Prenatal and perinatal exposure to Per- and polyfluoroalkyl substances (PFAS)-contaminated drinking water impacts offspring neurobehavior and development

Abstract

Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants ubiquitous in the environment and humans. In-utero PFAS exposure is associated with numerous adverse health impacts. However, little is known about how prenatal PFAS mixture exposure affects offspring’s neurobehavioral function. This study aims to determine the causal relationship between in-utero PFAS mixture exposure and neurobehavioral changes in Sprague-Dawley rat offspring. Dams were exposed via drinking water to the vehicle (control), an environmentally relevant PFAS mixture, or a high-dose PFAS mixture. The environmentally relevant mixture was formulated to resemble measured tap water levels in Pittsboro, NC, USA (10 PFAS compounds; sum PFAS =758.6 ng/L). The high-dose PFAS load was 3.8 mg/L (5000x), within the range of exposures in the experimental literature. Exposure occurred seven days before mating until birth. Following exposure to PFAS-laden water or the vehicle during fetal development, neurobehavioral toxicity was assessed in male and female offspring with a battery of motor, cognitive, and affective function tests as juveniles, adolescents, and adults. Just before weaning, the environmentally relevant exposure group had smaller anogenital distances compared to the vehicle and high-dose groups on day 17, and males in the environmentally relevant exposure group demonstrated lower weights than the high-dose group on day 21 (p < 0.05). Reflex development delays were seen in negative geotaxis acquisition for both exposure groups compared to vehicle-exposed controls (p= 0.009). Our post-weaning behavioral measures of anxiety, depression, and memory were not found to be affected by maternal PFAS exposure. In adolescence (week five) and adulthood (week eight), the high PFAS dose significantly attenuated typical sex differences in locomotor activity. Maternal exposure to an environmentally relevant PFAS mixture produced developmental delays in the domains of pup weight, anogenital distance, and reflex acquisition for rat offspring. The high-dose PFAS exposure significantly decreased typical sex differences in locomotor activity.

1. Introduction

Per- and polyfluoroalkyl substances (PFAS) are a class of over 10,000 manmade organic compounds used for a broad range of industrial and commercial applications since the 1940s (Birnbaum, 2018; Rappazzo et al., 2017). An exceptionally strong carbon-fluorine bond allows PFAS to resist degradation and persist in the environment; this has earned them the nickname “forever chemicals” (Birnbaum, 2018). The signature surfactant and degradation-resistant characteristics of PFAS have been harnessed in essential products—from nonstick cookware to firefighting foams—making them globally ubiquitous in natural and human environments (Birnbaum, 2018; Pelch et al., 2019). Consequently, the serum of virtually all Americans contains detectable quantities of PFAS, but exposure risk varies widely among communities and populations (Calafat et al., 2007; Khalil et al., 2016; Lewis et al., 2015). Only a handful of PFAS are well-studied, with two common legacy compounds—perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA)—predominating the literature.

Ingestion of contaminated drinking water and food is considered the primary PFAS exposure route for the general population (Fromme et al., 2009). A national monitoring effort has estimated the average sum content of 17 PFAS compounds from treated United States drinking water to be 19.5 ng/L (Boone et al., 2019). As of 2016, the United States Environmental Protection Agency (EPA) set a non-enforceable lifetime health advisory level for PFOS and PFOA in drinking water at 70 ng/L (U.S. Environmental Protection Agency, 2016a, 2016b). In March of 2023, the EPA has since proposed considerably lower enforceable Maximum Contaminant Levels (MCLs) for PFOS and PFOA of 4 ng/L which was anticipated to be finalized by the end of 2023 (U.S. Environmental Protection Agency, 2023). Further, a hazard index of 1.0 was concurrently proposed as the MCL for perfluorononanoic acid (PFNA), tridecafluorohexane-1-sulfonic acid (PFHxS), potassium perfluorobutanesulfonate (PFBS), and hexafluoropropylene oxide dimer acid (HFPO-DA; GenX) as a PFAS mixture (U.S. Environmental Protection Agency, 2023). In 2022, the EPA indicated PFAS as a priority contaminant for regulatory review under the Safe Drinking Water Act, as included in the fifth edition of the EPA’s candidate contaminant list (CC-5) (Drinking Water Contaminant Candidate List 5-Final, 2022).

In North Carolina, USA, PFAS levels far exceeding EPA recommendations, likely related to industrial effluent releases dating back to 1980, were discovered in the Cape Fear River Estuary and its tributaries (Hopkins et al., 2018; Wagner and Buckland, 2017). In the Haw River, a Cape Fear tributary and drinking water source for Pittsboro, North Carolina, monitoring data found sum PFAS concentrations up to 1,076 ng/L at drinking water intakes and 758.6 ng/L in finished household drinking water, which was the model used for our study (Table 1) (Barnes, 2019; Crute et al., 2022; Hall et al., 2023). PFAS contamination recorded in the Haw River surpasses typical exposures on a national and local scale (using the nearby city of Durham, North Carolina in the Neuse River Basin as a reference) by over 30-fold (Herkert et al., 2020). The presence of PFAS in Pittsboro drinking water is likely associated with observed PFAS serum concentrations in residents that were two to four times higher than the national average (Barnes, 2020; Duke University Nicholas School of the Environment, 2020). Elevated PFAS levels observed in blood and drinking water from Pittsboro resemble those observed in other PFAS-contaminated regions of North Carolina, such as the city of Wilmington (Calafat et al., 2019; Kotlarz et al., 2020; Wallis et al., 2023).

Table 1:

Experimental PFAS Exposures Based on Pittsboro, North Carolina Drinking Water [nanograms per liter]

PFAS Compound	Cas Registry Number	Low-dose Concentration	High-dose Concentration
PFHxA (Perfluorohexanoic acid)	307-24-4	261.4	1307000
PFPA (Perfluoropentanoic acid)	2706-90-3	158.4	792000
PFHpA (Perfluoroheptanoic acid)	375-85-9	144.7	723500
PFBA (Perfluorobutyric acid)	375-22-4	96.1	480500
PFOA (Perfluoro-n-octanoic acid)	335-67-1	48.2	241000
PFOS (Perfluorooctanesulfonic acid)	2795-39-3	15.4	77000
PFHxS (Perfluorohexane-1- sulfonic acid)	3871-99-6	9.6	48000
PFDA (Perfluorodecanoic acid)	335-76-2	9.0	45000
PFBS (Potassium Perfluorobutanesulfonate)	29420-49-3	7.9	39500
PFNA (Perfluorononanoic acid)	375-95-1	7.9	39500
Total		758.6	3793000

Studies at the cellular, organismal, and population levels have linked PFAS exposure to an extensive list of adverse health effects, from elevated cancer risks to immune dysfunction (Agency for Toxic Substances and Disease Registry (ATSDR), 2021). Fetuses and babies are particularly susceptible to long-term health consequences due to PFAS transfer via the placenta and breast milk, respectively (Apelberg et al., 2007; Austin et al., 2003; Kärrman et al., 2007). Using an experimental rat model and environmental context, a primary goal of this study was to determine the causative relationship between elevated PFAS exposure during pregnancy and disrupted neurobehavioral development in offspring.

