by J. V. Reistrup

Ten years ago, in the summer of 1997, a mysterious and changeable microbe dramatically made its presence known in the lower Chesapeake Bay--killing thousands of fish, affecting the health of people nearby and causing a public scare that cost the seafood industry tens of millions of dollars. It also touched off scientific controversy and political debate about the cause. The fish kills stopped after 1999 and never resumed, but the scientific argument raged on. 

Now there is reason to believe the solution to the most elusive secret of the outbreak is finally at hand: how and when this microscopic creature suddenly gives off a poison fatal to fish. Researchers have not only chemically identified the poison but also explained the off/on switch. It may turn on when trace metals commonly found in the surrounding waters--principally copper--produce the unstable molecules known as free radicals, making the   tiny microorganism defend itself against them.

The mystery opened when thousands of fish--mostly menhaden--in rivers and other tributaries in the lower Eastern Shore died in August 1997, their floating bodies marked by lesions, or open sores. The kills got so bad that the governors of Maryland and Virginia temporarily closed the lower Pocomoke River to commercial and recreational uses. Most troubling of all was the news that a dozen or so people--including researchers, watermen and state employees--had fallen ill after coming into contact with the nearby water. Symptoms included blisters, peeling skin and loss of memory.

The culprit was quickly identified (although this answer hasn't been completely accepted over the years) as Pfiesteria piscicida, one of many tiny creatures collectively known as plankton that are at the bottom of the estuarine food chain and upon which menhaden feed. The microbe was discovered in 1988 by researchers at North Carolina State University--JoAnn Burkholder and Ed Noga. Burkholder, a professor of applied aquatic biology, soon confirmed that pfiesteria (pronounced fis-TEER-ee-ah), or an organism like it, was in water samples collected during the fish kills on the Pocomoke River and other waterways in our region [see "The Bay Boater's Complete Guide to Pfiesteria," June 1998].

It appeared to researchers that excessive nutrients  in the water--particularly nitrogen and phosphorus--were part of the reason for the pfiesteria outbreaks. In North Carolina, hog farms were blamed for the runoff. On the Eastern Shore, fingers pointed at poultry operations. Thus, as happened after the 1962 publication of Silent Spring, the DDT alarm sounded by Rachel Carson, people who produce food for a living found themselves contending with defenders of the environment. Pfiesteria won the nickname "the cell from hell" and inspired a popular book with a title harking back to the plagues in the book of Exodus, And the Waters Turned to Blood, by Rodney Barker. People shied away from fish and shellfish generally when they heard about and saw pictures of the fish kills. The Bay's seafood industry took a hit of nearly $50 million.

Like Carson, Burkholder became the focus of the discussion--a heroine to some and a menace to others. In 1997, the prestigious American Association for the Advancement of Science awarded her the AAAS Scientific Freedom and Responsibility Award "in recognition of her dedicated and untiring efforts to focus public attention on the potential negative impacts of the microorganism Pfisteria piscicida on the marine environment."

Concerns about human health were soon eased. The U.S. Environmental Protection Agency has concluded that there has never been a case of illness from eating fish or shellfish exposed to pfiesteria or even any evidence of contaminated fish or shellfish coming on the market. Any human illnesses related to pfiesteria appear to be caused by toxins in the water around the affected fish, not by eating fish or shellfish, the EPA says. There were no such categorical statements, however, from the EPA or anyone else, about what was killing the fish.

Most vexing of all for researchers was the fact that pfiesteria "blooms" didn't always cause fish kills. Furthermore, Burkholder's findings at N.C. State had proven very difficult to reproduce in other laboratories. It's a basic rule of science that laboratory findings are meaningful only if they can be replicated. Researchers divided into at least two camps, those blaming pfiesteria for the kills and others who suggested at various times that this microorganism didn't produce any toxins, that it wasn't killing the fish but was merely feeding on them, and that something else entirely was to blame.

Some researchers attributed the fish kills to the fungus-like Aphanomyces invadans, a common water mold or oomycete. Since then, these researchers have published paper after paper piling up evidence for their theory. Last year, for example, they reported they had isolated the mold, cultured it in a petri dish, injected it into fish in the lab and watched the sores, or lesions, grow. This was enough to convince some that the mold was the culprit, but others point out, quite reasonably, that those tests prove only that the mold causes lesions, not that pfiesteria doesn't.

The most telling arguments made by Burkholder's scientific critics, though, were their reports that they couldn't get pfiesteria to produce the toxic effects she and her colleagues said it could. Her response has been that they weren't doing the tests right, but unsurprisingly the argument wasn't totally persuasive.

