Dimercaptopropane Sulfonate Chelation Affects In Vivo Hg and MeHg Distribution in Tissues and Urine of Prairie Voles (Microtus ochrogaster).

Methyl mercury cation (MeHg(+)) and divalent mercury (Hg(2+)) were quantified in urine, liver, kidney, and brain of prairie voles (Microtus ochrogaster) during a 12 week exposure to aqueous MeHg(+) at concentrations of 10, 100, and 1000 ng MeHg(+)/mL. Aqueous MeHg(+) exposures increased mercury accumulation in tissues of voles from each exposure group. Accumulation was greater within the higher two exposure groups. Similar [Hg(2+)] and [MeHg(+)] were determined within a given organ type before and after 2,3-dimercapto-1-propane sulfonate (DMPS) chelation. Similar correlations were seen for Hg(2+) and MeHg(+) concentrations in pre and post chelation urine. Post chelation urine more reliably predicted mercury species concentrations in tissues than did urine collected before chelation. These data demonstrate the utility of DMPS in noninvasive assessment of wildlife exposure to mercury, which may have utility in evaluating meta-population level exposure to hazardous wastes.

Accumulation and effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) exposure in the green anole (Anolis carolinensis).

Environmental contamination by energetic compounds is an increasing international concern, although little is known of their accumulation in and affect on wildlife. Reptiles are often good models for contaminants studies due to natural history traits that increase their potential for exposure. We report a study to assess accumulation and effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX, High Melting Explosive) in green anoles (Anolis carolinensis). Acute oral toxicity (LD(50)) was estimated to exceed 2000 mg/kg body weight in adult male and female anoles using a standard up-and-down method. Accumulation of HMX was assessed in adult females via dietary exposure and into eggs by two routes (directly from the soil and via maternal transfer). HMX readily accumulated into adult females in a dose-dependent manner and into eggs following both exposure pathways. However, total HMX in soil-exposed eggs was up to 40-times greater than those exposed via maternal transfer. Although there was a suggestion of an HMX-induced reduction in body weight in adult females, overall there were no effects observed over the 12 week exposure period. The only significant effect on eggs was a 50% reduction in hatching success for eggs exposed to 2000 mg/kg HMX in the soil during incubation. Growth and survival of hatchlings was not affected by HMX exposure. Our results demonstrate that HMX accumulates through the food chain and into eggs from the soil, but likely poses minimal threat to lizards except to hatching success in eggs incubated in soils with HMX levels near maximum environmental concentrations.

Liquid chromatography-tandem mass spectrometry analysis of 17α-trenbolone, 17ß-trenbolone and trendione in airborne particulate matter.

Trenbolone acetate (TbA) is a potent synthetic anabolic steroid that was approved by the FDA as a growth promoter in beef cattle in 1987. Given the endocrine-modulating activity of TbA and its metabolites in all vertebrates, a sensitive and reliable analytical method is needed to detect TbA and related residues in environmental matrices. We have developed a method that incorporates solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of the three major TbA metabolites (trendione, 17ß-trenbolone, 17α-trenbolone) in total suspended particulate matter (TSP) samples. Sample preparation involved pressurized liquid extraction followed by cleanup on solid-phase extraction cartridges. The procedure was optimized to obtain maximum recovery and minimum signal suppression/enhancement from matrix effects. Analytes were separated with a Phenomenex Gemini-NX C18 analytical column (150 mm × 2.0 mm, 3 µm particle size) using an aqueous methanol gradient at a flow rate of 0.2 mL/min. Column effluent underwent positive electrospray ionization (ESI). Two or more diagnostic product ions were acquired from analyte specific precursor ions for unambiguous confirmation and quantification. The method detection limit was 3.27-4.87 ng/g of particulate matter (PM). Method accuracy, determined with analyte recoveries, ranged between 68% and 117%, and method precision, expressed as relative standard deviation, was below 15% at spiked levels of 6.67, 33.3, and 167 ng/g PM. Analysis of TSP samples demonstrated the presence of the target species associated with PM in the vicinity of beef cattle feeding operations.

Mercury and methylmercury accumulation and excretion in prairie voles (Microtus ochrogaster) receiving chronic doses of methylmercury.

