Genetic status of the wood stork (Mycteria americana) from the southeastern United States and the Brazilian Pantanal as revealed by mitochondrial DNA analysis.

The wood stork (Mycteria americana) is a colonial wading bird that inhabits the Neotropical region from the southeastern United States (US) to northern Argentina. The species is considered to be endangered in the US due to degradation of its foraging and breeding habitat. In other parts of its range, such as in the Brazilian Pantanal region, breeding populations of this species appear to be stable. We compared the levels of genetic variability and population structuring of the US and the Pantanal breeding populations using mitochondrial DNA (mtDNA) control region sequences. Twenty-seven haplotypes were identified among 88 wood stork samples collected from eight breeding colonies in the US and eight in the Pantanal. Patterns indicative of heteroplasmy were observed in 35.3% of the mtDNA sequences that were examined. Significantly higher levels of haplotype diversity were observed in the Pantanal samples compared to those from the US, suggesting that during the last century, demographic declines or a recent evolutionary bottleneck reduced the levels of mtDNA variability of the US population. Analyses of genetic structuring revealed non-significant genetic differentiation between these regions, indicating that either the populations were only recently separated or that gene flow continues to occur at low levels. Haplotype network analysis indicated low current levels of gene flow between populations that were closely related in the past.

Clapper rails as indicators of mercury and PCB bioavailability in a Georgia saltmarsh system.

Clapper rails (Rallus longirostris) were used as an indicator species of estuarine marsh habitat quality because of their strong site fidelity and predictable diet consisting of mostly benthic organisms. Mercury (Hg) and the polychlorinated biphenyl (PCB) Aroclor 1268 concentrations were determined for sediments, crabs, as well as clapper rail adults and chicks collected from salt marshes associated with the LCP Superfund site in Brunswick, Georgia. Home ranges were established for adult rails, and sediment and crab samples were taken from each individual's range. The study was designed to minimize the spatial variability associated with trophic transfer studies by choosing an endpoint species with a potentially small home range and specifically sampling its foraging range. The mean home range for clapper rails was 1.2 ha with a median of 0.28 ha. Concentrations of Hg and Aroclor 1268 were shown to increase with each trophic level. Transfer factors between media followed the same pattern for both contaminants with the highest between fiddler crabs and clapper rail liver. Hg and PCB transfer factors were similar between sediment to fiddler crab and fiddler crab to muscle, however the PCB transfer factor from fiddler crabs to liver was over twice as large as for Hg. PCB congener profiles did not significantly differ between media types.

Raccoon (Procyon lotor) as a bioindicator of mercury contamination at the U.S. Department of Energy's Savannah River Site.

Raccoons (n = 95) were collected from the U.S. Department of Energy's Savannah River Site (SRS) and from public hunting areas. Raccoons were collected near a stream drainage system (Steel Creek delta) and a former reactor-cooling reservoir (pond B) that received inputs of mercury-contaminated Savannah River water. Mercury concentrations were determined for hair, liver, kidney, muscle, and spleen tissues. Samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS) and cold vapor atomic absorption spectroscopy (CVAAS). Raccoons were also collected from a natural stream floodplain system (Upper Three Runs/Tinker Creek) located upstream of Hg use and storage areas and near coal ash basins. These samples were compared to samples collected from off-site hunting areas near the SRS. Hg concentrations between internal tissues were significantly correlated. Hair did not correlate well with internal tissue and was a weak indicator of raccoon exposure to Hg. Nonetheless, raccoons are potentially good indicators of Hg contamination because tissue concentrations were higher in raccoons from areas with known Hg input than in those from reference areas, and muscle biopsies can be used as biomarkers for contaminant exposure. Muscle Hg concentrations ranged from 0-0.14 ppm from nearby hunting grounds, indicating that people hunting in designated areas near the SRS are at negligible risk for Hg consumption from raccoon meat. Several raccoons collected from the SRS had muscle Hg concentrations at or near the FDA action level for seizing commercial fish due to mercury contamination of 1.0 ppm. Though Hg action levels for wild game have not been proposed, it is clear that some SRS raccoons had Hg levels that warrant concern if these areas would be open to public hunting. Last, 64 raccoons from this study had Hg concentrations that were considered elevated by the U.S. FWS standard (> 1.1 ppm) of ecosystem health for one or two tissues (hair, liver, or kidney), and 17 had high concentrations for most or all tissues.

