At first, it looks like a fishing expedition. But the Styrofoam cooler graduate student Cynthia Gill carries is not stocked with bait and beer, and she is carrying a long-clawed hammer rather than a fishing pole. Our quarry is long-toed salamanders, and our hunting ground is a small, ice-covered pond about 10 miles northwest of Pullman. The hammer is for breaking the ice.

Our haul that day is 68 male salamanders and six females, and it’s not an easy catch. Although the animals are confined by plastic traps, they are slippery beasts to grab and hold onto, especially with ice-cold hands. But they are lovely–shiny and dark in color, some with bright olive green stripes down the center of their backs. Salamanders are amphibians, and there is worldwide concern about an apparent steep drop in their numbers. Amphibian populations, even entire species, have declined, some to extinction. The Costa Rican golden toad hasn’t been seen since 1989.

“Amphibians are the canaries in the coal mine for the environment,” says Paul Verrell, Gill’s advisor and an associate professor in the School of Biological Sciences at washington State University and the Center for Reproductive Biology, a research unit based at WSU and the University of Idaho.

Most amphibian species spend part of their lives in the water and part on land and are exposed to contaminants in both places. Their soft, permeable skins also make them especially vulnerable to contaminants. Most are predators, and if environmental chemicals accumulate or concentrate as they move up through the food chain, they will hit predators the hardest.

The problem comes in deciphering just what these amphibian “canaries” may be telling us, for their decline undoubtedly has many causes. The most obvious is habitat loss due to our expanding human population. Non-native predators and competitors, introduced on purpose or arriving by accident, also play an important role. But there are declines in places where neither of these have happened–”mystery declines”–that could be due to one or a combination of toxic chemicals, UV radiation, global climate change, or disease, says Andrew Storfer, assistant professor of biological sciences.

Determining cause-and-effect relationships is difficult at best, controversial for certain–especially when we’re looking at chemicals. Even if laboratory studies show that a chemical has negative effects on an animal, it’s difficult to correlate that information with exposure in the animal’s natural environment–or even to know what a relevant environmental exposure level might be.

Although early research on the effects of chemicals on wildlife and humans concentrated on testing for cancer-causing properties, more and more research now is focused on looking for developmental and reproductive abnormalities. This change comes in part out of concern for the wild species, since in some fish and wildlife species there is strong evidence that chemicals found in the animals’ habitats may cause these problems. But we are primarily concerned about the possible effects on humans.

Obviously, humans as a species need not yet worry about reproductive fitness or species survival. However, work aimed at determining if and how environmental chemicals cause abnormalities in our reproductive systems should lead to better a understanding of those systems and help foster the discovery of safer, more effective and affordable methods of controlling our own population.

Verrell’s work with salamanders such as those Gill and I collected that cold March day is aimed at understanding their reproductive biology, their sexual behavior in particular. He would like to understand why some individuals are more successful reproducers than others. He also is involved in a wide-ranging project at the Center for Reproductive Biology, a research unit based at Washington State University and the University of Idaho. One of the center’s programs is aimed at investigating how environmental toxins, especially those that disrupt the endocrine system, affect reproduction. (See sidebar.)

The endocrine system helps regulate the body’s activities via hormones, extremely sensitive chemical messengers that are secreted by specialized cells and travel throughout the body in the bloodstream. The timing of their secretion and delivery is critical and carefully orchestrated. While adult mammals have feedback mechanisms that can cope with at least some variation in hormone levels, those mechanisms may not be active during embryonic development, a time at which it is thought animals are most susceptible to the effects of endocrine disrupters.

Of particular interest to the Center for Reproductive Biology is the hormonal regulation of the basic processes of reproduction. Estrogen-like hormones primarily regulate the female reproductive system, and androgenic hormones primarily regulate the male system. Both, however, play at least some role at critical times in the development of the reproductive system of the opposite sex.