Methylmercury cation (MeHg) and divalent mercury (Hg++) accumulation in liver, kidney, and brain were quantified in prairie voles (Microtus ochrogaster) at 0, 3, 6, and 12 weeks during chronic exposure to aqueous MeHg. Dose groups received deionized water or aqueous solutions containing 9, 103, or 920 ng MeHg/ml. Our study presents temporal patterns of Hg++ and MeHg concentrations in organ tissues and makes inter-tissue comparisons at each time point to illustrate the accumulation and distribution of Hg species during the study. MeHg was accumulated in tissues for 3 weeks and then concentrations plateaued. Mercury accumulated in brain, liver, and kidney to average concentrations of 510 ng/g, 180 ng/g, and 3400 ng/g, respectively. MeHg and Hg++ concentrations were roughly equivalent in liver, kidney, and urine. MeHg concentrations in brain tissue were 2 to 20 times the concentrations of Hg++. Regression analysis was also used to demonstrate the utility of urinalysis as an indicator of Hg++ and MeHg concentrations in organ tissue (p < 0.001).

Evidence that miRNAs are different from other RNAs.

An examination of 513 known pre-miRNAs and 237 other RNAs (tRNA, rRNA, and mRNA) revealed that miRNAs were significantly different from other RNAs (p < 0.001). miRNA genes were less conserved than other RNA genes, although their mature miRNA sequences were highly conserved. The A+U content of pre-miRNAs was higher than non-coding RNA (p < 0.001), but lower than mRNAs. The nucleotides in pre-miRNAs formed more hydrogen bonds and base pairs than in other RNAs. miRNAs had higher negative adjusted minimal folding free energies than other RNAs except tRNAs (p < 0.001). The MFE index (MFEI) was a sufficient criterion to distinguish miRNAs from all coding and non-coding RNAs (p < 0.001). The MFEI for miRNAs was 0.97, significantly higher than tRNAs (0.64), rRNAs (0.59), or mRNAs (0.65). Our findings should facilitate the prediction and identification of new miRNAs using computational and experimental strategies.

Relationship between DDE concentrations and laying sequence in eggs of two passerine species.

Passerine eggs make useful biomonitors of environmental pollutants. Among passerines, it is not known whether organochlorine contaminants in eggs within the same clutch are independent observations or follow a laying order effect. Intraclutch variation of DDE (1,1-dichloro-2,2-bis[(p-chlorophenyl)]ethylene) concentrations was studied in eggs collected from prothonotary warblers (Protonotaria citrea) and European starlings (Sturnus vulgaris) nesting on National Priority List sites in lower Alabama and central Colorado, respectively. All 209 eggs collected for this study contained detectable levels of DDE. Mean concentration of DDE across all prothonotary warbler eggs (mean 8.71 microg/g +/- 1.19, n = 20) was almost two orders of magnitude greater than mean concentrations of DDE in all starling eggs (mean 0.70 microg/g +/- 0.06, n = 189). In both species, there was a large amount of variability among individual eggs of the same clutch and no significant relationship between laying order and DDE concentration. Variation among eggs laid in the same sequential order was high and effectively masked any potential trends in laying order effect. We hypothesized that the variability was caused by the spatial heterogeneity of DDE on our study sites, the nature of egg development within a female passerine, or a combination of these factors. Investigators focusing on lipophilic contaminants should exercise caution when making inferences about contaminant concentrations in an entire clutch of passerine eggs after the collection and analysis of a single egg because our data show that DDE levels in a single egg collected for analysis do not consistently reflect DDE levels in the eggs remaining in the nest.

Mercury in Morelet's crocodile eggs from northern Belize.

