Journal Pre-proof

Long-term impact of embryonic exposure to
ethinylestradiol and clotrimazole on behavior and
neuroplasticity in zebrafish (Danio rerio).

M. Blanc-Legendre, L. Guillot, L. Chevalier, C. Malleret, K. Le Menach, P. Pardon, H. Budzinski, F. Brion, S. Sire, P. Coumailleau, T.D. Charlier, E. Pellegrini, X. Cousin

<figure><img src="image_1.png" /></figure>

PII: S1382-6689(24)00232-1

DOI: https://doi.org/10.1016/j.etap.2024.104592

Reference: ENVTOX104592

To appear in: *Environmental Toxicology and Pharmacology*

Received date: 4 July 2024

Revised date: 10 October 2024

Accepted date: 21 November 2024

Please cite this article as: M. Blanc-Legendre, L. Guillot, L. Chevalier, C. Malleret, K. Le Menach, P. Pardon, H. Budzinski, F. Brion, S. Sire, P. Coumailleau, T.D. Charlier, E. Pellegrini and X. Cousin, Long-term impact of embryonic exposure to ethinylestradiol and clotrimazole on behavior and neuroplasticity in zebrafish (Danio rerio), *Environmental Toxicology and Pharmacology*, (2024) doi:https://doi.org/10.1016/j.etap.2024.104592

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**Long-term impact of embryonic exposure to ethinylestradiol and clotrimazole on
behavior and neuroplasticity in zebrafish (Danio rerio).**

Blanc-Legendre M.<sup>a*</sup>, Guillot L.<sup>b*</sup>, Chevalier L.<sup>b</sup>, Malleret C.<sup>b</sup>, Le Menach K.<sup>c</sup>, Pardon P.<sup>c</sup>, Budzinski H.<sup>c</sup>, Brion F.<sup>d</sup>, Sire S.<sup>a</sup>, Coumailleau P.<sup>b</sup>, Charlier T.D.<sup>b,e</sup>, Pellegrini E.<sup>b</sup>, Cousin X.<sup>a#</sup>
* First co-authors

# Corresponding author

<sup>a</sup> MARBEC, University of Montpellier, CNRS, Ifremer, IRD, INRAE, Palavas-les-Flots, France. Electronic address: xavier.cousin@ifremer.fr

<sup>b</sup> University of Rennes, Inserm, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, Rennes, France

<sup>c</sup> University Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, 33600 Pessac, France

<sup>d</sup> Ecotoxicologie des substances et des milieux, Parc ALATA, INERIS, 60550 Verneuil-en-Halatte, France.

<sup>e</sup> University of Rennes, ImPACcell Platform, Biosit, Rennes, France.

**Abstract**

Estrogen receptors (ER) are widely expressed in the brain of many species and experimental results highlighted the role of estradiol in neuronal plasticity and behavior. Consequently, the brain is therefore a prime target for endocrine disrupting chemicals (EDCs) interacting with estrogen signaling. Very little is known about the late effects of early disruption of estrogen signaling by EDCs. We focused on: ethinylestradiol (EE2; ER agonist) and clotrimazole (inhibitor of key steroidogenesis enzymes, including aromatases). Zebrafish eleutheroembryos were exposed (0-5 days) and then raised normally until adulthood. Several behavioral tests were performed in adults, then cell proliferation and dopaminergic neurons were quantified in several brain regions using immunostaining. Overall, a developmental
exposure to EDCs stimulates cell proliferation in the dorsal telencephalon. At environmentally-
relevant concentrations, male fish exposed to EE2 exhibited increased activity levels and
decreased social behavior, posing a potential risk to population balance and health.

Abbreviations

ER Estrogen receptor; EDC Endocrine-Disrupting Chemical; E2 17β-estradiol; EE2 17α-ethinylestradiol; CLO Clotrimazole; SC Solvent Control; NC Negative Control; hpf hours post-fertilization; dpf days post-fertilization; PBS phosphate-buffered saline; PFA paraformaldehyde; EDTA ethylenediaminetetracetic acid; PCNA Proliferative Cell Nuclear Antigen; L-DOPA L-3,4-dihydroxyphenylalanine; TH Tyrosine hydroxylase; DAPI 4',6-diamidino-2-phénylindole; OB Olfactory Bulbs; Cantv commissura anterior pars ventralis; PPpa parvocellular preoptic nucleus anterior part; Hc caudal zone of periventricular hypothalamus; D dorsal telencephalic area or pallium; Dm medial zone of D; Dd dorsal zone of D; Dl lateral zone of D; Dp posterior zone of D; V ventral telencephalic area or subpallium; Vd dorsal nucleus of V; Vv ventral nucleus of V; TH-ir TH immunoreactive-neurons; PCNA-ir PCNA immunoreactive-cells; ANOVA analysis of variance; PCA principal component analysis.

**1. Introduction**

Estrogens are steroid hormones synthesized in many tissues and organs, and are involved in the control of reproductive and non-reproductive functions and behaviors such as copulatory behavior, parental behavior, aggressiveness, learning and memory, social behavior, as well as anxiety, depression (Albert and Newhouse, 2019; Krolick et al., 2018; Ogawa et al., 2020; Oka, 2023).

Amongst estrogens, 17β-estradiol (E2) is the most active hormone in vertebrates. E2 is derived from the aromatization of testosterone by the aromatase enzymes (Cyp19a1). E2 effects
can be mediated at the cellular level by nuclear estrogen receptors alpha (ERα) and beta (ERβ),
by GPER1 membrane estrogen receptors, or by mean of cross-talk between signaling cascades
activated by both nuclear and membrane estrogen receptors (Diotel et al., 2018). Estrogen
receptors are expressed in several brain regions, allowing E2 to affect neurogenesis and
neuroplasticity by modulating neural stem cell proliferation, newborn neurons migration,
differentiation, survival or synaptic neurotransmission (Diotel et al., 2018).

Amongst the various neurotransmitters and neuromodulators affected by circulating
estrogens, such as serotonin, nitric oxide and glutamate, numerous studies have shown that
dopaminergic system is also modulated by 17β-estradiol during development and adulthood.
Dopamine neurotransmission is known to underlie a wide range of behavioral processes
including control of locomotor activity, cognition, reward, attention, memory, anxiety and
hyperactivity (Iversen and Iversen, 2007). Importantly, the dopaminergic system is divided in
numerous subregions Previous studies have shown estrogens and xeno-estrogens exposure
modulates behavior through effects on different aspects of dopaminergic neurotransmission
such as dopamine synthesis, release and pre-and post-synaptic receptors expression (Murcia et
al., 2016; Krolick et al., 2018; Bertotto et al., 2019; Hu et al., 2022).

