Transsexual Frogs

A popular weed killer makes some frogs grow the wrong sex organs. Your drinking water may have 30 times the dose they're getting

By Elizabeth Royte, Catherine Ledner|Saturday, February 01, 2003


Tyrone Hayes stands out in the overwhelmingly white field of biology, and his skin color isn't the half of it. To use his own idiom, Hayes is several standard deviations from the norm. At the University of California at Berkeley, he glides around his lab wearing nylon shorts and rubber flip-flops, with a gold hoop in one ear and his beard braided into two impish points. Not counting his four inches of thick, upstanding hair, Hayes is just over five feet tall, with smooth features and warm eyes. He drives a truck littered with detritus human, amphibian, and reptilian. He keeps his pocket money in a baby's sock. "Hey, wassup?" he'll say to anyone, from the president of the United States on down. He can't help the informality, he says. "Tyrone can only be Tyrone."

A leopard frog destined for testing at the University of California at Berkeley. Endocrinologist Tyrone Hayes keeps 3,000 such frogs in his basement laboratory. Many of them have had sex problems due to the effects of the chemical atrazine.

    Hayes, 35, is a professor at Berkeley, where he has taught human endocrinology since 1994. His research centers on frogs, of which he keeps enormous colonies. Frogs make convenient study subjects for anyone interested in how hormones affect physical development. Their transformation from egg to tadpole to adult is rapid, and it's visible to the naked eye. With their permeable skin, frogs are especially vulnerable to environmental factors such as solar radiation or herbicides. That vulnerability has lately garnered Hayes more attention than his appearance ever has.
    The controversy began five years ago, when a company called Syngenta asked Hayes to run safety tests on its product atrazine. Syngenta is the world's largest agribusiness company, with $6.3 billion in sales of crop-related chemicals and other products in 2001 alone. Atrazine is the most widely used weed killer in the United States. To test its safety, Hayes put trace amounts of the compound in the water tanks in which he raised African clawed frogs. When the frogs were fully grown, they appeared normal. But when Hayes looked closer, he found problems. Some male frogs had developed multiple sex organs, and some had both ovaries and testes. There were also males with shrunken larynxes, a crippling handicap for a frog intent on mating. The atrazine apparently created hermaphrodites at a concentration one-thirtieth the safe level set by the Environmental Protection Agency for drinking water.
    The next summer Hayes loaded a refrigerated 18-wheel truck with 500 half-gallon buckets and headed east, followed by his students. He parked near an Indiana farm, a Wyoming river, and a Utah pond, filled his buckets with 18,000 pounds of water, and headed back to Berkeley. He thawed the frozen water, poured it into hundreds of individual tanks, and dropped in thousands of leopard-frog eggs collected en route. To find out if frogs in the wild showed hermaphroditism, Hayes dissected juveniles from numerous sites. To see if frogs were vulnerable as adults, and if the effects were reversible, he exposed them to atrazine at different stages of their development.
    Hayes published his first set of findings last April, in the Proceedings of the National Academy of Sciences. He published the second set in October, in Nature. Both times the media went a little crazy. The two studies showed equally dramatic results: 40 percent of male frogs were feminized; 80 percent had diminished larynxes. Wild frogs collected from areas with atrazine showed the same number of abnormalities. Could the chemical also affect humans? The beginning of an answer may be emerging. Workers at a Louisiana plant where atrazine is manufactured are now suing their employer, saying they were nine times as likely to get prostate cancer as the average Louisianan.


Tyrone Hayes hoists a jug of subjects in his Berkeley laboratory. He is so enamored of amphibians that he has even named his daughter, Kassina, after a genus of frog.

