Development of Mercury Analysis by NanoSIMS for the Localization of Mercury-Selenium Particles in Whale Liver.

Nanoscale secondary ion mass spectrometry (NanoSIMS) is a dynamic SIMS technique, which offers high spatial resolution allowing the mapping of chemical elements at the nanometer scale combined with high sensitivity. However, SIMS for mercury analysis is a challenging issue due to the low secondary ion yield and has never been done on NanoSIMS. The introduction of an rf plasma oxygen primary ion source on NanoSIMS enabled higher lateral resolution and higher sensitivity for electropositive elements such as most metals. In this paper, for the first time, mercury analysis by NanoSIMS was developed applying the new rf plasma O- ion source. All mercury isotopes could be detected as Hg+ secondary ions and the isotopic pattern corresponded to their natural isotopic abundances. Furthermore, Hg+ detection in HgSe nanocrystals has been investigated where polyatomic interferences from selenium clusters were identified and separated by high mass resolution (ΔM/M ≥ 3200). However, in the presence of selenium a strong matrix effect was observed, decreasing the Hg+ secondary ion yield. In addition, a detection of Se+ ions was possible, too. The newly developed method was successfully applied to nanoscale localization by chemical imaging of HgSe particles accumulated in the liver tissue of sperm whale (Physeter macrocephalus). This demonstrated the applicability of NanoSIMS not only for mercury detection in surface analysis but also for mercury mapping in biological samples.

Estimating the acute impacts of Arctic marine oil spills using expert elicitation.

Increasing maritime traffic in the Arctic has heightened the oil spill-related risks in this highly sensitive environment. To quantitatively assess these risks, we need knowledge about both the vulnerability and sensitivity of the key Arctic functional groups that may be affected by spilled oil. However, in the Arctic these data are typically scarce or lacking altogether. To compensate for this limited data availability, we propose the use of a probabilistic expert elicitation methodology, which we apply to seals, anatids, and seabirds. Our results suggest that the impacts of oil vary between functional groups, seasons, and oil types. Overall, the impacts are least for seals and greatest for anatids. Offspring seem to be more sensitive than adults, the impact is greatest in spring, and medium and heavy oils are the most harmful oil types. The elicitation process worked well, yet finding enough skilled and motivated experts proved to be difficult.

Selenium protein identification and profiling by mass spectrometry: A tool to assess progression of cardiomyopathy in a whale model.

Non-ischemic cardiomyopathy is a leading cause of congestive heart failure and sudden cardiac death in humans and in some cases the etiology of cardiomyopathy can include the downstream effects of an essential element deficiency. Of all mammal species, pygmy sperm whales (Kogia breviceps) present the greatest known prevalence of cardiomyopathy with more than half of examined individuals indicating the presence of cardiomyopathy from gross and histo-pathology. Several factors such as genetics, infectious agents, contaminants, biotoxins, and inappropriate dietary intake (vitamins, selenium, mercury, and pro-oxidants), may contribute to the development of idiopathic cardiomyopathy in K. breviceps. Due to the important role Se can play in antioxidant biochemistry and protein formation, Se protein presence and relative abundance were explored in cardiomyopathy related cases. Selenium proteins were separated and detected by multi-dimension liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS), Se protein identification was performed by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS), and Se protein profiles were examined in liver (n=30) and heart tissue (n=5) by SEC/UV/ICP-MS detection. Data collected on selenium proteins was evaluated in the context of individual animal trace element concentration, life history, and histological information. Selenium containing protein peak profiles varied in presence and intensity between animals with no pathological findings of cardiomyopathy and animals exhibiting evidence of cardiomyopathy. In particular, one class of proteins, metallothioneins, was found to be associated with Se and was in greater abundance in animals with cardiomyopathy than those with no pathological findings. Profiling Se species with SEC/ICP-MS proved to be a useful tool to identify Se protein pattern differences between heart disease stages in K. breviceps and an approach similar to this may be applied to other species to study Se protein associations with cardiomyopathy.

Adrenal responses of large whales: Integrating fecal aldosterone as a complementary biomarker to glucocorticoids.