The synthesis and action of E2 can be altered by numerous endocrine-disrupting
chemicals (EDCs) with estrogenic or anti-estrogenic activity and many of them are suspected
to interfere with brain structure and function. Among EDCs, 17α-ethinylestradiol (EE2), a very
active synthetic derivative of E2, is widely used around the globe. Present in almost all modern
formulations of combined contraceptive pills, EE2 is excreted with urine and feces and
therefore commonly found in the aquatic environment in the low to high ng/L range (Klaic and
Jirsa, 2022). EE2 is a potent ER agonist in teleost fish and induces biological effects similar to
those observed with endogenous. Amongst the different targets, EE2 strongly stimulates
*cyp19a1b* gene transcription in radial glial progenitor cells in fish (Brion et al., 2012). The gene *cyp19a1b* encodes brain aromatase (aroB) in fish and variation of its expression following exposure to estrogens or estrogen-mimetic compounds is likely to lead to local changes in estrogen production. Steroid-synthesizing enzymes, including aromatase, are common targets of azole compounds (Bhagat et al., 2021). Clotrimazole (CLO) belongs to the imidazole family and is commonly used as a broad-spectrum antimycotic drug. CLO inhibits aromatase activity in fish (Hinfray et al., 2006). As EE2, CLO is found in effluents from wastewater treatment plants and in surface water in the low to high ng/L range (Assress et al., 2020).

Zebrafish (*Danio rerio*) is a useful model for addressing ecotoxicological concerns and evaluating short and long-term effects of chemicals found in the aquatic environment. A growing number of studies are using this animal model to study the impact of EDCs on vertebrate physiology, particularly with regard to neurobehavioral developmental disorders (Legradi et al., 2018).

Previous studies showed that adult exposure to CLO or EE2 can interfere with fish endocrine systems and cause deleterious effects on behavior and/or physiology (Colman et al., 2009; Fenske et al., 2020; Bhagat et al., 2021; Melefa and Nwani, 2021). Other studies focused on exposure to EDCs showed effects on early nervous system development of zebrafish larvae (Nasri et al., 2021; Vaillant et al., 2020, Crowley-Perry et al., 2021; Bernardo and Connaughton, 2022). While the developmental period is highly sensitive to EDCs, there is only a very limited number of studies investigating the long-term effects of an embryo-larval exposure to EDCs in fish (Volkova et al., 2012; Horzmann et al., 2020, 2022).

In this study we hypothesized that early embryonic exposure to EDCs would disturb estrogen signaling during early life, affect brain development and modify some behavioral traits in adults. Zebrafish embryos were exposed for the first five days post-fertilization to EE2 or CLO. The initial rationale for using these two model molecules was therefore to have a positive
(EE2) and negative (CLO) consequence on brain aromatase and characterize the long-term consequences. The initial rationale for using these two model molecules was therefore to have a positive (EE2) and negative (CLO) consequence on brain aromatase and characterize the long-term consequences. Delayed effects were investigated in 3-month-old adults by analysis of behavior, cell proliferation and neuronal differentiation into dopaminergic neurons. We specifically focused on the dopaminergic cells as their various populations within the vertebrate brain are relatively well-described and their sensitivity to estrogen exposure is region-dependent. In addition, while long-term consequences of early, developmental exposure to estrogens (organizing effects) were described in mammals, there is no studies to our knowledge describing such a mechanism on dopaminergic neurons in zebrafish.

## 2. Materials and methods

### 2.1. Fish housing and exposure experiments

The *cyp19a1b-GFP* zebrafish transgenic line (Tong et al., 2009) was previously used to quantify brain aromatase expression and to measure direct behavioral effects in larvae following EE2 and CLO exposure (Blanc-Legendre et al., 2023). To be able to link early disruption to potential late effects, exposure was performed using the same transgenic line. In addition, the OECD test No. 250 to detect xenoestrogens is based on this specific transgenic zebrafish line, offering a unique opportunity to better assess hazards and risks linked to EDC exposure affecting aromatase. Fish were kept at the MARBEC Palavas Experimental Marine Platform (agreement D34121926 for animal experiments) in a dedicated room with photoperiod (14h:10h light:dark) and temperature controlled. Water was maintained at 27.0°C ± 1.0°C with an hourly automated supply of new water resulting in a daily renewal of 50 %. Fish were fed twice a day with dry food and once a day with newly hatched brine shrimps (*Artemia salina*). On the day before exposure, pairs of zebrafish were set in dedicated 1 L spawning boxes. In the morning, fertilized eggs were collected and assessed for quality. Only homogeneous spawns
with eggs ≤ 4 hours post-fertilization were kept for experiments. A mixture of approximately
90 eggs from 3-5 spawns were evenly divided into 4 glass crystallizers containing pre-warmed
exposure solutions. Exposures were carried out at the following final concentrations: 4 ng/L
EE2, 250 µg/L CLO, from 10 000x stock solutions in dimethylsulfoxide. Induction of GFP
expression upon exposure to EE2 is shown in supplementary Fig. S1. All chemicals were
bought from Sigma-Aldrich. Concentrations for EE2 and CLO were selected based on previous
work. An exposure of zebrafish larvae to 4 ng/L EE2 induces a strong increase in *cyp19a1b*
transcription as shown by GFP induction in the *cyp19a1b-GFP* transgenic line (Fig. S1).
Regarding CLO, concentration was chosen as a high concentration to inhibit AroB without
inducing acute toxicity (Blanc-Legendre et al., 2023. EE2 concentration can be found in the
environment while for CLO, concentration used in this study is several order of magnitude
above the one encountered in the environment. Concentrated solutions were diluted (1/10 000)
in ISO water according to ISO 7346-2 (1996) (294 mg/L CaCl2·2 H2O, 123.3 mg/L MgSO4·7
H2O, 63 mg/L NaHCO3, 5.5 mg/L KCl, pH 7.8). To account for a possible solvent effect, we
used both a solvent control (SC) consisting of dimethylsulfoxide with a final concentration of
0.01% (v/v) and a control without solvent, the negative control (NC). Crystallizers with
eggs/eleutheroembryos were incubated at 27 ± 1 °C. The medium was exchanged every 24 h
until 120 hours post-fertilization (hpf) to maintain stable concentrations over the course of the
experiment, and embryo development was monitored daily by microscopic observation. At 120
hpf, exposure solutions were removed and zebrafish larvae were rinsed in two successive ISO
water baths. In order to monitor stability of chemicals during the exposure protocol, 5 mL
samples were taken before medium renewal at 0, 24, 96 and 120 hpf in all tested conditions
(NC, SC, EE2, CLO). A total of 12 samples for EE2/CLO and 20-22 samples for SC/NC were
analyzed. Protocol for chemical analyses is described in Supplementary materials.
They were then kept for 10 days in 1 L aquariums (approximately 30 larvae per aquarium) progressively filled with system water, then transferred to 3 L inserts placed in 10 L tanks. At approximately 30 days post-fertilization (dpf), inserts were removed and fish were kept in 10 L tanks until the end of the experiment. The exposure protocol was performed in triplicate (for a total of 12 tanks per tested condition). Survival rate was > 90% independently of the treatment.

