# Bisphenol analogue exposure at low concentrations modifies heart-brain functions and transcriptomics in zebrafish larvae

Ieremias Chousidis, Sandrine Ellero-Simatos, Laurence Payrastre, Gaëlle Payros, Yannick Lippi, Marine Blaquière, Adèle Faucherre, Aurélien Drouard, Chris Jopling, Angelo G Torrente, et al.

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## To cite this version:

Ieremias Chousidis, Sandrine Ellero-Simatos, Laurence Payrastre, Gaëlle Payros, Yannick Lippi, et al. Bisphenol analogue exposure at low concentrations modifies heart-brain functions and transcriptomics in zebrafish larvae. Journal of Hazardous Materials, 2025, 498, pp.140004. 10.1016/j.jhazmat.2025.140004. hal-05304450

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# Bisphenol analogue exposure at low concentrations modifies heart-brain functions and transcriptomics in zebrafish larvae

Ieremias Chousidis<sup>a, ∗</sup>, Sandrine Ellero-Simatos<sup>b, †</sup>, Laurence Payrastre<sup>b</sup>, Gaëlle Payros<sup>b</sup>, Yannick Lippi<sup>b, †</sup>, Marine Blaquière<sup>a</sup>, Adèle Faucherre<sup>a, †</sup>, Aurélien Drouard<sup>a</sup>, Chris Jopling<sup>a, ‡</sup>, Angelo G. Torrente<sup>a, ‡</sup>, Nicola Marchi<sup>a, ‡</sup>

## HIGHLIGHTS

* Standardized LD₅₀ and toxicological assessment of BPS, BPF, BPAF, and BPA.

* Environmentally relevant bisphenols altered motor behavior in zebrafish larvae.

* Low-level bisphenol exposure modified heart-brain physiology and transcriptomics.

* Bisphenol analogues at low concentrations can promote extra-physiological adaptations.

## GRAPHICAL ABSTRACT

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## ARTICLE INFO

**Keywords:**

Bisphenol analogues

Environmental levels

Heart-brain functions

Neurophysiology

Transcriptomics

Zebrafish larvae

## ABSTRACT

Substituting bisphenol A (BPA) with analogues could mitigate environmental harm. By mimicking water contamination and using zebrafish larvae, we tested the hypothesis that low environmentally relevant concentrations of BPS, BPF, and BPAF, compared to BPA, elicit physiological and molecular modifications. We verified lethal doses (LD₅₀; BPAF > BPF > BPA > BPS) using an OECD-compliant protocol and reported the absence of morphological and muscle abnormalities, and hatching delays below the LD₅₀ at 5 days post-fertilization (dpf). We identified the lowest bisphenol exposure levels that provoke the most significant motor-swimming changes (100 ng/L BPS, 10 ng/L BPF, 100 ng/L BPAF, and 100 ng/L BPA). We asked whether the observed motor phenotype at low bisphenol levels could be underpinned by heart-brain electrophysiological adaptations. Using *in vivo* optical mapping, we found increased high-frequency, low-amplitude brain activity and, in the cases of BPF and BPAF, bradycardia. Within these specific low-exposure settings, transcriptomics identified eight genes involved in heart-brain functions, homeostatic and immune regulation, that were down-regulated by each bisphenol, namely *anxa1c*, *vwa10.1*, *zgc:172053*, *grna.2*, *ehf*, *gna14*, *ca15c*, *atp1a1a.5*. Low environmentally

<footnote><sup>a</sup> Institute of Functional Genomics, University of Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote><sup>b</sup> Toxalim, Université de Toulouse, INRAE, ENVT, EI-Purpan, Toulouse, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: Chris.Jopling@igf.cnrs.fr (C. Jopling), angelo.torrente@igf.cnrs.fr (A.G. Torrente), Nicola.Marchi@igf.cnrs.fr (N. Marchi).</footnote>
relevant concentrations of bisphenol analogues modify organ physiology and the transcriptome in zebrafish larvae. The implications for ecotoxicology and the one-health framework are discussed.

# 1. Introduction

Bisphenol A (BPA) is a well-documented environmental pollutant, commonly detected at ng/L concentrations in surface waters and drinking water sources [1–8], and is known to exert adverse effects on both ecosystems and human health [9–11]. Concerns over BPA exposure [9,12–14] have led to regulatory restrictions [15], prompting its replacement with structurally similar analogues, with Bisphenol S (BPS), Bisphenol F (BPF), and Bisphenol AF (BPAF) [16] being the most used in thermal paper, plastics, epoxy resins, and consumer products [1–8]. These substitutes were introduced to retain BPA's functionality [13,16] while reducing health and environmental risks. To serve as safer alternatives to BPA, bisphenol analogues are structurally modified by altering the bridging group or adding polar groups [17]. However, emerging evidence suggests that these analogues may be associated with adverse effects similar to those of BPA [18–22]. Bisphenol analogues have been proposed to induce metabolomic and transcriptomic changes in zebrafish larvae at low concentrations [23,24]. BPAF exposure has been shown to cause cardiomyocyte hypertrophy *in vitro* [25], oxidative stress, and behavioral abnormalities also at low doses [26–30]. BPF was linked to cardiotoxicity in zebrafish larvae and cardiac hypertrophy *in vitro* at low concentrations [31,32]. BPS can provoke similar adverse effects, although these have been examined at higher concentrations [33,34]. In general, this accumulating evidence supports the view that substituting BPA with its analogues is not without risk, underscoring the need for broad and standardized assessments of the potential physiological effects of these compounds.

