Regrettable Substitutes and the Brain: What Animal Models and Human Studies Tell Us about the Neurodevelopmental Effects of Bisphenol, Per- and Polyfluoroalkyl Substances, and Phthalate Replacements

In recent decades, emerging evidence has identified endocrine and neurologic health concerns related to exposure to endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA), certain per- and polyfluoroalkyl compounds (PFASs), and phthalates. This has resulted in consumer pressure to remove these chemicals from the market, especially in food-contact materials and personal care products, driving their replacement with structurally or functionally similar substitutes. However, these “new-generation” chemicals may be just as or more harmful than their predecessors and some have not received adequate testing. This review discusses the research on early-life exposures to new-generation bisphenols, PFASs, and phthalates and their links to neurodevelopmental and behavioral alterations in zebrafish, rodents, and humans. As a whole, the evidence suggests that BPA alternatives, especially BPAF, and newer PFASs, such as GenX, can have significant effects on neurodevelopment. The need for further research, especially regarding phthalate replacements and bio-based alternatives, is briefly discussed.


Introduction
Bisphenols, phthalate esters, and per-and polyfluoroalkyl substances (PFASs) have been used widely for industrial and manufacturing purposes for nearly a century [1,2].The term "endocrine-disrupting chemical" (EDC) was first introduced in the 1970s, when increasing evidence identified adverse reproductive and hormonal effects of these and other similarly-acting chemicals in humans and wildlife [3].Some of these EDCs, known as "legacy" EDCs, persist in the environment and continue to be found in tissues of humans and wildlife despite being decades removed from commercial production [4][5][6][7].Since then, public interest groups, regulatory bodies, and industry have pushed to ban or phase out the manufacture and use of certain chemicals.While this represents progress in terms of advancing toxicological safety standards to better address the unique properties of endocrine disruptors (which are not always identified via standard toxicology testing) and reducing human exposure to legacy EDCs, a concerning trend has emerged wherein these chemicals are replaced in consumer products or in manufacturing with sister compounds that are structurally or functionally similar [8][9][10].The result is a phenomenon termed "regrettable substitution", by which a compound is replaced with a substitute chemical that may later prove to have a similar or even stronger endocrine-disrupting capability-often after it has been released into the market.
The developing brain is a target of EDCs, with far-reaching consequences on an individual's mental and neurological health and wellness.The brain has high hormone sensitivity, especially as it undergoes rapid growth during critical periods of development including the prenatal period, infancy, and adolescence.During this time, steroid hormones such as androgens and estrogens organize the brain in a sexually dimorphic manner and later activate certain sex-specific brain processes [11][12][13].EDCs can interfere with this by altering the normal function of these and other hormones, acting as hormone mimics, blockers, or altering synthesis and transport [14][15][16][17][18].The disruption of hormone signaling during critical periods can have lasting effects on brain structure and function, potentially affecting later cognitive-behavioral function and increasing the risk of neurodevelopmental disorders such as autism spectrum disorder, attention deficit/hyperactivity disorder (ADHD), and/or learning disabilities in humans and animal models [19][20][21][22][23].
Legacy EDCs such as bisphenol A (BPA) and certain phthalate esters [i.e., butyl benzyl phthalate (BBP), (2-ethylhexyl) phthalate (DEHP), and dibutyl phthalate (DBP), among others] alter the development of the brain and lead to disrupted cognitive-behavioral function in adulthood in zebrafish, rodents, and humans [24,25].The same is true for legacy PFASs [26,27].As the brain is highly sensitive to hormones and serves as a central regulatory hub for many of the body's homeostatic functions, disrupted development has implications beyond cognitive and affective behavior, affecting a range of functions such as metabolic, sexual, and immunological health [15,18,28].This and other scientific evidence have inspired the phasing out of such chemicals; however, new-generation bisphenols, PFASs, and phthalate esters have emerged to take their place.Because the regulatory framework that allowed the mass use of legacy EDCs also grandfathered in new-generation substitutes, there is a real concern in the scientific community that "like has replaced like" and that new-generation substitutes do not represent a lessened toxicological burden.Rather, these substitutes lack the scientific, public, and regulatory scrutiny of their predecessors and represent a new cadre of EDCs whose toxicological profile has yet to be determined [8,[29][30][31].
This review collects available information on new-generation bisphenols, PFASs, and phthalate esters in the context of the developing brain.Because of the rise in bio-based alternatives such as bioplastics, we also discuss the limited evidence for their possible endocrinedisrupting activity.Here, we discuss the literature on neurodevelopmental disruption from several model organisms-zebrafish, rodents, and humans-focusing on cognitive and other neurobiological consequences.All articles discussed and cited were found on PubMed prior to March 2024 using combinations of the following search terms: bisphenol/perand polyfluoroalkyl substance/phthalate/bio-plastic AND neuro*/behavior/replacement AND rodent/mice/rat/zebrafish/human.Only studies using in vivo models (zebrafish, mice, rats, and humans) of single-chemical exposure are discussed.Studies that only examined legacy EDCs (e.g., BPA) and no replacement or substitute EDC (as discussed below) were excluded.We also excluded studies whose EDC exposures started after 30 dpf in zebrafish, P21 in rodents, and 13 years of age in humans, as we were interested in earlier periods of development.We verified that all included studies appeared to be appropriately powered and used proper controls.