Gestational PFAS exposure has been associated with developmental, neurocognitive, and behavioral impacts in population-level studies. In several human studies, elevated PFAS exposure during early developmental periods has been associated with impairments related to visual-motor abilities (Harris et al., 2018), IQ (Wang et al., 2015), externalizing behaviors (Høyer et al., 2018; Luo et al., 2020; Vuong et al., 2021), neuropsychological development (Niu et al., 2019), motor development (Donauer et al., 2015), executive function (Chen et al., 2013; Forns et al., 2015; Sucharew et al., 2012; Vuong et al., 2016), and attention (Hoffman et al., 2010; Lien et al., 2016; Vuong et al., 2021, 2018). Conversely, other reports detected no associations between PFAS exposure and changes in neurological outcomes (Liew et al., 2018; Stein et al., 2013; Vuong et al., 2016). Inconsistent results—likely due to unique exposure scenarios, measured endpoints, timelines, sample sizes, and populations among the studies—necessitate further investigation of the neurodevelopmental consequences of PFAS exposure (Zhuchen et al., 2023).

Additionally, an increasing number of recent epidemiological studies have modeled the neurobehavioral and developmental health effects of mixture exposures rather than single compounds. For instance, an increased serum 14-PFAS mixture burden in children has been related to greater odds of being diagnosed with autism spectrum disorder through regression analysis, although the authors emphasize their use of concurrent PFAS measurements as a common pitfall limiting the results (Oh et al., 2022). Further, elevated concentrations of a 25-PFAS mixture in second-trimester cord blood were associated with lower cognitive composite scores in children at two years of age (Reardon et al., 2023). In a ten-PFAS study, elevated gestational PFAS mixture levels were associated with decelerated weight and height growth trajectories up to two years old, and the effect was more pronounced in male participants (Gao et al., 2022). Increasing levels of eight PFAS in maternal plasma between 12 and 16 weeks’ gestation were associated with hyperactivity and inattention at six years old through multiple linear regression models, while cognition was unaffected (Xie et al., 2023). A separate logistic regression found that elevated levels of a 13-compound legacy PFAS mixture, but not a four-compound alternative PFAS mixture, in third-trimester serum were associated with higher odds of persistently low neuropsychological developmental trajectories (communication, gross motor, problem-solving) in offspring from three to 36 months old (Li et al., 2023). Finally, models of a 16-PFAS mixture in cord blood (nine legacy long-chain, five short-chain, two alternative) demonstrated that every mixture quartile increase yielded communication delays, particularly in six-month-old children, and the effect was primarily attributable to PFOS (Zhou et al., 2023). Still, some studies demonstrated developmental impacts only through individual compound associations, even in the setting of well-defined mixture exposures (Wang et al., 2023; Xie et al., 2022; Yao et al., 2022). Emerging mixture exposure studies will continue to provide a necessary understanding of the effects of multiple-PFAS exposures on childhood behavior and development.

Although cohort studies have demonstrated associations between PFAS exposure and many human health complications, their generalizability is limited by differences in the composition and magnitude of the PFAS contamination. Experimental studies using animal models are necessary to investigate a definitive relationship between in-utero PFAS exposure and the neurobehavioral effects seen in population analyses.

Central to this work, humans are exposed to a broad mixture of PFAS compounds rather than single compounds in isolation. However, most previous studies have focused on exposure to individual legacy PFAS compounds, primarily PFOS and PFOA (Supplemental Table S1). Although important, single-exposure experiments do not adequately represent the risks posed by environmentally relevant mixture compositions and require extrapolation with considerable uncertainty from high-dose to low-dose effects (Zhuchen et al., 2023).

In this study, preconception and pregnant rats consumed a Pittsboro-analog PFAS-containing drinking water mixture at an environmentally relevant level as well as a high-dose level (5000x). In neurotoxicology animal models, considerably higher doses are often administered to rodents than those measured in environmental studies for more sensitive detection of results and to account for faster PFAS clearance compared to humans. Offspring development was monitored post-birth, and a range of motor, affective, and cognitive outcomes was evaluated across juvenile, adolescent, and early adult life stages. This is the first study using a rat model to investigate the neurodevelopmental toxicity of a ten-compound mixture of legacy and emerging PFAS at levels mimicking human exposure through drinking water.

2. Methods

2.1. Setting & Subjects

The Sprague-Dawley rat model was selected for this study because of its predictive value in neurotoxicology for numerous chemicals—including heavy metals, pesticides, flame retardants, and PCBs—and its use in well-validated developmental neurotoxicity tests. Sprague-Dawley rats are widely used in assessing offspring effects of parental toxicant exposure. They offer an excellent neurobehavior model due to basic structural and functional similarities to the human brain and behavior (Ellenbroek and Youn, 2016; Teegarden, 2012). (Weber et al., 2011; Yoon and Barton, 2008).

Nulliparous reproductive age female Sprague-Dawley rats weighing between 200 and 250 grams (Charles Rivers Labs, Raleigh, NC, USA) at the time of arrival and their offspring comprised the subjects of this study. The rats were maintained in a vivarium on a reversed day-night light schedule (12:12 hours), and all behavioral assays were conducted during their active period under low ambient light conditions. All behavioral testing occurred between 8 a.m. and 5 p.m. Start times for each test were consistent across cohorts. Unless fasting was required for food-motivated behavioral tests, all animals were given ad libitum access to food (Laboratory Rodent Chow 5001, Purina) and water. The colony room temperature was maintained between 20 and 25 °C, and humidity did not exceed 65%. Animal welfare checks, temperature, and humidity were logged daily. All procedures were approved by the Institutional Animal Care and Use Committee of Duke University under protocol A214-20-11.

2.2. Exposures and Measures

Upon arrival, female rats were singly housed and given a minimum of seven days to acclimate before beginning the prenatal drinking water exposure. Each female rat was randomly assigned to an exposure group. Female rats received either deionized water (vehicle) as a control, a low-dose drinking water mixture to directly mimic concentrations measured in Pittsboro, North Carolina, or a high-dose mixture (Table 1). The mixture contained ten different PFAS compounds (PFHxA, PFPeA, PFHpA, PFBA, PFOA, PFOS, PFHxS, PFDA, PFNA, and PFBS). The PFAS mixture preparation was described in detail in our published article (Crute et al., 2022). The high-dose concentrations were selected based on a review of previous animal toxicity studies that demonstrated significant non-lethal exposure-attributable changes (Supplemental Table S1). In the high-dose group, we avoided exposures that have previously demonstrated drastic reproductive effects in rodents and, in turn, would yield results of limited value, such as miscarriage and persistent weight loss. Long-established guidelines in developmental toxicology suggest that high doses should be targeted below levels of maternal toxicity and should not be substantially higher than the minimal toxic dose (Iyer and Makris, 2022; U.S. Environmental Protection Agency Risk Assessment Forum, 1991). At most, high doses should only pose risks of slight toxic effects to dams so that most pregnancies are carried to term. Because developmental exposures can be embryolethal without causing birth defects, the range between doses at which adverse effects and fetal demise occurs is often very narrow (Iyer and Makris, 2022).

