Murky Waters

“You won’t have to worry about funding any more.” For any scientist, these are magical words. Independent researchers rarely receive such grand offers of unsolicited funding. Tyrone Hayes is one of the lucky few.

At the time, Hayes was already a noted amphibian developmental biologist in UC Berkeley’s integrative biology department. His patent for using frogs to screen potential environmental toxins caught the attention of EcoRisk Inc., who contracted him to sit on a panel of scientists in 1997.

The funding came from the agrochemical giant Syngenta, then Novartis. Atrazine, one of their most profitable products, had recently come up for reregistration with the Environmental Protection Agency (EPA). A small company of three or four employees, EcoRisk was essentially a financial conduit between Syngenta and independent researchers who could conduct the studies necessary for atrazine’s re-approval.

Professor Tyrone Hayes holding an African clawed frog (Xenopus laevis), the model organism used for his atrazine studies. Credit: Peg Skorpinski

Professor Tyrone Hayes holding an African clawed frog (Xenopus laevis), the model organism used for his atrazine studies.
Credit: Peg Skorpinski

Initially, Hayes was tasked with reviewing studies showing that atrazine did not have adverse effects in frogs. The paucity of data in the open literature made it an easy job. While sipping expensive whiskey at beachfront resorts, Hayes whipped up reports and wrote review papers with other members of the panel. His keen recommendations on an experimental proposal so impressed Syngenta that they asked him to perform the studies instead.

In those early days, Hayes was unconcerned with any potential conflicts of interest; the relationship was purely transactional. “I’ll do the experiment however you want. I’ll give you the results you want, and you go away. That was how I approached it,” says Hayes. “Until I did the experiment and got the results.”

Not expecting to find any effect, Hayes was surprised to discover quite the opposite: he saw a striking decrease in the size of the laryngeal muscles in male frogs raised in atrazine-containing water. The larynx, also known as the voice box, is crucial for reproduction. Smaller larynxes result in elevated pitch, and female frogs don’t like male sopranos. Most surprisingly, the effect was significant even at extremely low concentrations, down to one part per billion (ppb), one third of the EPA limit for atrazine in drinking water and roughly equivalent to half a teaspoon in an Olympic size swimming pool.

If correct, the results were exciting news. Hayes sent them along to EcoRisk, expecting to be praised for his good work. “Naively, I thought that would be what they wanted,” says Hayes. Instead, he got a chilly reception, marked by, “a series of efforts to get [him] to change the results.” A Syngenta scientist contacted him directly, suggesting that he normalize laryngeal size by the size of individual animals to make the effect go away. The company continued to ask for more and more experiments. Money slowed to a trickle.

When Hayes began to suspect that both EcoRisk and Syngenta were intentionally stalling publication of the results, he resigned from the panel so that he could continue the work. Hayes was contractually bound to ask EcoRisk for approval when publishing any research that had been funded by the company. Thus, he needed to reproduce the results to publish them independently. By that time, atrazine had already taken center stage in his lab. Without Syngenta funding, he had to scrape together money from disparate sources to pay for supplies. Dedicated undergraduates volunteered their time to perform the lab work.

As Hayes was cutting the strings of financial dependency, Syngenta and EcoRisk repeatedly offered to fly him out to their headquarters for negotiations. According to Hayes, the chairman of the panel wanted him to change dates on lab notebooks to allow EcoRisk to take retroactive control of his new data, presumably to indefinitely delay its publication. In return, Syngenta would shower Hayes with funding. Though he had only been paid about $250,000 for his twoand- a-half year stint with the company, they were hinting at figures around two million dollars to continue his research under their wing. Ron Kendall, the chairman of EcoRisk at the time, has not responded to requests for comment.

For any scientist, it would be a very tempting offer. Hayes’s refusal was the first shot in what would be become a nearly decade-long battle with Syngenta.

