# Endocrine effects of Imazalil on aromatase expression, vitellogenesis and ovarian histology using cyp19a1a-eGFP-casper transgenic zebrafish

Julie de Oliveira, Tu-Ky Ly, Edith Chadili, Violette Thermes, Antoine Mergot, Benjamin Piccini, Olivier Palluel, Hélène Budzinski, Karyn Le Menach, Patrick Pardon, et al.

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## To cite this version:

Julie de Oliveira, Tu-Ky Ly, Edith Chadili, Violette Thermes, Antoine Mergot, et al. Endocrine effects of Imazalil on aromatase expression, vitellogenesis and ovarian histology using cyp19a1a-eGFP-casper transgenic zebrafish. Aquatic Toxicology, 2025, 289, pp.107580. 10.1016/j.aquatox.2025.107580. ineris-05326837

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https://ineris.hal.science/ineris-05326837v1

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# Endocrine effects of Imazalil on aromatase expression, vitellogenesis and ovarian histology using *cyp19a1a-eGFP-casper* transgenic zebrafish

Julie De Oliveira<sup>a</sup>, Tu-Ky Ly<sup>a,b,c</sup>, Edith Chadili<sup>a</sup>, Violette Thermes<sup>d</sup>, Antoine Mergot<sup>d</sup>, Benjamin Piccini<sup>a</sup>, Olivier Palluel<sup>a</sup>, Hélène Budzinski<sup>e</sup>, Karyn Le Menach<sup>e</sup>, Patrick Pardon<sup>e</sup>, Rémy Beaudouin<sup>b,c</sup>, Xavier Cousin<sup>f</sup>, François Brion<sup>a</sup>, Nathalie Hinfray<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

transgenic zebrafish

aromatase

imazalil

endocrine disrupting chemicals

OECD TG230

## ABSTRACT

Considering the hazards and risks posed by endocrine disrupting chemicals (EDC) to organisms, there is a need to study their effects. To that end, transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, we used a newly developed transgenic zebrafish line (*cyp19a1a-eGFP-casper*) in the OECD 21-day fish assay (OECD TG 230) to provide additional mechanistic insight on Imazalil (IMZ; 1.9; 9.9 and 140.7 µg/L). After 21 days of exposure to IMZ, the circulating concentrations of 17-β-estradiol (E2) and vitellogenin decreased in females, reflecting the aromatase activities inhibition. Exposure to 140.7 µg/L of IMZ for 21 days also resulted in a change in the proportion of the different oocyte stages in the ovaries, with an accumulation of large oocytes in exposed females. In addition to the classical endpoints, *in vivo* GFP fluorescence was quantified in the ovaries during the time course of the exposure to follow gonadal aromatase expression. After seven days of exposure, ovarian aromatase expression increased in females exposed to medium and high concentrations of IMZ, persisting over the 21-day of exposure in fish from the highest concentration group and reflecting a compensatory response to the aromatase enzymatic activities inhibition. Results from the present study provided valuable information on the mode of action and the effects of IMZ in zebrafish. Transgenic zebrafish exposure to IMZ caused a cascade of responses consistent with effects reported for wild-type fish exposed to azole fungicides, both qualitatively and quantitatively. The *cyp19a1a-eGFP*-(*casper*) transgenic zebrafish lines, allowed *in vivo* monitoring of gonadal aromatase expression in a time- and concentration-dependent manner thereby demonstrating their relevance to provide complementary mechanistic information on aromatase in regulatory assays such as OECD TG 230.

## 1. Introduction

With the appearance of the endocrine disrupting chemicals (EDC) concept in the early 1990s, the scientific community's interest in this concern steadily increased. Due to their widespread origins, many EDC eventually end up in the aquatic environment via wastewater or water runoff and negatively interfere with the endocrine system of non-target aquatic organisms. In fish, exposure to EDC has been associated to reproductive disorders both at individual and population levels, such as

disturbed sex steroid concentrations, abnormal vitellogenin synthesis or skewed sex-ratios (*Jobling et al.*, 2002; *Noaksson et al.*, 2003; *Nash et al.*, 2004; *Kidd et al.*, 2007; *Sanchez et al.*, 2011; *Tetreault et al.*, 2011). At least in part, these adverse effects on fish reproduction may be a consequence of the disruption of steroidogenesis, a physiological process supported by several enzymes; and especially gonadal aromatase, namely Cyp19a1a, which is responsible for the synthesis of estrogens, key players of reproductive processes (*Ankley et al.*, 2002; *Villeneuve et al.*, 2007; *Villeneuve 2016*, *De Oliveira et al.*, 2020).

