John J. Johnston

Chemically mediated foraging preference of black bears (Ursus americanus)

The role of chemical constituents in the foraging behavior of black bears ( Ursus americanas ) was investigated using two field studies. Vascular tissue samples were collected from Douglas-fir ( Pseudotsuga menziesii ) trees recently foraged by black bears. Samples were extracted and analyzed by liquid and gas chromatography to determine carbohydrates and terpenes, respectively. Chemical data were subjected to correlation analyses and multiple regression to examine if they adequately describe observed foraging preferences. Free-ranging black bears also were offered a choice of four test diets that differed in content of carbohydrates and terpenes. Results indicated that forage preferences were based in part on chemical constituents in the forage. Black bears maximized intake of carbohydrates and minimized intake of terpenes. In multiple choice tests, free-ranging bears preferred a low-terpene diet to a high-terpene diet with identical carbohydrate content. Bears also preferred a high-carbohydrate diet to a low-carbohydrate diet with identical terpene content.

Toxicokinetics and coagulopathy threshold of the rodenticide diphacinone in eastern screech‐owls (Megascops asio)

In the United States, new regulations on second-generation anticoagulant rodenticides will likely be offset by expanded use of first-generation anticoagulant rodenticides. In the present study, eastern screech-owls (Megascops asio) were fed 10 µg diphacinone/g wet weight food for 7 d, and recovery was monitored over a 21-d postexposure period. By day 3 of exposure, diphacinone (DPN) was detected in liver (1.63 µg/g wet wt) and kidney (5.83 µg/g) and coagulopathy was apparent. By day 7, prothrombin time (PT) and Russells viper venom time (RVVT) were prolonged, and some individuals were anemic. Upon termination of exposure, coagulopathy and anemia were resolved within 4 d, and residues decreased to <0.3 µg/g by day 7. Liver and kidney DPN elimination occurred in 2 phases (initial rapid loss, followed by slower loss rate), with overall half-lives of 11.7 d and 2.1 d, respectively. Prolonged PT and RVVT occurred in 10% of the exposed owls with liver DPN concentrations of 0.122 µg/g and 0.282 µg/g and in 90% of the owls with liver concentrations of 0.638 µg/g and 0.361 µg/g. These liver residue levels associated with coagulopathy fall in the range of values reported in raptor mortality incidents involving DPN. These tissue-based toxicity reference values for coagulopathy in adult screech-owls have application for interpreting nontarget mortality and assessing the hazard of DPN in rodent-control operations. Diphacinone exposure evokes toxicity in raptors within a matter of days; but once exposure is terminated, recovery of hemostasis occurs rapidly.

Population-Based Fish Consumption Survey and Probabilistic Methylmercury Risk Assessment

ABSTRACT A fish consumption survey was developed and administered by telephone to 820 Wyoming fishing license holders. Survey respondents provided the frequency, species, and quantity of Wyoming-caught and store-bought fish consumed for license holder and household members. Deterministic and probabilistic methylmercury exposure distributions were estimated by multiplying fish consumption by species-specific mercury concentrations for each household member. Risk assessments were conducted for children, women of childbearing age, and the rest of the population by comparing methylmercury exposure distributions to levels of concern. The results indicate that probabilistic risk assessment likely provides a more realistic view of the risk to the study population. The results of this study clearly indicate that: (1) there is no level of fish consumption that is without risk of methylmercury exposure, (2) fish advisories may be warranted for children and women of childbearing age, and (3) that store-bought fish generally contribute more to methylmercury exposure than do Wyoming-caught fish.

TEMPORAL GENETIC VARIATION IN A COYOTE (CANIS LATRANS) POPULATION EXPERIENCING HIGH TURNOVER

Abstract Temporal genetic variation was examined in a coyote (Canis latrans) population that experienced intensive removal for several decades. The population experienced separate periods of nonselective and selective control, and comparisons were made between control methods. Analyses at 11 microsatellite loci revealed only subtle genetic differences between removal regimes when analyzed by year of birth or resident status. Numbers of alleles per locus (4–16) and expected heterozygosities (0.617–0.915) were high across groups and few 1st-order relatives were detected within groups. Coyote social structure and dispersal patterns appear to adequately maintain genetic variation and promote genetic homogeneity over relatively small geographic scales during periods of locally aggressive removal.