Recent studies have examined mercury accumulation in crocodilians. However, though most researchers have focused on tissue concentrations, few have examined mercury levels in crocodilian eggs. In July 1995, we analyzed mercury in 31 nonviable Morelet's crocodile ( Crocodylus moreletii) eggs collected from eight nests across three localities in northern Belize. All eggs were found to contain mercury. Based on an individual egg basis, mean concentration of mercury for all three localities was among the lowest reported for any crocodilian species. When localities were examined separately, mean concentrations for Laguna Seca and Gold Button Lagoon were comparable to those observed in other studies, and the mean for Sapote Lagoon was the lowest ever reported. Based on mean nest concentrations, mercury in eggs from Laguna Seca was approximately two- and tenfold higher than for Gold Button Lagoon and Sapote Lagoon, respectively. Variability in mercury concentrations among localities is likely the result of site-specific differences in mercury input, bioavailabilty, and bioaccumulation. Mercury concentrations were relatively uniform in eggs from the same nest and among nests from the same localities. The presence of mercury in Morelet's crocodile eggs suggests exposure in adult females, developing embryos, and neonates. However, crocodiles in these areas show no overt signs of mercury toxicity, and no indication of population decline is evident. A paucity of data on the effects of mercury on crocodilians precludes meaningful speculation as to the biological significance of tissue and egg concentrations. Controlled laboratory studies and long-term population monitoring are needed to address these questions.

Uptake and distribution of three PCB congeners and endosulfan by developing white leghorn chicken embryos (Gallus domesticus).

The distributions of PCB 105, 156, 189, and endosulfan in incubating, maternally exposed, viable white leghorn chicken eggs (Gallus domesticus) were investigated. Hens were subcutaneously injected every 4 days with a mixture of the above chemicals. One group of five eggs was removed from the incubator at each of 9, 14, and 19 days of incubation; dissected into three compartments (embryo, chorioallantoic membrane, and yolk + albumin); weighed; frozen; and then later analyzed for the dosing chemicals. Through 19 days of development (90% of incubation), greater than 70% of the total chemical mass in the whole egg remained within the yolk + albumin, whereas, depending on the chemical, 17% to 30% was absorbed by the embryo and 0.2% to 9% was transported into the chorioallantoic membrane. As a percentage of total PCB mass within the respective compartment, PCB 105 composition in the embryo and chorioallantoic membrane decreased significantly throughout development while PCB 156 and 189 composition increased significantly throughout development. Though endosulfan composition within any of the compartments was highly variable, it did not change significantly during development. The results of this study indicate that the majority of avian chick exposure to contaminants occurs posthatch as the chick continues to utilize the residual yolk.

Maternal transfer of contaminants: case study of the excretion of three polychlorinated biphenyl congeners and technical-grade endosulfan into eggs by white Leghorn chickens (Gallus domesticus).

Reported avian maternal transfer rates of organochlorine contaminants range from 1% to as much as 20% of maternal body burdens. However, to our knowledge, no investigation of factors governing maternal transfer has been reported. Here, we report an investigation of maternal transfer of 2,3,3',4,4'-pentachlorinated biphenyl (PCB 105), 2,3,3',4,4',5-hexachlorinated biphenyl (PCB 156), 2,3,3',4,4',5,5'-heptachlorinated biphenyl (PCB 189), and technical-grade endosulfan into eggs by white leghorn chickens (Gallus domesticus). Two experiments were performed to evaluate individual chemical excretion into eggs when hens were injected with each chemical individually (experiment one) or with a mixture of all four chemicals (experiment two). Each hen was injected subcutaneously every 4 d during a 21-d period with 100 microliters of the dosing solution during both experiments. The mass of each chemical excreted into the egg was compared among eggs and with the mass injected into hens to determine the influence of chemical structure (experiment one) and interaction (experiment two) on maternal transfer of those chemicals into eggs. Maternal transfer of PCBs was inversely related to congener chlorination. The congener mass in eggs, as a percentage of the mass injected into hens, was 0.42% for PCB 189, 0.54% for PCB 156, and 0.61% for PCB 105. In experiment two, absolute excretion of only PCB 189 and alpha-endosulfan into eggs was affected by the presence of other chemicals. Excretion of PCB 189 (0.51%) and alpha-endosulfan (0.03%) increased and decreased, respectively, compared with when they were individually injected into hens during experiment one. Lastly, much less of the more metabolically susceptible endosulfan (0.04-0.12% of the mass injected) was excreted into the egg relative to PCBs, despite being injected into the hens at concentrations comparable with those of PCBs, suggesting, at least in avian species, lower maternal transfer of more metabolically susceptible chemicals.

Nonlethal method for forensic evaluation of aldicarb exposure in wildlife.

