Cetacean Blubber Research Articles

Optimization and validation of a micro–QuEChERS method for phthalates detection in small samples of cetacean blubber

In this study, an innovative method was developed to detect and quantify phthalates in fresh cetacean blubber. An adaptation of the ammonium formate QuEChERS method was used and adapted as a micro-extraction for small quantities of samples. Significantly, this technique utilized minimal quantities of reagents and salts, with the additional implementation of rigorous Quality Assurance/Quality Control protocols to further reduce background contamination. To ensure the reliability of this method, comprehensive validation procedures were conducted, with a specific focus on two widely studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus). Determination coefficients (R2) for matrix-matched calibration were >0.93 with limits of quantifications (LOQ) of the method in the range of 5–10 ng/g. Mean recovery values were between 40 and 100 %. This novel methodology holds particular relevance for environmental research studies, offering the capability to detect emerging contaminants with minimal sample requirements. This aspect is particularly valuable in investigations that involve free-ranging animals and rely on biopsy sampling. It allows for the assessment of contaminant levels in healthy individuals within wild populations, enhancing our understanding of ecological impacts and potential conservation measures.•A micro-extraction adaptation of the ammonium formate QuEChERS method was developed and applied to a small quantity of fresh cetacean blubber to detect phthalates.•Small quantities of reagents and salts were used, and additional Quality Assurance/ Quality Control procedures were taken to further minimize background contamination.•Method validation was carried out for two cosmopolitan and extensively studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus).

Assessment of the presence of UV filters and UV stabilizers in stranded dolphin blubber

The presence of ultraviolet filters (UVFs) and stabilizers (UVSs) was evaluated for the first time in the common bottlenose dolphin (Tursiops truncatus). UVFs and UVSs are compounds of growing concern because their effects on the environment are not completely known. UVFs and UVSs are added to personal care products (PCPs), such as cosmetics and products related to sun care and once released to the aquatic ecosystem, marine organisms can bioaccumulate these substances.This work aimed to determine the presence of 12 UVFs and UVSs in cetacean blubber samples to assess the pollution to which these animals of the highest trophic chain levels are exposed due to human activity. Analytical determinations were carried out using a method based on microwave-assisted extraction combined with ultrahigh-performance liquid chromatography and tandem mass spectrometry detection. The developed method was successfully applied to determine the target compounds in the blubber tissues of five necropsied common bottlenose dolphins.Three of the 12 studied compounds, namely 2-ethylhexyl 2-cyano-3,3-diphenylprop-2-enoate (octocrilene, OC), 2-hydroxy-4-methoxybenzophenone (benzophenone 3, BP3) and 3-methylbutyl (E)-3-(4methoxyphenyl) prop-2-enoate (IMC), were detected in several samples. Of the identified compounds, OC was present in all the samples and at the highest concentration within the range from 52.61 ± 18.59 to 108.0 ± 11.32 ng·g−1.

Histomorphological stratification of blubber of three dolphin species from subtropical waters.

Blubber is a highly specialized and dynamic tissue unique to marine mammals and presents a reflection of the individuals' nutrition, environment, and life history traits. Few studies have investigated the histomorphology of cetacean blubber in subtropical environments. The aim of this study was to investigate the blubber histomorphology of three different dolphin species off the subtropical KwaZulu-Natal coast, South Africa, using adipocyte cell size, number, and density. Blubber tissue samples from the saddle area of 43 incidentally bycaught animals (fourSousa plumbea, 36 Tursiops aduncus, and threeDelphinus delphis) were used to compare cell parameters between blubber layers. Samples were divided into the upper third (corresponding to the superficial layer closest to the epidermis), middle third, and lower third (corresponding to the deep layer). For T. aduncus, factors potentially affecting blubber histomorphology, such as sex, age class, and season, were also assessed. Our results showed that no stratification was present in S. plumbea, which could be ascribed to the species' warmer inshore habitat, large body size, and apparent lower mobility. For T. aduncus and D. capensis, however, blubber stratification was determined, characterized by a gradual transition of cell size, number, and density between layers rather than clearly defined layers. Significant differences in adipocyte cell number and density were found for different sexes and age classes of T. aduncus. However, there were no significant differences between seasons, which was attributed to the small temperature differences between seasons. This study represents the first investigation of odontocete blubber histomorphology in subtropical waters. It is recommended that future studies investigate blubber lipid content, while also taking into consideration the reproductive status of the females and the temperature range of their study area. It is hoped that our results, in conjunction with histopathology and other health indicators, could assist in assessing health and body condition.

