Micro-distribution of arsenic in toenail clippings using laser ablation inductively coupled plasma mass spectrometry: implications for biomonitoring.

Toenails are a common monitoring tool for arsenic exposure, but the risk of external contamination of toenails has cast doubt on its usefulness. The main objective of this study is to investigate the micro-distribution of arsenic through the dorsoventral plane of nail clippings to understand endogenous vs exogenous sources. We used laser-ablation inductively coupled plasma mass spectrometry to measure arsenic through a dorsoventral cross-section of the nail plate collected from reference (N = 17) and exposed individuals (N = 35). Our main results showed (1) bulk toenail concentrations measured using ICP-MS in this study ranged from 0.54 to 4.35 µg/g; (2) there was a double-hump pattern in arsenic concentrations, i.e., dorsal and ventral layers had higher arsenic than the inner layer; (3) the double-hump was more pronounced in the exposed group (ventral: 6.25 µg/g; inner: 0.75 µg/g; dorsal: 0.95 µg/g) than the reference group (ventral: 0.58 µg/g; inner: 0.15 µg/g; dorsal: 0.29 µg/g) on average; (4) the distribution was, in part, associated with different binding affinity of nail layers (i.e., ventral > dorsal > inner); (5) most individuals in the higher exposure group showed > 25% contamination in ventral and dorsal nail layers; and (6) there were no statistically significant correlations between LA-ICP-MS arsenic with either bulk toenail arsenic or urine arsenic from the same individuals. Our results on micro-distribution and binding affinity provide insight into the impact of external contamination on arsenic concentrations and show how LA-ICP-MS can access the protected inner nail layer to provide a more accurate result.

Grizzly bear hair reveals toxic exposure to mercury through salmon consumption.

Mercury obtained from the diet accumulates in mammalian hair as it grows thus preserving a record of mercury intake over the growth period of a given hair segment. We adapted a microanalysis approach, using laser ablation inductively coupled plasma mass spectrometry, to characterize temporal changes in mercury exposure and uptake in wild and captive grizzly bears. Captive grizzlies fed diets containing known and varied amounts of mercury provided data to allow prediction of Hg ingestion rates in wild bears. Here, we show, for the first time, that 70% of the coastal grizzly bears sampled had Hg levels exceeding the neurochemical effect level proposed for polar bears. In a context where the international community is taking global actions to reduce Hg emissions through the "Minamata Convention on Mercury", our study provides valuable information on the exposure to mercury of these grizzly bears already under many threats.

Microchemistry of Single Hair Strands Below and Above the Scalp: Impacts of External Contamination on Cuticle and Cortex Layers.

External contamination of hair is the most significant challenge to it becoming an accepted matrix for monitoring endogenous metal exposure and nutritional deficiency. Here we use laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to quantify elemental concentrations in hair strands below and above the scalp in the cuticle and cortex layers to determine the extent of external contamination in a reference population. Evidence of hair strand contamination occurred for barium, calcium, iron, magnesium, and strontium in both the outer cuticle and the inner cortex layers, with increasing concentrations from root to tip. Aluminum, boron, copper, lead, and manganese showed significant contamination in the cuticle layer only, suggesting some protection of the inner cortex. Phosphorus and potassium decreased outside the scalp suggesting loss by washing, while chromium, mercury, selenium, sodium, titanium, and zinc showed no evidence of loss or external contamination above the scalp. The results clearly show that for most elements, hair chemistry above the scalp is unreliable for use in interpretation of endogenous exposures or deficiencies, and that the below-scalp portion provides a more accurate monitoring tool. This is the first paper to provide a reference range of elemental hair chemistry that is not impacted by the external environment.

Determination of Elemental Composition in Soft Biological Tissue Using Laser Ablation Inductively Coupled Plasma Mass Spectrometry: Method Validation.