Until now, physiological stress assessment of large whales has predominantly focused on adrenal glucocorticoid (GC) measures. Elevated GC concentrations in feces (fGC) are known to reflect stressful disturbances, such as fishing gear entanglement and human-generated underwater noise, in North Atlantic right whales (Eubalaena glacialis). However, there can be considerable variation in GC production as a function of sex and life history stage, which may confound the interpretation of fGC levels. Additionally, GC antibodies used in immunoassays can cross-react with other fecal metabolites (i.e., non-target steroids), potentially influencing fGC data. Here, aldosterone concentrations (fALD; aldosterone and related metabolites) were measured in fecal samples from right whales (total n=315 samples), including samples from identified individuals of known life history (n=82 individual whales), to evaluate its utility as a complementary biomarker to fGC for identifying adrenal activation. Concentrations of fALD were positively correlated with fGCs in right whales (r=0.59, P<0.001), suggesting concurrent secretion of these hormones by the adrenal gland. However, fALD levels were less influenced by concentrations of reproductive steroids in feces, minimizing the potential confounder of assay cross-reactivity in samples with highly skewed hormone ratios. Across different life history states for right whales, fALD concentrations showed similar patterns to those reported for fGC, with higher levels in pregnant females (35.9±7.6ng/g) followed by reproductively mature males (9.5±0.9ng/g) (P<0.05), providing further evidence of elevated adrenal activation in these groups of whales. The addition of fALD measurement as a biomarker of adrenal activation may help distinguish between intrinsic and external causes of stress hormone elevations in large whales, as well as other free-living wildlife species, providing a more comprehensive approach for associating adrenal activation with specific natural and anthropogenic stressors.

The biogeochemical role of baleen whales and krill in Southern Ocean nutrient cycling.

The availability of micronutrients is a key factor that affects primary productivity in High Nutrient Low Chlorophyll (HNLC) regions of the Southern Ocean. Nutrient supply is governed by a range of physical, chemical and biological processes, and there are significant feedbacks within the ecosystem. It has been suggested that baleen whales form a crucial part of biogeochemical cycling processes through the consumption of nutrient-rich krill and subsequent defecation, but data on their contribution are scarce. We analysed the concentration of iron, cadmium, manganese, cobalt, copper, zinc, phosphorus and carbon in baleen whale faeces and muscle, and krill tissue using inductively coupled plasma mass spectrometry. Metal concentrations in krill tissue were between 20 thousand and 4.8 million times higher than typical Southern Ocean HNLC seawater concentrations, while whale faecal matter was between 276 thousand and 10 million times higher. These findings suggest that krill act as a mechanism for concentrating and retaining elements in the surface layer, which are subsequently released back into the ocean, once eaten by whales, through defecation. Trace metal to carbon ratios were also higher in whale faeces compared to whale muscle indicating that whales are concentrating carbon and actively defecating trace elements. Consequently, recovery of the great whales may facilitate the recycling of nutrients via defecation, which may affect productivity in HNLC areas.

Methylmercury exposure and neurological outcomes in Taiji residents accustomed to consuming whale meat.

Methylmercury (MeHg) is a major environmental neurotoxicant that causes damage to the central nervous system. In Japan, industrial emission of MeHg has resulted in MeHg intoxication in Minamata and Niigata, the so-called Minamata disease. Humans are exposed to MeHg derived from natural sources, primarily fish and fish predators. Therefore, MeHg continues to be an environmental risk to human health, particularly in susceptible populations that frequently consume substantial amounts of fish or fish predators such as whale. This study aimed to investigate the health effects of MeHg exposure in adults. The subjects were 194 residents (117 males, 77 females; age 20-85 years) who resided in the coastal town of Taiji, the birthplace of traditional whaling in Japan. We analyzed hair for mercury content and performed detailed neurological examinations and dietary surveys. Audiometry, magnetic resonance imaging, and electromyography were performed to diagnose neurological defects. Whole blood mercury and selenium (Se) levels were measured in 23 subjects. The geometric mean of the hair mercury levels was 14.9 µg/g. Twelve subjects revealed hair mercury levels >50 µg/g (NOAEL) set by WHO. Hair mercury levels significantly correlated with daily whale meat intake. These results suggested that residents in Taiji were highly exposed to MeHg by ingesting MeHg-contaminated whale meat. Multivariate regression analysis demonstrated no significant correlations between hair mercury levels and neurological outcomes, whereas some of the findings significantly correlated with age. A significantly positive correlation between whole blood mercury and Se levels was observed and the whole blood mercury/Se molar ratios of all subjects were <1. These findings suggested that sufficient Se intake might be one of causes of the absence of adverse effects of MeHg exposure in this study.

