A reconstituted water medium was developed for use in the Frog Embryo Teratogenesis Assay--Xenopus (FETAX). FETAX solution was then tested on three compounds with known mammalian teratogenicity (ethanol, caffeine, and 5-fluorouracil) as was a non-teratogen (saccharin). The results obtained were then compared with results from tests on these compounds in two other media that had previously been used in the assay. Saccharin was not teratogenic. Ethanol and caffeine were weak and moderate teratogens, respectively. 5-fluorouracil was a strong teratogen. The results compare favorably with those obtained in mammalian studies. The amount of growth inhibition in embryos in the 96 h tests was positively correlated with the degree of teratogenicity of the compound. Final validation of FETAX will allow it to be used to screen and rank compounds for further testing and as a tool for studying the basic mechanisms of teratogenesis.

Development of a reconstituted water medium and preliminary validation of the frog embryo teratogenesis assay--Xenopus (FETAX).

Amphibians are considered reliable indicators of environmental quality. In the western United States, a general decline of frog populations parallels an apparent worldwide decline. The factors thought to be contributing to declines in frog populations include habitat loss, introduction of exotic species, overexploitation, disease, climate change, and decreasing water quality. With respect to water quality, agroecosystems use 80-90% of the water resources in the western United States, frequently resulting in highly eutrophic conditions. Recent investigations suggest that these eutrophic conditions (elevated pH, water temperature, and un-ionized ammonia) may be associated with frog embryo mortality or malformations. However, water quality criteria for frogs and other amphibians do not currently exist. Here, we briefly review data that support the need to develop water quality parameters for frogs in agroecosystems and other habitats.

The need for water quality criteria for frogs.

Significantly lower anuran species diversity and density was recorded in a vegetable growing area relative to upstream and downstream sites in the Holland River watershed, Ontario, Canada Egg hatching success and tadpole deformity rates of American toads (Bufo americanus americanus), green frogs (Rana clamitans melanota) and northern leopard frogs (Rana pipiens) eggs in water from field sites and control water were assessed Compared to the control and the upstream site, the total abnormality rate (unhatched eggs plus deformed tadpoles) was higher for American toads in water from the agricultural and downstream sites Total abnormality was higher in green frog eggs in water from the agricultural site and a downstream site Trace concentrations of organophosphorus pesticides were detected most often in agricultural zone water and sediments than in upstream and downstream sites Organochlorine pesticide residues, especially in agricultural zone samples, exceeded the no effect level guidelines for the protection of aquatic life Ammonia, phosphorus, particulates, BOD and TKN were highest in the agricultural zone Significant correlations between these parameters and anuran development suggest nutrient run-off as a causal or contributing factor in lower anuran diversity, density and reproductive success of American toads and green frogs in the site dominated by agriculture

Anuran Development, Density and Diversity in Relation to Agricultural Activity in the Holland River Watershed, Ontario, Canada (1990–1992)

Due to the use of a limited number of species and subchronic exposures, current ecological hazard assessment processes can underestimate the chronic toxicity of environmental contaminants resulting...

Chronic toxicity of environmental contaminants: sentinels and biomarkers.

Propiconazole is a fungicide used in a variety of agricultural applications. Preliminary studies had suggested that embryos of the crustacean Daphnia magna are particularly susceptible to the toxicity of this chemical. The goals of the present study were to define endpoints of daphnid embryonic development that could be routinely used to assess the embryo toxicity of chemicals and to characterize definitively the embryo toxicity of propiconazole to daphnids. Daphnid embryonic development was characterized into six readily distinguishable stages based on the degree of tissue differentiation. Embryonic development could be monitored either in the brood chamber of the maternal organism or using embryos removed from the brood chamber and incubated ex vivo. Standard toxicity assessment revealed that propiconazole elicited no significant adverse effects on daphnid survival or fecundity during a 21-d exposure to concentrations as high as 0.25 mg/L. Exposure to 0.25 mg/L propiconazole, however, caused a significant incidence of developmental abnormalities and embryonic death. Abnormalities were consistent with developmental arrest at later stages of embryonic maturation. Propiconazole elicited a steep concentration-response curve with respect to embryo toxicity, with a 10% and a 90% incidence of embryo toxicity measured at 0.50 and 0.82 mg/L, respectively. Direct exposure of embryos to propiconazole resulted in toxicity, though the incidence and characteristics of developmental abnormalities were not consistent with that observed during chronic exposures. However, maternal exposure to propiconazole followed by transfer of early embryos to propiconazole-free media resulted in embryo toxicity consistent with that observed during chronic exposure. These results indicate that propiconazole interferes with the later stages of daphnid embryonic development, and that this toxicity is manifested largely via maternal exposure to the fungicide.

The fungicide propiconazole interferes with embryonic development of the crustacean Daphnia magna

The FETAX (Frog Embryo Teratogenesis Assay — Xenopus) whole embryo bioassay has been developed to screen for environmental substances that cause birth defects. We have used this assay to test its effectiveness in working with actual water samples from the field. Tar Creek is contaminated by discharges from abandoned lead and zinc mines. In addition to high concentrations of zinc, iron and other metals, water samples are routinely low in pH and oxygen content. The pH values of three Tar Creek sample sites were below the established tolerance limits of the embryos. Therefore, one group of samples had no pH adjustment while a second group had the pH adjusted to 7.0. Two of the four sites contained agents that reduced embryonic growth and caused high rates of mortality. A third site contained teratogenic substances. We have determined that metal content is responsible, along with the low pH, for the biologic effects. We have identified high concentrations of toxic metals in Tar Creek by water analysis and were able to demonstrate, by removing metals via Chelex® 100 ion exchange chromatography, that the observed toxicity and teratogenicity were caused by metal ions.

We have concluded that FETAX is an excellent test for complex mixtures. Physicochemical parameters, such as pH, oxygen and metals content, can be altered and the effect of these changes on toxicity and teratogenicity determined using FETAX. The interaction of toxic substances and low pH are important when considering embryo survival and development.

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Detection of teratogenic substances in acidic mine water samples using the frog embryo teratogenesis assay--Xenopus (FETAX).

Effects of acid mine wastewater upon grand lake, neosho and spring rivers of northeastern oklahoma and effects of geological fractures and lineaments upon hydrologic flow of acid mine wastewater

Curt A. McCormick

Papers

Detection of teratogenic substances in acidic mine water samples using the frog embryo teratogenesis assay--Xenopus (FETAX).

Effects of acid mine wastewater upon grand lake, neosho and spring rivers of northeastern oklahoma and effects of geological fractures and lineaments upon hydrologic flow of acid mine wastewater