# The impact of an early exposure to 17α-ethynylestradiol on the physiology of the three-spined stickleback (Gasterosteus aculeatus) under current and future climatic scenarios

Jimmy Devergne, Arianna Servili, Sylvain Jodet, Titouan Brandicourt, Christophe Lebigre, Sophie Collet, Olivier Mouchel, Marie Lou Fleury, Sabine Roussel, Veronique Loizeau

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Jimmy Devergne, Arianna Servili, Sylvain Jodet, Titouan Brandicourt, Christophe Lebigre, et al.. The impact of an early exposure to 17α-ethynylestradiol on the physiology of the three-spined stickleback (Gasterosteus aculeatus) under current and future climatic scenarios. Aquatic Toxicology, 2025, 287, pp.107528. 10.1016/j.aquatox.2025.107528. hal-05244367

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# The impact of an early exposure to 17α-ethynylestradiol on the physiology of the three-spined stickleback (*Gasterosteus aculeatus*) under current and future climatic scenarios

Jimmy Devergne<sup>a, *</sup>, Arianna Servili<sup>a</sup>, Sylvain Jodet<sup>a</sup>, Titouan Brandicourt<sup>a</sup>,

Christophe Lebigre<sup>b</sup>, Sophie Collet<sup>a</sup>, Olivier Mouchel<sup>a</sup>, Marie Lou Fleury<sup>a</sup>, Sabine Roussel<sup>a</sup>,

Véronique Loizeau<sup>a</sup>

## ARTICLE INFO

### Keywords:

- Global change

- Xenoestrogens

- Multi-stress

- Survival

- Growth

- Sexual maturation

## ABSTRACT

Ocean warming and acidification are climate change related drivers that impact the physiology of marine organisms and their ability to cope with future environments. Marine ecosystems are also facing pollution from an ever-growing diversity of chemical contaminants, including endocrine disruptors. A common example is the 17α-ethynylestradiol (EE2), which can affect the endocrine regulation of fish and hence potentially impact their fitness. Thus, fish have to cope to multiple climatic and chemical stresses that can interact, influencing the overall impact on fish physiology. In this study, we investigated whether the direct and carry-over effect of early exposure to EE2 (15 ng.L⁻¹; one month during embryo-larval development) are modulated by the RCP8.5 scenario (+3°C; -0.4 pH unit). Five months post-contamination, we measured survival, growth and reproductive axis of prepubertal sticklebacks. Our findings revealed that the survival of juveniles, when exposed to EE2 during early development, is reduced under Current but not RCP8.5 scenario. Furthermore, under RCP8.5-EE2, a significantly lower body length was observed. Sex and tissue specific responses in terms of the expression profiles of genes related to development and sexual maturation was reported. Interestingly, significant interaction between RCP8.5 and EE2 was observed for the expression of ovarian aromatase (*cyp19a1a*), suggesting a long-lasting estrogenic effect under RCP8.5 scenario. Additionally, the skewed sex ratios and the presence of intersex individuals in both scenarios early exposed to EE2 suggested a feminization due to EE2, which could potentially disrupt sexual maturation and future reproduction. Hence, the early EE2 exposure had carry-over physiological effects on sticklebacks, and these effects can be modulated by the climate scenario. This underscores the importance of conducting long-term multi-stress studies to comprehensively understand the vulnerability on fish populations in future environments.

## 1. Introduction

The ocean has played a crucial role in regulating the Earth's climate by absorbing 93% of the excess of heat, generated by the greenhouse gases since 1970s, and by storing 28% of the CO₂ emissions since the beginning of the industrial era in 1750 (Gattuso et al., 2015). However, the regulating role of the oceans could not prevent the overall warming trend, and the increasing concentration of CO₂ has led to acidification (Gattuso et al., 2015). More specifically, sea surface temperature has risen by 0.7°C over the last century, while ocean pH has fallen by 0.1

(Canadell et al., 2021; Fox-Kemper et al., 2021). Model projections from Intergovernmental Panel on Climate Change (IPCC) indicate that by 2100 the oceans will be warmer by 1.4 to 3.4°C and experience additional acidification (ΔpH -0.2 to -0.4), depending on the region, habitat, and emission scenario (Kwiatkowski et al., 2020; Meinshausen et al., 2020). For instance, the Representative Concentration Pathway 8.5 (RCP8.5) scenario, corresponding to the more recent Shared Socio-economic Pathways (SSP) SSP3-7.0 and SSP5-8.5 models, predicts an average temperature increase of 2.9 and 3.5°C, respectively, relative to late 19th-century levels, with a pH decrease of 0.3 and 0.4

<footnote><sup>a</sup> Univ Brest, CNRS, IRD, Ifremer, UMR 6539, LEMAR, Plouzane, France</footnote>

<footnote><sup>b</sup> UMR DECOD (Ecosystem Dynamics and Sustainability), IFREMER, INRAE, Institut Agro, F-29280, Plouzané, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: jimmy.devergne@gmail.com (J. Devergne).</footnote>
unit in surface seawater by 2100 (Kwiatkowski et al., 2020).

Such major changes in the characteristics of the ocean raise questions about the ability of marine species to acclimate to future conditions, as these changes could affect survival, growth rates, reproductive success, and ultimately the dynamics of fish recruitment (Baag and Mandal, 2022). For many marine species, physiological responses to environmental change vary significantly between life stages, with the larval and pubertal stages being the most sensitive (Baag and Mandal, 2022; Dahlke et al., 2020; Pörtner et al., 2014; Przeslawski et al., 2015). To accurately quantify the impact of climate change on a species, it is necessary to consider prolonged exposures to environmental stressors that span different life stages.

In addition to warming and acidification, marine biota are also affected by the chronic and widespread pollution of the marine environment resulting from human activities. Aquatic organisms are exposed to an increasing number of chemical contaminants, including endocrine disruptors, which are likely to affect their fitness (Alava et al., 2017). These include a group of molecules with estrogenic activity: the xenoestrogens. The 17α-ethynylestradiol (EE2), used in most human contraceptive pills, is one of the most widely reported xenoestrogenic endocrine disruptors in surface waters worldwide, with concentrations ranging from 0.2 to 73 ng.L⁻¹ (Bhandari et al., 2015). In France, in the Seine and its estuary, the water concentrations range from 2 to 17 ng.L⁻¹ (Cargouet et al., 2004). Despite its short half-life in rivers (up to 6 days), the consequent and continuous discharge in the wastewater leads to a constant presence of this contaminant in surface waters (Adeel et al., 2017). Environmental concentrations of EE2 are known to induce tissue damage, feminisation and reproductive dysfunction (Jackson et al., 2019), behavioural changes (Saaristo et al., 2019), steroidogenesis disruption (Sridevi et al., 2015), and spawning inhibition (Cosme et al., 2015) in teleosts.

Following recommendations to assess the effects of multiple environmental stressors across the life cycle of fish (Alava et al., 2017; Kibria et al., 2021), a growing number of studies have begun to explore interactions between warming and EE2 contamination. Investigations on the combined effects of EE2 and warming in several fish species show that warmer waters exacerbate EE2's negative effects on survival, development, and reproduction. For instance, elevated temperatures (ΔT +3°C) combined with 1 ng.L⁻¹ EE2 exposure resulted in fewer viable offspring and increased developmental malformations across generations in *Menidia beryllina* (DeCourten and Brander, 2017). EE2 exposure has been shown to override the stimulatory effects of warmer water on gonad maturation in *Danio rerio* (ΔT +5°C, 4 ng.L⁻¹ EE2; Luzio et al., 2016), ultimately inhibiting maturation in this teleost. Furthermore, a study demonstrated a downregulation of genes involved in reproductive pathways in *Menidia beryllina* (ΔT +3°C, 1 ng.L⁻¹ EE2), including estrogen receptors 2 alpha (*esr2α*) and beta (*esr2β*) (DeCourten et al., 2019). One study suggests that warmer temperatures increase EE2 uptake, after 24 h at ΔT + 3°C and 100 ng.L⁻¹ EE2 exposure, in *fundulus heteroclitus* (Blewett et al., 2013). This indicates that temperature could modulate the observed effects of EE2 by increasing EE2 uptake. Taken together, these results suggest that future ocean warming may lead to more adverse effects of EE2 on fish physiology. Ocean acidification and endocrine disruptors have rarely been studied together on teleost. So far, only one study has examined their combined effect on juvenile Atlantic cod (*Gadus morhua*). This study revealed that combined exposure to the perfluorooctane sulfonic and high CO₂ levels had increased effects on sex steroid levels compared to high CO₂ levels alone, with transcription expression profiles indicating time-dependent interactive effects (Preus-Olsen et al., 2014). To our knowledge, no study has investigated the combined effects of EE2 and future ocean acidification on fish physiology, nor have any explored the multi-stress composed of EE2 exposure, acidification and warming. Studying the impact of ocean warming and acidification combined with exposure to a proxy for environmental estrogenic contamination will enable estimation of the physiological response of fish species to near future environments.