Legacy EDCs and New-Generation Substitutes 2.1. Bisphenols
Bisphenols are a group of chemicals made up of two phenol rings joined by an atom, often carbon (Figure 1a).They are used to make epoxy resins, acting as protective coatings in metal products, such as pipes and food cans.Bisphenols are also polymerized to make polycarbonate, a hard plastic found in everyday products, from electronics to food containers [32].The most well-known member of this group, bisphenol A (BPA), underwent selective bans around the world in the 2000s, especially in baby bottles, after research studies linked the chemical to cancers, diabetes, obesity, and reproductive and neurodevelopmental problems and attracting public outcry [32][33][34][35][36][37].The "BPA-free" label has since become near-ubiquitous.This label is deceptive, however, as it does not reveal that BPA has been replaced with newer analogs whose safety may not be thoroughly (PPARγ) [46].Any or all of these are potentially relevant to neurodevelopment and behavior as these receptors are expressed in the brain [47].Recently, certain G-proteincoupled receptors were identified as being activated by plastics including BPA and diethyl phthalate [48].Although the half-lives of bisphenols are short, their persistence in the environment yields detectable levels in most humans.The detection of BPA in humans is decreasing while the detection of BPA alternatives, such as BPF and BPS, is increasing [39].

Per-and Polyfluoroalkyl Substances
Per-and polyfluoroalkyl substances (PFASs) are a group of >9000 chemicals made up of a carbon-fluorine backbone and a polar functional group (Figure 1b).One of the most famous PFASs, polytetrafluoroethylene, known under its trademarked name, Teflon, is used as a nonstick coating for cookware.PFASs' nonstick, waterproof, and stain-repellent properties drove their wide utilization in food packaging, clothing (e.g., Gore-Tex), furniture (e.g., Scotchgard, Stainmaster treatments), and even fire-suppressing foams [49,50].Their thermal/chemical stability and amphiphilicity unfortunately also increase their persistence both in the environment and in the body [51,52].PFAS exposure has been linked with cancer, altered immune function, reproductive deficits, and other adverse health outcomes [53][54][55][56].PFAS interferes with the thyroid hormones and brain-derived neurotrophic factor and leads to an increase in oxidative stress and cell apoptosis, all of which are involved in neurodevelopment [31].Because of the wide body of research demonstrating the dangers of PFASs, PFOS, PFOA, and PFHxS were globally banned under the Stockholm Convention in 2009, 2019, and 2022, respectively [57].Although now members of the legacy chemical family, their persistence makes them a lasting part of the world's ecosystem.
Fluorinated chain length is one of the key factors considered regarding PFAS safety, as longer chain lengths are typically associated with higher toxicity and biomagnification [52, 58,59].Because of this, there has been a rise in short-chain alternatives (≤4 continuous fluorinated carbons), including GenX (hexafluoropropylene-oxide-dimer-acid) and ADONA (dodecafluoro-3H-4,8-dioxanonanoate) [60].Unfortunately, research indicates that these alternatives may not be any safer [55,56,61].In addition, long-chain PFAS concentrations continue to increase in living organisms, a trend that will likely continue thanks to their ubiquitousness, due to their accumulation in the food chain and their release from melting ice caps [61,62].In this review, we will focus on short-and long-chain PFASs that are not the legacy chemicals PFOS, PFOA, and PFHxS (see Table 1 for examples) and that are not as well evaluated and may prove to be "regrettable substitutions".

Phthalates
Phthalates (also referred to as ortho-phthalate diesters and phthalic acid esters) are esters of phthalic acid, 1,2-benzenedicarboxylic acid (Figure 1c).They are primarily used as plasticizers for cosmetic products, medical devices, toys, and food packaging.Phthalate exposure has been linked to effects on the neuroendocrine and reproductive systems and neurodevelopment, as well as to increases in cancer, asthma, diabetes, and other health concerns [37,63].Their effects on neurodevelopment are of special concern given the extensive phthalate exposure that premature and intensive care newborns are exposed to through medical tubing [64].In 2017, the United States banned eight phthalates from use in children's products, including toys.These included diisononyl phthalate (DINP), di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and diisobutyl phthalate (DIBP) [65].Advocacy groups petitioned the FDA to ban the use of phthalates in food packaging, but in 2022, this petition was denied [66].Efforts to create safer options resulted in the development of structurally similar groups, including isophthalates, terephthalates, and trimellitates, advertised as "non-phthalate alternatives".Structurally distinct alternatives now exist as well.The most common alternatives include Bis(2-ethylhexyl) terephthalate (DEHT), diethylhexyl adipate (DEHA), and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) [67].In this paper, we focus on alternatives to ortho-phthalates and their impacts on neurodevelopment (Table 1).However, there is little research assessing the endocrine-and neurodevelopmental-disrupting potential of phthalate replacements.Research studies do suggest that DINCH, for example, is not estrogenic or androgenic, although it can disrupt steroidogenesis [68,69].DINCH induces oxidative stress in some cell lines, causing potential disruptions in neurodevelopment [70,71].