Inside Berkeley's Valley Life Sciences building, Hayes approaches a set of double doors and lifts his thigh, doggy style, toward the wall. The doors respond to a security card in his pocket and swing wide onto an empty corridor. It's 7 a.m., but Hayes has been here since 4:30 this morning, when he came to "make water"—mix the chemical cocktails in which he's raising 3,000 leopard frogs in a crowded basement lab. He deftly shakes crickets—frog breakfast—from a plastic bag into dozens of tanks. On another shelf, tadpoles swim in one set of deli cups while metamorphs, which have both tails and legs, swim in another. Escaped crickets dart around the room. Strips of colored tape adorn each tank, each color denoting a particular mix of compounds. In this quadruple-blind experiment, neither Hayes nor his assistants know exactly what they're testing. Except for the notorious Red Yellow Red.
    We peek into the suspect tank. "They're not doing too well, are they?" Hayes says, brushing a cricket off his neck with a practiced flick. The frogs are listless. Their heads tilt at a creepy angle. "Everything we put in this mixture died within a week, except for frogs that have adapted to that environment. So I had to look it up." Red Yellow Red, the codebook said, is the brew that runs off a Nebraska cornfield in springtime. "These frogs took a month longer than average to metamorphose, and then they were smaller than average," Hayes says. "That's wrong: Usually a longer metamorphosis means a bigger frog." He dumps in another meal of crickets and delivers the kicker: "This mixture from the cornfield has a lower dose than what's in the drinking water there."
    The problem, Hayes knows, goes well beyond frogs that loiter near cornfields. According to James Hanken, a biologist at Harvard University who heads a task force on declining amphibian populations, "at least one-third to one-half of all living species of amphibian that have been examined in this regard are on their way down, and out." Researchers have offered a number of explanations for the die-off: attacks of parasites, exposure to radiation or ultraviolet light, fungal infections, climate change, habitat loss, competition with exotic species, and pesticides. Atrazine is used in more than 80 countries, primarily on corn and sorghum fields. By interfering with frog reproduction, Hayes wonders, could it be part of the problem?

In a park in Berkeley, Hayes demonstrates his frog-catching technique. As a teenager in Columbia, South Carolina, he used to take girlfriends frog-hunting. Now he takes students, piling his catch into a refrigerated truck.

    Atrazine is a synthetic chemical that belongs to the triazine class of herbicides. Its technical name is 2-chloro- 4-ethylamino- 6-isopropylamine- 1,3,5- triazine. In the United States, farmers apply around 60 million pounds of atrazine a year. Nearly all of it eventually degrades in the environment, but usually not before it's reapplied. The EPA permits up to three parts per billion of atrazine in drinking water. Every year, as waters drain down the Mississippi River basin, they accumulate 1.2 million pounds of atrazine before reaching the Gulf of Mexico.
    Like the smoke from factory chimneys, pesticides cross borders. Atrazine molecules easily attach to dust particles: Researchers have found it in clouds, fog, and snow. In Iowa the herbicide has been documented at 40 parts per billion in rainwater. According to the U.S. Geological Survey, atrazine contaminates well water and groundwater in states where the compound isn't even used. "It's hard to find an atrazine-free environment," Hayes says. In Switzerland, where it is banned, atrazine occurs at one part per billion, even in the Alps. Hayes says that's still enough to turn some male frogs into females.
    Hayes talks rapidly as he walks from the basement lab. He'll also talk rapidly as he drives to his children's school in an hour, as he eats at a nearby restaurant, and as he types e-mail. "I'll calm down after lunch," he promises. "Here's how we think it works. à Testosterone is a precursor to estrogen. In male frogs, it makes their voice boxes grow and their vocal sacs develop. But atrazine, in frogs, switches on a gene that makes the enzyme aromatase, which turns testosterone to estrogen. Normally, males don't make aromatase; it's silent. In these males, the estrogen induces the growth of ovaries, eggs, and yolk." We're at the double doors, and Hayes lifts his thigh again. "So you've got two things happening: The frog is demasculinized, and it's also feminized."
    And the females that get extra estrogen? "It wouldn't happen," Hayes says. "There's a feedback mechanism. The excess hormone would decrease stimulation of the ovary, which would then cut off its production of estrogen."
    Because hormones, not genes, regulate the structure of reproductive organs, vertebrates are particularly vulnerable to their environment during early development. Frogs are most susceptible just before they metamorphose. Unfortunately, that change occurs in the spring, when atrazine levels peak in waterways. "All it takes is a single application to affect the frog's development," Hayes says.
    Theo Colborn, a senior scientist with the World Wildlife Fund who has spent nearly 15 years studying endocrine-disrupting chemicals in the environment, calls Hayes's work a breakthrough. "At a time when other developmental biologists were taking a broad, traditional approach, he was taking long-term effects into consideration," she says. "No one had looked at the histology the way he has. Everyone was so hung up on limb deformities in frogs that they forgot about other effects. His work may explain why frogs are disappearing."