Comparative Analysis of Blood Transcriptome in the Yangtze Finless Porpoise (Neophocaena asiaeorientalis)

The Yangtze finless porpoise (Neophocaena asiaeorientalis) is the sole freshwater subspecies of Neophocaenaphocaenoides, and there is a lack of data on its transcriptome. In this study, we applied RNA-seq technology to assemble, de novo, a transcriptome and analyzed differential expressed genes (DEGs). About 6 Gb of clean data was generated for the Yangtze finless porpoise blood (n = 6) through de novo sequencing. In total, 151,211 unigenes were generated and a total of 119,039 of these unigenes (78.72%) were functionally annotated when searched for within the NCBI Nr, SwissProt, GO, COG, and KEGG databases. Diverse and extensive expressed gene catalogs were sampled for the Yangtze finless porpoise. DESeq2 was used to analyze the differential expression genes (DEGs) obtained from the assembled transcriptome. The results indicated that DEGs have close relationships with the Yangtze finless porpoise’s development, evolution and adaptation. Further, we found that genes involved in cetacean TAG synthesis might directly explain the molecular basis of cetacean blubber thickening. These transcriptome data will assist in understanding molecular mechanisms of Yangtze finless porpoise adaptation.

Small Blubber Samples (50 mg) Sufficient for Analyses of 10 Stress and Reproductive Steroid Hormones in Gray and Fin Whales via Liquid Chromatography Mass Spectrometry

Information on stress, reproductive fitness, and health is difficult to obtain in wild cetaceans but critical for conservation and management. The goal of this study was to develop a methodology requiring minimal blubber mass for analysis of reproductive and stress steroid hormones and, hence, suitable for cetacean biopsies. Blubber biopsies and samples were collected from free-ranging and stranded gray and fin whales. Steroid hormones were extracted from blubber samples as small as 50 mg using liquid-liquid extraction methodology developed to handle the high fat content of blubber. Samples were analyzed via liquid chromatography with tandem mass spectrometry for 10 hormones: aldosterone, androstenedione, cortisol, cortisone, corticosterone, 17β-estradiol, estrone, 17α-hydroxyprogesterone, progesterone, and testosterone. As part of the optimization, homogenization via bead beating and blade dispersion were compared, and the former found superior. To investigate optimal yet minimal tissue mass required, hormone panels were compared among paired 50, 150, and 400 mg samples, the latter two being commonly reported masses for hormone blubber analysis. Results indicated that 50 mg of blubber was suitable and sometimes superior. Additionally, significant differences in precision values were observed between species, possibly stemming from differences in blubber composition, and relevant to homogenization technique selection and calibration methods that use blubber matrix matches obtained from a species other than the study species. Based on recovery and precision values, our methodology was accurate and precise in the measurement of spiked known quantities for all 10 hormones, confirming the methodology capabilities in 50 mg blubber mass in both species. Altogether, and in our specific sample sets, all endogenous hormones, except corticosterone, were identified above the detection limit in 50 mg gray whale blubber samples while all endogenous hormones, except aldosterone, cortisone, estrone, and progesterone, were detected in 50 mg fin whale blubber samples. We present a robust methodology for the analysis of multiple reproductive and stress steroid hormones in minimal masses of cetacean blubber compatible with small biopsies. Finally, we identified statistically significant differences in corticosteroid concentrations between stranded and free ranging animals.

