Since 1989, evidence has been mounting of a global decline in the numbers of certain species of amphibians. Three years ago, biologists in Minnesota discovered a different problem in the amphibian community--large numbers of frogs with missing or deformed limbs. Subsequent research indicates the problem is widespread, including large areas of Minnesota, Wisconsin, New York, Vermont, and parts of Ontario and Quebec. Determined to track down the cause of these deformities, the Minnesota Pollution Control Agency (MPCA), based in St. Paul, has called on the NIEHS to collaborate on a research effort.
Unhoppy frogs
. Extra limbs are some of the deformities showing up in frogs in Minnesota, the cause of which is still unknown.
The first hint of trouble among frogs came in 1993, when a group of schoolchildren discovered frogs with malformed and missing limbs in a Minnesota farm pond, and posted their findings on the Internet. During the following two years, reports of deformities increased but were restricted to a few localities and one species, the northern leopard frog (
Rana pipiens
). In 1996, the number of reports and species involved increased tremendously. Six species of deformed frogs have been documented by the MPCA at more than 150 sites across 54 counties in Minnesota. The malformations found include missing or additional feet, legs, toes, and eyes, as well as musculoskeletal and urogenital defects. The incidence of abnormalities was as high as 60% among some frog populations. Abnormalities are rare among natural and laboratory-reared frogs--less than 0.05%. Thus, biologists have little doubt they are witnessing a genuine phenomenon.
Starting in late 1996, workshops were held by various government agencies to postulate mechanisms that could result in the deformities, review possible environmental causes, and identify research needs. Participants agreed that a national reporting center was needed to encourage standardization of the data being collected by federal, state, and university research groups. The National Reporting Center for Amphibian Malformities (NARCAM) has subsequently been established at the Northern Prairie Wildlife Research Center in Jamestown, North Dakota, which has an Internet site located at
http://www.npsc.nbs.gov/narcam
.
Scientists have advanced a number of theories to explain the deformities. Prime suspects include chemical contamination, increased ultraviolet (UV) radiation due to ozone depletion, parasitic infestation, or some combination of the three. Frogs spend a major portion of their lives in water, and thus may be particularly vulnerable to ill effects stemming from chemical contamination of water. Many of the sites in which deformed frogs have been discovered are close to agricultural fields that are intensively sprayed with pesticides and herbicides at certain times of the year. It has been suggested that one or more of the chemicals used, acting alone or in combination with other agents such as UV light, might be disrupting normal frog development.
"There are several prominent cases in this country in which chemical agents were known to cause gross anatomical deformities in wildlife," says Tim Kubiak, national water quality coordinator for the U.S. Fish and Wildlife Service. "In the western United States, high levels of selenium leaching into surface water caused defects in the eggs of shorebirds and fish. And in the Great Lakes, dioxin and dioxin-related chemicals are suspected of causing crossed bills among fish-eating birds. So there is certainly reason to explore a chemical connection with the frog deformities."
Stanley Sessions, a biology professor at Hartwick College in Oneonta, New York, has argued that a class of parasitic flatworms known as trematodes may be responsible for the deformities. Trematodes living in aquatic environments can induce deformities by boring into the skin of larval amphibians and causing cysts near the developing hind limbs. These cysts are hypothesized to disrupt normal development. However, parasitology information indicates that amphibians and parasites have long coexisted in equilibrium and that parasite populations tend to be very stable. Also, in recent testing, water samples from several sites where deformed frogs have been found from which all parasites had been removed still produced abnormalities in frogs.
Jim Burkhart
Photo credit: Steve McCaw, Image Associates, Inc.
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As of yet, there are insufficient data to establish any correlation between chemical contamination, or any other cause, and the incidence of abnormality. In the spring of 1997, the MPCA requested help from the NIEHS to establish a collaborative research effort to tease out associations between chemical exposures and malformations, to demonstrate similar effects under controlled laboratory experiments, and to identify the mechanisms by which the deformities are occurring. Jim Burkhart, head of the Alternative Systems Group within the NIEHS's Environmental Toxicology Program, is coordinating this effort. Researchers from the EPA, the National Wildlife Health Center in Madison, Wisconsin, the U.S. Geological Survey's Environmental and Contaminants Research Center in Columbia, Missouri, and the University of Minnesota are also participating.
