By Anna Maddison, Environmental Toxicology ‘21, Janaé Bonnell, Environmental Toxicology ‘22, Dr. Michele La Merrill

Authors’ Note: This literature review was conducted for the Office of Environmental Health Hazard Assessment in the California Environmental Protection Agency under a contract issued to Dr. Michele La Merrill. We wanted to understand the current research on the reproductive and developmental toxicity of PFHxS, PFBS, PFHxA, PFHpA, PFNA, PFDA, and ADONA to draw conclusions and make recommendations for future policy and research. It is important to understand the health effects of substances such as PFAS chemicals that are present in our food, water, and consumer products to develop regulatory standards that protect public health.

Abstract

Per- and polyfluoroalkyl (PFAS) chemicals are used in the production of many industrial processes and consumer goods, and they have been widely detected in humans and animals. PFOS and PFOA have been comprehensively studied and are being phased out of use, but there are other understudied PFAS chemicals with effects that should be considered in regulatory affairs regarding public health and safety. In this study, we focus on the reproductive and developmental effects of seven PFAS chemicals: perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorohexane sulfonic acid (PFHxS), 4,8-dioxia-3H-perfluorononanoic acid (ADONA), perfluorobutane sulfonic acid (PFBS), perfluoroheptanoic acid (PFHpA), perfluorodecanoic acid (PFDA). This literature review presents the observed reproductive and developmental effects of these chemicals on animal models, which can be used to help establish legislative priorities and draw attention to current gaps in published literature.

Keywords: PFAS, review, animal model, PFBS, PFHxS, PFHpA, PFHxA, PFDA, PFNA, ADONA

Introduction

Poly- and perfluoroalkyl (PFAS) chemicals are widespread synthetic chemicals that are highly mobile, persistent in the environment, and are known to bioaccumulate in humans and animals[1,2]. PFAS chemicals have extensive use due to their unique anti-wetting abilities, as well as their ability to act as a surfactant, a molecule that lowers the surface tension between two liquids[3]. These properties have led to their use in oil- and water-repellent textiles, coatings, and fire-retardant products. PFAS chemicals can also be found in drinking water, production facilities and industries, and many commercial household products[1,4]. Humans are mainly exposed to PFAS through their diet but are unable to metabolize these chemicals in their bodies[5,6]. 

These chemicals have found use in multiple industries for over 60 years[7]. PFAS production peaked in the 1990s, with PFOS and PFOA being the most popularly used PFAS chemicals. However, after studies linked these chemicals to adverse human health effects, including damage to the immune system and liver, reproductive and developmental harm, hormone disruption, and cancer, the United States had voluntarily phased out their use[1,5,2]. The industry has since shifted to favor PFASs with shorter chains, as these were believed to be less harmful to human health[7,8]. Although no association between chain length and toxicity has been proven, it is still being explored. 

To this end, perfluorohexane sulfonic acid (PFHxS), perfluorobutane sulfonic acid (PFBS), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and 4,8-dioxia-3H- perfluorononanoic acid (ADONA) have been increasingly used for industrial purposes as alternatives to PFOS and PFOA[6,8]. These PFAS chemicals have shorter carbon backbone chains, so it is believed they may have less negative health effects than PFOA and PFOS, but this has not been proven. However, their health effects are not as well-explored as those of PFOS and PFOA, thus necessitating new research into the long-term health impacts of exposure to these replacement PFAS chemicals.

Multiple epidemiological studies in humans have suggested that there is an association between PFAS exposure and adverse reproductive and developmental health impacts. After exposure during pregnancy, lower levels of thyroid hormones T3 and thyroxine have been observed in both pregnant women and fetuses, as well as adverse birth outcomes and behavioral effects on children exposed in-utero[6,9]. Some of these PFAS chemicals, such as PFNA, are being detected in both seminal plasma and breast milk of humans[10].

Individuals who are not fully developed may be more vulnerable to PFAS exposure and therefore, more at risk for any toxic effects. The purpose of this literature review is to survey reproductive and developmental effects of PFAS exposure to inform on potential literary gaps and future regulatory priorities. 

Methods

Search Procedure

The research for this literature review was conducted by systematically choosing articles discovered through the search engine PubMed. The initial database of articles was created from searching the abbreviated forms of the chemical names (PFHxS, PFBS, PFHxA, PFHpA, PFNA, PFDA, ADONA) in PubMed using the filter “Other Animals” (Table 1). Only papers in English were considered for inclusion. An exception was made for ADONA due to our search yielding an excess of papers written by an author by that name. Instead the search term “ADONA NOT adona [author]” was used. Even with the reduced search, many of the results did not include the PFAS chemical ADONA, and subsequently, were not included. 