Click on the image to enlarge (75k).
Whither Weed Killer?
A map of atrazine sales in the U.S. shows that the herbicide is heavily used on Midwestern farms. The shaded area is the leopard frog's natural range. Hayes collected 100 frogs for his research at each of the eight numbered locations.
Map by Matt Zang
Map adapted from Hayes, T., et al. Nature 419 (October 31, 2002): 895-896.

Hayes has always been fond of frogs. He grew up in a modest neighborhood of brick houses outside Columbia, South Carolina. The development had been drained of its marsh, but snakes, turtles, and amphibians abounded. Hayes followed them and learned their ways. As a teenager, he dug a pond in his backyard, hoping to breed turtles. He kept lizards. His father brought him boxes of National Geographic from houses in which he had installed carpet. The boy read them all. "Those magazines were the beginning of it," Romeo Hayes says. "Even then he knew he wanted to be a scientist." The television was always on in the Hayes household, even during meals, and Tyrone paid particular attention to the nature specials. When he began dating, he took girlfriends to the Congaree Swamp, nine miles away. The young women assumed he had other things in mind, but his motives were always the same: He wanted help catching frogs.
    The summer after sixth grade, Hayes taught himself to play basketball. "That was the only way I knew for blacks to get into college," he says. Through high school he wrestled, struggling with a hypothyroid condition to make weight. Entranced by the pop star Prince, he wore frilly shirts and velvet jackets, winning Best-Dressed Student five years in a row. "I wanted a hoop earring, but my mother forbade it," he says. Within days of arriving at college, he pierced his ear himself. (These days, Hayes wears a coat and tie to meetings. "But it's a real Men in Black kind of suit," one former student says. "And he wears a skullcap.")
    Geography and family circumstances narrowed expectations. Hayes's father had been the first on his side of the family to attend high school. Hayes had never heard of an academic scholarship; he had never known anyone who left South Carolina to go to school. But his high PSAT scores brought a sheaf of recruitment letters to his house. He wrote a personal statement about his interest in armadillo biology and mailed it to Harvard. It was the only school to which he applied. "I'd heard of it on Green Acres and figured it must be good," he says, without a trace of irony.
    Once on scholarship in Cambridge, Hayes thought he'd become a doctor. Then he began working with the biologist Bruce Waldman on kin recognition in toads. Waldman recognized Hayes's talent for asking challenging research questions and his skill in the field and the lab. He treated the freshman like a grad student. Soon Hayes was studying environmental effects on tadpole metamorphosis. "I realized what a person who enjoyed what I did might do for a living," Hayes says. "I saw the whole picture coming together."
    Still, nothing in his background had prepared him for Harvard's social and academic pressures. "Most blacks at Harvard were from private schools," Hayes says. "They knew what was going on. Their parents had gone to school there. They flew to Bermuda at spring break." Hayes felt out of place. He didn't join any campus groups and spent all his time in the laboratory. "It was the only place I felt at home," he says. "I had four finals to study for and didn't know how to organize my time. I couldn't get advice from my dad." His grades fell, and he was placed on academic probation. Hayes nearly dropped out at that point, but Waldman and Kathy Kim, the girlfriend he later married, persuaded him to stick it out. In 1989 he graduated with departmental honors and moved to Berkeley, where he earned his Ph.D. at the age of 24.
    "You think Tyrone is manic now, you should have seen him in those years," says Nigel Noriega, a research scientist in reproductive toxicology at the EPA. At Berkeley, Hayes's weight ballooned from 135 pounds to 260 pounds in six months. To get back into fighting shape, he ran 18 miles a day, often with an infant in a stroller. He went for days without sleep, then set the alarm to ring after just a few minutes. He was running a shape-shifting experiment on himself.
    He drove his students to the edge as well. Lab assistants, drawn in by his dynamism, became exhausted and depressed. "It was hard; we barely saw the light of day," says Roger Liu, who spent the better part of 10 years in Hayes's lab. The results of their experiments would be so far in the future that they lost sight of their goals. Still, they loved Hayes. "Tyrone treated undergrads like grad students and grad students like postdocs," Noriega says, echoing Hayes's assessment of Waldman. "You could ask him for anything." When Hayes found attendance flagging at his 6:30 a.m. lab meetings, he started baking, at 2 a.m., to lure students in. When he worried about his charges walking to the lab in the dark, he picked them up at 4 in the morning, shining a spotlight into their windows to wake them.
    From the outset, Hayes's lab attracted minority students and soon became far and away the most diverse in the department. The department of integrative biology is only 3 percent black and has produced just four black Ph.D.'s in its history. (Noriega is one.) Now nearly 20 percent of his lecture class is black. Hayes says he concentrates on selecting talented students who need nurturing. This semester's crop of researchers comes from Vietnam, India, Pakistan, Thailand, Tunisia, Mexico, Guatemala, Canada, and the United States.
    "Maybe minority students think they'll make some kind of connection with me," Hayes says, shrugging. Or maybe they appreciate his holistic approach to science. He often brings his two children—Tyler, 10, and Kassina, 7—into the lab with him, and he watches over his students with the same paternal eye. "The lab was like a family," Liu says. "Dad got pissed, the siblings fought, but we were happy."
    Last year, at the departmental graduation, the students gave Hayes a standing ovation. This past spring he won the College of Letters and Science's award for Distinguished Research Mentoring; a week later he won its Distinguished Teaching Award. "Tyrone reveals that science is inbred and flawed and political, just like art and music," Noriega says. "But he's still striving for its bright and shining truth. He lays all this out, and you see it's still worth it."
    Even after all the weirdness with Syngenta.