Forensic evaluation of aldicarb exposure is difficult due to the rapid hydrolysis and oxidation of the parent compound. Oxidation products-aldicarb sulfoxide and aldicarb sulfone-are commonly analyzed, but hydrolytic products-aldicarb nitrile, aldicarb nitrile sulfoxide, aldicarb nitrile sulfone-are infrequently analyzed even though they are the primary stable products of aldicarb degradation. Nitrile analyses provide an important avenue to verify aldicarb exposure or aldicarb-induced mortality. Our aproach allows lethal and sublethal exposure assessment. Extraction of samples with acetonitrile:water is followed by chromatographic determination. Sublethal exposure assessment utilizes excreta samples, which is nonlethal and requires holding animals in captivity for 12 h or less. Sublethal exposures of northern bobwhite Colinus virginianus to aldicarb can be identified with greater than 80% confidence for 6 h after dosing. By analyzing GI tracts, lethal exposures of bobwhite to aldicarb can be identified with greater than 90% confidence for 4 days post mortem and with 75% confidence for 8 days post mortem. Sublethal exposures to aldicarb was identified in greater than 80% of Peromyscus maniculatis for 6 h after dosing. Aldicarb and its transformation products were detected for 8 days post mortem in all mice that received aldicarb doses at or above the LD50.

Accumulation of DDT and mercury in prothonotary warblers (Protonotaria citrea) foraging in a heterogeneously contaminated environment.

Foraging areas of adult prothonotary warblers (Protonotaria citrea) were determined using standard radiotelemetry techniques to determine if soil concentrations of p,p'dichlorodiphenyltrichloroethane (p,p'DDT) and mercury in foraging areas could be used to predict contaminant levels in diets and tissues of nestling warblers. Adult warblers were fitted with transmitters and monitored for approximately 2 d while foraging and feeding 6- to 8-d-old nestlings. Foraging ecology data were integrated with contaminant levels of soil, diets, and tissues into a comprehensive analysis of geographic variation in contaminant exposure and uptake using linear regression. Concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) and mercury in nestling tissues varied considerably across the study site. Mean concentration of DDE was greater in eggs than all other tissues, with individual samples ranging from 0.24 to 8.12 microgram/kg. In general, concentrations of DDT in soil were effective in describing the variation of contaminants in adipose samples. Concentrations of mercury in soils accounted for 78% of the variation in kidney samples. This was the best relationship of any of the paired variables. All other relationships showed relatively poor predictive ability.

Bioaccumulation of polychlorinated biphenyls in ranid frogs and northern water snakes from a hazardous waste site and a contaminated watershed.

Livers of bullfrogs (Rana catesbeiana) from a polychlorinated biphenyl (PCB) contaminated watershed and hazardous waste site located in Pickens County, South Carolina, contained significantly higher concentrations of PCBs (2.33 and 2.26 ppm, respectively) than those from a reference site (0.05 ppm). Green frogs (R. clamitans) from the two contaminated sites also accumulated higher levels of PCBs (2.37 and 3.88 ppm, respectively) than those from the reference site (0.02 ppm). No temporal variation was observed in PCB concentrations of bullfrogs or green frogs from the contaminated sites between 1992 and 1993. Levels of PCBs in the livers of northern water snakes (Nerodia sipedon) were significantly higher in snakes from the contaminated watershed (13.70 ppm) than in those from the waste site (2.29 ppm) and two reference sites (2.50 and 1.23 ppm). When compared to frogs, significantly higher bioaccumulation occurred in water snakes from the contaminated watershed. No significant differences in PCB levels were found with respect to sex or body size (snout-vent length (SVL) or body mass) for frogs or snakes. PCBs were detected also in eggs of both frogs and snakes. Results of this study provide baseline data and document the bioaccumulation of PCB residues in frog and snake tissues; however, the significance of these tissue residues to reproduction, survival, growth/development, and population dynamics in contaminated habitats is unknown.

Relative distribution of polychlorinated biphenyls among tissues of neonatal American alligators (Alligator mississippiensis).