In this study we focused on the three-spined stickleback (*Gasterosteus aculeatus*), as a model species. This species is an euryhaline teleost that is widely distributed throughout the northern hemisphere. Its small size and short life cycle (sexual maturity at one year), combined with its ease of rearing in lab facilities has led to its use by the scientific community with extensive knowledge of its physiology and ecology (Blaker et al., 2022). This makes the species a good model for long-term studies. The Organisation for Economic Co-operation and Development (OECD) has also recognized this species as a good model for endocrine disruption studies, using vitellogenin and spiggin production as biomarkers of estrogenic or androgenic pollution (OECD, 2011). As a sentinel species for endocrine disruption, the stickleback has been extensively studied in the face of estrogenic contamination, particularly EE2. Elevated concentrations of EE2 increased growth in early life stages of this species, as well as the motor activity of the fish (100 ng.L⁻¹, Bell, 2004). Moreover, feminisation of stickleback was observed after early life EE2 contamination at 50 ng.L⁻¹ (Hahlbeck et al., 2004) and 30 ng.L⁻¹ but not 5 ng. L⁻¹ (Porseryd et al., 2019), showing partial or complete male sex reversal.

The individual effects of ocean warming and acidification have already been evaluated on stickleback physiology. Several studies have reported the effect of increased temperature on key physiological functions of the stickleback: under warmer conditions, growth in size and mass (Hani et al., 2018), as well as fecundity and egg diameter are lower, while egg development is accelerated (Kim et al., 2017b; Shama, 2015, 2017a). Moreover, higher temperatures have been shown to up-regulate metabolic and redox processes as well as the expression of several genes involved in growth and reproduction (Kim et al., 2017a). Studies on the effects of acidification on this species are still scarce and few studies have reported an impact of acidification on the reproductive axis, resulting in higher fecundity under acidification (Schade et al., 2014), but no effect on the egg survival and size (Glippa et al., 2017). As presented above, warming, acidification and EE2 contamination at environmental concentrations are all factors that, taken individually, are likely to impact the physiological traits of the stickleback and thus increase the vulnerability of its population. However, to our knowledge, the combination of these stressors remains unexplored.

In this context, we investigated whether the direct and carry-over effects of an early exposure to EE2 on survival, growth and reproductive axis of stickleback are modulated by the RCP8.5 climate scenario (ΔpH = -0.4; ΔT = + 3°C during six months). Individuals were issued of parents acclimated to the same climatic conditions (Current and RCP8.5) since their juvenile stage (Devergne et al., 2023). The EE2 concentration applied at the embryo-larval stage (15 ng.L⁻¹ during one month) was chosen according to the mean estrogenic environmental contamination observed in French rivers (Cargouet et al., 2004).

Using an integrative approach based on physiological, biochemical, and molecular analysis, this study aimed to assess the effects of this multi-stress on the growth and the development of the reproductive axis, in order to improve our knowledge of the vulnerability of fish species to predicted future environments. We hypothesised that the interaction between simulated climate change (RCP8.5) and exposure to EE2 at environmental concentrations would lead to more pronounced physiological impacts than those observed with each stressor alone.

## 2. Matériel and methods

This experimental study complied with French national law and EU Directive 2010/63/EU, as well as the European Commission recommendation 2007/526/EC on the care of animals used for scientific purposes. The experiment was performed in fish experimental facilities of the Ecophysiology and Life History Traits of Marine Organisms Unit (PHYTNESS) of Ifremer (agreement number B29-212-05, Brittany, France). The experimental design was previously approved by the French Ethics Committee for animal testing (CEFEA: Comité d'Éthique Finistérien en Expérimentation Animale, registering code C2EA-74) and
the French Ministère de l'Enseignement Supérieur de la Recherche et de l'Innovation (Authorization APAFIS #27,510, permit number 2,020,100,911,422,298_v3). Fish were handled by accredited personnel.

## 2.1. Fish husbandry and scenario set up

The F1 population used for this experiment was generated by *in vitro* fertilization (IVF) in June 2021, with gametes derived from a F0 population used in a previous experiment (Devergne et al., 2023). Briefly, F0 fish were acclimated for 7 months to the “Current” scenario, corresponding to the current seasonal variation in temperature and pH of the “Rade de Brest”, and to the “RCP8.5” scenario, with 3°C warming and 0.4 pH unit acidification as predicted by 2100 (Pörtner et al., 2014). Eggs were collected from 9 Current and 14 RCP8.5 females. Each clutch of eggs was fertilised with the sperm solution of 2 males from the same scenario and distributed among all F1 aquariums from the same climatic scenario. The IVF procedure was detailed in Devergne et al. (2023). The obtained offspring (F1) were reared into hatching trays inside 60 L rectangular glass tanks, under the same climatic scenario as their parents (n = 4 tanks per scenario). During the embryo-larval period, half of the tanks were exposed to EE2 contamination (Current-EE2, RCP8.5-EE2, n = 2 tanks per scenario) while the other halves remained uncontaminated (Current, RCP8.5, n = 2 tanks per scenario). Fish from all experimental treatments were fed *ad libitum* with nauplii artemia during the whole larval stage and 1 month after reaching the three-spine stage (two months of age). Afterwards, juveniles were fed with frozen sliced artemia and bloodworms (proportion of 1:4, Antinea, France), enriched twice a week with vitamins (Vitaligo, Geosane, France). At 70 days post-fertilisation (dpf), biodensities were adjusted to ~20 fish per tank by transferring fish from hatching trays to remain freely into the 60 L tanks (n = 2 tanks for Current-EE2 and RCP8.5, and n = 3 tanks for Current and RCP8.5-EE2 experimental treatments, from 71 dpf to 178

dpf). Tanks were filled with natural seawater pumped at 500 m from the coastline from a depth of 20 m, filtered as described previously (Devergne et al., 2023). In each tank, a flow rate of 30 L·h⁻¹ was set and an artificial light regime applied (Supplementary Table 1), following the natural seasonal variation of the photoperiod in the Bay of Brest (42 W halogen lamp, 55–60 lx). For the RCP8.5 climate scenario (RCP8.5 and RCP8.5-EE2), seawater was heated via a tungsten plate heat exchanger (Vicarb, France) and released into two 6 L header tanks from which CO₂ was diffused (Fig. 1). For pH regulation, a degassing column was placed within each header tanks to allow independent diffusion of carbon dioxide (CO₂). A WTW 3110 pH meter (Xylem Analytics Germany, Weilheim, Germany; with electrode: WTW Sentix 41, NBS scale) was used for daily temperature and pH measurements (Fig. 2). It was daily calibrated with NBS certified WTW technical buffers pH 4.01 and pH 7.00 (Xylem Analytics Germany, Weilheim, Germany). Total alkalinity was measured twice a month to estimate pCO₂ using the CO₂sys macro (Lewis and Wallace, 2012) with constants from Mehrbach et al. (1973) refit by Dickson and Millero (1987). The same pCO₂ values were then used in the macro to calculate total scale pH (Table 1), as described in Devergne et al. (2023). Weekly measurements of salinity and oxygen saturation were performed with a WTW LF 340 salinometer (Xylem Analytics Germany, Weilheim, Germany; with electrode: WTW Tetracon® 325/C, ‰ scale) and WTW Oxi 340 oximeter (Xylem Analytics Germany, Weilheim, Germany; with electrode: WTW CellOx 325, ‰ scale).

## 2.2. Xenoestrogen exposure

Contamination was carried out during the first 32 days (i.e. at the embryo-larvae stage), starting from fertilisation to the appearance of the third dorsal spine (Swarup, 1958). The 17α-ethynylestradiol (EE2, VETRANAL®, Sigma-Aldrich, 46,263–250MG, USA) was used at environmental concentrations (15 ng·L⁻¹). The solvent, dimethyl sulfoxide

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Experimental design at 70 days post-fertilization (dpf), with Current (light grey), Current-EE2 (grey), RCP8.5 (medium grey), and RCP8.5-EE2 (dark grey) experimental treatments. In the RCP8.5 scenario, warmer water was passed through the header tanks and was acidified by CO₂ diffusion, ensuring homogeneous mixing through the water flow. EE2 and its solvent were added using peristaltic pumps throughout the embryo-larval development stage. Black arrows indicate water flow.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Daily Seawater mean temperature (°C), from hatching (0 dph) to the prepubertal stage (178 dph) under the four experimental treatments: Current (light grey), Current-EE2 (medium grey), RCP8.5 (grey), and RCP8.5-EE2 (dark grey). Data are presented as means ± S.D. from 2–3 replicate tanks per treatment.</figcaption></figure>

<caption>**Table 1**</caption>

Average physico-chemical parameters of the seawater recorded twice a month and once for contamination during the experimental period (mean ± S.D.). TA: total alkalinity, pCO₂: partial pressure of CO₂, EE2: ethynylestradiol.