Long-Chain Chlorinated Paraffins Have Reached the Arctic

Chlorinated paraffins (CPs) were analyzed in marine wildlife from Greenland, Iceland, and the Swedish west coast. CPs up to C29 were detected in the samples from the Arctic, indicating long-range transport of an industrial chemical group with a 2-million-ton annual production volume. CP concentrations were generally higher in biota from the Swedish west coast. The Sweden/Arctic concentration ratios for very-short-chain (vSCCPs, C<10), short-chain (SCCPs, C10–13), medium-chain (MCCPs, C14–17), and long-chain CPs (LCCPs, C>17) in cetacean and bivalve species were in the range of 3.6–150, 5–29, 3–11, and 11–450, respectively. For the first time, fetal accumulation of four CP classes was found, whereby concentrations were 3.4 to 4.5 times lower in the fetus relative to the pregnant female minke whale. Cetacean blubber contained a higher proportion of higher chlorinated CPs compared to muscle tissue, while muscle contained higher lipid-normalized concentrations of longer chain CPs than blubber tissue. LCCPs predominated (52% of total CPs) in the muscle of a killer whale from Swedish waters, which is the first report where concentrations of LCCPs surpassed those of other CPs in marine mammals. This study shows that, like other CP classes, LCCPs are Arctic contaminants that may adversely affect biota in this remote region.

Stress and reproductive events detected in North Atlantic right whale blubber using a simplified hormone extraction protocol.

As studies quantifying steroid hormones in marine mammal blubber progress, methodological refinements may improve the utility and consistency of blubber hormone measurements. This study advances blubber extraction methodologies by testing a simplified extraction protocol that reduces time and complexity compared to a protocol widely used in cetacean blubber studies. Using blubber samples archived from remote biopsy (n = 21 live whales) and necropsy collection (n = 7 dead whales) of North Atlantic right whales (NARW; Eubalaena glacialis) of known life history states, we performed analytical and biological validations to assess the feasibility of measuring reproductive (testosterone, progesterone) and glucocorticoid (cortisol) hormones in blubber via enzyme immunoassay following the simplified extraction. Analytical validations (parallelism, accuracy, extraction efficiency, repeatability) showed the simplified extraction produced similar results to the extended protocol, offering a more efficient and consistent technique. In live, apparently healthy whales, blubber testosterone concentrations (mean ± SE) were significantly higher in males (2.02 ± 0.36ng/g) compared to females (0.81 ± 0.15ng/g). Blubber progesterone was highest in a confirmed pregnant female (60.3ng/g), which was 12-fold greater than the mean concentration of non-pregnant females (4.56 ± 0.88ng/g). Blubber cortisol concentrations in whales that died from anthropogenic causes averaged 5.31 ± 2.28ng/g, whereas most live, healthy whales had cortisol values below 1ng/g. Among living whales, a whale actively entangled in fishing gear had the highest blubber cortisol measurement (3.51ng/g), exhibiting levels similar to whales that died from acute entanglement (2.88 ± 0.42ng/g). Overall, the highest blubber cortisol concentration (18.0ng/g) was measured in a dead whale with a severe chronic entanglement, approximately 30-fold greater than mean blubber cortisol of apparently healthy whales (0.58 ± 0.11ng/g). The methodological approach presented here provides a reference for researchers interested in an alternative, streamlined technique for hormone extraction of cetacean blubber and contributes to the diverse tool set for stress and reproductive assessments of endangered NARWs.

