Spatial Variation in Mercury Accumulation in Bottlenose Dolphins (Tursiops spp.) in Southeastern U.S.A.

Bottlenose dolphins (Tursiops spp.) inhabit bays, sounds, and estuaries (BSEs) throughout the southeast region of the U.S.A. and are sentinel species for human and ecosystem-level health. Dolphins are vulnerable to the bioaccumulation of contaminants through the coastal food chain because they are high-level predators. Currently, there is limited information on the spatial dynamics of mercury accumulation in these dolphins. Total mercury (THg) was measured in dolphin skin from multiple populations across the U.S. Southeast Atlantic and Gulf of Mexico coasts, and the influence of geographic origin, sex, and age class was investigated. Mercury varied significantly among sampling sites and was greatest in dolphins in St. Joseph Bay, Florida Everglades, and Choctawhatchee Bay (14,193 ng/g ± 2196 ng/g, 10,916 ng/g ± 1532 ng/g, and 7333 ng/g ± 1405 ng/g wet mass (wm), respectively) and lowest in dolphins in Charleston and Skidaway River Estuary (509 ng/g ± 32.1 ng/g and 530 ng/g ± 58.4 ng/g wm, respectively). Spatial mercury patterns were consistent regardless of sex or age class. Bottlenose dolphin mercury exposure can effectively represent regional trends and reflect large-scale atmospheric mercury input and local biogeochemical processes. As a sentinel species, the bottlenose dolphin data presented here can direct future studies to evaluate mercury exposure to human residents in St. Joseph Bay, Choctawhatchee Bay, and Florida Coastal Everglades, as well as additional sites with similar geographical, oceanographic, or anthropogenic parameters. These data may also inform state and federal authorities that establish fish consumption advisories to determine if residents in these locales are at heightened risk for mercury toxicity.

Mercury accumulation and biomarkers of exposure in two popular recreational fishes in Hawaiian waters.

Mercury (Hg) exposure has not been examined in many recreational nearshore fish species that are commonly consumed around the Hawaiian Islands. Specific gene transcripts, such as metallothionein (MET) and thioredoxin reductase (TrxR), can be used to examine Hg exposure responses in aquatic organisms. This study measured total mercury (THg) in four species from two groups of Hawaiian nearshore fishes: giant trevally (Caranx ignobilis, n = 13), bluefin trevally (C. melampygus, n = 4), sharp jaw bonefish (Albula virgata, n = 2), and round jaw bonefish (A. glossodonta, n = 19). Total Hg accumulation and abundance profiles of MET and TrxR were evaluated for muscle, liver, and kidney tissues. Total Hg in round jaw bonefish and giant trevally tissues accumulated with length and calculated age. In round jaw bonefish tissues, mean THg was greater in kidney (1156 ng/g wet mass (wm)) than liver (339 ng/g wm) and muscle (330 ng/g wm). Giant trevally muscle (187 ng/g wm) and liver (277 ng/g wm) mean THg did not differ significantly. Fish species in this study were compared to commercial and local fish species with state and federal muscle tissue consumption advisories based on THg benchmarks developed by the U.S. Food and Drug Administration (FDA) and Environmental Protection Agency (EPA). Both bonefishes had mean muscle THg that exceeded benchmarks suggesting consumption advisories should be considered. MET transcript in round jaw bonefish kidney tissue and kidney THg exhibited a marginally significant positive correlation, while TrxR transcript in liver tissue negatively correlated with increasing liver THg. These results contribute to our understanding of Hg exposure associated health effects in fish.

Development of Kudzu (Pueraria Montana var. lobata) Reference Materials for the Determination of Isoflavones and Toxic Elements.

BACKGROUND: In collaboration with the Office of Dietary Supplements at the National Institutes of Health, the National Institute of Standards and Technology issued a suite of botanical matrix reference materials (RMs) and Standard Reference Material® (SRM) for determination of isoflavones and toxic elements in kudzu dietary supplement ingredients. OBJECTIVE: RM 8650 Pueraria montana var. lobata (Kudzu) Rhizome, SRM 3268 Pueraria montana var. lobata (Kudzu) Extract, and RM 8652 Kudzu-Containing Solid Oral Dosage Form were issued with values assigned for isoflavones (puerarin, daidzin, and daidzein), toxic elements (arsenic, cadmium, and lead), and selenium. METHODS: Isoflavone values were assigned using liquid chromatography with UV absorbance or mass spectrometry detection. Element values were assigned using inductively coupled plasma mass spectrometry and results from an interlaboratory comparison exercise. RESULTS: Mass fractions for puerarin were 32.2 ± 3.2 mg/g, 128 ± 13 mg/g, and 68.2 ± 6.9 mg/g in RM 8650, SRM 3268, and RM 8652, respectively. Arsenic increases from 156 ± 14 ng/g to 849 ± 83 ng/g and cadmium decreases from 348 ± 14 ng/g to 82.1 ± 4.9 ng/g from rhizome to extract. CONCLUSION: The kudzu RM/SRM suite complements previously issued soy-related SRMs with values assigned for isoflavones, which have been studied for their potential health benefits, and expands the analytical resource by providing values for puerarin, an isoflavone not found in soy. HIGHLIGHTS: The three new kudzurmaterials are for use in the determination of isoflavones, toxic elements, and selenium. For the isoflavones, these new kudzu materials provide higher levels of daidzin and daidzein than existing soy-related SRMs, and they provide a value for an isoflavone not in existing SRMs (puerarin). Toxic elements in RM 8650 and SRM 3268 provide new botanical matrixes for use by dietary supplement manufacturers for the verification of the safety of their raw materials.