## 2.2. Non-reproductive behavior

Behavioral tests were carried out at 105-110 dpf in a dedicated room with controlled temperature and photoperiod synchronized with the rearing room. A total of 104 individuals were included in the behavioral tests. Different behavioral traits were analyzed sequentially: shoaling (day 1), spatial memory (day 1), associative learning capacity by classical conditioning (day 2), exploration capacities (day 3), anxiety (day 4), sociability (day 4) and aggressiveness (day 5) were studied sequentially. Care was taken to randomize conditions (treatment and sex) to avoid bias due to e.g. time of the day for testing. Fish were kept in individual 1 L tanks located on a shelf with visual contact between neighbor tanks over the week of testing; with a full renewal of water every day and feeding once a day in the evening with freshly hatched artemia. For all tests, behavior of fish was video-recorded and individuals were automatically tracked using Ethovision software (Noldus®). Devices were located either above or next to infrared lighting and camera were equipped with infrared passing filters. Water in behavioral devices was changed between every run except when mentioned otherwise. Within the 5 minutes following the last behavioral test (aggressiveness test), fish were euthanized in 500 mg/L benzocaine solution, measured and weighed; and heads were sectioned and transferred to 4% paraformaldehyde (PFA) in 0.01M phosphate buffered saline (PBS) solution for immunostaining.
### 2.2.1. Shoaling

This test aims at measuring the cohesion of a shoal (Pham et al., 2012). A group of 6 fish (3 males and 3 females) was transferred to a narrow 3 L tank and their behavior was video-recorded (lateral view, DMK33GP1300e, The Imaging Source®) for 6 min, including 3 min for acclimation and 3 min for behavioral recording. Afterward, fish were removed and distributed into annotated individual 1 L tanks. Water was exchanged between each group and a total of 9 groups / condition were tested. Data were analyzed as individual traveled distance (mm) and average interindividual distance (mm).

### 2.2.2. Spatial memory

The y-maze (arm size 25 cm × 8 cm × 15 cm, length×width×height) is used to evaluate short term spatial memory (Cognato et al., 2012). The maze consists of three arms, each visually identifiable by fish by different symbols. The test was performed 3 h after the end of the shoaling test. During a first 5 min session, the individual fish was allowed to navigate between two arms, the third one being closed. After a 60 min interval, all three arms were made accessible and the fish was submitted to a second 5 min session. Since spatial memory is evaluated through the increase of time spent in the novel arm compared to initial exploration, no acclimation is performed whatever the session (Cognato et al., 2012). Behavior of fish was video-recorded (top view, DMK33GP1300e, The Imaging Source®). Data were analyzed as traveled distance (mm), number of alternations between arms, and time spent in the novel arm (sec).

### 2.2.3. Associative learning

This test aims at assessing the learning capacity of zebrafish using classical conditioning and was performed according to Zantiks® protocol with minor modifications (Valente et al., 2012). Fish were exposed to an unconditioned aversive stimulus (US, mild electric shock) associated with a conditioned visual stimulus (CS, visual cue checkerboard pattern as opposed
to a plain gray background). Test was performed in the Zantiks AD unit (Zantiks®). Visual
cues were presented on the integrated screen below the testing tank and aversive stimuli were
given by metal plates inserted at both ends of the testing tank. Fish were conditioned to avoid
the checkerboard pattern. One session first comprised of an acclimatation period of 40 min,
divided into 5-minute sequences of both patterns being displayed alternatively to one or the
other half of the tank. The last 10 minutes were recorded and analyzed to ensure the absence of
pattern preference. The acclimation was followed by the conditioning period of 9 US/CS
associations (90 sec total). Finally, a last 1 min probe period during which checkerboard and
plain gray patterns are displayed in each half of the screen. This period was used to evaluate
the preference of the fish to spend time outside the checkerboard pattern associated to the
electric shock during conditioning. Data were analyzed as time spent outside the checkerboard
area (sec), number of visits to the checkerboard area, and traveled distance (sec).

**2.2.4. Exploratory capacities**

A maze of 45 cm * 75 cm * 7.5 cm (L*1*h), divided into 4 corridors (Z-shape) and 16
virtual zones, was filled with water (Vignet et al., 2014). The fish was transferred to a safety
area initially isolated from the rest of the labyrinth by a closed door and covered by an opaque
plate (acclimation phase). After 2 min of acclimation, the door was opened and behavior of the
fish was recorded (top view, DMK33GP1300e, The Imaging Source®) for 5 min from the time
it went out of the start zone. Water was thoroughly aerated and mixed between each run and
totally renewed every 5 runs. Data were analyzed as time to exit the start zone (sec), distance
moved (mm), time spent in distal zone (sec), time to reach distal zone (sec), time to reach the
furthest area (zone 16, sec) and time spent in zone 16 (sec).
### 2.2.5. Anxiety and sociability

These two tests were carried out successively in the same device consisting in a narrow 3 L tank filled with 1.6 L of water. Dark gray walls lined the two ends of the tank. On one side the wall was removable and allowed, after its removal, the view of 4 conspecifics isolated by a transparent plexiglass wall. The novel tank diving test aims at evaluating the anxiety level of fish facing a novel environment by evaluation of their vertical positioning (Levin et al., 2007). A fish was transferred into the device described above, with the removable wall lowered and its behavior was recorded (lateral view, DMK33GP1300e, The Imaging Source®) for 5 min. Data were analyzed as distance traveled (mm), time spent in the virtual upper zone (sec) and number of visits to the upper zone.