Toward New Ecologically Relevant Markers of Health for Cetaceans

Multiple stressors caused by human-induced disturbances can affect the foraging opportunities of cetaceans, potentially depleting their energy stores, and ultimately impact survival and reproductive success. Currently, blubber thickness and lipid composition is used as measure of health and nutritional status in cetaceans. This assumes that blubber functions in the same manner as adipose tissue in terrestrial mammals. However, cetaceans have evolved to have thickened blubber which serves as thermoregulation, buoyancy and energy store. In addition, blubber is composed of several layers and regions that have different physiological functions. We currently lack a clear understanding of how blubber biology contributes to maintaining energy status in cetaceans and several studies show blubber thickness, and composition in some body regions, is an inappropriate measure of health. Before new markers of health can be identified, we need to understand how environmental stressors influence blubber biology and particularly unravel its complex signalling roles with other organs. Currently, we do not understand how changes in energy status drive changes in health in cetaceans and eventually population dynamics. This review synthesises recent developments in cetacean blubber biology to propose potential directions to develop novel cetacean health markers.

Liquid chromatography tandem mass spectrometry for the quantification of steroid hormone profiles in blubber from stranded humpback whales (Megaptera novaeangliae).

Marine mammal blubber is known to have quantifiable concentrations of steroid hormones and is increasingly chosen as a matrix for the detection of these reproductive and stress biomarkers. Steroid hormones act through complex cascades, often in concert, yet studies conducted on cetaceans have rarely measured more than two steroids simultaneously. Due to the role of steroid hormones in multiple physiological processes, and variability in concentration among individuals, data on single compounds are often difficult to interpret. Here a liquid chromatography tandem mass spectrometry method for the simultaneous analyses of multiple steroid hormones in cetacean blubber was validated and applied to samples from 10 stranded humpback whales (Megaptera novaeangliae). Progesterone, 17α-hydroxyprogesterone, testosterone, androstenedione, oestrone, oestradiol, cortisone, cortisol, corticosterone and 11-deoxycorticosterone were reliably (relative standard deviation on six replicates <15%) and accurately (recovery of an amended sample between 70% and 120%) quantified, but not 11-deoxycortisol. With the exception of progesterone, testosterone, oestradiol and cortisol, these compounds were quantified for the first time in humpback whales. Given that blubber is frequently collected from free-swimming cetaceans in ongoing research programs, the technique developed here could substantially strengthen understanding and monitoring of the physiological condition of these species.

From banana fields to the deep blue: Assessment of chlordecone contamination of oceanic cetaceans in the eastern Caribbean

In the French West Indies (Caribbean), the insecticide Chlordecone (CLD) has been extensively used to reduce banana weevil (Cosmopolites sordidus) infestations in banana plantations. Previous studies have shown high CLD concentrations in freshwater and coastal communities of the region. CLD concentrations, however, have not yet been assessed in marine top predators. We investigated CLD concentrations in cetacean blubber tissues from Guadeloupe, including Physeter macrocephalus, Lagenodelphis hosei, Stenella attenuata and Pseudorca crassidens. Chlordecone was detected in all blubber samples analysed, with the exception of four P. macrocephalus. Concentrations (range: 1 to 329 ng·g−1 of lipid weight) were, however, lower than those found in species from fresh and brackish water. Ecological factors (open ocean habitat), CLD kinetics, and cetacean metabolism (high or specific enzymatic activity) might explain low concentrations found in cetacean blubber. Future analyses that include internal organ sampling would help to confirm CLD levels observed in this study.

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.

First detection of seven phthalate esters (PAEs) as plastic tracers in superficial neustonic/planktonic samples and cetacean blubber

PAEs concentrations can serve as tracer of plastics ingestion by marine organisms.