<table><thead><tr><th rowspan="2">Experimental treatments</th><th rowspan="2">Salinity (%)</th><th rowspan="2">O₂ (%)</th><th rowspan="2">pH total</th><th rowspan="2">TA (µmol.kg<sup>-1</sup>sw)</th><th rowspan="2">pCO₂ (µatm)</th><th colspan="2">EE2 concentration (ng.L<sup>-1</sup>)</th></tr><tr><th>Min</th><th>Max</th></tr></thead><tbody><tr><td>Current</td><td>35.11 ± 0.90</td><td>97.41 ± 2.89</td><td>7.92 ± 0.02</td><td>2389.16 ± 49.51</td><td>579.28 ± 27.13</td><td>–</td><td>–</td></tr><tr><td>Current – EE2</td><td>35.10 ± 1.20</td><td>98.89 ± 0.77</td><td>7.92 ± 0.03</td><td>2414.32 ± 213.74</td><td>595.20 ± 67.96</td><td>22.00</td><td>24.30</td></tr><tr><td>RCP8.5</td><td>35.14 ± 1.01</td><td>97.86 ± 1.86</td><td>7.49 ± 0.07</td><td>2415.60 ± 118.72</td><td>1786.19 ± 311.81</td><td>–</td><td>–</td></tr><tr><td>RCP8.5 – EE2</td><td>35.27 ± 1.09</td><td>98.70 ± 0.80</td><td>7.52 ± 0.08</td><td>2365.36 ± 84.79</td><td>1599.48 ± 272.08</td><td>11.70</td><td>24.70</td></tr></tbody></table>

(DMSO, anhydrous ≥ 99.9%, Sigma-Aldrich, 276,855–2 L) was used at a final concentration < 0.001 % in all the tanks. This concentration is 10 times lower than the maximum solvent concentration of 0.1 mL·L⁻¹ (0.01 %) specified by the Organization for Economic Cooperation and Development (OECD, 2019, 2013) for aquatic test guidelines. Contamination with EE2 and DMSO (15 ng·L⁻¹, Current-EE2, RCP8.5-EE2) or with DMSO alone (Current, RCP8.5) was carried out by pumping a continuous flow into the respective tanks using multichannel peristaltic pumps (pump models 7520–47 and 7554–95, header model 7535–08, MasterFlexe®, Cole-Parmer Instrument Company, USA). The contaminated seawater flow was treated with an activated charcoal filter (DIS-POSORB®, MINIDISPOSORB™, Chemviron, France) before being discharged into the sewer. Contamination levels of seawater from each tank and at the outlet of the decontamination columns were verified, in triplicate, once during the contamination and once during the post-contamination phases. To measure the EE2 concentration in the seawater, a solid phase extraction was performed using Sep-Pak® C18 Plus Short Cartridges (WatersTM, US). Then the EE2 concentration was determined using an ELISA kit (Ecologiena®, Tokiwa chemical industries CO.LTD, Japan), according to the protocol described by Wernicke von Siebenthal et al. (2018), and measured using a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Finland), at the 450 nm wavelength (Table 1). The detection limit is represented by three times the standard deviation of the absorbance of the blank (LOD = 0.5 ng·L⁻¹) and the upper linearity limit is set by the supplier's recommendation at the last calibration point (3 µg·L⁻¹).

## 2.3. Egg quality

At 3 dpf, the fertilised eggs (n in Supplementary Table 6) were

photographed (NIKON, D7200, Japan) with a binocular magnifier (Discovery.V8 SteREO, ZEISS, Germany). Eggs diameter and the perivitelline index, both markers for egg quality (Hani et al., 2019; Sganga et al., 2022; Valdebenito et al., 2015), were measured individually using ImageJ software (Version 1.53e, Java 1.8.0_172, Schneider et al., 2012) as described in Devergne et al. (2023). Although the perivitelline index has not yet been correlated with developmental success in stickleback specifically, it is a widely used as a proxy for egg quality and is known to respond to environmental stressors such as ocean acidification (Sganga et al., 2022).

## 2.4. Biometrics and individual sampling

When individuals reached the juvenile stage (32 dpf), all fish of each tank were carefully transferred into water trays every two weeks and photographed (NIKON, D7200, Japan), before being returned into their respective tanks. The individual standard length was measured with the software ImageJ (Version 1.53e, Java 1.8.0_172; Schneider et al., 2012). After six months of exposure to the RCP8.5 or the Current scenario, a sampling was carried out to evaluate physiological indexes, sex ratio, histological analysis and gene expression profiles in prepubertal fish. Prior to sampling, fish were fasted for 24 h, captured, and transferred to an anaesthesia container with 0.125 g·L⁻¹ MS222. Subsequently, they were euthanized with a lethal concentration of 0.250 g·L⁻¹ MS222. Sex ratio, standard length, body mass and physiological indexes were estimated (Sartorius, MC BA 1000, Germany) as described in Devergne et al. (2023): Fulton index (K) = ((Mass of the fish in grams)/(Size of the fish in centimeters)³) × 100; Gonado-somatic index (GSI) = ((Mass of the gonads in grams)/(Mass of the whole body in grams)) × 100; Hepato-somatic index (HSI) = ((Mass of the liver in grams)/(Mass of the whole body in grams)) ×
100 (n in Supplementary Table 7). Fish survival was monitored throughout the experiment and reported as embryo-larval survival = (number of juveniles at the appearance of the third spine)/(number of stripped oocytes), to assess the direct effect of the multi-stress during early development, and as juvenile survival = (number of fish at 6 months of age)/(number of juveniles at the appearance of the third spine), to assess the multi-stress carry over effects of the early EE2 exposure in both scenarios.

## 2.5. Histological analysis of the gonads

To test for the potential presence of intersex individuals, the left gonad of fish was sampled (n in Supplementary Table 7) and fixed in 4% paraformaldehyde (PFA) for 24 h at 4°C. Tissues were dehydrated, impregnated, embedded, sliced and stained using the trichrome Prenant-Gabe method as explained in Devergne et al. (2023). The staining labelled the nucleus in purplish blue, the cytoplasm in red and the conjunctive tissue in green. The mounted slides were then analysed and photographed (EOS 700D, Canon, Japon) under a light microscope (AXIO observer Z1, ZEISS, Germany).

## 2.6. RNA extraction and purification

The brain and the right gonad (n in Supplementary Table 8) of fish were quickly removed, and the median region of the brain containing the hypothalamus was dissected. Tissues were stored in a RNA stabilization reagent (RNAlater, Qiagen, Hilden, Germany) at 4°C for 24 h and then stored at -20°C until extraction. Total RNA extraction and purification of the median brain was performed with the Nucleospin® RNAXS kit (Macherey Nagel, Germany) according to the supplier's protocol (Version January 2020/Rev.10) as previously described (Devergne et al., 2023). For the gonads, the RNA extraction was performed under chemical and mechanical lysis with Extract-All® (Qiagen, Germany) and Polytron® grinder (PT-MR 2100, Kinematic AG, Switzerland). Isolation of nucleic acids was done by phase separation with chloroform, followed by isopropanol to create a binding condition for purification. The isopropanol-RNA solution was purified with the Nucleospin® RNA kit (Macherey Nagel, Deutschland) according to the supplier's protocol (Version January 2020/Rev.10). Concentration and purity of total RNA extracted from brains and gonads were assessed with NanodropTM 2000 (Thermo Scientific Inc., Waltham, MA, USA) and RNA integrity using an Agilent Tapstation 4150 (Agilent Technologies Inc., Santa Clara, CA, USA). All samples analysed showed an integrity number (RIN) mean of 9.6 for the brain and 8.5 for the gonad. Samples were stored at -80°C until use for reverse-transcription quantitative PCR (RT-qPCR) analysis.