Metal levels in raccoon tissues: differences on and off the Department of Energy's Savannah River Site in South Carolina.

Levels of arsenic, cadmium, chromium, copper, lead, manganese, selenium, and strontium88 were examined in heart, kidney, muscle, spleen and liver of raccoons (Procyon lotor) from four areas on the Department of Energy's Savannah River Site (SRS), including near a former reactor cooling reservoir and a coal ash basin, and from public hunting areas within 15 km of the site. Mercury is mentioned briefly because it is discussed more fully in another paper. We test the hypotheses that there are no differences in metal levels between raccoons on SRS and off the SRS (off-site), and among different locations on the SRS. Although raccoons collected off-site had significantly lower levels of mercury and selenium in both the liver and kidney, there were few consistencies otherwise. There were significantly higher levels of cadmium in liver of on-site compared to off-site raccoons, and significantly higher levels of chromium and strontium88 in kidney of on-site compared to off-site raccoons. Copper and manganese were highest in the liver; cadmium, lead, mercury and selenium were highest in the liver and kidney; chromium was highest in the spleen and muscle; arsenic was highest in the heart, and strontium88 was slightly higher in the kidney than other organs. Where there were significant differences on site, chromium, manganese were highest in raccoon tissues from Steel Creek; arsenic, lead and selenium were highest in the Ash Basin; cadmium was highest at Upper Three Runs; and strontium88 was highest at Upper Three Runs and Steel Creek. The patterns were far from consistent.

Radiocesium in fish from the Savannah River and Steel Creek: potential food chain exposure to the public.

This study examined radiocesium (137Cs) levels in fish from the vicinity of the Department of Energy's Savannah River Site (SRS), a former nuclear weapons production facility in South Carolina. Fish from the Savannah River were sampled above (upstream), along, and below (downstream) the SRS, and from Steel Creek, a tributary that runs through the SRS. There was some off-site contamination of 137Cs in the Savannah River watershed due to low-level releases from past nuclear production on the SRS. The null hypotheses tested were that there would be no differences in 137Cs levels as a function of location along the river, and between species collected from the river and from Steel Creek on the SRS. For six of eight species of fish collected from the Savannah River, there were no differences in 137Cs levels in muscle from fish collected above, along, or below the SRS; exceptions were bowfin and shellcracker. Fish collected from Steel Creek had significantly higher levels (by about an order of magnitude) of 137Cs in muscle tissue than fish collected in the Savannah River. However, no fish from either Steel Creek or the Savannah River had 137Cs levels above the European Economic Community limit for fresh meat of 0.6 Bq/g. Lifetime cancer risk was calculated using the cancer slope factor of 3.2 x 10(-11)/pCi, and various fish consumption scenarios reflecting actual data from Savannah River fishermen. Using mean 137Cs concentrations and median fish consumption for 70 years for Black males-the group with the highest consumption-the excess lifetime risk associated with the eight species of fish in the Savannah River ranged from 9.0 x 10(-7) to 1.0 x 10(-5). The same calculation for fish from Steel Creek gave risk estimates from 1.4 to 8.0 x 10(-5). The 95% level for consumption by Blacks, however, was about 70 kg/year. Black fishermen consuming that amount of bass from Steel Creek would sustain a lifetime risk of 3.1 x 10(-4), whereas the same consumption of Savannah River bass would yield a risk estimate of 1.5 x 10(-5).

Unusual leg malformations in screech owls from a South Carolina Superfund site.