Determination of elemental concentrations in biological tissue is fundamental to many environmental studies. Analytical methods typically used to quantify concentrations in such studies have minimum sample volumes that necessitate lethal or impactful collection of tissues. Laser ablation inductively coupled mass spectrometry (LA-ICP-MS) has small sample volume requirements and offers environmental practitioners an opportunity to employ low-impact sample collection methods. Environmental applications of LA-ICP-MS are limited by the lack of validated methods, partly due to the need for dry samples and scarcity of matrix-matched certified reference materials (CRMs). This study validates an LA-ICP-MS method to determine concentrations of 30 elements in soft biological tissue (fish ovary and muscle). Tissue samples (median: 0.48 grams (g); inter-quartile range: 0.30 g to 0.56 g wet weight) were dehydrated, powdered, compressed into pellets (weighing approximately 0.03 g) and analyzed using LA-ICP-MS alongside three matrix-matched CRMs. The method yielded concentration determinations for CRM elements that were typically accurate to within 30% of theoretical concentrations, and precise (relative standard deviation <20%). These results were repeatable: accuracy rarely deviated from theoretical values by more than 20%, and precision rarely exceeded 33%. Determinations for biological samples were replicable irrespective of tissue (ovary or muscle). There was good linearity between analyte signal strength and theoretical concentration (median R2 ≥ 0.981 for all elements) across ranges typically encountered in environmental studies. Concentrations could not be consistently obtained (i.e., determined concentrations were typically below detection limits) for boron, vanadium, molybdenum, and cadmium in muscles, and arsenic in both ovaries and muscles; however, detection limits were sufficiently low for most environmental contexts. Further methodological refinement could include the incorporation of spiked standards to extend linear ranges, and fine-tuning instrument parameters to obtain smoother signal intensities for rare elements. The method presented promotes the use of low-impact sample collection methods while enabling high-quality determinations of elemental concentrations in biological tissues.

Persistent or not persistent? Polychlorinated biphenyls are readily depurated by grizzly bears (Ursus arctos horribilis).

Major pharmacokinetic processes influencing polychlorinated biphenyl (PCB) accumulation in mammals include uptake, biotransformation, respiration, and excretion. We characterized some of the factors underlying PCB accumulation/loss by evaluating PCB concentrations and patterns in pre- and posthibernation grizzly bears (Ursus arctos horribilis) and their prey. The PCB congeners with vicinal meta- and para-chlorine unsubstituted hydrogen positions consistently showed loss both before and during hibernation, supporting the idea of a dominant role for biotransformation. Retention of all other studied congeners relative to that of PCB 194 varied widely (from <1 to 100%) and was highly correlated with log octanol-water partition coefficient (p < 0.0001). A lack of loss for most of these other congeners during hibernation supports the notion that excretion (e.g., fecal or urinary) or lack of uptake during the feeding season underlies their lack of accumulation, because hibernating bears do not eat or excrete. We estimate that grizzly bears retain less than 10% of total PCBs taken up from their diet. Our results suggest that for grizzly bears, depuration of PCBs via biotransformation is important (explaining approximately 40% of loss), but that nonbiotransformation processes, such as excretion, may be more important (explaining approximately 60% of loss). These findings, together with the approximately 91% loss of the persistent PCB 153 congener relative to PCB 194 in grizzly bears, raise important questions about how one defines persistence of PCBs in wildlife and may have bearing on the interpretation of food-web biomagnification studies.

Effects of nonylphenol on rates of tail resorption and metamorphosis in Rana catesbeiana tadpoles.

Nonylphenol (NP) is a persistent, lipophilic, and toxic chemical that can be endocrine disrupting (estrogenic) at sublethal concentrations. Since amphibian metamorphosis is a hormone-driven process and a delicate balance of hormone levels is required for successful metamorphosis, exposure of larval amphibians to NP might disrupt metamorphic processes. This study tested whether NP exposure influenced rate of metamorphic progression and tail resorption in bullfrog (Rana catesbeiana) tadpoles. Premetamorphic bullfrog tadpoles were exposed for 7 d to one of 3 nominal concentrations of NP (234 microg/L, 468 microg/L, or 936 microg/L) with or without the addition of exogenous 3,3',5-triiodothyronine (T3). In the absence of exogenous T3, NP significantly increased the rate of tail growth (as measured by tail length) at 936 microg/L. There was no significant effect of NP alone on tail width, limb development, or the process of cranial transformation. When T3 was added to the treatments, increasing NP concentrations were associated with a significant decrease in the rate of cranial transformation, and at the highest dose, the rate of tail resorption was significantly lower than in the controls. Overall, NP had an inhibitory effect on the rate of bullfrog tadpole metamorphic progression and tail resorption.

