A seasonal study on the microbiomes of Diploid vs. Triploid eastern oysters and their denitrification potential.

Oyster reefs are hotspots of denitrification mediated removal of dissolved nitrogen (N), however, information on their denitrifier microbiota is scarce. Furthermore, in oyster aquaculture, triploids are often preferred over diploids, yet again, microbiome differences between oyster ploidies are unknown. To address these knowledge gaps, farmed diploid and triploid oysters were collected over an annual growth cycle and analyzed using shotgun metagenomics and quantitative microbial elemental cycling (QMEC) techniques. Regardless of ploidy, Psychrobacter genus was abundant, with positive correlations found for genes of central metabolism, DNA metabolism, and carbohydrate metabolism. MAGs (metagenome-assembled genomes) yielded multiple Psychrobacter genomes harboring norB, narH, narI, and nirK denitrification genes, indicating their functional relevance within the eastern oysters. QMEC analysis indicated the predominance of carbon (C) and nitrogen (N) cycling genes, with no discernable patterns between ploidies. Among the N-cycling genes, the nosZII clade was overrepresented, suggesting its role in the eastern oyster's N removal processes.

Tissue Distribution of Mercury in the Bodies of Wild American Alligators (Alligator mississippiensis) from a Coastal Marsh in Louisiana (USA).

Total mercury (THg) concentrations were measured in wild alligators inhabiting a coastal marsh in southern Louisiana, to determine the tissue distribution of THg among various body organs and tissue compartments. Concentrations of THg in claws and dermal tail scutes were compared to those in blood, brain, gonad, heart, kidney, liver, and skeletal muscle to determine if the former tissues, commonly available by non-lethal sampling, could be used as measures of body burdens in various internal organs. Mercury was found in all body organs and tissue compartments. However, overall, THg concentrations measured in alligators were below the FDA action level for fish consumption and were comparable to previous data reported from southwestern Louisiana. Our results suggest consumption of meat from alligators found in this region may be of little public health concern. However, the extended period of time between sampling (in this study) and the present-day highlight the need for continuous, additional, and more recent sampling to ensure consumer safety. Total mercury concentrations were highest in the kidney (3.18 ± 0.69 mg/kg dw) and liver (3.12 ± 0.76 mg/kg dw). THg levels in non-lethal samples (blood, claws, and dermal tail scutes) were positively correlated with all tissue THg concentrations (blood: R2 = 0.513-0.988; claw: R2 = 0.347-0.637, scutes: R2 = 0.333-0.649). Because THg concentrations from blood, claws, and scutes were correlated with those of the internal organs, non-lethal sampling methods may be a viable method of estimating levels of THg in other body tissues.

Precipitation influences on uptake of a global pollutant by a coastal avian species.

Climatic variation, including precipitation amounts and timing, has been linked to abundance and breeding success of many avian species. Less studied, but also of significance, is the consequence of climatic variability on the exposure and uptake of nutrients and contaminants by wildlife. The authors examined mercury (Hg) concentrations in nestling wood stork feathers in a coastal setting over a 16-yr period to understand the influence of rainfall amounts on Hg transfer by parental provisioning relative to habitat use, assuming differential bioavailability of Hg within freshwater and saltwater habitat types. Coastal Hg uptake by stork nestlings was linked to freshwater habitat use, as indicated by stable carbon isotope (δ(13)C) analyses. Cumulative rainfall amounts exceeding 220 cm in the 23 mo preceding the breeding seasons resulted in greater use of freshwater wetlands as foraging habitat and greater Hg accumulation by nestling storks.

Mercury concentrations in nestling wading birds relative to diet in the southeastern United States: a stable isotope analysis.

Mercury (Hg) is a ubiquitous environmental contaminant that is transferred trophically through aquatic and terrestrial food webs. To better understand the routes of Hg uptake in organisms that rely on both aquatic and terrestrial food resources, we analyzed feather and down samples from nestling wading birds of varying trophic positions in both inland and coastal colonies. We used stable nitrogen and carbon isotope analyses to evaluate trophic positions of individual species (δ(15)N) and differences in foraging habitat use (δ(13)C). Inland, aquatic species had higher trophic status than the single terrestrial species examined, and the expected positive relationship between δ(15)N and Hg content of feathers was observed. However, the same was not true for all species from coastal colonies. Feathers from species that primarily consumed saltwater prey were relatively high in δ(15)N value and low in Hg content, which is opposite of the trend expected due to Hg biomagnification in food chains. In contrast, coastal species foraging in freshwater or a combination of freshwater and saltwater habitats displayed greater Hg contents in feathers. The apparent differential use of the two aquatic systems (freshwater and saltwater) in coastal environments by wading bird species results in variations in δ(15)N values and Hg contents in nestling feathers not found in species associated with only freshwater systems.