The sociability test evaluates the propensity of the fish to spend time close to congeners. Two minutes after the end of the anxiety test recording (transition period with no behavioral recording), the wall was raised, thus allowing a view of the conspecifics and the behavior of the fish was recorded during 5 min (lateral view, DMK33GP1300e, The Imaging Source®). Data were analyzed as distance traveled (mm), number of visits to the social area (i.e. next to congeners), and time spent in the social area (sec).

### 2.2.6. Aggressiveness

The test analyzes the behavior of a fish facing its own reflection. An individual was transferred into a narrow 3 L tank where a mirror covers one extremity. During the first period of 2 min, an opaque door hid the mirror (acclimation). The door was automatically lifted and the behavior of the fish was recorded for 5 min (lateral view, DMK33GP1300). Data were analyzed as distance traveled (mm), number of visits to the mirror zone, and time spent in the mirror zone (sec).
## 2.3. Reproduction and sexual behavior

These tests were performed using other fish from the same batches. The test took place in a dedicated room with controlled temperature and photoperiod synchronized with the rearing room. The afternoon before the test started, one male and one female from the same condition were placed in a 1 L spawning box divided into two equal sections by a grid insert to separate individuals. A spawning area consisting of a circular insert (45 mm diameter) filled with glass marbles was placed in one corner of the spawning box on the female side. A maximum of nine couples from different exposure conditions were tested at the same time (for a total of 15 CLO, 23 EE2, 20 SC and 16 NC individual pairs across 9 independent trials). In the morning of the test, at light onset, dividers were removed and behavior of fish was recorded laterally for 3 h (DMK33GP1300e, The Imaging Source®). At the end of the recording, fish were transferred back to the rearing system and spawns were collected. In terms of behavior, total distance traveled (cm), time spent in the spawning area (sec), body contact time (sec) and average interindividual distance (cm) were analyzed. Besides, spawning successes were referenced and number of eggs as well as fertilization rate were counted for each spawn. Additional spawns were collected from paired reproduction events in the same conditions (without video-recording) for a total of 36 EE2, 31 CLO, 34 SC and 24 NC.

## 2.4. Sampling and brain sections

The fish heads (n=84 as SC: 20 males, 17 females; CLO :12 males, 11 females; EE2 12 males, 12 females) were kept for 48 hours at 4°C in 4% PFA in PBS 1X. The samples were washed 3 times for 5 minutes in 1X PBS, then incubated in a solution of ethylene diamine tetraacetic acid for decalcification (EDTA: 0.5 M and pH 7.5) for 14 days. The decalcified heads were then washed in PBS 1X and cryoprotected by immersion in 0.01M PBS solution containing sucrose, respectively at 15% for 3 to 24 hours then at 30% for at least 24 hours, at
4°C pending their inclusion in the mounting medium. The samples were then embedded in a cup filled with a mounting medium (NEG-50TM, ThermoFisher Scientific, USA). Heads were oriented under a binocular loupe and then frozen in their mounting medium by inserting the cup into a container containing isopentane at -60°C. Once frozen, the samples were stored at -80°C until further processing. 20 µm-thick coronal sections were then obtained using the cryostat Microm HM 560 (ThermoFisher Scientific, Lithuania) and mounted on Epredia™ SuperFrost Plus™ Adhesion Microscopes Slides 25x75x1mm (ThermoFisher Scientific, USA). The sections were placed on 3 slides successively to obtain 3 replicates per brain. They were then dried for at least 30 minutes on a RaymondALamb slide dryer, then stored at -20°C pending immunostaining.

**2.5. Double immunostaining TH/PCNA**

We performed a double immunofluorescence to label PCNA and Tyrosine Hydroxylase.

To study proliferative cells, we used a marker for cells in S phase, the Proliferative Cell Nuclear Antigen (PCNA). Tyrosine hydroxylase (TH) is a member of the aromatic amino acid hydroxylase family that catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). L-DOPA is processed into dopamine through a series of downstream enzymatic reactions. TH is therefore a commonly used marker for dopaminergic neuron identification. A double immunostaining was performed on males and females from each condition (same numbers as indicated above). It should be noted that we did not expect colocalization of the 2 markers within the same cells. Since there was no difference between NC and SC treatments for any of the behavioral parameters investigated (see below), we did not analyze NC brain and only SC samples are called Control. This analysis allows us to define whether EE2 and/or clotrimazole will affect neurogenesis or tyrosine hydroxylase expression compared to the control group treated with the vehicle. The double immunocytofluorescence was performed using antibodies against tyrosine hydroxylase (1/500 Polyclonal rabbit AB152, EMD Millipore
Corporation, USA), and PCNA (PCNA 1/100 clone PC10 mouse Monoclonal, DakoDenmark A/S, Denmark) as previously performed (Pellegrini et al., 2007).

Tissue sections were rinsed in 0.01M PBS 3 times for 10 minutes and subjected to antigen retrieval in sodium citrate buffer (pH = 6) at 80 °C for 30 min. Slides were rinsed in PBS 1X 3 times for 10 minutes, and then one time in a solution of PBS 1X with 0.2% triton and 0,5% milk powder for 45 minutes, to block non-specific binding. Tissue sections were incubated overnight at room temperature in PBS 0.01M containing 0.5% milk powder and anti-TH and anti-PCNA antibodies. The following day, tissue sections were washed two times in PBS 1X and one time in PBS 1X 0.1% triton. Then, they were incubated with secondary antibodies, anti-rabbit IgG (H+L) antibody conjugated to Alexa Fluor (GAR 633TM) and anti-mouse IgG (H+L) antibody conjugated to Alexa Fluor (GAM 594TM), diluted 1:500 (Invitrogen, ThermoFisher, USA) for 1h30. Slides were rinsed in PBS 1X 2 times for 10 minutes and one time in a solution PBS 1X with 0.1% Triton. Finally, slides were coverslipped using VECTASHIELD Vibrance Antifade with DAPI (H-1800-10, Eurobio Scientific) to allow visualization of cell nuclei. Slides were scanned using the Hamamatsu scanner at X20 resolution. Scanned sections were then observed and analyzed with NDP.view2TM (2.9.29, Hamamatsu Photonics K.K).