Using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to characterize copper, zinc and mercury along grizzly bear hair providing estimate of diet.

We enhanced an existing technique, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), to function as a non-lethal tool in the temporal characterization of trace element exposure in wild mammals. Mercury (Hg), copper (Cu), cadmium (Cd), lead (Pb), iron (Fe) and zinc (Zn) were analyzed along the hair of captive and wild grizzly bears (Ursus arctos horribilis). Laser parameters were optimized (consecutive 2000 µm line scans along the middle line of the hair at a speed of 50 µm/s; spot size=30 µm) for consistent ablation of the hair. A pressed pellet of reference material DOLT-2 and sulfur were used as external and internal standards, respectively. Our newly adapted method passed the quality control tests with strong correlations between trace element concentrations obtained using LA-ICP-MS and those obtained with regular solution-ICP-MS (r(2)=0.92, 0.98, 0.63, 0.57, 0.99 and 0.90 for Hg, Fe, Cu, Zn, Cd and Pb, respectively). Cross-correlation analyses revealed good reproducibility between trace element patterns obtained from hair collected from the same bear. One exception was Cd for which external contamination was observed resulting in poor reproducibility. In order to validate the method, we used LA-ICP-MS on the hair of five captive grizzly bears fed known and varying amounts of cutthroat trout over a period of 33 days. Trace element patterns along the hair revealed strong Hg, Cu and Zn signals coinciding with fish consumption. Accordingly, significant correlations between Hg, Cu, and Zn in the hair and Hg, Cu, and Zn intake were evident and we were able to develop accumulation models for each of these elements. While the use of LA-ICP-MS for the monitoring of trace elements in wildlife is in its infancy, this study highlights the robustness and applicability of this newly adapted method.

Validation of an amphibian sperm inhibition toxicological test method using zinc.

Analysis of sperm has been investigated as a possible method to examine the toxicity of environmental contaminants. The amphibian sperm inhibition toxicological test (ASITT) method examines the effects of contaminants on Xenopus laevis (African clawed frog) sperm motility and path trajectories. As part of a preliminary validation of the method, the effects of increasing divalent metal ion, zinc (Zn2+), on X. laevis sperm motility were examined. We hypothesized that Zn2+ concentration would have significant inhibitory effects on percent sperm motility, velocities, and trajectories. The Zn2+ was added to a control solution in concentrations from 0 to 1,417 microg/L. Sperm cells were videotaped at 30 frames per second under x 400 microscope, and percent motility was recorded and paths were mapped by marking the change in position of the sperm head over a period of 1 s. Sperm motility was categorized as progressive, hyperactivated, idle, or nonmotile, and velocities and trajectories were calculated on the basis of x,y coordinates. Increasing Zn2+ concentrations caused a significant exponential decay in percent total motility and progressive motility. Straight-line velocity increased with increasing Zn2+ concentrations. Overall, results suggest that Zn2+ may be interfering with cellular processes, such as cellular respiration, flagellar bending, or ion exchange, thereby inhibiting sperm motility.

Plant consumption by grizzly bears reduces biomagnification of salmon-derived polychlorinated biphenyls, polybrominated diphenyl ethers, and organochlorine pesticides.

The present study characterizes the uptake and loss of persistent organic pollutants (POPs) in grizzly bears (Ursus arctos horribilis) by sampling and analyzing their terrestrial and marine foods and fecal material from a remote coastal watershed in British Columbia, Canada. The authors estimate that grizzly bears consume 341 to 1,120 µg of polychlorinated biphenyls (PCBs) and 3.9 to 33 µg of polybrominated diphenyl ethers daily in the fall when they have access to an abundant supply of returning salmon. The authors also estimate that POP elimination by grizzly bears through defecation is very low following salmon consumption (typically <2% of intake) but surprisingly high following plant consumption (>100% for PCBs and organochlorine pesticides). Excretion of individual POPs is largely driven by a combination of fugacity (differences between bear and food concentrations) and the digestibility of the food. The results of the present study are substantiated by a principal components analysis, which also demonstrates a strong role for log KOW in governing the excretion of different POPs in grizzly bears. Collectively, the present study's results reveal that grizzly bears experience a vegetation-associated drawdown of POPs previously acquired through the consumption of salmon, to such an extent that net biomagnification is reduced.