Here, we used zebrafish larvae as an ecotoxicologically pertinent model [35–38], allowing us to examine the impact of bisphenol analogue BPS, BPF, and BPAF exposure at broad concentration-range levels, including those found in water [1–5,10,39], compared to BPA. We applied the OECD European Acute Toxicity Protocol (Directive 2010/63/EU). First, we calculated and cross-validated each bisphenols lethal dose (LD₅₀) against available literature data [21,27,33,40]. Below the LD₅₀, we assessed morphological, developmental, and motor-behavioral parameters following bisphenol exposures. From this screening and for each bisphenol, we identified the lowest concentrations that elicited robust motor-behavioral changes. At these specific low concentrations, we continued by examining the presence of cardiac and brain electrophysiological alterations and performing direct cross-comparisons among the four bisphenols. Finally, we provided the overlapping and distinct transcriptomic signatures that accompany these functional changes at low bisphenol concentrations. This multi-level experimental approach offers an integrated view of how low levels of BPS, BPF, and BPAF impact zebrafish larval physiology, offering an ecotoxicologically relevant comparison to BPA.

# 2. Materials and methods

## 2.1. Chemicals

Analytical-grade bisphenol A (BPA, 99 %, CAS 80–05–7), bisphenol S (BPS, 98 %, CAS 80–09–1), bisphenol F (BPF, 99 %, CAS 620–92–8), and bisphenol AF (BPAF, 99 %, CAS 1478–61–1) were purchased from Sigma-Aldrich (France). Each compound was separately dissolved in ethanol (70 %) and further diluted in 50 mL E3 culture medium to prepare 0.1 mg/mL stock solutions (EtOH concentration at each stock solution ≤ 0.1 % v/v). Stocks were stored at –20°C and renewed weekly to ensure stability. For each bisphenol analogue concentration, a corresponding vehicle ethanol control was prepared to match the ethanol content. Vehicle toxicity was assessed (n = 144/condition, triplicate),

showing no statistically significant differences between the E3 culture medium control and the vehicle control for all the tested parameters, including hatching rate, total length, swim bladder area, eye diameter, and somite length (Supporting Information, Table S1).

## 2.2. Zebrafish husbandry

Zebrafish were maintained under standardized conditions, and experiments were conducted in accordance with local approval (APAFIS#4054–2016021116464098 v5) and the European Communities directive 2010/63/EU. Sexually mature (3-month-old) zebrafish were used for spawning, and collected embryos were pooled in E3 culture medium. Larvae (maximum at 5 days post fertilization, dpf) were anesthetized and euthanized using Tricaine methane sulfonate (MS222, Sigma-Aldrich, France).

## 2.3. Toxicological, morphological, and functional screening of bisphenol analogue exposure

At 1.5 h post-fertilization (hpf), embryos were placed in 24-well plates (2 embryos per well, 2 mL solution per well). Exposures lasted 96 h, ending at 5 days post-fertilization (dpf). Solutions were renewed daily, and embryos were rinsed twice with E3 culture medium post-exposure to remove residual chemicals. The procedure was consistent with the OECD guidelines [41]. Experiments included (1) LD₅₀ determination and (2) assessment of sub-lethal effects at environmentally relevant and below-LD₅₀ concentrations. LD₅₀ values were determined by exploring the concentration range that induced 0–100 % lethality: BPS (N<sub>conc</sub>:6, N<sub>individuals</sub>:144/concentration in triplicate), BPF (N<sub>conc</sub>:8, N<sub>individuals</sub>:144/concentration in triplicate), BPAF (N<sub>conc</sub>:8, N<sub>individuals</sub>:144/concentration in triplicate), and BPA (N<sub>conc</sub>:10, N<sub>individuals</sub>:144/concentration in triplicate). LD₅₀ calculations, including 95 % confidence intervals (LD<sub>25</sub> and LD<sub>75</sub>), were performed using Probit regression analysis (chi-square test, Pearson goodness-of-fit test) in SPSS Statistics v29 (IBM Corp., NY, USA).

After LD₅₀ determination, embryonic malformations were examined below-LD₅₀, starting from environmentally relevant concentrations (10 ng/L–1000 µg/L) [1,3,39] for each bisphenol analogue. Morphological parameters, including total body length (TL, mm), swim bladder area (SWB, mm²), and eye diameter (ED, mm), were analyzed at 5 dpf using a stereoscope equipped with a camera (Olympus SZX16, ProgRes CapturePro 2.8.8 software) (n = 20/group, duplicate). Hatching rates (%) were also recorded (n = 144/concentration, duplicate). Muscle formation defects were further assessed via phalloidin staining, as described [42] (n = 10/group, duplicate). The effects of low-dose bisphenol analogues (below LD₅₀) were evaluated by functional screening of zebrafish larvae using two tests at the end of the exposure: (1) Basal Locomotor Activity (BLA) and (2) Vibrational Startle Response (VSR). Only zebrafish larvae exposed to low doses (10 ng/L, 100 ng/L, 1000 ng/L, 10 µg/L, 100 µg/L, 1000 µg/L) were assessed to reflect environmentally relevant concentrations. Prior to behavioral testing, larvae were screened to confirm the absence of morphological malformations after 96 h of exposure. This ensured that observed differences in swimming activity were not due to physical impairments. For the BLA test, larvae were subjected to alternating light-dark phases for 30 min (10 min dark, 10 min light, 10 min dark, see Fig. 3A), after a 10 min acclimation period in the dark. This protocol assessed the ability of larvae to adapt to baseline environmental conditions [43]. Each larva was individually placed into a 24-well plate and transferred to the DanioVision Observation Chamber (Noldus Information Technology, VA, USA) equipped with a temperature control unit. Locomotor activity
was tracked using an infrared camera (25 frames/sec, Basler acA1300-60gm, Basler Inc., PA, USA), and movement parameters (distance traveled [cm]) were analyzed (n = 48 for each condition, duplicate). The VSR test evaluated locomotor activity, sensory organ function, and stress-like behaviors [44]. A mechanical tapping stimulus was applied using a piston in the behavior recording system (DanioVision Tapping Device DVOC-004x/T, Noldus, The Netherlands). After a 10 min acclimation period in the dark, a 6 min video recording (60 frames/sec) was conducted in complete darkness. The tapping stimulus was delivered at the 3-minute mark (180 sec). The response was analyzed by measuring (1) the distance traveled (cm) within 3 min post-stimulus and (2) the latency to respond to the stimulus (time to first movement, sec) (n = 24 for each condition, duplicate, See Fig. 4A). Both behavioral tests (BLA and VSR) were conducted between 10:00 AM and 12:00 PM to account for circadian rhythms.

