas (Delphinapterus leucas) were sampled postmortem for blubber FA analysis and aged by counting the number of growth layer groups in teeth dentine. We found significant positive and negative relationships between some FAs and age. These relationships were stronger with outer blubber layer samples, the layer most accessible via biopsy, than with inner or middle layer samples, a pattern that is consistent with observed turnover rates and biological function across the blubber depth. The FA 12:0, 14:1n‐7, and 14:1n‐9 were promising correlates of age in belugas, allowing estimation of age with a precision of ±7–10 yr. Further work is required to determine the mechanisms underlying changes in FA composition with age and whether these mechanisms are stable through time and across populations.