Metamorphosis

Growing up poor in rural South Carolina, Hayes didn’t always have many educational resources at his disposal. Yet he seemed to possess a natural inquisitiveness about animals and an uncommon scientific talent. At an early age, he ran controlled experiments on color-changing lizards called anoles, built a concrete turtle pond in the backyard with his father, and observed tadpoles as they went through the stages of metamorphosis.

His precocity attracted the attention of Harvard University, which recruited him heavily. As an undergraduate, he had the opportunity to work with Bruce Waldman, a celebrated amphibian biologist. While working in Waldman’s lab, Hayes’s childhood love of frogs and his preference for self-driven education converged in his independent project, a study on the sexual differentiation of wood frogs in response to temperature fluctuations. This research led directly to a very successful graduate career at UC Berkeley. In less than four years, Hayes produced half a dozen publications and received multiple job offers upon the completion of his PhD.

Despite this success, his career was not without its share of challenges and conflicts. As an undergraduate, he was so discouraged by the rigidity of Harvard’s educational ethos and the elitist social atmosphere that he nearly dropped out. Only the strong encouragement of Waldman and Hayes’s future wife kept him going. Later, as the only African-American professor in UC Berkeley’s integrative biology department, he was perpetually assigned to the diversity committee while other faculty members sat on the committees that made crucial decisions on hiring, fees, and lab space assignments. He claims that within ten years, these decisions resulted in a pricing structure that overcharged him significantly for his animal facilities. When he found out and demanded to be placed on a different committee, he was rebuffed by the administration. According to Hayes, one dean even told him, “Well, you’re disagreeable, so they don’t want you on the committee.”

Hayes has since been appointed to other committees that make decisions directly impacting his work. “I’ve never been asked until this year to evaluate one of my colleagues,” he says, “but they’re all evaluating me.”

Atrazine’s origins

First registered in 1958 by a predecessor of Syngenta, atrazine is now blanketing America’s croplands and golf courses at a staggering rate of 80 million pounds per year, surpassed in quantity only by Monsanto’s Roundup. The favored herbicide of corn farmers across the Midwest, atrazine chokes the growth of broadleaf weeds by blocking photosynthesis. Certain plants like corn, sorghum, and grasses have natural pathways to break down the compound before it can wreak metabolic havoc. The selective toxicity of atrazine to weeds is crucial to its success.

At the time atrazine was developed, American attitudes and government involvement in environmental issues were at an inflection point. The publication of Rachel Carson’s Silent Spring in 1962 launched a nationwide panic about the health risks of the widely used insecticide DDT. These changing perspectives led to the federal government taking over the reins of environmental stewardship from the states, culminating in the establishment of the EPA in 1970.

Atrazine snuck onto the scene just before these events transpired. The Department of Agriculture, which was then tasked with registering pesticides, only required that they be as effective as advertised. The use of atrazine was authorized without fanfare and avoided regulatory scrutiny until 1996, when an amendment to the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) was passed, addressing concerns that previous government standards were not stringent enough. It stipulated that all pesticides registered before 1985 be reregistered, including atrazine.

A few bad eggs

Even as Syngenta was attempting to buy him out, Hayes was expanding his research beyond laryngeal shrinkage. Smaller voice boxes seemed to be the least of the problems facing male frogs exposed to atrazine. At concentrations as low as 0.1 ppb, the frogs began sprouting eggs in their testes. In some cases, they became fully hermaphroditic, with multiple testes and ovaries strung together in series. Having dissected over 100,000 frogs, Hayes had never seen these types of gonadal structures before.

When he looked in the field, Hayes and his group found that the frequency of gonadal abnormalities correlated with atrazine levels in the wild. In areas with atrazine concentrations as low as 0.2 ppb, up to 80 percent of the native leopard frogs tested had gonadal abnormalities.

The presence of these abnormalities could alter the sex ratio in wild frog populations, significantly reducing their reproduction rate. Hayes believes that environmental toxins like atrazine may be responsible for a well-established nationwide decrease in amphibian populations.