<footnote><sup>a</sup> INERIS, Ecotoxicology of substances and environment unit, Verneuil-en-Halatte, France</footnote>

<footnote><sup>b</sup> INERIS, Experimental toxicology and modeling unit, Verneuil-en-Halatte, France</footnote>

<footnote><sup>c</sup> INERIS, Université de Reims Champagne-Ardenne, Université Le Havre Normandie, Normandie Univ, UMR-I 02 SEBIO, 60550 Verneuil-en-Halatte, France</footnote>

<footnote><sup>d</sup> INRAE, Fish Physiology and Genomics Institute, Rennes, France</footnote>

<footnote><sup>e</sup> University of Bordeaux, LPTC, UMR EPOC, Bordeaux, France</footnote>

<footnote><sup>f</sup> MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, INRAE, Palavas, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: nathalie.hinfray@ineris.fr (N. Hinfray).</footnote>
Considering the hazards and risks EDC represent to human and aquatic organisms, there is a need to study their effects and to develop as well as to implement tests to account for the endocrine disrupting activity of chemicals. The Organization for Economic Cooperation and Development (OECD) has published a conceptual framework referencing the existing tests to that end (OECD 2018). In fish, two assays, the fish short term reproduction assay (TG 229) and the 21-days fish assay (TG 230), were designed to identify estrogenic, androgenic and aromatase inhibiting substances, using one of the three recommended fish species, i.e. fathead minnow (*Pimephales promelas*), medaka (*Oryzias latipes*) or zebrafish (*Danio rerio*) (OECD 2009b). In these test guidelines, at the end of the 21 days exposure period, the study of secondary sexual characteristics allows the detection of androgenic compounds (only in fathead minnow and medaka) and induction of vitellogenin (VTG), a protein produced by the liver under estrogen regulation, informs on the estrogenic potency of compounds. While aromatase inhibiting substances are highlighted indirectly by inhibitions of circulating VTG concentrations after 21 days, no direct measurement of gonadal aromatase expression/activity is performed to ensure that VTG inhibition observed are not the consequences of other mechanisms of actions such as androgenic or anti-estrogenic activity or liver toxicity. Thus, new models informing on the mode of action of chemicals should be developed to bring complementary information on gonadal aromatase in these 21-days exposure fish assays. To that end, transgenic fish expressing a reporter gene under the control of the gonadal aromatase promoter appear to be helpful and relevant biological models to provide additional information on the mode of action of chemicals on gonadal aromatase expression. In a previous study, we demonstrated the suitability of a transgenic *cyp19a1a-eGFP* zebrafish line to perform an OECD TG 229 assay with prochloraz, an azole fungicide often used as a model aromatase inhibiting compound. By following, time- and concentration-dependent changes in *in vivo* GFP fluorescence in ovaries, we provided novel mechanistic information on prochloraz effects (De Oliveira *et al.*, 2020). To further improve sensitivity of this assay, in the present study, we used a *cyp19a1a-eGFP* in a *Casper* genetic background. This background contains mutations in *nacre* and *roy* loci resulting in the absence of pigmentation in the fish (White *et al.*, 2008). We previously showed that there are no significant differences between these transgenic zebrafish and wild type zebrafish on different reproductive parameters including circulating VTG concentrations (De Oliveira *et al.*, 2021).

Azoles are antifungal chemicals used both in agriculture and medicine. They can also interact with numerous cytochrome enzymes in non-target organisms, including in vertebrate, and disrupt hormone synthesis and signaling (Draskau and Svingen 2022). Imazalil (IMZ) is an imidazole fungicide widely used to protect vegetable and fruit plantations against rot or in post-harvest treatments to increase the shelf life of products on the market. Residues of IMZ have been found in vegetables, fruits and fruit juices (few ng/g up to few µg/g); water (surface, ground and waste waters at concentrations ranging from several ng/L to a couple of µg/L) and aquatic organisms such as fish (several ng/g) (Castillo et al., 2000; Nougadere et al., 2012; Belenguer et al., 2014; Ccanccapa et al., 2016a; Ccanccapa et al., 2016b; Pascual Aguilar et al., 2017, OECD 2018, Pérez-Villanueva et al., 2022, Radulovic et al., 2023, Basij et al., 2025). Like most other azole fungicides, IMZ is a known inhibitor of fish aromatase activities in vitro (Monod et al., 1993; Hinfray et al., 2006). But only few information is available, especially on its endocrine disrupting potency towards fish. In female fathead minnow, IMZ inhibitory effect on aromatase activity led to a reduction in plasma 17β-estradiol (E2), vitellogenin (VTG) concentrations and reproductive output (Villeneuve et al., 2021, Villeneuve et al., 2023).

In that context, the aim of this study was to evaluate the relevance of using the *cyp19a1a-eGFP-casper* transgenic zebrafish line in regulatory assays and to acquire more information on IMZ endocrine effects in fish. A TG 230 assay was carried out with 3 concentrations of IMZ (3, 30, 300 µg/L nominal) followed by the measurement of the TG 230 endpoint

final circulating VTG concentration, enriched by the acquisition of additional parameters (time-dependent recording of gonadal aromatase expression (*in vivo* GFP fluorescence imaging); circulating steroid concentrations; gene expressions, histological analysis of ovaries).

## 2. Materials and methods

All experiments with animals were performed in accordance with Directive 2010/63/EU and approved by the French Animal care and experiment committee under the official license n°11177.

### 2.1. Chemical analysis

Water (about 10 ml) was sampled in the aquaria (2 aquaria per exposure condition) at 24, 48h and 7, 14 and 21 days of exposure; and stocked in amber glass bottles at -20°C until chemical analysis. Determination of IMZ concentrations in water was performed by LC/MS/MS as described in (Ly et al., 2025).

### 2.2. Transgenic zebrafish exposure to Imazalil

In this study, we used the *cyp19a1a-eGFP-casper* transgenic zebrafish line (De Oliveira et al., 2021). The *cyp19a1a-eGFP-casper* expresses an enhanced Green Fluorescent Protein (eGFP) under the control of the zebrafish ovarian aromatase promoter (*cyp19a1a*). This strain results from the crossbreeding of the transparent double-mutant zebrafish named casper (White et al., 2008) and the transgenic *cyp19a1a-eGFP* zebrafish (Hinfray et al., 2018).

