A new study from the University of Rochester finds that developmental exposure to a type of “forever chemical” known as PFHxA may lead to increased anxiety-related behaviors and memory impairments—but only in male mice. The chemical, once considered safer than older forms of PFAS, was found to enter the brain during early life and trigger long-lasting behavioral changes that persisted even after the chemical had cleared from the body. The findings were published in the European Journal of Neuroscience.
Perfluorohexanoic acid (PFHxA) belongs to a larger group of chemicals called per- and polyfluoroalkyl substances, or PFAS. These substances have been widely used for decades in everything from firefighting foams and nonstick cookware to waterproof fabrics and food packaging. Because they don’t naturally break down, they accumulate in the environment and in human and animal bodies—earning the nickname “forever chemicals.”
While older, long-chain PFAS like PFOA and PFOS have been linked to developmental and behavioral problems in both humans and animals, newer short-chain alternatives like PFHxA were introduced in the hope that they would pose fewer risks. But evidence is beginning to suggest that these replacements may not be as harmless as originally thought. PFHxA has already been detected in human serum and breast milk, and postmortem studies show it accumulates in the brain, especially in the cerebellum—a brain region involved in motor control and emotion regulation.
“There is a really thorough and rigorous body of research focusing on the effects of perfluorooctanoic acid (PFOA) and perfluorosulfonic acid (PFOS) exposures on brain development,” said study author Ania K. Majewska, a professor of neuroscience and senior author of the study.
“However, there isn’t as much research focusing on the effects of short-chain, next generation PFAS like perfluorohexanoic acid (PFHxA) despite the growing detection of them in contaminated waters and soils and in human tissue samples. We found this to be a huge gap of knowledge, and we were hopeful to begin the conversation around PFAS that have been historically less studied but to which we are all exposed to throughout our lives.”
The researchers used a well-established strain of lab mice and exposed pregnant females daily to PFHxA by feeding them specially prepared mealworms from the first day of pregnancy through the end of lactation. Two doses were tested: a low dose representing a human safety threshold proposed in France, and a high dose previously used by the U.S. Environmental Protection Agency in animal studies. A separate group received a chemical-free control feed.
After birth, offspring were monitored through different life stages. At various time points—postnatal day 1, day 21 (weaning), and day 90 (adulthood)—some animals were sacrificed to measure PFHxA levels in their brains and blood. The remaining mice underwent a range of behavioral tests to assess activity levels, anxiety-like behavior, memory, strength, coordination, and gait.
To minimize confounding factors, only one male and one female from each litter were included in behavioral tests. This helped ensure that any effects observed weren’t due to differences in maternal care or genetics within litters.
PFHxA was detected in the brains of exposed offspring as early as one day after birth, and levels were especially high by weaning. By adulthood (day 90), brain concentrations had returned to control levels, suggesting the chemical had been cleared. However, behavioral effects persisted, especially in male mice.
Males exposed to PFHxA showed a consistent pattern of reduced activity in an open-field test, a common way to assess exploratory behavior and anxiety. They spent less time moving and jumping, especially in the center of the testing area, which animals tend to avoid when anxious. Females did not show these same effects.
In an elevated plus maze, another test of anxiety, males exposed to the low dose entered closed and center areas of the maze more often—indicating increased stress or risk-avoidance—while those exposed to the higher dose spent more time frozen in the center. Again, females showed no significant behavioral differences.
Memory was also affected in males. In a novel object recognition test, male mice exposed to the higher dose spent less time investigating new objects, suggesting impairments in short-term memory or attention. There were no such effects in females.
“Perhaps it shouldn’t be too surprising, as neurotoxicant exposures often result in deficits in several behavioral domains, but we were surprised to find that the behavioral changes spanned multiple areas, including locomotor activity, anxiolytic behaviors, and memory,” Majewska told PsyPost. “This finding will hopefully inspire future research investigating the effects of PFHxA exposure on specific behavioral domains.”
Interestingly, PFHxA exposure did not impair strength or coordination in either sex, as measured by the hang test and an inverted screen challenge. Gait was mostly unaffected, although minor changes in stride timing were observed.
Taken together, these results suggest that PFHxA exposure during gestation and nursing affects brain function in male mice, leading to subtle but measurable changes in activity, stress responses, and memory.
“One of the key takeaways from this study is that PFHxA should receive the same level of research focus as older PFAS, as developmental exposure to PFHxA may cause similar male-specific behavioral effects,” Majewska said. “More research is needed on this pervasive PFAS.”
While the findings are suggestive, the authors caution that this was a preliminary study. Sample sizes were relatively small, especially for the brain PFHxA measurements, and behavioral outcomes were only assessed at specific time points. Larger studies examining behavior across development—and in both sexes—will be needed to confirm and expand on these results.
The study also did not directly measure how PFHxA may be altering the brain at a cellular or molecular level. Future work is planned to explore changes in gene expression and protein activity in brain regions linked to emotion and memory. The authors also noted that they did not examine how PFHxA might affect maternal behavior, which could indirectly influence offspring development.
Importantly, while the mouse model allows for precise control of exposure and behavior, extrapolating these findings to humans should be done with caution. Human development is more complex, and people are typically exposed to multiple PFAS chemicals at once, often over long periods.
Still, the fact that PFHxA was shown to enter the brain, influence behavior, and linger during early development—even though it later cleared from the body—raises questions about how seemingly short-lived exposures might shape long-term mental health.
“This study is a starting point suggesting that developmental PFHxA exposure can affect different behavioral domains, studies with larger sample sizes for both sexes and that address behavior at different time points and in different domains – social behaviors, for example – would allow us to determine how behavioral differences arise and evolve after exposure in a mouse model,” Majewska said.
This study is one of the first to demonstrate that PFHxA, a widely used short-chain PFAS, can alter brain function in mammals. Although the behavioral effects were modest, the sex-specific pattern—affecting only males—mirrors trends seen in many developmental disorders like autism and attention problems.
By identifying these effects in a controlled animal model, the researchers hope to provide a foundation for future work on how PFHxA acts in the brain. They also hope their findings will inform public health policies on the use, regulation, and disposal of short-chain PFAS chemicals.
As Majewska explained, “This publication focused on behavioral effects of gestational and lactational exposures to PFHxA, and we are currently working on understanding the effects of this exposure at the gene and protein level. A complete characterization of the developmental neurotoxic profile of this chemical will hopefully aid in the creation of policies around its use and disposal.”
The study, “Gestational and Lactational Exposure to Perfluorohexanoic Acid Affects Behavior in Adult Male Mice: A Preliminary Study,” was authored by Elizabeth C. Plunk, Katherine E. Manz, Andre Gomes, Kurt D. Pennell, Marissa E. Sobolewski, and Ania K. Majewska.