Recent papers have investigated the utility of chemical analysis of the chorioallantoic membrane (CAM) as a nonlethal indicator of avian and reptilian exposure to persistent environmental contaminants. This study was undertaken to evaluate the chemical distribution among fat, CAM, and residual yolk tissues of live neonatal American alligators (Alligator mississippiensis) to investigate the potential utility of CAM use as a nonlethal indicator of exposure to polychlorinated biphenyls (PCBs). CAMs were collected from neonatal alligators at hatch, whereas yolk sacs and fat tissues were taken from each animal at euthanization (3 weeks posthatch). All collected tissue samples were separately Soxhlet extracted and analyzed by GC/ECD for PCBs. Log normalized, individual PCB congener concentrations in CAMs were significantly correlated with concentrations in fat (r(2) = 0.62) and yolk (r(2) = 0.56) tissues. Coefficients of determination from comparisons of homologue group concentrations varied from 0.13 to 0.90. Residue levels in neonatal oviparous organism tissues may be qualitatively assessed through chemical analyses of CAM tissues removed from discarded eggshells.

Improved preparation of small biological samples for mercury analysis using cold vapor atomic absorption spectroscopy.

Concentrations of mercury in biological samples collected for environmental studies are often less than 0.1 microgram/g. Low mercury concentrations and small organ sizes in many wildlife species (approximately 0.1 g) increase the difficulty of mercury determination at environmentally relevant concentrations. We have developed a digestion technique to extract mercury from small (0.1 g), biological samples at these relevant concentrations. Mean recoveries (+/- standard error) from validation trials of mercury fortified tissue samples using cold vapor atomic absorption spectroscopy for analysis ranged from 102 +/- 4.3% (2.5 micrograms/L, n = 15) to 108 +/- 1.4% (25 micrograms/L, n = 15). Recoveries of inorganic mercury were 99 +/- 5 (n = 19) for quality assurance samples analyzed during environmental evaluations conducted during a 24 month period. This technique can be used to determine total mercury concentrations of 60 ng Hg/g sample. Samples can be analyzed in standard laboratories in a short time, at minimal cost. The technique is versatile and can be used to determine mercury concentrations in several different matrices, limiting the time and expense of method development and validation.

Extraction of aldicarb and its metabolites from excreta and gastrointestinal tissue.

Carbamate insecticide screens often include aldicarb and its oxidative metabolites, aldicarb sulfoxide (ASX), and aldicarb sulfone (ASN). The rapid hydrolysis and thermal cleavage of the C-N bond within the carbamate functional group of these compounds produces nitrile transformation products. Nitriles are primary transformation products from aldicarb, its sulfoxide, or its sulfone. However, these nitriles are infrequently monitored. The method reported used acetonitrile/water extraction and HPLC postcolumn derivitization to determine aldicarb, ASX, and ASN from avian excreta and from gastrointestinal (GI) tissue. Recoveries of aldicarb, ASX and ASN from excreta were of 79% +/- 5.4, 120% +/- 7.7, and 93% +/- 6.2, respectively. Recoveries from tissue were 70% +/- 5.0, 80% +/- 12.1, and 85% +/- 6.7, respectively. The same extraction procedure and a GC-FPD analysis were used to determine nitrile metabolites from the same tissues. Aldicarb nitrile, ASX nitrile, and ASN nitrile recoveries from excreta were 42% +/- 2.3, 65% +/- 3.6, and 79% +/- 3.3, respectively. Overall recoveries from tissue were 29% +/- 3.4, 72% +/- 8.3, and 83% +/- 11.4, respectively. Since aldicarb, ASX, and ASN are normally detectable in organ tissues for 1-2 days following exposure, determining the presence of nitrile cleavage products provides an important forensic tool for evaluating aldicarb exposures.

PCB concentrations of eggs and chorioallantoic membranes of loggerhead sea turtles (Caretta caretta) from the Cape Romain National Wildlife Refuge.

PCBs were found in eggs and chorioallantoic membranes (CAMs) of loggerhead sea turtles (Caretta caretta). Total PCB concentrations in CAMs were larger than concentrations in eggs. Total PCB concentrations in egg and CAM tissues were highly correlated (r2 = 0.782; p = 0.0001). Sums of PCB congeners within homologue groups were also correlated in the two tissues. Data from these turtle eggs indicate 1) PCB concentrations in CAMs represent PCB concentrations in whole eggs and 2) CAMs can be collected in a nonlethal manner to determine PCB concentrations in sea turtle hatchlings.

The decline of mink in Georgia, North Carolina, and South Carolina: the role of contaminants.

