Factors Affecting the Toxicity of Methylmercury Injected into Eggs

We developed a standardized protocol for comparing the sensitivities of the embryos of different bird species to methylmercury when methylmercury was injected into their eggs. During the course of developing this protocol, we investigated the effects of various factors on the toxicity of the injected methylmercury. Most of our experiments were done with chicken (Gallus domesticus), mallard (Anas platyrhynchos), and ring-necked pheasant (Phasianus colchicus) eggs, all of which were purchased in large numbers from game farms. A smaller amount of work was done with double-crested cormorant (Phalacrocorax auritus) eggs collected from the wild. Several solvents were tested, and corn oil at a rate of 1 μl/g egg contents was selected for the final standardized protocol because it had minimal toxicity to embryos and because methylmercury dissolved in corn oil yielded a dose–response curve in a range of egg concentrations that was similar to the range that causes reproductive impairment when the mother deposits methylmercury into her own eggs. The embryonic stage at which eggs were injected with corn oil altered mercury toxicity; at early stages, the corn oil itself was toxic. Therefore, in the final protocol we standardized the time of injection to occur when each species reached the morphologic equivalent of a 3-day-old chicken embryo. Although solvents can be injected directly into the albumen of an egg, high embryo mortality can occur in the solvent controls because of the formation of air bubbles in the albumen. Our final protocol used corn oil injections into the air cell, which are easier and safer than albumen injections. Most of the methylmercury, when dissolved in corn oil, injected into the air cell passes through the inner shell membrane and into the egg albumen. Most commercial incubators incubate eggs in trays with the air cell end of the egg pointing upward, but we discovered that mercury-induced mortality was too great when eggs were held in this orientation. In addition, some species of bird eggs require incubation on their sides with the eggs being rolled 180° for them to develop normally. Therefore, we adopted a procedure of incubating the eggs of all species on their sides and rolling them 180° every hour. Little has been published about the conditions of temperature, humidity, and the movements to which eggs of wild birds need to be subjected for them to hatch optimally under artificial incubation. Not unexpectedly, hatching success in an artificial incubator is generally less than what natural incubation by the parents can achieve. However, the survival of control embryos of most wild bird species was good (generally ≥ 80%) up to within 1 or 2 days of hatching when we incubated the eggs at 37.5°C (or 37.6°C for gallinaceous species) at a relative humidity that resulted in an approximate 15% to 16% loss in egg weight by the end of incubation and by incubating the eggs on their sides and rolling them 180°/h. To improve statistical comparisons, we used survival through 90% of incubation as our measurement to compare survival of controls with survival of eggs injected with graded concentrations of mercury.     

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).     

Embryotoxicity of weathered crude oil from the Gulf of Mexico in mallard ducks (Anas platyrhynchos)

Weathered crude oil in the Gulf of Mexico can result from oil spills such as the Deepwater Horizon incident that occurred on April 20, 2010 or from natural seeps. Adult waterbirds of the Gulf Coast region may become exposed to weathered crude oil while foraging, wading, or resting, and residues can then be transferred to nests, eggs, and hatchlings. Although the toxicity of many types of crude oil to avian embryos has been thoroughly studied, the effects of weathered crude oil on developing avian embryos are not well characterized. The objective of the present study was to examine embryotoxicity of weathered crude oil collected from the Gulf of Mexico in June 2010 using mallard ducks (Anas platyrhynchos) as a model species. Weathered crude oil was applied to fertilized mallard duck eggs by paintbrush in masses ranging from 0.1 to 99.9?mg on day 3 of incubation. Mortality occurred as early as day 7 and the conservatively derived median lethal application of weathered crude oil was 30.8?mg/egg (0.5?mg/g egg) or 30.7?μl/egg (0.5?μl/g egg). Body mass, liver and spleen mass, crown-rump and bill lengths, and frequency of deformities were not significantly different among hatchlings from oiled and control eggs. In comparison to published reports of fresh crude oil embryotoxicity, weathered crude oil was considerably less toxic. We conclude that avian toxicity varies according to the degree of crude oil weathering and the stage of embryonic development at the time of exposure. Results indicate bird eggs exposed to weathered crude oil from the Gulf of Mexico during summer 2010 may have had reduced hatching success.     