Mature transgenic zebrafish *cyp19a1a-eGFP-casper* (5–6 months old) were produced from our breeding stock at INERIS (Institut National de l'Environnement Industriel et des Risques, Verneuil-en-Halatte, France). 120 zebrafish (5 males and 5 females/aquarium) were randomly distributed in a continuous flow-through system containing 12 aquariums of 8 liters (3 aquariums per exposure condition). Each aquarium was equipped with an aeration system. The continuous flow-through system allows a permanent renewal of the water in the aquariums by gravity (6 times a day). Following water parameters were monitored throughout the experiment: temperature (27.3 ± 0.1°C), pH (7.7 ± 0.2), dissolved oxygen (94.2 ± 3.7%), conductivity (346.5 ± 28.8 µS/cm), ammonia (not detectable), nitrite (< 0.025) and nitrate (not detectable). Photoperiod was set at 14 h / 10 h light/dark. This study protocol was based on OECD 230 guideline (OECD 2009b). After a 7-day acclimation period, fish were exposed to solvent alone (DMSO, 0.01%; Sigma-Aldrich, Lyon, France, CAS 67-68-5, purity 99.5%) or three nominal concentrations of IMZ (3, 30 and 300 µg/L, nominal) for 21 days (Sigma-Aldrich, CAS 35554-44-0, purity 98%). Stock solutions were prepared every week in amber bottles and protected from the light during the exposure. The fish were fed with flake food (SDS400, Special Diets Services, Augy, France) in the morning and with newly hatched brine shrimp (Sep-Art, Ocean Nutrition Europe, Essen, Belgium) in the afternoon.

### 2.3. *In vivo* monitoring of gonadal GFP fluorescence

*In vivo* monitoring of gonadal GFP fluorescence was carried out at 0 (T0), 7 (T7), 14 (T14) and 21 days (T21) of exposure following the same protocol as presented in De Oliveira et al. (2020). Briefly, female zebrafish were anaesthetized in MS222 (200 mg/L) (Sigma-Aldrich, CAS 886-86-2) and photographed in a fluorescence camera system (IVIS Lumina II fluorescence imaging system, Perkin Elmer, Courtaboeuf, France) in both bright field and fluorescence modes. The fluorescence pictures, all taken with the same settings, were subjected to spectral unmixing using the Living Image software (Caliper Life Sciences, Hopkinton, Massachusetts, USA). GFP quantification was expressed as the Total Radiant Efficiency (number of photons per second normalized by the excitation light intensity, [p/s]/[µW/cm²]) divided by the gonad
area (cm²) and normalized to the mean value measured at T0 in the same group of fish to obtain fold-inductions. After imaging, each fish is placed in a tank with clean water, separated from the other fish, for about 10 min to ensure complete recovery, before returning to the original aquarium.

## 2.4. Samples collection

After 21 days of exposure, fish were anaesthetized with MS222 (200 mg/L) to measure the gonadal GFP fluorescence as described previously and then euthanized with ice-cold water (Wilson et al., 2009). Each fish was then measured, weighted, and 5 to 10 µl of blood were sampled by cardiac puncture and transferred to Microvette® pre-heparinized tubes (Sarstedt, Nümbrecht, Germany) containing a phosphate buffered saline buffer (PBS 1X, glycerol (10%) and PMSF (0.2 mM) at a ratio of 1:10 v:v) and stored at -80°C for subsequent circulating steroids and VTG concentrations analysis. Liver and gonads were also sampled and weighted to calculate gonado-somatic index (GSI= gonad weight [mg] / body weight [mg] x 100) and hepato-somatic index (HSI = liver weight [mg] / body weight [mg] x 100). The liver and one gonad of each fish were stored in RNAlater® (Sigma-Aldrich) at 4°C overnight and then at -80°C for subsequent gene expression analysis. The remaining gonads were fixed in modified Davidson's fixative (22% v/v formaldehyde 37-40%; 11.5% v/v glacial acetic acid; 33% v/v ethyl alcohol 95%; 33.5% v/v distilled water) for 48 h at room temperature and then stored at 4°C in ethanol 70% until histological processing.

## 2.5. Gene expression analysis

RNA isolation, complementary DNA synthesis and qPCR were performed as described in De Oliveira et al. (2020). The only modification was the hybridization/elongation temperature which was set at 60°C for esr1, esr2a, esr2b, fshr and lhcggr genes. All primers and probes used in this study (cyp19a1a, esr1, esr2a, esr2b, fshr, lhr) are summarized in the Table 1.