Biomagnification of polychlorinated biphenyls in a harbor seal (Phoca vitulina) food web from the Strait of Georgia, British Columbia, Canada.

Polychlorinated biphenyl (PCB) biomagnification was characterized in a harbor seal food web in the Strait of Georgia, British Columbia, Canada. Trophic magnification factors (TMFs) for PCBs averaged 3.6, with a range of 0.7 to 9.4. The TMFs for individual congeners correlated with log K(OW) (r(2) = 0.56, p < 0.001), reflecting the role that physicochemical properties play in driving the biomagnification of PCBs in marine food webs. However, TMFs differed among PCB structure activity groups, clearly indicating an additional role for metabolic transformation of certain PCBs. The known feeding preferences of harbor seals enabled the calculation of trophic level-adjusted biomagnification factors (BMF(TL)) for PCBs in this species, which averaged 13.4 and ranged from 0.2 to 150.6. Metabolic transformation in seals explained some of the variation in congener-specific biomagnification, with lower BMF(TL) values for PCB congeners with meta- and parachlorine unsubstituted positions. Principal components analysis revealed the distinct roles played by trophic level, log K(OW), and metabolic transformation in explaining the notable differences in PCB patterns among harbor seals, their pups, and their prey. In the present study, the authors estimate there to be approximately 76 kg of PCBs in the biota of the Strait of Georgia, of which 1.6 kg is retained by harbor seals.

Hibernation-associated changes in persistent organic pollutant (POP) levels and patterns in British Columbia grizzly bears (ursus arctos horribilis).

We hypothesized that depleted fat reserves in grizzly bears (Ursus arctos horribilis) following annual hibernation would reveal increases in persistent organic pollutant (POP) concentrations compared to those present in the fall. We obtained fat and hair from British Columbia grizzly bears in early spring 2004 to compare with those collected in fall 2003, with the two tissue types providing contaminant and dietary information, respectively. By correcting for the individual feeding habits of grizzlies using a stable isotope-based approach, we found that polychlorinated biphenyls (sigmaPCBs) increased by 2.21x, polybrominated diphenylethers (sigmaPBDEs) increased by 1.58x, and chlordanes (sigmaCHL) by 1.49x in fat following hibernation. Interestingly, individual POPs elicited a wide range of hibernation-associated concentration effects (e.g., CB-153, 2.25x vs CB-169, 0.00x), resulting in POP pattern convergence in a PCA model of two distinct fall feeding groups (salmon-eating vs non-salmon-eating) into a single spring (post-hibernation) group. Our results suggest that diet dictates contaminant patterns during a feeding phase, while metabolism drives patterns during a fasting phase. This work suggests a duality of POP-associated health risks to hibernating grizzly bears: (1) increased concentrations of some POPs during hibernation; and (2) a potentially prolonged accumulation of water-soluble, highly reactive POP metabolites, since grizzly bears do not excrete during hibernation.

Persistent organic pollutants in British Columbia grizzly bears: consequence of divergent diets.

Nitrogen and carbon stable isotope signatures in growing hair reveal that while some British Columbia grizzly bears (Ursus arctos horribilis) rely entirely on terrestrial foods, others switch in late summer to returning Pacific salmon (Oncorynchus spp.). Implications for persistent organic pollutant (POP) concentrations and patterns measured in the two feeding groups of grizzly bears were profound. While the bears consuming a higher proportion of terrestrial vegetation ("interior" grizzlies) exhibited POP patterns dominated bythe more volatile organochlorine (OC) pesticides and the heavier polybrominated diphenyl ethers (PBDEs: e.g., BDE-209), the bears consuming salmon were dominated by the more bioaccumulative POPs (e.g., DDT, chlordanes, and BDE-47). The ocean-salmon-bear pathway appeared to preferentially select for those contaminants with intermediate partitioning strength from water into lipid (log Kow approximately 6.5). This pattern reflects an optimum contaminant log Kow range for atmospheric transport, deposition into the marine environment, uptake into marine biota, accumulation through the food web, and retention in the bear tissues. We estimate that salmon deliver 70% of all OC pesticides, up to 85% of the lower brominated PBDE congeners, and 90% of PCBs found in salmon-eating grizzly bears, thereby inextricably linking these terrestrial predators to contaminants from the North Pacific Ocean.