The bountiful fields of the U.S. are awash in atrazine. Some 36 million kilograms of the odorless, white powder are applied on farms to control grassy weeds. Some 225,000 kilograms of the herbicide fall with the rain each year, sometimes up to 1,000 kilometers from the source. All that atrazine may be having another effect: turning male frogs female.



As described in the March 1 Proceedings of the National Academies of Sciences, biologist Tyrone Hayes of the University of California, Berkeley, and his colleagues exposed 40 African clawed frogs (Xenopus laevis) to 2.5 parts per billion (ppb) of atrazine in a water solution continuously for three years—a level below the three ppb allowed in drinking water by the U.S. Environmental Protection Agency (EPA). As a result, 30 of the frogs were chemically castrated, incapable of reproducing, among other impacts. And four of the treated frogs actually turned female, going so far as to mate with other males and produce viable eggs despite being genetically male.



Whereas another four of the treated frogs apparently resisted atrazine's effects, the rest "lacked male reproductive behavior, had reduced male features, and severely reduced sperm and low fertility," Hayes says. The key may be aromatase, a protein that spurs the production of the female hormone estrogen, causing originally male gonads to become ovaries and whose production is spurred by atrazine. Plus, the researchers used frogs bearing only the ZZ sex chromosomes of male African clawed frogs. In previous studies "if we got hermaphrodites, there was no way to know if they were males with ovaries or females with testes," Hayes says. "By using all ZZ males we were assured that any hermaphrodites or females were indeed sex-reversed males." Frogs follow the ZZ (male), ZW (female) sex determination scheme rather than the more familiar XX (female), XY (male) pattern in humans.



Hayes has a long history studying atrazine, starting in the 1990s with research funded by its maker, now known as Syngenta, that first raised the prospect that the herbicide might be a interfering with the natural hormones of animals, including humans. A barrage of studies on such endocrine disruption have followed—some confirming that amphibians such as frogs are suffering from an atrazine onslaught, others finding no effect, and some even finding evidence of reduced sperm count in men from agricultural regions of the U.S.



"The proposed findings are incredibly important," says biologist Jason Rohr of the University of South Florida in Tampa, who has also studied atrazine but was not involved in this research.



This study is not likely to be the last word in the controversy, however. Critics, such as biologist Werner Kloas of Humboldt University in Berlin, charge that samples may have been contaminated by endocrine disruptors like bisphenol A (BPA) leaching from plastic containers or introduced during screening. He also questions the single exposure level, lack of measurement of female hormone levels in the affected frogs, and the use of ethanol in the water solution "as they claim atrazine is easily water soluble and it is better to avoid any solvent if feasible."



This study also went through an unorthodox form of peer review, in which the paper was edited for publication by another professor at U.C. Berkeley, rather than an unaffiliated scientific reviewer as is more common. "Such a paper should not have become accepted for publication," Kloas charges.



For his part, Kloas in the past reviewed atrazine's effects for Syngenta and found no impact on African clawed frogs at up to 100 micrograms per liter of atrazine and no genetic impact at 25 micrograms per liter. He says he currently has "no running experiment with Syngenta or any further chemical company."



In their native habitats, African clawed frogs do not appear to be suffering from the herbicide. "Atrazine has been used widely in South Africa for the past 45 years and our studies showed that Xenopus are doing equally fine in agricultural and nonagricultural areas," says zoologist Louis du Preez of North-West University in South Africa. "If atrazine had these adverse effects on Xenopus in the wild, surely we would have picked it up by now."



Nevertheless, the countries of the European Union have banned atrazine because of its ability to contaminate water. "I personally prefer our European habit to use the precautionary principle concerning environmental chemicals to phase out persistent compounds," Kloas says.



And Rohr's research has found that atrazine could exert indirect effects: At levels found in water throughout the U.S., it increases northern leopard frogs' susceptibility to potentially deadly parasites. Herbicides like atrazine are now found in 57 percent of U.S. streams, according to the U.S. Geological Survey.



The EPA, for its part, announced yet another review of the herbicide last October because of human health concerns, after declaring the chemical treatment safe in 2006. Ultimately, atrazine may be silencing the amphibian calls of the wild: "It leads to them not croaking," Hayes says, noting that atrazine may be contributing to the global decline in amphibians, along with chytrid fungus. "Atrazine increases aromatase and/or estrogen production in zebra fish, goldfish, caimans, alligators, turtles, quail and rats. So this is not just a frog problem."