## 2.6. Vitellogenin ELISA

Analysis of circulating VTG concentrations were performed on whole blood. The technique of indirect competitive ELISA using a liquid handling robot Freedom EVO® (Tecan, Männedorf, Switzerland) was

<table><caption>**Table 1**
Summary of the primers used for the qPCR.</caption><thead><tr><th>Gene Name</th><th>Gene ID</th><th>Primers</th><th>probe</th><th>ref</th></tr></thead><tbody><tr><td>cyp19a1a</td><td>30390</td><td>forward primer<br>5'-atcagacaccatcgaccac-<br>3',<br>reverse primer<br>5'-acaacaggacccagaccaaa-<br>3'</td><td>probe #85 (Roche<br>LifeScience, cat n°<br>04689097001)<br>from the universal<br>probe library</td><td>(De<br>Oliveira<br>et al.,<br>2020)</td></tr><tr><td>esr1</td><td>259252</td><td>TaqMan® Gene Expression assay®<br>Dr03093579_m1</td><td></td><td>(Elersek<br>et al.,<br>2021)</td></tr><tr><td>esr2a</td><td>317734</td><td>TaqMan® Gene Expression assay®<br>Dr03074408_m1</td><td></td><td>/</td></tr><tr><td>esr2b</td><td>317733</td><td>TaqMan® Gene Expression assay®<br>Dr03150586_m1</td><td></td><td>(Elersek<br>et al.,<br>2021)</td></tr><tr><td>fshr</td><td>195820</td><td>TaqMan® Gene Expression assay®<br>Dr03425643_m1</td><td></td><td>/</td></tr><tr><td>lhcggr</td><td>402920</td><td>TaqMan® Gene Expression assay®<br>Dr03203190_s1</td><td></td><td>/</td></tr></tbody></table>

employed, as described in our previous study (De Oliveira et al., 2021).

## 2.7. Circulating steroid concentrations analysis

After VTG analysis, remaining blood was used for steroid measurements. For females blood samples, 100 µL of ultrapure water was added to each sample to allow extraction of steroids using an OASIS® HLB plate (µElution Plate 30 µm, Waters SAS, Saint-Quentin-en-Yveline, France) with a vacuum SPE system (Extraction plate manifold for Oasis 96-well plates, Waters SAS) as described by De Oliveira et al. (2020). Extracted steroids were pooled (n=3 individuals/pool; n = 5 pools/conditions) and resuspended in 100 µL PBS. E2 was quantified in accordance with the manufacturer's instructions using the E2 immunoassay kit (Cayman Chemical Company, Ann Arbor, Michigan, USA).

For males blood samples, 150 µL of ultrapure water was added to dilute samples and no extraction was realized as described in (De Oliveira et al., 2021). In accordance with the manufacturer's instructions, 11-ketotestosterone (11KT) concentrations were quantified individually in males blood using the 11KT immunoassay kit (Cayman Chemical Company).

## 2.8. Histological analyses

After fixation in modified Davidson's fixative, ovaries were dehydrated in ethanol, cleared in isopropanol and finally, embedded in paraffin. To explore the effects of IMZ on oogenesis, paraffin sections (5-7 µm) were performed, only for DMSO and the highest IMZ concentration groups, on nine females' gonads per condition (three per replicate) and analysed using both classical and computerized histological analyses.

### 2.8.1. Classical histological processing and analysis

Sections were performed at four different levels into the gonad, separated at least by 500 µm and stained with hematoxylin-eosin. One section at each level was analyzed for every individual. All the oocytes of the section were counted and sorted following a simplified version of Selman's classification (Selman et al., 1993): pre-vitellogenic oocyte (PO, d=7-140 µm), cortical alveoli (CA, d=140-340 µm), vitellogenic and mature oocytes (VO/MO, d=340-750 µm), post-spawning follicle (PSF) and atretic oocyte (AO). In each individual, the number of oocytes of the four sections analyzed were added for each category of oocytes. Final data are presented as mean ± SEM of the nine individuals analyzed in each exposure condition.

### 2.8.2. Computerized histological analysis

Sections were performed at three different levels into the ovary, separated at least by 500 µm. One or two sections at each level was analyzed for every individual. Sections were stained with the Methyl Green dye (MG) (40 µg/ml, 323829, Sigma-Aldrich) in PBS at room temperature for 15 min in the dark. Whole-section images were acquired with a slide scanning nanozoomer (HAMAMATSU, Shizuoka, Japan) on the H2P2 facility of the University of Rennes. Images were processed automatically using deep learning-based tools in an appropriate workflow adapted from Lesage et al. (2023). Images were first automatically denoised using the Noise2Void Fiji plug-in (n2v-0.8.6) (Krull et al., 2019) with a specifically trained model. Next, images were segmented using Cellpose (v0.6.1) with the cyto pre-trained model (Stringer et al., 2021). The masks obtained were then post-treated for filtration, and for individual oocyte and surface area measurements. The segmentation quality was visually validated for each image. No manual corrections were made. All steps were performed using the open-source FIJI software (version 2.9.0/1.53t) (Schindelin et al., 2012) with the exception of Cellpose segmentation, which was run with the Anaconda3-2021.11 distribution of python.

<footnote>* TaqMan® Gene Expression assays are predesigned assays containing both primers and probe and developed by ThermoFisher scientific</footnote>
## 2.9. Data analysis

Normality of data and homogeneity of variances were verified using Shapiro-Wilk's and Bartlett's tests. Non-normal data were analyzed with a Kruskall-Wallis test followed by a Dunn's test, to determine differences between groups. For normal data, an analysis of variance (ANOVA) followed by a Post-Hoc test (Tukey's test) was performed. Different letters on the graphs represent significant differences, as explained in the captions of each Fig.. Data were expressed as means ± standard error (SD) or standard error of the mean (SEM) as indicated in the legend of each Fig. and table.