Environmental Toxicity Studies Using Chickens as Surrogates for Wildlife: Effects of Injection Day

Domestic chicken embryos are frequently used for avian developmental toxicity studies of polyhalogenated aromatic hydrocarbons, which are often injected into eggs with oil-based vehicles. Injection times range from immediately prior to incubation (embryonic day zero, E0) to after 4 days of incubation (E4) and beyond. Because the majority of organogenesis in chicken embryos occurs during the first 4 days of development, injection after E0 may miss critical, sensitive, developmental periods. We evaluated whether differences in the day of vehicle administration would lead to differences in standard measures of embryotoxicity. We assessed embryotoxicity using mortality, organ somatic indices, teratogenesis, and behavior in hatchling chickens developmentally exposed to a high volume (1.0 l/g egg) of corn oil vehicle, which was injected into the airsac at E0 or E4. The E0 vehicle group had 76.5% higher overall embryo mortality, embryos died earlier in development, and hatchlings took more than two times longer to right in a righting reflex test compared to the E4 vehicle group. Other behavioral results demonstrated that hatchling chickens from the E0 vehicle group performed differently from their respective no-inject controls, whereas hatchling chickens from the E4 vehicle group did not. The bursal somatic index differed statistically by injection day and weighed 23.7% more in the E0 vehicle group than the E4 vehicle group. These results suggest that the embryonic day of contaminant injection is an important consideration, particularly when using a high volume of vehicle to evaluate developmental toxicity of a contaminant on embryo mortality or behavior.     

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.     

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.     

Environmental Toxicity Studies Using Chickens as Surrogates for Wildlife: Effects of Vehicle Volume

Domestic chicken embryos are frequently used for avian developmental toxicity studies of polyhalogenated aromatic hydrocarbons, which are often injected into eggs with oil-based vehicles. The volume of toxicant and vehicle injected ranges from relatively low volumes (0.1–0.5 l/g egg) to relatively high volumes (1.0 l/g egg and above). Previous research from our laboratory suggested that high volumes of vehicle oil may disrupt normal growth of chicken embryos, possibly from hypoxia-like effects. This analysis explored the potential effects of vehicle volume on developing chicken embryos. We assessed standard measures of mortality, organ growth, body growth, and behavior from chickens developmentally exposed in ovo prior to incubation to low (0.1 l/g egg) or high volumes (1.0 l/g egg) of corn oil injected into airsacs or to no injection. The chickens receiving high volumes of oil showed increases in overall embryonic mortality and early embryo mortality compared to chickens receiving low volumes of oil or no injection. The chickens receiving high volumes of oil showed decreased activity during righting reflex, running time, visual discrimination, and olfactory aversion tests, and increased activity during an open-field activity test compared to chickens receiving low volumes of oil or no injection. Somatic endpoints do not appear to be affected by high volume injections. These results suggest that high volumes of vehicle injected into airsacs of eggs may lead to hypoxia-like conditions that increase embryonic mortality and disrupt simple behaviors. However, some effects of volume may diminish when injections are performed later in incubation.     

Embryotoxic Thresholds of Mercury: Estimates from Individual Mallard Eggs

Eighty pairs of mallards (Anas platyrhynchos) were fed an uncontaminated diet until each female had laid 15 eggs. After each female had laid her 15th egg, the pair was randomly assigned to a control diet or diets containing 5, 10, or 20 μg/g mercury as methylmercury until she had laid a second set of 15 eggs. There were 20 pairs in each group. After the second set of 15 eggs, the pair was returned to an uncontaminated diet, and the female was permitted to lay another 30 eggs. For those pairs fed the mercury diets, the even-numbered eggs were incubated and the odd-numbered eggs were saved for possible mercury analysis. Mercury in the even-numbered eggs was estimated as the average of what was in the neighboring odd-numbered eggs. Neurological signs of methylmercury poisoning were observed in ducklings that hatched from eggs containing as little as 2.3 μg/g estimated mercury on a wet-weight basis, and deformities were seen in embryos from eggs containing about 1 μg/g estimated mercury. Although embryo mortality was seen in eggs estimated to contain as little as 0.74 μg/g mercury, there were considerable differences in the sensitivity of mallard embryos, especially from different parents, with some embryos surviving as much as 30 or more μg/g mercury in the egg. Received: 9 February 2002/Accepted: 12 June 2002     