Table 1: Contains a list of searches and number of results yielded for each one.

Study Selection

The titles and abstracts of each article were scanned for relevance to reproductive toxicity. This includes in vitro studies on isolated cells, different toxicological effects based on sex, responses affecting reproductive organs, and developmental effects in offspring. To limit the scope, this excluded human studies, bioconcentration studies that did not specify reproductive organs or unique developmental patterns, wildlife studies, and studies focused on other organ systems not in the context of reproduction or development. Additionally, we chose to exclude studies which used a mixture of chemicals or in which the PFAS chemical was analyzed as a metabolite of a parent chemical. This was due to the fact that it is uncertain which chemical actually caused an effect. Relevant articles were examined in depth to determine significant toxicological effects of the PFAS chemicals. The number of relevant articles in comparison to the total articles which arose from our search can be seen in Table 2. Other notable responses, from papers which included reproductive effects, were denoted in our accompanying spreadsheet. Due to time constraints, the results for PFDA were summarized with less depth and were instead included as an appendix. 

Table 2: Contains the number of resulting studies chosen for inclusion in this review.

Results

PFNA

Seventeen studies contained relevant reproductive and/or developmental effects of PFNA exposure. All of these studies except for one, which dosed Xenopus laevis (African clawed toad, African claw-toed frog or the platanna), examined the effects of PFNA exposure on Danio rerio (zebrafish), Rattus (rats), or Mus (mice). These studies are compiled in Supplementary Table 1 (ST1).

PFNA exposure in Danio rerio caused embryo disfigurement and altered motion patterns, as well as other reproductive and developmental effects. In recently fertilized eggs, PFNA exposure resulted in an increase in malformation rate[6], specifically, in the rate of ventricular edema[19]. Studies also showed correlation between PFNA exposure and locomotion: Danio rerio exposed as embryos travelled less distance [11][13][6], their activity level increased[24] [13], they bumped into the mirror more[13], their startle response and burst activity increased[6],  the amount of time they spent in the middle of the water, and their velocity changed[13][11]. Additionally, exposure to PFNA caused reproductive effects such as an increase in the number of opaque embryos[19][, a decrease in hatching rate[19][15], a decrease in the number of eggs in females[15], and an increase in yolk sac area[11]. Observed developmental effects included a decrease in body length[13], abnormally enlarged follicles in the thyroid, and increased T3 hormone levels[18]. 

In Mus and Rattus, the most observed reproductive effects occurred in the testis. In Rattus, cell viability in Sertoli[12] and testicular[23] cells decreased, DNA damage in testicular cells increased[23], vacuoles formed in the Sertoli cells[12], the number of germ cells that degenerated or were TUNEL-positive increased[12][20], and the percentage of apoptotic cells in the testis increased[20]. In Mus, intratesticular and serum levels of testosterone, testicular glucose level, and testicular lactate concentration decreased[14][10]. Also in Mus, seminiferous tubules had intraepithelial vacuolation where small cavities formed in the tubules, marginal condensation of chromatin in round spermatids, and giant cells and exfoliation of germ cells in the lumen of the tubules[14]. Reproductive effects in mammals exposed to PFNA included reduced prenatal and postnatal survival[16], maternal weight decreased[16][25], and lower birth weight and higher blood pressure in pups[25]. Additionally, there was a sex-specific difference in that serum concentration of PFNA decreased more rapidly in adult females than males[17]. Finally, the developmental effects of PFNA exposure in Mus who had not experienced puberty included a decrease in weight, an increase in absolute and relative liver weight, and hepatocellular hypertrophy[14][10].

In Xenopus laevis, PFNA exposure resulted in increased embryo mortality and malformations including stunted tadpole length, multiple edemas, gut miscoiling, microcephaly in which the offspring had abnormally small heads, and skeletal kinking[22].

