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If Mother Nature has her way, the snapping turtle eggs in the incubator to Kathy Patnode's right will hatch as males, those on the left will hatch as females.
"Temperature determines their sex," says Patnode, a DNR wildlife toxicologist. "A nest temperature of 73.5 degrees would develop males. If it heats up to 83.5, hormones would trigger changes causing the embryonic cells to differentiate as females." She carefully digs a nest for each egg in a small jar before arranging it on a tray destined for the incubator.
But Patnode suspects Mother Nature has been fooled.
She hypothesizes the mother turtle that laid along a contaminated river was likely exposed to environmental pollutants. These contaminants may have scrambled the natural chemical messages directing embryo cells to develop male or female sex organs. As a result, Patnode expects the hatchlings could exist in a state of sexual limbo.
"If the PCB contaminants acts like an estrogen, turtles incubated at the male temperature may develop abnormal testes that contain female ovary cells," Patnode said. "If they act like a male hormone, turtles incubated at the female temperature might develop abnormal ovaries."
Patnode is among a growing number of researchers investigating whether environmental pollutants are interfering with the hormonal, or endocrine, systems that orchestrate growth, sexual development and regulate other internal functions in humans and wildlife. These compounds, which include industrial chemicals such as PCBs, formaldehyde, mercury, pesticides, and even some plastics, may mimic hormones and disrupt the body's subtle systems that produce, use or breakdown these chemical signals.
The results, some scientists hypothesize, might explain cancers, reproductive problems, immune system problems and even behavioral changes.
DNR toxicologist Mike Meyer has been trapping and taking blood samples from 400 loons on northern lakes where fish contain the highest recorded levels of mercury. He marks the birds and monitors their nest every year to compare their reproduction rates with loons from lakes where fish have lower mercury levels.
Candy Schrank, DNR fisheries toxicologist, is similarly sampling yellow perch from Lake Michigan and examining sex organs, hormone levels and other reproductive measures as part of a broader study to identify possible causes of a mysterious, precipitous crash in the perch population since the late 1980s.
Other DNR wildlife health studies are investigating if hormonal disruption can explain why mink populations haven't recovered since a mid-1970s crash or why contaminated stretches of some rivers have no mudpuppies while nearby rivers support healthy numbers of these freshwater salamanders.
Public and Congressional interest in the issue has exploded in recent years, fueled in part by sensational, but conflicting, reports of dramatic drops in human sperm counts, and by scientific speculation that exposure to environmental chemicals may cause rising breast cancer and infertility rates. The publication in 1996 of "Our Stolen Future: Are We Threatening our Fertility, Intelligence and Survival?" further fanned the flames. The book, by Theo Colborn, senior scientist with the World Wildlife Fund who received her zoology doctorate from University of Wisconsin-Madison, galvanized some environmental groups and politicians. Vice President Al Gore wrote the foreword likening the book to Rachel Carson's "Silent Spring," but some scientists and reviewers criticized it as long on theories and short on proof.
The endocrine system is one of two chemical messenger systems in humans and wildlife. The other, the central nervous system, serves as the communications network linking the senses, the environment, and other parts of the body to the outside world. Eyes, ears, and other senses relay news of outside stimuli to the brain, which sends rapid-fire messages along neural paths to tell the appropriate tissues how to respond.
In contrast, the endocrine system functions as the body's internal communications network and releases its messages over seconds, minutes, hours, days and years. Within cells, these messages help maintain constant temperature and acidity levels, as well as keeping steady concentrations of glucose, fatty acids and other dissolved substances. More broadly, these chemical messages regulate growth, maturation, sex differentiation, reproduction, pigmentation, and behavior.
The pituitary, the thyroid, the pancreas, the testes in males and the ovaries in females are all endocrine glands which produce and release hormones to the bloodstream. Hormones find their way to intended receptors on cell surfaces or within cells, bind to them and trigger specific responses in these target cells.