## 2.7. RT-qPCR analysis

The reverse transcription (RT) was carried out with the iScript™ cDNA Synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) and the ThermalCycler T100™ (Bio-Rad Laboratories Inc.) on all RNA samples with positive and negative (without enzyme) reaction according to the manufacturer's protocol. Then, cDNA was stored at -20°C until qPCR. The qPCR analysis performed on the brain targeted genes associated with the neuroendocrine regulation of the sexual maturation: Gonadotropin Releasing Hormone 2 and 3 (gnrh2, gnrh3) neuropeptides involved in the release of pituitary gonadotropins (Muñoz-Cueto et al., 2020); cyp19a1b, the brain form of aromatase, an enzyme converting androgens to estrogens specifically into the fish brain (Piferrer and Blázquez, 2005); Kisspeptin 2 (kiss2) a key upstream trigger of gnrh secretion (Mechaly et al., 2013); and Gonadotropin-Inhibitory Hormone (gnih) a neuropeptide that regulates gnrh and pituitary gonadotropins release in teleosts (Muñoz-Cueto et al., 2020; Paullada-Salmerón et al., 2019).

Two other genes regulating the stress axis and development were studied in the brain: respectively Corticotropin-releasing hormone

(crhβ) coordinating the hypothalamic-pituitary-interrenal stress axis in teleost (Flik et al., 2006); and the thyrotropin-releasing hormone (trh) an upstream regulator of the thyroid-stimulating hormone secretion in vertebrates (Janz, 2000).

For the gonadal gene expression study, genes related to estrogenic regulation were assessed: cyp19a1a, the aromatase converting androgens to estrogens into the gonads (Piferrer and Blázquez, 2005); estrogen receptor 1 (esr1), estrogen receptor 2a (esr2a), estrogen receptor 2b (esr2b) which are nuclear estrogens receptors respectively α, β1 and β2 subtypes mediating 17β-estradiol effects in target tissues (Tokarz et al., 2015). The primers were specially designed for this project (Table 2) with the primer 3Plus software (Untergasser et al., 2007).

The qPCRs were performed with the CFX96 Touch Real-Time PCR Detection system (Bio-Rad Laboratories Inc.) and SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad Laboratories Inc.) as explained previously (Devergne et al., 2023). For each sample the reaction was carried out in duplicate with 1/20 dilution for brain cDNA and 1/10 for gonads cDNA. The high-resolution melting curves produced by the protocol were used to verify the amplification of a single product in each reaction. The Gene Expression Module of the CFX Manager program (Bio-Rad Laboratories Inc.) was used to automatically determine the related quantification cycle (Cq) value for each reaction. For the brain, ribosomal protein l8 (rpl8), ribosomal protein l13a (rpl13a), and for the gonad, follicle-stimulating hormone receptor (fshr), luteinizing hormone receptor (lhr), were used as reference genes in the ΔΔCt method to normalize the relative quantity of transcripts. For housekeeping genes, no significant variations in Cq values were found between experimental treatments.

## 2.8. Statistical analysis

The data analysis and graphical representation were performed using the R software (R Core Team, 2018). The significance threshold was set to p-values < 0.05 (see Supplementary Table 1).

To test the effect of climatic scenario and EE2 contamination on perivitelline index data a two-way ANOVA was run since the normality of data (Shapiro-Wilk test) and the homogeneity of variance (Bartlett test) were met. For egg diameter data, neither the normality nor the homogeneity of the variance (Levene's test) were met. Therefore, we ran Welch's heteroscedastic F test, followed by a Games-Howell post-hoc test with Tukey's correction method.

To determine the effects of the multi-stress on fish survival and sex ratio a non-parametric Pearson's chi-squared test was performed on the four experimental treatments (Current, Current-EE2, RCP8.5, RCP8.5-EE2) followed by pairwise Pearson's chi-squared test with Holm's correction.

To evaluate the effect of the climatic scenario and EE2 contamination on fish size throughout the 6 months of the experiment, a linear mixed effects model was performed (package "nlme" (Pinheiro and Bates, 2000)) using the "climatic scenario" (Current vs RCP8.5), the "xenoestrogen contamination" (non-EE2 vs EE2), and the "time" as fixed factors and the rearing tank identification code as random factor. To account for the non-independence of the time effect of consecutive measurements, the model considered an autoregressive process with correlation structure of order 1 (AR1). This test was followed by the emmeans function for the post-hoc test with a Tukey correction.

Because males and females might respond differently to the estrogenic contamination, we decided to analyse the data separately for each sex at the 6-month sampling. For each sex, the standard length, body mass, physiological indexes and gene expression profiles were analysed by a linear mixed-model using the package "lmer" (Bates et al., 2015) with the climatic scenario (Current vs RCP8.5) and the xenoestrogen contamination (non-EE2 vs EE2) as fixed factors and the tanks as a random factor, after graphically checking residual normality and homogeneity of variance. The Anova function was applied to obtain an Analysis of Deviance Table for the fixed factors (Type II Wald chi-square
<table><caption>**Table 2**

Primers used in the qPCR analysis of the median brains and gonads.</caption><thead><tr><th>Targets</th><th>Genes</th><th>Forward primer</th><th>Reverse primer</th><th>GenBank accession number</th></tr></thead><tbody><tr><td rowspan="10">Brain</td><td>rpl8</td><td><sup>5</sup>GTGAAGGACATCATCCACGA<sup>3</sup></td><td><sup>5</sup>CTCCTCCACACAGCAGATGA<sup>3</sup></td><td>ENSGACT00000002668.1</td></tr><tr><td>rpl13a</td><td><sup>5</sup>CGCCCTACGACAAGAGGAAG<sup>3</sup></td><td><sup>5</sup>CTCTGCCACCTTGGTCAACT<sup>3</sup></td><td>ENSGACT000000012382.1</td></tr><tr><td>gnrh2</td><td><sup>5</sup>TGTGTTGGAGCTCAGCTGTC<sup>3</sup></td><td><sup>5</sup>CTCTGGCTAAGGCATCCAAA<sup>3</sup></td><td>ENSGACT000000011943.1</td></tr><tr><td>gnrh3</td><td><sup>5</sup>GTTGGTGGTCCAGGTCACTC<sup>3</sup></td><td><sup>5</sup>CTCTCTTGGGTCTGGGCACT<sup>3</sup></td><td>ENSGACT000000012668.1</td></tr><tr><td>cyp19a1b</td><td><sup>5</sup>ATACCCGGTCCGTGCTTCT<sup>3</sup></td><td><sup>5</sup>CCCGAATCTGGCTGTGTAGT<sup>3</sup></td><td>ENSGACT000000007929.1</td></tr><tr><td>kiss2</td><td><sup>5</sup>GGGTCAGTTCTTTCTACGCTCA<sup>3</sup></td><td><sup>5</sup>AATGTAGCGTTTCCCAAAGC<sup>3</sup></td><td>KT202354.1</td></tr><tr><td>Gnih</td><td><sup>5</sup>AGAGCTTCCGCATACTCTCG<sup>3</sup></td><td><sup>5</sup>ATGTTGTGGGTCGAACTGGT<sup>3</sup></td><td>KT202315.1</td></tr><tr><td>crhβ</td><td><sup>5</sup>ACCGTGATTCTGCTAGTTGC<sup>3</sup></td><td><sup>5</sup>CATGGATGAAGATGGGAAAG<sup>3</sup></td><td>XM_040167877.1</td></tr><tr><td>trh</td><td><sup>5</sup>GAAGACGGCGGAGACGAG<sup>3</sup></td><td><sup>5</sup>CGTCCAAGTATGGTTCATCC<sup>3</sup></td><td>ENSGACG000000002980</td></tr><tr><td rowspan="7">Gonad</td><td>cyp19a1a</td><td><sup>5</sup>CCTACTACACCAAAGCCCTGA<sup>3</sup></td><td><sup>5</sup>TAGCTGAGAAAGACCCGCAT<sup>3</sup></td><td>XM_040167072.1</td></tr><tr><td>esr1</td><td><sup>5</sup>GGCATGATGAAAGGAGGTGT<sup>3</sup></td><td><sup>5</sup>TTGTCGTTGTCGTTGTCGC<sup>3</sup></td><td>NM_001267672.1</td></tr><tr><td>esr2a</td><td><sup>5</sup>TTCAAGAGGAGCATCCAAGG<sup>3</sup></td><td><sup>5</sup>TCCAGTCAACGGCTTACTC<sup>3</sup></td><td>XM_040160794.1</td></tr><tr><td>esr2b</td><td><sup>5</sup>CAACCAATCAATGCACCATC<sup>3</sup></td><td><sup>5</sup>TGCTTCACCCGTACTACTT<sup>3</sup></td><td>LC006094.1</td></tr><tr><td>Fshr</td><td><sup>5</sup>CCTGACACACATCCACCAAG<sup>3</sup></td><td><sup>5</sup>TATGCCGAGTGGTCTTTACC<sup>3</sup></td><td>XM_040176746.1</td></tr><tr><td>ahr</td><td><sup>5</sup>CGTTTCTGAATGCCACCTCT<sup>3</sup></td><td><sup>5</sup>GACTTTATCTCGGGTGACGC<sup>3</sup></td><td>XM_040179510.1</td></tr></tbody></table>

tests). For post-hoc test, emmeans function with the Tukey correction was used (Lenth, 2016). To find the most parsimonious model, stepwise backward selections were carried out in all analysis.