## 2.4. *In vivo neuro and cardiac electrophysiological screening by optical mapping*

At the end of each treatment (5 dpf), zebrafish larvae were incubated in blebbistatin (8.5 µM prepared in E3 culture medium) (Selleckchem, Cologne, Germany) for 30 min to prevent muscle contractions. Larvae were then incubated in the dark for 40 min with the voltage-sensitive probe Di-4-Anepps (9 µM prepared in E3 culture medium) (AAT Bioquest, Pleasanton, CA, USA). Larvae were immobilized in 35 mm glass-bottom petri dishes using low-melting agarose (LMA, Sigma-Aldrich, France) and positioned laterally for cardiac recordings and dorsally for cerebral recordings, as shown in our previous work [45]. To maintain viability and prevent muscle contractions through the recordings, 2 mL

of E3 culture medium with blebbistatin (8.5 µM) was added to each petri dish to ensure viability throughout the recordings. Each 35 mm petri dish was positioned under the microscope setup within a warming chamber (QE-1, Warner Instruments, Holliston, MA, USA), connected to a dual automatic temperature controller (TC-344B, Warner Instruments, Holliston, MA, USA), and maintained at a constant temperature of 28°C.

Cardiac and cerebral recordings acquisitions (30 sec.) were performed with an acquisition frequency of 500 frames/sec using a 150 W halogen lighting system (SciMedia, Costa Mesa, CA, USA) as an excitation source coupled with a 531/50 nm excitation filter, a 580 nm dichroic mirror, and a 580 long-pass emission filter. The SDF PLAN FLUOR 0.3X stereo microscope objective mounted as an eyepiece lens (Olympus, France), the Plan Apo 5.0 X/0.50 LWD mounted as magnification lens (Leica, France) and a MiCAM03 optical camera with a sensor of 17.6 × 17.6 mm (SciMedia, Costa Mesa, CA, USA), completed the optical mapping system allowing to work with a field of view of 1.05 × 1.05 mm corresponding to 256 × 256 pixels (as described in S. Micou et al., 2023 [45]). BV Workbench software (ver 2.7.2, SciMedia, Costa Mesa, CA, USA) was used to perform the acquisitions. Cardiac depolarization was recorded as fluorescence change in the ventricular region, corresponding to cyclical heart rate traces (See Fig. 5A). Brain depolarization was recorded by capturing the cumulative fluorescent signal (See Fig. 6A). Fluorescent signals deriving from the heart were extracted and analyzed using ClampFit 11.3 (Molecular Devices, LLC, San Jose, CA, USA). The effects of bisphenol analogues were assessed at the cardiac level by analyzing heart rate (beats per minute, BPM) and the coefficient of variability of the interval between two heartbeats (coefficient of variability, % CV), recorded as optical action potentials in the ventricular region (n = 15/condition). For brain activity, data were

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Verifications of LD₅₀ values for bisphenol analogues (BPS, BPF, BPAF) and BPA. Toxicity curves and lethal concentrations were determined for A) BPS, B) BPF, C) BPAF, and D) BPA. Toxicity assays (LD₅₀ values) and confidence limits (LD₂₅ and LD₇₅) were calculated based on cumulative mortality at the end of the experiment. The LD₅₀ values were assessed using Regression Probit analysis (the chi-square test, Pearson goodness of fit test, and 95 % confidence interval).</figcaption></figure>
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(caption on next page)
**Fig. 2.** Absence of hatching, morphological, and muscle abnormalities after exposure to BPS, BPF, BPAF, and BPA at sub-LD₅₀. A) Examples of measurements for morphological assessment: Total body length (TL, µm), swimming bladder (SWB, µm²), eye diameter (ED, µm) of zebrafish larvae at 5dpf. A1) Lateral views of trunk somites in zebrafish larvae stained using phalloidin. Dashed lines with arrows indicate the length of a typical muscle fiber measured. Table (B1-B3): no statistically significant changes were observed. As shown in Supplemental Table 1, controls with matching ethanol content and E3 exhibit no statistically significant changes in all the tested parameters. Experiments were conducted in triplicate (n = 10/group). Screening hatching rates and gross morphological parameters at sub-LD₅₀ and environmentally-relevant bisphenol analogues concentrations for B) BPS, B1) BPF, B2) BPAF, and B3) BPA (1-way ANOVA, Dunnett's multiple comparisons tests, p < 0.05). For morphology measurements, data are reported as means ± SD (experiments conducted in duplicate, n = 20/group). Cumulative hatching rates consecutively assessed at 72, 96, and 120 hpf are expressed as hatched eggs / total × 100 (experiments conducted in triplicate, n = 120/group; 2-way ANOVA, Dunnett's multiple comparisons test, p < 0.05). Muscle measures were reported as means of somite length (µm, n = 10/group, duplicate; 2-way ANOVA, Dunnett's multiple comparisons test, p < 0.05).

exported to OriginPro software to perform Fourier Transform (FTT, OriginPro 2018, Northampton, MA, USA) from whole-brain signals (N = 20/condition) to examine low-amplitude voltage frequency patterns. To minimize cardiac signal interference, frequencies between 2–3 Hz were removed. We specifically assessed the FT frequency range between 0.2–4 Hz, 4.1–7 Hz, and 30–80 Hz [46], not exceeding 80 Hz, to allow robust calculations (Nyquist sampling law [47]) within the available sampling rate (500 frames/s). The recordings were visually inspected for high-amplitude spike activities, and artifacts were excluded. Y-axis unit in brain activity plots derives from the mathematical FT elaboration, which indicates the relative abundance of low-amplitude waves in the targeted frequency ranges and for the executed recording time. This solution analyzes the frequency range of activities emerging from excitable cells [48].