After a seven-day minimum acclimation period, female rats were given ad libitum access to their respective drinking water mixtures. Drinking water mixtures were supplied through 455 mL glass bottles with rubber stoppers. Water consumption was measured by bottle weight every three days when the water was replenished. Bottles were washed weekly. Following seven days of exposure to the mixture, female rats entered a five-day mating period during which a male was placed into each female’s home cage. After five days, the males were removed. The experiment was replicated four times. In each replication, referred to as cohorts, four females were exposed to one of the three drinking water mixtures and mated with unexposed male rats, making 12 dams and 12 males per cohort. 48 dams in total were mated during the study course.

Before birth, pregnancy was confirmed by dam weight. Drinking water exposure continued throughout pregnancy, and drinking water mixtures containing PFAS were removed on the day each dam gave birth. Drinking water consumption data for dams in each cohort and exposure group are presented in Supplemental Table S2A. Estimated PFAS consumption (ng PFAS/kg rat) is also presented on a weekly basis in Supplemental Table S2B to facilitate comparison with controlled administration studies. No statistically significant differences in water intake were observed between groups.

On postnatal day (PND) 1, litters were culled to a recommended uniform size (eight pups) to minimize differences in nutrition, as larger litter size is associated with restricted pup growth in Sprague-Dawley rats (Dubovický et al., 2008; Fiorotto et al., 1991; Reddy and Donker, 1964). Litters were culled with the goal of an even offspring sex distribution (four males and four females per litter) to allow for observation of sex-specific exposure responses. One control litter and one high-dose litter had four pups or fewer and did not have both sexes available for behavioral testing. These two small litters were culled on PND 1. Reflex and growth measurements were obtained for all pups from sufficiently sized litters, and they were weaned at three weeks of age. Approximately four males and four females underwent growth and reflex testing for each of the 12 control litters, 16 low-dose litters, and 11 high-dose litters. For post-weaning behavioral testing, however, one male and one female were randomly selected from each litter. 78 total offspring represented each of the 39 litters for behavioral assays. The experimental timeline (Figure 1) and time of day of experimentation were held constant across cohorts. The order in which animals performed each behavioral assay was randomized each time.

Figure 1:

Experimental timeline for pre- and post-weaning behavioral testing.

2.3. PFAS Measurements in Pup Serum Samples

When pups from large litters were culled on PND 1, blood samples were collected and frozen at −80 °C, and serum PFAS levels were measured. Because individual pups did not produce sufficient blood volume for separate analyses, a combined serum analysis was performed with pups from the same sex, cohort, and exposure (Supplemental Table S4A).

All PFAS analyses occurred at the Research Triangle Institute (Research Triangle Park, NC, USA) following the methods described in Kotlarz et al. (2020). Ten PFAS compounds (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS, PFOS) were measured in serum using solvent extraction combined with liquid chromatography/tandem mass spectrometry (LC-MS/MS) on a SCIEX Triple Quad 7500 and an ExionLC AC UHPLC (Shimadzu hardware 9,000 PSI). See Supplemental Methods for additional details regarding serum analysis.

2.4. Pregnancy Outcomes

Maternal weight was tracked weekly, beginning at prenatal exposure and ending at weaning. Dam weight was used for pregnancy confirmation and dosing quality control as it is a useful indicator of toxicity in animal models (Johnson, 1981). Dam weights are presented in Supplemental Table S3. In addition to gestational weight, measured pregnancy outcomes included pregnancy rates after a five-day mating window, litter sizes, sex ratios, and pup survival rates for each exposure group.

2.5. Pre-weaning Growth and Reflex Development Measurements

Although somatic growth and maturation are not direct measures of neurobehavior, they are necessary for assessing complex developmental processes during early postnatal life stages (Dubovický et al., 2008). Birth to weaning development was tracked through body weight and anogenital distance for individual pups on PND 1, 2, 4, 7, 10, 14, 17, and 21. Anogenital distance, measured using a digital caliper, was defined as the distance from the anus to the urethra opening. All pups from each viable litter underwent two reflex tests, and scores from each pup were averaged to provide one representative data point per sex, per litter at each time point. Neonatal righting reflex and negative geotaxis assessments were conducted sequentially over the PND 2–6 and 7–13 periods, respectively. Pre-weaning tests included all pups from each litter.

2.5.1. Righting Reflex

Reflex tests were used to evaluate offspring’s motor development and coordination (Dubovický et al., 2008). The righting reflex was measured on PND 2, 4, and 6. Pups were individually placed on their back in a supine position on a clean surface. The time it took for the pup to return to the prone position was recorded. 30-second attempts were allotted for pups to complete the righting response, and an inability to demonstrate the reflex within three attempts was recorded as a failure (input as maximum possible time).

2.5.2. Negative Geotaxis

The negative geotaxis reflex was evaluated on PND 7, 9, 11, and 13. The testing apparatus consisted of a 45-degree mesh slope made of metal. Pups were individually placed facing down the incline and, using vestibular cues, the pups reflexively turned to face up the slope. The latency for each pup to turn 180⁰ and face upward was recorded. Pups were allotted 60 seconds to complete the righting response, and an inability to show the response within three attempts was recorded as a failure and input as the maximum possible time.

2.6. Post-Weaning Behavioral Testing

After weaning (three weeks of age), the offspring completed a battery of behavioral tests to evaluate motor function, affective response, and cognition. The battery has been used repeatedly in prior research to measure the behavioral effects of chronic maternal toxicant exposures (Cauley et al., 2018; Hawkey et al., 2020). In addition to broad measures of neurotoxicity and overall health, specific tests targeting activity, habituation, depressive and anxiety-like behaviors, and memory were conducted. Rats were tested in a random order within each test, and scoring was blinded for all tests.

2.6.1. Elevated Plus Maze

At four weeks of age, male and female rats from each litter were individually observed in the elevated plus maze (Med Associates, St Albans, VT, USA), which assesses anxiety-like behavior versus risk-taking behavior. Prey species, such as rats, have an unconditioned tendency to avoid open spaces with elevated predation risks and prefer areas bordered by walls (Walf and Frye, 2007). The rats’ movements were measured in a plus-shaped maze with two “closed arms” that had walls bordering them and faced one another across a central hub, and “open arms” in the remaining two positions. See Supplemental Methods for additional details and maze photographs (Supplemental Figure S1).