A transgenic strain of the nematode Caenorhabditis elegans as a biomonitor for heavy metal contamination.

Metallothionein (MT), a protein involved in metal regulation and detoxification, has been used widely as a biomarker of metal exposure. In the present study, a transgenic strain of the free-living soil nematode Caenorhabditis elegans was developed using the C. elegans MT-2 (mtl-2) promoter to control the transcription of green fluorescence protein (GFP) reporter. Response of this transgenic system to Cd, Hg, Cu, Zn, Ni, Pb, and As exposure in aquatic media was tested by quantifying GFP expression after 24 h of exposure. Response in Cd-spiked soil was tested in a similar manner. The mtl-2 transcription also was measured using real-time reverse transcription-polymerase chain reaction to gain a mechanistic understanding of the transgene expression. Green fluorescence protein is induced by Cd, Hg, Cu, and Zn in a time- and concentration-dependent manner; mtl-2 transcription is consistent with the GFP response. The minimum concentrations of Cd, Hg, Cu, and Zn that induce GFP response are 2- to 1000-fold lower than concentrations affecting traditional endpoints, such as lethality or behavioral change. The system responds to Cd in soil in a similar manner. Neither Ni nor Pb induces GFP, and neither induces mtl-2 transcription. Arsenic does not induce GFP, yet an increase in mtl-2 transcription was found, suggesting that As may interfere with GFP signaling. This mtl-2::GFP transgenic bioassay represents an alternative approach to quantify, both easily and quickly, a surrogate of MT in response to metal exposure (e.g., Cd, Hg, Cu, and Zn) in a variety of environments and potentially may be used for quantitative or semiquantitativebiomonitoring of metal contamination in soils and aquatic systems.

Transgenic lambda medaka as a new model for germ cell mutagenesis.

To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated lambda transgenic medaka, a small fish that carries the cII mutation target gene, as a new model for germ cell mutagenesis. Mutations in the cII gene in progeny derived from ethyl-nitrosourea (ENU)-exposed males were readily detected. Frequencies of mutant offspring, proportions of mosaic or whole body mutant offspring, and mutational spectra differed according to germ cell stage exposed to ENU. Postmeiotic germ cells (spermatozoa/late spermatids) generated a higher frequency of mutant offspring (11%) compared to premeiotic germ cells (3.5%). Individuals with cII mutant frequencies (MF) elevated more than threefold above the spontaneous MF (3 x 10(-5)) in the range of 10(-4) to 10(-3) were mosaic mutant offspring, whereas those with MFs approaching 1 x 10(-2) were whole body mutant offspring. Mosaic mutant offspring comprised the majority of mutant offspring derived from postmeiotic germ cells, and unexpectedly, from spermatogonial stem cells. Mutational spectra comprised of two different mutations, but at identical sites were unusual and characteristic of delayed mutations, in which fixation of a second mutation was delayed following fertilization. Delayed mutations and prevalence of mosaic mutant offspring add to growing evidence that implicates germ cells in mediating processes postfertilization that contribute to genomic instability in progeny. This model provides an efficient and sensitive approach to assess germ cell mutations, expands opportunities to increase understanding of fundamental mechanisms of mutagenesis, and provides a means for improved assessment of potential genetic health risks.

Using trace element concentrations in Corbicula fluminea to identify potential sources of contamination in an urban river.

We used the biomonitor, Corbicula fluminea, to investigate the contributions of trace elements associated with different point sources and land uses in a large river. Trace elements were analyzed in tissues of clams collected from 15 tributary streams draining five land use or point source types: agriculture, forest, urban, coal-fired power plant (CFPP), and wastewater (WWTP). Clams from forested catchments had elevated Hg concentrations, and concentrations of arsenic and selenium were highest (5.0+/-0.2 and 13.6+/-0.9 microg g(-1) dry mass (DM), respectively) in clams from CFPP sites. Cadmium concentrations were significantly higher in clams from urban and CFPP sites (4.1+/-0.2 and 3.6+/-0.9 microg g(-1) DM, respectively). Non-metric multidimensional scaling (NMS) of tissue concentrations in clams clustered at CFPP and forest/agriculture sites at opposite ends of the ordination space, and the distribution of sites was driven by Cu, Zn, Cd, and Hg.

Coselection for microbial resistance to metals and antibiotics in freshwater microcosms.