Endocrine disrupters interfere with the normal interactions of the hormones and their targets. They may do so by preventing the interaction (anti-estrogenic or anti-androgenic) or by mimicking the interactions, and producing effects at inappropriate, usually earlier, times.

It’s clear that some wildlife populations have developmental and reproductive system abnormalities that may be related to endocrine-disrupting chemicals. The most well-known are the alligators of Florida’s Lake Apopka. A variety of endocrine-related abnormalities were reported following a large chemical spill into the lake. The majority of male alligators were found to be feminized, and the females, super-feminized. In addition, hatching rates for alligator eggs were substantially lower than normal.

Some of the chemicals that are believed to affect animals in their natural environments have been shown to cause similar effects in laboratory animals. Some chemicals that have been linked to developmental and reproductive system abnormalities in wildlife already have been identified and banned. For other chemicals there are no cause-and-effect data.

In humans, evidence points to a number of problems related to fertility and reproduction. The Environmental Protection Agency (EPA) says that the rates of breast, prostate, and testicular cancer have risen, and that male fertility has been reduced. Although the endocrine system seems a logical place to look for causes, the EPA also says that except in the case of DDT/DDE, DES, and dioxin, it has not been shown that endocrine disrupters are causing problems for humans. The National Academy of Sciences says that even these chemicals have not been shown to affect humans. DDT/DDE, DES, and dioxin have all been banned in the United States or are heavily regulated.

It also is important to note that many if not all of the environmental estrogens, except DES, are much, much less active in our bodies than the natural estrogens we produce.

Verrell first became interested in endocrine disrupters as possible tools for helping to dissect various aspects of reproductive behavior. If a disrupter knocked out sperm production, then it might be possible to determine the effects of courtship on reproductive success without the associated complications of an actual mating. But he soon realized that these compounds were interesting in their own right. After Rolf Ingerman, professor of biological sciences at U I, found that methoxychlor appears to partially paralyze salamander larvae, Verrell wondered whether it would make them more vulnerable to predation. While predation does not directly affect reproduction nor directly involve the endocrine system, it does have an impact on species survival. A dead salamander larva is unlikely to contribute to its species’ survival.

The answer to Verrell’s question is “yes,” at least if the predators are juvenile dragon flies, voracious natural predators of salamander larvae. When both the salamander larvae and the juvenile dragon flies are exposed to me
thoxychlor, a widely used pesticide, in a concentration similar to what might be found at certain times of year in streams or wetlands, the salamander larvae show more sensitivity than the dragon flies. That suggests that larvae in areas treated with methoxychlor are more likely to be eaten than larvae in other areas.

However, since estrogen alone does not produce the same results, it seems unlikely that this particular effect of methoxychlor is due to its endocrine disrupter properties, says Verrell.

In reality, there is little known about the effects of methoxychlor or vinclozolin–or any other endocrine disrupter–on amphibians, says Verrell. Given the concern about their declining populations, he finds that a bit puzzling. But it is known that when male long-toed salamanders like those he studies are exposed to estrogen, there are more dead cells in their testis than in those of unexposed animals. Dead cells make neither sperm nor natural androgens. Since methoxychlor has estrogenic properties, it might have the same effect, but those experiments haven’t been done yet–in amphibians. Similar ones have, however, been done in fish, on embryonic rainbow trout.

Joseph Cloud, professor of biological sciences at U I, has found that methoxychlor does not feminize embryonic rainbow trout or cause it to reverse sex. If that also is the case for chinook salmon, then scientists will have to find an alternative explanation for what’s happening to some of the Columbia River chinook.