In 1995, the discovery of leg malformations in several screech owl (Otis asio) nestlings and in their female parent at a Department of Energy (DOE) Superfund site in South Carolina prompted an investigation into the nature of the observed abnormalities. Surviving nestlings and the female parent were transferred to a captive screech owl breeding colony at the USGS Patuxent Wildlife Research Center, Laurel, MD. The malformed female parent and her offspring were each mated with normal owls from the colony for 3 yr. Matings of the malfored female produced five malformed and six normal owls; all owls produced by matings of normal offspring were normal. Malformed offspring were euthanized when it became apparent that their physical distress precluded survival under normal conditions of colony care. Euthanized owls were necropsied and examined for skeletal development. Detailed descriptions of eight malformed owls are presented. Results of the matings indicated that the leg mafformations were caused by a genetic trait in the female parent that was heterozygous dominant. The characteristic was lethal except in occasional mild manifestations and resembled an extreme form of a dominant abnormality previously described for domestic fowl called duplicate polydactyly. Other reports of skeletal abnormalities in wild birds and potential environmental causes of genetic mutations at the DOE Superfund site are presented. Other studies performed at the DOE Superfund site do not implicate elevated (above background) ionizing radiation from 137Cs, the dominant radionuclide where the owls were captured, as the cause of the mutation. The cause of this genetic abnormality remains unknown.

Metals and metallothionein in the liver of raccoons: utility for environmental assessment and monitoring.

The relationship between metallothionein levels and concentrations of several metals and radionuclides was examined in liver tissues of raccoons (Procyon lotor, n = 47) from the Department of Energy's Savannah River Site in South Carolina to determine the applicability of metallothioneins as an initial screening device for exposure assessment in free-living mammals and environmental monitoring. Using a fluorescent marker and a cell sorter to measure metallothionein, a significant positive correlation was found across animals between levels of metallothioneins and concentrations of selenium (Pearson's r = .30), mercury (Pearson's r = .3 1), and copper (Pearson's r = .30) in liver tissue. Arsenic, cobalt, silver, thallium, and tin were below detection limits in most or all liver samples. Other metals, including cadmium, chromium, radiocesium (137-Cs), copper, lead, manganese, strontium, and vanadium, showed only weak and nonsignificant correlations with metallothionein. Concentrations of mercury were correlated with concentrations of selenium (Pearson's r = .73), manganese (Pearson's r = .56), and strontium (Pearson's r = .57). In an a posteriori test, there was a still unexplained positive correlation between mercury (Pearson r = .56), selenium (Pearson r = .54), and radiocesium (Pearson's r = .38) concentrations and background cellular autofluorescence, and a negative correlation of strontium with the latter (Kendall tau = -.38). Background cellular autofluorescence may represent a generalized cellular stress response, or a yet unidentified biomarker. To better understand which metals contribute to the induction of metallothionein, principle component analysis (PCA) was performed. The first three principle components explained 78% of the variance, with highest loadings being from mercury and radiocesium. Metallothionein levels did not correlate well with the principal components from the metals and radiocesium, while autofluorescent background levels tended to correlate better.

Foraging ecology of the endangered wood stork recorded in the stable isotope signature of feathers.

Down feathers and regurgitant were collected from nestling wood storks (Mycteria americana) from two inland and two coastal breeding colonies in Georgia. The stable isotopic ratios of carbon (13C/12C) and nitrogen (15N/14N) in these materials were analyzed to gain insights into the natal origins of juvenile storks and the foraging activities of adults. Down feathers differed in δ13C between inland and coastal colonies, having average isotopic values that reflected the sources of carbon fixed in biomass at the base of the food web. Feathers from the inland colonies differed between colonies in δ15N, while those from the coastal colonies did not. These patterns primarily reflected the foraging activities of parent storks, with individuals capturing differing percentages of prey of distinct trophic status at each colony. Collectively, the carbon and nitrogen isotopic signatures of feather keratin were used to distinguish nestlings from each colony, except for instances where storks from different colonies foraged in common wetlands. The stable isotopic composition of food items in regurgitant was used to reconstruct the trophic structure of the ecosystems in which wood storks foraged. Predicted foraging activities based on the isotopic composition of keratin were generally consistent with the percentage of prey types (freshwater vs. saltwater and lower trophic level vs. upper trophic level consumer) observed in regurgitant, except for the coastal colony at St. Simons Island, where the δ13C of feathers strongly suggested that freshwater prey were a significant component of the diet. This inconsistency was resolved by aerial tracking of adults during foraging excursions using a fixed-wing aircraft. Observed foraging activities supported interpretations based on the stable isotope content of feathers, suggesting that the latter provided a better record of overall foraging activity than regurgitant analysis alone. Observed foraging patterns were compared to the predictions of a statistical model that determined habitat utilization based on habitat availability using a geographic information system (GIS) database. Observed foraging activities and those predicted from feathers both suggested that some adult storks preferred to feed their young freshwater prey, even when saltwater resources were more accessible in the local environment. This conclusion supports the contention that wood stork populations are sensitive to changes in the distribution of freshwater habitats along the southeastern coastal plain of the United States.

