In-lab, low-dose atrazine frog study suggests similar field effects

Low part-per-billion (ppb) doses of atrazine disrupt the sexual development of frogs in laboratory experiments, with gonadal alterations starting at 0.1 ppb, say developmental endocrinologist Tyrone Hayes, of the University of California, Berkeley, and colleagues. The observed effects occur at atrazine concentrations frequently found in the environment, and such exposures may account for gonadal abnormalities observed in frogs in the wild, Hayes says.

The findings, presented at the Society of Environmental Toxicology and Chemistry annual meeting in Baltimore last November, suggest that the herbicide apparently modifies the steroid hormones balance in frogs at a sensitive time in their development.

The study performed by Hayes’s group involved dosing African clawed frog larvae (Xenopus laevis) from hatching until metamorphosis. At doses as low as 1 ppb, they observed laryngeal muscle size reduction in 80% of exposed males. Twenty percent of dosed males developed into hermaphrodites having both testes and ovaries. Atrazine exposure at these levels did not affect mortality, developmental rates, or time to metamorphosis in the experiments.

Hayes proposes that atrazine affects frog sexual development because it increases levels of aromatase, an enzyme that converts testosterone to estrogen, and results in partial male frog feminization. The affected males have high levels of aromatase in their gonadal tissues and have blood plasma testosterone levels that are unusually low—comparable to levels typically found in female frogs. As mentioned in a recent article appearing in this journal (Environ. Sci. Technol. 2000, 34 (19), 415A), the suggested mechanism has been investigated previously, in 1997 by Louis Guillette and co-workers, who found that atrazine induced aromatase activity in male hatchling alligators, and in 2000 by Thomas Sanderson and co-workers, who found that atrazine could induce aromatase activity and modify hormonal balance in cell line in vitro experiments.

“This is an intriguing set of observations,” says EPA scientist Gary Ankley, who heads the toxic effects research team at EPA’s Duluth, Minn., research center. “The effects observed by Hayes are more subtle than the sort of endpoints, such as gross malformations (multiple or missing arms and legs), that we usually look for in the field. As a result, it’s quite possible that these effects could have been missed by other researchers,” Ankley says.

Because his laboratory experiments indicate that X. laevis males are vulnerable to atrazine exposure when they are larvae, Hayes compared the time when wild frogs spawn with when atrazine levels in surface waters are highest and found that the two coincide—surface water concentrations are highest at the time of the first heavy spring rains, which is also the time when frogs spawn, suggesting that wild frogs could at that time be getting a sufficient dose delivered to cause adverse effects.

Last summer, Hayes and co-workers traversed the U.S. West and Midwest and found intersex wild frogs at sites associated with atrazine contamination in Iowa, Nebraska, and Illinois. At these sites, they found male Leopard frogs (Rana pipiens), whose testes contained oocytes, eggs that should only be found in female frogs. They did not find intersex frogs in Utah or Wyoming.

Hayes doesn’t know whether the intersex effects in the wild are caused by exposure to atrazine or another contaminant. “The intersexes are correlated with atrazine use patterns, regardless of habitat,” he says. Hayes’ group is currently working on a controlled lab study to determine whether atrazine produces this effect in R. pipiens. “We do know that it is not a normal part of development for the species, as animals in the lab do not develop this way,” he observes.

Atrazine (2-chloro-4-ethylamino-6-isopropylamine-1,2,5-triazine), with an annual usage of 60 million pounds in 1993, is the most commonly used herbicide in the United States and is also used in more than 80 countries. Despite its widespread presence in U.S. surface waters, a 1996 ecological risk assessment led by Keith Solomon, director of the Centre for Toxicology,University of Guelph, Ontario, estimated that the herbicide does not pose a risk to the aquatic environment (Environ. Sci. Technol. 1996, 30 (5), 110A). Atrazine producer Syngenta, formed by the merger of Novartis Agribusiness and Zeneca Agrochemicals has, since 1997, been sponsoring an independent, multimillion-dollar research program into ecological endocrine effects, and a special review of atrazine begun by EPA in 1994 because of concerns about the health risks to people and the environment, is expected to conclude soon. —REBECCA RENNER