Bacterial resistances to diverse metals and antibiotics are often genetically linked, suggesting that exposure to toxic metals may select for strains resistant to antibiotics and vice versa. To test the hypothesis that resistances to metals and antibiotics are coselected for in environmental microbial assemblages, we investigated the frequency of diverse resistances in freshwater microcosms amended with Cd, Ni, ampicillin or tetracycline. We found that all four toxicants significantly increased the frequency of bacterioplankton resistance to multiple, chemically unrelated metals and antibiotics. An ampicillin-resistant strain of the opportunistic human pathogen Ralstonia mannitolilytica was enriched in microcosms amended with Cd. Frequencies of antibiotic resistance were elevated in microcosms with metal concentrations representative of industry and mining-impacted environments (0.01-1 mM). Metal but not antibiotic amendments decreased microbial diversity, and a weeklong exposure to high concentrations of ampicillin (0.01-10 mg l-1) and tetracycline (0.03-30 mg l-1) decreased microbial abundance only slightly, implying a large reservoir of antibiotic resistance in the studied environment. Our results provide first experimental evidence that the exposure of freshwater environments to individual metals and antibiotics selects for multiresistant microorganisms, including opportunistic human pathogens.

Biological half-life and oxidative stress effects in mice with low-level, oral exposure to tritium.

Tritium ((3)H) may enter the environment from human activities, particularly at production, processing, or waste storage sites such as the Department of Energy's Savannah River Site, a former nuclear production facility in South Carolina. Understanding the dynamics and potential adverse effects of tritium in exposed organisms is critical to evaluating risks of tritium releases at such sites. Previous studies estimated the biological half-life of tritium in mice to be approximately 1.13 d; however, these laboratory studies were not conducted under environmentally realistic conditions. In this study, designed to be more representative of environmental exposure, mice were allowed to drink water containing tritium (activity about 300 Bq/ml) for a period of 2 wk. The induction of oxidative stress from tritium exposure was evaluated by comparing the activities of antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase) in exposed and control mice. From this experiment, the biological half-life of tritium was determined to be 2.26 +/- 0.04 d, almost double previous estimates. While positive controls (x-ray irradiated mice) showed responses in antioxidant enzyme activity, there was no indication of oxidative stress induction in mice exposed to tritium at this concentration.

Elevated microbial tolerance to metals and antibiotics in metal-contaminated industrial environments.

To test the hypothesis that industrial metal contaminants select for microorganisms tolerant to unrelated agents, such as antibiotics, we analyzed metal and antibiotic tolerance patterns in microbial communities in the intake and discharge of ash settling basins (ASBs) of three coal-fired power plants. High-throughput flow-cytometric analyses using cell viability probes were employed to determine tolerances of entire bacterioplankton communities, avoiding bias toward culturable versus nonculturable bacteria. We found that bacterioplankton collected in ASB discharges were significantly more tolerant to metal and antibiotic exposures than bacterioplankton collected in ASB intakes. Optical properties of microorganisms collected in ASB discharges indicated no defensive physiological adaptations such as formation of resting stages or excessive production of exopolymers. Thus, it is likely that the elevated frequency of metal and antibiotic tolerances in bacterioplankton in ASB discharges were caused by shifts in microbial community composition, resulting from the selective pressure imposed by elevated metal concentrations or organic toxicants present in ASBs.

Shifts in relative tissue delta15N values in snowy egret nestlings with dietary mercury exposure: a marker for increased protein degradation.

Shifts in tissue nitrogen isotope composition may be a more sensitive general indicator of stress than measurement of high-turnover defensive biomolecules such as metallothionein and glutathione. As a physical resource transmitted along the trophic web, perturbations in protein nitrogen metabolism may also help resolve issues concerning the effects of contaminants on organisms and their consequential hierarchical linkages in ecotoxicology. Snowy egret nestlings (Egretta thula) fed mercury-contaminated diets of constant nitrogen isotope composition exhibited increased relative delta15N values in whole liver (p = 0.0011) and the acid-soluble fraction (ASF) of the liver (p = 0.0005) when compared to nestlings fed a reference diet. When nitrogen isotope data were adjusted for the source term of the diet, liver mercury concentrations corresponded with both whole liver relative 15N enrichment (r2 = 0.79, slope 0.009, p < 0.0001) and relative 15N enrichment in the acid-soluble fraction of the liver (r2 = 0.85, slope 0.026, p < 0.0001). Meanwhile, significant differences were not observed in hepatic levels of the metal-binding peptides metallothionein and glutathione despite a nearly 3-fold difference in liver mercury content. Because increases in tissue delta15N values result from increased rates of protein breakdown relative to synthesis, we propose that the increased relative liver delta15N values reflect a shift in protein metabolism. The relationship between ASF and mercury was significantly stronger (p < 0.0001) than that for whole liver, suggesting that the relationship is driven by an increase in bodily derived amino acids in the acid-soluble, free amino acid pool.