PFHxA

Five articles on reproductive and/or developmental effects of PFHxA, which showed statistically significant responses, were found to be relevant to our study. These studies are compiled in Supplementary Table 2 (ST2).  Each of the five papers studied a different animal: Danio rerio, Daphnia magna, anuran Xenopus laevis, Mus, and Brachionus calyciflorus. In both Danio rerio and anuran Xenopus laevis embryos, a decrease in body length was observed in the animals exposed to PFHxA[26][28]. In Daphnia magna, reproductive output increased with chronic exposure to PFHxA and mobility decreased with acute exposure to PFHxA[27]. In the Mus, indirect exposure in utero to PFHxA resulted in pups taking longer to open their eyes, a decrease in their body weight, and a lower ratio of liver to body weight[29]. Xenopus also saw liver effects in the form of swollen livers in tadpoles[28. In Brachionus calyciflorus, the rate of population increase decreased, and the mictic ratio, the ratio between eggs that require fertilization and those that do not, and egg size increased[30].

PFHxS

Using the methods described above, fifteen articles examining the effects of PFHxS on Mus, Rattus, Gallus gallus domesticus, and Danio rerio were within the scope of our literature review. These studies are compiled in Supplementary Table 3 (ST3). In the mammals, notable, visible reproductive and developmental effects were observed. In general, male mammals dosed with PFHxS seemed to present more deviations from the control opposed to females. For example, when PFHxS was orally administered or injected via IV, male Rattus took a significantly longer time to expel it from their plasma than female Rattus[38]. Other instances of this trend were apparent when male pups displayed increased anogenital distance[35] and decreased weight gain[36] at more concentrations than females. Other effects of PFHxS in mammals included decreased activity[32] and increased liver and thyroid weight[35] in pups who were respectively dosed neonatally and exposed indirectly in utero as well as directly for two weeks. In Gallus gallus domesticus, PFHxS egg injections decreased pipping success, which is the chick’s ability to break themselves out from their shell, and decreased the mass of embryos[37]. Finally, in Danio rerio embryos an EC50 value was calculated to be 84.5μM using the concentration at which they died or suffered from an adverse effect, defined to be non-inflated swim bladder, pericardial or yolk sac edemas, or scoliosis, as a reference[34]. Altered motion patterns and body lengths were also observed[6][26]. 

ADONA

Only one article was found that showed reproductive effects of ADONA. This study is shown in Supplementary Table 4 (ST4). Pregnant Rattus norvegicus were exposed to ADONA via oral administration which resulted in a decrease in pup weight, maternal food consumption while pregnant, maternal weight gain, and the number of pups that survived[40]. A higher dosage was abandoned after two days due to death, significant body weight loss, reduced food consumption, decreased activity, dehydration, coldness to touch, pale extremities, rales or rattling sounds in the lungs, ungroomed coat, urine-stained fur, and ptosis or drooping eyelids in the pregnant Rattus[40].

PFBS

Seventy-nine articles resulted from a PubMed search on perfluorobutane sulfonic acid (PFBS) using the above-described methods. Of those seventy-nine articles, fifteen were deemed to be within the scope of this review. These studies are compiled in Supplementary Table 5 (ST5). These studies investigated the effects of PFBS on Mus musculus (mice), Oryzias melastigma (marine medaka), Caenorhabditis elegans, Danio rerio, Rattus norvegicus (rats), Xenopus laevis, and C. riparius (harlequin flies). A multitude of adverse reproductive effects was observed as a result of PFBS exposure. Multiple studies demonstrated that PFBS affects hormone levels in both ICR Mus and Oryzias, generally including a decrease in estrogen levels in all animals and a testosterone decrease in males[7][43]. One study noted that males were experiencing estrogenic changes and females were experiencing antiestrogenic changes as a result of PFBS exposure[43]. PFBS exposure also leads to alterations in levels of follicle-stimulating hormone and progesterone[7][43]. In several egg-producing organisms studied, it was found that overall egg production was significantly decreased[42][43] and that this effect could also occur after several generations of exposure to PFBS[48]. In both Mus and Oryzias, significant decreases in uterus and ovary size were recorded in both exposed organisms[43][9]. These species also had significant changes in the number of follicles and oocytes in various stages after exposure[7][43][9]. Lastly, organisms were observed to have decreased length after exposure to PFBS, an effect that was usually more prominent in males[3][48][49]. 