Ovaries, for instance, produce estrogen which travels in the bloodstream to the uterus, where it binds to a receptor and triggers tissue that lines the womb to grow and prepare for a possible pregnancy. Estrogen levels also trigger the developing female reproductive tract, secondary sexual characteristics, spur bone growth and maintain a healthy heart.
The system also has a negative feedback loop which monitors hormone levels and signals the ovaries – and other glands producing hormones – to slow down, speed up or stop. In this way, the system protects the body from too much of a good thing: a woman's lifetime exposure to estrogen, for instance, is the most well-charted risk factor for breast cancer and endometrial cancer.
Such feedback loops protect adults from modest fluctuations in natural hormones. Some scientists believe the endocrine system has evolved over time to handle natural estrogen-like substances produced in plants such as wheat, soybeans, beets, cherries, clover and apples. Synthetic chemicals were introduced too recently and too rapidly for the body to adapt, they say.
Although wildlife and humans ingest infinitesimally small amounts of these chemicals, some contaminants build up in fatty tissues. The contaminant may beat the real hormone to its receptor, preventing normal chemical signals in the body. Once in the womb or at the breast, the critical amount of a hormone-mimicking chemical at a critical time may throw a biological curveball to the developing fetus or newborn. The results – some of which may not surface until the child has reached adolescence – can be profound. Problems in sexual development and reproduction, the immune system, behavior changes, or neurological development may be attributed to endocrine disruption in the formative years.
Endocrine disruption is not a new problem, but scientists are only now unraveling its secrets, says Dr. Henry Anderson, Chief of the State Division of Public Health.
The Greeks described diabetes, an endocrine system disease, but they didn't understand how it occurred. Understanding more about endocrine mechanisms has beneficial uses. For example, birth control pills adjust hormone levels to prevent pregnancies. For the most part, scientists and government regulators examining endocrine disruption have focused their studies on preventing cancer.
"Now we've advanced the understanding that the endocrine system is vulnerable," Anderson said. "It can be altered from exposure to chemicals at levels below our traditional limits to protect people from cancer and gross reproductive effects."
Another new wrinkle, Anderson said, is concern about thousands of chemical compounds that have not been tested for adverse effects.
"There are 70,000 chemicals out there in the environment, and 20,000 of them are still being manufactured," he said. "Pesticides receive more rigorous testing before they are marketed, But at best, fewer than 100 other chemicals have been fully tested, so it is a realistic hypothesis that some of the other compounds have endocrine-disrupting effects."
He serves on an Environmental Protection Agency committee that is developing screening and testing guidelines for endocrine-disrupting chemicals. The group's recommendations are due in January.
Scientists who believe endocrine disruption is more than theory build their case on a handful of human studies in which people were exposed to large amounts of chemicals in prescribed drugs or as a consequence of an environmental spill or accident. Other fieldwork provides what EPA describes as "compelling evidence" that endocrine disruption has occurred in snails, oysters, alligators, other reptiles, and birds such as gulls and eagles.
The strongest proof that hormone-like compounds can disrupt human endocrine systems lies in the experience hundreds of families had with DES. Diethylstilbestrol used to be prescribed in the 1940s, 1950s and 1960s for pregnant women with a history of miscarriages to help them carry babies to full term.
The drug eventually proved to have the opposite effect on many of the expectant mothers. By the time that proof was conclusive, daughters born to these mothers had developed higher rates of a rare vaginal cancer, various genital tract abnormalities, and abnormal pregnancies.
Another key finding was UW-Madison researcher Richard Peterson's discovery in the early 1990s that the male offspring of pregnant laboratory rats exposed to low doses of dioxin developed reproductive problems.
In "Our Stolen Future" Colborn writes that Petersen's work "hit with the shock of an unanticipated asteroid" because it illustrated that low levels of contaminants – similar to those found in the environment rather than the large doses often used in lab experiments – had adverse effects. Dioxin was not just a potential cancer-causer, but could also disrupt the endocrine system and cause development and reproduction problems.
