John B. French

Effects of methylmercury on reproduction in American kestrels.

Sixty breeding pairs of captive American kestrels (Falco sparverius) were exposed to a range of sublethal dietary concentrations of mercury (Hg), in the form of methylmercuric chloride, and their subsequent reproduction was measured. Egg production, incubation performance, and the number and percent of eggs hatched decreased markedly between 3.3 and 4.6 mg/kg dry weight of Hg (1.2 and 1.7 mg/kg wet wt), in the diet. The number of fledglings and the percent of nestlings fledged were reduced markedly at 0.7 mg/kg dry weight (0.3 mg/kg wet wt) and declined further between 2 and 3.3 mg/kg dry weight (0.7 and 1.2 mg/kg wet wt). Dietary concentrations of >or=4.6 mg/kg dry weight (1.7 mg/kg wet wt) were associated with total fledging failure. The estimated decline in fledged young per pair (24%, Bayesian regression) for kestrels consuming 0.7 mg/kg dry weight (0.3 mg/ kg wet wt) raises concerns about population maintenance in areas subject to high inputs of anthropogenic Hg. Mercury concentrations in 20 second-laid eggs collected from all groups were related to dietary concentrations of Hg, and the Hg concentrations in 19 of these eggs were related to eggs laid and young fledged. Concentrations of Hg in eggs from the highest diet group (5.9 mg/kg dry wt; 2.2 mg/kg wet wt) were higher than egg concentrations reported for either wild birds or for captive birds (nonraptors) fed dry commercial food containing 5 mg/kg methylmercury. Accumulation ratios of Hg from diets to eggs were higher than those reported for feeding studies with other species.

Experimental Infection of a North American Raptor, American Kestrel (Falco sparverius), with Highly Pathogenic Avian Influenza Virus (H5N1)

Several species of wild raptors have been found in Eurasia infected with highly pathogenic avian influenza virus (HPAIV) subtype H5N1. Should HPAIV (H5N1) reach North America in migratory birds, species of raptors are at risk not only from environmental exposure, but also from consuming infected birds and carcasses. In this study we used American kestrels as a representative species of a North American raptor to examine the effects of HPAIV (H5N1) infection in terms of dose response, viral shedding, pathology, and survival. Our data showed that kestrels are highly susceptible to HPAIV (H5N1). All birds typically died or were euthanized due to severe neurologic disease within 4–5 days of inoculation and shed significant amounts of virus both orally and cloacally, regardless of dose administered. The most consistent microscopic lesions were necrosis in the brain and pancreas. This is the first experimental study of HPAIV infection in a North American raptor and highlights the potential risks to birds of prey if HPAIV (H5N1) is introduced into North America.

Toxic effects of dietary methylmercury on immune function and hematology in American kestrels (Falco sparverius)

Fifty-nine adult male American kestrels (Falco sparverius) were assigned to one of three diet formulations including 0 (control), 0.6, and 3.9 µg/g (dry wt) methylmercury (MeHg). Kestrels received their diets daily for 13 weeks to assess the effects of dietary MeHg on immunocompetence. Immunotoxic endpoints included assessment of cell-mediated immunity (CMI) using the phytohemagglutinin (PHA) skin-swelling assay and primary and secondary antibody-mediated immune responses (IR) via the sheep red blood cell (SRBC) hemagglutination assay. Select hematology and histology parameters were evaluated to corroborate the results of functional assays and to assess immunosuppression of T and B cell-dependent components in spleen tissue. Kestrels in the 0.6 and 3.9 µg/g MeHg groups exhibited suppression of CMI, including lower PHA stimulation indexes (p = 0.019) and a 42 to 45% depletion of T cell-dependent splenic lymphoid tissue (p = 0.006). Kestrels in the 0.6 µg/g group exhibited suppression of the primary IR to SRBCs (p = 0.014). MeHg did not have a noticeable effect on the secondary IR (p = 0.166). Elevation of absolute heterophil counts (p < 0.001), the heterophil to lymphocyte ratio (p < 0.001), and total white blood cell counts (p = 0.003) was apparent in the 3.9 µg/g group at week 12. Heterophilia, or the excess of heterophils in peripheral blood above normal ranges, was apparent in seven of 17 (41%) kestrels in the 3.9 µg/g group and was indicative of an acute inflammatory response or physiological stress. This study revealed that adult kestrels were more sensitive to immunotoxic effects of MeHg at environmentally relevant dietary concentrations than they were to reproductive effects as previously reported.

Mercury in the blood and eggs of American kestrels fed methylmercury chloride.