Congenital abnormalities in nickel poisoning in chick embryos

Experiments were conducted to evaluate the teratogenic potential of nickel chloride given to developing chick embryos. Nickel chloride was dissolved in saline and injected into chicken eggs at dosages ranging from 0.02 to 0.7 mg per egg. The injections were made into the air sacs of eggs at days 0, 1, 2, 3, and 4 of incubation. Control eggs were injected with an equivalent volume of saline (0.1 ml per egg); in all, 840 chicken eggs were used. All embryos were examined on day eight. The following malformations were observed: exencephaly, everted viscera, short and twisted neck, short and twisted limbs, microphthalmia, hemorrhage, and reduced body size. The dose-response relationship was observed in all of the groups tested. The toxicity and teratogenicity of nickel chloride was the highest in the embryos treated at day 2. The results of the present study indicate that nickel chloride is teratogenic.     

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 (LC₅₀). Based on the dose–response curves and LC₅₀s, 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 LC₅₀ was 1.79 μg/g mercury on a wet-weight basis. Other species we categorized as also exhibiting relatively low sensitivity to injected methylmercury (their LC₅₀s were 1 μg/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 LC₅₀s were greater than 0.25 μg/g mercury but less than 1 μg/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 LC₅₀s were less than 0.25 μg/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 LC₅₀s derived from our egg injections are useful for ranking the sensitivities of various species but are not identical to the LC₅₀s that would be observed if the mother bird had put the same concentrations of mercury into her eggs; the LC₅₀s of maternally deposited methylmercury would be higher.     

Chromium poisoning and chick embryogenesis

The effects of chromium on chick embryos were analyzed in the present study. Chromium trioxide was dissolved in saline and injected into embryonating chicken eggs at doses ranging from 0.002 to 0.05 mg per egg. The injections were made into the air sacs of eggs at Days 0, 1, 2, 3, and 4 of incubation. Control eggs were injected with an equivalent volume of saline (0.1 ml per egg). In all, 840 chicken eggs were used for this study. All the embryos were examined at Day 8. The following malformations were observed: short and twisted limbs, microphthalmia, exencephaly, short and twisted neck, everted viscera, edema, and reduced body size. Most of the embryos showed unilateral and bilateral limb defects. The results of the present study indicate that chromium trioxide has tendency to cause malformations in the chick embryos tested.     

Reproductive and transgenerational effects of methylmercury or Aroclor 1268 on Fundulus heteroclitus

This research determined the potential for methylmercury or Aroclor 1268 to disrupt reproduction and sexual differentiation in Fundulus heteroclitus. The research determined whether fish that are exposed to mercury or Aroclor 1268 survive and successfully reproduce; whether offspring of exposed fish hatch, survive, produce eggs, and fertilize them; and whether the second-generation offspring of exposed fish hatch and survive. Fundulus heteroclitus were exposed to mercury or Aroclor 1268 via contaminated food. Endpoints evaluated included survival, growth, fecundity, fertilization success, hatch success, larval survival, sex ratios, and the prevalence of gonadal abnormalities. In general, polychlorinated biphenyls were highly bioavailable and accumulated well through feeding. The only statistically significant effect observed as a result of treatment with Aroclor 1268 was an increase in growth in the offspring of exposed fish. Mercury was accumulated in a dose-dependent fashion via food exposures. Exposure to mercury in food increased mortality in male F. heteroclitus, which possibly occurred as a result of behavioral alterations. Increased mortality was observed at body burdens of 0.2 to 0.47 microgram/g. Offspring of F. heteroclitus fed mercury-contaminated food were less able to successfully reproduce, with reduced fertilization success observed at egg concentrations of 0.01 to 0.63 microgram/g, which corresponds with parent whole-body concentrations of 1.1 to 1.2 micrograms/g. Offspring of exposed fish also had altered sex ratios, with treatment at moderate concentrations producing fewer females and treatment at the highest concentration producing more females than expected. Alterations in sex ratios were observed at concentrations of less than 0.01 microgram/g in eggs or between 0.44 and 1.1 micrograms/g in parents. Offspring of mercury-exposed fish also had increased growth in moderate treatments, when egg concentrations were less than 0.02 microgram/g, or when parent whole bodies contained 0.2 to 0.47 microgram/g. In summary, exposure to mercury reduced male survival, reduced the ability of offspring to successfully reproduce, and altered sex ratios in offspring. Both direct effects on exposed fish and transgenerational effects were observed.     