## 2.5. RNA sequencing and analysis

A pool of n = 50 zebrafish larvae (5 dpf) constituted one sample. The experimental conditions included four vehicle control groups corresponding to varying ethanol content for each bisphenol analogue concentration tested: one group exposed to 100 ng/L BPA, one group exposed to 100 ng/L BPS, one group exposed to 10 ng/L BPF, and one group exposed to 100 ng/L BPAF. Vehicle (Etoh) controls were paired with their respective bisphenol treatment groups, and comparisons were conducted accordingly. Thus, each bisphenol condition was directly compared to its matched vehicle control. Each condition was analyzed in six replicates, resulting in a total of 48 samples and a total of 2000 larvae. Total RNA was extracted using Trizol (Invitrogen) and sequenced at the GeT-TRiX platform at the TOXALIM Institute in Toulouse, France. For each of 48 samples, RNAseq libraries were constructed from 1000 ng of total RNA at the GeT-TRiX facility (GénoToul, Génopole Toulouse Midi-Pyrénées) using Illumina® Stranded mRNA Prep kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. Briefly, mRNAs were captured, then fragmented, converted to double-stranded cDNAs, and sequencing adaptors were ligated. Libraries were amplified with ten cycles of PCR. The yield and the quality of the libraries were assessed using D1000 ScreenTapes on TapeStation 4200 (Agilent Technologies, Santa Clara). The libraries were then pooled into equimolar concentrations and transferred to INRAE PGTB sequencing facility for sequencing. The library pool was loaded into one P4 Flow Cell on Illumina NextSeq 2000 using 2 × 50 bp paired-end read sequencing mode with NextSeq 2000 P4 XLEAP-SBS Reagent kit (100 Cycles) at INRAE PGTB sequencing facility. Bioinformatics treatments of sequencing data were performed by the GeT-TRiX facility (GénoToul, Génopole Toulouse Midi-Pyrénées) using the computing resources of genotoul bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo Genotoul). The analysis pipeline was executed with Nextflow v24.10.0 [49] and processed using nf-core/rnaseq v3.16.0 [50] of the nf-core collection of workflows [51]. Reads were aligned to the Zebrafish genome reference GRCz11 (assembly GCA_000002035.4). Biostatistics analyses were performed under R 4.3.0 (R Core Team, 2023), edgeR [52], limma [53], and Bioconductor packages [54]. Briefly, the raw count table was filtered to eliminate undetected genes using filterByExpr function with min.count = 10. Normalization factors were calculated using TMM method. Then, the common, trended, and tagwise negative

binomial dispersions were estimated using the robust estimateDisp function. The experimentation dates were included in the design matrix as a fixed blocking factor. A negative binomial generalized log-linear model was fitted to count data with the quasi-likelihood (QL) methods using glmQLFit function to moderate the genewise QL dispersion [55]. Pairwise comparisons between biological conditions were extracted using specific contrasts with glmQLFtest function to identify differential expression. A correction for multiple testing was applied using Benjamini-Hochberg procedure [56] to control the False Discovery Rate (FDR). Genes with FDR ≤ 0.05 were considered to be differentially expressed between conditions. Hierarchical clustering was applied to the samples and the differentially expressed genes using 1-Pearson correlation coefficient as distance and Ward's criterion for agglomeration. The clustering results are illustrated as a heatmap of expression signals. Full details, including quality control metrics, alignment statistics, and count data, are publicly available in the GEO database under accession number GSE304009. Enrichment analysis of Gene Ontology (GO) Biological Processes and KEGG pathways (more than 2 genes) was applied to differentially expressed gene lists for each comparison using ShinyGO software (FDR cutoff 0.05, -log10(P)> 1, https://bioinformatics.sdstate.edu/go/).

## 2.6. Statistical analyses

For LD₅₀ determination, we followed Probit Analysis using SPSS Statistics Package (v.28, IBM Corp., NY, USA). Further analyses were performed using GraphPad Prism 10.4.1. When the data fulfilled normal distribution, we applied parametric tests such as one-way ANOVA followed by Dunnett's multiple comparisons test. Otherwise, Kruskal-Wallis (non-parametric) followed by Dunn's multiple comparisons test was applied (p < 0.05). Results are presented using violin plots. The cumulative hatching rate was analyzed using two-way ANOVA followed by Dunnett's multiple comparisons test (p < 0.05). Asterisks indicate statistical differences compared to the control group (CTRL): * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).

# 3. Results

## 3.1. Toxicological and morphological validations of bisphenol exposure

First, we verified that our exposure conditions and outcomes were consistent with available literature, enabling subsequent analyses at selected exposure levels. The dose-response toxicity of bisphenols followed a sigmoidal curve, as determined by Regression Probit Analysis (Fig. 1A–D). The LD₅₀ values ranked as follows: BPAF (1.28 mg/L), BPF (2.11 mg/L), BPA (8.71 mg/L), and BPS (16.02 mg/L) (Table 1). Vehicle controls did not induce statistically significant toxicity (see Methods and Supporting Information, Table S1). This analysis aligns with available datasets [21,27,33,40], confirms the validity of our exposure conditions, and provides a systematic comparison of the LD₅₀ for the four bisphenols under standardized and unified experimental settings. Below each of the bisphenols' LD₅₀ value (from 10 ng/L to 1000 µg/L with 10-fold increments), including environmentally relevant concentrations, morphological alterations were absent (Fig. 2A). Hatching rates, total length, swim bladder area, and eye diameter showed no statistically
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Environmentally relevant exposure to BPS, BPF, BPAF, and BPA alters locomotor activity patterns in zebrafish larvae at 5dpf. A) Protocol utilized to identify locomotor activities and changes. CTRL refers to E3. As shown in Supplemental Fig. 1, controls with matching ethanol content and E3 exhibit no statistically significant changes in motor behavior. B-E) Locomotion outputs obtained during the 30 min tracking and according to light/dark conditions for BPS, BPF, BPAF, and BPA tested below LD₅₀ from 10 ng/L to 1000 µg/L with 10-fold increments (n = 48 zebrafish each condition, executed in duplicate; non-parametric test, Kruskal-Wallis, Dunn's multiple comparisons test, p < 0.05). F) Examples of motor-activity of selected low and environmentally relevant concentrations of bisphenols.</figcaption></figure>