2.6.2. Figure-8 Locomotor Activity Maze

Motor activity is known to be a sensitive test that can “unmask” functional deficits and detect perturbations in central nervous system function (Bowers et al., 2016; Graham et al., 2018). Observations were made in the figure-8 maze in adolescence (five weeks of age) and adulthood (eight weeks of age) to assess locomotor activity and habituation (Reiter, 1983). This apparatus consists of an enclosed figure-8-shaped locomotor chamber, with two additional short arms extending from the center. Rats were individually placed in the maze and allowed to move freely for one hour while activity and habituation were quantified by photobeam crossings. See Supplemental Methods for additional details and maze photographs (Supplemental Figure S2).

2.6.3. Novelty Suppressed Feeding

At six weeks of age, rats’ feeding behaviors were assessed in a novel environment to measure fear responsivity. Novel, bright, and open environments induce an unconditioned mild stress response in rodents (hyponeophagia), which suppresses appetite (Samuels and Hen, 2011). Fasted rats were individually presented with 12 food pellets in a new cage and unfamiliar room. Latency to begin eating, number of eating bouts, total time spent eating, and post-test pellet weight were recorded to characterize eating behavior. For quality control, rats were equivalently assessed in their home cage in the familiar colony room environment one week of testing, with the weight of food consumed serving as the primary measure. See Supplemental Methods for additional details.

2.6.4. Novel Object Recognition

At seven weeks of age, the novel object recognition test was performed to assess rats’ attention, cognitive function, and non-spatial memory (Mathiasen and DiCamillo, 2010). In a low-motivational state (no food involvement), rats will investigate two identical objects for the same amount of time (baseline). Subsequently, when discriminating between a new and familiar object, rats display greater interest in and preferentially engage with the novel object. After two prior habituation sessions with no objects, rats were placed into the testing environment to freely investigate two randomly assigned identical objects for ten minutes and then returned to their home cages. One hour later, rats were presented with one familiar object and one new object. The side of the novel object was counterbalanced to overcome any potential side bias. Trained experimenters blindly scored videos of each session and recorded the total time rats spent investigating each object. Familiar versus novel object investigation times served as the recognition index. The investigation was defined as sniffing, licking, or chewing on the objects; incidental contact or climbing on the objects while sniffing elsewhere was not considered investigation. See Supplemental Methods for additional details.

2.6.5. Forced Swim Test

At nine weeks of age, rats were individually placed into a clear glass cylinder half-filled with 25 °C water for 300 seconds. The time rats spent immobile was recorded by trained experimenters during the session to represent depression-like behavior (Yankelevitch-Yahav et al., 2015). Immobility is defined as passive floating beyond what is necessary to keep the head above water (with at least three paws not moving). Subjects were removed from the water and dried before returning to their home cages.

2.7. Statistical Analysis

A multivariate analysis of variance (ANOVA) was used to assess the main effects of PFAS exposure on dependent variables. Any significant outcomes were followed by a two-tailed post hoc Dunnett’s test for multiple comparisons. Individuals from each dam served as the unit of variance for main effects. In developmental neurotoxicology, maintaining subject independence can be challenging when dealing with multiparous species (Vorhees and Williams, 2021). It is recommended to use litter as the unit of variance to combat this, which this study accomplishes by using one value per sex to represent the entire litter (Vorhees and Williams, 2021). Therefore, to control for commonly observed litter effects and similarities in maternal care, sex-stratified litter averages were analyzed for pre-weaning developmental assessments rather than treating same-sex siblings as statistically independent. After weaning, one male and one female represented each litter in the behavioral battery. Between-subjects variables included sex, litter, and treatment, while within-subjects variables included time block and object familiarity, as relevant to each test. Results were sex-stratified only if sex was found to interact with exposure. A non-parametric Kendall’s tau correlation coefficient was obtained to determine the relationship between weight and behavioral outcomes. Results for all statistical tests were considered significant if p < 0.05, and ANOVA results are presented as (F([df], [residual df])=[F-value]; p=[p-value]). Summary statistics are reported as means ± standard error.

3. Results

3.1. Pregnancy Outcomes

No significant PFAS effects on maternal weight gain were observed in either exposure group compared to the control. Pregnancy outcomes by dam exposure are presented in Table 2. No significant associations were found between gestational PFAS exposure and the number of successful pregnancies, average litter size, sex ratio, or pup mortality. The number of pregnancies was highest in the low-dose group (16 pregnancies) compared to the high-dose (12 pregnancies) and control (13 pregnancies) groups. Across four cohorts, 41 out of 48 females became pregnant and gave birth. However, two litters were not included in subsequent developmental testing due to their small size (four pups or less).

Table 2:

Pregnancy outcomes stratified by exposure. N= 13 litters (control), 16 litters (low-dose), and 12 litters (high-dose).

	Number of Pregnancies	Pregnancy Rate (out of 16 target litters per exposure)	Average Litter Size (pups/litter)	Average Sex Ratio (males/females ± standard error)	Total Pup Mortality Rate (per litter)	Average Day of Birth (during 5-day window)	Average Maternal Weight Gain During Pregnancy (g)
Control	13	81%	11.42	1.13 ± 0.25	0.31	3.08	94.73
Low-dose	16	100%	12.00	0.91 ± 0.11	0.13	2.75	103.77
High-dose	12	75%	12.15	0.90 ± 0.10	0.00	2.67	102.03

3.2. Growth and Development Outcomes

Maternal exposure to PFAS-contaminated drinking water did not lead to significant changes in the ontogeny of the righting reflex (data presented in Supplemental Figure S3 and Supplemental Table S5). In the case of negative geotaxis, however, a main effect for PFAS × day was observed (F(6,78)= 3.12, p= 0.009; Figure 2), suggesting that maternal PFAS exposure significantly influences righting reflex acquisition at a particular time point during the measurement period. More specifically, a post hoc Dunnett’s test revealed that regardless of sex, both the low-dose and high-dose PFAS-exposed pups took more time to demonstrate the reflex (higher average latency) compared to the control pups on PND 7 (p < 0.05). In the control group, pups of both sexes took an average of 22.3 ± 1.9s to complete the reflex task on PND 7, whereas the low-dose group took 33.0 ± 2.8s and the high-dose took 34.7 ± 2.5s to display the reflex. Increased reflex latency in both exposure groups compared to controls was observed on PND 9, 11, and 13 as well, but the differences in reflex timing became smaller and were not significant (Supplemental Table S6). These data demonstrate delays in negative geotaxis reflex development among PFAS-exposed pups.

Figure 2: Negative geotaxis reflex ontogeny (PND 7, 9, 11, 13) stratified by sex and maternal exposure group. N= 12 litters (control), 16 litters (low-dose), and 11 litters (high-dose).

A PFAS × day main effect was observed (p= 0.009), suggesting that maternal PFAS exposure significantly influences righting reflex acquisition on a particular day. Further, low-dose and high-dose PFAS-exposed had higher average latency compared to the control pups on PND 7 (p < 0.05).