Although some runs of wild salmon appear far from threatened, other runs of wild salmon and steelhead on the Columbia River are teetering on the brink of extinction. Thinking that reproductive problems might be part of the cause, James Nagler, associate professor of zoology at U I, with the help of Gary Thorgaard, professor in WSU’s School of Biological Sciences, investigated whether the chinooks’ genotypes–the content of their chromosomes–were the same as their phenotypes–how they looked or behaved. The two had the necessary tool, a marker that recognizes a portion of the Y chromosome, and an appropriate location, the Hanford Reach area of the Columbia, home of one of the river’s only two healthy runs of chinook. Y chromosomes usually define the male animals of a species. Males have one each of the X and Y chromosomes, while females have two X chromosomes. Thorgaard found that 84 percent of the fish that were functioning as females and laying eggs actually carried the marker, implying that they had a Y chromosome and were in fact genotypically male. Yet all of the female fish from the nearby Priest Rapids hatchery were genotypically normal females with two X chromosomes. The Hanford Reach fish and Priest Rapids hatchery fish share essentially the same habitat throughout life once they’ve hatched and headed down the Columbia River.

Several possible explanations for the anomaly were proposed, some of which were discounted. Since the hatchery fish were derived from Hanford Reach fish and the two runs appear to be otherwise genetically identical, it seems unlikely that the bit of the Y chromosome recognized by the marker has moved to another chromosome only in the Hanford Reach fish. It also seems unlikely that radiation is to blame. The dosages the fish might have received would have been small, and radiation usually sterilizes fish rather than changing their sex.

The main difference between the two populations was the water that they were raised in–the Columbia for the wild fish, well water for the hatchery fish. Since incubation temperature can affect the sex of developing embryos of several species of cold-blooded vertebrates, it’s unfortunate that the temperatures of these two water sources during the fish egg incubation are not known.

The story becomes more interesting with data from the following two years. The marker continued to be found on functionally female fish from the Hanford Reach, though the incidence decreased. Female Priest Rapids hatchery fish carrying the marker also have been found, as have female fish from farther down the Columbia.

Sex reversal does appear to occur naturally in trout and salmon, but the frequency is low, says Thorgaard. In some fish species, natural sex reversal is thought to be a means of maximizing offspring. Larger females produce more eggs, but even small males can mate. Conversely, if a dominant male fish dies or there are few males, a female might change into a male and still mate. Sex reversal happens in the laboratory if male chinook or other male salmonids are exposed to estrogenic hormones or estrogenic endocrine disrupters at a critical time near hatching. This type of manipulation has been used as a hatchery management tool for some years.

Several estrogenic compounds can be detected throughout the year in the Columbia. Some undoubtedly come from agricultural chemicals, some from upstream pharmaceuticals, and some from pregnant women who live upstream. The latter secrete estrogen in large amounts. It is not known if the concentration in the river of any one of these compounds is sufficient to cause sex reversal, nor how they may or may not work together to affect the endocrine system of the fish.

“We really have no idea of . . . what might be going on out there,” says Thorgaard. But we know that a similar study in California’s Sacramento River suggests that the same thing has happened there. And we know that the city of Richland gets its drinking water from the Columbia River.

At this time, there doesn’t appear to be a problem with respect to the numbers of functioning male and female fish in the Hanford Reach. But that could change. Since each parent in a mating gives one of its chromosomes to an offspring, the mating of an XY female and an XY male should produce fertilized eggs that are YY as well as XY and XX. While it’s unknown at this time if YY chinook can survive, it is known that YY rainbow trout and coho salmon do and that they are sexually viable. If a YY mates with any kind of female, whether XX or XY, all the offspring would be male, for all would have a Y chromosome. An all-male population will have difficulty finding anyone to mate with.

Nagler and Thorgaard currently are looking for YY males in the Hanford Reach as well as considering possible causes for the genotype-phenotype anomaly. Given the close association between endocrines in both the development and functioning of the reproductive system, it certainly seems reasonable to investigate endocrine disrupters as a cause. But it’s difficult to determine where any disrupters might come from–pharmaceuticals, chemicals, or natural sources. And it’s difficult to connect even a known endocrine disrupter to what we see happening in the environment, says Allan Felsot, professor of entomology and environmental toxicologist at WSU Tri-Cities.