Modeling the utility of binaural cues for underwater sound localization.

The binaural cues used by terrestrial animals for sound localization in azimuth may not always suffice for accurate sound localization underwater. The purpose of this research was to examine the theoretical limits of interaural timing and level differences available underwater using computational and physical models. A paired-hydrophone system was used to record sounds transmitted underwater and recordings were analyzed using neural networks calibrated to reflect the auditory capabilities of terrestrial mammals. Estimates of source direction based on temporal differences were most accurate for frequencies between 0.5 and 1.75 kHz, with greater resolution toward the midline (2°), and lower resolution toward the periphery (9°). Level cues also changed systematically with source azimuth, even at lower frequencies than expected from theoretical calculations, suggesting that binaural mechanical coupling (e.g., through bone conduction) might, in principle, facilitate underwater sound localization. Overall, the relatively limited ability of the model to estimate source position using temporal and level difference cues underwater suggests that animals such as whales may use additional cues to accurately localize conspecifics and predators at long distances.

Concentrations of the genotoxic metals, chromium and nickel, in whales, tar balls, oil slicks, and released oil from the gulf of Mexico in the immediate aftermath of the deepwater horizon oil crisis: is genotoxic metal exposure part of the deepwater horizon legacy?

Concern regarding the Deepwater Horizon oil crisis has largely focused on oil and dispersants while the threat of genotoxic metals in the oil has gone largely overlooked. Genotoxic metals, such as chromium and nickel, damage DNA and bioaccumulate in organisms, resulting in persistent exposures. We found chromium and nickel concentrations ranged from 0.24 to 8.46 ppm in crude oil from the riser, oil from slicks on surface waters and tar balls from Gulf of Mexico beaches. We found nickel concentrations ranged from 1.7 to 94.6 ppm wet weight with a mean of 15.9 ± 3.5 ppm and chromium concentrations ranged from 2.0 to 73.6 ppm wet weight with a mean of 12.8 ± 2.6 ppm in tissue collected from Gulf of Mexico whales in the wake of the crisis. Mean tissue concentrations were significantly higher than those found in whales collected around the world prior to the spill. Given the capacity of these metals to damage DNA, their presence in the oil, and their elevated concentrations in whales, we suggest that metal exposure is an important understudied concern for the Deepwater Horizon oil disaster.

Circulation of whale-bone artifacts in the northern Pyrenees during the late Upper Paleolithic.

The importance of coastal resources in the late Upper Paleolithic of western Europe has been reevaluated in recent years thanks to a growing body of new archeological evidence, including the identification of more than 50 implements made of whale bone in the Magdalenian level of the Isturitz cave (western Pyrenees). In the present study, the assemblages of osseous industry from 23 Magdalenian sites and site clusters in the northern Pyrenees were investigated, systematically searching for whale-bone implements. The objective of this research was to determine if, and how, tools and weapons of coastal origin were circulated beyond Isturitz into the inland, and if similar implements existed on the eastern, Mediterranean side of the Pyrenees. A total of 109 whale-bone artifacts, mostly projectile heads of large dimensions, were identified in 11 sites. Their geographic distribution shows that whale bone in the Pyrenean Magdalenian is exclusively of Atlantic origin, and that objects made of this material were transported along the Pyrenees up to the central part of the range at travel distances of at least 350 km from the seashore. This phenomenon seems to have taken place during the second half of the Middle Magdalenian and the first half of the Late Magdalenian, ca. 17,500-15,000 cal BP (calibrated years before present). The existence of a durable, extended coastal-inland interaction network including the circulation of regular tools is thus demonstrated. Additionally, differences between the whale-bone projectile heads of the Middle Magdalenian and those of the Late Magdalenian document an evolutionary process in the design of hunting weapons.

Comparison of the ability of chondroitin sulfate derived from bovine, fish and pigs to suppress human osteoclast activity in vitro.

Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 µg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed.

Echolocation in Blainville's beaked whales (Mesoplodon densirostris).