Contaminant exposure and biomarker responses in spectacled eiders (Somateria fischeri) from St. Lawrence Island, Alaska.

Effects of chemical contaminant exposure may be contributing to the decline of spectacled eiders (Somateria fischeri) nesting in coastal areas of western Alaska. We evaluated chemical exposure and potential effects in 20 male eiders collected near St. Lawrence Island, Alaska. Analytes included metals, trace elements, chlorinated organics, and (137)Cesium ((137)Cs). Effects of contaminant exposure were evaluated using histopathology and biochemical measures of porphyrin profiles, cytochrome P450 activities, and metallothionein (MT) concentrations. Copper, cadmium, and selenium concentrations were elevated in spectacled eiders when compared to literature values for other marine birds. Only a few samples had trace concentrations of chlorinated organic compounds. Muscle (137)Cs levels were all below the average minimum quantifiable concentration of 0.079 Bq/g. No histopathological lesions were associated with elevated contaminant concentrations in liver, kidney, or testes. Protoporphyrin was found in highest concentration in both the liver and kidneys, followed by coproporphyrin and uroporphyrin, respectively. Hepatic uroporphyrin concentrations correlated significantly to hepatic arsenic concentrations. Mean activities of hepatic EROD, MROD, BROD, and PROD were consistent with other avian species. Comparisons of cadmium/MT ratios from this study to published literature ratios in seven marine avian species suggest that, although adult male spectacled eiders have elevated liver concentrations of certain MT-inducing metals, their MT concentrations are not as strongly induced as would be predicted based on literature values. Despite elevated metal concentrations, the apparent good health of the St. Lawrence Island birds suggests that should these contaminants be a factor in population declines, they likely act by decreasing fecundity or survival of young rather than via direct health impacts on adult male spectacled eiders.

Lack of demonstratable effects of pollutants on cyt b sequences in wood ducks from a contaminated nuclear reactor cooling pond.

The effects of low levels of radiation on DNA mutation rates are largely unknown for free-living vertebrates. In this study we investigated the effects of contamination from cooling ponds at a nuclear production facility in South Carolina on the mutation rates in mitochondrial DNA in wood ducks (Aix sponsa). Specifically, we sequenced a 433-bp portion of the cytochrome b gene from 18 female-offspring pairs of wood ducks from contaminated ponds and 2 female-offspring pairs from control ponds. Very low haplotype diversity was observed overall, and no case of mutation between female and offspring could be satisfactorily documented. This suggests that the levels of radioactive contamination in these cooling ponds have little effect on the mutation rate of mitochondrial DNA in these waterfowl and that mitochondrial DNA may not be as sensitive an indicator as previously anticipated.

A risk assessment for consumers of mourning doves.

Recreational and subsistence hunters and anglers consume a wide range of species, including birds, mammals, fish and shellfish, some of which represent significant exposure pathways for environmental toxic agents. This study focuses on the Department of Energy's (DOE's) Savannah River Site (SRS), a former nuclear weapons production facility in South Carolina. The potential risk of contaminant intake from consuming mourning doves (Zenaida macroura), the most popular United States game bird, was examined under various risk scenarios. For all of these scenarios we used the mean tissue concentration of six metals (lead, mercury, cadmium, selenium, chromium, manganese) and radiocesium, in doves collected on and near SRS. We also estimated risk to a child consuming doves that had the maximum contaminant level. We used the cancer slope factor for radiocesium, the Environmental Protection Agencies Uptake/Biokinetic model for lead, and published reference doses for the other metals. As a result of our risk assessments we recommend management of water levels in contaminated reservoirs so that lake bed sediments are not exposed to use by gamebirds and other terrestrial wildlife. Particularly, measures should be taken to insure that the hunting public does not have access to such a site. Our data also indicate that doves on popular hunting areas are exposed to excess lead, suggesting that banning lead shot for doves, as has been done for waterfowl, is desirable.

