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Frogs: Clues to how weed killer may feminize males

Study turns up new perturbations in multiple hormone systems

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Atrazine, a widely used agricultural herbicide, not only can alter hormone levels in developing frogs, but also perturb their physical development — and lead to an excess number of females, researchers report. Their new findings may help explain observations reported by a number of other research groups that at least in frogs, fairly low concentrations of atrazine can induce a feminization — or demasculinization.

Industry scientists have charged that lab studies with non-native frogs are poor models of what to expect in the environment. (Among such studies is one conducted by Berkeley scientists and reported yesterday in a paper that will appear online this week in the Proceedings of the National Academy of Sciences.)

With such criticisms in mind, a Canadian team did its best to model atrazine’s likely impacts in the wild. They now report a host of new subtle and potentially deleterious impacts. The atrazine concentrations triggering these changes — up to 1.8 micrograms per liter of water (also referred to as parts per billion) — is a value that has been measured in surface waters. It’s also well below Canada’s recommended drinking water limit of 5 ppb for this pollutant.

The findings appear in Environmental Health Perspectives (published online, ahead of print).

Valérie Langlois, Amanda Carew and Vance Trudeau of the University of Ottawa teamed up with colleagues at Environment Canada and Health Canada (two federal agencies) to study atrazine’s effects on young northern leopard frogs (Rana pipiens). They collected fertilized eggs from local ponds and incubated them until they hatched. Then they transplanted little tadpoles into mesocosms — 378-liter polyethylene tanks of water that were left outdoors to weather the elements.

The biologists supplied each pool with 150 pollywogs, together with leaves, twigs, and other debris to simulate a natural pond. They also threw in tadpole snacks — water fleas (Daphnia magna) acquired from a nearby creek.

Clean groundwater was added to one mesocosm. The researchers doctored with atrazine the groundwater added to two more — either with 0.1 or 1.8 µg/l of the chemical.

Rain water entering the tanks was analyzed to see if it added any additional atrazine (it didn’t) and supplemental atrazine was added to the herbicide-treated mesocosms a couple times to keep treatment concentrations in them fairly uniform over several months.

Even at the relatively low and environmentally relevant concentrations of atrazine present in the higher-treatment group, a number of fairly surprising changes occurred, Trudeau notes. For instance, there was an apparent 20 percent increase in the number of females within this group. Ordinarily the sex ratio averages roughly 50:50. In fact, there was a surfeit of apparent males in all of the mesocosms — except the one with the higher concentration of atrazine.

The supposition, Trudeau says, is that some males in the 1.8 µg/l treatment pool were in fact genetic males that merely gave the outward appearance of being female. That’s what the Berkeley group found in some new studies, including one reported yesterday (and based on exposures in the same ballpark as those in the Ottawa study).

Unfortunately, Trudeau notes, easy genetic fingerprinting of frogs — at least from a gender point of view — remains unavailable. So his group had no way of confirming the number of true females versus sex-reversed males (bearing the internal and external appearance of females).

Animals in the higher-atrazine exposure group also developed a 2.5-fold increase in their brains of receptors for estrogen — a change that could render these frogs more susceptible to the effects of their own internally produced estrogen or to effects of any of the hormone’s environmental mimics.

Aggravating the animals' hormonal perturbations, notes Caren Helbing of the University of Victoria in British Columbia, was an observed change in the activity of a liver enzyme responsible for converting testosterone to another androgen, 5-beta-dihyrdotestosterone. Although the role of this enzyme, called 5-beta reductase, is under investigation, the Ottawa scientists show females produce more of this enzyme than males, she explains. This obvious sex difference was abolished in atrazine-exposed animals. “So when you compare girl frogs to boy frogs, you can normally tell them apart by how much of this enzyme is around,” Helbing notes. But the Ottawa team now shows that "atrazine is removing another thing that differentiates a boy [frog] from a girl.”

Finally, animals in the atrazine treatment groups were slower to metamorphose. After a while, the researchers just gave up waiting for the hold-outs to do so and ended their experiment. Around 90 percent of the animals raised in clean water successfully transformed from pollywogs to hopping frogs. In contrast, no more than half from even the lowest atrazine-exposure group did.

"So even our low dose is having a clear effect on metamorphic success," Trudeau points out. "And I would consider that quite important, because if you have 50 percent less metamorphosing, you're likely to have more [young] picked off [by predators]."

One explanation for reduced metamorphic success, Trudeau notes, is a change in thyroid hormone levels that his team witnessed in atrazine-exposed animals. This change is one that particularly intrigued Helbing. “Thyroid hormone is what causes the legs to form and a tadpole to metamorphose into a frog,” she explains. If thyroid hormone values aren’t normal, metamorphosis may not occur on time if at all.

Helbing also expressed concern that what the Ottawa group saw in frogs may indicate risks to other species as well. “The ways that our bodies — frogs and people — respond to hormone signals is very, very similar,“ she says. So something that changes levels of estrogen receptors or alters thyroid hormone levels in frogs may exhibit a related change in people. At a minimum, she argues, such findings “are certainly a wakeup call that we'd better look.”

The new Ottawa study also points to another issue — the value of getting out of the lab, argues Louis Guillette, a herpatologist and wildlife endocrinologist at the University of Florida. “When you do a study in a real-world mesocosm,” he says, “you’ll find some of the same effects that have shown up in the lab. But you’ll also find some new and different things because you won’t have the same water, the same temperatures, or even the same genetics within a studied species.” All can alter how animals — like people — will respond to their environment.

He’s also not surprised to see that atrazine appears to have effects through multiple pathways, such as increasing estrogen sensitivity, altering testosterone metabolism and changing thyroid hormone levels. Guillette concludes: “I think it’s a really powerful statement that even in a real-world scenario, where you have all of these natural variables and you have [low] ecologically relevant concentrations, you can still see atrazine’s having an effect.”

Citations

Langlois, V.S., ... and V.L. Trudeau. 2010. Low Levels of the Herbicide Atrazine Alters Sex Ratios and Reduces Metamorphic Success in Rana pipiens Tadpoles Raised in Outdoor Mesocosms. Environmental Health Perspectives (in press). DOI: 10.1289/ehp.0901418

Hayes, T.B., et al. 2010. Atrazine Induces Complete Feminization and Chemical Castration in Male African Clawed Frogs (Xenopus laevis). Proceedings of the National Academy of Sciences (in press). DOI: 10.1073/pnas.0909519107
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