Scientists who reported discovering feminized fish in the Pacific Ocean off Southern California now say the findings were based erroneously on samples accidentally contaminated by researchers.

The 2005 discovery received worldwide attention because it was the first and only time that intersex animals – a phenomenon when males exposed to hormone-like pollutants have ovary-like testes that grow eggs – were found in the ocean. At the time, the half-male, half-female flatfish were linked to massive sewage outfalls off Los Angeles and Orange counties.

But when scientists at the Southern California Coastal Water Research Project returned to the same locations to test several hundred more fish, they didn’t find any egg-growing males. So last November they retested the original fish samples, and reread the pathology report, and realized that the eggs found on the males were not actually inside their testes.

Steven Bay, head toxicologist at the government-funded research institution, said they apparently were “stray eggs.” Some of the females’ eggs apparently dropped onto the male fish tissues. Bay said he is unsure whether the cross-contamination occurred on the boat, where the turbot and sole were collected and dissected off Los Angeles, Palos Verdes Peninsula and Huntington Beach, or in the laboratory.

“What happened, when we interpreted the report, we weren’t careful in reading the pathologist’s words,” Bay said. “The problem was in my group not interpreting the report with as much caution as they should have. The hints were there.”

Other scientists who do similar work with fish said they were surprised by the mistake. It’s a sign that the tissue dissection or processing, perhaps most likely in the field, was “sloppy.”

“If the knife was not cleaned between samples and people were sloppy then they may have contaminated the testes samples. I guess the best thing to do in this situation is to accept that this is what happened. Very unfortunate,” said Karen Kidd, a biology professor and Canada Research Chair at University of New Brunswick who researches hormone disruption in freshwater fish.

Emerging over the past 20 years, research that connects hormone-mimicking pollutants to feminizing effects in wildlife and people around the world has been highly controversial.

Joseph Gully, supervising environmental scientist at the Los Angeles County Sanitation Districts, called the mistake “a bit of a disaster.”

Gully said the false feminized fish “triggered a lot of concern and a lot of effort” by the local sewage agencies, which were so worried that they funded a $750,000 follow-up study of 600 more fish in 2006. “We couldn’t figure out why [intersex fish] were there one year and not the next. It is somewhat encouraging that we don’t have the results we first thought we had.”

Altered hormones in ocean fish
Despite the error, the Southern California scientists said their new peer-reviewed, published research confirms that bottom-dwelling ocean fish are highly exposed to hormone-disrupting chemicals off Los Angeles and Orange counties. Other more subtle signs of altered hormones were confirmed – including reduced estrogen levels in male turbot near the sites where wastewater is discharged.

Every day, billions of gallons of treated sewage from the two counties are discharged into the ocean via three outfalls, or ocean pipelines, that extend two to five miles offshore. The discharge includes women’s natural estrogen as well as manmade, hormone-like substances from birth control pills and consumer products.

The fish were collected in 2003 a few miles offshore in multiple locations as part of research to determine whether ocean life is harmed at the outfalls. At the time, 11 of 64 male turbot and sole caught near the outfalls were marked positive as having eggs, while none were found farther away. The locations fit the hypothesis that the estrogenic pollutants in the wastewater could alter fish sex organs. “All of them were near an outfall site so the pattern we saw made sense,” Bay said.

 The scientists originally discussed their findings of feminized fish at a 2005 toxicology conference in Baltimore, and the Los Angeles Times published a story on it then. “At the time, we felt these were real findings,” Bay said.

“It was depressing” to find the error, Bay said. “But thank god we didn’t write a paper on it.” Since the data were preliminary, the scientists hadn’t submitted anything to a journal for publication. The authors acknowledged the error in December in a report in the journal Environmental Toxicology and Chemistry.

Bay said the mistake came when his team saw in the pathology report that 11 male samples were checked positive for eggs but failed to notice the eggs’ location; the pathologist had noted the eggs were outside the testes. When the same slides were retested three months ago, three of the turbot and all six English sole had no eggs, and two other turbot had “minimal testis-ova,“ likely due to contamination during dissection or processing, Bay said.

Errors "a rarity"
Founded in 1969, the Southern California Coastal Water Research Project is a public agency funded by the U.S. Environmental Protection Agency, state environmental agencies and county and city sanitation agencies. It is well known and its science is highly regarded, although they are not specialists in hormone disruption. The private Virginia-based lab, Experimental Pathology Laboratories, that analyzed the tissues is considered among the most experienced animal pathology labs in the country. The company's pathology supervisor, contacted by EHN, declined to comment, citing client privacy.

Steve Weisberg, executive director of the Southern California institute, called the errors “a rarity."

Elsewhere, feminized fish have been discovered, and confirmed, in many rivers, estuaries and lakes contaminated by sewage treatment plants or industries, including ones in England, Canada, the Mediterranean Sea and the Potomac River.

During its expanded testing, the Southern California scientists found no intersex fish out of 373 males caught in 2006. However, they did find subtle changes in their hormones.

For example, male hornyhead turbot had very high estrogen levels, higher than the females, regardless of where they were caught. It may be a natural oddity of the species. But both male and females near the outfalls had about half the estrogen found in the ones near uncontaminated sites, according to their December report. Anti-estrogenic chemicals near the outfalls could be the cause, the report says. Stress and thyroid hormones also were different in the fish near the outfalls, indictating an effect on their physiology.

However, hornyhead turbot, chosen for study because they are bottom-dwellers and are fairly sedentary, are abundant, and there seem to be no overall effects on their populations or reproductive organs. “We know that populations of the fish seem to be healthy. We don’t see declines,” Bay said.

Kidd agreed after reviewing their report.

“Our overall impression is that the fish [off Southern California] are being exposed to contaminants and that there are some effects on hormone production in male and female fish,” Kidd said. “However, the conclusions of the report make sense and there is no strong overall evidence of endocrine disruption in turbot at the wastewater-impacted sites.”

The discovery of no gender-bending fish is not that surprising, Kidd said, since that “can be an extreme effect... My gut feeling is that it wouldn't happen very often and it would be species-specific."

“Intersex isn’t the only canary in the coal mine, so to speak,” she said, adding that there could be other signs that their reproductive systems are unhealthy. Scientists do not know much about what altered hormones do to fish fertility. "Any changes in the exposed population, we should be concerned about," she said.

Gully said the money for the second study “wasn’t wasted” because it revealed a lot of new data. But he said it was paid for by residents of the two counties through sewage fees “during a time of budget consciousness.”

The cross-contamination “generated a flurry of concern and stimulated a lot of good research. But that’s a good thing,” Bay said.

This article originally ran at Environmental Health News, a news source published by Environmental Health Sciences, a nonprofit media company.