First-generation offspring from exposed parents were observed to have changes in hormone levels that would affect growth and development, generally including increases in luteinizing hormone[43][9] and significant alterations in T3 concentration[3][9]. Reduced estrogen levels and elevation of thyroid-stimulating hormone were also observed[9]. The length of diestrus in Mus and Rattus was also significantly increased after parental exposure to PFBS[9][8]. Significant decreases in uterus and ovary size were recorded in the offspring of exposed Mus and Oryzias[43][9]. Mus offspring were also observed to have significant changes in the number of follicles and oocytes in various stages[9]. Furthermore, organismal body weight was observed to be influenced by parental PFBS exposure, although it both increased and decreased in separate experiments[3][8][48]. A study exploring the influence of maternal and paternal dosage on Oryzias offspring observed greater negative impacts on offspring related to paternal exposure to PFBS rather than maternal exposure, such as swimming hyperactivity[44]. Offspring of exposed parents also experienced deformities, such as increased tail and craniofacial malformations in Danio rerio[49]. Lastly, it was observed that PFBS exposure delayed both the age of vaginal opening[9] and preputial separation in Mus and Rattus with parents exposed to PFBS[8].

Of the fifteen studies included, only two focused on the multigenerational effects of PFBS on organismal reproduction and development. PFBS was observed to affect T4 hormone concentration in F2 generation Oryzias after a life-cycle exposure during the F0 generation[3]. In the same species, both the weight of F2 generation eggs, as well as their lipid and protein content, were significantly increased after ancestral exposure during the F0 generation development[43]. 

PFHpA

A search of PubMed using the above methods yielded thirty results for the chemical PFHpA. Of these thirty results, two were found to be within the scope of this review. These studies are compiled in Supplementary Table 6 (ST6). These reports studied the effects of multiple chemicals, including PFHpA, on Danio rerio and Xenopus laevis via exposure in solution.  Both studies are described here due to the limited number of papers. The lowest-observed-adverse-effect levels in the Menger[6] and Kim[28] studies were found to be 89 µM and <0.25 mM (250 μM) renewed daily, respectively. The findings in these studies suggest that PFHpA is both a possible teratogen negatively affecting the embryo or fetus and a developmental toxicant in multiple animal species[28]. A dose-response relationship was exhibited regarding PFHpA dosage and rates of both malformations and mortality in Xenopus embryos[28]. Observed malformations included reduction of body length in 24% of Xenopus tadpoles dosed with 1,000 µM of PFHpA as embryos, as well as enlarged, abnormal livers[28]. However, the observation of reduced tadpole body length was only noted at a dosage of 1,000 µM of PFHpA, which is comparable to the LC50 value in this study, found to be 942.4 µM[28]. There was also evidence of potential behavioral effects in developing organisms. For example, Danio rerio exposed to PFHpA had a significant decrease in swimming distance recorded at the highest dosage[6]. No studies listed in PFHpA searches investigated the reproductive hazard of the chemical.

PFDA

Of the two hundred articles listed for PFDA on PubMed using the above search methods, sixteen studies were considered relevant to be included within this report. These sixteen studies are summarized in Appendix 1 (A1) and a subset of them is highlighted here. Both the viability and maturation of pig oocytes was shown to be negatively impacted by exposure to PFDA[50]. The thyroid was shown to be affected in multiple studies, with thyroid hormone levels being both decreased[59][61][62]  and increased[57] in different reports. PFDA also adversely affected the sexual organs of exposed organisms, including the seminal vesicle[56] and seminiferous tubules[60]. In rats, hamsters, and guinea pigs, degeneration of the seminiferous tubules was observed following PFDA exposure[60].

Outside of the 184 remaining studies not included in Appendix 1, three studies stated that PFDA may be estrogen-like in its action. However, these three papers did not contain health endpoints that were within the scope of this report, such as gene expression and carcinogenesis[64][65][66]. These papers are referenced within the sources of this report but were not included within the review of reproductive and developmental health, nor were they included within Appendix 1 (A1). 

Discussion

While the toxicity of PFBS, PFHxS, PFHpA, PFHxA, PFDA, PFNA, and ADONA, which are used as substitutes for PFOS and PFOA, is still being investigated, the current body of scientific research suggests that exposure to these PFAS chemicals poses potential reproductive and developmental risk to an organism.

In several species, PFNA exposure resulted in several effects that could interfere with normal procreation and aging systems. Observed deviations from control groups included embryo malformation, offspring mortality, altered behavioral patterns, morphological abnormalities, weight changes, and different hormone levels. There was evidence to suggest that males retain more PFNA than their female counterparts, and several studies suggested that male sex organs were impacted by PFNA exposure. Relative to the other PFAS chemicals studied in this paper, PFNA had a high amount of significant papers with reproductive and developmental effects. This may suggest that it poses a higher risk than some of the other chemicals, but it should be considered in the context that PFNA also had a higher number of search results than the other chemicals and therefore, may just be more well studied. A limitation in these findings that should be considered is the apparent lack of corresponding research on female sexual organs. Also, several effects were not observed to have a dose-dependent response, while others were only seen at lower concentrations. 