significant changes following exposure at this concentration range. (See Fig. 2, Tables B-B3). Furthermore, examination of phalloidin immunostaining ruled out the presence of muscle deformities at 5dpf (Fig. 2A1, Tables B-B3). Vehicle controls did not elicit significant changes in the morphological parameters analyzed (Supporting Information, Table S1).

### 3.2. Identification of the lowest bisphenol analogue concentrations eliciting motor-behavioral changes

From the morphological study (Fig. 2, Table 1), we next examined whether the bisphenol analogues can induce changes in motor behavior. Fig. 3A shows the paradigm utilized to test the ability of zebrafish larvae (5 dpf) to adapt to changing environmental light and dark stimuli upon exposure to bisphenols. Exposure to vehicle controls (EtOH) did not result in statistically significant motor changes compared to E3 medium (Fig. S1). Hypoactivity, as measured by reduced distance traveled, was observed during one or both dark phases following exposure to 100 ng/L and 100 µg/L BPS, respectively (Fig. 3B). A comparable pattern of hypoactivity was observed under dark conditions following exposure to 10 ng/L, 1000 ng/L, or 10 µg/L BPF (Fig. 3C). Swimming activity remained unaffected during light conditions after BPF exposure (Fig. 3C). When examining BPAF, larvae showed hypoactivity during one or both dark periods after exposure to 100 ng/L, 100 µg/L, and 1000 µg/L (Fig. 3D). Hyperactivity occurred during light conditions after exposure to 100 µg/L BPAF and during the second dark condition at 10 µg/L BPAF (Fig. 3D). Importantly, larvae exhibited a generalized pattern of hypoactivity in response to all BPA concentrations tested (Fig. 3E). Fig. 3F provides motor-trace examples for each bisphenol, showing the low concentrations eliciting significant changes.

Next, we examined the startle reflex using mechanical tapping as an external stimulus. Fig. 4A illustrates the experimental paradigm used. We observed no statistically significant difference in the latency to the first movement after tapping across all tested concentrations of BPS, BPF, and BPAF (Fig. 4B, C, and D). Importantly, larvae exhibited reduced latency after exposure to BPA, including at ng/L concentrations (Fig. 4E). We report that exposure to BPS, BPAF, and BPA modifies the distance traveled after tapping (Fig. 4B1, D1, and E1). These datasets confirm the adverse effects of BPA and identify the lowest bisphenol analogue concentrations, with environmental relevance, that elicit motor-behavioral changes.

### 3.3. Neuro and cardiac electrophysiological adaptations at low levels of bisphenols exposure

We asked whether the reported motor-behavioral alterations at low exposure levels could be underpinned by changes in organ physiology, specifically in the brain and heart. Guided by the behavioral screening in Fig. 3B-D, we focused on the lowest bisphenol analogue concentrations that provoked robust hypoactivity in motor-behavioral activity, specifically 100 ng/L BPS, 10 ng/L BPF, 100 ng/L BPAF, and cross-compared to 100 ng/L BPA (Fig. 3E). Optical mapping [45] was used to image the spatiotemporal and anatomical propagation of the cardiac electrical impulse *in vivo* in a non-invasive manner (Fig. 5). Compared to controls (CTRL E3 medium), we report significant alterations in heart rate following exposure to BPAF (100 ng/L) and BPF (10 ng/L; Fig. 5C), whereas the coefficient of variability of heart rate remained unaffected, ruling out cardiac arrhythmias (Fig. 5D). Next, we examined neuronal activity [45] in the forebrain over time following the chosen exposure conditions compared to controls (CTRL E3 medium; Fig. 6A-B). With the protocol adopted, we did not observe abnormal spike activity indicative of pathological neuronal firing (*not shown*). By computing spectrogram analyses (see Methods), we report no significant changes in the abundance of the low wave frequency spectrum (delta 0.2–4 Hz; theta 4–7; Figs. 6C and 6D). The abundance of high-frequency waves (gamma, 30–80 Hz) was increased after exposure to BPAF, BPF, and BPA (Fig. 6E), indicating neuronal functional adaptations in these conditions.
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Patterns of vibrational startle response triggered by sub-LD₅₀ exposure to BPS, BPF, BPAF, and BPA. A) Protocol utilized to examine reflex modifications potentially triggered by bisphenol analogue exposure, tested below LD₅₀ from 10 ng/L to 1000 µg/L with 10-fold increments. CTRL refers to E3. B-E) Latency to first movement after tapping for BPS, BPF, BPAF, and BPA. B1-E1) Total distance moved for 3 min after tapping with exposure to BPS, BPF, BPAF, and BPA. For the latency dataset, we report means ± SEM, n = 24 for each condition, executed in duplicate, non-parametric test, Kruskal-Wallis, Dunn's multiple comparisons test, p < 0.05.</figcaption></figure>

Taken together, these results reveal specific cardio and neuro-physiological changes that emerge in a bisphenol-specific manner at low concentrations.