For computerized histological analysis, distribution of the logarithm of the oocyte size was analyzed using linear mixed-effects models using the lmer function in the lme4 package for R cran (R Core Team, 2022). Concentration of IMZ was considered as a fixed effect; tank and individual fish were considered as random effects; fish being nested in tank. Model used was: oocyte.size ~ Conc + (Conc | Tank) + (Conc | Tank: Fish)). Frequency (probability densities) of the oocyte diameter for each individual was computed using 40 classes from 10 to 800 µm on the cumulated data of the different sections.

# 3. Results

## 3.1. IMZ concentrations in water

Water was sampled from aquaria of each condition of exposure. The actual concentration of IMZ in DMSO control aquaria was below the limit of quantification (LQ of 0.002 µg/L). In the IMZ exposed groups, the actual concentrations were 1.9 ± 0.5; 9.9 ± 1.6 and 140.7 ± 26 µg/L (mean ± SD) for the nominal concentrations of 3, 30 and 300 µg/L, respectively.

## 3.2. Fish biometrical parameters

No mortality was observed neither during the acclimation period nor during the exposure period. Whatever the condition of exposure, no significant difference was observed on body length, weight, gonads weight, GSI and HSI of females and males. (Table 2).

## 3.3. *In vivo* monitoring of gonadal GFP fluorescence, expression of gonadal aromatase and circulating steroid concentrations

In the *cyp19a1a-eGFP-casper* transgenic line, like in the *cyp19a1a-eGFP* line, gonadal GFP fluorescence is only detected in females

<caption>**Table 2**</caption>

**Biometrical parameters of solvent (DMSO 0.01%) and IMZ-exposed zebrafish.** GSI: gonadosomatic index. HSI: hepato-somatic index. IMZ: imazalil. Data are means ± SD (n = 14 - 15).

<table><thead><tr><th>Sexes</th><th>Parameters</th><th>DMSO</th><th>IMZ 1.9 µg/L</th><th>IMZ 9.9 µg/L</th><th>IMZ 140.7 µg/L</th></tr></thead><tbody><tr><td rowspan="7">Females</td><td>Body length (mm)</td><td>34.2 ± 1.5</td><td>35.3 ± 2.3</td><td>35.1 ± 1.7</td><td>35.9 ± 1.9</td></tr><tr><td>Weight (mg)</td><td>404.8 ± 73.4</td><td>448.4 ± 90.0</td><td>424.0 ± 81.5</td><td>454.0 ± 76.9</td></tr><tr><td>Gonads Weight (mg)</td><td>52.0 ± 27.8</td><td>61.0 ± 29.4</td><td>53.6 ± 26.9</td><td>58.8 ± 15.4</td></tr><tr><td>GSI (%)</td><td>12.6 ± 5.1</td><td>13.2 ± 4.6</td><td>12.3 ± 4.6</td><td>13.2 ± 2.7</td></tr><tr><td>HSI (%)</td><td>2.8 ± 0.7</td><td>2.9 ± 0.6</td><td>2.7 ± 1.2</td><td>2.4 ± 0.7</td></tr><tr><td>Body length (mm)</td><td>35.5 ± 0.8</td><td>35.3 ± 1.0</td><td>36.3 ± 1.1</td><td>34.9 ± 1.3</td></tr><tr><td>Weight (mg)</td><td>416.2 ± 34.8</td><td>396.0 ± 39.7</td><td>415.6 ± 40.0</td><td>375.5 ± 31.1</td></tr><tr><td rowspan="3">Males</td><td>Gonads Weight (mg)</td><td>6.1 ± 1.7</td><td>5.3 ± 2.1</td><td>5.7 ± 1.7</td><td>6.0 ± 2.9</td></tr><tr><td>GSI (%)</td><td>1.2 ± 0.6</td><td>1.2 ± 0.5</td><td>1.3 ± 0.4</td><td>1.6 ± 0.8</td></tr><tr><td>HSI (%)</td><td>1.5 ± 0.4</td><td>1.2 ± 0.5</td><td>1.3 ± 0.4</td><td>1.2 ± 0.4</td></tr></tbody></table>

(Fig. 1A), while GFP levels in male gonads were below detection limits. Thus, in the present study, gonadal GFP fluorescence levels were quantified *in vivo* every seven days during the exposure period solely in *cyp19a1a-eGFP-casper* females (Fig. 1B).

No significant variations were shown between T0 and T21 for DMSO-exposed females and the low concentration of IMZ (Fig. 1B). For medium and high concentrations of IMZ, *in vivo* GFP fluorescence in ovaries was significantly induced from T7 compared to their own T0. This significant induction persisted at T14 and T21 in females exposed to the highest concentration but was no longer significant in females exposed to the medium concentration (Fig. 1B). Comparison of *in vivo* gonadal GFP fluorescence between DMSO and IMZ groups at T21 revealed a significant induction in females exposed the highest concentration of IMZ (Fig. 1B). This result was confirmed by qPCR at T21 (Fig. 1C). qPCR results also showed a significant induction of *cyp19a1a* gene expression in ovaries of fish exposed to the medium concentration compared to the DMSO group (Fig. 1C).

At the end of the experiment, circulating steroids (11KT in males and E2 in females) concentrations were quantified in fish blood samples. In fish exposed to 140.7 µg/L of IMZ, a slight decrease (42%) was observed in circulating E2 concentrations compared to DMSO (Fig. 1D). In males, no significant differences were observed between DMSO and exposed fish for 11KT (Fig. 1E).