In its February 1997 review of scientific literature, the Environmental Protection Agency found no conclusive evidence that levels of pollutants commonly found in the environment are disrupting human hormone systems. However, there was an ample list of harmful effects observed in aquatic life and wildlife that may be attributed to such exposure.
Female marine snails, for example, are now commonly found with male genitalia, an effect linked to exposure to chemicals used in marine paints on ship hulls. Female fish downstream from Florida pulp and paper mills had developed male sex organs and tried to mate with normal females or each other. In perhaps the best known example of endocrine disruption, alligators living in Lake Apopka, Florida's fourth largest lake, had significantly lower sperm counts and poorer reproductive success after exposure to organochlorine pesticides. The EPA report cites case after case of birds and fish suffering thyroid problems; mammals and shellfish suffering decreased fertility; organisms developing the opposite sexual organs, and birds and mammals whose immune systems have been weakened by exposure to environmental pollutants.
Evidence linking such adverse health effects back to a particular chemical has been more elusive. Many chemical and non-chemical factors like parasites, viruses and habitat destruction could contribute to the findings.
In addition, researchers often don't know if the number and nature of deformities they are observe in wildlife are unusual because their understanding of the endocrine system's workings are so recent, notes DNR's Patnode.
"We're asked to judge whether the changes in hormone balances we are seeing is 'abnormal' when we're still trying to elucidate all the normal structures and conditions of the endocrine system," she said.
Patnode will run a series of tests on the turtle hatchlings during the next few years to assess their overall health and development. After the turtles hatch, she and technician Barb Bodenstein will nestle the turtles onto a bed of wet sphagnum moss for a few weeks, then transfer them to water trays, and finally to a winter home at the Poynette Game Farm.
The scientists will euthanize some of the turtles, analyze their tissues for pathogens and bacteria, check blood for abnormal hormone levels, analyze enzyme levels in turtle livers, and examine reproductive tissues for abnormalities. They'll release the rest of the turtles to the wild in the spring after implanting a microchip the size of a rice grain in their legs so Patnode can track the turtles in coming years and monitor their health and PCB exposure. It's a long process – turtles don't start reproducing until they are eight years old and can live up to 50 years.
The battery of tests and the monitoring will help Patnode gauge the turtles' overall health and test her hypothesis – that endocrine disruption has occurred affecting the hatchlings' sexual development and reproductive ability.
She'll still have to answer the big question.
"Even if we find six ovarian cells in a turtle's testes, what does that mean to the turtle?" Patnode says rhetorically. "Will he be unable to produce functioning sperm? And what does that mean to the turtle population's ability to survive if 10 percent of the male turtles have these abnormal testes?"
Such studies have been common in Wisconsin and other Great Lakes states in recent years because the region has documented a number of cases of dead and deformed fish and wildlife in the 1960s and 1970s, before PCBs and DDT were banned, says DNR toxicologist Mike Meyer.
The Environmental Information Center, a Washington D.C.-based advocacy group, issued a report last June contending that the Great Lakes region legally released to the environment more "endocrine-disrupting" chemicals than any other region in the nation – 17 million pounds in 1995. And most of these chemicals likely remained in the area. Many DNR scientists following the issue said the group's list includes chemicals which scientists don't agree are endocrine disruptors.
Meyer said that most of the wildlife populations across the state are increasing or maintaining their own even though some species in some areas show elevated levels of synthetic chemicals. Regionwide, chemical use and release to the environment continues to drop. "We do have some hot spots – Green Bay, the Sheboygan River area, Superior – and I'm still concerned about the industrial Wisconsin River," he said.
In his seven-year study, Meyer has found that bald eagles living on highly industrialized stretches of a handful of rivers – the middle stretch of the Wisconsin River, the Fox, the Menominee and the Mississippi – had elevated PCB levels in their blood yet reproduced normally. But only six pairs of eagles near Green Bay, which analysis showed had high PCB and DDE levels in their eggs, reproduced at lower rates than eagles living elsewhere in the state.
Also, eggs taken from the nests of osprey with elevated dioxin levels in their blood that were hatched in non-contaminated nests hatched at the same rate as eggs removed from clean sites and subsequently hatched in contaminated nests.