Here we use sound and movement recording tags to study how deep-diving Blainville's beaked whales (Mesoplodon densirostris) use echolocation to forage in their natural mesopelagic habitat. These whales ensonify thousands of organisms per dive but select only about 25 prey for capture. They negotiate their cluttered environment by radiating sound in a narrow 20° field of view which they sample with 1.5-3 clicks per metre travelled requiring only some 60 clicks to locate, select and approach each prey. Sampling rates do not appear to be defined by the range to individual targets, but rather by the movement of the predator. Whales sample faster when they encounter patches of prey allowing them to search new water volumes while turning rapidly to stay within a patch. This implies that the Griffin search-approach-capture model of biosonar foraging must be expanded to account for sampling behaviours adapted to the overall prey distribution. Beaked whales can classify prey at more than 15 m range adopting stereotyped motor patterns when approaching some prey. This long detection range relative to swimming speed facilitates a deliberate mode of sensory-motor operation in which prey and capture tactics can be selected to optimize energy returns during long breath-hold dives.

Influence of mercury and selenium chemistries on the progression of cardiomyopathy in pygmy sperm whales, Kogia breviceps.

More than half of pygmy sperm whales (Kogia breviceps) that strand exhibit signs of cardiomyopathy (CMP). Many factors may contribute to the development of idiopathic CMP in K. breviceps, including genetics, infectious agents, contaminants, biotoxins, and dietary intake (e.g. selenium, mercury, and pro-oxidants). This study assessed trace elements in K. breviceps at various stages of CMP progression using fresh frozen liver and heart samples collected from individuals that stranded along US Atlantic and Gulf coasts between 1993 and 2007. Standard addition calibration and collision cell inductively coupled plasma mass spectrometry (ICP-MS) were employed for total Se analysis and pyrolysis atomic absorption (AA) was utilized for total Hg analysis to examine if the Se/Hg detoxification pathway inhibits the bioavailability of Se. Double spike speciated isotope dilution gas chromatography ICP-MS was utilized to measure methyl Hg and inorganic Hg. Immunoblot detection and colorimetric assays were used to assess protein oxidation status. Data collected on trace elements, selenoproteins, and oxidative status were evaluated in the context of animal life history and other complementary histological information to gain insight into the biochemical pathways contributing to the development of CMP in K. breviceps. Cardiomyopathy was only observed in adult pygmy sperm whales, predominantly in male animals. Both Hg:Se molar ratios and overall protein oxidation were greater in males than females and increased with progression of CMP.

Resolution of the effects induced by W → F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F.

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.

Ecosystem service tradeoff analysis reveals the value of marine spatial planning for multiple ocean uses.

Marine spatial planning (MSP) is an emerging responsibility of resource managers around the United States and elsewhere. A key proposed advantage of MSP is that it makes tradeoffs in resource use and sector (stakeholder group) values explicit, but doing so requires tools to assess tradeoffs. We extended tradeoff analyses from economics to simultaneously assess multiple ecosystem services and the values they provide to sectors using a robust, quantitative, and transparent framework. We used the framework to assess potential conflicts among offshore wind energy, commercial fishing, and whale-watching sectors in Massachusetts and identify and quantify the value from choosing optimal wind farm designs that minimize conflicts among these sectors. Most notably, we show that using MSP over conventional planning could prevent >$1 million dollars in losses to the incumbent fishery and whale-watching sectors and could generate >$10 billion in extra value to the energy sector. The value of MSP increased with the greater the number of sectors considered and the larger the area under management. Importantly, the framework can be applied even when sectors are not measured in dollars (e.g., conservation). Making tradeoffs explicit improves transparency in decision-making, helps avoid unnecessary conflicts attributable to perceived but weak tradeoffs, and focuses debate on finding the most efficient solutions to mitigate real tradeoffs and maximize sector values. Our analysis demonstrates the utility, feasibility, and value of MSP and provides timely support for the management transitions needed for society to address the challenges of an increasingly crowded ocean environment.

The chemical forms of mercury and selenium in whale skeletal muscle.

