The most extensive and detailed study of bone changes found in malformed frogs to date shows that both time- and location-specific environmental events may influence the development of these malformations, according to a paper to be published in the journal Teratology. The study revealed that similar malformations are occurring in frogs collected at the same time from a particular site, indicating that tadpoles at specific sites have received the same type of environmental insult at the same developmental stage. The data represent 180 frogs collected at 16 sites in three states -- Maine, Minnesota and Vermont -- over two years of study and is the result of a large multi-agency effort involving the USGS National Wildlife Health Center, University of Wisconsin Department of Anatomy, NIH-National Institute of Environmental Health Sciences, Minnesota Pollution Control Agency, Vermont Agency of Natural Resources, and the U.S. Fish and Wildlife Service. Key to accomplishing this multi-agency work was a Memorandum of Understanding between NIEHS and USGS Since middle-school students on an outing discovered large numbers of deformed frogs in a Minnesota pond in 1995, the dramatic increase in recent reports of malformed frogs has drawn the attention of scientists and the public. Malformed frogs are now documented in 44 states, in 38 species of frogs and 19 species of toads, with estimates of deformities as high as 60 percent in some local populations. Scientists now agree that current numbers of reported malformations exceed any norm and that the situation warrants urgent attention. These die-offs and deformities of amphibians around the globe are of great concern because amphibians are good barometers of significant environmental changes that may go initially undetected by humans. The study, Hind Limb Malformations in Free-Living Northern Leopard Frogs (Rana pipiens) from Maine, Minnesota and Vermont Suggest Multiple Etiologies, will be released in the journal Teratology, said lead author Dr. Carol Meteyer, a wildlife pathologist at the USGS National Wildlife Health Center in Madison, Wisc. The study, said Meteyer, contributes to the ongoing multidisciplinary analyses of frog malformations and presents the range, complexity and recurring patterns found in hind limb malformations in frogs that have survived to metamorphosis -- the transformation from tadpole to frog -- at several different locations in the United States. "These malformations are the result of environmental factors affecting the frog limb development during early tadpole stages, and this study shows the wide variety of responses the developing limb can have to these factors," Meteyer said. "The precision and patterns of some of these malformations are striking." For example, the wildlife pathologist said, two frogs from Minnesota in 1998 were missing a single middle bone from each toe on both feet. Frogs from the study site in Maine and two sites in Minnesota had bones duplicated in their hind limbs, primarily in the form of multiple toes. In addition, the research revealed that although multiple -- or extra -- limbs were found in only 5 percent of the frogs in the study, all frogs with multiple limbs came from the two sites in Minnesota and the site in Maine where frogs were also afflicted with multiple toes. No frogs with multiple limbs, said Meteyer, came from the 13 other sites with malformed frogs, although the frogs at some of these other sites also exhibited site-specific deformity patterns. In Vermont, for example, all of the 65 malformed frogs the researchers examined were missing all or a portion of their hind legs, but none had multiple toes or limbs. Of the Vermont frogs missing entire limbs, x-rays revealed that 73 percent were also missing bones in the hip, providing evidence that a predator had not removed the limbs, but that developmental errors were to blame. "The wide geographic occurrence and the variety of malformations that occur in free-living frogs," said Meteyer, "suggest that both site-specific and time-dependent influences may be involved in the development of these malformations. Since similar malformations are occurring in frogs collected at the same time from a particular site, it seems that tadpoles at a specific site have received the same type of developmental insult at the same stage of development." The paper also discusses the varied current hypotheses proposed as causes of malformations in light of the x-ray data presented in this paper. Several hypotheses are being considered to explain the causes of malformations, including chemical contamination; infection with metacercariae, a kind of immature parasitic worm; exposure to the sun^s ultraviolet rays; and physical trauma. Based on the x-ray data presented in the Teratology study, Meteyer and her colleagues feel that none of the current hypotheses adequately address the full range of malformations. "The detailed descriptions and classification presented in this paper will contribute to the foundation for further multidisciplinary research designed to provide insights into the causes of malformations in free-living frogs," Meteyer said. Scientists at the NIH^s National Institute of Environmental Health Sciences are currently attempting to identify potential malformation-causing agents in the environment where deformations occur and are exploring their potential role in this complex problem. Dr. Jim Burkhart, the biochemist responsible for the NIEHS efforts to evaluate whether agents present in the water may be contributing to the increase in frog malformations, said, "The likelihood of multiple causes demonstrated by this work clearly parallels some of our efforts to evaluate possible water-borne factors by analysis of water and sediments. We have not identified single causes of deformities - however, we have identified mixtures with the capacity to disrupt development of amphibians and many other species at several sites that are a part of this study." Additionally, the USGS National Wildlife Health Center and the University of Wisconsin^s Department of Anatomy/NIEHS Center for Developmental, Molecular and Toxicology are performing extensive examinations on wild-caught tadpoles to more clearly delineate the developmental causes of frog malformations.