The UC Berkeley scientists also showed that male leopard frogs raised in laboratory tanks contaminated with atrazine develop egg cells in their testes and essentially turn into hermaphrodites. These sexual abnormalities were observed at atrazine levels as low as 0.1 parts per billion (ppb), 30 times lower than the current allowable limit for atrazine in drinking water set by the Environmental Protection Agency.
These findings, added to earlier evidence that atrazine demasculinizes two other species of frog, suggest that the herbicide could be a factor in the decline of frogs and other amphibians in the United States and around the world, the authors say. Atrazine has been used on crops since 1956 and currently is the most widely used herbicide in the nation.
"These studies clearly indicate that atrazine is detrimental to amphibians," said study author Tyrone Hayes, associate professor of integrative biology at UC Berkeley.
At the least, he said, atrazine is altering amphibian populations in large areas of the United States. His field studies show that frogs seem to adapt, since leopard frogs (Rana pipiens) - the most common frogs in the Midwest - are often abundant in some of the corn growing areas region where atrazine is used most. Atrazine may feminize male tadpoles and turn them into female frogs, he said, or it may render some males infertile. Alternatively, atrazine may favor tadpoles that delay sexual differentiation until after they've turned into frogs and leave the contaminated water.
"Atrazine is potentially destroying biodiversity," said Hayes, now engaged in studies to determine the ultimate fate of these feminized tadpoles. "In my opinion, this is an unacceptable risk."
Hayes and his colleagues sampled leopard frog tadpoles in eight separate ponds, ditches, rivers and streams in the Midwest during the summer of 2001 and found feminized male frogs at every site with measurable levels of atrazine. The current laboratory detection limit is 0.1 parts per billion (ppb).
The sites were scattered through the Corn Belt and beyond, including in Utah, Wyoming, Nebraska and near the Iowa-Illinois border. Several ponds chosen as controls because they are in nonagricultural areas also had measurable levels of atrazine and feminized frogs, while only one, in Utah, had neither detectable atrazine nor affected frogs.
The site with the highest concentration of feminized frogs was along the North Platte River in Wyoming. There, 92 percent of male frogs showed sex reversal. This area of Wyoming reports little use of atrazine, but the river is fed by streams that carry runoff from Colorado farms, which do use significant amounts of the herbicide.
Earlier this year, Hayes and his colleagues reported that a common laboratory frog, the African clawed frog (Xenopus laevis), became demasculinized when raised in lab tanks with concentrations of atrazine of 0.1 ppb or higher. The current allowable limit set by the Environmental Protection Agency for atrazine in drinking water is 3 ppb, and the proposed chronic exposure limit for aquatic life is 12 ppb.
The new experiment, reported in the Oct. 31 issue of Nature, is a repeat of these experiments using the leopard frog . The new report also contains a summary of Hayes' survey of Midwestern frog ponds.
A field study by another group showed similar gonadal abnormalities in atrazine-exposed northern cricket frogs (Acris crepitans).
Just as in Hayes' earlier experiment, the male tadpoles developed female characteristics in their sex organs, or gonads. Up to 29 percent of males developed female egg cells (oocytes) in their testes, becoming hermaphrodites. In one experiment, more than a third of the male frogs exposed to 0.1 ppb atrazine showed under-developed testes.
Details of the experiment are reported in the November issue of the journal Environmental Health Perspectives (EHP).
"The testes in these male frogs are obviously not functioning normally, because if they were, egg cells would not be able to grow in them," Hayes said. "Some testes are so invaded by ovary cells it looks like they are converted, and technically, they could be considered ovaries."
Hayes suspects that atrazine boosts the activity of an enzyme, aromatase, that converts male sex hormones, or androgens, to female hormones, or estrogens. The lowered androgens and increased estrogens allow egg cells to grow within the testes, which is normally impossible. Atrazine's effects on aromatase have been demonstrated in fish, reptiles and mammals, but not yet in amphibians.
Atrazine is so widespread that it can be found far from agricultural areas and even in rainwater and snow. At one site in Nebraska, Hayes found that rain and tap water contained enough atrazine to disrupt normal male development in amphibians.
"The current data raise concern about the effects of atrazine on amphibians in general and the potential role of atrazine and other endocrine-disrupting pesticides in amphibian declines," the authors wrote in their EHP article.
The leopard frog studies are supported by various private grants and by the National Science Foundation. Hayes' coauthors are former or current undergraduate students - Kelly Haston, Mable Tsui and Cathryn Haeffele - postdoctoral fellow Anhthu Hoang and research associate Aaron Vonk.
Hayes is a member of UC Berkeley's Health Sciences Initiative, a broad-based effort to bring the physical and biological sciences together to tackle health problems of the 21st century.