Throughout the manuscript, the effect of the RCP8.5 climate scenario is referred as “scenario”, and the effect of EE2 exposure as “contamination”.

## 3. Results

### 3.1. Egg quality

A significant effect of the multi-stress on the egg diameter was observed (W<sub>3, 16.29</sub> = 12.2, *p* < 0.01, Supplementary Table 2): the egg diameter was lower under the RCP8.5-EE2 treatment compared to the Current treatment (t = 6.19, *p* < 0.01; Fig. 3A). Moreover, eggs exposed to the RCP8.5 climatic scenario showed a higher perivitelline index compared to eggs exposed to the Current scenario (F<sub>1, 36</sub> = 4.60, *p* = 0.04, Fig. 3B, Supplementary Table 2). There was no significant effect of contamination alone or in interaction with the climatic scenario (respectively F<sub>1, 36</sub> = 0.77, *p* = 0.39; F<sub>1, 36</sub> = 0.01, *p* = 0.99, Fig. 3B, Supplementary Table 2).

### 3.2. Survival and development

Embryo-larval survival was different among the experimental treatments ($\chi^2$ = 13.49, *p* < 0.01; Table 3 and supplementary Table 2).

Direct EE2 contamination in the Current scenario (Current-EE2) did not impact embryo-larval survival compared to the uncontaminated Current treatment ($\chi^2$ = 0.68, *p* = 0.81; Table 3 and supplementary Table 2). EE2 also had no effect on the survival in the RCP8.5 scenario (RCP8.5-EE2), compared to the uncontaminated RCP8.5 treatment without EE2 contamination ($\chi^2$ = 4.87, *p* = 0.11; Table 3 and supplementary Table 2). Embryo-larval survival was significantly reduced in the RCP8.5 experimental treatment compared to the Current and Current-EE2 treatments ($\chi^2$ = 7.03, *p* = 0.04; $\chi^2$ = 12.18, *p* < 0.01 respectively; Table 3 and supplementary Table 3). Additionally, the time to hatching post-fertilisation was one day shorter in the RCP8.5 scenario compared to the Current scenario with respectively 8 and 9 day post-fertilisation (dpf). Survival among juveniles varied significantly between the experimental treatments ($\chi^2$ = 14.03, *p* < 0.01; Table 3 and supplementary Table 2). Juvenile survival was lower in the Current-EE2 experimental treatment ($\chi^2$ = 12.59, *p* < 0.01; Table 3 and supplementary Table 3) compared to the Current treatment. No carry over effect of EE2 was observed on juvenile survival in the RCP8.5 scenario ($\chi^2$ = < 0.01, *p* = 1; Table 3 and supplementary Table 3). The sex ratio of juveniles was significantly different among the experimental treatments ($\chi^2$ = 19.12, df = 3, *p* < 0.01): fewer proportion of males was observed in the Current-EE2 and RCP8.5-EE2 compared to the Current treatment (respectively, *p* < 0.01, *p* = 0.02, Table 4 and supplementary Table 2 & 4).

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Boxplots representing the diameter (A) and the perivitelline index (B) of eggs at 3 days post-fertilisation under Current (light grey), Current-EE2 (grey), RCP8.5 (medium grey), and RCP8.5-EE2 (dark grey) experimental treatments (n = 14 Current, 8 Current-EE2, 10 RCP8.5, 8 RCP8.5-EE2). The 10th and 90th percentiles are represented by the whiskers, the 25th and 75th percentiles by the boxes, the median values by horizontal lines and outliers by points. Different letters indicate statistically significant differences between boxes in A and scenarios in B (*p* < 0.05).</figcaption></figure>
<caption>**Table 3**</caption>

Embryo-larval survival and juvenile survival in the experimental treatments. Different letters indicate statistically significant differences between experimental treatments (*p* < 0.05).

<table><caption>**Table 4**</caption><thead><tr><th></th><th>Current</th><th>Current-EE2</th><th>RCP8.5</th><th>RCP8.5-EE2</th></tr></thead><tbody><tr><td><strong>Embryo-larval survival</strong><br><em>number of juveniles at the appearance of the third spine</em><br><em>number of stripped oocytes</em></td><td><strong>62</strong><br><em>305</em><sup>a</sup></td><td><strong>64</strong><br><em>272</em><sup>a</sup></td><td><strong>35</strong><br><em>292</em><sup>b</sup></td><td><strong>84</strong><br><em>459</em><sup>ab</sup></td></tr><tr><td><strong>Juvenile survival</strong><br><em>number of fish at 6 months of age</em><br><em>number of juveniles at the appearance of the third spine</em></td><td><strong>57</strong><br><em>62</em><sup>a</sup></td><td><strong>42</strong><br><em>64</em><sup>b</sup></td><td><strong>25</strong><br><em>35</em><sup>ab</sup></td><td><strong>62</strong><br><em>84</em><sup>ab</sup></td></tr></tbody></table>

Proportion of males in the experimental treatments. Different letters indicate statistically significant differences between experimental treatments (*p* < 0.05).

<table><thead><tr><th></th><th>Current</th><th>Current-EE2</th><th>RCP8.5</th><th>RCP8.5-EE2</th></tr></thead><tbody><tr><td><em>Number of males</em></td><td><strong>35</strong><br><em>57</em><sup>a</sup></td><td><strong>8</strong><br><em>40</em><sup>b</sup></td><td><strong>8</strong><br><em>24</em><sup>ab</sup></td><td><strong>19</strong><br><em>57</em><sup>b</sup></td></tr><tr><td><em>Total population</em></td><td></td><td></td><td></td><td></td></tr></tbody></table>

### 3.3. Growth and individual physiological indexes

An effect of scenario (F₁, 6 = 9.04, *p* < 0.01), contamination (F₁, 6 = 11.9, *p* < 0.01), time (F₈, 42 = 9.04, *p* < 0.01), and interaction between scenario and time (F₈, 42 = 2408, *p* = 0.01) was observed for standard length (Fig. 4, Supplementary Table 2). Fish under RCP8.5-EE2 experimental treatments had significantly lower standard lengths than those under Current treatment, from the fourth month of exposure until the end of the experiment (*p*₁₂₀-dph = 0.01; *p*₁₃₉-dph = 0.03; *p*₁₅₂-dph = 0.01; Supplementary Table 5).

After 6 months of multi-stress (Table 5), no effect of scenario (F₁, 66 = 0.19), contamination (F₁, 66 = 0.65), or the interaction between scenario and contamination (F₁, 66 = 0.29) was observed for male standard length and mass (Supplementary Table 2). However, in RCP8.5 scenario, males had a higher GSI compared to the Current scenario (F₁, 65 = 5.22, *p* = 0.02; Supplementary Table 2) and those exposed to EE2 contamination had lower K and HSI compared to those unexposed (respectively F₁, 65 = 4.48, *p* = 0.03; F₁, 41 = 4.88, *p* = 0.03 Supplementary Table 2). For females (Table 5), no effect of scenario, contamination, nor their interaction was observed on the standard length, body mass, GSI, and HSI (Supplementary Table 2). However, females showed a lower K index in

the RCP8.5 scenario compared to the Current scenario (F₁, 103 = 9.61, *p* < 0.01; Supplementary Tables 2).

### 3.4. Histological analysis of the gonads

Histological analysis of the gonads showed that neither sex reached the final maturation. No aberrant structural modifications, such as necrosis, fibrosis, or dysplasia, were noted within the gonadal tissues in any sex and experimental treatments. Nevertheless, oocytes were observed in the testis of 2 individuals: one (out of 5 males analysed) was identified in the Current-EE2 experimental treatment (Fig. 5C), and another one (out of 9 males analysed) in the RCP8.5-EE2 treatment (Fig. 5D).

