Gender-bending Water

Drive south on Calgary’s Macleod Trail. Catch the left onto 194th Ave SE and follow it down into the valley toward the Blue Devil golf course. Before you get there, turn right onto an unnamed road and drive to the end. That low hill you pass off to the left just before the road ends may look like no more than—what, weirdly gentle toboggan runs? As for the facility just ahead, its circular tanks, like oversize above-ground swimming pools, give it away as a sewage plant. This spot is a scientific jewel? A lab unique not only in Alberta or even all of Canada, but the entire world?

Yup.

Only here can certain kinds of research be conducted under those rarest of conditions: a natural setting completely controlled to the minutest detail by scientists. For the quest being undertaken here, new tools are imperative. To the scientists who spent the summer of 2014 inaugurating this unobtrusive facility, it may even be a breakthrough weapon in a two-decade-long pursuit of a will-o’-the-wisp guerrilla army that many hold responsible for a global epidemic of foreboding reproductive weirdness. Welcome to a world of deformed penises, hermaphrodite bears, intersex fish and missing boys.

Something strange has been happening to the male of the species—ours and others—and suspicion centres on the vast spectrum of chemicals that leak into the environment from a million points across the 21st century economy. But the suspect molecules operate in concentrations of one in a million or more other molecules. “Smoking gun” scientific proof of any one compound’s direct impact on a human life is proving elusive. And here is where this out-of-the-way hillside shines.

Those “toboggan runs” are, in fact, the centrepiece of a $38.5-million laboratory, a joint initiative between the City and the University of Calgary. Its grammatically awkward name is “Advancing Canadian Wastewater Assets,” which sounds better spoken aloud as an acronym: ACWA, an obvious play on “aqua.” Twelve 350 m artificial streams, in which riffles alternate with quieter pools, lie in two sets of facing J-shaped pairs. They are home to an expanding community of insect, plant and algal life, and are fed from large pipes emerging from one of the two utilitarian buildings at the top of the constructed hill. The water from these pipes can be doctored to precisely calibrated mixtures of almost anything. Every inch of the streams is accessible to researchers. For the biologists and other specialists trying to discern how a minute trace of the wrong chemical in the water can totally mess up a young minnow’s sex life—and may be doing the same to human boys—acquiring ACWA is a bit like getting their own Large Hadron Collider, that vastly expensive installation beneath the French–Swiss border built to sniff out the semi-mythical subatomic Higgs boson.

Welcome to a world of deformed penises, hermaphrodite bears, intersex fish and missing boys.

Admittedly, the targets they’re chasing here are larger, relatively speaking, than the boson, but they are equally invisible and a lot more frightening. They run a chemical gamut from incidental residues from petroleum production to highly evolved pesticides and even the supposedly benign ingredients in skin cream. They also include perfectly natural chemicals. What they all have in common is the ability to trigger the endocrine system, that finely tuned chemical network that organizes the development and ongoing bodily functions of every form of complex life.

Natural hormones—estrogen, testosterone, androgen—are examples. But the human body continuously secretes scores of others as well, to control every aspect of life, from organizing fetal cells to timing the onset of puberty. It is an artifact of evolution that the chemical codes in this signalling system are largely the same among all vertebrates, that is, all animals with spines: frogs, minnows, us. But in the last few decades, hundreds of thousands of new chemicals have been invented and put to use that closely mimic that natural signalling code. When those chemicals enter a body, its processes easily mistake the stray synthetic signal for the real thing, setting off biological mayhem. For that reason, these contaminants are known collectively as EDCs, for “endocrine disrupting chemicals.”

A startling number of these escape into the environment wherever humans or their livestock congregate. Birth-control pills are concentrated hormones; not all of their active EDCs are metabolized on use. They, other pharmaceuticals, and cosmetics washed off in the shower collect in municipal wastewater streams. Sewage treatment removes many, but not all of them. Some of the most potent EDCs are literally spread over the ground: Herbicides in wide farm and home use rely on endocrine disruption for their effect. More EDCs reach Alberta’s rivers, especially in the south, from animal feedlots. Cattle (as well as pigs and chickens) not only release natural hormones of their own but often receive additional EDCs in growth stimulants or veterinary drugs. Residual amounts pass through into their manure, which is usually disposed of on nearby fields. It is the equivalent of the entire population of Canada living in this province—without benefit of sewage treatment.

Some EDCs, like polychlorinated biphenyls (PCBs), can persist in the environment for decades. Most begin to break down quickly. Even so, enough hang around to turn up in virtually every Alberta waterway tested—as well as in most other lakes and rivers across the country. And even their breakdown may not entirely end their threat. Fragments of decayed EDCs have been found to reassemble in the wild, raising the unsettling prospect of the unscripted mixing and matching of powerful molecular snippets into entirely new compounds. Familiar or novel, they’re believed to be responsible for some decidedly strange stuff.