Feasibility of using humpback whale blubber to measure sex hormones

This study encompasses a series of interrelated experiments regarding the appropriate handling of samples and the interpretation of measurements of estradiol (described here for the first time in cetacean blubber), progesterone, and testosterone from both live and dead humpback whales. The experiments determined the effects on hormone levels of the following parameters: the state of decomposition of the blubber, the location on the body, the depth of the blubber layer, and the mass of the analyzed sample. The decomposition of carcasses for up to six days (144h) after death of the animal under natural conditions increased the levels of all three hormones. The dorsal fin presented higher levels of testosterone than other locations. The outer layer of blubber in decomposing samples exhibited higher values of progesterone and estradiol than the middle and inner layers and also exhibited a greater amount of extracted lipids. A lack of adjustment for relative volumes of extract and solvent led to an inverse relationship between hormone level and sample mass; smaller samples (25–50mg) exhibited higher levels of hormones than did larger ones (50–300mg). Certain data adjustments are proposed to minimize the effect of sample mass on hormone measurement, including the use of an alternative mass unit (amount of extracted lipid). The methodological approaches presented here contribute to the better standardization of this emerging technique and thereby facilitate the comparison of hormone levels among different cetacean populations and species.

Beyond thermoregulation: metabolic function of cetacean blubber in migrating bowhead and beluga whales.

The processes of lipid deposition and utilization, via the gene leptin (Lep), are poorly understood in taxa with varying degrees of adipose storage. This study examines how these systems may have adapted in marine aquatic environments inhabited by cetaceans. Bowhead (Balaena mysticetus) and beluga whales (Delphinapterus leucas) are ideal study animals-they possess large subcutaneous adipose stores (blubber) and undergo bi-annual migrations concurrent with variations in food availability. To answer long-standing questions regarding how (or if) energy and lipid utilization adapted to aquatic stressors, we quantified variations in gene transcripts critical to lipid metabolism related to season, age, and blubber depth. We predicted leptin tertiary structure conservation and assessed inter-specific variations in Lep transcript numbers between bowheads and other mammals. Our study is the first to identify seasonal and age-related variations in Lep and lipolysis in these cetaceans. While Lep transcripts and protein oscillate with season in adult bowheads reminiscent of hibernating mammals, transcript levels reach up to 10 times higher in bowheads than any other mammal. Data from immature bowheads are consistent with the hypothesis that short baleen inhibits efficient feeding. Lipolysis transcripts also indicate young Fall bowheads and those sampled during Spring months limit energy utilization. These novel data from rarely examined species expand the existing knowledge and offer unique insight into how the regulation of Lep and lipolysis has adapted to permit seasonal deposition and maintain vital blubber stores.

Contributions of allochthonous inputs of food to the diets of benthopelagic fish over the northwest Mediterranean slope (to 2300 m)

The contributions of allochthonous inputs of food (food falls, plastics and other anthropogenic remains) in the diets of large fish (6 teleosteans, 3 sharks) were analyzed for depths between 500 and 2300m in the deep Balearic basin (western Mediterranean). The analyses were based on gut contents. The identification was based on a multi-analytic approach, comprising morphological features (including morphometric analysis) and molecular genetics (DNA barcoding method). Remains of a number of anthropogenic, inorganic materials (microplastic fibres, plastic bags and cartons) appeared regularly in the guts of deep-sea fish (e.g., in Trachyrhynchus scabrus and Mora moro), though always at low occurrence (9.1% of fish at most) and negligible weights (< 2%W of diet). In our sampling, covering an area of ca. 12km2, large food falls contribute only a little to fish diets by weight, W, e.g., in shark diets they represented 4.5%W for Centroscymnus coelolepis and 11%W for Galeus melastomus. However, the importance of food falls (e.g., cetacean blubber and carcharhinid shark remains) was substantial locally (up to 70.8%W of C. coelolepis diet) particularly near canyons. The arrival of livestock remains (beef flesh, goat ribs and vertebrae) was shown by molecular analyses to contributed to deep-sea shark diets (ca. 5.5%W) comparably to natural food falls. These remains, which originate from human activity, may locally alter the food webs of oligotrophic environments like that of the deep Mediterranean. Food falls of both natural and anthropogenic origin were mainly found in fish collected close to canyon axes. The only cetacean fall documented in the deep Balearic Basin was also near a canyon, the carcass of a small (ca. 1.2m) striped dolphin, Stenella coeruleoalba, collected in a haul at 1750m off Barcelona.