The work of a handful of other independent researchers was also coming to light, confirming atrazine’s deleterious effects and offering up a possible mechanism. One paper detailed gonadal abnormalities in response to atrazine (albeit at a single, much higher dose) in the same species of frog studied by Hayes’s research group, Xenopus laevis. A native of Africa, Xenopus is a widely used model organism due to simple requirements for growth in captivity and because the frog’s response to cancer-causing agents is similar to that of mammals and as the number of cases related to cancer are growing by the day as per Roundup cancer lawsuit, it’s high time a solution to this fatal condition be found soon. Of importance to regulatory agencies, native North American frog species showed a similar response outside the lab. In cricket frogs, researchers found intersexed frogs in areas contaminated with atrazine.

Results from the laboratory of Martin van den Berg at the University of Utrecht hinted at a possible mechanism for the gender-bending effects of atrazine. Using cultured mammalian cells, he showed that atrazine induced the expression of aromatase, the enzyme responsible for converting testosterone into estradiol. Induction of this enzyme in frogs was hypothesized to be responsible for increased estradiol production, leading to the feminization that Hayes observed. Since estradiol is well known to cause feminization in frogs when they are exposed to it at early developmental stages, the hypothesis was plausible. However, it remains unproven in frogs.

These studies establish a general consensus that atrazine can affect vertebrate sexual development, but the magnitude of these effects and the concentration at which they occur varies substantially. To date, no other research group has reported reproducing Hayes’s results at the extraordinarily low atrazine concentrations he used, although few have tried.

As these results were emerging, the Syngenta-sponsored EcoRisk panel published paper after paper claiming to demonstrate that atrazine had no effect. According to Hayes, the EPA, and other scientists, much of this work was crippled by poor technique. In a field study, no systematic procedures were in place to control for how the animals were treated while en route from the field to the laboratory for testing. Over half of the animals died before maturation during one set of experiments. In one study purporting to measure aromatase activity, the positive controls failed. The text of another claimed no effect of atrazine on gonadal development, while the corresponding figure and statistics showed the opposite.

Data flooding

The simmering conflict between Hayes and EcoRisk in the scientific literature came to a head in the first of two meetings of the EPA’s Scientific Advisory Panel (SAP) in June 2003. In light of Hayes’s research, the Natural Resources Defense Council (NRDC) forced the EPA to evaluate the effect of atrazine on amphibians, using settlement terms from a lawsuit it had previously won against the agency.

The panel, a collection of prominent independent scientists, was tasked with advising the EPA on how to interpret the science on atrazine. They found Hayes’s results on hermaphroditism cause for alarm. “I’ve cut up…more [frogs] than my children would ever want me to. And I’ve never seen eggs in the testes,” says Professor Darcy Kelley, the lead discussant of the 2003 SAP and a renowned expert on frog sexual differentiation at Columbia University.

Additionally, they were skeptical of the results from the EcoRisk scientists and questioned them at length about their methodologies. “It was very disturbing that the people Syngenta hired to look into this couldn’t replicate some of the most basic things that people in amphibian biology know,” says Kelley.

The EcoRisk scientists were the only other scientists who repeated Hayes’s experiments directly. In spite of their public stance on atrazine, they wrote in private emails to Tyrone that they agreed with his conclusions about atrazine’s ability to disrupt gonadal formation in frogs. “Atrazine is bad news,” wrote one member. “There is no denying” the effect, wrote another.

Still, they published numerous papers asserting the opposite, giving the impression that Hayes’s results were anomalous, even though no fully financially independent investigator had corroborated the EcoRisk results. Of the 17 studies submitted for review by the SAP, 11 were funded by Syngenta.