Body weight gain and anogenital distance were monitored from birth to weaning, and age-specific exposure interactions were observed. For body weight, there was a significant PFAS × sex × day interaction (F(14, 189)= 3.54, p= 0.001; Figure 3A and Supplemental Table S5), indicating that maternal PFAS exposure affects pup weight differently between sexes at specific time points. This observation prompted further analysis of the PFAS exposure effects on pup weight during the pre-weaning period. A Dunnett’s test of the simple main effects on each measurement day revealed a sex-specific effect of exposure on pup weight between the exposure groups for PND 21 (p < 0.05; Figure 3B). Specifically, the average weight of male pups in the low-dose group on PND 21 (57.41 ± 1.34g) was significantly lower than those in the high-dose groups (61.89 ± 2.34g; p < 0.01). Low-dose males also weighed less than males in the control group (59.19 ± 2.33g), but the weight difference was not significant. Female pups did not exhibit the same growth patterns (Supplemental Table S5). Consequently, the sex difference in weight typically seen between males and females was diminished in the low-dose group. After weaning, week four measurements showed that male offspring weight in the low-dose group recovered when they were on a solid diet only.

Figure 3: A) Pup weight gain (PND 1 – 21) stratified by sex and maternal exposure group; B) Sex-stratified pup weight gain on PND 21 only; N= 12 litters (control), 16 litters (low-dose), and 11 litters (high-dose).

A significant PFAS × sex × day interaction (p= 0.001) showed that maternal PFAS exposure affects pup weight differently by sex at different times. A sex-specific effect of exposure on pup weight between the exposure groups was revealed on PND 21 (p < 0.05). When compared to the control group, the same trend was present but not statistically significant.

A significant PFAS × day interaction was also seen for anogenital distance (F(14, 189)= 1.88; p=0.031; Figure 4A and Supplemental Table S5). PFAS exposure was associated with permutations in anogenital distance at a specific time point (PND 17). In contrast to weight, there were no significant PFAS × sex interactions for AGD. Analysis of the simple main effects of PFAS on each day was conducted to follow up on this interaction. On PND 17, a significant effect of PFAS exposure on anogenital distance (F(2, 27)= 4.35; p= 0.023; Figure 4B) across both sexes was detected with an ANOVA. Namely, the low-dose group demonstrated a trend of smaller anogenital distances (10.82 ± 0.23mm) compared to the high-dose group (11.40 ± 0.31mm) and the control group (11.20 ± 0.23mm). There were no significant additional findings revealed by the more conservative post hoc Dunnett’s test. When weight was adjusted for in an additional ANOVA for AGD, the PFAS × day interaction was not significant, suggesting that AGD varies directly with body size and is unlikely to indicate feminization in this case. Taken together, the stunted acquisition of the righting reflex in the low-dose group—coupled with the weight and anogenital distance deficits—represents considerable developmental delays, named as such because the effects were transient rather than permanent.

Figure 4: A) Pup anogenital distance (PND 1 – 21) stratified by sex and maternal exposure group; B) Sex-stratified pup anogenital distance on PND 17 only; N= 12 litters (control), 16 litters (low-dose), and 11 litters (high-dose).

A significant PFAS × day interaction was noted for anogenital distance (p=0.031), meaning anogenital distance varied by study day. There was a significant effect of PFAS on PND 17 (p= 0.023). Specifically, anogenital distances were smaller in the low-dose group compared to the high-dose group and control groups.

3.3. Post-weaning Behavioral Outcomes

3.3.1. Figure-8 Locomotor Activity Maze

When the locomotor activity was evaluated during adolescence and adulthood in the figure-8 maze, a sexually differentiated effect of maternal PFAS exposure was observed (Figure 5 and Supplemental Table S8). There was a significant PFAS × sex interaction in a combined analysis of both life stages (F(2, 27)= 4.62, p= 0.019). Namely, in adolescents and adults, activity in the maze was altered by maternal PFAS exposure compared to controls in a variable manner between sexes. Typically, females are more active than males in the figure-8 test. Accordingly, sex was a main effect in this experiment, meaning activity differed between males and females independent of PFAS exposure, time block, or cohort (F(1, 27)= 7.12, p= 0.013). However, in the case of PFAS-exposed offspring, the normal sex difference between adult males and adult females was significantly attenuated in the high-dose group compared to the controls according to a Dunnett’s test (p < 0.05). In females, motor activity decreased with PFAS exposure, whereas motor activity increased with exposure in males. A similar trend was observed in the high-dose group during adolescence, but the post hoc analysis did not support additional statistically significant findings. Time block was also a main effect, demonstrating that activity levels varied during the session, which is an expected trend that is necessary to demonstrate habituation (F(1, 297)= 98.97, p < 0.001). Accordingly, PFAS exposure did not yield any significant effects on habituation (PFAS × time block or PFAS × sex × time block).

Figure 5: A) Figure-8 maze total locomotor activity (mean ± standard error) in adolescent (5 weeks of age) and adult (8 weeks of age) offspring; B) Sex difference in Figure-8 maze total locomotor activity; N= 12 litters (control), 16 litters (low-dose), and 11 litters (high-dose); 1 male and 1 female per litter.

There was a significant PFAS × sex interaction in adults and adolescents (p= 0.019). Maze activity was altered by maternal PFAS exposure in a variable manner by sex. In PFAS-exposed offspring, the normal sex difference between adult males and adult females was significantly attenuated in the high-dose group compared to the controls (p < 0.05).

3.3.2. Additional Behavioral Tests

Maternal PFAS exposure was not associated with any significant changes in measures of anxiety and risk-taking, fear response, memory, or depression as measured by the elevated plus maze, novelty-suppressed feeding, novel object recognition, and forced swim assays, respectively. See Supplemental Results for additional details.

3.4. Serum Accumulation

PND 1 PFAS serum measurements demonstrated the most readily accumulated compounds to be PFHxS, PFOS, PFDA, PFHpA, PFNA, and PFOA (Figure 6). All compounds of the PFAS mixture were detected in the serum of pups in the low-dose and high-dose exposure groups. Precise levels of PFBA, PPFPeA, PFHxA, and PFBS could not be reliably reported because serum measurements fell below the lower limit of quantification. Detection frequencies and summary statistics are reported in Supplemental Table S4.

Figure 6:

Log-scale mixture concentrations and sex-stratified pup serum accumulation (PND 1) of each PFAS compound.

4. Discussion

In this study, maternal exposure of rats to PFAS via drinking water—at environmentally relevant concentrations and 5000-fold the environmental concentration which was in the dose range of previous experimental studies—was associated with developmental changes in the F1 generation including offspring weight, anogenital distance, reflex ontogeny, and locomotor activity. Measures of non-spatial memory, attention, anxiety, and depression-like behaviors were unaffected by developmental PFAS exposure at both doses. Thus, the consequences of exposure were relatively specific, and affective and memory-like behaviors were spared from long-term effects.