Zebrafish
Zebrafish are useful models for studying neurodevelopment and neurotoxicity.Thanks to their small size, high breeding rate, and short life cycle, they are a high-throughput screening tool that enables researchers to predict the potential neurotoxicity of chemicals in mammals [72].
The neural signaling systems of zebrafish are homologous to humans, including the GABAergic, dopaminergic, and glutamatergic systems.The presence of a blood-brain barrier allows for the assessment of chemical entry into the brain.Through immunostaining techniques that allow for the direct assessment of neural growth and death, in addition to the widespread analysis of gene expression patterns, zebrafish are also useful for understanding the mechanisms of neurotoxicity.
Multiple zebrafish behavioral endpoints can be used to understand the neurobehavioral effects of endocrine-disrupting chemicals.Behaviors measured include embryonic spontaneous movements and larvae locomotion, where changes can indicate developmental motor-neural toxicity.Selderslaghs et al. looked at a wide set of known neurotoxic and non-neurotoxic chemicals and found that 9/10 of the chemicals tested demonstrated developmental neurotoxicity concordance between mammal studies and zebrafish studies assessing embryonic and larvae movement.Newer studies are also adapting classical rodent assays to zebrafish models, including the open-field assay, T-maze, and social tests to assess more complex behaviors such as anxiety, learning, reward preference, and sociability [73][74][75].
For neurodevelopmental research, zebrafish embryos are placed in well plates filled with the chemical of choice in DMSO, or another solvent.The liquid exposure media will commonly be changed throughout the duration of the exposure, which can range from a couple of hours to days.Zebrafish often undergo behavioral and molecular testing at numerous developmental stages (Figure 2a).

Rodents
As mammals, rodents share more similarities than zebrafish to humans when it comes to brain structure, physiology, and behavior [76,77].Unlike humans, rats can undergo experimentation, allowing for the causality of the effects of EDC exposures to be determined.Homologous endocrine systems in rodents and humans also allow for the analysis of the sexually dimorphic effects of toxicants on neurodevelopment.
A wide array of tests in rodents aim to assess human-relevant behaviors, including assays for anxiety, learning and memory, and social and sexual behaviors.Many of these tests show concordance with human toxicological effects [78].Well-established antibodies and probes allow scientists to look at specific brain regions, cell types, and markers.Advances in genetic and transcriptomic analysis in rodents also aid in understanding the

Rodents
As mammals, rodents share more similarities than zebrafish to humans when it comes to brain structure, physiology, and behavior [76,77].Unlike humans, rats can undergo experimentation, allowing for the causality of the effects of EDC exposures to be determined.Homologous endocrine systems in rodents and humans also allow for the analysis of the sexually dimorphic effects of toxicants on neurodevelopment.
A wide array of tests in rodents aim to assess human-relevant behaviors, including assays for anxiety, learning and memory, and social and sexual behaviors.Many of these tests show concordance with human toxicological effects [78].Well-established antibodies and probes allow scientists to look at specific brain regions, cell types, and markers.Advances in genetic and transcriptomic analysis in rodents also aid in understanding the effects and potential mechanisms of toxicants on the central nervous.
Rodent chemical exposures are most typically administered via oral consumption (e.g., feeding a wafer or other edible treated with the chemical, or gavage, the latter which is more stressful), drinking water treated with the chemical, or via injection.For perinatal exposures, this will often consist of administering the chemical to the dam during gestation and lactation rather than subjecting developing rodents to injections or gavage.The offspring then undergo behavioral and molecular testing at a variety of selected ages from early postnatal life through adulthood (Figure 2b).

Humans
Studies assessing the effect of toxicants on humans are nearly always correlational, with the exception of known chemical spills or certain industrial settings.Although researchers do their best to include confounding variables, such as maternal education, smoking, and socio-economic status, the nature of this research means causal effects of toxicants cannot be elucidated in the general population.
For human research studies, blood or urine samples may be collected from the mother during pregnancy, from the µMbilical cord at birth, and/or from the child.Amniotic fluid can also be used as a proxy of exposure.For neurodevelopmental assessments, the children undergo a test of choice (e.g., IQ, autism spectrum disorders, ADHD assessments, social behaviors), and a statistical analysis is conducted to explore whether higher chemical concentrations are associated with scores on the given tests (Figure 2c).