### 3.5. Genes expression profiles

#### 3.5.1. Median brain

The gene expression profiles in the median brain revealed different patterns between females and males (Fig. 6). A scenario effect was observed in males with a down-regulation of *gnrh3*, *trh*, and *gnih* expression for juveniles from RCP8.5 scenario compared to the Current scenario (respectively for *gnrh3*, *trh*, and *gnih* expression: F₁, 32 = 13.20, *p* < 0.01; F₁, 32 = 6.34, *p* = 0.01; F₁, 32 = 8.93, *p* < 0.01; Supplementary Table 2). No significant effect of the contamination or the interaction between scenario and contamination was reported (Supplementary Table 2). No significant variations in expression between scenario, contamination or their interaction were observed for the other studied genes in males (Supplementary Table 2). For females, no effect for scenario, contamination or the interaction between scenario and contamination was reported for the gene expression profiles studied

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Juvenile sticklebacks mean standard length per tanks from 22 dph until 2 weeks before the end of the experiment. Experimental treatments include Current (light grey), Current-EE2 (medium grey), RCP8.5 (grey) and RCP8.5-EE2 (dark grey) (n tanks = 2–3 Current, 2 Current-EE2, 2 RCP8.5, 2–3 RCP8.5-EE2). Different letters indicate statistically significant differences between boxes within the same time point (*p* < 0.05).</figcaption></figure>
<caption>**Table 5**</caption>

Stickleback standard body length, body mass, Fulton's index (K), gonado-somatic index (GSI) and hepato-somatic index (HIS) after 6 months of experiment (females n = 15–22 Current, 19–32 Current-EE2, 13–16 RCP8.5, 19–38 RCP8.5-EE2, males n = 21–35 Current, 5–8 Current-EE2, 8 RCP8.5, 15–19 RCP8.5-EE2). Data are presented as mean ± S.D. Different letters indicate statistically significant differences between scenarios or contamination levels (p < 0.05).

<table><thead><tr><th>Sex</th><th>Experimental treatments</th><th>Standard length (mm)</th><th>Mass (g)</th><th>K</th><th>GSI</th><th>HSI</th></tr></thead><tbody><tr><td rowspan="4">♀</td><td>Current</td><td>44.73 ± 4.06</td><td>1.14 ± 0.30</td><td>1.26 ± 0.14</td><td>2.11 ± 0.68</td><td>5.62 ± 1.73</td></tr><tr><td>Current – EE2</td><td>45.12 ± 4.93</td><td>1.25 ± 0.39</td><td>1.32 ± 0.15</td><td>2.01 ± 0.58</td><td>5.24 ± 1.46</td></tr><tr><td>RCP8.5</td><td>45.25 ± 4.31</td><td>1.10 ± 0.39</td><td>1.17 ± 0.25</td><td>1.97 ± 0.58</td><td>4.93 ± 2.21</td></tr><tr><td>RCP8.5 – EE2</td><td>42.53 ± 5.90</td><td>0.97 ± 0.39</td><td>1.21 ± 0.16</td><td>1.49 ± 0.81</td><td>5.14 ± 1.53</td></tr><tr><td rowspan="4">♂</td><td>Current</td><td>47.83 ± 4.66</td><td>1.46 ± 0.40</td><td>1.31 ± 0.16</td><td>1.33 ± 0.57</td><td>5.53 ± 1.41</td></tr><tr><td>Current – EE2</td><td>49.38 ± 2.62</td><td>1.50 ± 0.31</td><td>1.23 ± 0.15</td><td>1.07 ± 0.29</td><td>4.89 ± 0.90</td></tr><tr><td>RCP8.5</td><td>46.88 ± 3.80</td><td>1.47 ± 0.46</td><td>1.39 ± 0.10</td><td>1.52 ± 0.38</td><td>6.25 ± 1.42</td></tr><tr><td>RCP8.5 – EE2</td><td>45.95 ± 4.08</td><td>1.29 ± 0.31</td><td>1.31 ± 0.09</td><td>1.68 ± 0.75</td><td>4.81 ± 1.11</td></tr></tbody></table>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Histological sections of testis from sticklebacks reared under Current (A), Current-EE2 (C), RCP8.5 (B) and RCP8.5-EE2 (D) experimental treatments (n = 7 Current, 5 Current-EE2, 3 RCP8.5, 9 RCP8.5-EE2). Prenant-Gabe trichrome staining: nucleus marked by hematoxylin (purplish blue), cytoplasm marked by eosin Y (pink), connective tissue marked by light green (green). The black arrows show oocytes. Scale bars = 100 µm. Pictures have been edited to increase the brightness (+20 %).</figcaption></figure>

(Supplementary Table 2).

### 3.5.2. Gonads

In the ovaries (Fig. 7), an effect of the scenario was observed for the gene expression profiles of *esr1* and *esr2b* (respectively: F₁, 35 = 8.97, *p* < 0.01; F₁, 31 = 7.33, *p* < 0.01; Supplementary Table 2), with the RCP8.5 scenario leading to an upregulation of these estrogen receptors compared to the Current scenario. An effect of EE2 contamination was observed for *esr1* and *esr2a* expression (respectively F₁, 35 = 20.08, *p* < 0.01; F₁, 35 = 5.75, *p* < 0.01; Supplementary Table 2), with a down-regulation of *esr1* and *esr2a* expressions in the EE2 exposed treatments compared to the unexposed treatments. The expression profile of *cyp19a1a* was the only one showing a significant interaction between scenario and contamination (F₁, 35 = 7.77, *p* < 0.01). In the testis (Fig. 7), a significant contamination effect was observed with an up-

regulation of *esr2a* expression in EE2 exposed treatments compared with those unexposed (F₁, 18 = 7.77, *p* = 0.01; Supplementary Table 2). No effect of scenario, contamination nor interaction was observed for other gene expression profiles (*p* > 0.05; Supplementary Table 2).

### 4. Discussion

Overall, this study suggests that EE2 effects on parameters related to survival, growth, and sex determination/reproductive axis in stickleback could be modulated by the tested climatic scenarios. The first results also suggest that this climatic modulation depends on both life stage and sex of the fish. Nevertheless, only limited interactions between EE2 contamination and the climate scenario were observed across the physiological parameters measured. Although, some trends suggest an increased vulnerability in fish exposed early to multi-stress treatment,
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Boxplots representing relative expression of *gnrh3* (A), *gnrh2* (B), *gnih* (C), *kiss2* (D) and *cyp19a1b* (E), *trh* (F), and *crh* (I) in the median brain of sticklebacks after 6 months of experiment. Experimental treatments include Current (light grey), Current-EE2 (medium grey), RCP8.5 (grey) and RCP8.5-EE2 (dark grey) (females *n* = 10–13 Current, 10–12 Current-EE2, 8–11 RCP8.5, 9–10 RCP8.5-EE2, males *n* = 11–13 Current, 3–4 Current-EE2, 6–7 RCP8.5, 8–9 RCP8.5-EE2). The 10th and 90th percentiles are represented by the whiskers, the 25th and 75th percentiles by the boxes, the median values by horizontal lines and outliers by points. Different letters indicate statistically significant differences between scenarios (*p* < 0.05).</figcaption></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

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

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

(caption on next page)
**Fig. 7.** Boxplots representing relative expression of *esr1* (A), *esr2a* (B), *esr2b* (C), and *cyp19a1* (D) in the gonads of sticklebacks after 6 months of experiment. Experimental treatments include Current (light grey), Current-EE2 (medium grey), RCP8.5 (grey) and RCP8.5-EE2 (dark grey) (females *n* = 7–8 Current, 9 Current-EE2, 10 RCP8.5, 5–8 RCP8.5-EE2, males *n* = 3–7 Current, 2 Current-EE2, 3 RCP8.5, 4–6 RCP8.5-EE2). The 10th and 90th percentiles are represented by the whiskers, the 25th and 75th percentiles by the boxes, the median values by horizontal lines and outliers by points. Different letters indicate statistically significant differences between boxes (in A for females and B for both sexes) or scenarios (in C for females) (*p* < 0.05).

highlighting the need for further investigation through complementary analyses.