American kestrels (Falco sparverius) were fed diets containing methylmercury chloride (MeHg) at 0, 0.6, 1.7, 2.8, 3.9, or 5.0 µg/g (dry wt) starting approximately eight weeks before the onset of egg laying. Dietary treatment was terminated after 12 to 14 weeks, and unhatched eggs were collected for Hg analysis. Blood samples were collected after four weeks of treatment and the termination of the study (i.e., 12-14 weeks of treatment). Clutch size decreased at dietary concentrations above 2.8 µg/g. The average total mercury concentration in clutches of eggs and in the second egg laid (i.e., egg B) increased linearly with dietary concentration. Mercury concentrations in egg B were approximately 25% lower than in the first egg laid and similar in concentration to the third egg laid. Mercury concentrations in whole blood and plasma also increased linearly with dietary concentration. Total Hg concentrations in June blood samples were lower than those in April, despite 8 to 10 weeks of additional dietary exposure to MeHg in the diet. This is likely because of excretion of Hg into growing flight feathers beginning shortly after the start of egg production. The strongest relationships between Hg concentrations in blood and eggs occurred when we used blood samples collected in April before egg laying and feather molt.

A physiologically based toxicokinetic model for methylmercury in female American kestrels

A physiologically based toxicokinetic (PBTK) model was developed to describe the uptake, distribution, and elimination of methylmercury (CH(3)Hg) in female American kestrels. The model consists of six tissue compartments corresponding to the brain, liver, kidney, gut, red blood cells, and remaining carcass. Additional compartments describe the elimination of CH(3)Hg to eggs and growing feathers. Dietary uptake of CH(3)Hg was modeled as a diffusion-limited process, and the distribution of CH(3)Hg among compartments was assumed to be mediated by the flow of blood plasma. To the extent possible, model parameters were developed using information from American kestrels. Additional parameters were based on measured values for closely related species and allometric relationships for birds. The model was calibrated using data from dietary dosing studies with American kestrels. Good agreement between model simulations and measured CH(3)Hg concentrations in blood and tissues during the loading phase of these studies was obtained by fitting model parameters that control dietary uptake of CH(3)Hg and possible hepatic demethylation. Modeled results tended to underestimate the observed effect of egg production on circulating levels of CH(3)Hg. In general, however, simulations were consistent with observed patterns of CH(3)Hg uptake and elimination in birds, including the dominant role of feather molt. This model could be used to extrapolate CH(3)Hg kinetics from American kestrels to other bird species by appropriate reassignment of parameter values. Alternatively, when combined with a bioenergetics-based description, the model could be used to simulate CH(3)Hg kinetics in a long-term environmental exposure.

Toxic effects of dietary methylmercury on immune system development in nestling American kestrels (Falco sparverius)

This study evaluated the effects of dietary methylmercury (MeHg) on immune system development in captive-reared nestling American kestrels (Falco sparverius) to determine whether T cell-mediated and antibody-mediated adaptive immunity are targets for MeHg toxicity at environmentally relevant concentrations. Nestlings received various diets, including 0 (control), 0.6, and 3.9 µg/g (dry wt) MeHg for up to 18 d posthatch. Immunotoxicity endpoints included cell-mediated immunity (CMI) using the phytohemagglutinin (PHA) skin-swelling assay and antibody-mediated immune response via the sheep red blood cell (SRBC) hemagglutination assay. T cell- and B cell-dependent histological parameters in the spleen, thymus, and bursa of Fabricius were correlated with the functional assays. For nestlings in the 0.6 and 3.9 µg/g MeHg groups, CMI was suppressed by 73 and 62%, respectively, at 11 d of age. Results of this functional assay were correlated with T cell-dependent components of the spleen and thymus. Dose-dependent lymphoid depletion in spleen tissue directly affected the proliferation of T-lymphocyte populations, insofar as lower stimulation indexes from the PHA assay occurred in nestlings with lower proportions of splenic white pulp and higher THg concentrations. Nestlings in the 3.9 µg/g group also exhibited lymphoid depletion and a lack of macrophage activity in the thymus. Methylmercury did not have a noticeable effect on antibody-mediated immune function or B cell-dependent histological correlates. We conclude that T cell-mediated immunosuppression is the primary target of MeHg toward adaptive immunity in developing kestrels. This study provides evidence that environmentally relevant concentrations of MeHg may compromise immunocompetence in a developing terrestrial predator and raises concern regarding the long-term health effects of kestrels that were exposed to dietary MeHg during early avian development.

Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.