Mercury accumulation and loss in mallard eggs

Female mallards (Anas platyrhynchos) were fed diets containing 5, 10, or 20 ppm mercury as methylmercury chloride. One egg was collected from each bird before the start of the mercury diets and 15 eggs were collected from each bird while it was being fed mercury. The mercury diets were then replaced by uncontaminated diets, and each female was allowed to lay 29 more eggs. Mercury levels in eggs rose to about 7, 18, and 35 ppm wet-weight in females fed 5, 10, or 20 ppm mercury, respectively. Mercury levels fell to about 0.16, 0.80, and 1.7 ppm in the last egg laid by birds that had earlier been fed 5, 10, or 20 ppm mercury, respectively. Higher concentrations of mercury were found in egg albumen than in yolk, and between 95 and 100% of the mercury in the eggs was in the form of methylmercury.     

The type B brevetoxin (PbTx-3) adversely affects development, cardiovascular function, and survival in Medaka (Oryzias latipes) embryos

Brevetoxins are produced by the red tide dinoflagellate Karenia brevis. The toxins are lipophilic polyether toxins that elicit a myriad of effects depending on the route of exposure and the target organism. Brevetoxins are therefore broadly toxic to marine and estuarine animals. By mimicking the maternal route of exposure to the oocytes in finfish, we characterized the adverse effects of the type B brevetoxin brevetoxin-3 (PbTx-3) on embryonic fish development and survival. The Japanese rice fish, medaka (Oryzias latipes), was used as the experimental model in which individual eggs were exposed via microinjection to various known concentrations of PbTx-3 dissolved in an oil vehicle. Embryos injected with doses exceeding 1.0 ng/egg displayed tachycardia, hyperkinetic twitches in the form of sustained convulsions, spinal curvature, clumping of the erythrocytes, and decreased hatching success. Furthermore, fish dosed with toxin were often unable to hatch in the classic tail-first fashion and emerged head first, which resulted in partial hatches and death. We determined that the LD(50) (dose that is lethal to 50% of the fish) for an injected dose of PbTx-3 is 4.0 ng/egg. The results of this study complement previous studies of the developmental toxicity of the type A brevetoxin brevetoxin-1 (PbTx-1), by illustrating in vivo the differing affinities of the two congeners for cardiac sodium channels. Consequently, we observed differing cardiovascular responses in the embryos, wherein embryos exposed to PbTx-3 exhibited persistent tachycardia, whereas embryos exposed to PbTx-1 displayed bradycardia, the onset of which was delayed.     

Egg incubation position affects toxicity of air cell administered polychlorinated biphenyl 126 (3,3',4,4',5-pentachlorobiphenyl) in chicken (Gallus gallus) embryos

The avian egg is used extensively for chemical screening and determining the relative sensitivity of species to environmental contaminants (e.g., metals, pesticides, polyhalogenated compounds). The effect of egg incubation position on embryonic survival, pipping, and hatching success was examined following air cell administration of polychlorinated biphenyl (PCB) congener 126 (3,3',4,4',5-pentachlorobiphenyl [PCB 126]; 500-2,000 pg/g egg) on day 4 of development in fertile chicken (Gallus gallus) eggs. Depending on dose, toxicity was found to be up to nine times greater in vertically versus horizontally incubated eggs. This may be due to enhanced embryonic exposure to the injection bolus in vertically incubated eggs compared to more gradual uptake in horizontally incubated eggs. Following air cell administration of PCB 126, horizontal incubation of eggs may more closely approximate uptake and toxicity that has been observed with naturally incorporated contaminants. These data have implications for chemical screening and use of laboratory data for ecological risk assessments.     

Selenium impacts on razorback sucker, Colorado River, Colorado II. Eggs

Effects on hatching and development of fertilized eggs in adult razorback sucker (Xyrauchen texanus) exposed to selenium in flooded bottomland sites near Grand Junction, Colorado, were determined. After 9 months exposure, fish were collected and induced to spawn and eggs collected for inorganic element analyses. A 9-day egg study was conducted with five spawns from Horsethief ponds, six spawns from Adobe Creek channel, and four spawns from North Pond using a reference water and site waters. Selenium concentrations in eggs were 6.5 microg/g from Horsethief, 46 microg/g from Adobe Creek, 38 microg/g from North Pond, and 6.0 microg/g from brood stock. Eggs from young adults had a smaller diameter and higher moisture content than brood stock. There were no differences among the four sources in viability, survival, hatch, hatchability, or mortality of deformed embryos or larvae. Adobe Creek larvae had more deformed embryos in eggs held in site water than held in reference water. There were significant negative correlations between selenium concentrations in adult muscle plugs and percent hatch, egg diameter, and deformities in embryos. Results from this study suggest that selenium contamination in parts of the upper basin of the Colorado River should be a major concern to recovery efforts for endangered fish.     