Rodents as receptor species at a tritium disposal site.

New methods are being employed on the Department of Energy's Savannah River Site to deal with the disposal of tritium, including the irrigation of a hardwood/pine forest with tritiated water from an intercepted contaminant plume to reduce concentrations of tritium outcropping into Fourmile Branch, a tributary of the Savannah River. The use of this system has proven to be an effective means of tritium disposal. To evaluate the impact of this activity on terrestrial biota, rodent species were captured on the tritium disposal site and a control site during two trapping seasons in order to assess tritium exposure resulting from the forest irrigation. Control site mice had background levels of tritium, 0.02 Bq/mL, with disposal site mice having significantly higher tritium concentrations, mean=34.86 Bq/mL. Whole body tritium concentrations of the mice captured at the disposal site were positively correlated with tritium application and negatively correlated with precipitation at the site.

Adverse effects of ecologically relevant dietary mercury exposure in southern leopard frog (Rana sphenocephala) larvae.

Southern leopard frog (Rana sphenocephala) larvae were exposed to experimental diets supplemented with aufwuchs from control and mercury-enriched mesocosms combined in proportions intended to mimic mercury concentrations and speciation in aufwuchs observed from aquatic systems contaminated by atmospheric deposition. Observations on rates of mortality, malformation, and larval growth and development were made for 254 d. Increased incidence of mortality, malformation, and changes in growth and development were observed at concentrations that reflect the highest concentrations expected in the amphibian diet from atmospheric deposition (1,500-3,300 ng Hg/g dry wt). The results of this study are probably more ecologically realistic than results obtained from previous studies of aqueous mercury toxicity and suggest that dietary mercury exposure in habitats contaminated primarily by atmospheric deposition has the potential to cause adverse effects in amphibian larvae.

Dietary mercury exposure and bioaccumulation in southern leopard frog (Rana sphenocephala) larvae.

Aufwuchs was collected from three reservoirs, a constructed wetland used for groundwater treatment, and mercury (Hg)-enriched mesocosms to examine the relationship between inorganic Hg and methylmercury concentrations in the diet of tadpoles. Four diets were then formulated with Hg-enriched aufwuchs to concentrations that bracketed those of Hg observed in aufwuchs from the field and reported in the literature from sites contaminated by atmospheric deposition. The diets were fed to southern leopard frog tadpoles in the laboratory for the entire larval period (60-254 d). Metamorphs and tadpoles were analyzed for inorganic Hg and methylmercury contents by gas chromatography-cold-vapor atomic fluorescence spectrophotometry. Methylmercury concentration increased with total Hg concentration in aufwuchs, but the proportion of methylmercury to inorganic Hg decreased with increasing total Hg concentration. In the feeding experiment, there was an inverse relationship between Hg exposure concentration and the bioaccumulation factor for each Hg species. We concluded that neither methylmercury nor inorganic Hg in aufwuchs is highly bioavailable to tadpoles and that bioaccumulation is not well explained by a simple partitioning model. This suggests that bioaccumulation factors as currently used are not the best predictors of dietary Hg bioaccumulation.

A microscaled mercury saturation assay for metallothionein in fish.

A mercury (Hg) saturation assay for measuring metallothionein (MT) in fish liver was modified by optimizing binding conditions to minimize the mercury and tissue consumed. The revised method uses stable Hg at low concentrations instead of 203Hg. At the reduced Hg concentrations used, MT concentrations in livers homogenized in saline appeared to increase systematically with dilution in both bluegill sunfish (Lepomis macrochirus) and largemouth bass (Micropterus salmoides). This error suggested a binding limitation due to sulfhydryl oxidation or competition for and removal of mercury by non-MT proteins. Homogenizing tissues in trichloroacetic acid (TCA) eliminated the interference. To further evaluate the method, the protocol was tested in the laboratory and field. Metallothionein in bluegill injected with 0.6 mg/kg zinc chloride increased at a rate of 0.03 nmole MT/g liver/h (r2 = 0.53, p = 0.001). Linearity improved when data were corrected for protein content (r2 = 0.74, p < 0.0001). Metallothionein levels in bluegill from a coal ash-contaminated environment were significantly increased over that of hatchery-reared sunfish (F = 20.17, p = 0.0003). The microscaled procedure minimizes concerns related to radioisotope use and waste generation while retaining the high sensitivity of the 203Hg assay.

