Katherine R. Stebbins

Oxidative stress response of Forster's terns (Sterna forsteri) and Caspian terns (Hydroprogne caspia) to mercury and selenium bioaccumulation in liver, kidney, and brain

Bioindicators of oxidative stress were examined in prebreeding and breeding adult and chick Forsters terns (Sterna forsteri) and in prebreeding adult Caspian terns (Hydroprogne caspia) in San Francisco Bay, California. Highest total mercury (THg) concentrations (mean ± standard error; µg/g dry wt) in liver (17.7 ± 1.7), kidney (20.5 ± 1.9), and brain (3.0 ± 0.3) occurred in breeding adult Forsters terns. The THg concentrations in liver were significantly correlated with hepatic depletion of reduced glutathione (GSH), increased oxidized glutathione (GSSG):GSH ratio, and decreased hepatic gamma-glutamyl transferase (GGT) activity in adults of both tern species. Prefledging Forsters tern chicks with one-fourth the hepatic THg concentration of breeding adults exhibited effects similar to adults. Total mercury-related renal GSSG increased in adults and chicks. In brains of prebreeding adults, THg was correlated with a small increase in glucose-6-phosphate dehydrogenase (G-6-PDH) activity, suggestive of a compensatory response. Brain THg concentrations were highest in breeding adult Forsters terns and brain tissue exhibited increased lipid peroxidation as thiobarbituric acid-reactive substances, loss of protein bound thiols (PBSH), and decreased activity of antioxidant enzymes, GSSG reductase (GSSGrd), and G-6-PDH. In brains of Forsters tern chicks there was a decrease in total reduced thiols and PBSH. Multiple indicator responses also pointed to greater oxidative stress in breeding Forsters terns relative to prebreeding terns, attributable to the physiological stress of reproduction. Some biondicators also were related to age and species, including thiol concentrations. Enzymes GGT, G-6-PDH, and GSSGred activities were related to species. Our results indicate that THg concentrations induced oxidative stress in terns, and suggest that histopathological, immunological, and behavioral effects may occur in terns as reported in other species.

Mercury and Drought Along the Lower Carson River, Nevada: III. Effects on Blood and Organ Biochemistry and Histopathology of Snowy Egrets and Black-Crowned Night-Herons on Lahontan Reservoir, 2002–2006

A 10-year study (1997–2006) was conducted to evaluate reproduction and health of aquatic birds in the Carson River Basin of northwestern Nevada (on the U.S. Environmental Protection Agency Natural Priorities List) due to high mercury (Hg) concentrations from past mining activities. This part of the study evaluated physiological associations with blood Hg in young snowy egrets (Egretta thula) and black-crowned night-herons (Nycticorax nycticorax), and organ biochemistry and histopathological effects in snowy egrets on Lahontan Reservoir (LR) from the period 2002–2006. LR snowy egret geometric mean total Hg concentrations (µg/g ww) ranged from 1.5 to 4.8 for blood, 2.4 to 3.1 liver, 1.8 to 2.5 kidneys, 1.7 to 2.4 brain, and 20.5 to 36.4 feathers over these years. For night-herons, mean Hg for blood ranged from 1.6 to 7.4. Significant positive correlations were found between total Hg in blood and five plasma enzyme activities of snowy egrets suggesting hepatic stress. Histopathological findings revealed vacuolar changes in hepatocytes in LR snowy egrets as well as correlation of increased liver inflammation with increasing blood and tissue Hg. Hepatic oxidative effects were manifested by decreased hepatic total thiol concentration and glutathione reductase activity and elevated hepatic thiobarbituric acid-reactive subatances (TBARS), a measure of lipid peroxidation. However, other hepatic changes indicated compensatory mechanisms in response to oxidative stress, including decreased oxidized glutathione (GSSG) concentration and decreased ratio of GSSG to reduced glutathione. In young black-crowned night-herons, fewer correlations were apparent. In both species, positive correlations between blood total Hg and plasma uric acid and inorganic phosphorus were suggestive of renal stress, which was supported by histopathological findings. Both oxidative effects and adaptive responses to oxidative stress were apparent in kidneys and brain. Vacuolar change and inflammation in peripheral nerves were found to correlate with blood and tissue Hg. Hg-associated effects related to the immune system included alterations in specific white blood cells and lymphoid depletion in the bursa that were correlated with blood and tissue Hg. When the number of plasma variables that differed between young snowy egrets from the LR site and the reference site were compared between wet and drought years, over twice as many variables were affected during drought years. This resulted in many more variables correlating with blood total Hg during dry than during wet years, suggesting the combination of drought and Hg was more stressful than Hg alone. Drought may have exacerbated Hg-related effects as reported previously for overall productivity. This relationship was not evident in black-crowned night-herons, although data were more limited.