Bisphenols and Rodents
Research assessing the influence of early-life single-BPA-analogue exposure in rodents is limited to BPAF, BPAP, BPF, and BPS.All four of these chemicals demonstrate adverse neurodevelopmental and behavioral effects (Table 3).
Early-life BPAF, BPAP, BPF, and BPS exposure resulted in decreased time or number of entries in the center of the open field and open arms of the elevated plus maze in males [103][104][105][106][107] and females [104][105][106]108,109]. BPS produced the least consistent results of the four bisphenols, with multiple studies finding no effect on behavior in these anxiety-related tests [107,[109][110][111][112]. Tests of depression-like behaviors, including the novelty-suppressed feeding test (food-deprived animal chooses between a dark chamber or light chamber with food), tail suspension test, forced swim test, and sucrose preference test indicate effects in both sexes, but primarily males, exposed to BPAF [103,104,108].In one study, BPF exposure increased immobility in female mice in the forced swim test [106], while a study with 1/200th of the dosage found no effect in the tail suspension and forced swim tests [111].No effects of early BPS exposure were observed on depression-like behaviors [107,111].Multiple studies also assessed the effect of early BPA analogue exposure on learning and memory.BPAF and BPAP in both sexes affected long-term and spatial memory [103][104][105]113].Meanwhile, a decrease in olfactory short-term memory was found with early BPF exposure [114], another study at a similar dose found no impact on cognition and memory [111].Early BPS exposure produced no identifiable effects on memory in another study [111].Social behavior alterations were also present after perinatal exposure to BPAF [104], BPAP [105], BPF [111], and BPS [111,112], in addition to altered maternal behavior after BPS exposure.
All of the above bisphenol analogues have been shown to influence neurodevelopment through decreasing synapse and spine densities [113].Alterations in gene expression and the transcriptome related to neuron differentiation, synaptic signaling, and organization were also observed [104,105,114].Studies also demonstrate the effects of specific neurotransmitters, including dopamine and serotonin [109,115], as well as the disruption of hormone systems important for neurodevelopment, such as the estrogen system [110] and 5α-reductase, an enzyme important for hormone metabolism [115].Evidence suggests microglia and oxidative stress may be drivers in these neurodevelopmental disruptions caused by bisphenols [105,113].↓ Time in center of open field at all doses in females ↑ Latency to feeding at all doses, total intake at 4 mg/kg, and ↓ total intake at 0.4 mg/kg in females in novelty-suppressed feeding test No effect on the latency to feeding, but ↓ total intake in males at all doses in novelty-suppressed feeding test ↑ Immobility time at all doses but no effect on latency to immobility in females in the tail suspension test ↓ Immobility time at 4 mg/kg and latency to immobility at 0.4 mg/kg in males in tail suspension test ↑ Floating time at 4 mg/kg for females in forced swim test.No effect of floating time in males or latency to floating in males or females.
↓ Sucrose preference for females in 0.  All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.

Bisphenols and Humans
The literature on human studies is based on epidemiological and population studies relating exposure or body burden to outcomes.A wide variety of BPA analogues have been considered, although most bisphenol replacements have had levels of detection that were too low for further analysis [118].Because of this, most studies to date have only assessed the relationship between perinatal and childhood exposure to BPAF, BPF, and BPS on measures of cognition and behavior, and results have varied, no doubt due to differences in the populations, methodology of behavioral testing, and other experimental factors (Table 4).While early BPAF exposure was associated with lower levels of social development in girls [119], no associations were found with IQ, scores of perceptual reasoning, verbal comprehension, gross and fine motor skills, adaptive skills, and language [119,120].Early BPF exposure was linked to lower IQ, perceptual reasoning, and verbal comprehension in boys [120,121] and higher ADHD rating scores in both sexes (although higher in girls) [122].Other studies found no association between BPF exposure and IQ and measures of infant neurodevelopment [123,124].Early BPS exposure was correlated with increases in ADHD rating scores in both sexes [122], an increase in emotionally reactive behaviors in girls [125], and a decrease in psychomotor development in boys [123].Multiple studies found no correlations with early BPS exposure and IQ, mental development, and other measures of cognition [118][119][120][121]123,124,126,127].Again, the disparities in results can likely be attributed to the different characteristics of the populations studied.All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.

