FEATURE
Amphibian Deformities Continue to Puzzle Researchers Studies are under way to discover the cause of widespread malformations in wild amphibians. CHARLES W. SCHMIDT | n the summer of 1995, a group of schoolchilI dren discovered several frogs with malformed or missing legs in a Minnesota farm pond, unwitI tingly becoming the first to uncover what has : | since emerged as one of the most perplexing ecological issues of the decade. Since the initial discovery, amphibian deformities have been reported at more than 170 locations in Minnesota, as well as in Wisconsin, Ohio, Vermont, Missouri, California, Iowa, Kansas, South Dakota, and Quebec and Ontario, Canada. The widespread and sudden occurrence of the deformities has initiated a flurry of activity among the amphibian research community and state and federal agencies. Several workshops and symposia— including meetings sponsored by EPA last September in Duluth, Minn., and in Shenandoah National Park in Virginia this spring—have raised many research questions. Scientists wonder whether the increase in amphibian deformities indicates a serious environmental problem. Although inadequate baseline data have constrained their attempts to quantify increases in the numbers of deformed amphibians, many researchers have little doubt that they are witnessing a genuine phenomenon. "I've observed literally thousands of Minnesota frogs over the last 20 years," said David M. Hoppe, professor of biology at the University of Minnesota. "Up until 1995 I had only seen two with macroscopic limb defects. In 1996,1 saw over 200." Thus far, the deformities appear to be concentrated in a limited number of species, primarily the Northern leopard frog, the mink frog, and the green frog. Deformities in wood frogs, bullfrogs, gray tree frogs, spring Pacific tree frogs, long-toed salamanders, and American toads have also been reported (1). Given the issue's potential importance, state and federal agencies and universities are defining the questions that need to be answered and scrambling to find research money. An estimated $1 million has been requested this year from EPA, the Na-
tional Institute of Environmental Health Sciences (NIEHS), the U.S. Geological Survey (USGS), and the states for field surveys and associated laboratory analyses. Major multi-institution collaborative studies are under way in Minnesota and Vermont, and EPA and die National Park Service (NPS) are developing an environmental monitoring program to assess amphibian populations in national parks. No consensus on possible causes Although no consensus has yet been reached regarding any single cause of the deformities, many researchers attribute them to chemical pollutants, increased UV radiation from ozone depletion (particularly UV-B radiation in the range of 295-330 nanometers), parasitic infestations, or some combination of the three. One possibility is that photomodification of chemicals could change their toxicological properties and thus affect development. According to EPA toxicologist Gerald Ankley, changes in UV light also offer an intriguing explanation for the somewhat random nature of locations where deformities have been observed, which range from state forests to wetlands affected by agricultural practices (2). One focus of research is an assessment of deformity distribution, a difficult task given the effects of differences in data reporting, minimal baseline data, insufficient sample sizes, and regional and temporal differences in climate. Sam Droege, a USGS staff scientist in Laurel, Md., speculates that these factors will likely preclude drawing any conclusions about trends in the data for at least five years. "Unfortunately, virtually no one has conducted any largescale regional surveys," he said. "Most studies to date have focused on specific geographic locations. What we have to do now is look at the big picture." However, Droege emphasized mat such efforts will be arduous without a coordinated effort to standardize methods for data collection and reporting. To this end, researchers at the USGS Northern Prairie Sci-
3 2 4 A • VOL. 31, NO. 7, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
0013-936X/97/0931-324A$14.00/0 © 1997 American Chemical Society
Vermont Deformed: Rama pipiens 10/9/96
In Minnesota, the first place where significant frog deformities were seen, frogs appeared with missing, additional, or malformed limbs (at right). During 1996, large numbers of deformed leopard frogs (above) also were found in Vermont leading scientists to speculate about possible causes for the abnormalities. (Courtesy Minnesota Pollution Control Agency and Vermont Department of Environmental Conservation)
ence Center in North Dakota and the Mid-Continent Ecology Division of EPA's National Health and Environmental Effects Laboratory in Duluth, Minn., have proposed a national reporting center for amphibian deformities that will be located on the World Wide Web. Ultimately, the proposed reporting center would enable anyone who had observed a deformed amphibian to contribute key information— species, type of deformity, location, and habitat type to a data repository that could be used to establish correlates with specific land-use patterns and identify areas for future research and field investigations. Researchers already are communicatinsj some of this information on the North American Amphibian Monitoring Program's Web site (http7/wwwim nbs gov/naamp3) Environmental contaminants are gaining the most attention as the potential cause of amphibian deformities, particularly following the recent publication of a paper by Martin Ouellet (McGill University, Quebec) in the Journal of Wildlife Diseases (3)) Ouellet reported missing limbs in 12% of a group of 853 metamorphosing amphibians from 14 agricultural sites exposed to pesticide runoff, as com-
pared with a 0.7% deformity rate in a group of 271 animals from 12 control sites. However, the study results were highly variable, and differences between control and pesticide-contaminated habitats were not statistically significant. Search for chemical mechanism There are many ways in which environmental chemicals might interfere with normal animal development. According to Joseph Tietge, an EPA biologist who has become the agency's de facto lead on the amphibian deformities issue, the retinoic acid (RA) pathway is the most studied of all potential chemical mechanisms. RA is a metabolite of vitamin A that plays an important role in limb formation in many species. According to Kenneth Muneoka, a biology professor at Tulane University in New Orleans, scientists have long known that exogenously applied RA results in either supernumery (extra limbs) or reduction (missing limbs) deformities, depending on the developmental stage at the time of exposure. "Similarities between laboratory-induced deformities and those observed in the field suggest that VOL. 31, NO. 7, 1997/ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS » 3 2 5 A