### 3.4. Transcriptomic signatures associated with low levels of bisphenol exposures

Lastly, we performed a comparative bulk RNA-seq analysis to determine whether exposing larval zebrafish to bisphenol analogues at the studied low concentrations (100 ng/L BPS, 10 ng/L BPF, 100 ng/L BPAF, and cross-compared to 100 ng/L BPA) induces transcriptomic changes. All bisphenols induced both down- and up-regulation of transcripts, as illustrated in Fig. 7A. The complete lists of differentially expressed genes (DEGs) for each exposure condition are provided in the

**Supporting Information (Tables S2 and S3).** First, we examined whether common genes were dysregulated as a consequence of the selected bisphenol exposures. Under these conditions, we report that eight transcripts (*anxa1c*, *vwa10.1*, *zgc:172053*, *grna.2*, *ehf*, *gna14*, *ca15c*, *atp1a1a.5*) were significantly down-regulated by each of the bisphenols, while no genes were commonly up-regulated (Fig. 7A2). The down-regulated genes are involved in cardiac or neuronal functions (*grna.2*; granulin a, tandem duplicate 2, *gna14*; guanine nucleotide-binding protein - G protein, alpha 14, and *atp1a1a.5*; ATPase Na+/K+ transporting subunit alpha 1a, tandem duplicate 5), acid-base homeostasis and immune regulations (*ca15c*; carbonic anhydrase XV a, *vwa10.1*; von Willebrand factor A domain containing 10, tandem duplicate 1, *anxa1c*; annexin A1c). We also performed direct pairwise comparisons of each bisphenol analogue with BPA, as shown in the Venn diagrams in
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Narrowing down to low and environmental bisphenol concentrations: cardiac physiological adaptations. A) Zebrafish larvae in a lateral position, exposing the heart charged with a voltage-sensitive dye (red indicates depolarization) and a time series of the voltage depolarization signal across heart regions. The image at time 0 corresponds to the beginning of atrial depolarization and the end of ventricular depolarization. B) Action potentials (APs) recorded in the atrial and ventricular regions of the zebrafish heart. C) Quantification of cardiac APs rate and D) coefficient of variability (interval between two APs) in zebrafish exposed to the selected low concentrations (see Fig. 3; 100 ng/L BPS, 10 ng/L BPF, 100 ng/L BPAF, and 100 ng/L BPA) of bisphenol analogues (n = 20 for each condition, non-parametric test, Kruskal-Wallis, Dunn's multiple comparisons test, p < 0.05).</figcaption></figure>

Fig. 7A1 and detailed in the lists of common transcript changes provided in Tables S4 and S5. Briefly: BPS (100 ng/L) vs. BPA (100 ng/L) indicates 13 common up-regulated DEGs and 31 common down-regulated DEGs (Tables S4 and S5); BPF (10 ng/L) vs BPA (100 ng/L) indicates 1 common up-regulated DEGs and 58 common down-regulated DEGs (Table S4 and S5); BPAF (100 ng/L) vs. BPA (100 ng/L) indicates 1 common up-regulated DEGs and 8 common down-regulated DEGs (Tables S4 and S5). Finally, KEGG and GO analyses identified putative common dysregulated pathways (up-regulation in cell metabolism and down-regulation of immune and defense mechanisms) delineated by three or more genes, in the comparisons of each bisphenol analogue with BPA (Fig. 7B–D). Taken together, our data highlight the existence of transcriptomic changes that are common, or unique, across all the bisphenol analogues and BPA, emerging at low levels of exposure when hypoactivity is reported.

# 4. Discussion

## 4.1. Comparative analysis of the physiological effects of BPS, BPF, and BPAF versus BPA

Our results indicate that BPS, BPF, and BPAF, introduced as safer alternatives to BPA, can cause extra-physiological cardiac, neuronal, and transcriptomic adaptations at low exposure levels in zebrafish larvae. The presence of low levels of bisphenols in the ecosystem is a growing concern due to their potential to negatively impact biological functions. While high-dose toxicity is well-documented, increasing

evidence suggests that environmentally relevant concentrations can interfere with endocrine, behavioral, and developmental processes [21, 33, 40, 57, 58]. These biologically significant effects may accumulate over time, threatening ecosystem stability [16]. Therefore, understanding the impacts of low-dose bisphenol exposure is important for accurate risk assessment and the advancement of environmental regulatory policies, with potential extensions to human health.

In the absence of morphological or muscle malformations, bisphenol analogues induced hypoactivity at low concentrations (100 ng/L BPAF; 10 ng/L BPF; 100 ng/L BPS) under dark conditions. The reported low-dose effects appear to follow a non-monotonic pattern (Fig. 3), as documented among endocrine-disrupting chemicals [23,24,59]. All larvae, regardless of exposure modality, responded to a tap stimulus, ruling out sensory organ impairment [44,60]. BPA exposure caused dose-dependent post-stimulus hypoactivity, while BPAF and BPS induced hyperactivity at specific concentrations. These non-physiological patterns could indicate signs associated with stress responses, as previously hypothesized [26,27,58,61–65]. As the concentrations tested in this study encompass environmentally relevant values (ng/L ranges; [33–35]), our results underscore the potential ecotoxicological risks associated with exposure to bisphenol analogues and highlight the necessity to identify conditions that may contribute to frailty and adverse outcomes.

