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An experiment that started last month in St. Cloud State University's Aquatic Toxicology Lab expands upon 2012 studies that determined fish might be affected by pharmaceuticals entering rivers and lakes through wastewater treatment plant effluents.

Details of the 2012 study, led by lab director and St. Cloud State anatomy professor Heiko Schoenfuss, appeared in this month's scientific journal Environmental Toxicology & Chemistry.

Using larval and adult fathead minnows, it looked at individual and combined exposure to sleep aids, muscle relaxants, painkillers and antidepressants — chemicals previously detected in the environment. Juvenile fish didn't get as big or escape as fast. Adult females had larger livers. Adult males didn't defend their nests as rigorously and produced a chemical protein, plasma vitellogenin, associated with female egg production.

The work is part of a long-term U.S. Geological Survey effort to determine the effects of emerging contaminants, inform water-resources managers, and, ultimately, help develop effective water management practices.

Schoenfuss' related research dates back 20 years.

The experiments that started Nov. 18 will expose three generations of fathead minnows to three concentrations of a 10-chemical mix — including prescription drugs, BPA, (bisphenol A, an industrial chemical used to make some plastics), surfactants (used in cleaners to cut grease), DEET (mosquito repellant) and hormones (naturally produced in humans).

That particular cocktail was based on what's found in a typical stream flowing into the Great Lakes. The experiment also includes one control group, and one group with exposure to ethanol only. (The latter is included as a control for ethanol, because the chemical mixes are dissolved in ethanol.)

Each set of conditions is replicated 20 times.

"In the environment, we know that compounds show up as very complex mixtures," Schoenfuss said, standing among three rows of 16-liter tanks where 104 pairs of first-generation minnows swam, water ran and monitors tracked.

"These are complex-mixture experiments, but they are all stemming from that original question of what do these pharmaceuticals do in the environment, what is the adverse effect that fish may experience from being exposed to pharmaceuticals and other compounds in the environment," Schoenfuss said.

Samples of the chemical concentrations, measured in parts per trillion, are mailed weekly to the USGS' National Water Quality Lab in Denver, where Edward Furlong of USGS' methods research and development program, has helped formulate and analyze the latest chemical cocktail for the SCSU experiments.

"We want to understand how (the chemicals) are transported and transformed in the environment and, ultimately, are there effects that are displayed," Furlong told the 

The current experiment runs through early August, and involves another SCSU professor, two graduate students and four or five undergrads. It requires attention 24 hours a day, seven days a week. It requires six to eight hours of work a day.

The $250,000 cost includes chemicals and analysis. Funding comes from the U.S. Fish & Wildlife Service, the National Science Foundation, the Minnesota Natural Resources Trust Fund and the Metropolitan Water Reclamation District of Greater Chicago. Collaborators include the U.S. Fish & Wildlife Service and the U.S. Geological Survey.

Because the results affect both regulatory agencies and industry, researchers aim to secure balanced funding — from agencies, from industry and from academic sources.

Grad student Lina Wang, 23, of Mankato, set up the system, which is organized in three well-labeled rows. She is pursuing a master's degree in biology with a focus on ecology and natural resources.

"Our hope is to be able to compare an adult exposure to an entire life-cycle exposure," Wang said. "When we go into the environment, most of these fish are most likely being exposed to these chemicals throughout their entire life."

A second lab space contains the work of grad student Troy Lehto, 27, of Cokato, who is pursuing a master's of science with a focus on cellular and molecular biology. He's studying the effects of pharmaceuticals on larvae, one drug at a time.

"The ultimate question from the perspective of a toxicologist is, do we need to worry about the compounds being produced in the environment," Schoenfuss said.

The chemicals include sulfamethoxaole (an antibiotic), methocarbamol (a muscle relaxant), temazepam (a sleep aid), diclofenac (an anti-inflammatory), a control group and a mix of all four drugs.

The experiments Lehto is working on are behavioral — they consider how the chemicals affect the ability of young fish to feed and avoid predators. Gene expression is another part of the study. Analysis takes place about 21 days after hatching, when the minnows are about a half-inch long and weigh about one-third of a gram.

With three generations of adult fish, study will focus on the number of eggs produced, the number fertilized, the number of offspring, any changes in the adults' organs, bloodwork, age of sexual maturity, survival rate and sex ratio. Gene, blood and tissue analysis takes place in the Aquatic Toxicology Lab.

"Just like a physician would ask what other drugs are you taking, in order to truly understand the environmental impact of these pharmaceuticals, we have to look at them in the context of all of the other pharmaceuticals and other non-prescription pollutants that you would find in an aquatic environment," Schoenfuss said.

"It's our intent to get closer to the environmental question. The first question is: Does this compound have an effect by itself? But then we have to put it into the context: When combined, will the individual effect be exacerbated, will it be moderated? We really don't know. These are very, very complex mixtures. And we don't have any theoretical tools at this point to predict how a compound would act in mixture of that sort without running these experiments," Schoenfuss said.

The experiment that started Nov. 18 will be conducted entirely in the lab, and then followed with field studies on an as-yet-undetermined river that is a tributary to the Great Lakes.

An AP Exchange feature by Ann Wessel, St. Cloud Times

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An experiment that started last month in St. Cloud State University's Aquatic Toxicology Lab expands upon 2012 studies that determined fish might be affected.

Experiment looks at effect of pharmaceuticals on fish

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