A retrospective analysis of honey bee (Apis mellifera) pesticide toxicity data.

Current USEPA ecological risk assessments for pesticide registration include a determination of potential risks to bees. Toxicity data are submitted to support these assessments and the USEPA maintains a large database containing acute and chronic toxicity data on adult and larval honey bees (Apis mellifera), which USEPA considers a surrogate for Apis and non-Apis bees. We compared these toxicity data to explore possible trends. This analysis indicated a significant correlation between acute contact and oral median lethal dose (LD50) values for adult honey bees (ρ = 0.74, p <0.0001). Using default EPA modeling assumptions, where exposure for an individual bee is roughly 12x lower through contact than through ingestion, the analysis indicates that the oral LD50 is similarly if not more protective of the contact LD50 for the majority of pesticides and modes of action evaluated. The analysis also provided evidence that compounds with a lower acute toxicity for adults through contact and oral exposure pathways may still be acutely toxic for larvae. The acute toxicity of herbicides and fungicides was higher for larvae relative to oral and contact toxicity for adult honey bees for the same compounds and the no observed adverse effect level (NOAEL) from chronic toxicity studies were lower for larvae relative to adults, indicating increased sensitivity of larvae. When comparing 8-day LD50 values between single dose larval acute studies to those derived from repeat dose 22-day larval chronic toxicity studies, the LD50 values derived from chronic studies were significantly lower than those from acute toxicity tests (Z = -37, p = 0.03).

Hormesis associated with a low dose of methylmercury injected into mallard eggs.

We injected mallard (Anas platyrhynchos) eggs with methylmercury chloride at doses of 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 µg mercury/g egg contents on a wet-weight basis. A case of hormesis seemed to occur because hatching success of eggs injected with 0.05 µg/g mercury (the lowest dose) was significantly greater (93.3%) than that of controls (72.6%), whereas hatching success decreased at progressively greater doses of mercury. Our finding of hormesis when a low dose of methylmercury was injected into eggs agrees with a similar observation in a study in which a group of female mallards was fed a low dietary concentration of methylmercury and hatching of their eggs was significantly better than that of controls. If methylmercury has a hormetic effect at low concentrations in avian eggs, these low concentrations may be important in a regulatory sense in that they may represent a no-observed adverse effect level (NOAEL).

A comparison of the teratogenicity of methylmercury and selenomethionine injected into bird eggs.

Methylmercury chloride and seleno-L-methionine were injected separately or in combinations into the fertile eggs of mallards (Anas platyrhynchos), chickens (Gallus gallus), and double-crested cormorants (Phalacrocorax auritus), and the incidence and types of teratogenic effects were recorded. For all three species, selenomethionine alone caused more deformities than did methylmercury alone. When mallard eggs were injected with the lowest dose of selenium (Se) alone (0.1 µg/g), 28 of 44 embryos and hatchlings were deformed, whereas when eggs were injected with the lowest dose of mercury (Hg) alone (0.2 µg/g), only 1 of 56 embryos or hatchlings was deformed. Mallard embryos seemed to be more sensitive to the teratogenic effects of Se than chicken embryos: 0 of 15 chicken embryos or hatchlings from eggs injected with 0.1 µg/g Se exhibited deformities. Sample sizes were small with double-crested cormorant eggs, but they also seemed to be less sensitive to the teratogenic effects of Se than mallard eggs. There were no obvious differences among species regarding Hg-induced deformities. Overall, few interactions were apparent between methylmercury and selenomethionine with respect to the types of deformities observed. However, the deformities spina bifida and craniorachischisis were observed only when Hg and Se were injected in combination. One paradoxical finding was that some doses of methylmercury seemed to counteract the negative effect selenomethionine had on hatching of eggs while at the same time enhancing the negative effect selenomethionine had on creating deformities. When either methylmercury or selenomethionine is injected into avian eggs, deformities start to occur at much lower concentrations than when the Hg or Se is deposited naturally in the egg by the mother.

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.

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.

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.

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 Forster's 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 Forster's 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 Forster's 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 Forster's 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 Forster's tern chicks there was a decrease in total reduced thiols and PBSH. Multiple indicator responses also pointed to greater oxidative stress in breeding Forster's 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.

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.

Enhanced reproduction in mallards fed a low level of methylmercury: an apparent case of hormesis.