Trace element proxies and stable isotopes used to identify water quality threats to elkhorn coral (Acropora palmata) at two national parks in St. Croix, USVI.

Biological impairments have been documented on reefs at two national parks in St. Croix, USVI. Although several water quality parameters have been out of compliance with USVI criteria, whether these parameters or other pollutants are responsible for coral health impacts is unknown. Trace elements quantified in sediment showed four sites at SARI, which is closer than BUIS to settlements and land-derived anthropogenic outflows, had Cu mass fractions above sediment quality guidelines for invertebrate toxicity. Trace elements were also analyzed in the skeleton of threatened elkhorn coral, Acropora palmata, to evaluate potential exposure. Heavy metals (Pb, Zn) were significantly greater in coral skeleton at SARI than BUIS. Cu, Pb, and Zn may be impacting coral health in these parks. Potential anthropogenic sources of these metals were revealed by the coral tissue stable isotope levels (δ13C and δ15N). These findings provide a framework for determining heavy metal impacts on these invaluable reefs.

Comparison of Primary Laser Spectroscopy and Mass Spectrometry Methods for Measuring Mass Concentration of Gaseous Elemental Mercury.

We present a direct comparison between two independent methods for the measurement of gaseous elemental mercury (GEM) mass concentration: isotope dilution cold-vapor inductively coupled plasma mass spectrometry (ID-CV-ICP-MS) and laser absorption spectroscopy (LAS). The former technique combined with passive sorbent tube sampling is currently the primary method at NIST for mercury gas standards traceability to the International System of Units (SI). This traceability is achieved via measurements on a mercury-containing reference material. The latter technique has been recently developed at NIST and involves real-time measurements of light attenuation caused by GEM, with SI traceability based in part on the known spontaneous emission lifetime of the probed 6 1S0-6 3P1 intercombination transition of elemental mercury (Hg0). Using a steady-flow Hg0-in-air generator to produce samples measured by both methods, we use LAS to measure the sample gas and in parallel we collect the Hg0 on sorbent tubes to be subsequently analyzed using ID-CV-ICP-MS. Over the examined mass concentration range (41 µg/m3 to 287 µg/m3 Hg0 in air), the relative disagreement between the two approaches ranged from (1.0 to 1.8)%. The relative combined standard uncertainty on average is 0.4% and 0.9%, for the LAS and MS methods, respectively. Our comparison studies help validate the accuracy of the ID-CV-ICP-MS primary method as well as establish the LAS technique as an attractive alternative primary method for SI-traceable measurements of GEM.

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.

Avian mercury exposure and toxicological risk across western North America: A synthesis.

Methylmercury contamination of the environment is an important issue globally, and birds are useful bioindicators for mercury monitoring programs. The available data on mercury contamination of birds in western North America were synthesized. Original data from multiple databases were obtained and a literature review was conducted to obtain additional mercury concentrations. In total, 29219 original bird mercury concentrations from 225 species were compiled, and an additional 1712 mean mercury concentrations, representing 19998 individuals and 176 species, from 200 publications were obtained. To make mercury data comparable across bird tissues, published equations of tissue mercury correlations were used to convert all mercury concentrations into blood-equivalent mercury concentrations. Blood-equivalent mercury concentrations differed among species, foraging guilds, habitat types, locations, and ecoregions. Piscivores and carnivores exhibited the greatest mercury concentrations, whereas herbivores and granivores exhibited the lowest mercury concentrations. Bird mercury concentrations were greatest in ocean and salt marsh habitats and lowest in terrestrial habitats. Bird mercury concentrations were above toxicity benchmarks in many areas throughout western North America, and multiple hotspots were identified. Additionally, published toxicity benchmarks established in multiple tissues were summarized and translated into a common blood-equivalent mercury concentration. Overall, 66% of birds sampled in western North American exceeded a blood-equivalent mercury concentration of 0.2 µg/g wet weight (ww; above background levels), which is the lowest-observed effect level, 28% exceeded 1.0 µg/g ww (moderate risk), 8% exceeded 3.0 µg/g ww (high risk), and 4% exceeded 4.0 µg/g ww (severe risk). Mercury monitoring programs should sample bird tissues, such as adult blood and eggs, that are most-easily translated into tissues with well-developed toxicity benchmarks and that are directly relevant to bird reproduction. Results indicate that mercury contamination of birds is prevalent in many areas throughout western North America, and large-scale ecological attributes are important factors influencing bird mercury concentrations.