Maternal PFAS exposure led to transient disruptions to expected growth late in the pre-weaning period, classified as developmental delays. Delays presented in a dose-dependent manner for both weight and anogenital distance and a sex-dependent manner for weight. The nonmonotonic effects suggest a complex dose-response relationship between PFAS exposure and offspring growth. The growth effects were not present at earlier neonatal ages but emerged over time, indicating that growth restriction may appear later, even in the absence of low birth weight. The decreased anogenital distances for both sexes in the low-dose group emerged on PND 17 and were resolved by PND 21. Further, the lower body weight seen in low-dose males on PND 21 disappeared by four weeks of age after the offspring were weaned. The transient weight deficits may stem from differences in dam milk production relative to offspring demand or other dam and offspring lactational tendencies, which resolve when the offspring switch to solid food, but there is insufficient evidence to define an underlying mechanism at this time.

The low- and high-dose exposure groups exhibited increased latency in the negative geotaxis test. This effect was not sexually differentiated, and PFAS-exposed offspring achieved control performance levels by the end of testing (PND 13). Although subjects eventually acquired the reflex, poor performance at the beginning of the testing sequence suggests delays in motor development. This observation is consistent with those reported by Cheng et al. (2013), where mice exposed to drinking water with 10.0 μg/mL of PFOA from gestational day (GD) 1 through PND 21 experienced delayed negative geotaxis acquisition compared to controls. Similar to the results presented here, the righting reflex was unaffected despite changes in negative geotaxis performance (Cheng et al., 2013). However, righting reflex development was altered in an experiment by Luebker et al. (2005) where rats were exposed to PFOS for six weeks prior to mating through the end of lactation. Offspring in the 1.6 mg/kg/day group displayed righting reflex delays, but the same trend was not seen at lower doses of 0.1 and 0.4 mg/kg/day (Luebker et al., 2005). Further, in a six-day gestational exposure study, the offspring of rats exposed to 6.0 mg/kg/day of PFOS from GD 12–18 showed increased righting reflex latency (Fuentes et al., 2007). In contrast, following a 28-day oral gavage with up to 150 mg/kg/day of PFBA, righting reflex in rats was unaffected (Butenhoff et al., 2012). Prior investigations show that gestational PFAS exposure can influence reflex development, but so far these reports mainly speak to the impacts of single-compound legacy PFAS exposures (Agency for Toxic Substances and Disease Registry (ATSDR), 2021).

Given that PFOA and PFOS make up a small proportion (8–9%) of PFAS in the administered mixture, the developmental changes observed here are likely impacted by other, less well-characterized PFAS. Approximately 75% of the exposure consisted of PFHxA, PFPA, and PFHpA, none of which have been tested previously in this model. Despite its low mixture concentrations relative to other compounds, PFOS proved highly accumulative in pup serum, likely attributable to its long half-life (Figure 6). PFHxS, PFDA, PFHpA, PFNA, and PFOA were also readily accumulated in serum. Future research is needed to determine whether neonatal reflex deficits are associated with specific compounds included in this mixture or generated by interactions or joined effects within the mixture.

After offspring were weaned, measures of anxiety, depression, and memory were unaffected by maternal PFAS exposure at both doses. Altered neurobehavioral trends were revealed in the domain of locomotor activity. The complex locomotor activity and habituation trends measured in the figure-8 maze are reflective of nervous system development and maturation and are relevant to human attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder phenotypes (Bowers et al., 2016; Dougnon and Matsui, 2022). We observed increased activity following toxicant exposure in males and depressed activity in females. The locomotor activity changes not only persisted into adulthood, but they were also strengthened.

The typical baseline difference in male and female activity was not seen across the adolescent and adult life stages for the high-dose group. The sex-specific activity trends are indicative of endocrine-mediated PFAS effects, toxicokinetic differences by sex, or effects on brain systems that underlie typical sex differences in these behaviors (Bowers et al., 2016). Elimination of sex-related locomotor activity patterns has been previously observed in the case of other pollutant exposures, such as benzo[a]pyrene and organophosphate pesticides (Aldridge et al., 2005; Hawkey et al., 2019; Levin et al., 2010; Roegge et al., 2008). The potential for PFAS exposure to eliminate typical sex differences and for those changes to persist into adulthood is concerning. In humans, neurodevelopmental disorders such as ADHD and ADHD-associated comorbidities are differentially detected among males and females in the general population (May et al., 2019; Ottosen et al., 2019), and exposures that alter sex differences may contribute to shifts in the prevalence and nature of clinical symptoms of these disorders.

Locomotor trends following PFOA and PFOS exposure have been studied in rodents with variable results. Sobolewski et al. (2014) described significantly increased locomotor activity in adult male mice compared to controls after 0.1 mg/kg PFOA exposure from GD 7 through weaning. Following 1.0 and 0.3 mg/kg/day oral PFOS exposure from GD 0 through PND 20, Butenhoff et al. (2009) noted transient hyperactivity and reduced habituation in male rats on PND 17 but not PND 13, 21, or 61. Onishchenko et al. (2011) reported the opposite trend wherein gestational exposure to 0.3 mg/kg of PFOS from GD 0 until birth led to decreased locomotor activity in male mouse offspring in novel environments. Multiple prior studies have observed locomotor activity trends that were not specific to one sex. Reardon et al. (2019), demonstrated hyperactivity in juvenile rats following 1.0 mg/kg PFOS exposures from GD 1 through weaning. In a neonatal exposure study, single 21.0 μmol/kg PFOA and PFOS doses at PND 10 led to hyperactivity in mice at four months of age (Johansson et al., 2008). The results presented from this study augment a growing body of evidence on the influence of developmental PFAS exposure on locomotor activity.

The influence of exposure to emerging PFAS compounds on activity has garnered less attention compared to PFOA and PFOS. Following administration of 9.2 mg/kg/day PFHxS to mice on PND 10, increased spontaneous motor activity was observed (Viberg et al., 2013). In contrast, three-month exposures to 200 mg/kg/day of PFHxA or 500 mg/kg/day of NaPFHx did not affect the results of a functional observational battery or locomotion (Klaunig et al., 2015; Loveless et al., 2009). Other rodent studies noted no change in motor activity following PFBS exposure up to 600 mg/kg/day for 90 days, PFBA exposure up to 150 mg/kg/day for 28 days, or a single exposure to 50 mg/kg of PFDA (Butenhoff et al., 2012; Kawabata et al., 2017; Lieder et al., 2009). In zebrafish larvae, overall activity was diminished following PFBS, PFOS, PFNA, and PFHpA exposures compared to controls, but active swimming speed was increased with PFDA, PFBS, and PFOS exposures (Huang et al., 2023; Ulhaq et al., 2013). Hyperactivity arose in zebrafish exposed to PFOS, PFOA, and PFNA (Jantzen et al., 2016). Prior findings suggest the potential for emerging PFAS contaminants to impact locomotor activity, as observed in this study, but variable results across exposure scenarios welcome continued investigation.

Besides sex, several factors may influence behavioral outcomes, such as weight, age, social isolation, handling frequency, and other individual differences (Bogdanova et al., 2013). To control for these potential confounders, animals were tested at the same age, handled with equal frequency, and doubly housed across all cohorts. Weight could not be controlled, as animals had ad libitum access to food. In humans, low birth weight is associated with increased risks for childhood behavioral issues (Kelly et al., 2001). However, there were no significant differences in birth weight among the exposure groups. Hence, the effects of exposure on birth weight are unlikely to be responsible for observed differences in behavioral outcomes. Elevated PFAS exposure in human cohort studies is also associated with childhood overweight and obesity (Geiger et al., 2021). In this study, no significant trends were observed between in-utero PFAS exposure and post-weaning weights.