"We [the NIEHS] have a very broad base of scientific expertise that can be brought to bear on this issue, including environmental chemistry, different laboratory models, retinoid receptor models, and endocrine disrupter models," Burkhart says. "Our role is to determine cause and effect, and any possible relevance to human health."
The possibility that whatever is causing deformities in frogs may also be linked to human health is indicated by a 1996 study by University of Minnesota researchers (
EHP
104[4]:394-399). The study showed an increased incidence of abnormality and chromosomal aberration among children born to pesticide appliers in Minnesota. In western Minnesota, a major wheat, sugar beet, and potato growing region, the increase was particularly pronounced for children conceived in the spring, the time of heaviest pesticide application.
Field work in the MPCA/NIEHS collaboration began during the frog breeding season in July 1997. On 30 September 1997, the MPCA and the NIEHS held a joint press conference to convey results of preliminary studies. Water and sediment samples were collected from affected and unaffected ponds in Minnesota and tested using the Frog Embryo Teratogenesis Assay:
Xenopus
, known as FETAX. The assay involves exposing developing embryos of
Xenopus laevis
to toxins for four days and observing any resulting malformations. Although this is not the same frog species as those observed with naturally occurring malformations in Minnesota, at concentrations above 50%, water from the ponds with a high incidence of malformed frogs induced a high rate of malformations in the FETAX as well. There is good agreement for birth defects between the FETAX and rodent bioassays, which are often used as the basis for regulatory decisions concerning environmental contaminants and human health. Water from ponds that did not contain malformed frogs did not induce effects in the FETAX. However, preliminary findings suggest that similar effects are induced by groundwater and well water collected at residences near the affected ponds, prompting further concern for human health. The residents have been advised of the findings and offered the option of having the state provide bottled water as a precaution until more is known. George Lucier, director of the Environmental Toxicology Program at the NIEHS, says, "We know that something in the water, including groundwater, is extraordinarily potent in malforming frogs. We now need to determine if people are at risk."
The goals of the MPCA/NIEHS project call for establishing continuity in the field observations taken over the past three years, and characterizing the frequency and nature of the malformations in frog larval stages and metamorphs. The researchers will continue to conduct chemical analyses of water and sediment from test sites where deformed animals have previously been observed, as well as from sites containing no deformed frogs, and attempt to determine the identity of the causative agent or agents.
An approach to studying the affected waters in standardized rodent teratogenicity assays is being considered, and an evaluation of human health effects associated with living in the affected areas is planned in collaboration with state and federal epidemiologists. Says Burkhart, "The exciting thing about this is that, within Minnesota, we have a set of circumstances that provides a basis for study. With numerous sites containing confirmed deformities, we have at least some opportunity to determine cause and effect."
John Manuel
Anabaena circinalis
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The toxic dinoflagellate
Pfiesteria piscicida
may have captured the spotlight along the coast of North Carolina and Maryland, but this nefarious creature is not the only one of its kind. Worldwide, toxic aquatic organisms responsible for such maladies as red tides, cholera outbreaks, and seafood poisoning have surfaced with increasing frequency and geographic distribution during the past 20 years. Such surfacings have brought more questions than answers, questions that an assemblage of scientists recently tried to answer at the NIEHS Workshop on Hazardous Marine/Freshwater Microbes and Toxins, held 26-27 August 1997 at the North Carolina Biotechnology Center in Research Triangle Park, North Carolina.
Prorocentrum lima
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Top
Pfiesteria
expert JoAnn Burkholder, an associate professor of aquatic botany at North Carolina State University in Raleigh, unveiled new research findings from August experiments in which she and her colleagues isolated and purified a water-soluble toxin from the fish-killing organism
Pfiesteria
. Her collaborative work with the NIEHS and the National Marine Fisheries Service has also produced the first reliable test for detecting low levels of the toxin in water samples in the laboratory. This reporter gene assay will now be tested in the field. Also, Burkholder and Karen Steidinger of the Florida Department of Environmental Protection confirmed that at least four
Pfiesteria
-like organisms occurring from Alabama to Delaware have been identified, although only
P. piscicida
has been formally named.