Tyrone Hayes exposes the offspring of African clawed frogs to estrogen to investigate the effects of atrazine. In male frogs, he has found, the herbicide switches on a gene that makes aromatase, an enzyme that turns testosterone to estrogen. Some male frogs eventually grow ovaries, eggs, and yolk.

Like all chemical companies, Syngenta has to have its products tested for safety before the EPA will approve them. The company came to Hayes in 1997 because he had experience with hormones and amphibians: He had developed an assay in which frogs exposed to estrogen mimics turned from green to red. "This was a chance to use my research," Hayes says. "Also, not that many labs are set up to travel and collect eggs, establish a colony, and breed. I had a big lab, with lots of people willing to move 3,000 frogs from tanks to deli cups."
    Hayes says that when he informed Syngenta about atrazine's negative low-dose effects in August 2001, the company treated his data like a hot potato. "They told me, 'That's not what you were contracted to do. We don't acknowledge your work,'" Hayes says. "I sent them all my raw data, and they FedExed it back to me." Ronald Kendall, an environmental toxicologist at Texas Tech University and a leader of Syngenta's atrazine-testing panel, insists that Hayes told the team only about the frogs' shrunken larynxes, not their hermaphroditism: "We didn't learn about gonadal effects until a hormone meeting late in November." Rather than keep quiet about his findings, Hayes quit his contract and repeated his experiments. The week before he was scheduled to share his data with the EPA, he received 500 computer viruses.
    After Hayes quit his contract, Syngenta funded some of Kendall's colleagues at Texas Tech to replicate the work. They produced almost no hermaphrodites at the atrazine levels Hayes had tested. The lab conditions in Texas differed from conditions in the Berkeley basement. For example, the Texas experimenters raised their frogs in glass instead of plastic tanks, at higher population densities, and at cooler temperatures, and they fed them differently. "But if the effect is robust, as Hayes claims it is, you should still be able to see it under slightly different conditions," says James Carr, a comparative endocrinologist on the Texas Tech team.
    Hayes accused the Texas team of raising unhealthy frogs in tanks with uncontrolled atrazine levels. "Their animals were underfed and overcrowded," he says. "How can you tell if their gonads are deformed if the animals don't develop properly?" In response, the Texas team crafted an 18-page defense, to which Hayes responded with 22 pages of his own. The Texas team says it was difficult to compare the health of their animals with the health of Hayes's because he didn't report hatching success, mortality, survivorship, and other data. Hayes responds: "They've had all my information on protocols and SOPs since 1999. They signed off on this work. They even visited my lab."
    While the scientists sparred, workers at Syngenta's atrazine plant in St. Gabriel, Louisiana, stole the spotlight when their cancer rates became public. At least 14 of 600 employees who'd been at the plant for more than 10 years had developed prostate cancer—a rate nine times as high as that of the general statewide population.
