Research shows effects of chemicals downstream

Karen EldridgeBlount, Our Town Youth

He ultimately will be concerned with how pharmaceuticals affect his patients, but Christian Carlton ’24 is enrolling in medical school with an impressive understanding of one drug’s larger impact on the environment and the life that the environment supports.

Carlton, a Rockwood, Tennessee, native who graduated on May 4 with a bachelor’s degree in science, majored in biology and completed a Senior Study entitled “Effects of Carbamazepine on the Behavior and Coloration of Siamese Fighting Fish (Betta Splendens).” He chose the topic in spring 2023 after consultation with his advisor, biology Professor Dr. Drew Crain.

One of the distinctive features of a Maryville education, the Senior Study requirement calls for students to complete an independent research and writing project that is guided by a faculty supervisor. Such in-depth research is an impressive endeavor by any Scot, made even more so in Carlton’s case, given that he was also an infielder with the MC baseball team, which recently won both the Collegiate Conference of the South regular season and tournament championships.

“[In medicine], you have to eventually use some of these pharmaceuticals,” Carlton said of his research choice. “And it’s interesting to learn how, through human excretion, some of them enter and stay in water systems and affect other living things that are not just humans. They may help a human but eventually hurt some other form of animal out there. Other studies could lead to better water management practices that could filter out these drugs and chemicals.”

Crain said multiple studies have been done on the presence and levels of pharmaceuticals in streams, rivers and lakes, but the effects of those drugs in specific species are relatively unknown.

Carbamazepine (CBZ), an anticonvulsant used to treat bipolar disorder and epilepsy, was chosen for the study because it is “one of the most abundantly found chemicals in waterways in every corner of the globe,” Carlton wrote in his abstract. And many animals, large and small, that live in water runoffs positive for CBZ are absorbing the drug into their bodies. Pharmaceuticals like CBZ are not filtered out by water treatment plants because of their small molecular size.

Carlton chose Betta fish (also known as Siamese fighting fish) for his study because they live in the wild in many affected areas and act similarly whether in their natural habitat or in controlled settings like laboratories. The fish, characterized by their vibrant colors and natural aggression, are commonly sold as pets for domestic fish tanks.

Conducting a paired study

Under the supervision of Crain, Carlton designed a paired study that would test CBZ’s effect on the vibrancy and behavior of the fish. He acquired 10 fish (five females and five males) and 10 tanks. During a 20-day period, he had all 10 fish spend equal time in both control tanks without CBZ and experimental exposure tanks with 1.10µg/L of the drug.

“All received both control and treatment,” Crain explained. “A paired study is a way to increase the statistical power, whenever you have a limited sample size, and control for that individual and the natural variation. At the beginning of the study, we had five fish that were not exposed to anything, and we measured their responses. Halfway through, we exposed them and measured responses. The other half started off exposed, and then didn’t get exposed for the rest of the study.”

Betta fish are known to be aggressive, with males typically exhibiting more aggressive behavior than females. Carlton measured aggressiveness by holding a mirror up to the tank. If the fish, suspecting a competitor was near, flared their gills or arched their bodies, Carlton assessed points for each behavior. He timed the experiment with the mirror, recording behavior every 20 seconds. Baseline measurements without the CBZ were measured against results in the experimental tanks.

“The data showed that the aggression of the fish was not significantly affected by carbamazepine, but females had higher levels of aggression than the males, which was not an expected outcome,” he said.

Carlton assessed vibrancy of the fish by taking photos of them throughout the study and comparing them to the photo of each fish taken at the start.

“Their color decreased in vibrancy, in most cases. Some changes were pretty dramatic,” he said. “I rated it from -5 to +5, with 0 being no change. I definitely got a lot of -2’s and a few -3’s.

“But in my study, I wrote that the color change could have been induced by the stresses in their environment,” Carlton continued. “The tanks didn’t have any rocks or plant life to make it homey, so they could have been very stressed out.”

However, Crain pointed out, the vibrancy in their bodies and fins returned once they were not exposed to CBZ.

Broader implications

While Carlton’s study did not present theories for the impact to humans, the research did open his mind to the broader implications.

“If there are small amounts of pharmaceuticals in water that have not been removed through the treatment process, then whatever water that the animals are living in, we’re also getting some of that, too,” he said. “So, we’re taking small doses of everyone’s medicine at times.”

Coursework in upper-level Biology classes like animal physiology, genetics and ecology and evolution helped inform this revelation, as well as prepare him to conduct the research.

“The general knowledge of the anatomy and physiology of many species of animals allowed me to have a clear understanding of what was taking place in the fish themselves over the course of the study,” he explained.

Studying watershed systems in another course, he became familiar with water treatment plants and how other countries are dealing with pharmaceuticals in water. Some use plants and other natural filtration processes while others use chemicals.

“I know the European water treatment plants are much more stringent as far as removing pharmaceuticals from the water,” Crain said, adding that United States is beginning to address this issue.

“PFAS [Per- and polyfluoroalkyl substances] is a substance in Teflon coating that has just been regulated this spring. It’s getting into the waterways, and it’s harmful — a carcinogen — so the EPA has regulated the amount allowable in drinking water, and water treatment plants are starting to say ‘We’re going to have to remove a lot of things in the future that we’re not removing right now.’ In years past, the operation has been ‘Let’s remove the bacteria. Let’s make sure there are no viruses in it. Let’s get rid of all the sediment.’ In the future, more removal will be occurring.”

Carlton is also aware of the need for more research to determine how carbamazepine breaks down.

“The studies I read found that about 3% of the total mass of carbamazepine that is excreted is the same chemical formula or makeup as the original carbamazepine that is ingested. So, a lot of the body metabolizes it into smaller different parts or pieces of the carbamazepine and secrets it out,” he explained. “It might seem like it’s breaking down so it’s not harmful anymore, but we really don’t know what it’s breaking down into and if that could be harmful.”

Next steps

In June, Carlton will report for anatomy bootcamp at the Lincoln Memorial University-DeBusk College of Osteopathic Medicine in Harrogate, Tennessee. The first day of medical school there will begin in late July.

“My mom is a family medicine doctor, so I’ve always been interested in family medicine because you get to treat nearly everything,” Carlton said. “But if there’s something that I eventually want to specialize in, then I’ll go down that route.”

He knows writing prescriptions will be in his future, and while recommending drugs that treat disease and improve the health of his patients will be his top priority, Carlton said he will remain interested in what is happening “downstream.”

He said he is thankful for the experience that required him to read more scientific studies, design an experiment and collect data. He said he expects to do more of that as a medical student and then as a doctor.

Acknowledging the limitations of his study in sample size and time, Carlton said he hopes future students will choose to build on his research and findings. Crain believes they will.

“I am extremely proud of the research that Christian conducted,” the professor said.

“I expect future Senior Study students to continue to examine the effects of carbamazepine on fish behavior, and Christian’s foundational study is a great launching point for these future research projects.”

Karen Eldridge is executive director of marketing & communications at Maryville College.


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