Single‐Point Calibration with a Non‐linear Detector: Carbohydrate Analysis of Conifer Needles by Hydrophobic Interaction Chromatography–Evaporative Light‐Scattering Detection (HIC–ELSD)

Abstract An analytical method employing hydrophobic interaction chromatography (HIC) combined with parallel detectors, provided a quantitative method for the determination of soluble carbohydrates in conifer needle tissues. Evaporative light‐scattering detection (ELSD), in parallel with atmospheric pressure chemical ionization mass spectrometry (APCI‐MS), yielded excellent quantitative and spectral data. Non‐linear detector responses of the ELSD were overcome by performing a simple exponential transformation of the detector response data. The transformation allowed for single‐point calibrations that yielded quantitative results with excellent accuracy and precision. Method recovery and precision were determined from maltose‐fortified conifer tissues. Maltose recovery was 101.7% [relative standard deviation (RSD) = 9.74%] for homogenized Douglas‐fir (Pseudotsuga menziesii) needle tissue fortified at 1.01 mg/g and 107.8% (RSD = 2.47%) for tissue fortified at 10.1 mg/g. Maltose recovery from fortified western redcedar (Thuja plicata) tissues (7.60 mg/g) was 93.0% (RSD = 3.0%). The method limit of detection (MLOD) was 0.24 mg/g for maltose in Douglas‐fir. The method was employed for the quantitative analysis of soluble carbohydrates in Douglas‐fir needles, collected from new and previous‐years growth for 7 weeks after the emergence of new growth (bud break). Needles collected from dormant trees were also analyzed. The concentrations of fructose and glucose were consistently greater in previous‐years growth vs. new and dormant growth.

Quantitation of digitoxin, digoxin, and their metabolites by high-performance liquid chromatography using pulsed amperometric detection

Abstract A novel detection method for the cardiac glycosides digitoxin, digoxin, and their metabolites is described. Both isocratic and gradient water-acetonitrile elutions were employed with an octadecylsilane column. The effluent was monitored by integrated pulsed amperometry in an alkaline medium (pH > 12) by adding sodium hydroxide post-column. Using isocratic elution, peak responses were linear from 10 to 70 ng, with lower limits of detection between 4 and 15 ng. This method allows easy modification of well-established high-performance liquid chromatographic systems, as well as comparable sensitivity and greater selectivity than UV detection.

EVALUATION AND SIGNIFICANCE OF TETRACYCLINE STABILITY IN RABIES VACCINE BAITS

Tetracycline is widely used as a biomarker for bait consumption by wildlife; tetracycline is incorporated into bones and teeth and can be detected by fluorescence microscopy several weeks postconsumption. During 2003, the United States Department of Agriculture distributed more than 10 million tetracycline-containing rabies-vaccine baits to control the spread of wildlife vectored rabies to humans, pets, and livestock. To estimate the percentage of target species consuming the baits, raccoons and skunks were collected in baited areas and teeth were analyzed for the presence of the biomarker. Several incidents of low biomarker detection rates prompted an investigation of the stability of the biomarker in the baits. Baits were collected at several points along the manufacturing and distribution chain. Baits were analyzed for free and polymer-bound tetracycline and the less active isomer epitetracycline. Results indicated that a portion of the tetracycline was converted to epitetracycline. Additionally, significant quantities of both compounds were trapped in the polymer, which is homogeneously distributed throughout the bait. The results of this study suggest that approximately 40% of the target quantity of tetracycline was unavailable for absorption. This situation could contribute to low biomarker detection rates and suggests that formulation modification should be considered.

Absorption and excretion of 14C-perfluorooctanoic acid (PFOA) in Angus cattle (Bos taurus).