Brown adipose tissue in cetacean blubber.

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.

Rope trauma, sedation, disentanglement, and monitoring‐tag associated lesions in a terminally entangled North Atlantic right whale (Eubalaena glacialis)

AbstractA chronically entangled North Atlantic right whale, with consequent emaciation was sedated, disentangled to the extent possible, administered antibiotics, and satellite tag tracked for six subsequent days. It was found dead 11 d after the tag ceased transmission. Chronic constrictive deep rope lacerations and emaciation were found to be the proximate cause of death, which may have ultimately involved shark predation. A broadhead cutter and a spring‐loaded knife used for disentanglement were found to induce moderate wounds to the skin and blubber. The telemetry tag, with two barbed shafts partially penetrating the blubber was shed, leaving barbs embedded with localized histological reaction. One of four darts administered shed the barrel, but the needle was found postmortem in the whale with an 80º bend at the blubber‐muscle interface. This bend occurred due to epaxial muscle movement relative to the overlying blubber, with resultant necrosis and cavitation of underlying muscle. This suggests that rigid, implanted devices that span the cetacean blubber muscle interface, where the muscle moves relative to the blubber, could have secondary health impacts. Thus we encourage efforts to develop new tag telemetry systems that do not penetrate the subdermal sheath, but still remain attached for many months.

Vascularization of Air Sinuses and Fat Bodies in the Head of the Bottlenose Dolphin (Tursiops truncatus): Morphological Implications on Physiology

Cetaceans have long been considered capable of limiting diving-induced nitrogen absorption and subsequent decompression sickness through a series of behavioral, anatomical, and physiological adaptations. Recent studies however suggest that in some situations these adaptive mechanisms might be overcome, resulting in lethal and sublethal injuries. Perhaps most relevant to this discussion is the finding of intravascular gas and fat emboli in mass-stranded beaked whales. Although the source of the gas emboli has as yet to been ascertained, preliminary findings suggest nitrogen is the primary component. Since nitrogen gas embolus formation in divers is linked to nitrogen saturation, it seems premature to dismiss similar pathogenic mechanisms in breath-hold diving cetaceans. Due to the various anatomical adaptations in cetacean lungs, the pulmonary system is thought of as an unlikely site of significant nitrogen absorption. The accessory sinus system on the ventral head of odontocete cetaceans contains a sizeable volume of air that is exposed to the changing hydrostatic pressures during a dive, and is intimately associated with vasculature potentially capable of absorbing nitrogen through its walls. The source of the fat emboli has also remained elusive. Most mammalian fat deposits are considered poorly vascularized and therefore unlikely sites of intravascular introduction of lipid, although cetacean blubber may not be as poorly vascularized as previously thought. We present new data on the vasculature of air sinuses and acoustic fat bodies in the head of bottlenose dolphins and compare it to published accounts. We show that the mandibular fat bodies and accessory sinus system are associated with extensive venous plexuses and suggest potential physiological and pathological implications.

Complex contaminant exposure in cetaceans: A comparative E‐Screen analysis of bottlenose dolphin blubber and mixtures of four persistent organic pollutants