4.1. Potential Mechanisms

Sex differences in response to PFAS exposure could be due to variable toxicokinetics, effects on hormonal systems, or signaling pathway disruptions. Emerging evidence points to PFAS as endocrine disruptors, formally defined as compounds that alter endocrine function and create adverse health effects in intact organisms, their progeny, or populations (Solecki et al., 2017). Perturbations in hormone homeostasis have been implicated in many of the adverse health effects associated with PFAS exposure, particularly in the domains of development and reproduction. For instance, both short- and long-chain PFAS have been shown to interfere with the molecular systems, synthesis, distribution, and secretion of sex hormones and thyroid hormone (Mokra, 2021). Like natural hormones, endocrine disruptors can produce considerable effects at low environmentally relevant doses, known as the “low-dose hypothesis” (Vandenberg, 2014). Endocrine disruptors have also demonstrated nonmonotonic dose response curves—like those seen in this study—in humans, animals, and cell lines (Vandenberg, 2014). The mechanism of nonmonotonic dose responses may be congruent with that of the low-dose effects, or it could be due to distinct mechanisms involving cytotoxicity, receptor downregulation/desensitization, complex cell- and tissue-specific molecular interactions, receptor selectivity, and competition, or negative feedback loops (Vandenberg, 2014). Nonmonotonic tendencies are increasingly reported but still debated in toxicology due to the lack of a well-understood mechanism.

Thyroid hormone disruption is one potential pathway for resulting effects. Strong in vivo evidence has shown associations between short- and long-chain PFAS exposure and disruptions of thyroid hormone secretion and the hypothalamic-pituitary-gonadal axis (Mokra, 2021). In humans, an 11,588-child German cohort study with 527 ADHD cases demonstrated thyroid-stimulating hormone (TSH) levels were inversely proportional to the likelihood of an ADHD diagnosis (Albrecht et al., 2020). A Norwegian 405-case 1092-control study corroborated the finding in newborns of associations between low TSH and elevated ADHD risk (Villanger et al., 2020). Thyroid hormone’s critical role in neurodevelopment and its vulnerability to PFAS impacts offers additional insight into potential mechanisms for the observed changes in locomotor activity.

PFAS have demonstrated considerable endocrine disruption by impacting steroidogenesis and interacting with nuclear hormone receptors (Mokra, 2021). Through in vitro and in silico models with the HeLa cell line, PFOA and PFOS have acted as androgen receptor antagonists (Di Nisio et al., 2019). PFHxS, PFOS, and PFOA have demonstrated estrogen receptor affinity across various cell lines (Kjeldsen and Bonefeld-Jørgensen, 2013). Additionally, MCF-7 BUS human breast cancer cell line models showed PFBA to act as an estrogen receptor antagonist (Li et al., 2020). Further, PFBA, PFHxA, and PFHpA downregulated estradiol-mediated genes, demonstrating that some PFAS compounds disrupt the expression of endogenous estrogen-mediated genes. Dampened endogenous estrogen receptor signaling could underlie part of the sexually differentiated hyperactivity responses in exposed rats.

In amphibians, Foguth et al. (2020) studied the effects of exposure to a five-PFAS mixture containing 4.0 ppb PFOS, 3.0 ppb PFHxS, 1.25 ppb PFOA, 1.25 ppb PFHxA, and 0.5 ppb PFPeA. After 30 days, exposed groups demonstrated a significant decrease in glutamate and 5-hydroxytryptamine (5-HT) neurotransmitter levels. Later in development, at Gosner stage 46 (the final step of leopard frog metamorphosis), the 5-HT and glutamate deficits disappeared, but acetylcholine levels were significantly increased. This study corroborates the finding that the neurochemical effects of PFAS mixture exposure will change across different stages of development. Transient effects on the serotonergic system and excitatory neurotransmitters may explain the delayed but ultimately acquired, reflexes seen in this study.

Sexually differentiated PFAS toxicokinetics are another potential factor that may influence sex differences in biological outcomes vulnerability. For instance, the transformation of PFOA to PFOA-containing lipids is reported to occur more readily in the livers of male rats (Vanden Heuvel et al., 1991). Further, rapid PFOA urine excretion by female rats is likely due to hormonally modulated active renal tubular secretion (Nakayama et al., 2005). However, differences in PFAS clearance by sex have been noted in mature animals and require further investigation during early life stages. In addition to the less efficient elimination of various PFAS compounds, males may face higher in-utero exposure (Björvang and Mamsen, 2022). Following incidental in-utero PFAS exposure, Mamsen et al. (2019) observed that human pregnancies with male fetuses demonstrated higher placental accumulation of PFOA, PFOS, and PFNA compared to females. PFAS compounds are likely transferred in a sexually differentiated manner during pregnancy. Sex-dependent toxicokinetics, resulting in altered levels or duration of early-life exposures, may contribute to the distinct or even contradictory patterns of impairment displayed by male and female rats in select tests.

4.2. Human Correlates

We have not encountered any human cohort studies in which the primary measured exposure included the main components (PFHxA, PFPA, PFHpA, and PFBA) of our mixture or the compounds most accumulated in serum (PFHxS, PFOS, PFDA, PFHpA, PFNA, and PFOA) which underscores the value of this study. In studies during which PFOS and/or PFOA were primary exposure constituents, elevated exposures were associated with worsened visuomotor abilities (Harris et al., 2018), decreased IQ scores (Wang et al., 2015), and increased rates of ADHD in children (Hoffman et al., 2010; Vuong et al., 2021). In studies with PNFA acting as a primary exposure, externalizing behavioral difficulties and increased rates of ADHD were observed (Vuong et al., 2021). In the Shanghai Birth Cohort study of ten PFAS compounds in maternal plasma, negative correlations between motor scores and PFNA, PFUnDA, and PFHpA exposures were noted in two-year-old children (Luo et al., 2022). In the Taiwan Birth Panel Study, prenatal exposure to PFOS alone was negatively associated with gross motor development in two-year-old children (Chen et al., 2013). Sex-specific neurodevelopmental outcomes were also seen in human studies. In another Shanghai Cohort analysis that examined PFHxS, PFOS, PFOA, PFNA, PFDA, and PDUdA as the main exposures, worsened interpersonal social skills were seen in girls, while boys did not demonstrate increased risks for developmental issues (Niu et al., 2019). The sex-specific effects observed only in females contrast with the male developmental delays in this study. The precise genetic and hormonal mechanisms underlying the variable developmental effects resulting from PFAS exposure have yet to be elucidated (Niu et al., 2019).