“We make a mistake when we equate hazardous residues in the environment and their risk of adverse effect,” says Felsot.

Identifying substances that are hazardous in laboratory experiments is just the first step in assessing their risk in the environment. After that comes dose-response assessment, exposure assessment, and risk characterization. All have to be completed in order to relate what is learned in the laboratory to wildlife and humans.

Establishing a dose-response relationship involves characterizing the relationship between the amount of compound and how often it causes an adverse effect and how strong that effect is. Exposure assessment involves determining how much of a hazardous compound actually is in the air, water, plants, or other places, and then whether those amounts are relevant for how an animal might be exposed to them. Exposure assessment also involves determining how reactive the compound is, how long it remains in the environment, and what happens to it as it degrades.

The final step in the process is risk assessment. Interestingly, this has a non-scientific element that is determined by what we as a society define as safe, says Felsot. The EPA may take the highest dose of a pesticide that
produces no effect in experimental animals and call that the allowable exposure. Then it builds in a safety factor that at present may be so high that the allowable exposure for humans is 1/100 of that animal dose.

While this process cannot guarantee that there won’t be unforeseen problems with a chemical in the future, it does offer some protection. Unfortunately, no such process offers protection from pharmaceuticals or naturally produced hormones that pass through wastewater treatment facilities.

Richard Bull, professor of environmental science at WSU Tri-Cities, has spent a large part of his scientific career involved with various aspects of drinking water and water treatment. Among his concerns is a worry that the focus on pesticides may result in our not looking in other places that contribute more important endocrine-active compounds–such as wastewater. “In all probability, if there are environmental impacts, these are the estrogens that are important,” he says. And there are a lot of them. They come from pharmaceuticals such as birth control pills and from hormones secreted by pregnant women and other animals. The metabolites of natural estrogens are likely to remain in the water column, he says, while chemicals like methoxychlor that are not highly water soluble are more likely to become attached to sediments in the water and be removed during drinking water treatment.

A second concern comes from the increasing dependence upon municipal wastewater as a drinking water source that is being contemplated in many parts of the country, including southern California. A third is related to the effects of intensive agriculture, an issue here in Washington. Intensive agriculture is affecting the environment right now, and there may be human effects down the line, he says. Effluents from feedlots contain biologically active amounts of androgens that are released slowly from androgen pellets attached to the ears of cattle to increase growth. In dairy farms, there are high concentrations of natural estrogens.

On the other hand, Bull is concerned that the focus on endocrine disruption as a cause of reproductive problems will result in our ignoring other explanations. If there has been a decline in human male fertility as some suggest, there will be multiple causes, and the focus on endocrine disrupters may bias research that is needed to identify other causes, he says. He feels that the focus should be on reproductive and developmental effects. “This broader approach will catch the endocrine disrupters if they’re responsible,” he says.

Kim’s work deals with the effects of environmental chemicals on the embryo during its development, but, as is the case for Verrell, the effects she studies are not directly related to endocrine disruption. They are, however, directly related to reproduction, for she focuses on substances critical for sperm production: vitamin A and the molecules it interacts with in specific cells within the testis.

Kim’s work has shown that vitamin A binds or attaches to a specific molecule in some types of testis cells. The molecule, a receptor, then moves into the cell’s nucleus. Once there, it binds to the DNA and leads to a change in the expression of genes and the making of proteins.

Phthalates are known to be toxic to testicular cells, to be endocrine disrupters, and to cause liver cancer. Kim is interested in how they affect the interaction of vitamin A and its receptor. She has found that phthalates interfere with the movement of the receptor into the nucleus. If pregnant female rats are fed phthalates for just five days during a specific time of embryonic development, then the mature male offspring will have empty or partly empty testes.

Preliminary results suggest that the effects of endocrine disrupters also may be passed to the next generation. When both the male and female in a mating have been exposed to methoxychlor as embryos, their offspring may have problems. Over generations, a population living in a small area regularly exposed to methoxychlor would have difficulty remaining viable, says Skinner.