"It's plausible that endocrine disruption could be occurring," said Meyer, who represents the Department of Natural Resources on an EPA work group investigating the issue, "but [the work group believes] it's a phenomenon found primarily in highly contaminated areas. There's no widespread phenomenon in the Great Lakes region or nationally. The hypothesis is plausible, and perhaps the right measurements have just not been made."
The endocrine disruption theory – and how regulatory agencies such as the DNR and EPA should respond to it – has scientists and policymakers choosing sides in recent years. They agree that much more research is needed, but that's about the only thing they agree on. The debate and the evidence haven't spurred changes in the way we regulate pollutants.
Colborn calls for worldwide negotiations and agreements to stop producing and using compounds like DDT and PCBs that persist in the environment and act as endocrine disruptors.
She also advocates pulling other chemicals from the marketplace which have been shown to change hormonal levels until those effects are proven to be trivial.
She stressed changing manufacturing processes to avoid using these chemicals or developing less hazardous synthetic substitutes.
Scientists at the other end of the spectrum argue that we shouldn't worry. Humans are exposed to such large amount of natural hormones in the foods we eat – wheat, oats, soybeans, potatoes and coffee, just to name a few – that manmade chemicals that mimic substances like estrogen are hardly cause for concern.
Patnode for one, falls in between. She thinks there are compounds regulators need to be concerned about because they are causing the most serious problems and are most difficult for humans and wildlife to eliminate from their bodies. Many compounds, like PCBs and DDT, have known consequences and are banned in the United States, but are still produced and used elsewhere and persist in the global environment. Patnode believes any broad-brush ban on synthetic chemicals is premature and may backfire. If research reveals certain chemicals have been wrongly implicated, then industry and the public will be increasingly skeptical and less cooperative, she says.
"The Chicken Little approach doesn't work," she said. "You need to have well-substantiated evidence that compound X or mixture XY has this effect at levels present in the environment, and that the disruption harms the individual and the population. We don't have that information now for many environmental contaminants."
Already, a backlash seems to be building against endocrine disruption hypotheses. "Another Enviro-Scare Debunked" screamed a headline in the August 20, 1997, Wall Street Journal. "Forget the Frenzy" said the Milwaukee Journal Sentinel about the same time. The stories detailed retractions in July of a study printed in the magazine Science that purported to show that chemicals which had weak estrogenic effects on their own were up to 1,600 times more active when combined. The Tulane researchers withdrew that study after they – and a host of other scientists who had rushed out to test the compounds – were unable to replicate the results.
But that too, illustrates an extreme position.
There are concerted efforts to provide some solid scientific answers.
EPA's Office of Research and Development includes endocrine disruptors as a high priority research issue, and President Clinton's National Science and Technology Council is coordinating research efforts by the EPA and other government agencies. The National Academy of Sciences is conducting a more extensive review of the scientific literature. And last September, the EPA and U.S. Dept. of Health and Human Services announced they were creating a federal research center dedicated to protecting children's health from environmental threats.
In the face of the uncertainty, Anderson and Patnode recommend that the public follow some simple steps to minimize the environmental risks we are aware of as the research continues: use chemicals sparingly and carefully, wash fruits and vegetables before preparing them, follow advice offered in fish consumption advisories and eat a variety of foods so no one food group forms too large a part of one's diet. Like handling so many of life's unknowns, minimizing problems from endocrine disruptors may include heeding a bit of your mother's advice: all things in moderation.
Patnode will continue her work, tracking turtles and examining how chemical contaminants may affect animals.
"We've done a good job giving people warnings and information to judge whether they want to eat fish and wildlife that may be contaminated," Patnode said. "It's time to concentrate on how these contaminants may affect wildlife itself. If we can determine what level of contamination causes subtle health effects, then we can give better guidance on how clean the environment needs to be to protect animals as well as people."
Lisa Gaumnitz tracks environmental trends and events as DNR's public affairs manager for the Division of Enforcement and Science programs.