PFAS and Zebrafish
A wide array of PFAS alternatives cause behavioral and neurodevelopmental effects in zebrafish (Table 5).Commonly studied alternatives for the legacy chemicals PFOA, PFOS, and PFHxS include PFBS, PFHpA, 6:2 FTS, 6:2 FTSA, Gen-X, PFBA, PFHpS, and PFNA.Embryonic exposure to all of these chemicals, except 6:2 FST, caused alterations in larvae locomotion and photomotor (movement in response to light) response [129][130][131][132][133][134][135][136].In terms of neurodevelopmental effects, studies showed that early-life GenX and PFHxA exposure caused an alteration in genes related to neuron differentiation and growth [129,136].PFBA exposure led to an increase in the enrichment of pathways associated with neurological disorders and neurodevelopment [132].All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.
Truong et al. compared 139 PFASs and assessed larvae and embryonic photomotor response, a measure of neurodevelopmental toxicity.Interestingly, they found that the average mass of the compound, number of fluorinated carbons, functional head groups, and physicochemical properties (e.g., vapor pressure) were not predictive of behavior.They did find that less volatile PFASs were associated with alterations in these neurodevelopmental tests, although the implications for non-aquatic models are unknown.Rericha et al. similarly tested 58 PFASs and found no conclusive association between chain length and neurotoxicity as well, with alterations to the behavior observed among a variety of chain lengths and subclasses.Gaballah et al., who explored the effects of chain length in five aliphatic sulfonic acid PFASs, found that although chain length was generally associated with neurotoxicity, exceptions exist, bringing into question the notion that smaller chain length PFASs are safer.

PFAS and Rodents
Only limited studies have assessed the influence of new-generation PFAS on rodent neurodevelopment and behavior.In fact, we only found two studies that evaluated the effects of the perinatal administration of GenX on thyroid hormone during gestation, which is essential for the proper development of the brain (Table 6) [143,144].Early GenX exposure altered placental thyroid hormone levels, potentially disrupting offspring neurodevelopment [143,144].All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.

PFASs and Humans
Many PFASs measured in human subjects fall below the level of detection, making analysis difficult.PFASs with concentrations above the level of detection include 6:2Cl-PFESA, EtFOSAA, MeFOSAA, PFBS, PFDA, PFDeA, PFDoA, PFHpA, PFHpS, PFNA, and more (Table 7).PFBS, PFDeA, PFDoA, PFNA, and PFUnDA have demonstrated an inverse relationship with IQ or other measures of cognition [145][146][147][148][149][150].Associations between early PFAS exposure and autism spectrum disorder (ASD) or deficits in social behavior were observed with 6:2 Cl-PFESA, PFBS, PFDA, PFDoA, and PFNA [146,[151][152][153][154]. Positive correlations have also been seen with early PFAS exposure and ADHD, specifically with exposure to PFDA, PFNA, and PFUnDA [128,155].Deficits in cognition, social behavior, and other behavioral alterations were found with a variety of PFASs from different subclasses and with different chain lengths, including short-chain PFASs such as PFBS.Concurrently, many studies found null correlations with the chemical discussed above and measures of cognition, ASD, ADHD, and more.Differences in findings may be a result of variations in levels of exposure, participant demographics, and the timing of exposures measured.
It is surprising that some studies observed apparent beneficial relationships between PFAS exposure and neurodevelopment and behavior.Harris et al. found that early-life PFNA, EtFOSAA, and MeFOSAA exposure were associated with higher visual-motor scores.They hypothesized that PFASs at specific concentrations may be neuroprotective through their agonist activity on peroxisome proliferator-activated receptor gamma (PPAR-γ), leading to reduced inflammation [156][157][158].Luo et al. also found that PFBS, PFDoA, and PFUnDA were positively associated with social-emotional and/or adaptive scores [146].Skogheim et al. found positive correlations between PFDA, PFNA, PFHpS, and PFUnDA with verbal working memory.Enright et al. found that early exposure to PFNA, PFDeA, and PFUdA was associated with better visual attention [159].Finally, PFNA was positively associated with visual-spatial and working memory, in addition to vocabulary scores [160][161][162].While the seemingly beneficial effects of certain PFASs are notable, it is important to consider that these same chemicals can (and do) have adverse effects on other biological systems.
PFDA Gestation (<22 weeks) and 6.6-10.9years Range Plasma (ng/mL): Prenatal: NA Childhood: <0.1-1.9 ↑ Association with childhood 0.5-1.9ng/mL quartile and parent-rated total difficulties, internalizing scores (emotional and peer problems), and externalizing scores (hyperactivity and conduct problems)   ↑ Association with childhood 2.4-25.7 ng/mL and parent-rated total difficulties, internalizing (emotional and peer problems), and externalizing scores (hyperactivity and conduct problems) ↑ Association with 1.1-1.5 ng/mL childhood quartiles and teacher-rated total difficulties and the 1. 6   All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.