The available human literature does not directly correlate to our study’s particular exposure circumstances. We did not observe any effects on anxiety, depression, or memory, as may be expected from cohort studies. We observed only two vulnerable areas in the domains of hyperactivity and reflex development. Accordingly, unique exposure profiles and associated altered cognitive outcomes cannot be extrapolated to the PFAS chemical class in its entirety. The effects of understudied PFAS compounds in our mixture warrant further analysis in human studies.

4.3. PFAS Chemical Mixture Dose

The significant developmental effects in both the environmentally relevant dose and the high dose suggest that the range between the lowest observed effect concentration and the lowest lethal dose is very large. Even at 5000x environmental levels, we were unable to find an intolerable dose at which exposed rats experienced elevated mortality rates or severe morbidity. Furthermore, similar behavioral effects were observed in the reflex and hyperactivity tests, irrespective of exposure dose. Thus, comparable health risks could be expected across populations with vastly different exposure scenarios. Rather than studying overt toxicity at extreme doses, the consequences of different human exposures may be best understood through dose-response studies. For example, growth curves could be compared across a wide range of mixtures to elucidate the toxicologic basis for differences in pup size between the high- and low-dose groups while ruling out survivorship bias or compensatory mechanisms.

4.4. Study Considerations, Strengths, and Limitations

The unique chemical properties of PFAS give rise to dual lipophilicity and hydrophobicity (known as amphiphilicity), leading not only to environmental persistence and bioaccumulation in food webs but also accumulation in breast milk and maternal transfer to offspring during nursing (Ding et al., 2020; Mondal et al., 2014). Although neonatal exposure during nursing is low relative to the maternal gestational exposure period, it is important to acknowledge that a variable perinatal component influences the results outside of prenatal exposure.

The study design has several technical strengths. First, the dose range used for PFAS exposure was appropriate for rats (according to the cited rodent literature) and environmentally relevant for affected populations. The EPA strongly recommends a high-dose or positive control group because it allows for more sensitive detection of results (Maurissen and Marable, 2005). Further, the low-dose drinking water exposure directly mimics a primary human exposure pathway, aiding in animal-to-human extrapolation. Finally, the use of a mixture, which includes both legacy and emerging contaminants, represents realistic human exposure scenarios in Pittsboro, North Carolina, and other sites more effectively than single-chemical exposures (Monosson, 2005).

Although drinking water consumption best represents the human exposure scenario, some inherent associated variability exists. Specifically, between-subjects differences in intake due to weight (i.e., larger animals demonstrating higher total water consumption) may lead to different levels of accumulation. However, drinking water administration has strengths over gavage as oral gavage has been found to bypass critical interactions with the oral mucosa, cause complications such as esophageal perforation, and induce stress in subjects (Vandenberg et al., 2014). Further, our data complement observations found in other PFAS-exposure studies using gavage (see Supplemental Table S1).

Toxicokinetic data from the dams could not be obtained and thus poses limitations. Serum PFAS levels were measured in culled pups one day after birth following drinking water exposure cessation on PND 0 (Supplemental Table S4). Blood was not drawn from dams to avoid significant additional stress during pregnancy and nursing that could affect birth outcomes. In serum from litters exposed to the low-dose mixture, 10 of 10 PFAS compounds from the drinking water mixture were detected with 100% frequency, and 6 of 10 compounds were measured above the lower limits of quantification (LLOQ) (Supplemental Table S4B). In the low-dose group, serum sum PFAS concentrations measured 7 .975 ng/mL. By contrast, 7 of 10 compounds were detected with 100% frequency in control serum samples, and only 3 of 10 PFAS compounds were measured above the LLOQ. In the high-dose serum, 10 of 10 PFAS compounds were detected with 100% frequency, and 9 of 10 compounds were measured above the LLOQ. High-dose group sum PFAS levels were 1306.678 ng/mL. As previously observed in our rabbit mixture study, short-chain PFAS were appreciated in smaller quantities and detected less frequently above the LLOQ (Crute et al., 2022). Notably, the most readily accumulated compounds in PND 1 serum of exposed pups were long-chain PFAS (PFOS, PFDA, and PFNA), which are known to have longer half-lives in humans and rodents than short-chain alternatives (Mokra, 2021).

It is important to note that the pup serum data are a snapshot of PFAS burden at PND 1, but do not necessarily reflect exposures over the full course of gestation. The PND 1 measurements are likely to represent the serum levels of long-chain PFAS over time more accurately than short-chain PFAS with faster kinetics. Thus, we emphasize the observed effects in the context of the toxicant exposures themselves (high-dose and low-dose) rather than serum levels. If biological outcomes were to be related exclusively to body burden, only persistent toxicants would be implicated despite the potential for lasting effects from exposure to toxicants with fast kinetics during vulnerable developmental periods. An animal study with controlled exposures allows us to discern the effects of short- and long-dwelling toxicants alike.

Overall, pups in the low-dose exposure group demonstrated slightly lower serum levels than those observed in human studies. Among infants less than one year old in the United States, the National Health and Nutrition Examination Survey found mean sum PFAS levels of 27.01ng/mL and 51.59 ng/mL for the 95^th percentile (Kirk et al., 2022). In a cohort of adults with a median age of 58 in Pittsboro, North Carolina, the average serum levels of PFHxA, PFHpA, PFOA, PFNA, PFDA, and PFOS summed to 23.94 ng/g (Hall et al., 2023). PFBA, PFPeA, PFBS, and PFHxS were not reliably detected (Hall et al., 2023). Sum PFAS levels falling below those of human populations are likely reflective of the short exposure period. Even so, the low dose was still able to produce appreciable developmental effects.

5. Conclusion

Using a rat model, our results are the first to report neurobehavioral and developmental toxicity by comparing an environmentally relevant PFAS mixture dose to a much higher dose modeled after previous laboratory PFAS studies. In this study, offspring growth was measured by weight and anogenital distance. Reflexes were assessed through negative geotaxis and righting reflex acquisition. Hyperactivity was measured by the adult and adolescent figure-8 maze. Anxiety was assessed by the novelty suppressed feeding and elevated plus maze tests. Attention and memory were assessed by the novel object recognition assay. Finally, depression-like behavior was evaluated through the forced swim test. This battery gives a broad overview of potentially affected domains to be correlated with findings in future animal studies.

PFAS-exposed offspring in this experiment demonstrated transient sex-specific and sex-independent neonatal growth attenuations, sex-independent reflex developmental delays, and lasting altered hyperactivity patterns compared to unexposed offspring. Our findings contribute to a greater understanding of the health effects associated with PFAS contamination in communities like Pittsboro, North Carolina. This animal model study demonstrates a cause-and-effect relationship between developmental PFAS exposure and selective delays in growth and reflexes, as well as a diminution of typical sex differences in activity with two doses representing a wide exposure range. Further studies to improve understanding of the long-term effects of developmental PFAS exposure, the underlying mechanisms, and interactions between PFAS and co-exposures to other toxicants are warranted.

Abbreviations

PFAS

Per- and polyfluoroalkyl substances

Data Sharing

The study data are available upon request.