Jon D. Klimstra

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.

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.

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.

Toxicity of methylmercury injected into eggs when dissolved in water versus corn oil

In a previous study, the embryotoxicity of methylmercury dissolved in corn oil was compared among 26 species of birds. Corn oil is not soluble in the water-based matrix that constitutes the albumen of an egg. To determine whether the use of corn oil limited the usefulness of this earlier study, a comparison was made of the embryotoxicity of methylmercury dissolved in corn oil versus water. Mallard (Anas platyrhynchos) and chicken (Gallus gallus) eggs were injected with methylmercury chloride dissolved in corn oil or water to achieve concentrations of 0, 0.2, 0.4, 0.8, and 1.6 µg/g mercury in the egg on a wet weight basis. Hatching success at each dose of mercury was compared between the two solvents. For mallards, 16.4% of the eggs injected with 1.6 µg/g mercury dissolved in water hatched, which was statistically lower than the 37.6% hatch rate of eggs injected with 1.6 µg/g mercury dissolved in corn oil, but no differences in hatching success were observed between corn oil and water at any of the other doses. With chicken eggs, no significant differences occurred in percentage hatch of eggs between corn oil and water at any of the mercury doses. Methylmercury dissolved in corn oil seems to have a toxicity to avian embryos similar to that of does methylmercury dissolved in water. Consequently, the results from the earlier study that described the toxicity of methylmercury dissolved in corn oil to avian embryos were probably not compromised by the use of corn oil as a solvent.

Intraperitoneal injections as a possible means of generating varied levels of methylmercury in the eggs of birds in field studies

The ideal study of the effects of methylmercury on the reproductive success of a species of bird would be one in which eggs contained mercury concentrations ranging from controls to very heavily contaminated, all at the same site. Such a study cannot be realized at a Hg-contaminated area or under laboratory conditions but could be achieved by introducing methylmercury into breeding females and allowing them to deposit Hg in their eggs. Female mallards (Anas platyrhynchos) were intraperitoneally injected with solutions of methylmercury chloride dissolved in corn oil, propylene glycol, dimethyl sulfoxide, mineral oil, Olestra, Crisco, lard, hard paraffin, and a combination of hard and soft paraffin. In some cases, egg laying was delayed, as a result of either the solvent itself (in the case of Olestra, Crisco, and lard) or the highest concentration of methylmercury chloride (500 microg/g) in some of the solvents. Mercury in eggs ranged from a control level (<0.1 microg/g) to approximately 14 microg/g on a wet weight basis, which more than covers the range of concentrations reported in wild bird eggs. Mercury concentrations in a series of eggs from the same female declined mostly as a result of excretion of Hg in prior eggs and not because of the length of time since the injection. Intraperitoneal injections hold promise in field studies in which one would like to study the reproductive effects of a wide range of methylmercury levels in the eggs of a wild bird and under the natural conditions that exist in the field.