Relationships between mercury body concentrations, standard metabolic rate, and body mass in eastern mosquitofish (Gambusia holbrooki) from three experimental populations.

Eastern mosquitofish (Gambusia holbrooki) were sampled from three experimental populations (two Hg-exposed populations and one reference population) to determine whether transgenerational exposure (lifelong exposure of multiple generations) to Hg adversely affects standard metabolic rate (SMR). Mosquitofish subjected to lifelong Hg exposure accumulated significant concentrations of Hg in their tissues compared to fish from the reference population (mean: 3.89-4.13 vs 0.08 microHg/g wet mass, respectively). Less than 10% of the variability in Hg tissue concentrations could be explained by fish body mass, likely because of the short life span and/or dietary habits of this species. Despite the high body burdens of Hg in exposed fish, we found no significant difference in SMR among individuals from Hg-exposed or reference populations. Our findings contrast recent laboratory work describing elevated SMR in mosquitofish exposed to 100 microg/L dissolved inorganic Hg for 48 h. To account for contrasting results between studies, we hypothesize that acute exposure to dissolved inorganic Hg damages gill epithelium, resulting in increased metabolic rate, but that lifelong Hg exposure via trophic uptake of methyl mercury does not affect fish respiratory structures. Alternative hypotheses include the possibility that G. holbrooki is a species that can tolerate high body burdens of Hg or that more than four years of genetic isolation during Hg exposure (8-12 generations) resulted in selection for Hg-tolerant or -resistant individuals.

Potential risk to wood storks (Mycteria americana) from mercury in Carolina Bay fish.

Carolina bays are freshwater wetlands that serve as important feeding habitats for the endangered wood stork (Mycteria americana). Water levels in these bays fluctuate greatly and tend to be acidic and rich in dissolved organic carbon (DOC), factors that favor mercury (Hg) methylation and bioaccumulation in fish. To assess potential risks to wood storks consuming mercury contaminated fish in bays, we sampled fish from 10 bays on the Savannah River Site (SRS), South Carolina, an area with documented use by wood storks. Whole body mercury concentrations in 258 fishes of three species (Erimyzon sucetta, Acantharchuspomotis and Esox americanus) commonly consumed by wood storks were determined. Risk factors for nestlings and free-ranging adults were calculated using published no and lowest observable adverse effect concentration (NOAEC and LOAEC) values for birds. Fish from higher trophic levels and those from wetlands with relatively shallow maximum depths and fluctuating water levels were more likely to exceed NOAEC and LOAEC values. Calculation of exposure rates of nestling wood storks indicated they are at highest risk during the first 10 days of the nestling period. These calculations suggest that there is potential concern for wood storks foraging in relatively shallow bays with fluctuating water levels, even though there is no obvious local source of mercury to these wetlands.

Frequency distributions of 137Cs in fish and mammal populations.

We collected fish and mammals in several radioactively contaminated locations in the Chornobyl Exclusion Zone and analyzed them for 137Cs content. Frequency distributions were built for populations of channel catfish, yellow-necked mice and bank voles. We combined our data with similar data from several other studies to demonstrate the relationship between the standard deviations and means of 137Cs of fish and mammal populations. The frequency distributions of 137Cs in populations of fish and mammals are not normal, as indicated by the strong relationship between standard deviation and mean. Distributions for mammals are more skewed than those for fish. Fish and mammals probably use their environments in fundamentally different ways. The highest concentrations and thus greatest risks are therefore confined to relatively few individuals in each population.

Mercury in bald eagle nestlings from South Carolina, USA.

Bald eagles (Haliaeetus leucocephalus) may be at risk from contaminants in their diet and young birds may be particularly sensitive to contaminant exposure. To evaluate potential risks from dietary mercury exposure to eagle nestlings in South Carolina (USA), we surveyed mercury concentrations in 34 nestlings over two breeding seasons (1998 and 1999). Samples were also obtained from several post-fledging eagles in the region. Nestling feather mercury ranged from 0.61-6.67 micrograms Hg/g dry weight, nestling down mercury from 0.50-5.05 micrograms Hg/g dry weight, and nestling blood mercury from 0.02-0.25 microgram Hg/g wet weight. We did not detect significant differences in tissue mercury between nestlings from coastal and inland regions in contrast to some other studies of piscivorous birds. Mercury concentrations were much higher in the post fledging birds we sampled. Our data show that nestling eagles in South Carolina are accumulating mercury, and that concentrations in older birds may exceed regulatory guidelines.