Breeding pairs of mallards (Anas platyrhynchos) were fed a control diet or a diet containing 0.5 microg/g mercury (Hg) in the form of methylmercury chloride. There were no effects of Hg on adult weights and no overt signs of Hg poisoning in adults. The Hg-containing diet had no effect on fertility of eggs, but hatching success of eggs was significantly higher for females fed 0.5 microg/g Hg (71.8%) than for controls (57.5%). Survival of ducklings through 6 d of age was the same (97.8%) for controls and mallards fed 0.5 microg/g mercury. However, the mean number of ducklings produced per female was significantly higher for the pairs fed 0.5 microg/g Hg (21.4) than for controls (16.8). Although mercury in the parents' diet had no effect on mean duckling weights at hatching, ducklings from parents fed 0.5 microg/g Hg weighed significantly more (mean = 87.2 g) at 6 d of age than did control ducklings (81.0 g). The mean concentration of Hg in eggs laid by parents fed 0.5 microg/g mercury was 0.81 microg/g on a wet-weight basis. At this time, one cannot rule out the possibility that low concentrations of Hg in eggs may be beneficial, and this possibility should be considered when setting regulatory thresholds for methylmercury.

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.

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.

Reproduction in mallards exposed to dietary concentrations of methylmercury.

The purpose of this experiment was to use mallards (Anas platyrhynchos) tested under controlled conditions to determine how much harm to reproduction resulted from various concentrations of mercury in eggs. Breeding pairs of mallards were fed a control diet or diets containing 1, 2, 4, or 8 microg/g mercury, as methylmercury chloride. Mean concentrations of mercury in eggs laid by parents fed 0, 1, 2, 4, or 8 microg/g mercury were 0.0, 1.6, 3.7, 5.9, and 14 microg/g mercury on a wet-weight basis. There were no signs of mercury poisoning in the adults, and fertility and hatching success of eggs were not affected by mercury. Survival of ducklings and the number of ducklings produced per female were reduced by the 4 and 8-microg/g dietary mercury treatments (that resulted in 5.9 and 14 microg/g mercury in their eggs, respectively). Ducklings from parents fed the various mercury diets were just as heavy as controls at hatching, but by 6 days of age ducklings whose parents had been fed 4 or 8 microg/g mercury weighed less than controls. Because we do not know if absorption of mercury from our diets would be the same as absorption from natural foods, the mercury concentrations we report in eggs may be more useful in extrapolating to possible harmful effects in nature than are the dietary levels we fed. We conclude that mallard reproduction does not appear to be particularly sensitive to methylmercury.

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.

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 (microg/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.

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.

Species differences in the sensitivity of avian embryos to methylmercury.

We injected doses of methylmercury into the air cells of eggs of 26 species of birds and examined the dose-response curves of embryo survival. For 23 species we had adequate data to calculate the median lethal concentration (LC(50)). Based on the dose-response curves and LC(50)s, we ranked species according to their sensitivity to injected methylmercury. Although the previously published embryotoxic threshold of mercury in game farm mallards (Anas platyrhynchos) has been used as a default value to protect wild species of birds, we found that, relative to other species, mallard embryos are not very sensitive to injected methylmercury; their LC(50 )was 1.79 microg/g mercury on a wet-weight basis. Other species we categorized as also exhibiting relatively low sensitivity to injected methylmercury (their LC(50)s were 1 microg/g mercury or higher) were the hooded merganser (Lophodytes cucullatus), lesser scaup (Aythya affinis), Canada goose (Branta canadensis), double-crested cormorant (Phalacrocorax auritus), and laughing gull (Larus atricilla). Species we categorized as having medium sensitivity (their LC(50)s were greater than 0.25 microg/g mercury but less than 1 microg/g mercury) were the clapper rail (Rallus longirostris), sandhill crane (Grus canadensis), ring-necked pheasant (Phasianus colchicus), chicken (Gallus gallus), common grackle (Quiscalus quiscula), tree swallow (Tachycineta bicolor), herring gull (Larus argentatus), common tern (Sterna hirundo), royal tern (Sterna maxima), Caspian tern (Sterna caspia), great egret (Ardea alba), brown pelican (Pelecanus occidentalis), and anhinga (Anhinga anhinga). Species we categorized as exhibiting high sensitivity (their LC(50)s were less than 0.25 microg/g mercury) were the American kestrel (Falco sparverius), osprey (Pandion haliaetus), white ibis (Eudocimus albus), snowy egret (Egretta thula), and tri-colored heron (Egretta tricolor). For mallards, chickens, and ring-necked pheasants (all species for which we could compare the toxicity of our injected methylmercury with that of published reports where methylmercury was fed to breeding adults and was deposited into the egg by the mother), we found the injected mercury to be more toxic than the same amount of mercury deposited naturally by the mother. The rank order of sensitivity of these same three species to methylmercury was, however, the same whether the methylmercury was injected or maternally deposited in the egg (i.e., the ring-necked pheasant was more sensitive than the chicken, which was more sensitive than the mallard). It is important to note that the dose-response curves and LC(50)s derived from our egg injections are useful for ranking the sensitivities of various species but are not identical to the LC(50)s that would be observed if the mother bird had put the same concentrations of mercury into her eggs; the LC(50)s of maternally deposited methylmercury would be higher.