Around the world, wildlife biologists have been disturbed by parallel trends toward gender confusion in scores of species. Nearly half of Florida’s cane toads studied turned out to be hermaphrodites; so did a smaller but still unexpected percentage of Canadian polar bears (in a study one imagines was undertaken with extreme caution). One British summary reported abnormal testes in fish, turtles, bears, sea lions, whales and panthers. Otherwise-male fish, birds and amphibians were producing proteins that normally precede egg-laying in females.

That has been the case in Alberta among long-nosed dace, a kind of minnow that lives in the Bow, Oldman and Red Deer rivers. Downstream from where treated municipal waste enters the river, or from concentrated livestock feeding operations, not only have “male” fish shown signs of preparing for ovulation, they are hard to find at all. In some river pools, female minnows outnumber males by about 10 to 1. Populations upstream of the waste releases show the normal, roughly 1:1, sex ratio.

A similarly global, if much less dramatic, shift in the traditional human birth ratio of roughly 105 baby boys to every 100 girls, is a sign to many researchers that EDCs are messing with human reproductive norms as well. Examining records back to the 1930s, researchers Theo Colborn and Lynne Carroll concluded in 2007 that human “fertility, reproductive success and male/female live birth ratio are declining” all across the industrialized world. In Canada, they reported that “[a] clear shift…began to appear in 1970.” By the mid-1990s that drift had cut the male edge in birth numbers by nearly half, from five extra boys per 100 girls, to only two to three.

On this count Alberta may have dodged a chemical bullet: Birth ratios have remained stable here, at about the traditional 105 boys to 100 girls, since 1989. Across Canada, however, the male share of births has continued to fall. The implication: Hundreds of Canadian infants who might once have been born as boys are entering life as girls. On a global basis, the number of “missing” boys is believed to have surpassed a quarter million per year.

That’s not the end of unsettling parallels between what’s being seen in the wild and what’s turning up in delivery rooms. Researchers in several countries have documented an increase in cases of undescended testicles and of hypospadias—a defect in which the opening of the urethra is located not at the tip of the penis but partway down or even at the base of the shaft. Hypospadias occurs in some 110 births a year in Alberta, a number that’s up by 20 per cent since 1997 (the condition is correctable by outpatient surgery, usually before the age of two). Sperm production has also dropped off by more than a third (41 per cent) in the last half century among men in industrial countries. Testicular and prostate cancer rates, on the other hand, have doubled since the 1970s. “The basic male toolkit,” as one researcher quipped, “is under threat.”

The weirdness also extends to those with XX chromosomes. The World Health Organization in 2012 drew suspected links between EDCs in the environment and rising world rates of “endocrine-related” cancers of the breast, the endometrium (lining of the womb) and ovaries; earlier onset of breast development; difficulties conceiving; and “adverse pregnancy outcomes.”

In the US, controversy has swirled around the outsize figure of California biologist Tyrone Hayes. Hayes brought attention to himself when, at the request of the manufacturer of a product called atrazine, he began exploring its influence on his specialty, the health of frogs. Atrazine is one of the most widely used agricultural herbicides in the US and has long been approved; Hayes expected simply to confirm its clean bill of safety. What he discovered shocked him: As many as 20 per cent of tadpoles exposed to atrazine at levels well below US guidelines developed into frogs with multiple gonads (as many as six sets in one frog) or were hermaphrodites with both testes and ovaries. Subsequent research by others confirmed those effects, and similar ones on salmon, caiman alligators and rats. Then Hayes drew a provocative conclusion. In a paper co-written with more than a score of other scientists, Hayes presented evidence that “the effects of atrazine… are consistent across all vertebrate classes… Atrazine demasculinizes male gonads… in… fish, amphibians, reptiles, and mammals… These effects are strong (statistically significant), consistent across vertebrate classes, and specific.”

And mammals. In other words, the gender-bending seen in frogs probably occurs in humans too. There is some evidence to back up the proposition. Missouri men who lived in rural areas where atrazine was heavily used had poorer sperm quality than men in urban areas. French babies exposed to atrazine weighed on average five ounces less than babies not exposed. Boys born in parts of Texas where atrazine is present showed a “modest but consistent” higher likelihood of genital defects. Hayes’s claims set off a race among laboratories to test his findings. It also attracted a coordinated effort by atrazine’s maker—Swiss-based Syngenta—to discredit him. Cut from different cloth than the average lab rat—he wears a diamond ear stud and for public appearances affects a style reminiscent of soul singer James Brown—Hayes responded in part by firing back at the company in rhyming, rap-style emails. When these became public in 2010, his campaigning suffered a setback.