Teratogenic effects of injected methylmercury on avian embryos

Controlled laboratory studies with game farm mallards (Anas platyrhynchos) and chickens (Gallus gallus) have demonstrated that methylmercury can cause teratogenic effects in birds, but studies with wild species of birds are lacking. To address this need, doses of methylmercury chloride were injected into the eggs of 25 species of birds, and the dead embryos and hatched chicks were examined for external deformities. When data for controls were summed across all 25 species tested and across all types of deformities, 24 individuals out of a total of 1,533 (a rate of 1.57%) exhibited at least one deformity. In contrast, when data for all of the mercury treatments and all 25 species were summed, 188 deformed individuals out of a total of 2,292 (8.20%) were found. Some deformities, such as lordosis and scoliosis (twisting of the spine), misshapen heads, shortening or twisting of the neck, and deformities of the wings, were seldom observed in controls but occurred in much greater frequency in Hg-treated individuals. Only 0.59% of individual control dead embryos and hatchlings exhibited multiple deformities versus 3.18% for Hg-dosed dead embryos and hatchlings. Methylmercury seems to have a widespread teratogenic potential across many species of birds.

Evidence of Weak Contaminant-Related Oxidative Stress in Glaucous Gulls (Larus hyperboreus) from the Canadian Arctic

Environmental contaminants are transported over great distances to Arctic ecosystems, where they can accumulate in wildlife. Whether contaminant concentrations in wildlife are sufficient to produce adverse effects remains poorly understood. Exposure to contaminants elevates oxidative stress with possible fitness consequences. The glaucous gull (Larus hyperboreus), an Arctic top predator, was used as a bioindicator for investigating relationships between contaminant levels (organochlorines and polychlorinated biphenyls [OC/PCB], mercury [Hg], and selenium [Se]) and measures of oxidative stress (glutathione [GSH] metabolism and lipid peroxidation) in Canadian Arctic ecosystems. Contaminant levels were low and associations between contaminant exposure and oxidative stress were weak. Nevertheless, glutathione peroxidase activity rose with increasing hepatic Se concentrations, levels of thiols declined as Hg and OC/PCB levels rose, and at one of the two study sites levels of lipid peroxidation were elevated with increasing levels of hepatic Hg. These results suggest the possibility of a deleterious effect of exposure to contaminants on gull physiology even at low contaminant exposures.

Predicting mercury concentrations in mallard eggs from mercury in the diet or blood of adult females and from duckling down feathers

Measurements of Hg concentrations in avian eggs can be used to predict possible harm to reproduction, but it is not always possible to sample eggs. When eggs cannot be sampled, some substitute tissue, such as female blood, the diet of the breeding female, or down feathers of hatchlings, must be used. When female mallards (Anas platyrhynchos) were fed diets containing methylmercury chloride, the concentration of Hg in a sample of their blood was closely correlated with the concentration of Hg in the egg they laid the day they were bled (r2 = 0.88; p < 0.001). Even when the blood sample was taken more than two weeks after an egg was laid, there was a strong correlation between Hg concentrations in female blood and eggs (r2 = 0.67; p < 0.0002). When we plotted the dietary concentrations of Hg we fed to the egg-laying females against the concentrations of Hg in their eggs, the r2 value was 0.96 (p < 0.0001). When the concentrations of Hg in the down feathers of newly hatched ducklings were plotted against Hg in the whole ducklings, the r2 value was 0.99 (p < 0.0003). Although measuring Hg in eggs may be the most direct way of predicting possible embryotoxicity, our findings demonstrate that measuring Hg in the diet of breeding birds, in the blood of egg-laying females, or in down feathers of hatchlings all can be used to estimate what concentration of Hg may have been in the egg.

Rapid increases in mercury concentrations in the eggs of mallards fed methylmercury

To determine how quickly breeding birds would have to feed in a mercury-contaminated area before harmful concentrations of mercury, as methylmercury, built up in their eggs, we fed female mallards (Anas platyrhynchos) a control diet or diets containing 0.5, 1, 2, 4, or 8 microg/g mercury (on what was close to a dry weight basis) as methylmercury chloride for 23 d. After 18 d on their respective mercury diets, the eggs of mallards fed 0.5, 1, 2, 4, or 8 microg/g mercury contained 97.8, 86.0, 89.9, 88.9, and 85.9%, respectively, of the peak concentrations reached after 23 d. Depending on the dietary concentration of mercury, no more than approximately a week may be required for harmful concentrations (0.5-0.8 microg/g, wet weight) to be excreted into eggs.

