News — Triphenyl phosphate (TPhP), a chemical used in many fire retardants as well as nail polish, has been shown to be harmful to humans and aquatic species at relatively high levels, but in their work, a team of Bates College researchers used an exposure level a thousand times less concentrated than those from other studies and found it led to “worrisome” impacts on developing zebrafish.
Bates Associate Professor of Biology Larissa Williams and three recent graduates of the Maine liberal arts college have completed the first examination of the effect of TPhP exposure in everyday life, a level known as “environmentally relevant concentrations.” They’ve discovered that everyday exposure to TPhP is harmful to zebrafish embryos and larvae — and, potentially, to human beings as well.
It’s known that exposure to triphenyl phosphate (TPhP), a chemical used in fire retardants and nail polish, is harmful to humans and aquatic species at relatively high levels, and the U.S. Environmental Protection Agency has been in recent years.
The Bates research tested exposure levels on zebrafish as measured in parts per billion, rather than the levels most researchers have focused on, which is parts per million. “We were looking at exposure at the nanomolar level, a thousand times less concentrated than other studies had reported,” says Williams. The decision to look at the possible impacts of TPhP in lower concentrations came about in 2021, when Williams was working with one of her thesis students, Ben Schmandt ’22.
“Ben looked into the literature and found the level at which TPhP is found in surface waters,” Williams says. “So that’s where we started, at the nanomolar level, a thousand times less than the micromolar level that everyone else has been working on.”
“With toxicants, if you throw enough at [an organism], there will be an effect. But that doesn’t make it relevant. That’s why this research is particularly exciting.”
Spanning three years, the Bates research project studied TPhP exposure on zebrafish, a species that shares around 80 to 85 percent of its DNA with humans, making it an effective model for research on the effects of toxicants on both aquatic species and humans.
The findings, published as the cover article in the May 2024 issue of the journal Toxics, their paper, “,” send a clarion call for more research, Williams says. “We know that TPhP is a ubiquitous environmental contaminant that many organisms, including humans, are exposed to. Now we know that it is likely a developmental toxicant at environmentally relevant levels. That’s worrisome. It’s critical to understand the implications of that exposure.”
The team discovered that parts-per-billion TPhP exposure harmfully affected developing zebrafish through multiple biological pathways, though the exact harmful mechanism is still not fully understood. TPhP-exposed zebrafish larvae were stunted in size and suffered from heart swelling (pericardial edema), both of which are also seen at higher exposure levels. They also suffered from oxidative stress, causing cell damage within hours of exposure.
In addition to Schmandt, who is now a research associate in the Claussnitzer Lab at the Broad Institute, Williams’ coauthors are Bates alumni Gabrielle Smart ’23, now in veterinary school at Washington State, and Mfon Diduff ’24, each of whom contributed to the research while working in the Williams lab as students.
The research, conducted in Williams’ lab at the Bonney Science Center, involved exposing zebrafish larvae to TPhP over five days, a sufficient span to measure the chemical’s effects on this fast-growing species. Zebrafish grow from larva to adult in just 90 days, and are transparent. “At the age of 48 hours, zebrafish have produced hemoglobin, so we can see the heart,” Williams says.
The Bates researchers photographed and measured the size of the larvae by looking at them under a stereoscope in Williams lab. The team then measured heart rates manually, observing each beat. Using a stopwatch, the researcher counts the number of heartbeats within a 30-second interval. The researchers also checked for swelling around the heart and scored it on a scale from 1 (severe swelling) to 5 (normal). And, to see if the chemical affected blood circulation, they used another microscope to capture heart movement and blood flow in the larvae.
For the Williams lab, the next chapter in the research is to zero in on how and when TPhP does what it does to developing animals.
All developing animals, whether zebrafish larvae or human fetuses, “are exposed to a lot of different chemicals,” she explains. “It's really the timing of that exposure that really matters, what we call the window of susceptibility.”
Alcohol is a familiar example. “Alcohol in a human fetus is highly toxic very early in pregnancy, but becomes less toxic as the fetus develops. If there is exposure early on, it has a much larger impact than later on. Many chemicals work that way. Part of what we do in our lab is to figure out that window.”
With a new grant from Maine’s IDeA Network of Biomedical Research Excellence (INBRE), the lab will do more precise toxicant testing at different stages of zebrafish development. “We were able to show that chronic exposure over the entire developmental period impacted the," Williams said. "Now what we want to know is, as the heart's developing and you add a toxicant, how does that change the trajectory of development?”
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