The discrepancies in the open literature meant that the 2003 panel could not conclude that atrazine had an effect on amphibians at ecologically relevant levels. Instead, they offered a set of recommendations for further studies, specifying the appropriate ranges of atrazine doses, the inclusion of native species, the elucidation of a mechanism, and the use of flow-through water tanks to ensure water quality. In response to these recommendations, the EPA called in a study to address the lingering issues about atrazine’s effects on frogs. The requested study was required to adhere to the “good laboratory practices” (GLPs), a set of agency-wide quality control standards that ensure the reproducibility and reliability of research data. For the proposed amphibian studies, the specific GLP standards were tailored to the experimental recommendations of the SAP for atrazine.

Not only are flow-through tanks prohibitively expensive for independent researchers, the GLP also required a large number of replicates and dose levels. The only party with the means to conduct the study was the registrant, Syngenta, which stood to lose millions of dollars as a result of regulatory action. They commissioned two large studies at separate contract labs, which were collectively known as the data call-in (DCI) studies. The results came back negative.

When the EPA’s second scientific advisory panel met in 2007 to discuss the results of the Syngenta-funded studies, the fix was in. Tom Steeger, the EPA scientist in representing the agency before the panel, steered the proceedings carefully. When panel members suggested that the DCI results were insufficient, Steeger pushed back, often restating their comments in such a way as to diminish any need for further investigation. He dismissed all the published results from both Hayes and EcoRisk, saying that the open scientific literature “cannot hope to compete” with the Syngenta-sponsored DCI study. The panel did explicitly recommend some further investigation: a study on native frog species, and a re-examination of the microscope slides from the first study by different pathologists. Neither of these studies was conducted.

Instead, the EPA let the issue go. As of April 2010, the EPA still considers the amphibian issue closed. In the end, the EPA decided that there was no cause for concern based solely upon the results of a single industry-sponsored study.

Atrazine use in the United States is concentrated in heavy agricultural areas, mainly in the Midwest. Credit: USGS

Atrazine use in the United States is concentrated in heavy agricultural areas, mainly in the Midwest.
Credit: USGS

The case isn’t closed

But, according to many scientists, the case is still open. Robert Denver, a prominent endocrinologist at the University of Michigan and a member of both panels, said at the 2007 meeting that the DCI study did not “fully test” Hayes’s hypotheses. According to Denver, the flow-through tanks used in the DCI studies do not “mimic the characteristics of exposure that are encountered in nature.” Although the 2003 SAP recommended it for water quality issues, its applicability was controversial because developing frogs are known to avoid running streams. The EPA also overlooked the fact that no native species were tested.

Most strikingly, the crucial positive control, estradiol, failed. The scientists performing the DCI study could not get 100 percent feminization with estradiol, maxing out at 70 percent. “Anyone can come to my lab and we could get 100 percent females right now—to get incomplete feminization is a red flag,” says Kelley. A few members of the panel were concerned that the strain of Xenopus used was naturally resistant to hormone disruption.

The DCI study was only designed to address the narrow hypothesis that atrazine can cause malformed gonads in frogs. The panel was not asked to address whether atrazine is an endocrine disruptor, a legally defined class of chemicals believed to interfere with the human endocrine system. This category includes two heavy hitters of environmental toxicology: DDT and the PCBs (polychlorinated biphenyls), which are a class of highly toxic industrial chemicals. Because these toxins are associated with a wide variety of developmental defects and cancers, the classification of atrazine as an endocrine disruptor would present a major obstacle to reregistration.

“The question of whether or not atrazine affects this strain or that strain of frogs actually isn’t all that important,” says Hayes. The main questions are broader, he argues: “Is atrazine an endocrine disruptor? Do we see consistent effects of atrazine across vertebrate classes? And the answer is yes.”

The NRDC, whose lawsuit brought about the 2003 and 2007 SAPs, also strenuously objects to the EPA’s narrow line of inquiry. “The question we wanted looked at,” said an NRDC representative at the 2007 panel “is atrazine’s potential effects on endocrine disruption.” To Hayes, the NRDC, and others, it appeared that the EPA had skirted the real issue.