Phthalate Alternatives and Zebrafish
Relatively little research exists looking at the effects of phthalate replacement on neurodevelopment, although what is published suggests that exposure to phthalate replacements can have adverse effects (Table 8).Early ATBC and DINCH exposure led to alterations in locomotion, while ATEC exposure did not [186,187].Changes in gene expression related to neurodevelopment were seen in zebrafish exposed to ATBC, ATEC, and DINCH [186][187][188].Effects were also seen in transcriptomics related to thyroid hormone pathways in DGD, DINCH, and GTA, although no effects on thyroid hormone levels were observed [189].All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.

Phthalate Alternatives and Rodents
We were only able to identify one study assessing the effects of perinatal phthalate replacement exposure on neurodevelopment and behavior in rodents.Lee et al. observed that exposure to DEHA from GD 15-P21 at ~480-12,000 ppm led to a decrease in hypothalamic granulin precursor protein (grn) mRNA in males and p130 mRNA in females, genes related to brain sexual differentiation.The study also found an effect on sexual behavior, with fewer intromissions and ejaculations by males exposed to the chemical [190].

Phthalate Alternatives and Humans
Human studies examining the effects of phthalate replacements on neurodevelopment are, to our knowledge, limited to DEHTP, DINCH, and DEHT (Table 9).DEHTP exposure during gestation was associated with a decrease in adaptive and cognitive skills in boys, along with a decrease in communication scores in girls [191].Early DINCH exposure, however, did not have an association with IQ [192].Gestational exposure to DINCH and DEHT was associated with alterations in thyroid hormones, which could have implications for neurodevelopment [118,193].[193] All effects were statistically significant at p < 0.05.↓ refers to a decrease while ↑ refers to an increase in the EDC group(s) compared to a control.

Bisphenols
Evidence suggests that all BPA analogues pose health risks when it comes to neurodevelopment.BPAF appears to be a particular chemical of concern, with the most adverse effects seen in zebrafish, while BPS causes the fewest disturbances to neurodevelopment.These findings are consistent with evidence suggesting BPAF has high toxicity, teratogenicity, and estrogen receptor binding ability [195,196].The toxicity level rankings for top bisphenols are BPAF > BPA > BPF > BPS while the estrogenicity rankings are BPAF > BPA = BPF > BPS [195,196].Rodent and human studies are not as consistent, however.For both, even lower-toxicity bisphenols, such as BPS, caused or were associated with some neurodevelopmental disturbances.BPS, however, did produce null results in many studies.Differences in results between studies may be caused by differences in dosing and timing of EDC exposure in addition to differences in when and how measures (behavioral and molecular) were taken.
In human studies, authors often excluded bisphenols that were below their defined level of detection.Although this could suggest that these chemicals may be of lesser concern, evidence shows that BPA substitutes are on the rise [39].Furthermore, the limit of sensitivity of certain assays does not mean that lower exposures are biologically relevant.This demonstrates the need for additional assay development and further monitoring of exposures in humans.It is evident that efforts be put forth to reduce bisphenol exposure overall.

PFASs
As previously mentioned, the chain length of PFAS is one of the main factors considered regarding toxicity.Comparative studies, however, found that chain length has little to no association with neurodevelopmental endpoints in zebrafish [131,135].A wide array of PFASs caused neurotoxicity in zebrafish, including GenX, a common short-chain PFAS substitute.While rodent research is lacking, only showing disruption in maternal thyroid levels, human association studies revealed often contradictory findings: PFASs were associated with mixed effects on measures of neurodevelopment and in many cases no effect, possibly due to differing timing of exposure, exposure concentration, and socioenvironmental factors.These findings point to the complexity of endocrine disruptor research and the importance of considering multiple chemicals in epidemiological studies.Furthermore, many endocrine disruptors follow non-monotonic dose responses [197].Overall, findings demonstrating disruptions in neurodevelopment caused by PFASs at low concentrations, together with limitations in predicting endocrine-disrupting activity based on structural features or length, illustrate the importance of avoiding the regrettable substitution cycle.

Phthalates
Although research assessing the effects of phthalate substitutes on neurodevelopment is sparse, some studies suggested detrimental effects on neurodevelopment.The zebrafish studies pointed to disruptions in behavior and gene expression in the brain, while human studies found associations with cognition and thyroid hormone levels.A wider body of research is needed to determine whether and how phthalate replacements pose a risk to the developing brain.