Perfluoroalkyl substances (PFASs), such as perfluorooctanoic acid (PFOA), are environmentally persistent industrial chemicals often found in biosolids. Application of these biosolids to pastures raises concern about the accumulation of PFOA in the edible tissues of food animals. Because data on the absorption, distribution, metabolism, and excretion (ADME) of PFOA in cattle were unavailable, a study was conducted to determine pharmacokinetic parameters following a single oral exposure (1 mg/kg body weight of (14)C-PFOA) in four Lowline Angus steers. Radiocarbon was quantified in blood, urine, and feces for 28 days and in tissues at the time of slaughter (28 days) by liquid scintillation counting (LSC) or by combustion analysis with LSC with confirmation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (14)C-PFOA was completely absorbed and excreted (100.7 ± 3.3% recovery) in the urine within 9 days of dosing. The plasma elimination half-life was 19.2 ± 3.3 h. No (14)C-PFOA-derived radioactivity was detected in edible tissues. Although PFOA was rapidly absorbed, it was also rapidly excreted by steers and did not persist in edible tissues, suggesting meat from cattle exposed to an acute dose of PFOA is unlikely to be a major source of exposure to humans.

Analysis of Nitrosamines in Cooked Bacon by QuEChERS Sample Preparation and Gas Chromatography-Tandem Mass Spectrometry with Backflushing.

Nitrites are added as a preservative to a variety of cured meats, including bacon, to kill bacteria, extend shelf life, and improve quality. During cooking, nitrites in the meat can be converted to carcinogenic nitrosamines (NAs), the formation of which is mitigated by the addition of antioxidants. In the past, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) monitored NAs in pan-fried bacon, but FSIS terminated monitoring of NAs in the 1990s due to the very low levels found. FSIS recently chose to conduct a risk assessment of NAs in cooked bacon to determine if current levels warrant routine monitoring of NAs again. To meet FSIS needs, we developed, validated, and implemented a new method of sample preparation and analysis to test cooked bacon for five NAs of most concern, which consist of N-nitroso-dimethylamine, -diethylamine, -dibutylamine, -piperidine, and -pyrrolidine. Sample preparation was based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach and analysis by gas chromatography-tandem mass spectrometry. Ruggedness was improved markedly in the analysis of the complex fatty extracts by backflushing the guard column, injection liner, and half of the analytical column after every injection. Validation results were acceptable with recoveries of 70-120% and <20% RSDs for the five NAs, with a reporting limit of 0.1 ng/g. NA concentrations in 48 samples were all <15 ng/g, with most <1 ng/g and many <0.1 ng/g. Also, microwave cooking of bacon gave slightly lower concentrations overall compared to pan-frying.

Distribution and Excretion of Perfluorooctane Sulfonate (PFOS) in Beef Cattle (Bos taurus)

Perfluorooctane sulfonate (PFOS), a perfluoroalkyl surfactant used in many industrial products, is present in industrial wastes and in wastewater treatment plant biosolids. Biosolids are commonly applied to pastures and crops used for animal feed; consequently, PFOS may accumulate in the edible tissues of grazing animals or in animals exposed to contaminated feeds. There are no data on the absorption, distribution, and excretion of PFOS in beef cattle, so a 28-day study was conducted to determine these parameters for PFOS in three Lowline Angus steers given a single oral dose of PFOS at approximately 8 mg/kg body weight. PFOS concentrations were determined by liquid chromatography-tandem mass spectrometry in multiple tissue compartments. The major route of excretion was in the feces (11 ± 1.3% of the dose, mean ± standard deviation) with minimal PFOS elimination in urine (0.5 ± 0.07% of the dose). At day 28 the mean plasma concentration remained elevated at 52.6 ± 3.4 μg/mL, and it was estimated that 35.8 ± 4.3% of the dose was present in the plasma. Plasma half-lives could not be calculated due to multiple peaks caused by apparent redistributions from other tissues. These data indicate that after an acute exposure PFOS persists and accumulates in edible tissues. The largest PFOS body burdens were in the blood (∼36%), carcass remainder (5.7 ± 1.6%), and the muscle (4.3 ± 0.6%). It was concluded that PFOS would accumulate in edible tissues of beef, which could be a source of exposure for humans.