Researchers from the NIEHS Marine and Freshwater Biomedical Sciences Center in Miami, Florida, reported on the isolation of a lipid-soluble fraction that causes dermal necrosis in fish. This work is a collaboration between the Miami center and Ed Noga at North Carolina State University.
Alexandrium tamarense
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Meanwhile, other presenters described an array of syndromes traced to other marine and freshwater algae and some toxins that are responsible for deleterious effects on people and animals. Severe and even fatal reactions in human seafood consumers have resulted from the neurotoxins delivered in paralytic, neurotoxic (associated with red tide organisms), and amnesiac shellfish poisonings as well as ciguatera fish poisoning. Diarrhetic shellfish poisoning, common in areas surrounding intensive marine aquaculture operations, is traced to okadaic acid, a potent toxin that inhibits the secretion of protein enzymes key to regulating the body's metabolism and cell function. Also, toxins from blue-green algae have caused liver damage in humans and animals. Unlike such foodborne bacteria as salmonella and
E. coli
, which can be removed through proper cooking, most of the toxins discussed at the workshop are particularly insidious because they are heat-stable.
Gymnodinium breve
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Daniel Baden, director of the NIEHS Marine and Freshwater Biomedical Sciences Center in Miami, gave the first of several in-depth talks at the workshop on the chemical properties and structural workings of waterborne toxins and how they attack the nervous systems and cellular functions of target organisms. "These are really elegant studies, down to the molecular level," said John Pritchard, chief of the NIEHS laboratory of pharmacology and chemistry, during a roundtable discussion. "I think the challenge for us is to take this kind of information and . . . use this increased detailed understanding to develop either therapeutic strategies or increased-sensitivity assays--specific assays for specific toxins--to identify the particular players that are involved in environmental health issues."
Dinophysis fortii
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Participants pinpointed a variety of theories for the worldwide surge in hazardous aquatic pathogens and harmful algal blooms. Keynote speaker Rita Colwell, president of the University of Maryland Biotechnology Institute in College Park and former president of the American Association for the Advancement of Science, outlined the connection between increases in global temperature and unusual El NiƱo events and the opportunistic growth and spread of organisms such as
Vibrio cholerae
, which thrive on ubiquitous zooplankton. Colwell also examined the role of ocean currents and seagoing vessels that collect and discharge ballast water in transporting these organisms.
Other culprits in increased reported algal and dinoflagellate activity include coastal eutrophication caused by polluted runoff and rainfall, and possibly even an escalation in reporting of events due to new methods of detection and scrutiny. Baden stressed, however, that each organism has different ecological requirements, and general statements about the relationship of pollution to algal and dinoflagellate activity cannot be made.
Chattonella antigua
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A consensus of the workshop was that the scientific community has made great strides in understanding the acute effects of marine and freshwater toxins on human and animal species at the molecular level. But more research is needed to examine chronic effects such as accumulation of toxins and immunosuppression that may result from long-term exposure. More epidemiological studies would help, said Baden, citing the link between exposure to brevetoxins from algal blooms and respiratory effects. "If red tides occur every year on the west coast of Florida, which they do, is there chronic respiratory disease of some kind?" Baden said. "Well, the populations of west coast areas are skewed towards retirement-age individuals, so many are already predisposed to respiratory disease. Because such people are, in a sense, compromised before they ever even inhale, research into synergistic effects between these environmental chemicals and human health is a pressing issue."
Gymnodinium mikimotoi
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Researchers at the workshop also expressed the need for a better understanding of the causative role of these organisms in their natural environment, better monitoring of environmental indicators prior to epidemics and epizootics, and the development of detection methods that have inexpensive, reliable field applications. Better cooperation between scientists and communication with the public were also deemed essential. "There needs to be more dialogue between scientists, policy makers, and public health officials to achieve a better linkage between research priorities and the development process of public policy," said NIEHS director Kenneth Olden in a challenge to the audience, which included public health officials from East Coast states and the federal government, food safety specialists, physicians, policy makers, environmentalists, and educators.
Baden said the meeting helped advance an accord. "The beauty," he said, "was that we brought people together at this particular conference that are interested in the ocean and now are wading in to the shore. We got the people on land who are interested in human health issues walking down to the shore. And today they shook hands at the beachhead."
Carla Burgess
Photo credit: Daniel Baden
Last Update: October 21, 1997