Great Lakes Article:

National Academy of Sciences reports on atrazine as the cause of many frog mutations

05/07/2002

Hayes, TB, A Collins, M Lee, M Mendoza, N Noriega, AA Stuart, and A Vonk. 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide, atrazine, at low ecologically relevant doses. Proceedings of the National Academy of Sciences (US) 99:5476-5480.

Hayes et al. demonstrate that at exposure levels far beneath those found in the lakes, rivers, streams, drinking water and even rainwater, atrazine causes frogs to mature with multiple, mixed gonads and to become demasculinized. These effects occurred at exposure levels 10,000 - 30,000 times beneath levels previously identified as non-toxic to frogs.

Atrazine's impact on frogs appears to be caused by this herbicide's ability to promote the conversion of testosterone to estrogen via activity of the enzyme aromatase. This mechanism is found not just in frogs, but other vertebrates as well, including mammals. Their study raises important, and as yet unanswered, questions about the possible role of atrazine in world-wide frog population declines, and about the potential for atrazine to affect human health via the same enzymatic mechanism.

Atrazine is one of the most widely and heavily used agricultural chemicals in the world. Each year, American farmers alone apply 60 million pounds to U.S. farmland to prevent growth of weeds competing with corn and other crops. The US EPA considers short-term exposure to 200 ppb acceptable for people and allows 3 ppb atrazine in drinking water. In rainwater in regions where atrazine is applied, atrazine contamination has been measured up to 40 ppb. It has even been found at 1 ppb in rainwater in regions where atrazine is not used. Hence it is virtually certain that frogs (and people) living in the real world are regularly exposed to atrazine at levels many times greater than what Hayes et al. report is sufficient to harm frogs.

What did they do? Hayes et al. used two different experimental designs to study the effect of atrazine on the laboratory rat of the amphibian world, the African Clawed Toad Xenopus laevus. In both designs, Hayes took great care to make sure that the scientists measuring the exposed animals were unaware of which treatment they had received. Only after all the measurements were taken did a coding system reveal what treatment each animal experienced.

The sample sizes used in the experiments were quite large (90 animals per treatment); these experiments were repeated four times; and experiments with atrazine at these low levels were replicated 51 times in Hayes' lab with similar results.

Experiment 1: expose developing tadpoles to different levels of atrazine and examine individuals for morphological effects after metamorphisis. The tadpoles were exposed to concentrations of atrazine from 0.01 ppb to 25 ppb.

Experiment 2: expose adults directly to 25 ppb atrazine and measure testosterone and estrogen levels. Adults were used because they found it not possible to obtain enough serum from tadpoles to do the hormonal assays.

What did they find?
Experiment 1: When exposed as tadpoles to as little as 0.1 part per billion atrazine, individuals mature with hermaphroditic deformities in the reproductive tract. Normal adult frogs have two gonads, either two testes if male or two ovaries if female. Between 16% and 20% of animals treated with atrazine at 0.1 ppb and above had either two many gonads and/or mixtures of ovaries and testes. This is not normal. Hayes et al. comment that they had never seen these effects in 6 years of study of Xenopus involving over 10,000 animals.

The figures below, from Hayes et al. shows the deformed reproductive tract of one hermaphroditic frog. This particular individual has three testes and three ovaries. The microscopic photograph on the left shows the entire complex of the animals gonads and kidneys. The microphotograph on the right shows cross-sections through gonadal material at places specified by the arrows on the left. T= testis; O= ovary.

When tadpoles were exposed to atrazine at 1 ppb and above, Hayes et al. noted a demasculinization of the adults secondary sexual characteristics, specifically a reduction in the size of the males' larynges (vocal chords). Normally male larynges are larger than female. The atrazine-treated males had larynges that were intermediate in size between normal male and normal female larynges. There was no effect on the female larynges size.

Experiment 2: Adult males exposed to 25 ppb atrazine showed a 10-fold decrease in testosterone compared to controls. This effect is highly significant statistically. Treated male testosterone levels were indistinguishable from control females.   from Hayes et al.

What does it mean?
This research eliminates any doubt about atrazine as an endocrine disruptor at extraordinarily low levels of exposure. It puts endocrine disruption high onto the list of plausible factors contributing to frog population declines. And because of the apparent mechanism of action, via enhancement of aromatase conversion of testosterone to estrogen, it raises important concerns about atrazine disrupting the hormonal control of development in other organisms, including humans.

As Hayes et al. summarize in their paper, atrazine contamination is extremely widespread , including, via atmospheric transport, in rainwater in regions where atrazine is not used for agricultural crops. The level of atrazine sufficient to cause reproductive abnormalities in Xenopus—hermaphroditism—is 1/30th of the level allowed by the US EPA in drinking water:

Frogs in many places in the world are undoubtedly exposed to atrazine at these levels. It remains to be determined whether other frog species are as sensitive to atrazine as is Xenopus, and whether the effects are similar. Hayes' work should reinvigorate efforts to understand the contribution of chemical exposure to frog declines. While research has strongly implicated infection by a chytrid fungus as an important factor driving frog extinctions, other causes, including contaminants and the introduction of exotic species, remain plausible contributors to what is most likely a multi-factoral process. And given the ability of some contaminants to reduce disease resistance, it is not implausible to hypothesize that contaminants are involved in the rapid pace at which amphibians have succumbed to the fungal infection. No research has assessed the impact of low-level atrazine exposure on the development of frog immune competency.

This research by Hayes et al. also points out the crucial need to carry out experiments assessing toxicological impacts at environmentally-relevant levels. Prior work had seemingly dismissed atrazine as an endocrine disruptor affecting frogs, but the research used exposures that were 10,000 to 30,000 times higher than the dosage found by Hayes et al. to produce developmental disruption:

"Reported teratogenesis,growth inhibition, and mortality in amphibians in response to atrazine were not considered environmental concerns because
of the high doses required to produce these effects. Effects in the current study, however, occurred at levels 10,000 times lower than the dose required to produce effects in amphibians in these previous studies. Allran and Karasov (2001) reached the conclusion that atrazine was probably not a significant factor in amphibian declines based on their studies of toxicity, deformities, and effects on feeding and ventilation in leopard frogs that did not produce noticeable effects below 3 ppm. The current data show that negative effects on sex differentiation occur at doses 30,000 times lower than effective doses reported by Allran and Karasov."