We assessed the impact of EE2 and CLO on PCNA on proliferation and dopaminergic neurons in the forebrain regions that contains numerous periventricular proliferative areas and several dopaminergic nuclei involved in social and reproductive behaviors and suggested to be targeted by estrogens and estrogen mimetic molecules. The different brain regions were characterized using DAPI labeling and the nomenclatures from the topological atlas "Neuroanatomy of the Zebrafish brain" (Wullimann, Rupp and Reichert, 1996). Four brain regions were investigated: the olfactory bulbs (OB), the telencephalon from the most rostral part to the commissura anterior pars ventralis (Cantv), the parvocellular preoptic nucleus
anterior part (Ppa), and the caudal zone of periventricular hypothalamus (Hc). In the telencephalon, quantification of proliferative activity (PCNA-ir) was performed in the dorsal telencephalic area or pallium D: Dm (medial zone of D), Dd (dorsal zone of D), Dl (lateral zone of D) and Dp (posterior zone of D). Quantification of TH immunoreactive-neurons (TH-ir) was performed in the ventral telencephalic area or subpallium V: Vd (dorsal nucleus of V) and Vv (ventral nucleus of V). To identify different dopaminergic neuronal populations in different brain areas of adult zebrafish, we used the nomenclature for dopaminergic populations previously described (Panula et al., 2010): A16a in OB, A16p in the ventral telencephalon, A15 in Ppa and A10 in Hc.

## 2.6. Statistical analysis

### 2.6.1. Behavior

Raw data were exported as Excel files and analyses were performed using R (v 4.3.1) via RStudio (v2021.09.4+494). Statistical analyses were first performed on individual variables. Since there was no difference between NC and SC treatments for any of the measured variables (data not shown), NC and SC individuals were merged and further called Controls. Two-way (sex, treatment) ANOVA followed by Tukey's post-hoc tests were performed on data following normal distribution as evaluated via quantile-quantile graphics. Otherwise, Kruskal-Wallis test followed by Dunn's post-hoc tests were used on sex-specific datasets to evaluate effect from the treatment. In order to get further insight into how behavior is affected by the treatments, Principal Component Analysis (PCA) was performed on the entire behavioral dataset (104 observations vs 20 variables) (package factoextra). Significance of the PC axes and of the contributions of the different variables to the PC axes was checked using the PCAtest package (Camargo, 2022). Only significant PC axes and variables showing significant loadings on these axes were considered for interpretation of the data (Camargo, 2022). In order to identify specific differences between treatments, two-way (sex, treatment) ANOVA (alpha =
0.05) followed by Tukey's post-hoc test was performed. An adjusted p-value ≤ 0.05 was considered as a statistically significant difference.

### 2.6.2. Immunoreactive cells quantification

Immunoreactive cells were quantified on one of the 3 replicates for each sample, i.e. in 20-micrometer coronal sections separated by 40 micrometers. The average number of PCNA-ir cells and TH-ir cells was established in two consecutive sections in each region, except in the telencephalon where three sections were considered, between the end of the olfactory bulbs and the beginning of the commissura anterior pars ventralis. Two-way (sex, treatment) ANOVA (alpha = 0.05) were performed in GraphPad Prism™ (v. 9.5.1), for each brain area, and for each immuno-labeling separately. Overall, our results showed no significant interaction between sex and treatment. We then relaunched a two-way ANOVA without interaction. In cases where a significant effect of treatment was observed, a Tukey's post-hoc test was applied. An adjusted p-value ≤ 0.05 was considered as a statistically significant difference.

## 3. Results

### 3.1. Chemical analysis

Analysis of concentrations of EE2 (2.94±0.44 ng/L) and CLO (222.69±25.10 µg/L) in water revealed adequate and stable exposure of embryos, and absence of significant contamination in negative and solvent control conditions (Table S1).

### 3.2. Behavior results

Overall, we observed significant effects that were dependent on the behavioral context, the sex of the fish and the molecule used for exposure at the embryonic stage (see Table S2 for all results). During the shoaling test, no effect on shoal cohesion was observed (Fig. 1A). Nonetheless, regardless of sex, fish exposed to EE2 swam more than control fish ($F_{(2, 207)} = 5.33; p=0.0056$) (Fig. 1B). In the y-maze, fish exposed to EE2 tended to swim more ($F_{(2, 91)} =$
2.60; p=0.080) (Fig. 1D). In addition, EE2-exposed males showed a higher level of alternations between arms of the maze compared to control males (p=0.016) (Fig. 1C). Distance traveled by fish in the classical conditioning test was increased after treatment to CLO regardless of sex ($F_{(2, 83)} = 7.96; p=0.00069$) (Fig. 1E). All other variables remained unaffected when analyzed individually (Table S2).

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
Fig. 1: A) Average interindividual distance (mm) in the shoaling test; B) average distance traveled by fish in the shoaling test; C) number of arm alternations in the y-maze; D) Distance traveled in the y-maze; E) distance traveled during classical conditioning test. Results are shown as mean ± SEM for control fish and fish that were submitted to embryonic exposure to EE2 or CLO. #: p<0.1; *: p<0.05; **: p<0.01; ***: p<0.001 versus control (within-sex) group.

We further performed an integrative analysis using PCA to identify converging
behavioral differences between treatments that could give further insight into characterizing the
phenotype regardless of the behavioral context. PCA showed that treatments significantly
separate along the PC1 axis, explaining 16.2% of the total variance. Variables related to
swimming activity and exploration (i.e. distance traveled in the sociability test, distance
traveled in the y-maze test, distance traveled in the z-maze test, distance traveled in the novel
tank test, latency to reach distal zones in the z-maze) negatively correlate with PC1, while
sociability positively correlate with PC1 (time spent near congeners and frequency of visits to
the congener area) (Fig. S2 and S3). Two-way (sex, treatment) ANOVA analysis on PC1 scores
reported a main treatment effect ($F_{(2, 92)} = 6.00$; p=0.0036) and a main sex effect ($F_{(1, 92)} = 7.68$
; p=0.0068). Post-hoc analysis confirmed the significant effect in EE2-exposed male fish
(p=0.0069) while the effects in females failed to be significant (Fig. 2). CLO-exposed males
showed the same trend as EE2-exposed males (p=0.1) (Fig. 2). Overall, according to
contributing variables, EDC-exposed male fish showed a hyperactive behavior associated with
reduced sociability.
<figure><img src="image_7.png" /><figcaption>Fig. 2: Behavioral score of adult zebrafish females and males (mean ± SEM) after exposure to EE2, CLO, or Control obtained from Principal Component Analysis. Mean ± SEM; ***: p<0.001. #: p=0.1 versus control within-sex group.</figcaption></figure>