But the Albertan who’s best qualified to judge Hayes’s science, newly retired University of Calgary herpetologist Anthony Russell (he literally wrote the book: The Amphibians and Reptiles of Alberta, published in 2000), finds nothing to fault in Hayes’s basic findings. “Tyrone is a very flamboyant figure” who makes tamer scientists uncomfortable, Russell says. But when it comes to frogs, Hayes “knows his stuff. The controversy comes because he’s projected it [atrazine’s gender-bending effects] to human populations.”

Which raises the big question hovering over the science of EDCs, the one Calgary’s new multi-million dollar experimental streams were built to help answer: Are EDCs putting human sexuality in the blender along with that of frogs and minnows?

Here is what we know. That certain molecules interfere with the human endocrine system is beyond scientific dispute. Imagine that system as like the modern communications web that connects and coordinates every part of a community or business. Because it operates by making molecule-to-molecule bonds with cell receptors, it takes only a relative handful of such molecules to complete their signal. When nearly identical molecules—natural or man-made—enter the body, they result in the biological equivalent of a plague of dropped calls and wrong connections. With the normal hormonal command structure disrupted, bodily processes turn on or off at the wrong time, organs get confused about what they’re meant to do. Laboratory frogs develop both testes and ovaries.

In the last few decades, hundreds of thousands of chemicals have been invented. When they enter a body, they can set off biological mayhem.

We know as well that many EDCs are present in what we naively still call “nature.” Atrazine is effectively ubiquitous in US groundwater. In 25 of 28 US cities tested, tap water contained pharmaceuticals from mood stabilizers to anticonvulsants. Philadelphia’s taps flowed a virtual cocktail of more than 50 drug traces.

And here? Alberta Environment has sampled water from several spots along the North Saskatchewan and Bow rivers in alternating years for a decade (a task now transferred to the new Alberta Environmental Monitoring, Evaluation and Reporting Agency). The sampled water has revealed everything from the familiar—acetaminophen (Tylenol) and ibuprofen (Advil)—to compounds largely unknown beyond specialist circles. Among those that have turned up repeatedly:
– Nonylphenol tetraethoxylate: used in dish detergents, paints and lube oils, packaging and personal care products.
– Sulfamethoxazole: a “sulfa drug” antibiotic often used to treat urinary tract infections.
– Gemfibrozil: prescribed to raise “good” cholesterol in blood and lower “bad.”
– Galoxolide: a synthetic musk used in colognes and perfumes for its “clean, sweet, musky, flowery, woody odour.”
– Benzoylecgonine: a primary metabolite from cocaine use.

Separately, Environment Canada found pesticide or herbicide residues in wetlands, rivers and reservoirs across the prairies. Four compounds appeared in more than 80 per cent of samples: 2,4-D, MCPA (2-methyl-4-chlorophenoxyacetic acid), clopyralid and dicamba. All combat various broadleaf weeds using endocrine-like chemicals that speed up or otherwise disrupt growth until the plants die. The first two chemicals appeared in every wetland and 99 per cent of reservoir water tested. The survey also looked for atrazine, finding it in none of the wetlands but more than a quarter of prairie water reservoirs.

We also know that EDCs are affecting wildlife. Research in Alberta and beyond has clearly associated gender defects and skewed sex ratios in aquatic or amphibian species with a heightened presence of EDCs. The feminizing effect in long-nosed dace has been found as well in fathead minnows downstream from Lethbridge. Similar effects were found in a Saskatchewan creek whose flow in winter is mainly treated wastewater from the city of Regina.

One of the clearest demonstrations that EDCs from pharmaceuticals can impact aquatic life occurred in the early 2000s at the country’s closest equivalent to ACWA’s experimental streams: the Experimental Lakes Area of northern Ontario. There, scientists manipulated conditions in entire lakes. At Lake 260, Karen Kidd and her associates monitored the population of fathead minnows while injecting EE2 (17α-ethynylestradiol), a synthetic hormone used in birth-control pills, into its water until concentrations reached those typically found downstream from waste treatment plants. They did this for three years. In year one, male fathead minnows began showing signs of feminization. In year two, the fathead minnow population collapsed. By year three, Kidd says, the disruption of male function “basically resulted in the near extinction of a fish species in the lake.”

After the experimenters stopped adding EE2 to Lake 260’s water, its fathead minnows rebounded. To Kidd, that is further evidence, along with similar accounts of species that have recovered after toxins were removed from their environment, that endocrine contaminants are affecting wildlife.

Could the EDCs turning frog princes into princesses be doing the same thing to human males?