Embryopathic effects of cyclophosphamide

Cyclosphosphamide, dissolved in saline, was injected into the air sac of white Leghorn chick eggs in dose levels of 0.005, 0.007, 0.010, 0.012, 0.015, and 0.017 mg per egg. Eggs received a single injection of cyclophosphamide on Days 0, 1, 2, or 3 of incubation. Control eggs were injected with an equivalent volume of saline (0.1 ml per egg). In all 904 chicken eggs were used for this study. Surviving embryos were sacrificed when they reached 11 days of incubation. The LD₅₀ values for Days 1, 2, and 3 were 0.017, 0.007, and 0.012 mg per egg, respectively. The overall incidence of abnormal embryos for Days 0, 1, 2, and 3 were 7, 6.3, 12, and 22%, respectively. Abnormalities such as reduced body size, everted viscera, short and twisted limbs, eye defects, abnormal beak, and short and twisted neck were commonly seen in survivors no matter when exposed to cyclophosphamide. The teratogenicity of cyclophosphamide was noted to be the highest in the embryos treated on Day 3. The present study has demonstrated that cyclophosphamide is toxic and teratogenic during the period of early organogenesis in the chick embryos.     

Selecting Optimal Eggs and Embryonic Developmental Stages of Fathead Minnow (Pimephales promelas) for Early Life-Stage Toxicity Tests

Aquaculture research has indicated that fish embryo hatching success and larval survival can sometimes be predicted by embryo characteristics, such as blastomere cleavage patterns. An analogous strategy of individual assessment of spawned eggs could also be used to improve the quality of toxicity tests using early life-stages of fish where control-group survival determines experimental validity. Here we explored whether a simple method of assessing fathead minnow eggs and embryos for abnormalities could predict hatch success, and larval size at hatch, as indicators of embryo larval quality. Embryos were classified according to both their developmental stage and the presence of any abnormalities: uneven blastomere cleavage, atypical embryo size or shape, and the presence of inclusions in the yolk. Clutch size and fertilization rate did not predict embryo larval quality. Fewer abnormalities in embryos with ≤32 cells correlated with longer larvae at hatch. Normal embryos were more likely to hatch successfully than abnormal embryos of the same clutch, but because abnormality rates were generally low, much of the variation in hatch success could not be attributed to visible embryo malformations. Blastomere symmetry may be a useful selection criterion in embryos <3 h postfertilization. Where toxicant exposures early in embryonic development are not required or possible, hatch success could be increased by using older embryos that have survived gastrulation. Purposeful selection of embryos with at least two blastomeres, blastomere symmetry, and few inclusions can improve control survival and improve the quality of any generated (sub)lethality data. In our laboratory, application of the egg-selection criteria significantly improved control group hatch success increasing it from a mean of 84.4 to 94.2 %.     

Effects of low dietary levels of methyl mercury on mallard reproduction

Summary Mallard ducks were fed a control diet or a diet containing 0.5 ppm or 3 ppm mercury (as methylmercury dicyandiamide). Health of adults and reproductive success were studied. The dietary level of 3 ppm mercury had harmful effects on reproduction, although it did not appear to affect the health of the adults during the 12 months of dosage. Ducks that were fed the diet containing 0.5 ppm mercury reproduced as well as controls, and ducklings from parents fed 0.5 ppm mercury grew faster in the first week of life than did controls.The greatest harm to reproduction associated with the diet containing 3 ppm mercury was an increase in duckling mortality, but reduced egg laying and increased embryonic mortality also occurred.During the peak of egg laying, eggs laid by controls tended to be heavier than eggs laid by ducks fed either level of mercury; however, there seemed to be no eggshell thinning associated with mercury treatment.Levels of mercury reached about 1 ppm in eggs from ducks fed a dietary dosage of 0.5 ppm mercury and between 6 and 9 ppm in the eggs from ducks fed 3 ppm mercury.