Since the 1960s, mink (Mustela vision) populations in Georgia, North Carolina, and South Carolina have declined, especially in the coastal plain. A prior study suggested that the decline may stem from environmental contaminants. Based on water quality data from each state, we identified 17 substances potentially related to the decline: aldrin, dieldrin, endrin, DDD, DDE, DDT, PCBs, chlordane, alpha-BHC, toxaphene, dibenzofuran, copper, chromium, cadmium, lead, arsenic, and mercury. Mink livers were analyzed for PCB and organochlorine pesticides, and kidneys and femurs were analyzed for metals. Reference sample concentrations from piedmont, mountain, and foothill locations were compared to state coastal plain totals and counties. PCBs for Georgia, dieldrin for South Carolina, and endrin and aldrin for North Carolina were significantly higher than the piedmont reference group. Liver PCB concentrations were higher than those known to cause mink reproductive dysfunction. Mercury concentrations were significantly higher in coastal plain mink from all three states and were in the range of those known to cause impacts to reproduction, growth, and behavior to wild mink. It is unknown what concentrations of cyclodienes cause reduced reproduction or other physiological effects in mink, but the levels reported here probably indicate background concentrations that do not contribute to the decline.

Chronic polychlorinated biphenyls exposure on three generations of oldfield mice (Peromyscus polionotus): effects on reproduction, growth, and body residues.

Effects of chronic dietary exposure to low levels of PCBs (polychlorinated biphenyls) on reproduction, growth and whole body burdens were investigated in three generations of Peromyscus polionotus. Mated pairs were maintained on a diet containing 5 mg/kg PCBs (Aroclor 1254) for 12 months, beginning exposure as young adults; matched controls received a similar diet without PCBs. Offspring were maintained on the parental regime and paired at maturity with non-siblings in the same group. In first and second generation offspring, birth and weaning weights were significantly lower in PCB-exposed animals; in the second generation, there were also significantly fewer mice born/month, longer intervals prior to birth of the first litter, and decreased survival to weaning (25 days) among exposed mice. Whole body residue of PCBs increased significantly with each generation of exposure. This study clearly shows that chronic exposure to PCBs at a dosage of 5 mg/kg depressed fertility, growth and survival in Peromyscus, and that these effects were amplified through multigenerational exposure.

Chlorinated contaminants in chorio-allantoic membranes from great blue heron eggs at Whidbey Island Naval Air Station.

Chorio-allantoic membranes (CAMs) were collected and analyzed for chlorinated hydrocarbons as part of a wildlife toxicology demonstration project at Naval Air Station (NAS) Whidbey Island, Washington, USA. Concentrations of DDT, DDE, DDD, Aroclor 1254, and Aroclor 1260 were found at concentrations below 0.4 ppm for 13 of 14 samples. The low correlations among DDT and its metabolites in CAMs suggest herons are not being exposed to a consistent source of these compounds. Comparison of chlorinated hydrocarbon data for CAMs from three Puget Sound heron colonies, NAS Whidbey, Samish Island and Dumas Bay, indicates contaminant burdens in herons from NAS Whidbey and Samish Island are significantly lower than burdens in herons from Dumas Bay.

Toxicological foundations of ecological risk assessment: biomarker development and interpretation based on laboratory and wildlife species.

Ecological risk assessments based on chemical residue analysis and species demographics tend to ignore the bioavailability and bioaccumulation of the chemicals of concern. This study describes the incorporation of mechanistically based biomarkers into an ecological risk assessment of a poly-cyclic aromatic hydrocarbon (PAH)-contaminated site. A combination of soil residue analysis, tissue residue analysis, biomarkers in one-site trapped animals and biomarkers in animals confined to enclosures was used. In particular, the use of captured deer mice (Peromyscus maniculatus) for these studies is compared to the use of laboratory-raised deer mice placed in enclosures. This study indicates that the higher degree of variability in the responses of wild deer mice make the use of enclosure studies advantageous. Positive control studies performed by dosing laboratory-raised deer mice with the same PAHs as found on the site were used to validate this approach. These studies indicate that immune suppression occurred at PAH concentrations an order of magnitude below those required for the induction of ethoxyresorufin-O-dealkylase activity.