Mercury in alligators (Alligator mississippiensis) in the southeastern United States.

Mercury methylation may be enhanced in wetlands and humic-rich, blackwater systems that crocodiles and alligators typically inhabit. Given their high trophic level and long life-spans, crocodilians could accumulate significant burdens of Hg. Our objectives were to survey Hg concentrations in alligators from several areas in the southeastern United States to test their utility as sentinels of Hg contamination, to examine relationships among Hg concentrations in various tissues and to look for any differences in tissue Hg concentrations among locations. We measured total Hg concentrations in alligators collected in the Florida Everglades (n = 18), the Okefenokee National Wildlife Refuge, Georgia (n = 9), the Savannah River Site (SRS), South Carolina (n = 49) and various locations in central Florida (n = 21), sampling tissues including blood, brain, liver, kidney, muscle, bone, fat, spleen, claws and dermal scutes. Alligators from the Everglades were mostly juvenile, but Hg concentrations in tissues were high (means: liver 41.0, kidney 36.4, muscle 5.6 mg Hg/kg dry wt.). Concentrations in alligators from other locations in Florida were lower (means: liver 14.6, kidney 12.6, muscle 1.8 mg Hg/kg dry wt.), although they tended to be larger adults. Alligators from the Okefenokee were smallest and had the lowest Hg concentrations (means: liver 4.3, kidney 4.8, muscle 0.8 mg Hg/kg dry wt.). SRS alligators had the greatest size range and intermediate Hg levels (means: liver 14.9, muscle 4.8 mg Hg/kg dry wt.). At some locations, alligator length was correlated with Hg concentrations in some internal organs. However, at three of the four locations, muscle Hg was not related to length. Tissue Hg concentrations were correlated at most locations however, claw or dermal scute Hg explained less than 74% of the variation of Hg in muscle or organs, suggesting readily-obtained tissues, such as scutes or claws, have limited value for non-destructive screening of Hg in alligator populations.

Tissue mercury concentrations in alligators (Alligator mississippiensis) from the Florida Everglades and the Savannah River Site, South Carolina.

Mercury pollution is a serious problem in some areas of the southeastern United States. Due to biomagnification, long-lived predators should have high Hg concentrations in affected areas. American alligators(Alligator mississippiensis) are important predators in many southwestern wetlands, but little information is available on Hg concentrations in this species. We collected tissues from alligators inhabiting two sites in the Everglades, Florida (n = 18) and a manmade reservoir in South Carolina (Par Pond; n = 44), all with documented histories of Hg contamination, and analyzed them for total Hg. Mean concentrations in kidney, liver, muscle, and dermal scutes of alligators from the Everglades (expressed as mg Hg/kg dry mass +/- S.E.M.) were 36.42 +/- 5.23, 41.09 +/- 5.90, 5.57 +/- 0.47, and 5.83 +/- 1.04, respectively. Concentrations in liver, muscle, and scutes from Par Pond alligators were 17.73 +/- 2.56, 4.08 +/- 0.46, and 4.58 +/- 0.63,respectively. Blood from Par Pond alligators contained 2.20 +/- 0.38 mg Hg/kg wet mass. Mercury concentrations did not differ among sexes at any location. Tissue Hg levels did not differ significantly between Everglades locations,but were lower in Par Pond. In Everglades alligators, Hg concentrations in all tissues were positively correlated, as were tissue Hg and total length. Only total length and scute Hg were correlated in Par Pond alligators. Regression revealed a significant relationship between muscle and scute Hg concentrations in Everglades alligators, but not Par Pond alligators. Alligators living in polluted areas can accumulate substantial concentrations of Hg, but relationships among Hg concentrations in specific tissues may vary with location or age and size of the animals sampled.

Metal levels in mourning doves from South Carolina: potential hazards to doves and hunters.