PFHxA only had five relevant studies and each used a different species as test subjects. Additionally, there were four studies, two used in the results of PFHxA and two not used due to a lack of significant results, which also did their experiments with other PFAS chemicals, and in comparison, individuals dosed with PFHxA had milder, if any, effects. This suggests that PFHxA may have lower reproductive and developmental toxicity than other PFAS chemicals in this paper. This could be taken into consideration when assessing the risk of exposure associated with this chemical. 

PFHxS, similar to PFNA, had more toxic effects in males than females. Unlike the studies using PFNA, a majority of these did test both males and females in the same conditions. Exposed males retained a higher serum concentration of PFHxS for longer and displayed changes in anogenital distance and weight at a larger range of dosage concentrations than females.

There was one relevant paper that addressed the reproductive and developmental effects of ADONA exposure. This, coupled with the potential conflict of interest presented by the funding,  makes it impossible to draw any conclusions regarding the toxicity of this chemical. Even so, effects were observed from exposure to ADONA. These included changes in weight, mortality rate, and food consumption. To further understand the reproductive and developmental toxicity of ADONA, further research is necessary. 

Multiple species demonstrated adverse reproductive and developmental health outcomes after exposure to PFBS. Alterations to hormone levels and morphological abnormalities were observed following PFBS dosage. Also, PFBS was seen to potentially influence hormonal sex changes in exposed fish. Several studies emphasized the impacts PFBS exposure may have on offspring. The above changes were seen in offspring after parental exposure, as well as alterations to egg development and the size of female reproductive organs. It was also noted that paternal exposure to PFBS tends to more negatively impact the health of offspring than maternal exposure does. Compared to other chemicals included  within this report, PFBS had a relatively high number of studies that met the requirements for inclusion. 

Only two relevant studies were found regarding PFHpA, each using a different test species. These studies investigated the developmental effects of PFHpA on organisms exposed as embryos or tadpoles. Findings suggested that PFHpA may be teratogenic and toxic to developing organisms based on resulting morphological abnormalities and behavioral alterations in exposed organisms. However, no studies were found to investigate the reproductive impacts of PFHpA exposure, leading to a gap in the available research on this chemical.

The studies included on PFDA cover its effect on cell viability, hormone levels, and sex organs, as well the potential developmental toxicity and teratogenicity of PFDA exposure. However, within these studies, there are limitations. Multiple studies did not include information on the age at which organisms, usually Rattus, were dosed, making it more difficult to fully comprehend the effects of PFDA on stages of animal growth. Compared to several of the chemicals included in this paper, PFDA has a relatively higher number of papers that met the criteria for inclusion. Studies have been conducted on this chemical since the 1980s, far preceding work on some of the other chemicals in this report. Furthermore, while the effects of PFDA on hormones and the thyroid have been investigated in several studies, little work appears to have been done on the multigenerational and offspring effects of PFDA. Lastly, five of the sixteen studies investigated the effects of PFDA only on males, leaving further information on female-specific effects to be desired. Both multigenerational and female-specific studies could be considered potential avenues for further research.

Overall, these PFAS chemicals have demonstrated that they may have potential for reproductive and developmental health effects, and their effects on humans should be investigated to ascertain human risk. 

Conclusion

The objective of the present study was to compile and summarize the toxic effects of different PFAS chemicals on the reproduction and development of animals in controlled settings. While there are variations in the species used, as well as the method in which they were exposed and the concentration tested, exposure to all seven PFAS chemicals impacted aspects of development, and six had effects on reproduction. These effects should be considered in designing additional research on the outcomes of PFAS exposure and in assessing the risk of these chemicals on living organisms.

Acknowledgements

This work was conducted under California Environmental Protection Agency, Office of Environmental Health Hazard Assessment contract 17-E0024. The authors appreciate the support of Dr. David Furlow, professor of Neurobiology, Physiology, and Behavior and University Honors Program Director at University of California, Davis, and Dr. Sarah Elmore at the Office of Environmental Health Hazard Assessment. 

Abbreviations

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