### 3.3. Reproduction

We observed significant differences in behavior of fish exposed to EE2 in the reproduction test. As already shown by the above analyses, EE2-exposed fish swam more (treatment effect *F*(2, 147) = 9.48; p=0.00013), and they also spent more time in the spawning area (treatment effect *F*(2, 147) = 4.01; p=0.020) and in body contact (*F*(2, 147) = 7.08; p=0.0012) (Fig. 3). On the other hand, average reproductive success rate was similar between treatment groups (66.7% for EE2, 68.0% for CLO, 65.8% for Control). However, there was a treatment effect (*F*(2, 84) = 4.04; p=0.021) on spawn size that revealed that CLO-exposed pairs spawned significantly more eggs compared to Control pairs (multiple comparison post-hoc test, p=0.036) while there was only a tendency for EE2-exposed pairs (p=0.088) (Fig. S4). Fertilization rates remained unaffected (Fig. S4).
<figure><img src="image_8.png" /></figure>

Fig. 3: Effects of EE2 and CLO treatment on reproductive behavior of exposed zebrafish compared to controls: A. distance traveled during reproduction; B. time spent in spawning area (sec); C. body contact time (sec). Mean ± SEM; *: p<0.05; **: p<0.01; ***: p<0.001 vs Control group.

**3.4. Effects of EE2 and CLO on brain proliferation**

We evaluated proliferation by counting PCNA immuno-reactive cells (PCNA-ir cells) in four brain areas: olfactory bulbs, dorsal telencephalon or pallium, parvocellular preoptic nucleus anterior part, and caudal zone of periventricular hypothalamus. Representative coronal brain sections showing PCNA-ir cells are shown in Fig. 4.
<figure><img src="image_9.png" /><figcaption>Fig. 4: Coronal brain sections showing PCNA and TH immunoreactivity in representative sections of the four brain regions. The cross-sectional levels are shown in the illustration above</figcaption></figure>
the image A. PCNA and TH immuno-reactive cells (respectively in red and yellow) in sections counterstained for DAPI (in blue) in the olfactory bulbs (A,A'), in the dorsal telencephalic area (medial Dm, dorsal Dd, lateral Dl and posterior Dp zone; B,B'), in parvocellular preoptic nucleus, anterior part Ppa (C,C') and in the caudal zone of periventricular hypothalamus Hc (D,D').

In the dorsal telencephalon (Dm, Dd, Dl, Dp), PCNA-ir cell number was affected by the treatment ($F_{(2,35)}=11.49$) and was significantly higher in EE2-treated (p<0.0001) and CLO-treated fish (p=0.044) compared to controls (Fig. 5B). In terms of magnitude, the increase in PCNA-ir cells was more pronounced in EE2-exposed (+15%) than in CLO-exposed (+8%) fish. There was no significant effect of treatment, sex or treatment-sex interaction on cell proliferation in the other regions (Fig. 5 A, C and D).
<figure><img src="image_10.png" /></figure>

Fig. 5: Quantification of PCNA in the four investigated brain areas : A. Olfactory bulbs, OB, n=63; B. Dorsal telencephalic area, Tel, n=39; C. Parvocellular preoptic nucleus, anterior part, Ppa, n=28; D. Caudal zone of periventricular hypothalamus, Hc, n=39. Mean ± SEM; *: p<0.05; ****: p<0.0001 versus control group.

**3.5. Effects of EE2 and CLO on dopaminergic neurons**

Tyrosine hydroxylase (TH) immunofluorescence was used to identify dopaminergic neuronal populations in different brain areas of adult zebrafish named as above (Fig. 4). In the
olfactory bulbs (OB), TH-immunoreactive (TH-ir) cells correspond to the population A16a (Fig. 4 A,A'). In the ventral telencephalon (Vv, Vd), another group of neurons, A16p, is present. Caudally, the group identified as A16p shifts dorsally and laterally to the dorsal nucleus of the ventral telencephalic area (Vd) (Fig. 4 B,B'). The dopaminergic nucleus corresponds to A15 in the parvocellular preoptic nucleus, anterior part (Ppa) (Fig. 4 C,C') and A10 in the caudal zone of periventricular hypothalamus (Hc) (Fig. 4 D, D').

A significant main treatment effect ($F_{(2,34)}=7.56; p=0.003$) was identified in the Hc (A10) with a higher number of dopaminergic neurons in CLO-treated fish compared to controls independently of the sex (Fig. 6D). Interestingly, we found a significant main effect of sex in OB with a higher number of dopaminergic neurons in A16a in males than in females ($F_{(1,41)}=7.97; p=0.007$), with no difference between treatments (Fig. 6A). No significant difference in the number of TH-ir cells was observed in the telencephalon (A16p), and in the Ppa (A15) (Fig. 6 B and C).
<figure><img src="image_11.png" /><figcaption>Fig. 6: Quantification of TH-ir cells in the four investigated brain areas: A. Olfactory bulbs, OB, (A16a); B. Dorsal and ventral nucleus of ventral telencephalon, Tel, (A16p); C. Parvocellular preoptic nucleus, anterior part, Ppa, (A15); D. Caudal zone of periventricular hypothalamus, Hc, (A10). Mean ± SEM; **: p<0.01 versus control group.</figcaption></figure>

Discussion

A large number of EDCs interfere with estrogen signaling, disturb steroid balance and disrupt the development of brain structures, compromising many behaviors including motor
and associative functions (Frye et al., 2012). In this work, we investigated the long-term impact
of a short 5-days early exposure to EE2, a powerful estrogen receptor agonist or CLO, a
chemical known to inhibits steroidogenic enzyme activity, in particular aromatase. Our results
showed that both treatments induce persistent effects on behavior and neurogenesis in adult
zebrafish, hence reinforcing the idea that early exposure to synthetic chemicals can induce
organizational and functional alterations.