## 3.4. Circulating VTG concentrations

Circulating VTG concentrations were quantified in females and males fish blood samples for each condition at the end of the exposure period. In males, no significant difference was observed whatever the IMZ concentration (data not shown). In females, a concentration-dependent decrease of VTG levels was observed in fish exposed to IMZ compared to DMSO (Fig. 2).

## 3.5. Histological analysis

Computerized histological analysis of the ovaries showed that exposure to the highest concentration of IMZ (140.7 µg/L) led to alterations in the frequency of the different oocyte sizes compared to the DMSO females. After 21 days of exposure, a decrease of the small sizes oocytes and an increase in large sizes oocytes frequencies were observed in the ovaries of IMZ-exposed females (Fig. 3A,B). Classical histological analysis confirmed the computerized histological analysis, showing reduced proportions of pre-vitellogenic oocytes and post-spawning follicles, but an increased proportion of vitellogenic/mature oocytes and of atretic oocytes (from 0.1 to 0.7%) in IMZ-exposed females ovaries (Fig. 3C,D). Our results suggest that computerized histological analysis could be a relevant user-independent approach in the evaluation of chemical histological effects.

## 3.6. Estrogen and gonadotropins receptors genes expressions

In addition to ovarian aromatase, the expression of other genes of interest was studied: *esr1*, *esr2a* and *esr2b* in liver; and *lhcgr* and *fshr* in gonads.

In males, *lhcgr* and *fshr* expressions in the testes as well as *esr1*, *esr2a* and *esr2b* in the liver, were not disturbed by IMZ, regardless of the concentration (data not shown). In female livers, no significant modulation of *esr1*, *esr2a* and *esr2b* genes expressions was noticed (Fig. 4A). In female gonads, only *fshr* expression was induced in fish exposed to the highest concentration of IMZ after 21 days of exposure while no effect on *lhcgr* was highlighted (Fig. 4B).

# 4. Discussion

While the endocrine disrupting effects of different azole fungicides have been well studied in fish, with prochloraz and fadrozole often used
A

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

B

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

C

<figure><img src="image_6.png" /></figure>

D

<figure><img src="image_7.png" /></figure>

E

<figure><img src="image_8.png" /></figure>

**Fig. 1.** Effects of exposure to Imazalil on *in vivo* ovarian GFP fluorescence and steroid concentrations in *cyp19a1a-eGFP-casper* zebrafish. (A) Left: Picture of *in vivo* fluorescence of gonadal GFP in a female from the DMSO control group. Autofluorescence is in red and GFP fluorescence in green. Right: brightfield picture of the same female fish as in A. Contrast and luminosity have been modified to ensure a proper visibility of the organs. Head of the fish is on the right and tail on the left. Ov: ovary, in: intestine (B) Fold induction of *in vivo* gonadal GFP fluorescence in *cyp19a1a-eGFP-casper* transgenic female zebrafish exposed to DMSO or IMZ over 21 days, measured each week and compared to their own T0. n = 13–15. (C) Fold induction of *cyp19a1a* gene expression in ovaries of female fish exposed to IMZ for 21 days. Measurements were performed by qPCR. n = 13–15 for each condition. (D) Estradiol concentrations in the blood of females (n = 4-5 pools of 3 females per exposure condition). (E) 11-Ketotestosterone (11KT) concentrations in the blood of males (n = 5-9 individuals per exposure condition). DMSO: control groups. Data are means ± SEM. Significant differences are represented by different letters between times of exposure for each condition (B) or different exposure conditions (C, D, E) (p < 0.05).
<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Effects of a 21-days exposure to Imazalil on circulating vitellogenin (VTG) concentrations in *cyp19a1a-eGFP-casper* zebrafish females. DMSO: control groups. Data are means ± SEM (n = 11-15 individuals per exposure condition). Significant differences are represented by different letters (p < 0.05).</figcaption></figure>

for reglementary assays (OECD 2009a, OECD 2009b), data on the effects
of IMZ are very scarce. Recently, endocrine disrupting effects of IMZ
were assessed in fathead minnows and inhibitory effects on aromatase
activity leading to reductions in plasma E2 concentrations, VTG con-
centrations, and reproductive output were reported (Villeneuve et al.,
2023). In the present study, cyp19a1a-eGFP-casper transgenic zebrafish
were exposed to IMZ with two aims: i) to inform on the endocrine dis-
rupting effects of IMZ in fish, using OECD TG 230, ii) to evaluate the
relevance of the cyp19a1a-eGFP-casper transgenic zebrafish line to bring
complementary information on gonadal aromatase time course expres-
sion without increasing the number of fish needed for this reglementary
assay.

Endocrine effects of IMZ

At the end of the 21-days exposure to IMZ, the classical endpoint of
the OECD TG230, VTG, was measured in *cyp19a1a-eGFP-casper* females.
A concentration-dependent decrease in circulating VTG concentrations
was observed in exposed females, with significant differences at the
medium (9.9 µg/L) and highest (140 µg/L) concentrations of IMZ
compared to controls. A significant decrease (42%) in circulating E2
concentrations was also measured in females exposed to the highest IMZ
concentration. These results are consistent with the recently reported
effects of IMZ in fathead minnows (Villeneuve *et al.*, 2023). And, albeit