The same might well be the case for some birds. Preliminary work by Hubert Schwabl, associate professor of zoology at WSU, Victor Eroschenko, professor of zoology at UI, and post-doctoral researcher Rosemary Strasser suggests that endocrine disrupters reduce the reproductive success of Japanese quail. They do so by interfering with the development of the female reproductive tract and of the part of the brain that deals with reproductive function.

The researchers treated developing quail eggs with methoxychlor and one of its breakdown products at concentrations similar to what can be found in the eggs of wild birds. They found that the treated eggs didn’t hatch as well as those that weren’t treated, either hatching later or not at all. Interestingly, in the latter case, the embryos were alive and looked normal at the time they should have begun the hatching process, says Strasser. Female birds hatched from the treated eggs were smaller and lighter than those from untreated eggs. Many failed to lay eggs when they matured, and upon examination showed a second oviduct not seen in untreated birds. The eggs of those that did lay were uniformly medium in size. Normal, untreated birds lay eggs in a range of sizes. The eggs of treated birds also had weaker shells, one of the primary reasons that the use of DDT was severely reduced in the United States and that the bald eagle almost became extinct.

Male birds hatched from the treated eggs looked physically normal but didn’t act that way. They had a markedly reduced interest in mating with females, whether measured as attempts to mount or actual copulations. When presented with a normal male, they showed none of the normal aggression or “tough guy” routine, says Strasser. Given a choice, the treated birds actually preferred being alone and having nothing to do with either sex of bird. In another experiment, normal males actually were likely to mount the treated males, suggesting that the latter showed some feminine characteristics.

Gill and I collected our salamanders from that iced-over pond north of Pullman during their breeding season, at which time they leave their usual home, burrow on land, and venture down to local ponds or slow-moving streams. (If you think ice water seems an inauspicious place for a cold-blooded animal to mate, I agree.) That the males arrive first, perhaps staking out their bit of watery turf before the females arrive, was confirmed by Gill’s data. She set out her traps on February 2 but didn’t catch her first female until the 23rd.

Sexing the salamanders is sort of easy–superficially. During the breeding season–or at least when they’re ready to mate–the males swell at the base of their tales, around the orifice common to their reproductive and digestive systems. The females do not. In addition, a female that has mated is liable to be considerably plumper and wider than a male, says Gill. There also may be differences in the shape of their tails.

I thought I had the sexing thing down pat the first day out, was less sure by the end of the second.

Once mating has occurred, the eggs are deposited in the water and the females head back to their burrows. The eggs hatch, and the larvae spend part of the summer in the pond, then change into juveniles and stay on land until they reach breeding age and size.

We also caught a frog the second time we went out, along with 29 male and 11 female salamanders.

I was glad to see the frog, another amphibian whose numbers have declined worldwide and among which all kinds of odd physical abnormalities have been reported. This one looked normal, and, as is the case with the dichotomy between the healthy and endangered runs of salmon, suggests that answers to many questions about the decline in amphibians and other species will not come easy.

Even if a chemical causes deformities or death in the laboratory, even if it does so at concentrations relevant to those in the environment, what that means is unclear. It’s not the amount in the laboratory or the environment that matters, but the amount that gets to the right place within the animal. That animals seem much more susceptible as embryos makes it even more difficult, and I think this embryonic vulnerability is perhaps what scares us most. The effects of any substance that causes developmental disruption in an embryo are apt to be much greater than the effects of the same substance on adult animals.

It seems reasonable that we should temper our reactions to press reports of this or that laboratory finding with the awareness that much more needs to be done to determine whether the finding has relevance to the “real world.”

How scientists will sort out the mix of possible endocrine disrupting substances in the environment is hard to predict. No chemical, pharmaceutical, or natural hormone is out there alone–it truly is a soup of unknown makeup.

Gary Thorgaard’s observation bears repeating: We really don’t know what’s going on out there.