Popular Weed Killer Demasculinizes Frogs, Disrupts Their Sexual Development

In an article in the April 16 issue of Proceedings of the National Academy of Sciences, University of California, Berkeley, developmental endocrinologist Tyrone B. Hayes, associate professor of integrative biology, and his colleagues report that atrazine at levels often found in the environment demasculinizes tadpoles and turns them into hermaphrodites - creatures with both male and female sexual characteristics. The herbicide also lowers levels of the male hormone testosterone in sexually mature male frogs by a factor of 10, to levels lower than those in normal female frogs.

As Hayes later discovered, many atrazine-contaminated ponds in the Midwest contain native leopard frogs with the same abnormalities.

"Atrazine-exposed frogs don't have normal reproductive systems," he said. "The males have ovaries in their testes and much smaller vocal organs," which are essential in calling potential mates.

It is unclear whether these abnormalities lead to reduced fertility. Hayes now is trying to determine how the abnormalities affect the frogs' ability to produce offspring.

"The use of atrazine in the environment is basically an uncontrolled experiment - there seems to be no atrazine-free environment," Hayes said. "Because it is so widespread, aquatic environments are at risk."

Because the herbicide has been in use for 40 years in some 80 countries, its effect on sexual development in male frogs could be one of many factors in the global decline of amphibians, he added.

The findings come at a time when the EPA is re-evaluating allowable levels of atrazine in drinking water, which stand today at 3 parts per billion (ppb), and has drafted new criteria for the protection of aquatic life, limiting four-day average exposures to 12 ppb. Hayes found hermaphroditism in frogs at levels as low as 0.1 ppb. Even with today's limits, levels of 40 ppb atrazine have been measured in rain and spring water in parts of the Midwest, while atrazine in agricultural runoff can be present at several parts per million.

The herbicide also contaminates drinking water supplies in many communities in the Midwest, leading some environmental groups to voice concern about its effect on children, infants and the fetus. France, Germany, Italy, Sweden and Norway are among countries that have banned the use of atrazine.

"This is very important and elegant work," said Theo Colborn, PhD, a senior scientist at the World Wildlife Fund and an internationally recognized expert on endocrine disrupting chemicals. "Tyrone's work demonstrates the need to do research on the safety of chemicals in the field where the animals live and at the levels to which they are exposed. The changes he found in the gonads were not discovered with the traditional high-dose atrazine experiments used in the past. In addition, microscopic examination of the internal organs of the frogs is required to detect the hidden effects from low-dose exposure."

To date, atrazine's effects on mammals and amphibians have been tested only at large doses, not at doses commonly found in the environment.

In their journal article, Hayes and his colleagues write, "The effective doses in the current study ... demonstrate the sensitivity of amphibians relative to other taxa, validate the use of amphibians as sensitive environmental monitors/sentinels, and raise real concern for amphibians in the wild."

Hayes doubts that atrazine has such severe effects on humans, because the herbicide does not accumulate in tissue and humans don't spend their lives in water like frogs do. Nevertheless, the effects of atrazine on frogs could be a sign that the herbicide is subtly affecting human sex hormones, too, interfering with androgens, such as testosterone, that control male sex characteristics.

Some studies in cell culture point to a possible biochemical explanation for the observed effects on amphibian sex organs. John P. Giesy, a professor of zoology at Michigan State University in East Lansing, and his colleagues found last year that, at large doses, atrazine ups production of the enzyme aromatase, which converts androgen hormones to estrogen hormones. Extrapolating these results from mammalian cells to amphibians, Hayes argues that atrazine could feminize male frogs by promoting the conversion of male hormones to female hormones. The lowered androgens would interfere with voice box development, while increased estrogens would promote ovaries within the testes.

More than 60 million pounds of the herbicide were applied last year in the United States alone. Manufacturer Syngenta estimates that farmers use the herbicide to control weeds on about two-thirds of all U.S. corn and sorghum acreage. On average, it improves corn yield by slightly more than four percent. The compound is generally considered safe, however, because it quickly decomposes in the environment and, being water soluble, is quickly excreted from the body.

Aquatic life, however, swim and breed in atrazine-contaminated field runoff. Though previous studies showed deformities and abnormalities in adult amphibians only at very high doses, no one had looked in detail at hormone levels in frogs or at effects on tadpoles, the larval stage of frogs.

Prodded by the EPA, Syngenta approached Hayes, an expert on amphibian hormones, to find out if atrazine disrupts sex hormones in amphibians. He has developed several very sensitive assays to detect chemicals that affect hormones, including a test for estrogen-like chemicals that might induce human breast cancer.

Though Hayes initially received funds from Syngenta for the studies, all the current published studies were conducted independent of Syngenta.

For his laboratory tests, he used the African clawed frog, Xenopus laevis, a popular research subject that, like many frogs, is very sensitive to hormones that mimic the effect of their own sex hormones. If raised in a pond with estrogen, for example, all Xenopus tadpoles turn into females. In the presence of androgens, frogs grow larger voice boxes, or larynges.

In laboratory experiments at various concentrations of atrazine, using two separate populations of frogs raised in three separate tanks - experiments replicated 51 times - they found atrazine to affect the sexual development of frogs at concentrations of 0.1 ppb and higher. That is 30 times lower than the allowable limit of 3 ppb in drinking water and 120 times lower than the proposed chronic exposure limit for aquatic life, 12 ppb.

At these concentrations, as many as 16 percent of the animals had more than the normal numbers of gonads - including one animal with six testes - or had both male and female organs (testes and ovaries). No control animal had such abnormalities.

Also, while normal males at metamorphosis have larger vocal organs than females, the organs of more than 80 percent of males exposed to 1 ppb or more of atrazine were smaller than average.

Sexually mature males showed a 10-fold decrease in testosterone levels, bringing them below levels found in normal females. This suggests that atrazine acts by disrupting the synthesis of sex hormones, which could also explain the smaller larynges and abnormal gonads.

"... the current data raise new concerns for amphibians with regard to atrazine," the researchers wrote in their paper. "If such effects occur in the wild, exposed animals would suffer impaired reproductive function."

In fact, Hayes and his colleagues subsequently conducted a reconnaissance of atrazine-contaminated ponds in the Midwest to see if such reproductive abnormalities occur in frogs in the wild. They turned up many native leopard frogs (Rana pipiens) with similar problems, and are now testing captured animals to determine whether these changes are due to atrazine.

"Atrazine is obviously affecting frogs," Hayes said. "We have shown serious effects on their sexual development. We need to ask the questions, 'What are the environmental costs of using atrazine? What diversity have we lost?'"

Hayes conducted the study with the help of recent PhD recipient Nigel Noriega, research associate Aaron Vonk, and former or current undergraduate students Atif Collins, Melissa Lee, Magdelena Mendoz and A. Ali Stuart, all of whom are listed as coauthors of the paper.

The studies were supported by the National Science Foundation.