Credit: Katie Berry, Marek Jakubowski

Credit: Katie Berry, Marek Jakubowski

It’s not just about the science

There are millions of chemicals on the market, and each can potentially affect human health and the environment in numerous ways. How to decide which of these chemicals are safe and which may cause lasting damage requires exceptional triage. This is the job we entrust to the EPA.

“Necessarily, not all chemicals can be investigated as thoroughly as many scientists would like,” says Dr. Kelley. More studies can always be done, but the line must be drawn somewhere. As a government agency, the EPA’s decisions on where to draw those regulatory lines are influenced by the prevailing political atmosphere. Atrazine’s reregistration began during the George W. Bush administration, which had all but declared war on regulation in general and the EPA in particular. Budgets were being cut, and the already attenuated requests for regulatory action were met with blanket rejections by political appointees. EPA officials may have decided that the Bush administration would never regulate atrazine based on amphibian evidence alone.

Under Lisa Jackson, the Obama appointed EPA Administrator, the atrazine case has resurfaced. The reevaluation was called largely in response to atrazine’s association with cancer in humans, neglecting recent progress on the amphibian front.

Very recent work by Hayes has shown that with enough atrazine, some male frogs become completely female. In behavioral studies, these feminized genetic males mated successfully with real males to produce viable offspring. These results indicate that atrazine’s ecological impact may be larger than previously thought, but have not yet been officially evaluated by the EPA. Conflicting statements on separate sections of the EPA website leave it unclear whether these recent studies will ever be considered in future reregistration decisions.

Hayes has shown that a genetic male frog exposed to atrazine (below) can develop egg-filled ovaries and mate with an unexposed male (above) to produce viable offspring. Credit: Adapted from Tyrone Hayes

Hayes has shown that a genetic male frog exposed to atrazine (below) can develop egg-filled ovaries and mate with an unexposed male (above) to produce viable offspring.
Credit: Adapted from Tyrone Hayes

Is atrazine worth it?

Certainly in the case of atrazine, the EPA’s stance seems to be that a positive burden of proof is required before regulatory action can be taken. To be pulled off the shelves, atrazine must be proven to have acutely adverse effects on human health and the environment.

Contrast that with the approach of European regulatory agencies, which do not necessarily require that pesticides be shown to have toxic effects. Atrazine has been banned throughout Europe for more than five years, solely because its half-life in water systems is extremely long, between 10 and 200 days, making it impossible to keep pesticide levels below 0.1 ppb in ground and drinking water, the European limit across the board.

Even in the United States, Syngenta has been fighting a long, hard battle on numerous fronts for atrazine, which comes as no surprise given the profits it generates. Yet, Syngenta is not atrazine’s only defender; farmers have long been voting for it with their wallets, and with good reason. According to analyses from different sources, a ban on atrazine would cost the corn industry anywhere from $350 million to $1.6 billion yearly, a two to six percent loss per bushel. Farm lobbyists flock to every EPA meeting to defend atrazine, claiming that alternative weed-killers could plausibly have even worse effects. Although Europe’s ban on atrazine has resulted in no losses in crop yields, farmers there have shifted to a cocktail of other herbicides whose ecological effects are largely unknown. Who knows if there even exists a good alternative that’s both effective and environmentally friendly?

Apparently Syngenta does. Ten years ago, they requested EPA registration for an herbicide called mesotrione (tradename Callisto). Recent studies have shown that mesotrione is just as effective on broadleaf weeds as atrazine, if not more so. Moreover, mesotrione has low toxicity and is rapidly degraded in soil and water by microorganisms. The only downside is that it is more expensive, but according to an analysis by Dr. Frank Ackerman, an economist at Tufts University, the additional cost constitutes less than one percent of the market value of corn. Individual corn farmers are unlikely to make this switch without regulatory pressure; for the average 1,000-acre farm, a switch would cost between $3,000 and $7,000 per year.