Human exposure to potentially neurotoxic methylmercury species is a public-health concern for many populations worldwide. Both fish and whale are known to contain varying amounts of methylmercury species. However studies of populations that consume large quantities of fish or whale have provided no clear consensus as to the extent of the risk. The toxicological profile of an element depends strongly on its chemical form. We have used X-ray absorption spectroscopy to investigate the comparative chemical forms of mercury and selenium in fish and whale skeletal muscle. The predominant chemical form of mercury in whale is found to closely resemble that found in fish. In the samples of skeletal muscle studied, no involvement of selenium in coordination of mercury is indicated in either whale or fish, with no significant inorganic HgSe or HgS type phases being detected. The selenium speciation in fish and whale shows that similar chemical types are present in each, but in significantly different proportions. Our results suggest that for equal amounts of Hg in skeletal muscle, the direct detrimental effects arising from the mercury content from consuming skeletal muscle from whale and fish should be similar if the effects of interactions with other components in the meat are not considered.

Audiogram of a stranded Blainville's beaked whale (Mesoplodon densirostris) measured using auditory evoked potentials.

Quantifying and understanding the impact of anthropogenic sound on marine mammals has been the focus of many researchers both in laboratory settings as well as in the field. This study presents the audiogram of a sub-adult Blainville's beaked whale that stranded in Hawaii. The hearing measurements were conducted using the non-invasive auditory brainstem response technique. A total of 11 sinusoidally amplitude modulated tones were tested ranging from 5.6 to 160 kHz. The audiogram data indicated that the region of best hearing was found between 40 and 50 kHz with thresholds below 50 dB. This frequency range partially overlaps with the frequency modulated upsweep that Blainville's beaked whales have been reported to use during echolocation. These results match the frequency range obtained from the hearing measurements of a Gervais' beaked whale previously tested using contact acoustic stimulation and emphasize the importance of obtaining rapid hearing measurements on live stranded animals to improve the understanding of poorly known species.

Silver speciation in liver of marine mammals by synchrotron X-ray absorption fine structure and X-ray fluorescence spectroscopies.

The chemical form of Ag in the livers of five species of marine mammals was examined using X-ray absorption fine structure (XAFS) and X-ray fluorescence (XRF) spectroscopies. The XAFS analysis suggested that Ag(2)Se was present in the livers of the Franciscana dolphin (Pontoporia blainvillei), Dall's porpoise (Phocoenoides dalli), and Baird's beaked whale (Berardius bairdii), whereas Ag(2)S was present in the livers of the striped dolphin (Stenella coeruleoalba) and pygmy killer whale (Feresa attenuata). XRF spectroscopy results revealed that the distribution patterns of Ag and Se in a thin section of the liver of the Franciscana dolphin were the same; this also implied that Ag was associated with Se in the liver. Thus, the interaction of Ag with Se or S may offer significant protection against the toxicity of Ag in marine mammals. The formation of either Ag(2)Se or Ag(2)S might depend on the Hg levels in the liver. Ag(2)Se was observed in liver samples with relatively high Ag/Hg ratio, whereas liver samples with low Ag/Hg ratio contained Ag(2)S.

The unicorn and the narwhal: a tale of the tooth.

From as far back as the early Roman era, detailed descriptions and artistic images of the unicorn have been imagined. In Europe, the Mediterranean and the Far East, this creature became accepted as real. Although such an animal had never actually been seen, it was said to have the appearance of a horse, the beard and cloven hooves of a goat, the tail of a lion, and a single, spear-like horn protruding from the center of its forehead. The unicorn myth originated shortly after unidentified horn-like objects, from six to ten feet long, began to appear in ancient European marketplaces. Physicians quickly ascribed outlandish healing properties to these horns, pulverizing them for the treatment of various diseases. In 1638, a Danish zoologist challenged these claims and provided evidence that the so-called "unicorn horn" was actually the tusk (tooth) of the narwhal from the Arctic seas. However, his findings remained unheeded for the next 100 years. This paper explains how the myth of the unicorn and the purported powers contained within the substance of its magical horn evolved from the ages-old spoils of the Arctic fishing trade, where the narwhal was primarily speared for food and secondarily, for the export value of its ivory tusk. This unusually long, pointed and protruding single tooth, which breaks through the upper lip and the left jaw of each male narwhal, became a highly valuable commodity over the following centuries, due to its believed supernatural origins and curative qualities.