Bio-Based Alternatives-Are They Any Safer?
As concerns grow over the environmental and safety repercussions of petroleum-based plastics and plasticizers, bio-based materials have emerged.These include chemicals made from plants, materials intended to degrade naturally, or both.Although they are marketed as safer alternatives, little to no research exists to support or refute this [198].We were able to find one report that investigated the effects of polylactic acid microplastic (PLA BioMP) exposure in zebrafish larvae.de Oliveria et al. found that 3 and 9 mg/L of PLA BioMPs decreased larvae total locomotion, increased locomotion in the peripheral zone, and increased time spent immobile in the open-field test.They also found a decrease in acetylcholinesterase activity, suggesting a neurotoxic effect of the PLA microplastics [199].A possible concern is the potential use of soy or other phytoestrogen-based alternatives, which could act on estrogen receptors and alter the developing brain [200].Thorough testing should be conducted prior to the wide use of bio-based alternatives to ensure minimal effects on neurodevelopment.The use of plants known to act on hormone receptors should also be limited when making bio-based materials.

Conclusions and Recommendations for the Future
Legacy and new-generation EDC exposure can have developmental effects on the brain, causing functional changes in humans (Figure 3).As we continue to assess the safety of everyday chemicals and put forward important policy and legislation that restrict the use of EDCs, it is vital that we do not allow companies and corporations to substitute harmful chemicals with equally or more harmful alternatives.Toxicologists and endocrinologists play a key role in formulating guidelines that can help predict and/or identify potential harmful effects of commercial chemicals, and confirmation of the absence of endocrine-mediated and other harmful biological effects should be a prerequisite prior to their circulation in the world.Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

44 Figure 2 .
Figure 2. (a) Zebrafish, (b) rat, and (c) human models of neurodevelopmental exposure to EDCs.In a and b, EDC exposures are controlled and typically given through prenatal and/or postnatal exposure.In rats, this typically ends at or before weaning at P21.Note that the procedures in mice and rats are similar.In humans, exposures will vary across individual subjects.hpf = hours postfertilization; dpf = days post-fertilization; GD = gestational days; P = postnatal days; YO = years old (created with BioRender.com(accessed on 5 April 2024)).

Figure 2 .
Figure 2. (a) Zebrafish, (b) rat, and (c) human models of neurodevelopmental exposure to EDCs.In a and b, EDC exposures are controlled and typically given through prenatal and/or postnatal exposure.In rats, this typically ends at or before weaning at P21.Note that the procedures in mice and rats are similar.In humans, exposures will vary across individual subjects.hpf = hours post-fertilization; dpf = days post-fertilization; GD = gestational days; P = postnatal days; YO = years old (created with BioRender.com(accessed on 5 April 2024)).

Figure 3 .
Figure 3. Neurodevelopmental effects of legacy and new-generation endocrine disruptors (created with BioRender.com(accessed on 13 June 2024)).Author Contributions: Conceptualization, E.M.-G., E.N.H. and A.C.G.; writing-original draft preparation, E.M.-G.; writing-review and editing, E.M.-G., E.N.H. and A.C.G.All authors have read and agreed to the published version of the manuscript.Funding: E.M.-G. is supported by the Paglia Post-Baccalaureate fellowship from Carleton College, E.N.H. by NIH F32 ES034257, and A.C.G. by NIH R35 ES035024.

Table 2 .
Neurodevelopmental and behavioral effects of non-BPA bisphenols in zebrafish.
Larvae locomotion at all doses ↑ Time spent in well center at 8.5 µg/L

Table 3 .
Neurodevelopmental and behavioral effects of non-BPA bisphenols in rodents.
4 mg/kg test No effect on short-term or long-term memory in the novel object recognition test Time in center of open field at both doses in males No effect on locomotion in open field ↑ Latency to feeding at both doses in males and ↓ latency to feeding at 0.4 mg/kg in females in the novelty-suppressed feeding test ↓ Sucrose preference in males at 0.4 mg/kg ↑ Immobility in tail suspension test in males at both doses ↑ Floating time in forced swim test in females at 4 mg/kg ↓ Long-term memory at both doses for males and 0.4 mg/kg for females in novel object recognition test.No change seen in short-term memory.↓ Freezing time in both long-term and short-term memory in contextual fear conditioning test in males at both doses Time spent in target quadrant of MWM in males at all doses, indicating impaired spatial memory ↓ Number of quadrant crossing of MWM in females at 34 mg/kg, indicating impaired spatial memory No effect on hippocampal neuronal damage ↓ Number of intersections in CA1 and DG neurons at all doses in males and only at 34 mg/kg in DG neurons in females ↓ Spinal density in dendrites at 3.4 and 34 mg/kg in males No effect on spinal density in females ↓ PSD-95 at 3.4 and 34 mg/kg and Synapsin-1 at all doses in males; PSD-95 at 34 mg/kg in females ↓ Hippocampal ERα in males at all doses and ERβ in females at all doses No effect on hippocampal ERβ in males and ERα in females ↑ Latency to first immobility time and immobility in the tail suspension test for males ↓ Time with novel object in novel object recognition test for males ↓ Sociability for males ↑ Alterations in transcriptome related to synaptic signaling, organization, and structure, neurotransmitters, and neuron development for males (females not tested) Entries and velocity in center for both males and females distance traveled in center for males in the open-field test.No effect on time in center ↓ Time in open arms for females in the elevated plus maze ↑ Number of marbles buried for males and females in marble-burying test ↓ Time spent with novel objects in males but not females.↓ Preference index for novel object in both males and females in novel object recognition test ↓ Time spent with mouse compared to empty cage and novel mouse compared to familiar mouse in three-chamber test in both males and females ↓ Surviving neurons in male and female CA1 and DG ↑ Alterations in transcriptome related to astrocytes, microglia, neurons, oligodendrocytes, and pathways associated with Parkinson's and neurodegeneration.↑ Expression of C1qc, Ctss, and Iba1 (genes related to microglia) Neurogenesis and corpus collosum thickness at 200 µg/kg No effect on oligodendrogenesis, oligodendrocyte differentiation, or myelination ↑ Alterations in transcriptome related to brain development, neuron fate development, neuron differentiation at both doses, in addition to myelination and oligodendrogenesis at 200 µg/kg No alterations in genes related to intracellular TH, important for brain development ↓ Entries/time in center and locomotion in open field for both males and females ↓ Open-arm time and locomotion in EPM for males ↑ Marble burying in MBT for males ↑