Cetaceans are federally protected species that are prone to accumulate complex mixtures of persistent organic pollutants (POPs), which individually may exert estrogenic or antiestrogenic effects. In the present study it was assessed whether contaminant mixtures harbored by cetaceans are estrogenic or antiestrogenic using a comparative approach. Interactions of antiestrogenic and estrogenic compounds were first investigated with the E-Screen assay using a mixture of four POPs (dichlorodiphenyldichloroethylene [4,4'-DDE], trans-nonachlor, and polychlorinated biphenyls [PCBs] 138 180) prevalent in cetacean blubber. Estrogenic/antiestrogenic activity was determined for the individual compounds and their binary, tertiary, and quaternary combinations. Significantly different responses were observed for the various POP mixtures, including enhanced estrogenic and antiestrogenic effects and antagonistic interactions. These results were then compared to the concentrations and estrogenic/antiestrogenic activity of contaminant mixtures isolated directly from the blubber of 15 bottlenose dolphins (Tursiops truncatus) collected from five U.S. Atlantic and Gulf of Mexico locations. The lowest observed effect concentrations (LOECs) determined for 4,4'-DDE (20 µmol/L), PCB 138 (20 µmol/L), PCB 180 (21 µmol/L), and trans-nonachlor (3 µmol/L) in the E-Screen were greater than estimated dolphin blood concentrations. Although estimated blood concentrations were below the LOECs, significant estrogenic activity was detected in diluted dolphin blubber from Cape May, NJ and Bermuda. Positive correlations between blubber estrogenicity and select POP concentrations (ΣDDTs, ΣPBDEs, ΣHCB, Σestrogenic PCBs, Σestrogenic POPs) were also observed. Collectively, these results suggest that select bottlenose dolphin populations may be exposed to contaminants that act in concert to exert estrogenic effects at biologically relevant concentrations. These observations do not necessarily provide direct evidence of endocrine disruption; however, they may indicate an environmental source of xenoestrogenic exposure warranting future research.

Production of mixed halogenated congeners of the natural product heptachloro-1′-methyl-1,2′-bipyrrole (Q1) by photolysis in the presence of bromine

Halogenated 1′-methyl-1,2′-bipyrroles (MBPs) are a class of marine halogenated natural products that have been detected in biota from all over the world. However, structures and standards of many mixed chlorinated/brominated MBPs are not available. For this reason, the known 2,3,3′,4,4′,5,5′-heptachloro-1′-methyl-1,2′-bipyrrole (Q1 or MBP-79) was UV-irradiated in the presence of bromine with the goal of inducing a chlorine → bromine exchange. A few drops of bromine were added to a solution of Q1 and 10 mL of either CH 2Br 2, CH 2Cl 2, or CHCl 3. The experiments were performed both at room temperature and elevated temperature for 30 min. At least four out of five possible bromohexachloro-1′methyl-1,2′-bipyrroles (BrCl 6-MBPs), at least seven out of 13 possible Br 2Cl 5-MBPs, as well as traces of Br 3Cl 4-MBPs and Br 4Cl 3-MBPs were obtained in this way. Selective fragment ions in the GC/ECNI-MS spectra as well as electrophilic bromination of hexachloro-MBP solutions were used to verify the structures of the BrCl 6-MBP isomers. The BrCl 6-MBPs eluted from DB-5-like columns in the order of 4′-bromo-2,3,3′,4,5,5′-hexachloro-1′-methyl-1,2′-bipyrrole (Br-MBP-76), which co-eluted with 3′-bromo-2,3,4,4′,5,5′-hexachloro-1′-methyl-1,2′-bipyrrole (Br-MBP-78), followed by 2-bromo-3,3′,4,4′,5,5′-hexachloro-1′-methyl-1,2′-bipyrrole (Br-MBP-75), 3-bromo-2,3′,4,4′,5,5′-hexachloro-1′-methyl-1,2′-bipyrrole (Br-MBP-77), and 5′-bromo-2,3,3′,4,4′,5-hexachloro-1′-methyl-1,2′-bipyrrole (Br-MBP-74). These BrCl 6-MBPs were also detected in a sample of cetacean blubber from Australia, but the abundance pattern was different. While Br-MBP-76/Br-MBP-78 dominated in the cetacean, irradiation of Q1 (MBP-79) in the presence of bromine led to high proportions of Br-MBP-75. The suitability of the UV-induced Cl → Br exchange was confirmed by the Br-assisted UV-irradiation of pentachloroanisole (PCA). This experiment produced at least two bromotetrachloro- and three dibromotrichloroanisoles, the last eluting in each case being the most relevant. Thus, this method is most likely generally suited for the production of mixed-halogenated aromatic organohalogen compounds which are not readily obtainable by synthesis.