Hayes, T, K Haston, M Tsui, A Hoang, C Haeffele and A Vonk. 2002. Feminization of male frogs in the wild. Nature 419: 895 - 896. Hayes, T, K Haston, M Tsui, A Hoang, C Haeffele and A Vonk. 2003. Atrazine-Induced Hermaphroditism at 0.1 PPB in American Leopard Frogs (Rana pipiens): Laboratory and Field Evidence. Environmental Health Perspectives 111: 111:568-575. Press
coverage In April 2002, Tyrone Hayes and his coworkers published astonishing findings showing that atrazine causes tadpoles of the classic "laboratory rat" of the frog world, the African clawed toad Xenopus laevus, to become hermaphroditic as adults. The most remarkable aspect of this finding was the extraordinary low levels at which the impact occurs, 0.1 parts per billion, an exposure level not only astoundingly low but also one that is exceeded almost ubiquitously in regions where atrazine is used. Indeed, as Hayes et al. reported in that paper, rainwater away from regions of atrazine use can sometimes carry 1 ppb... ten times the amount they found sufficient to cause hermaphroditism in 20% of exposed animals. With these two new papers, Hayes et al. answer affirmatively two important questions raised by their earlier work: are other species of amphibians similarly sensitive to atrazine, and can the effects be observed in wild populations of frogs where atrazine is used? Their new laboratory experiments demonstrate conclusively that atrazine causes hermaphroditism in leopard frogs also. And their field survey transecting the US from Iowa to Utah, reveals hermaphroditic leopard frogs across a broad swath of the natural distribution of this species, but only in areas where atrazine can be detected (which is almost all places they surveyed). What did they do? Hayes et al. conducted a laboratory experiment with leopard frogs and then carried out a field survey: The lab experiment: Hayes et al. exposed leopard frog tadpoles to atrazine at two different levels in their water, 0.1 parts per billion and 25 ppb, and compared the percentage of animals that matured as hermaphrodites in these treatments vs. a control group that was not exposed to atrazine. Each exposure group contained 30 animals. The experiment was repeated completely three separate times. The field survey: Hayes's field team sampled young leopard frogs that had matured earlier that spring at a series of eight sites across the US mid-west, from Iowa to Utah (see map below). The sites were chosen based on USGS data on atrazine sales so that the survey included areas of high and no or little atrazine use, while also being within the natural range of the leopard frog. At each site, they collected 100 animals, which they preserved and took back to their laboratory at the Univ. of California, Berkeley, for dissection.
The figure overlays a depiction of the natural distribution of leopard frogs (light pink) within the US upon a map indicating the amount of atrazine use (kilograms per square kilometer) based on within-county atrazine sales. It also shows the location of 8 sites where Hayes et al. collected frogs and water for analysis (numbers 1-8) To avoid expectation biases, Hayes' team used a double-blind system that gave no clue as to the origin of the animal while before all histological analysis was completed. At each site they also took water samples, which they sent to two different laboratories for analysis to determine levels of atrazine and a series of other pesticides reported to be used at or near the collection sites. The labs were not told of the samples' origins. Limits of atrazine detection of the labs analyses was 0.1 ppb. What did they find? The laboratory experiment revealed that male leopard frogs are extremely sensitive to atrazine exposure during metamorphosis from tadpole to adult. Many of the males exposed to both very low and low levels of atrazine (0.1 and 25 ppb) had abnormalities in their reproductive tracts. For example, 29% of the 0.1 ppb-treated animals and 8% of the animals treated with 25 ppb displayed varying degrees of sex-reversal. No cases of sex-reversal were seen in the controls. While only one individual control animal out of all examined had under-developed testes, 36% and 12% of the males treated with 0.1 and 25 ppb atrazine, respectively, showed under-developed testes (gonadal dysgenesis). This was seen in only 2 control animals. The exposed males that had undergone almost complete sex-reversal had gonads almost completely filled with oocytes. This was never seen in controls.
The field surveys found reproductive abnormalities in wild leopard frogs comparable to those seen in the experimentally-treated frogs, except at the one site where measured atrazine levels were below 0.2 ppb, where no abnormalities were found.
Hayes et al. found no simple relationship between atrazine levels and the likelihood of gonadal abnormalities, except that abnormalities occurred everywhere except the site with the lowest atrazine leve (site 1). The site with the highest level of measured atrazine (site 6) did not have the greatest proportion of abnormalities. These inconsistencies suggest several possible interpretations. Most importantly, water sampling and frog collection occurred a month or more after tadpole exposure to atrazine... the scientists had to wait until after metamorphosis to find maturing individuals for dissection. There is no way using Hayes's existing data to work backward from the samples collected to determine actual exposure levels prior to metamorphosis. The one site with extremely low atrazine levels in an area with almost no atrazine use is the one site with no gonadal abnormalities. All other sites had atrazine levels well above the range shown by the experiments to induce abnormalities. All of these sites had male frogs with gonadal abnormalities. What does it mean? The lab studies confirm that male gonadal development in leopard frogs can be disrupted by extremely low levels of atrazine. The field studies reveal widespread gonadal abnormalities in regions where atrazine contamination is within the range shown by the laboratory studies to disrupt development. This does not prove with certainty that the effects observed in wild leopard frog populations are caused by atrazine, but it is strong circumstantial evidence. The case will be strengthened--or refuted--by further studies that measure atrazine levels prior to metamorphosis, analogous to the experimental design. The fact that atrazine contamination is so widespread complicates the practicalities of executing this design. The case is also complicated by the possibility that regular exposure to atrazine over many frog generations may have led to development of pesticide resistance. This hypothesis would predict that populations in the regions of most prolonged and intense atrazine use should have lower proportions of gonadal abnormalities than those with lesser and shorter traditions of use. If atrazine use histories can be established for different sites, this could be approached experimentally through laboratory experiments with animals from those sites. Hayes' work raises significant but unanswered questions about the role of atrazine in contributing to frog population declines. As they describe, the timing of atrazine use in crops coincides with the period of tadpole growth revealed by the experiments to be sensitive to atrazine exposure. The sites where tadpoles mature, moreover, are often precisely in the drainage wetlands that are the principal recipients of atrazine run-off from crops. Perhaps paradoxically, Hayes et al. note that juvenile frogs were abundant at the study sites. Clearly some reproduction is occurring. One possibility, mentioned above, is that pesticide resistance has evolved. Hayes et al. conclude:
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