Mercury risk to avian piscivores across western United States and Canada.

The widespread distribution of mercury (Hg) threatens wildlife health, particularly piscivorous birds. Western North America is a diverse region that provides critical habitat to many piscivorous bird species, and also has a well-documented history of mercury contamination from legacy mining and atmospheric deposition. The diversity of landscapes in the west limits the distribution of avian piscivore species, complicating broad comparisons across the region. Mercury risk to avian piscivores was evaluated across the western United States and Canada using a suite of avian piscivore species representing a variety of foraging strategies that together occur broadly across the region. Prey fish Hg concentrations were size-adjusted to the preferred size class of the diet for each avian piscivore (Bald Eagle=36cm, Osprey=30cm, Common and Yellow-billed Loon=15cm, Western and Clark's Grebe=6cm, and Belted Kingfisher=5cm) across each species breeding range. Using a combination of field and lab-based studies on Hg effect in a variety of species, wet weight blood estimates were grouped into five relative risk categories including: background (<0.5µg/g), low (0.5-1µg/g), moderate (1-2µg/g), high (2-3µg/g), and extra high (>3µg/g). These risk categories were used to estimate potential mercury risk to avian piscivores across the west at a 1degree-by-1degree grid cell resolution. Avian piscivores foraging on larger-sized fish generally were at a higher relative risk to Hg. Habitats with a relatively high risk included wetland complexes (e.g., prairie pothole in Saskatchewan), river deltas (e.g., San Francisco Bay, Puget Sound, Columbia River), and arid lands (Great Basin and central Arizona). These results indicate that more intensive avian piscivore sampling is needed across Western North America to generate a more robust assessment of exposure risk.

Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics.

Trace Element Concentrations in Liver of 16 Species of Cetaceans Stranded on Pacific Islands from 1997 through 2013.

The impacts of anthropogenic contaminants on marine ecosystems are a concern worldwide. Anthropogenic activities can enrich trace elements in marine biota to concentrations that may negatively impact organism health. Exposure to elevated concentrations of trace elements is considered a contributing factor in marine mammal population declines. Hawai'i is an increasingly important geographic location for global monitoring, yet trace element concentrations have not been quantified in Hawaiian cetaceans, and there is little trace element data for Pacific cetaceans. This study measured trace elements (Cr, Mn, Cu, Zn, As, Se, Sr, Cd, Sn, Hg, and Pb) in liver of 16 species of cetaceans that stranded on U.S. Pacific Islands from 1997 to 2013, using high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) (n = 31), and direct mercury analysis atomic absorption spectrometry (DMA-AAS) (n = 43). Concentration ranges (µg/g wet mass fraction) for non-essential trace elements, such as Cd (0.0031-58.93) and Hg (0.0062-1571.75) were much greater than essential trace elements, such as Mn (0.590-17.31) and Zn (14.72-245.38). Differences were found among age classes in Cu, Zn, Hg, and Se concentrations. The highest concentrations of Se, Cd, Sn, Hg, and Pb were found in one adult female false killer whale (Pseudorca crassidens) at concentrations that are known to affect health in marine mammals. The results of this study establish initial trace element concentration ranges for Pacific cetaceans in the Hawaiian Islands region, provide insights into contaminant exposure of these marine mammals, and contribute to a greater understanding of anthropogenic impacts in the Pacific Ocean.

Certification of Total Arsenic in Blood and Urine Standard Reference Materials by Radiochemical Neutron Activation Analysis and Inductively Coupled Plasma - Mass Spectrometry.

A newly developed procedure for determination of arsenic by radiochemical neutron activation analysis (RNAA) was used to measure arsenic at four levels in SRM 955c Toxic Elements in Caprine Blood and at two levels in SRM 2668 Toxic Elements in Frozen Human Urine for the purpose of providing mass concentration values for certification. Samples were freeze-dried prior to analysis followed by neutron irradiation for 3 h at a fluence rate of 1×1014cm-2s-1. After sample dissolution in perchloric and nitric acids, arsenic was separated from the matrix by extraction into zinc diethyldithiocarbamate in chloroform, and 76As quantified by gamma-ray spectroscopy. Differences in chemical yield and counting geometry between samples and standards were monitored by measuring the count rate of a 77As tracer added before sample dissolution. RNAA results were combined with inductively coupled plasma - mass spectrometry (ICP-MS) values from NIST and collaborating laboratories to provide certified values of (10.81 ± 0.54) µg/kg and (213.1 ± 0.73) µg/kg for SRM 2668 Levels I and II, and certified values of (21.66 ± 0.73) µg/kg, (52.7 ± 1.1) µg/kg, and (78.8 ± 4.9) µg/kg for SRM 955c Levels 2, 3, and 4 respectively. Because of discrepancies between values obtained by different methods for SRM 955c Level 1, an information value of < 5 µg/kg was assigned for this material.