The effects of CLO or EE2 treatment were first evaluated on a large panel of adult
behaviors. Taken together, we showed that an early exposure to EE2 induced hyperactivity
combined with reduced sociability, an effect that was observed in adult male fish specifically.
Interestingly, treatment with CLO revealed a similar trend. As far as we know, no previous
research investigated delayed effects of a 5-day exposure to EE2 or CLO on behavioral traits.
A few studies looked at the prolonged effect of developmental exposure to EE2 (30-40 days).
Results showed that EE2 increased anxiety and sociability of zebrafish in a shoaling test after
an exposure of zebrafish for 80 days followed by a 40 or 120 day depuration period (Volkova
et al., 2015; Porseryd et al., 2017). In another study, maternal developmental exposure to EE2
resulted in an increase in anxiety in adult guppies (Volkova et al., 2012). This contrasts with
the absence of changes in levels of anxiety observed in the present work, highlighting the impact
of the timing and duration of exposure periods. Most observed behavioral defects were related
to increase in activity with no major effect observed on learning performances (either classical
conditioning or spatial memory). However, the observed decrease in sociability revealed by the
PCA analysis and the changes in reproductive behaviors may be related to some social
cognition disruptions.
In parallel, we also investigated the effects of developmental EDC exposure on reproductive behavior and spawns. As above for non-reproductive behavior, we observed a significant hyperactive behavior in fish exposed to EE2 (but not CLO) that is in line with what was observed in a non-reproductive context, and led to heightened reproductive behavior: pairs spent more time in the spawning area and in body contact. These behavioral changes observed in EE2-exposed pairs have limited influence on spawn sizes. On the contrary, despite no change in reproductive behavior, CLO-exposed pairs laid significantly more eggs. To our knowledge, this is the first time that the reproductive effects of a 5-day embryonic exposure are investigated in adults and there is no comparable evidence of early organizational impact of estrogens on the central nervous system regulating reproduction in zebrafish as well as on the function of developmental neurosteroidogenesis for brain organization.

In this study we focused on the dopaminergic system that plays a key role in a range of behaviors and can be dysregulated by EDCs (Bertotto et al., 2019; Hu et al., 2022). Although functions associated with dopaminergic nuclei are not yet fully defined in fish, the dopaminergic system in the zebrafish brain appears to be relatively conserved and similar to mammals (Panula et al., 2010; Yamamoto and Vernier, 2011). In our results the number of dopaminergic neurons in the caudal hypothalamus was significantly higher in CLO-treated fish compared to control, independently of the sex. These dopaminergic nuclei could be involved in swimming behaviors and sensorimotor reactions (Barrios et al., 2020). This observation which is specific to CLO-treated fish may explain why they are specifically more active than other fish in some context (e.g. classical conditioning). In our experimental conditions, EE2 treatment did not affect the number of dopaminergic cells. We also noted that the number of dopaminergic neurons in olfactory bulbs was significantly higher in males compared to females,
independently of the treatment (control, EE2 and CLO). The olfactory system plays a key role
in animal survival and detection of odor molecules mediates many behaviors such as foraging,
mating, social behavior, and danger assessment (Calvo-Ochoa et al., 2019, Kermen et al. 2013).

Data have shown dopaminergic metabolism changes in the olfactory/telencephalon in winner-
type behavioral tests (Teles et al., 2013). Previous studies performed in rat and human also
observed a sex difference in the number of dopaminergic neurons in the olfactory bulbs and a
correlation between the number of dopaminergic neurons and the olfactory performance
(Alizadeh et al., 2015). In fish, dopaminergic neurons in olfactory bulbs are also thought to be
involved in olfaction modulation, but their role is still unclear (Korshunov et al., 2020). In
zebrafish, ablation of dopaminergic neurons in the olfactory bulbs impairs the ability of
zebrafish to detect repulsive stimulus and increase freezing behavior (Godoy et al., 2020). The
greater number of dopaminergic cells detected in male olfactory bulbs strongly suggests that
zebrafish brains display sex-specific differences in terms of dopamine neurotransmission and
function. In our study, early exposure to EE2 had a male-specific impact on social behavior,
raising the question of whether this observation is mechanistically linked to sex differences in
olfactory dopaminergic neuron number (Kermen et al., 2013).

In general, sex differences are found in different range of behaviors as well as in cerebral
structures and metabolic activity in adult zebrafish (Ampatzis et al., 2012; Philpott et al., 2012).

In addition, numerous studies have observed a sex-specific impact of steroids on
neurobehavioral aspects (Ampatzis et al., 2012, Volkova et al., 2015). In zebrafish, there is no
evidence for major brain sexualization, and gonad differentiation occurs from an early ovary-
like structure to ovary or testis between 21 and 45 dpf. Thus, within the scope of our study, we
could hypothesize that early neurophysiological changes, induced by EDCs, could lead to
different behavioral alterations in both females and males. Sensitivity of males toward EDC
exposure may be due to intrinsic neurophysiological differences between sexes and/or from the
distinct function of estrogens and the estrogenic pathway in both sexes.

We also investigated the impacts of early exposure to EDCs on adult brain proliferative
activity and we reported a significant increase in cell proliferation after both CLO and EE2
treatments in the dorsal telencephalon specifically, independently of the sex. The potential
mechanisms by which EE2 and CLO promote proliferation in the telencephalon are not fully
understood. Previous publications however reported that estradiol acts on cells proliferation by
up-regulation of genes involved in cell cycle (Cyclin D1, PCNA, mitogen-activated kinases),
trough epigenetically mechanisms or activation of growth factors (Kovac et al., 2020; Denley
et al., 2020). Transcriptomic analysis of whole brains from zebrafish (larvae) or guppy (adults)
exposed to EE2 highlight changes in the level of genes regulating transcription processes
(Porseryd et al., 2017; Saaristo et al., 2017). Regarding CLO impact on proliferation, to the best
of our knowledge, there are no references on the mechanisms used by clotrimazole to regulate
cell proliferation in brain tissue. It should be noted that most mechanisms of steroid regulation
of neurogenesis were studied following acute or chronic exposure to estrogens, but the long-
term regulation, following withdrawal of steroid did not receive much attention, especially in
fish. The molecular and cellular mechanisms underlying the long-term impact of CLO and EE2
on cell proliferation in the adult telencephalon is not clear but could be associated with early
organization of stem cell microenvironment, including via modification of perineuronal nets
associated with parvalbumin-expressing neurons. Further research based on transcriptomic
analysis could help to get a better understanding of the underlying molecular pathways driving
cell proliferation in EE2 and CLO-treated animals. Previous studies showed that adult teleost
brains are characterized by strong proliferative activity in many niches along the ventricular
edge and that, over time, newborn cells move away from the ventricles and differentiate into
neurons (Grandel et al., 2006; Pellegrini et al., 2007; Furlan et al., 2017). An increase in cell
proliferation is of particular interest as the dorsal telencephalon is involved in the processing of
various cognitive functions and was suggested to provide a form of plasticity necessary to adapt
the brain to changes in behavior (Mazzitelli-Fuentes et al., 2022). Different parts of the
telencephalon are known to be involved in social and motor behaviors, in particular the dorsal
telencephalon, a region homologous to the amygdala and hippocampus of mammals (Geng and
Peterson 2019). Our results suggest that increased neurogenesis in the dorsal telencephalon
(medial and lateral) may be linked to the behavioral alterations we described here
(hyperactivity, reduced sociability) but the causality needs to be investigated. Excessive
proliferation of neural cells, as well as hyperactivity and reduced sociability evidenced in this
work, echo features that are associated with autism spectrum disorders (ASD) in different
models and in humans. In particular, two recent studies performed in zebrafish larvae showed
a link between these typical behavioral manifestations and proliferation of neural cells upon
exposure to estrogenic EDCs, hence suggesting that the long-term effects we observed at the
cellular and behavioral level may be functionally linked (Chen et al., 2018; Bai et al., 2023).