A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /><figcaption>**Fig. 3.** Effects of a 21-days exposure to 140.7 µg/L of Imazalil (IMZ) on ovarian histology in *cyp19a1a-egFP-casper* transgenic zebrafish. (A) Frequency of the different classes of oocyte sizes. N=8-9 individuals per exposure condition. Medians of probability densities are plotted (histogram has a total area of one). Grey zones represent the 95% confidence intervals (Light grey and dark grey for DMSO and IMZ groups respectively). 40 regular classes were used to compute the frequency of the oocyte diameter. (B) Representative sections of ovaries of females from control group (DMSO) and IMZ-exposed group, used for frequency analysis. (Left) Sections stained with methyl green and scanned with a nanozoomer. Contrast and luminosity have been modified. (Right) Overlay of ovarian sections and cellpose masks. Scale bars: 500µm (C) Percentage of the different types of oocytes in the ovaries. DMSO: control group. PO: pre-vitelogenic oocytes, CA: cortical alveoli, VO/MO: vitellogenic and mature oocytes, PSF: post-spawning follicle and AO: atretic oocytes. Data are means ± SD (n = 9 individuals per exposure condition). * represents statistically significant differences (p < 0.05). (D) Representative histological sections of ovaries of females from control group (DMSO) and IMZ-exposed group. Haematoxilin-eosin staining. Scale bars: 200 µm.</figcaption></figure>

C

<figure><img src="image_12.png" /></figure>

D

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>Fig. 4. Effects of a 21-days exposure to Imazalil on different genes in *cyp19a1a-eGFP-casper* zebrafish female livers (A) and gonads (B). (A) Expression of estrogen receptors genes in liver. (B) Expression of *fhsr* and *lhcgr* genes in the ovaries. Data are means ± SEM. (n = 10 -15 individuals per exposure condition). Significant differences are represented by different letters (p < 0.05).</figcaption></figure>

aromatase activity inhibition was not directly demonstrated in the present study, the observed effects aligns with the ability of IMZ to inhibit *in vitro* aromatase activities (Hinfray et al., 2006) and potentially other cytochrome P450 enzymes activities (USEPA, 2021). By inhibiting aromatase activities, IMZ exposure led to a decrease in circulating E2 concentrations followed by a reduction in VTG synthesis, a suite of responses consistent with the already reported effects of azole fungicides in different fish species (Sun et al., 2007, Ankley et al., 2009, Skolness et al., 2011, Skolness et al., 2013, Dang et al., 2016, De Oliveira et al., 2020, Villeneuve et al., 2021) and the adverse outcome pathway (AOP) described for aromatase inhibition in fish (Villeneuve 2016, Doering et al., 2019, Ly et al., 2025).

The *cyp19a1a-eGFP-casper* transgenic zebrafish line also enables *in vivo* quantification of *cyp19a1a* expression in females by measuring the dynamic of ovarian GFP fluorescence throughout the exposure period. After seven days of exposure, ovarian GFP fluorescence increased in females exposed to medium and high concentrations of IMZ, persisting over the 21-day of exposure in females from the highest concentration group. These observations are consistent with the qPCR results showing increased *cyp19a1a* expression in the medium and high IMZ groups at the end of the 21-day exposure and a significant induction of *fshr* – a key receptor in the feedback regulation of steroidogenesis – at the highest concentration of IMZ. In previous studies with other azole aromatase inhibitors (i.e. propiconazole, prochloraz and letrozole), up-regulation of *cyp19a1a* and *fshr* transcripts has also been reported as a common response to aromatase activity inhibitions (Ankley et al., 2009, Liu et al., 2011, Skolness et al., 2013, Villeneuve et al., 2021). These transcripts inductions evidenced the compensatory mechanism taking place in the functioning of the Hypothalamus-Pituitary-Gonadal axis to restore E2 circulating concentrations diminished by the aromatase activity inhibitions (Ankley and Villeneuve 2015).

Another notable effect of the highest concentration of IMZ is the histological alteration of the ovaries as compared to females from the solvent control group. Exposure to the highest concentration of IMZ for 21 days resulted in a change in the proportion of the different oocyte stages in the ovaries, with a decrease of small oocytes (pre-vitellogenic oocytes) and an increase of larger oocytes (vitellogenic and mature oocytes) in IMZ-exposed females compared to the DMSO controls. In fathead minnows, exposure to IMZ reduces fecundity of females by reducing both the number of spawning events and the average number of eggs produced per female per day (Villeneuve et al., 2023), suggesting that in our study, the increase in large oocytes in exposed females was probably due to an accumulation of oocytes from reduced egg-laying

capacity. Indeed, almost no post-spawning follicles were observed in the females exposed to the highest concentration of IMZ compared to the control females.

## Relevance of the *cyp19a1a-eGFP-casper* transgenic zebrafish model

In a previous study, we showed no critical differences in the behavioral (novel tank and sociality assays) and reproductive physiology (VTG concentrations, number of eggs laid by females and viability of eggs) of the zebrafish *cyp19a1a-eGFP-casper* line compared to the wild-type AB strain under control conditions (De Oliveira et al., 2021). While not addressed in the work by De Oliveira et al. (2021), the circulating E2 levels measured in the transgenic line in the present study (around 3.5 ng/ml for control females) are consistent with concentrations reported in the literature for wild-type zebrafish ranging, non-exhaustively, from 1.5 to 4 ng/ml (Heiden et al., 2006, Ji et al., 2013, Shi et al., 2015, Peng et al., 2019, Haque et al., 2022). We also demonstrated that the *cyp19a1a-eGFP* transgenic zebrafish line responded similarly to wild-type zebrafish, both qualitatively and quantitatively, when exposed to prochloraz, another azole fungicide (De Oliveira et al., 2020).