Spatial and temporal trends of persistent organic pollutants and mercury in beluga whales (Delphinapterus leucas) from Alaska.

Remote locations, such as the Arctic, are often sinks for persistent contaminants which can ultimately bioaccumulate in local wildlife. Assessing temporal contaminant trends in the Arctic is important in understanding whether restrictions on legacy persistent organic pollutants (POPs) have led to concentration declines. Beluga whale (Delphinapterus leucas) tissue samples were collected from two subpopulations (Cook Inlet, Alaska and the eastern Chukchi Sea) between 1989 and 2006. Several POPs (polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethane and related compounds (DDTs), chlordanes, hexachlorocyclohexanes (HCHs), chlorobenzenes, mirex, polybrominated diphenyl ethers (PBDEs) and semi-quantitatively hexabromocyclododecanes (HBCDs)) were measured in 70 blubber samples, and total mercury (Hg) was measured in 67 liver samples from a similar set of individuals. Legacy POPs (PCBs, chlordanes, DDTs, and HCHs) were the predominant organic compound classes in both subpopulations, with median concentrations of 2360ng/g lipid for Σ80PCBs and 1890 ng/g lipid for Σ6DDTs. Backward stepwise multiple regressions showed that at least one of the four independent variables (subpopulation, sampling year, sex, and animal length) influenced the POP and Hg concentrations. ΣPCBs, ΣDDTs, Σchlordanes, Σchlorobenzenes, mirex, and Hg were significantly higher in belugas from the eastern Chukchi Sea than from the Cook Inlet (p≤0.0001). In contrast, Σ8PBDE and α-HBCD concentrations were significantly lower in belugas from the eastern Chukchi Sea than from the Cook Inlet (p<0.0001). Significant temporal increases in concentrations of Σ8PBDE and α-HBCD were observed for both subpopulations (p≤0.0003), and temporal declines were seen for ΣHCHs and Σchlorobenzenes in eastern Chukchi Sea belugas only (p≤0.0107). All other POP and Hg concentrations were stable, indicating either a lagging response of the Arctic to source reductions or the maintenance of concentrations by unregulated sources. Sex and length also significantly influenced some concentrations, and these findings are discussed.

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.

Establishing baseline levels of trace elements in blood and skin of bottlenose dolphins in Sarasota Bay, Florida: implications for non-invasive monitoring.

Several major unusual mortality events occurring in recent years have increased the level of concern for the health of bottlenose dolphin populations along the United States Atlantic and Gulf of Mexico coasts. Trace element concentrations were examined in a population of free-ranging dolphins in Sarasota Bay, Florida, in order to develop a benchmark for future comparisons within and between populations. Whole blood (n=51) and skin (n=40) samples were collected through capture and release health assessment events during 2002-2004. Samples were analyzed for Al, V, Cr, Mn, Cu, Zn, As, Se, Rb, Sr, Mo, Cd, and Pb by inductively coupled plasma mass spectrometry (ICPMS) and Hg via atomic fluorescence spectrometry (AFS). Trace element concentrations (wet mass) in skin were 2 to 45 times greater than blood, except Cu was approximately 1.5 times higher in blood. Statistically strong correlations (p<0.05) were found for V, As, Se, Rb, Sr, and Hg between blood and skin demonstrating that these tissues can be used as effective non-lethal monitoring tools. The strongest correlation was established for Hg (r=0.9689) and concentrations in both blood and skin were above the threshold at which detrimental effects are observed in other vertebrate species. Female dolphins had significantly greater Hg concentrations in blood and skin and Pb concentrations in skin, relative to males. Calves exhibited significantly lower V, As, and Hg concentrations in blood and V and Hg concentrations in skin, relative to other age classes. Rubidium and Cu concentrations in skin were greatest in subadults and calves, respectively. In blood, V, Zn, and As concentrations were significantly greater in winter, relative to summer, and the opposite trend was observed for Rb and Sr concentrations. In skin, Cu and Zn concentrations were significantly greater in winter, relative to summer, and the opposite trend was observed for Mn, Rb, Cd, and Pb concentrations. The baseline concentrations and trends established in this study will serve as a benchmark for comparison and aid in sampling design for future monitoring of this population and other coastal bottlenose dolphin populations.