Taken together, our results show that early exposure to either EE2 or CLO triggers, to
some extent, comparable long-term impacts at both social behavioral and cellular levels, which
may seem surprising. Indeed, while EE2 was demonstrated to strongly stimulate aromatase
expression, CLO was reported to inhibit aromatase activity (Blanc-Legendre et al., 2023).
Overall, our results show that the behavioral changes observed are not systematically associated
with the same cellular alterations, some being common to both treatments (e.g. increased cell
proliferation in the telencephalon) and others being specific to one of the two treatments (e.g.
increased number of dopaminergic neurons in the caudal hypothalamus in individuals treated
with CLO). Given the opposite property of these two molecules to interfere with aromatase,
more contrasting effects may have been expected, with specific behavioral and cellular patterns
specific to each of the substances. Our results strongly suggest that behavioral and cellular
effects of EE2 and CLO probably result from a much more complex mechanism than a single
modulation of aromatase activity. Indeed, the effect of a compound will depend on the context
of exposure. In our experiments, it is important to mention that effects of CLO on neural
proliferation and differentiation were identified at a concentration that interfere with AroB
activity but is above environmental inputs estimated at 1-100 ng/L (Assress et al., 2020), while
EE2 effects on neurophysiology and behavior were observed at environmentally relevant
concentrations (Klaic and Jirsa, 2022). These concentrations of EDCs were based on their
ability to influence aromatase expression (Blanc-Legendre et al, 2023). However, subsequent
effects on enzyme activity and local concentrations of metabolites, as substrate(s) for
aromatase, were not analyzed. Moreover, endocrine disrupting chemicals often affect several
pathways, interfering with each other and therefore leading to effects resulting from these
complex interferences (Moens et al., 2006; Villeneuve et al., 2009). As mentioned above, EE2
is a strong ER agonist. It is therefore possible that EE2 effects result from *cyp19a1b* gene
induction in radial glial cells but also from a direct action on other cell targets. As for CLO, it
was shown to activate pregnane X receptor (PXR), a ligand-dependent transcription factor that
is functionally expressed in the brain of adult zebrafish (Bainy et al., 2013) and shown to be
actively transcribed in embryos in the telencephalon and diencephalon from the 36th hour of
development (Bertrand et al., 2007). Thus, specific effects of CLO could result from the
modulation of aromatase and/or from the direct activation of the PXR signaling cascade.

## Conclusion

The present work shows that a 5-day developmental exposure to EE2 or CLO has persistent effects on behavior and neurophysiology in zebrafish. Effects were dependent on the treatment, however not as contrasted as initially hypothesized. The results support that EE2 exposure promotes neurogenesis in the dorsal telencephalon that could partly be linked to the observed hyperactive behavior associated with reduced sociability in EE2-exposed male fish. Besides, an increase in dopaminergic neurons, possibly involved in a range of swimming behaviors, was significant in the caudal hypothalamus after CLO treatment only. Changes in behaviors observed after an early exposure to EDCs such as hyperactivity and decreased sociability can negatively affect long-life individual fitness and social hierarchies. Considering that the concentration of EE2 that was used can be found in surface waters across the globe, these results emphasize the risk caused by environmental estrogenic EDCs on neurodevelopmental disorders in vertebrates.

## Funding

This work was supported by the French National Research Agency (FEATS project; ANR-19-CE34-0005-05).

## Ethical statement

This work has been performed in agreement with the Directive 2010/63/EU, and received specific approval of the Ministry of Research under the project authorization number APAFIS
#32568-2021072622109117 v3 Animal experiments were performed at MARBEC Palavas
Experimental Marine Platform which has agreement D34121926 for animal experiments.

CRediT authorship contribution statement

**Mélanie Blanc-Legendre** : Methodology, Investigation, Data curation, Formal analysis, Writing - original draft, Visualization. **Laetitia Guillot-Cloarec** : Methodology, Investigation, Data curation, Formal analysis, Writing - original draft, Visualization. **Léa Chevalier** : Investigation. **Cassandra Malleret** : Methodology, Investigation, Writing - review & editing.

**Karine Le Menach** : Investigation. **Patrick Pardon** : Investigation. **Hélène Budzinski** : Investigation. **François Brion** : Funding acquisition, Conceptualization. **Sacha Sire** : Investigation. **Pascal Coumailleau** : Methodology, Writing – review & editing. **Thierry. D. Charlier** : Funding acquisition, Conceptualization, Methodology, Writing – review & editing.

**Elisabeth Pellegrini** : Conceptualization, Methodology, Investigation, Writing – review & editing, Supervision. **Xavier Cousin** : Funding acquisition, Conceptualization, Methodology, Investigation, Writing – review & editing, Supervision.

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

The authors thank Stéphane Lallement for his help with fish maintenance. Chemical analyses
were performed using the technical facilities from the PLATINE mass spectrometry platform
at UMR 5805 EPOC. Alexis Chazeaubeny is acknowledged for technical support on chemical
analyses.
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**Declaration of interests**

☐ 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.

☑ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Cousin Xavier reports financial support was provided by French National Institute for Agricultural Research INRAE. Charlier Thierry reports financial support was provided by Research Institute for Environmental and Occupational Health. Budzinski Helene reports financial support was provided by University of Bordeaux. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Highlights**

* 17α-ethinylestradiol (EE2) and clotrimazole are two EDCs capable of interfering with brain aromatase activity.

* We evaluated the long-term effects of early exposure of zebrafish to EE2 or clotrimazole.

* EE2 induces hyperactivity and decreases social behavior in adult male fish.

* EE2 and clotrimazole increase neural cells proliferation in the dorsal telencephalon.

* Underlying mechanisms appear to rely on complex interactions.