## 4.2. Electrophysiological effects at low levels of bisphenol exposure

Informed by the motor-behavioral screening, we probed neuronal
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Narrowing down to low and environmental bisphenol concentrations: neuronal physiological adaptations. A) Example of zebrafish larvae in the dorsal position to expose the targeted brain region (indicated in transparent red) and a time (100 ms) series of voltage signal dynamics (red/yellow indicates depolarization) as captured by *in vivo* microscopy. B) Example of Fourier Transform (FT; F/Hz) of brain voltage activity. Quantifications of brain voltage activities for the frequency intervals C) 0.2–4, D) 4–7 and E) 30–80 Hz in zebrafish exposed to the selected low concentrations (see Fig. 3; 100 ng/L BPS, 10 ng/L BPF, 100 ng/L BPAF, and 100 ng/L BPA) of bisphenol analogues (n = 20 zebrafish larvae for each condition; Non-parametric test, Kruskal-Wallis, Dunn's multiple comparisons test, p < 0.05).</figcaption></figure>

<table><caption>**Table 1**
Lethal doses for the bisphenols tested.</caption><thead><tr><th></th><th>Bisphenol S<br>(N<sub>individuals</sub> =<br>144 in triplicate<br>for each<br>concentration,<br>N<sub>conc:6</sub>)</th><th>Bisphenol F<br>(N<sub>individuals</sub> =<br>144 in triplicate<br>for each<br>concentration,<br>N<sub>conc:8</sub>)</th><th>Bisphenol AF<br>(N<sub>individuals</sub> =<br>144 in triplicate<br>for each<br>concentration,<br>N<sub>conc:8</sub>)</th><th>Bisphenol A<br>(N<sub>individuals</sub>=<br>144 in triplicate<br>for each<br>concentration,<br>N<sub>conc:10</sub>)</th></tr></thead><tbody><tr><td>LD<sub>50</sub></td><td>16.024 mg/L</td><td>2.113 mg/L</td><td>1.28 mg/L</td><td>8.71 mg/L</td></tr><tr><td>LD<sub>75</sub></td><td>23.849 mg/L</td><td>12.274 mg/L</td><td>5.195 mg/L</td><td>15.17 mg/L</td></tr><tr><td>LD<sub>25</sub></td><td>10.767 mg/L</td><td>0.364 mg/L</td><td>0.315 mg/L</td><td>5.002 mg/L</td></tr></tbody></table>

and cardiac electrophysiological patterns in zebrafish larvae exposed to bisphenol analogues at the lowest concentrations that provoked robust motor-behavioral changes. As a result, the concentrations examined differ between molecules. Our results indicate that BPA and BPS do not exert a significant effect on cardiac activity. However, exposure to BPF (10 ng/L) and BPAF (100 ng/L) resulted in bradycardia. The relevance of these findings may extend to human health, for instance, in the case of pre-existing cardiac vulnerabilities, which could potentially be exacerbated by low-level exposure to BPF or BPAF. Our findings provide new insights, extending previous reports of bisphenol-induced bradycardia

to lower exposure concentrations [18,31,66].

At the neuronal level, we report that exposure to the selected low concentrations of bisphenol analogues, with the exception of BPS, was associated with an increased abundance of high-frequency (30–80 Hz) wave activity in the forebrain. These oscillations and gamma activity have been associated with increased neuronal excitability, which may reflect stress-related signatures [30,57,67,68]. However, the exact pathophysiological and neurobiological significance of these findings remains to be determined in this model [46]. The adaptations reported here are coherent with datasets relative to low-level pesticides, as we previously reported [45]. We acknowledge that these neurophysiological modifications are quantitatively smaller than those observed in a disease model (not shown). Importantly, evidence from pre-clinical and human studies suggests a potential link between adverse neurocognitive trajectories and alterations in low- and high-frequency waves [30,57, 68]. In summary, our findings reveal subtle neurophysiological adaptations to low levels of bisphenol exposure, except for BPS, in zebrafish larvae that occur in the absence of structural alterations [69,70].

### 4.3. Transcriptomic responses at low levels of bisphenol exposure

In line with the motor-behavioral, cardiophysiological, and neurophysiological assessments, we performed RNA-seq analysis (Tables S2 to
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Transcriptomic signatures following exposure to the selected low concentrations of BPS, BPF, and BPAF, with cross-comparison to BPA. A) Summary of DEGs for each of the selected bisphenol concentrations. A1-A2) The Venn diagram and the Table show the overlapping eight genes commonly down-regulated for all bisphenols at the concentration tested. B-D) Venn diagrams illustrating the DEGs for each bisphenol analogue as compared to BPA (see Tables S2 to S5 for complete gene lists). B1-C1) Bar plots display commonly enriched biological pathways defined by the shared genes (KEGG and GO analyses provided significant results for up-regulated and down-regulated genes, respectively), with color indicating significance (−log₁₀FDR) and dot size representing the number of genes involved (n ≥ 3) for each comparison. P adjusted < 0.05, significantly enriched pathways ranked by normalized enrichment score were presented; -log10(P)> 1.</figcaption></figure>
S5) to determine whether the selected low concentrations of BPS, BPF, or BPAF elicit transcriptomic signatures comparable to, or distinct from, those induced by the selected BPA condition (Fig. 7, Tables S2-S5) and potentially underpinning the physiological phenotypes observed. We report a list of eight genes that were consistently down-regulated following exposure to each bisphenol at low concentrations. We identified *anxa1c*, *vwa10.1*, *zgc:172053*, *grna.2*, *ehf*, *gna14*, *ca15c*, *atp1a1a.5*. *Anxa1c* encodes Annexin A1c, an anti-inflammatory protein that plays a central role in inflammation resolution in vertebrates and mammals [71–74]. This gene is important for cardiac repair after myocardial infarction, as mice lacking *anxa1* exhibit reduced heart function post-injury [74]. *Grna.2* (granulin a) is involved in neuronal development and survival in zebrafish; its loss can cause neuron, sensory hair cell, and retinal defects [75]. *Gna14*, a G-protein α-subunit, mediates GPCR-dependent phospholipase C-β (PLCβ) neuronal signaling [76] and modulates cardiac vascular tone via endothelial and smooth muscle cells [77]. *Atp1a1a.5*, a Na+/K+ ATPase α-subunit isoform in teleosts, is involved in cardiac myocyte excitability, contractility, and ion homeostasis [78–80]. Mutations in *atp1a1a* or pharmacological inhibition result in bradycardia in zebrafish larvae [81]. *Ca15c* is involved in acid-base regulation by catalyzing the reversible hydration of carbon dioxide in zebrafish [82], and *vwa10.1* encodes a protein containing von Willebrand factor A domains, which is predicted to function in extracellular matrix organization or cell adhesion processes [83]. We also performed pairwise comparisons of each bisphenol analogue with BPA (see Tables S5-S6 for complete gene lists). For instance, *hmgcra* was significantly up-regulated following exposure to BPS or BPA at 100 ng/L. This gene is implicated in steroid hormone biosynthesis, within a variety of biological processes, including neural and cardiac functions [84–86]. Previous research into the effects of BPA on neural development indicates that maternal low-dose exposure results in an up-regulation of the melanovate pathway, which could result in neurodevelopmental disorders [87,88]. Indeed, defective cholesterol metabolism associated with increased HMGCR activity has recently been associated with attention-deficit hyperactivity disorder (ADHD) [89]. Finally, KEGG and GO provided pathways outlined by three or more genes, in particular, up-regulation in cell metabolism (*pmm1*, *sc5d*, *hmgcra*; common between BPS 100 ng/L and BPA 100 ng/L) and down-regulation of immune or defense mechanisms (*nos2a*, *mpeg1.2*, *mpeg1.1*, *irg1l*, *ccl20b*, *ccl20a.3*; common between BPF 10 ng/L and BPA 100 ng/L). Although function and targeted testing are necessary for each gene, these data uncover potential links between transcriptomic changes and motor-behavioral or heart-brain adaptations at the specific low bisphenol concentrations.