Often the early life stages, such as embryos and larvae, are the most vulnerable to environmental variations and stressors (Przeslawski et al., 2015). In this study, two markers of egg quality (diameter and perivitelline index) were assessed. The perivitelline space is an important marker of egg quality because it helps to maintain the embryo's physiological environment during early development (Finn, 2007; Sganga et al., 2022). Furthermore, for an equivalent egg size, a higher perivitelline index indicates a lowest yolk content and therefore a reduction in the resources allocated to the embryo, which may affect subsequent developmental rates. Under multi-stress treatment (RCP8.5-EE2), the perivitelline index was higher and the egg diameter was smaller, indicating limited resources for the embryos. Consistent with this, we also observed a shorter duration of the embryonic stage (before hatching) in the RCP8.5 scenario, in agreement with the results obtained by Shama (2015) for this species under warmer conditions. These two observations, the higher perivitelline index and the shorter time to hatch in the RCP8.5 scenario, could explain the low embryo-larval survival in this experimental treatment. Previous stickleback studies did not reveal any impact of acidification alone on embryo and larval survival (Glippa et al., 2017). Conversely, a thermal stress (i.e. a temperature of 21°C), corresponding to the summer temperature of the RCP8.5 scenario in the present study, has been described as stressful for European temperate water stickleback larvae (Hani et al., 2019). Unexpectedly, eggs showing the lowest quality (RCP8.5-EE2), based on the egg quality markers investigated (egg diameter, perivitelline index, and time to hatch), did not exhibit a lower survival. However, the alteration of the embryonic development (suggested by lower egg diameter and higher perivitelline index), likely due to ion regulation stresses (Johansson et al., 1981), although not directly impacting mortality, could subsequently result in a physiological trade-off in growth, as observed in prepubertal fish at the end of the experiment.

At the juvenile stage, fish exposed to Current scenario combined with EE2 have the lowest survival rates. The better survival of juveniles in the RCP8.5 scenario may be attributed to a possible selection of the most resilient individuals occurring at the embryo-larval stage. In addition, several studies suggest that higher temperatures increase the activity of enzymes responsible for detoxification (Madeira et al., 2016; Pimentel et al., 2015), which could enhance the degradation and clearance of pollutants. If this is the case, fish acclimated to RCP8.5 and contaminated during the embryo-larval stage may have eliminated EE2 residues more rapidly during the post-contamination period than fish acclimated to lower temperatures. To confirm or refute this hypothesis, it would have been necessary to quantify EE2 levels in stickleback target accumulation organs, such as the gall bladder, liver, or whole body (Blewett et al., 2013).

Five-months after contamination, the standard length of fish in RCP8.5 scenario was lower than in the Current scenario. This reduced growth was also observed in fish exposed to EE2 during the embryo-larval stage, regardless of the climatic scenario, with this effect being amplified in the RCP8.5-EE2 treatment. In a previous study (Devergne et al., 2023), it was observed that, to reach the equivalent adult size and mass of control scenario, sticklebacks subjected to RCP8.5 scenario required a greater amount of food. The hypothesis is that sticklebacks subjected to higher temperature and lower pH have increased metabolic costs, resulting in additional energy requirements, that are compensated when food is not limiting (Devergne et al., 2023). In the present study, fish were fed the same amount of food in the four experimental

treatments, which did not allow for this possible compensation of metabolic cost in the RCP8.5 scenario, resulting in lower growth and lower fish size than under Current scenario. Xenoestrogens, and specifically EE2 contamination have been reported in fish species to reduce the growth (Länge et al., 2001; Zha et al., 2007). For instance, the exposure at environmental concentrations (around 25 ng.L⁻¹) during the early life led to lower body length and weight in *Oreochromis niloticus* (Shved et al., 2008) and *Danio rerio* (Van den Belt et al., 2003). Shved et al. (2008) hypothesized that the growth axis was directly impacted by EE2, resulting in lower mRNA expression of the *insulin growth factor 1* in the brain, gonads and liver, and of the *growth hormone* in the pituitary. It would be interesting to check whether the growth axis, and particularly the *insulin growth factor 1* signaling, would have been also disrupted in EE2 treated sticklebacks in the present study. In contrast, Bell (2004) showed accelerated growth throughout the life cycle of sticklebacks contaminated with EE2 at higher concentration (100 ng.L⁻¹). The author's hypothesis was that steroids can have anabolic effects and improve early growth by increasing food conversion efficiency and leading fishes to reach adult size faster. This highlights that the effects of EE2 exposure on fish growth can vary with life stage, EE2 concentration and the duration of the exposure/recovery.

The effect of the EE2 exposure on sex ratio of stickleback appeared independent of the climatic scenario. Indeed, the contamination of EE2 during the embryo-larval stages led to a higher proportion of females in both climatic scenarios. Xenoestrogenic endocrine disruptors, with EE2 being a preeminent example, are widely recognized for their capacity to induce sex ratio imbalances in fish populations, even at environmental concentrations (Länge et al., 2001; Maunder et al., 2007; Porseryd et al., 2019; Shved et al., 2008). Two plausible hypotheses can be formulated to explain the alteration of the sex ratio observed in this study: 1) an increase in the number of females through feminization of genotypic males, and/or 2) a selective mortality of males. The difficulty in accurately determining the phenotypic sex of immature sticklebacks, coupled with the fact that the genetic sex of fish was not determined in this study, did not allow us to confirm or refute these hypotheses. Yet, the presence of intersexes (ovo-testis) within the two contaminated experimental treatments ("Current-EE2" and "RCP8.5-EE2") showed that an early exposure to EE2 at environmental concentration of 15 ng.L⁻¹, is actually able to feminize, at least partially, stickleback testis, thus impacting the phenotypic sex of fish. Similarly, previous studies using equivalent levels of contamination in stickleback showed intersexes in their populations (Hahlbeck et al., 2004; Maunder et al., 2007) with persistence of these effects even after several months post-contamination (Porseryd et al., 2019). Furthermore, it has been shown that the larval period is a window of sensitivity for xenosettrogens, during which the phenotypic sex of sticklebacks can be altered (Hahlbeck et al., 2004). The presence of oocytes within the testes in fish can impact gonadal endocrine activity and alter plasma sex steroid profiles (Jobling et al., 2002a; Maunder et al., 2007), thereby disrupting steroidogenesis in intersex fish which could ultimately lead to reduced fertility, as shown in intersex roach (*rutilus*) (Jobling et al., 2002b). However, we have to remind that the intersex data collected in the present study are qualitative and derived from a small sample size (few individuals). This excludes any appreciation of the severity of the intersex effect of EE2 early exposure. Taken together, the results in this study suggest that the climate scenario did not modulate the effect of EE2 on sex determination in sticklebacks. It is known that the sex of the fish plays a role in the accumulation of EE2. Females *Pimephales promelas* showed lower uptake of EE2 and lower accumulation in the gills
compared to males (Blewett et al., 2014). This sex-dependent accumulation may potentially contribute to a greater vulnerability of males to the adverse effects of EE2 exposure. However, data from the present study only indicate a more pronounced impact of EE2 on the HSI of males. Gene expression analysis of the neuroendocrine system and estrogenic pathways in the gonads did not show a higher impact of EE2 at least in prepubertal males. At the sampling time, at the end of the present experiment, the GSI values showed in males were low in all treatments indicating that testis are still immature (Sokolowska and Kulczykowska, 2006), with RCP8.5 scenario's values significantly lower than Current scenario's ones. Similarly, the histological analysis of male gonads carried out in this study, confirmed the early stage of spermatogenesis at the sampling time in all the experimental treatments, as expected for prepubertal sticklebacks during the winter period. This prevents the possibility to assess the real impact of the multi-stress on sexually mature fish.

The EE2 effects observed on the sex ratio, the presence of intersex individuals, as well as the qualitative limitations of the histological approach to detect possible modulation by the climate scenario, highlighted the need for a complementary level of analysis. Therefore, we studied the expression levels of genes involved in the neuroendocrine control of reproduction and development at the central level in prepubertal fish. Interestingly, no persistent effects of EE2 exposure on the neuroendocrine control of sexual maturation and development were observed in this study in any scenario and sex. An important effect of the climatic scenarios was shown at least in males. A general trend was observed suggesting a downregulation in the expression profile of the studied genes in the median brain of males acclimated to RCP8.5 scenario compared to the Current scenario. The expression of *gnrh3*, *gnih* (main regulators of maturation (Shao et al., 2019) and *trh* (acting on the thyroid axis, to drive development and growth (Blanton and Specker, 2007), was significantly downregulated in males in the RCP8.5 scenario. Surprisingly, no significant impact on the expression levels of these genes was detected in females exposed to the same climatic scenario. These highlights the sex-specific nature of the impact of environmental stressors on the brain gene profiles in sticklebacks. Interestingly, in our previous study where we evaluated the impact of the combined acidification and warming on adult sticklebacks, we observed the opposite effect (Devergne et al., 2023). The expression level of genes involved in the neuroendocrine control of reproduction showed a general trend of upregulation as well as a higher GSI in males under RCP8.5 scenario (Devergne et al., 2023). The different maturation stage of males (fully mature in the previous study and prepubertal in the present one) could be the reason of the opposite effect of the climatic stress on central expression level and GSI. Another explanation could be related to transgenerational plasticity, a process through which parental environmental experiences shape offspring traits, enabling acclimation to stressors without genetic evolution (Salinas et al., 2013). Indeed, unlike fish employed in the previous study (FO), sticklebacks of the present study (F1) are the offspring of parents already acclimated to the same climatic scenario, in terms of temperature and pH levels. In sticklebacks, several studies have reported cases of transgenerational plasticity in response to environmental conditions, such as temperature (Shama, 2017b, 2015; Shama et al., 2014; Shama and Wegner, 2014) or CO₂ levels (Schade et al., 2014), affecting traits like early-life growth and survival.