chemical pollutants present in the environment may be acting either as retinoic-acid-signaling mimics or antagonists," he said. However, Muneoka cautions that this possibility has not yet been confirmed by experiments using environmental chemicals in amphibian models. "The most compelling similarities between the amphibian deformities and those produced in the laboratory were elicited in developmental studies of rodents administered retinoic acid in utero," he said. "To my knowledge, there are no environmental chemicals that have yet been shown to act via an RAmediated pathway." If environmental chemicals can affect RA pathways, it will be difficult to explain why amphibians specifically would be harmed, given how common the pathway is across species. "If these chemicals are acting via an RA-mediated pathway, then why aren't we seeing deformities in birds and fish—even plants?" asked Stanley Sessions, a biology professor at Hartwick College in Oneonta, N.Y. "The identification of environmental chemicals that could potentially interact with the retinoic acid receptor can be evaluated using in vitro models," said Tietge, adding mat EPA is refining such models in collaboration with university researchers. Some researchers who support a biological rather than a pollution-related cause for the deformities think that infestation by a class of parasitic flatworms, known as trematodes, is the most plausible explanation. Trematodes living in aquatic habitats cause deformities by boring into die skin of larval amphibians and forming cysts near the developing hind limbs. These cysts are hypothesized to disrupt normal development. Massive infestations by the trematode Manodistomum syntomentera were identified by Sessions as the likely cause of additional limbs in frog populations {Hyla regilla) and in salamanders {Ambystoma macrodactylum) from a pond in Santa Cruz Calif (4) Sessions, who is convinced that trematode activity explains the multiple limbs in wild amphibians, produced this condition experimentally. He implanted resin beads into the developing limb bud regions of lab-raised frogs (Xenopus laevis) (4). Because these "artificial cysts" were biologically inactive, the procedure helped confirm that the formation of additional limbs was likely related to mechanical obstructions in limb bud tissues ratiier than to potentially toxic effects of trematode secretions. "You always see supernumery defects in these cases, and the extra limbs develop abnormally due to the regulatory adjustments the cells are making in that process" he said. However, researchers at the Minnesota Pollution Control Agency and the Vermont Department of Environmental Conservation note that the deformities have involved missing rather than extra limbs. Sessions believes that animals with missing limbs may well be nothing more than the victims of injury or attempted predation by other animals. In terms of population levels, "the last few years have been very good for these species," said Sessions. "The percentage of animals with missing limbs hasn't changed; it's just that the overall populations are increasing, so it seems as if there are more [deformities]." 3 2 6 A • VOL. 31, NO. 7, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
Into the field this year The primary focus of ongoing field worlc is to test the hypothesis that chemicals are indeed at the heart of the deformities. Researchers in Vermont plan to characterize amphibian deformities, record habitat variables, and assess the prevalence of deformities at 30 locations during die 1997 field season. In 1996, deformity rates of 5-23% were observed in populations of Vermont leopard frogs. The Vermont Department of Environmental Conservation, the Vermont Agency of Natural Resources, the U.S. Fish and Wildlife Service, USGS's National Wildlife Health Center (NWHC), and Middlebury College are among the institutions collaborating on the Vermont study. The Minnesota Pollution Control Agency, NIEHS, NWHC, EPA, and the University of Minnesota will evaluate the condition of Minnesota frogs. According to Kathryn Converse of NWHC, scientists will focus on sites from which deformed frogs were collected in 1996. They will a t t e m p t to correlate abnormalities in frogs collected this year with chemical concentrations in tissues, sediments, and water. Additionally, internal and external examinations of the frogs will b e m a d e to characterize deformities. Changes in their tissues will be examined microscopically, and any bacteria, viruses, or parasites they carry will be isolated and identified. EPA and NPS are collaborating on the Demonstration Intensive Sites Project (DISPro), which will develop a large environmental monitoring program using existing research facilities at 12 national parks. Under DISPro, the species diversity and deformity rates among populations of amphibians will be assessed at the sites, which are scattered around the country. The installation of UV-B monitoring stations at each location is under way. Given the preliminary nature of deformity studies and the need to more clearly define causes, industry groups are not yet directly involved in this research. Ann Mason of the Chemical Manufacturers Association (CMA) said her organization will closely monitor this year's field research programs. "We haven't really developed a good hypothesis yet," she said. "If there is a chemical linkage, then CMA will become more closely involved." The notion long held by biologists—that amphibians are sentinels of environmental degradation, analogous to the proverbial "canaries in the coal mine"— has instilled a sense of urgency about resolving the deformities problem. Many questions are still unanswered, and ascertaining the status of amphibian populations and the extent of the deformities will pose a challenge for some time.
References (1) Tietge, J. E. Presented at the Third Annual Meeting of the North American Amphibian Monitoring Program, Nov. 14, 1996-Feb. 14, 1997, on the World Wide Web (http: //www.im.nbs.gov/naamp3/papers/59df.html). (2) Ankley, G. T. Presented at the Third Annual Meeting of the North American Amphibian Monitoring Program, (http: //www.im.nbs.gov/naamp3/papers/deformuv.html) (3) Ouellet, M. et al. /. Wildl. Dis. 1997, 33, 95-104. (4) Sessions, S.; Ruth, S. /. Exp. Zool. 1990, 254, 38-47. Charles W. Schmidt is a staff scientist with McLaren/ Hart Environmental Engineering Corporation in Portland, Maine.