In the present study, exposure of this novel transgenic zebrafish line, *cyp19a1a-eGFP-casper*, to various concentrations of IMZ led to effects consistent with the known mode of actions of azole fungicides in wild-type fish, namely reductions in circulating E2 and VTG concentrations in females. The biological responses observed also align with the AOP describing the biological links between aromatase inhibition and adverse effects on fish reproduction (Villeneuve 2016, Doering et al., 2019). Quantitatively, exposure of transgenic zebrafish to 140.7 µg/L IMZ resulted in a 42% inhibition of circulating E2 concentration and a 95% decrease of circulating VTG concentration, consistent with the quantitative predictions and measurements reported for wild-type zebrafish exposed to fadrozole (Doering et al., 2019). Eventually, the *cyp19a1a-eGFP-casper* transgenic zebrafish responds to aromatase activity inhibition with a sensitivity comparable to wild-type zebrafish.

This novel *cyp19a1a-eGFP-casper* transgenic line also enables the detection of EDC effects on gonadal aromatase expression in a concentration- and time-dependent manner and in a non-invasive way, thereby providing complementary mechanistic insight to the classical endpoints of the TG230 assay without increasing the number of animals needed. The detection of IMZ effects on aromatase expression as early as seven days suggests that for some substances with short-term effects during a TG230 assay, the exposure duration could be shortened. Indeed,
cyp19a1a gene expression perturbations, whether the cause or the consequence, are closely linked to aromatase activity perturbations, and therefore might be reliable endocrine indicators of potential changes in VTG levels. In comparison to the *cyp19a1a-eGFP* transgenic zebrafish line (Hinfray et al., 2018, De Oliveira et al., 2020), the *cyp19a1a-eGFP-casper* line used in this study presents an absence of pigmentation of skin allowing for precise *in vivo* determination of the ovarian area and avoiding interferences from pigments with *in vivo* GFP fluorescence measurements.

Altogether, the *cyp19a1a-eGFP-casper* transgenic zebrafish line demonstrated to be relevant tool for studying the endocrine disrupting potency of chemicals in existing regulatory assays, as it exhibits technical features allowing a concentration- and time-dependent measurement of aromatase expression *in vivo* and a sensitivity to aromatase inhibition comparable to wild-type zebrafish.

## 5. Conclusion

In the present work, originality of the approach resides with both the use of computerized histological analysis and of the transgenic *cyp19a1a-eGFP-casper* zebrafish line to study the effect of IMZ. Computerized histological analysis is an interesting user-independent analysis approach in the evaluation of chemical histological effects. Moreover, the *cyp19a1a-eGFP-casper* line proved to be a relevant mechanism-based tool to evaluate the endocrine disrupting effects of compounds, with the advantage of being devoid of pigments that could interfere with the *in vivo* fluorescence quantification. Whether in a Casper genetic background or not, the *cyp19a1a-eGFP*-(*casper*) transgenic zebrafish lines, allowed *in vivo* monitoring of gonadal aromatase expression in a time- and concentration-dependent manner thereby demonstrating their relevance to provide complementary mechanistic information on aromatase in regulatory assays such as OECD TG 230. Such approach is in line with the “refinement” of the 3Rs principles in animal experimentation as it provides more mechanistic insight without increasing animal number.

Results from the present study also provided valuable information on the mode of action and the effects of IMZ in fish. Indeed, transgenic zebrafish exposure to IMZ caused a cascade of responses physiologically linked to each other (decrease in E2 and VTG circulating concentrations, compensation mechanism on *cyp19a1a* and *fshr*, alteration of oogenesis). Those physiological responses are consistent with responses reported for wild-type fish exposed to IMZ and other azole fungicides, and with the previously described AOP on aromatase activity inhibition in fish. Such data could relevantly support hazard assessment for endocrine active chemicals in non-mammalian vertebrate models and might also be relevant to inform human health hazard assessment toward a pathway-based approach.

### Data availability

All data will be made available on request.

### CRediT authorship contribution statement

**Julie De Oliveira:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Tu-Ky Ly:** Writing – review & editing, Investigation. **Edith Chadili:** Investigation. **Violette Thermes:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Antoine Mergot:** Investigation. **Benjamin Piccini:** Investigation. **Olivier Palluel:** Investigation. **Hélène Budzinski:** Writing – review & editing, Methodology, Investigation. **Karyn Le Menach:** Methodology, Investigation. **Patrick Pardon:** Investigation. **Rémy Beaudouin:** Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Data curation. **Xavier Cousin:** Writing – review & editing, Supervision, Methodology, Conceptualization. **François Brion:** Writing – review & editing, Funding acquisition, Conceptualization.

**Nathalie Hinfray:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### 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.

### Acknowledgements

The authors would like to thank the H2P2 facility of the University of Rennes for the loan of the slide scanning nanozoomer (HAMAMATSU) and the Platine facility of the University of Bordeaux for chemicals analysis. This work was supported by the French National Research Program for Environmental and Occupational Health of ANSES [AIDEZ project n°EST-2016/1/189, GinFiz project n°EST-2020/01/133] and by the French Ministry of Ecological Transition [P190 program].

### Data availability

Data will be made available on request.

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