A simplified method for correcting contaminant concentrations in eggs for moisture loss.

We developed a simplified and highly accurate method for correcting contaminant concentrations in eggs for the moisture that is lost from an egg during incubation. To make the correction, one injects water into the air cell of the egg until overflowing. The amount of water injected corrects almost perfectly for the amount of water lost during incubation or when an egg is left in the nest and dehydrates and deteriorates over time. To validate the new method we weighed freshly laid chicken (Gallus gallus) eggs and then incubated sets of fertile and dead eggs for either 12 or 19 d. We then injected water into the air cells of these eggs and verified that the weights after water injection were almost identical to the weights of the eggs when they were fresh. The advantages of the new method are its speed, accuracy, and simplicity: It does not require the calculation of a correction factor that has to be applied to each contaminant residue.

Factors related to the artificial incubation of wild bird eggs

Attempts to artificially incubate the eggs of wild birds have failed in many respects in duplicating the success of natural incubation. As part of a larger study we had the opportunity to artificially incubate the eggs of 22 species of birds (three domestic and 19 wild species). We report the successes and failures associated with artificial incubation of these eggs. Moisture loss varied widely, not only for Orders of birds but for similar species within an Order. Overall hatching success and success through to 90% of incubation varied for different Orders and for similar species. Humidity and temperature are critical elements in the artificial incubation of wild bird eggs and must be closely monitored throughout incubation to ensure the best possible chance of hatching. Even when these elements are addressed, artificial incubation still cannot duplicate the success of incubation by the parent.

Interactions between methylmercury and selenomethionine injected into mallard eggs

Methylmercury chloride and seleno-L-methionine were injected separately or in combinations into mallard eggs (Anas platyrhynchos), and embryo mortality and teratogenic effects (deformities) were modeled using a logistic regression model. Methylmercury was injected at doses that resulted in concentrations of 0, 0.2, 0.4, 0.8, and 1.6 µg/g Hg in the egg on a wet weight basis and selenomethionine at doses that resulted in concentrations of 0, 0.1, 0.2, 0.4, and 0.6 µg/g Se in the egg, also on a wet weight basis. When selenomethionine and methylmercury were injected separately, hatching probability decreased in both cases. However, when methylmercury was injected at 1.6 µg/g in combination with selenomethionine at 0.2 µg/g, the presence of the methylmercury resulted in less embryo mortality than had been seen with 0.2 µg/g Se by itself, but it increased the number of deformed embryos and hatchlings. Selenomethionine appeared to be more embryotoxic than equivalent doses of methylmercury when injected into eggs, and both injected methylmercury and selenomethionine were more toxic to mallard embryos than when deposited naturally in the egg by the mother. The underlying mechanisms behind the interactions between methylmercury and selenomethionine and why methylmercury appeared to improve hatching probability of Se-dosed eggs yet increased deformities when the two compounds were combined are unclear. These findings warrant further studies to understand these mechanisms in both laboratory and field settings.

A nonlethal microsampling technique to monitor the effects of mercury on wild bird eggs

Methylmercury is the predominant chemical form of mercury reported in the eggs of wild birds, and the embryo is the most sensitive life stage to methylmercury toxicity. Protective guidelines have been based mainly on captive-breeding studies with chickens (Gallus gallus), mallards (Anas platyrhynchos), and ring-necked pheasants (Phasianus colchicus) or on field studies where whole eggs were collected and analyzed and the effects of the mercury were measured based on the reproductive success of the remaining eggs. However, both of these methods have limitations. As an alternative, we developed a technique that involves extracting a small sample of albumen from a live egg, sealing the egg, returning the egg to its nest to be naturally incubated by the parents, and then relating the hatching success of this microsampled egg to its mercury concentration. After first developing this technique in the laboratory using chicken and mallard eggs, we selected the laughing gull (Larus atricilla) and black-necked stilt (Himantopus mexicanus) as test subjects in the field. We found that 92% of the microsampled laughing gull eggs met our reproductive endpoint of survival to the beginning of hatching compared to 100% for the paired control eggs within the same nests. Microsampled black-necked stilt eggs exhibited 100% hatching success compared to 93% for the paired control eggs. Our results indicate that microsampling is an effective tool for nonlethally sampling mercury concentrations in eggs and, as such, can be used for monitoring sensitive species, as well as for improving studies that examine the effects of mercury on avian reproduction.