The endocrine system’s hormonal codes date from early in life’s evolution. As a result, “all vertebrates have similar sex hormone receptors,” observes Gwynne Lyons, who summarized the literature on EDC effects for CHEM Trust, a UK non-profit. In other words, the chemical codes that provide the prenatal blueprint for building a proper penis, or that signal a girl’s breasts to begin swelling at puberty, are pretty much the same ones that every other critter with a backbone uses, from frogs to fathead minnows to, yes, polar bears.

It seems plausible. And yet, unequivocal proof that nonylphenol tetraethoxylate, or 2,4-D, or anything else left over in the water, produced this stillbirth or that deformed genitalia or the cluster over there of breast or testes cancers is frustratingly elusive.

“Because wildlife species and humans share some similarities in their endocrine system, there is potential for chemicals to impact humans as they do for wildlife,” says Kidd, who was a lead editor of the 2012 WHO study. “There’s all the evidence of increases in endocrine cancers and diseases of the endocrine system or disorders as well. But linking that to a specific chemical exposure, knowing what their contribution is to human diseases, is really difficult.”

It’s not for lack of trying. Hundreds of thousands of potentially disruptive molecules out there, some known, others unimagined, all invisible, occur in vanishingly tiny amounts in water.
Galoxolide, the synthetic musk perfume, has been found in the Bow River in concentrations as high as 0.19 micrograms per litre—roughly the equivalent of a teaspoon in a municipal water tower. Most contaminants clock in at even less. Then again, if the foreign molecule bonds to a human cell’s receptors at just the right time, it takes only a little to create “a non-linear response,” as the researchers say. Of course, scientists can’t just subject people to suspect compounds at critical moments such as conception or puberty and monitor their response in the lab. Instead they have to infer causal connections from studying events in the outside world. And that world is, in scientific terms, a “noisy” place. Isolating one influence from among the unfathomable variety of others, and proving its presence at a time and place when its effect may have occurred, makes needles in haystacks look like child’s play.

Dr. R. Brian Lowry should know. Nearing retirement, the Calgary epidemiologist made his own contribution to Alberta’s science assets: a central record of birth defects extending back to the 1980s. To that record we owe the knowledge that Alberta’s boys share in the global epidemic of hypospadias, and that compared to other places, more Alberta infants are born missing parts of limbs. We also know that certain birth defects have turned up in clusters, suggesting perhaps an environmental influence. Lowry has spent much of his otherwise illustrious 50-year career in a generally fruitless search for an explanation for these clusters.

His voice betrays a certain wistfulness as he describes another one that got away: microtia, a deformity in which the outer ear fails to develop. “We had an interesting cluster a few years back. Four cases, all born in the same month, in the same hospital, [delivered] by the same obstetrician. I investigated them all and examined the parents and looked into their occupations and their risk factors and found nothing to correlate these four. And then the numbers fell away again.” Evidence of a correlation has slipped through his fingers repeatedly.

And here, finally, is where ACWA’s research director, U of C biologist Leland Jackson, has high hopes for his unprepossessing “toboggan runs” in southeast Calgary. Each of its dozen streambeds can be fed independently with water from a selection of sources. Early in 2014 they were gurgling with water straight from the Bow River. But they can also be fed in whole or part from the treated effluent released by the neighbouring Pine Creek Wastewater Treatment plant (a gold-plated facility commissioned in 2010), or from a second, small-city-size treatment plant that is part of ACWA’s laboratory and which is designed to test next-generation approaches to sanitizing sewage, or with some combination of these laced with any contaminant of interest to researchers. The facility can host up to two experiments at a time, each running in three “control” and three “test” channels for greater reliability. EDCs aren’t the only interest. ACWA has a lineup of researchers also interested in whether trace antibiotics in municipal sewage are breeding new drug-resistant “superbugs,” as well as engineers eager to test ideas for addressing those and other problems.

But the prospect of bringing new scientific insight to the elusive mechanisms that give EDCs their power keeps Jackson excited to come to work. His lab conducted the early research that associated municipal and feedlot effluent with girls-only schools of long-nosed dace. Now he wants to close that last step in the logic chain, to get to “understanding how to translate what’s happening to long-nosed dace, to people.” ACWA’s streams will let him probe for cause-and-effect connections between precisely known contaminants and sex biases, feminization and other effects in test fish. Once those are established, anatomical and cellular examinations may at last reveal, down to the precise molecular address or genetic switch, just what is going on.

Until then, Jackson says, Albertans “should continue to question what’s in the water, what fish like long-nosed dace are telling us about effects on people.” But they needn’t stay awake nights worrying about what’s coming out of their tap. Not yet, anyway. “I drink City of Calgary water,” he laughs. “I drink it and I like it and I don’t worry about what’s going to happen.”

Chris Wood is co-author of Down the Drain: How We Are Failing to Protect Our Water Resources (Greystone)