A decade ago, when fish started unexpectedly dying in parts of the Potomac River basin, biologists investigating the kills found something they didn't expect: Female eggs in the testes of male fish.

For the past decade, they have worked to unravel the story of what's causing "intersex" fish, a phenomenon that has turned up in 100 percent of smallmouth bass sampled in some areas of the Shenandoah and in 50 –75 percent of the those tested at a number of other sites in the Potomac basin.

In fish caught in more pristine locations outside the watershed, the intersex abnormality typically isn't seen in more than 10–15 percent of smallmouth bass.

Intersex is the presence of characteristics of both sexes in a single organism. In this case, immature eggs were found in the testes of smallmouth bass. Another indicator of exposure is the presence in the blood of male fish of vitellogenin, which is a precursor of egg yolk and is normally found only in the blood of egg-laying female fish.

Biologists recently published a summary report of their findings in the Potomac River basin and, while there are many chapters left to write, the report offers new insights about the problem.

For instance, previous research from other places tended to link intersex characteristics in fish to nearby wastewater treatment plant discharges.

While that relationship does exist in the Potomac basin, the prevalence of intersex fish tended to be highest in subbasins with large amounts of agricultural land and high animal densities.

Meanwhile, the severity of the intersex abnormality — such as more eggs in a male fish's testes — was generally higher in samples taken from areas with significant wastewater dischargers, basins with large amounts of agricultural land and areas with high densities of agricultural animals or poultry houses.

Vicki Blazer, the U.S. Geological Survey scientist who wrote the report, said any link between intersex fish and agriculture may stem from the fact that runoff from farms is highest in the spring, which is also when fish are being spawned.

"We seem to see more evidence that there is something that is probably initiating the intersex when the fish are really little," Blazer said. "It may be that ag is more of a player simply because of timing. The little fish are at their most sensitive stage."

The researchers also noted that the severity of problems — or those male fish that had the most pronounced female characteristics — correlated to long-term exposure.

Outside the Potomac basin, intersex fish have been found, often at high rates, at some National Wildlife Refuges around the Bay, and at eight sites surveyed in the Susquehanna River basin.

Of the fish studied, intersex seems to be most prevalent in smallmouth and largemouth bass, although it has occasionally been seen in other species.

Intersex is caused by endocrine disruptors, which affect the hormone systems in fish, animals and humans. They are found in products intended to have biological effects, such as hormones or other feed supplements fed to livestock, or medicines taken by humans. They enter water bodies primarily from excrement. Other chemicals, such as herbicides, are also suspected of contributing to the problem.

Blazer said the next step in the research is to identify specific chemicals, or groups of chemicals, that trigger the intersex phenomena.

One question that remains elusive is whether the high prevalence of intersex fish affects overall fish populations. Research in other areas suggests it can, but Blazer said there is no evidence that is happening in the Potomac.

But, she said, intersex fish have often been common in fish involved in large die-offs in both the Potomac and Susquehanna basins. That makes Blazer suspicious that chemicals triggering intersex are likely also suppressing the immune systems of fish, making them more susceptible to disease and fish kills.

"Intersex is not causing the fish kills," she said. "What we see in these fish kills is multiple bacteria, parasites, viruses and lots of stuff in those fish. And to me, that indicates immunosuppression."

"It's an indicator that there is something wrong in the system."

While most pollution control efforts concerning the Bay are focused on nitrogen and phosphorus, many biologists say chemical contaminants may be posing a more direct threat to human and aquatic life.

The impacts of chemical contaminants on fish in the watershed will get more attention later this year when the EPA releases a new report on the issue, part of an obligation under a federal Chesapeake Bay strategy approved in 2010. That strategy also calls for the development of specific chemical contaminant reduction goals in 2013.

The summary report, "Indicators of Reproductive Endocrine Disruption in Fish in the Chesapeake Bay Watershed," is available on the USGS Chesapeake Bay website, http://chesapeake.usgs.gov.