Here Today, Gone Today
Pfiesteria, a dinoflagellate, is a microscopic creature that is fairly common to estuarine waters and seems to thrive when they are polluted. It has been reported to have a highly complex life cycle, sometimes behaving like a plant and sometimes like an animal. Most of these forms are non-toxic, feeding on algae and bacteria in the water and sediments under it. Pollution may encourage the growth of the algae upon which it feeds.

But sometimes and somehow, pfiesteria appears to change to a form that gives off deadly toxins in the presence of fish, particularly schooling fish like menhaden--perhaps triggered by secretions from the fish or their excrement in the water. It might emit one toxin that stuns the fish, while others might break down fish skin tissue and cause the lesions. And as the fish are incapacitated, the pfiesteria cells might feed on their tissues and blood, either killing them outright or leaving them susceptible to fatal infections.

Reproducing this effect proved elusive, however, in part because toxic outbreaks typically last no more than a few hours. Afterward, the theory goes, pfiesteria cells revert to their benign forms and their toxins in the water break down. But by that time the damage has been done, and deaths among fish weakened by the toxins may persist for days. Such highly reactive, unstable substances are a "chemist's nightmare" to try to characterize, says Burkholder, who was trained in other fields, zoology and botany. That's why chemist Peter Moeller of the Charleston, S.C., marine laboratory of NOAA's National Ocean Service, plus a team of researchers at other institutions, got the assignment to find a toxin. "A lot of people have found it and then lost it," he said in a telephone interview. He said his team came up with the basic question, "What if this toxin was around for only a short period of time, and then disappeared, because it was so unstable?"

Last January, after nine years of research, his team said they had finally been able to keep the fatal toxin stable long enough to study it, in part by working under red lights in the laboratory so that natural white light wouldn't kill it. But they had only two to five days to examine each sample of the toxin before it began to decompose and become inactive.

A key finding was that the results from different tests--mass spectrometry and nuclear magnetic resonance spectroscopy among them--didn't match up. One set of results, for example, showed the toxin was three times heavier than the other test predicted. The researchers concluded there was something that changed the chemical characteristics, Moeller said. They concluded the something could be metal molecules that occur naturally in estuaries. And the switch appeared to be flipped when the metal molecules included free radicals--unstable because they intermittently swap electrons in a process known as "redox." It happened that all the fish kills took place in dark, murky, slow waters--which are rich not only in organisms but also in metals.

The Moeller team got guidance on naturally occurring free-radical toxins from David Newman of the National Cancer Institute, who has been researching natural products that can be turned into therapies. The Moeller team's findings were reported in the February 2007 issue of the American Chemical Society journal Environmental Science & Technology. They reported that for the first time they had been able to isolate and characterize labile (that is, readily changeable) and toxic copper complexes isolated from pfiesteria. And they said they killed fish with the stuff, time and again. "I invited any and all to my labs . . . and I could demonstrate that we're killing fish," Moeller says.

In one of many different tests, Moeller's team put sheepshead minnows in three separate jars of seawater: one containing only pfiesteria, one with the suspect molecules of metal and one with both pfiesteria and the metals. The minnows in the container with both pfiesteria and metals began dying within hours; the other fish were fine.

Burkholder sees vindication for herself and the many colleagues who blamed pfiesteria for the fish kills. She sees the Moeller team's chemical findings as the "smoking gun" which proves pfiesteria produces toxins that cause fish death.

There are still gaps: Moeller and his team worked in the lab, not in the field; they used a purified toxin, not a naturally occurring one; they used sheepshead minnows; they used ocean water from the Gulf Stream rather than brackish estuarine water. So they're still a ways from proving this toxin is to blame for killing menhaden a decade ago in the tributaries of the lower Bay and the coastal waters of North Carolina.

"Could it? Yes. Is it? I don't know," Moeller says. "I won't go any further than to say that I have found a toxic compound that is associated with Pfiesteria piscicida."

Take That, Copper
Moeller wonders whether pfiesteria produces its toxins as a defense mechanism to ward off the copper, which is poisonous to many organisms. Actually, he says, it might be more accurate to describe such chemicals as "metal regulators" that just happen to be toxic to other life, like menhaden.

But finding the toxins in the wild "would be a big issue," Moeller says. "You don't see them until they fall apart." So you would have to be able to predict when this sequence would occur--tough to do--and then sample the water and analyze it. All this would take continued multidisciplinary research, he says, perhaps focusing on organisms other than pfiesteria because that little creature has become so controversial among scientists.

Won't that take at least another decade? "Oh yeah. Oh yeah."