## 4.4. Study limitations and perspectives

Our study provides a snapshot of the impact of bisphenol analogues on zebrafish physiology and transcriptomics. Longer-term studies are necessary to understand whether these adaptations are persistent or reversible. Our methodology may mask individual variability within a population that contains both susceptible and resilient zebrafish. Nevertheless, resilient individuals may be more prevalent in wildlife environments than inbred laboratory animals. This framework requires investigation to establish unequivocal translational relevance to ecosystems. Furthermore, testing environmental water will provide important information, and this paradigm could constitute a logical extension of our research. Here, as an initial step, we aimed to examine the impact of specific bisphenols in the absence of other contaminants that may also be present in natural water bodies. A continuation of this research project could also encompass the examination of the effects of bisphenol mixtures to explore potential additive or synergistic toxicity. We acknowledge that BPS exposure at 100 ng/L induced significant behavioral changes without accompanying alterations in the measured cardiac or brain wave parameters. We can provide two views in support of this outcome, first the possibility exists that cell-specific adaptations

cannot be captured using the whole-organ optical mapping; second, we here report down-regulation of *swap70*, which in mice plays a role in oligodendrocyte progenitor cell development and neural plasticity could be potentially linked to the motor behavior modifications observed. [90]. Moreover, the absence of cardiac modifications after exposure to BPS at 100 ng/L may be, in part, associated with the up-regulation of *g0s2*, which has been shown in zebrafish to be involved in cardiac protection [91].

Here, we specify that our primary focus is on examining the lowest bisphenol concentrations that provoke significant motor-behavioral changes. Nevertheless, we also examined the effects of two specific higher concentrations of bisphenols (specifically BPF at 1000 ng/L and BPAF at 100 µg/L) that also induced motor-behavioral modifications and are below the LD₅₀ (see Fig. 3). Although we did not aim to study chemical potency or dose-response for gene regulation, we found that higher levels of these two bisphenols provoked transcriptomic changes similar to those observed with BPA at lower levels (see Tables S2-S5 for complete gene lists and comparisons). These two higher bisphenol concentrations also impacted brain neurophysiology (see Fig. S2 and S3 for extended results). It remains uncertain whether the gene expression changes are causative of the physiological and behavioral alterations or if they represent a consequence of a stress response to bisphenol exposure.

## 5. Conclusions

Altogether, our study identifies the lowest concentration for each bisphenol analogue that, without morphological changes, elicits motor-behavioral and heart-brain physiological modifications, along with transcriptomic dysregulation involving a set of specific genes that are commonly affected [16,33]. These results provide new evidence that bisphenol analogues can elicit biologically significant responses at sublethal concentrations, underscoring the continuous need to reassess risk, also within a one-health framework.

### Environmental Implication

This study examines the risks associated with bisphenol analogues as compared to BPA upon short-term exposure in zebrafish larvae. At low concentrations, we show that these compounds can alter locomotion, neuro-cardiac physiology, and gene expression. The observed effects raise concerns about the widespread release of bisphenol analogues into aquatic environments and highlight the need for comprehensive monitoring to mitigate their ecological impact, with extension to one-health frameworks.

### CRediT authorship contribution statement

**Adèle Faucherre:** Writing – review & editing, Methodology, Investigation. **Marine Blaquière:** Writing – review & editing, Methodology, Investigation. **Yannick Lippi:** Writing – review & editing, Investigation, Data curation. **Gaelle Payros:** Writing – review & editing, Investigation. **Laurence Payrastre:** Writing – review & editing, Investigation. **Sandrine Ellero-Simatos:** Writing – review & editing, Investigation, Data curation. **Ieremias Chousidis:** Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation. **Marchi Nicola:** Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Angelo G. Torrente:** Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Chris Jopling:** Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Aurélien Drouard:** Writing – review & editing, Methodology, Investigation.
Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Acknowledgments

This work was supported by ANSES Optofish. We thank the IGF zebrafish platform ZEFIX-ZebKO. We thank Claire Naylies for the preparation of the RNAseq libraries at GeT-TRiX, Zoé Compagnie, and Prescilia Alves-Gomes for the sequencing of libraries at INRAE PGTB sequencing facility. We are grateful to the Genotoul Bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo Genotoul) for providing computing resources for RNAseq bioinformatics and biostatistics analyses.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jhazmat.2025.140004.

Data Availability

Data will be made available on request.

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