Table 3 .
Cont.Time spent with novel mouse in three-chamber test ↓ Sniffing of conspecific in open field No effect on anxiety as measured in open field and elevated plus maze No effect on depressive behavior as measured in the tail suspension test and the forced swim test No effect on locomotion and motor learning as measured in rotarod test No effect on cognition and memory as measured in novel object test Time in center of open field for females ↑ Time spent in closed arm of EPM for both females and males ↑ Time immobile in forced swim test for females No effect on the open field ↑ Infanticide, pup neglect, and improper pup care carried out by exposed female offspring at 2 µg/kg ↓ Time spent in nest at both doses ↑ Nest building at 200 µg/kg No effect on time spent grooming, nest size ↓ Latency for pup retrieval No effect on ERα in the MPOA and TH in the VTA Time spent with novel mouse in three-chamber test ↓ Sniffing of conspecific in open field No effect on anxiety as measured in open field and elevated plus maze No effect on depressive behavior as measured in the tail suspension test and the forced swim test No effect on locomotion and motor learning as measured in rotarod test No effect on cognition and memory as measured in novel object test Time and entries in open arms of males at both doses ↑ High-fat diet consumption at both doses in males and at 10 µg/kg in females No effect on high-sugar diet consumption Open-arm time and ↑ locomotion in EPM in females but not males No effect on time spent in center of open field for males and females, ↓ latency to first entry into center for males ↑ Serotonin neurons in DRV and serotonin fractional area in DR and DRV in males and in serotonin fractional area in DRD in females (DR = Dorsal Raphe Nucleus, DRD = dorsal region of DR, DRV = ventral region of DR).No effects in the Median Raphe Nucleus.Time spent with familiar mice and ↑ locomotion in social test

Table 4 .
Associations between BPA-analogues and neurodevelopment and behavior in humans.

Table 5 .
Cont.Larvae photomotor behavior in the dark period at 2.49 and 34.3 µM, and in the light at 2.5 µM ↓ at 16.07 and 100 µM Larvae photomotor behavior at 2.46, 6.32, 34.3, and 73.3 µM in the dark period and ↑ at 2.46 and 16.07 µM in the light period No effect on embryonic photomotor behavior and larvae startle response Larvae startle response at 0.54 µM ↓ Larvae photomotor behavior at 33.95 µM in the dark period and at 2.46 and 33.95 µM in the light period ↑ in the light period at 0.97 and 72.56 µM No effect on embryonic photomotor behavior

Table 5 .
Cont.Larvae photomotor behavior at 15.91, 33.95, and 97 µM in the dark period and 6.26-33.95µM in the light period ↓Larvae photomotor behavior in the light period at 0.95 µM No effect on embryonic photomotor behavior and larvae startle response Larvae photomotor behavior at 10 µM in the dark period, 35 µM at light period, and ↑ at 10 µM in the light period.No effect on embryonic photomotor behavior and larvae startle response No effect on embryonic photomotor behavior and larvae startle response ↓ Larvae photomotor behavior in dark period at 52.8 µM and in light period at 0.96 and 2.44 µM

Table 7 .
Associations with non-PFOA, -PFOS, and -PFHxS PFASs and neurodevelopment and behavior in humans.Association with gross motor and adaptive skills in boys ↓ Association with social score ↓ Association with TSH and FT4 hormones No association with fine motor and language domain scores

Table 8 .
Neurodevelopmental and behavioral effects of phthalate alternatives in zebrafish.

Table 9 .
Associations with phthalate alternatives and neurodevelopment and behavior in humans.