    Had Syngenta inadvertently tested atrazine on humans? Studies of farm laborers who worked with the compound showed rates of certain cancers double to eight times the national average, but those exposures were intermittent and not exclusive, because workers handle many types of chemicals. In St. Gabriel, atrazine represented 80 percent of the plant's production, and it was made year-round. Atrazine dust covered the walls and floors, countertops and lunch tables.
    Hayes's frog data were alarming, but they probably wouldn't have persuaded the EPA to ban atrazine. The cancer findings may. This past summer, the Natural Resources Defense Council persuaded the agency to launch a criminal investigation of Syngenta for suppressing data on the herbicide's potential risks to the environment and to human health. The EPA has since extended the deadline for its atrazine review. In addition to the cases in Louisiana, laboratory studies have linked atrazine to hormonally responsive cancers in humans and lab animals. Studies have also suggested that it disrupts the production of hormones such as testosterone, prolactin (which stimulates the production of breast milk), progesterone, estrogen, and the thyroid hormones that regulate metabolism.
    Nonetheless, Hayes doesn't jump to condemn atrazine. He says he hasn't studied humans, but it is unlikely they'd be affected because atrazine doesn't accumulate in tissues the way DDT does. Others aren't so sure. "Why would anyone think these pesticides wouldn't affect us?" the World Wildlife Fund's Theo Colborn says. "No matter the species, we all have similar signaling systems in our bodies, similar chemical reactions. That's why we've always tested drugs on animals." Human kidneys filter atrazine, and humans don't spend a lot of time swimming in pesticide-laced water, the way frogs do. But human fetuses do live in water.
    "Our big concern is pregnant females," Colborn says. "There have been enough studies on farm families to show that babies conceived in the spring, when runoff is highest, have far higher rates of birth defects than babies conceived at other times." But what component of the runoff is toxic and at what levels? That may be impossible to say, because scientists don't run lethal-dose experiments on humans. Faced with this uncertainty, how cautious should we be? When pressed, Hayes says that if his wife was pregnant, he'd advise her against drinking water from much of the Midwest—his children too. "If there's a .01 percent freak chance that something could happen, why take that chance?"

The mystery of amphibian decline continues to intrigue Hayes. He believes a combination of many different effects may stress frogs' immune systems and that atrazine may be a part of it.
    He dreams of testing his ideas with the perfect field experiment, one without unquantifiable variables, and he knows just where he'd enact it. "We'd go to Biosphere 2, in Arizona," he says. "We'd bring in all our own air, our own water. We'd set up farm plots with corn. We'd bring in our own frogs, study every compound and its impact on the corn, the corn pests, the nontarget organisms." There's a gleam in Hayes's eyes. The thought of all those animals, the long hours, the phalanx of tired graduate students—it all makes his blood rise. "Nobody knows what these compounds actually do," he says. "I want to figure it out from beginning to end."




A consumer fact sheet on atrazine from the EPA: www.epa.gov/safewater/dwh/c-soc/atrazine.html.

More from the Natural Resources Defense Council: www.nrdc.org/health/pesticides/natrazine.asp.
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