Most game birds are found in lower trophic levels, but since such birds are harvested and consumed by humans, there is a particular need to assess their contaminant levels. In this paper, we report concentrations of mercury, lead, cadmium, selenium, manganese, and chromium in the breast feathers, liver, and muscle of mourning doves (Zenaida macroura) collected at a partially drawn-down, contaminated reactor-cooling reservoir (Par Pond) on the Department of Energy's Savannah River Site in South Carolina and at nearby agricultural fields managed as dove hunting areas. We test the hypothesis that the levels in doves are not harmful to either dove populations or humans. We also tested the simultaneous effects of collection location, year (1992, 1993), and dove age-class (hatch-year vs after hatch-year) on heavy metal and selenium levels. For all three tissues, mercury levels were nondetectable at all locations. Lead was highest in tissues from agricultural fields with prior histories of dove hunting activities. Doves at those fields were likely ingesting lead shot to a greater degree than at the recently drawn-down reservoir which was closed to public access and hunting. For other metals, Par Pond doves had equally high or higher tissue levels. For all metals, levels in doves from South Carolina were generally within the lower range of those reported in the literature, suggesting that these metals were likely to pose no health problems to these doves. Except for lead and selenium, metal levels in dove muscle that we observed were well below reference metal doses established for human intake. Lead and selenium, at the levels described here, would only be a problem if a child (not an adult) ate 120 g of dove meat every day of the year. Thus, we conclude that meat from these doves, if consumed by hunters, would not pose a risk.

Sources of error associated with sample collection and preparation of nucleated blood cells for flow cytometric analysis.

Analysis of cellular DNA content by flow cytometry has been used to detect genetic changes associated with exposure to environmental contaminants. In lower vertebrates, nucleated red blood cells can be collected for analysis without harm to the animal. Because erythrocytes sampled from an individual should have identical amounts of DNA, the coefficient of variation (CV) around the G0/G1 peak should be small. Increases in CV can indicate genetic aberrations, but may also be caused by sample handling and preparation or problems with instrumentation. To increase confidence in associating increases in CV with external causes, artifactual changes in CV due to sample treatment and instrument parameters should be identified and minimized. We assessed the effects of various sampling and handling protocols on the CV of nucleated blood cells collected from largemouth bass (Micropterus salmoides). We also compared the distribution of cells among the G0/G1, S, and G2/M phases of the cell cycle to see whether these were affected by sampling or treatment protocols. Groups of 7 fish were bled on 7 consecutive days, and blood from each fish was analyzed by flow cytometry when freshly collected, and after freezing for 1 hour or 10 days. The same fish were bled again over a consecutive 7-day period, and the experiment was repeated. CV and cell cycle distribution were not affected by our freezing protocol. Repeat sampling from the same individual did not affect CV, but altered the distribution of cells in the cell cycle, suggesting increased hemopoiesis in response to blood sampling.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric DNA analysis of ducks accumulating 137Cs on a reactor reservoir.

The objective of this study was to detect red blood cell (rbc) DNA abnormalities in male, game-farm mallard ducks as they ranged freely and accumulated 137Cs (radiocesium) from an abandoned nuclear reactor cooling reservoir. Prior to release, the ducks were tamed to enable recapture at will. Flow cytometric measurements conducted at intervals during the first year of exposure yielded cell cycle percentages of DNA (G0/G1, S, G2 + M phases) of rbc, as well as coefficients of variation (CV) in the G0/G1 phase. DNA histograms of exposed ducks were compared with two sets of controls which were maintained 30 and 150 miles from the study site. 137Cs live wholebody burdens were also measured in these animals in a parallel kinetics study, and an approximate steady-state equilibrium was attained after about 8 months. DNA histograms from 2 of the 14 contaminated ducks revealed DNA aneuploid-like patterns after 9 months exposure. These two ducks were removed from the experiment at this time, and when sampled again 1 month later, one continued to exhibit DNA aneuploidy. None of the control DNA histograms demonstrated DNA aneuploid-like patterns. There were no significant differences in cell cycle percentages at any time point between control and exposed animals. A significant increase in CV was observed at 9 months exposure, but after removal of the two ducks with DNA aneuploidy, no significant difference was detected in the group monitored after 12 months exposure. An increased variation in the DNA and DNA aneuploidy could, therefore, be detected in duck rbc using flow cytometric analysis, with the onset of these effects being related to the attainment of maximal levels of 137Cs body burdens in the exposed animals.