Why Syngenta is still fighting the battle for atrazine is less clear. When asked about the relative benefits of both herbicides, the company simply maintains that “when used according to the labels, both products are friendly to the environment.”

He just keeps coming back

Hayes reached a dead end with the EPA. Compared to most scientists, the EPA held a drastically different philosophy on what constituted adequate scientific evidence. Reproducibility was key, and no one but Hayes found a positive effect at such low atrazine levels in frogs. Thus, the EPA overlooked Hayes’s singular positive results in favor of the DCI study’s “no effect” finding, which better satisfied their particular data standards.

Convinced of the EPA’s “hidden agenda” favoring Syngenta, Hayes took his show on the road. He gave presentations on his work highlighting how atrazine feminizes amphibians at public health, endocrinology, and toxicology conferences, as well as legislative hearings and other public venues.

Then, things started to take a turn for the bizarre. Two Syngenta employees, a scientist named Timothy Pastoor and a public relations representative named Sherry Duvall Ford, began following him to these events. According to Hayes, they had a predilection for mischief. “At the American Public Health Association, they handed out fliers…and actually disrupted one of my talks,” he says. “They called a fire emergency when I was lecturing in Sydney, Australia.”

At the Illinois State House, during preliminary hearings into whether the state should pursue legal action over atrazine use, Pastoor allegedly physically threatened Hayes. “Next time you give a talk, I’m going to have some of my boys [come after you],” Pastoor said, according to Hayes. He says he has informed the FBI about these threats. To this day, his number is unlisted and his lab is always locked, as if on high alert. Ford and Pastoor have not responded to requests for comment.

In response to goading emails from Pastoor and Ford, Hayes responded in kind, with much creative flourish. His responses are filled with speculations on the nature of life, science, and truth, as well as quotes, poems, and often-explicit rap lyrics.

The conflict had moved beyond the scientific arena. “High-minded scientific discourse was still going on,” says Hayes. “But if you come to intimidate me and make comments about my wife for example… that’s a different kind of conversation.” He apologizes for, “offending some people that I care about, but I said what I meant.”

Syngenta, however, filed an ethics complaint several months ago with UC Berkeley against Hayes over emails they claimed to be, “not only aggressive, unprofessional and insulting, but also salacious and lewd.” With Pastoor’s and Ford’s names blacked out, the one-sided transcript of the emails was released by the company as ammunition in its attacks on Hayes’s work. These emails are available for download at the company website. Hayes claims that the emails from the Syngenta employees were equally offensive, but declined to release them, citing legal concerns.

Hayes made no serious threats and it is hard to see what he wrote as anything worse than unprofessional. Although the racy emails may have damaged his credibility in some circles, they mostly just kicked up a storm of public attention about atrazine. In the past year, many articles have been appearing in outlets ranging from Gawker to the New York Times.

Hayes’s level of involvement in the atrazine case far exceeds what most scientists could ever imagine committing to the societal implications of their work. In the midst of heavy teaching and research loads, who has the time to fly around the world to deliver politicized invectives against a company and compose elaborate emails and poems for a pair of its hired guns?

He could have let the matter go at any time. Why didn’t he just pull out?

The easy answer is that he is a selfless advocate, devoted to getting the truth out about a dire environmental hazard. His presentations and writings highlight a desire to speak out for the underdog, be it powerless frogs or the largely Hispanic agricultural laborers who bear the brunt of atrazine exposure in the fields. While these reasons do contribute to Hayes’s outrage, it is also fueled by something deeper.

When asked why he continued to study atrazine after EcoRisk withdrew its funding, Hayes says it is a question he has been rethinking lately. While he says he, “cares about public health and environmental health and all that stuff,” he also realizes that Syngenta’s offer angered and offended him. “You can’t buy me,” he says. “You can’t pay me enough money to be dishonest.”

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