We analyzed the expression patterns of genes related to reproduction also at the peripheral level, to specifically test whether the early EE2 exposure could differentially alter the endocrine regulation of the estrogenic signaling in gonads depending of the climatic scenario. In females, the early exposure to EE2 in Current and RCP8.5 scenario resulted in a reduction of *esr1* and *esr2a* expressions. This seems in line with a previous hypothesis of disruption of androgen-estrogen balance in females (Maunder et al., 2007), that would cause a negative feedback and the inhibition of estrogen receptor expression at gonad level. Surprisingly, this effect lasted several months post-contamination. The

effect of EE2 on the gonadal expression of *cyp19a1a* depended on the climatic scenario. EE2 may have amplified the rise of *cyp19a1a* expression levels usually observed at warmer temperatures and probably due to the epigenetic regulation (Voisin et al., 2016). Altogether, this may suggest a long-term effect on ovarian endocrine regulation under climate change condition, although further studies are needed to determine whether such effects involve epigenetic mechanisms. Interestingly, the effect of the scenario alone in females was not significant, even on gonadal aromatase (*cyp19a1a*) expression profile. However, *cyp19a1a* is known to be a thermo-sensitive enzyme (Alix et al., 2020), exhibiting reduced activity *in vivo* and *in situ*, as well as lower expression levels, under high temperature (Miranda et al., 2013). In this study, the temperature experienced at sampling (16°C) was probably low enough not to affect the *cyp19a1a* expression in stickleback gonads. The expression patterns of the three estrogen receptors (*esr1*, *esr2a*, and *esr2b*) were similar across experimental treatments. In teleost the influence of rising temperature on estrogens receptors expression is dependent on various factors such as receptor type, tissue studied, species, sex, developmental stage, and thermal intensity (Bock et al., 2021). In this study, under the RCP8.5 scenario, there was an increase in the expressions of *esr1* and *esr2b* of females, compared to Current scenario. The increase in estrogens receptors expression in females may compensate for the potential decreased affinity of estrogens for their receptors at high temperatures, as demonstrated in other teleost species (Watts et al., 2005). In males, the early exposure to EE2, in Current and RCP8.5 scenarios, induced an upregulation of the *esr2a* expression profile. For instance, previous studies have reported that *esr2a* gene plays a crucial role in the reproductive processes of female fish (Kayo et al., 2019; Lu et al., 2017), and the knockout of both *esr2a* and *esr2b* resulted in the interruption of follicle development and masculinisation (Lu et al., 2017). This is in line with the partial feminization of the testes observed in the present study after an early exposure to EE2.

No alteration of the gene expressions profile of testes under the RCP8.5 scenario was observed, making males more resilient to environmental condition than females at this level of regulation.

## 5. Conclusion

This study tested whether the physiological effects of an exposure to xenestrogen, at environmental concentration (15 ng.L⁻¹) and during early life stages of fish, are modulated by a predicted climatic scenario (+3°C; -0.4 pH unit). We investigated the impact of these multiple stressors on survival, growth and sexual maturation of prepubertal juvenile sticklebacks. The main outcomes observed are summarized in the

<caption>**Table 6**</caption>

Summary of main effects observed for RCP8.5 Scenario, EE2 contamination, and their interaction on the key parameters/ endpoints studied. Symbols: ↑ = significant increase; ↓ = significant decrease; – = no effect observed. Abbreviations: Fulton's index (K), gonado-somatic index (GSI) and hepato-somatic index (HIS).

<table><thead><tr><th>Physiological parameters</th><th>Scenario (RCP8.5 vs Current)</th><th>EE2 (EE2 vs non EE2)</th><th>Scenario × EE2</th></tr></thead><tbody><tr><td>Egg quality</td><td>↑ perivitelline index</td><td>–</td><td>↓ egg diameter in RCP8.5-EE2</td></tr><tr><td>Survival &amp; development</td><td>↓ embryo-larval survival; ↓ time to hatch</td><td>↓ Male sex ratio; ↓ juvenile survival</td><td>↓ juvenile survival in Current-EE2</td></tr><tr><td>Growth &amp; biometric indexes</td><td>↓ growth in standard length; ↑ male GSI; ↓ female K</td><td>↓ growth in standard length; ↓ male K and HSI</td><td>↓ growth in standard in RCP8.5-EE2</td></tr><tr><td>Phenotypic sex</td><td>–</td><td>↑ intersex incidence</td><td>–</td></tr><tr><td>Endocrine control of maturation and estrogenic system</td><td>↓ male brain <i>gnrh3</i>, <i>trh</i>, <i>gnih</i>; ↑ ovarian <i>esr1</i>, <i>esr2b</i></td><td>↓ ovarian <i>esr1</i>, <i>esr2a</i>; ↑ testis <i>esr2a</i></td><td>↑ ovarian <i>cyp19a1a</i> in RCP8.5-EE2</td></tr></tbody></table>
**Table 6.** Our results first revealed that early developmental stages exhibited lower egg quality under multi-stress treatment, potentially limiting embryonic resources. The experimental data indicated that impact of the EE2 early contamination on juvenile survivals depended on the climatic scenario. Furthermore, long-term effect of the multi-stress treatment (RCP8.5-EE2) were also observed on growth, leading to lower body length at prepubertal stage. Regarding the impact on the reproductive axis, the only significant interaction between the EE2 contamination and the scenario was evident for the expression level of *cyp19a1a* in the ovaries, suggesting a carry-over of the estrogenic effect in the RCP8.5 scenario. This also suggests a higher endocrine disruptor potential of EE2 early exposure in predicted future environments. The reproduction, development and growth on fish are globally more resilient to endocrine disruption at Current scenario. Interestingly, the impact of the early estrogenic exposure and the climatic stress on the physiological indexes are sex specific, at least in prepubertal fish (where the phenotypic sex was identified). In the tested experimental treatments, the climatic scenario has a higher impact in the central control of the reproductive axis in males compared to females. Whereas, the early EE2 contamination preferentially affects the gene expression in gonads, with an overall upregulation in males and downregulation in females. Furthermore, the observed skewed sex ratio, associated with the presence of intersexes in both Current-EE2 and RCP8.5-EE2, clearly indicated a feminization process of the population due to EE2 contamination. However, further quantitative investigation would be needed to strengthen the conclusion that this effect is independent of the climate scenario. Altogether, this could suggest a disruption in sexual maturation and future reproductive success, with consequent potential impact of population dynamic (Kidd et al., 2007).

In conclusion, the exposure to a contamination of EE2 during the embryo-larval stage can have long-term effects on stickleback physiology, even after several months post-contamination. This impact can be modulated by the climatic scenario, depending on the physiological endpoints considered. In other words, climate change can modify the physiological response of fish to well-known endocrine disruptors, impacting the resilience of species to predicted future environments.

# 6. Project information

**Supplementary Materials:** The following supporting information can be found in the online version.

Supplementary Table 1: Simulated seasonal variations of the photoperiod in the Bay of Brest

Supplementary Table 2: Statistical tests. Asterisks indicate statistical difference between scenarios.

Supplementary Table 3: Pearson chi-square test with pairwise proportion test on survival for embryo-larval stage and juvenile stage since the formation of the third spine. *p*: *p*-value; *p*.adj: adjusted *p*-value using Holm method. Asterisks indicate statistical difference between scenarios.

Supplementary Table 4: Pairwise proportion test on sex ratio at the end of the experiment. *p*: *p*-value; *p*.adj: adjusted *p*-value using Holm method. Asterisks indicate statistical difference between scenarios.

Supplementary Table 5: Least-Squares Means pairwise comparison test of the stickleback growth in size. SE: standard error, *p*-value: adjusted *p*-value using Tukey method. Asterisks indicate statistical difference between scenarios.

Supplementary Table 6: Sample size table of eggs used for the estimation of the diameter and perivitelline index.

Supplementary Table 7: Sample size table of individuals used for the estimation of the physiological indexes and histology analysis.

Supplementary Table 8: Sample size table of individual brain and gonads used for the gene expression profiles analysis.

# Funding

This work was supported by the French national programme EC2CO-DYCOVI through the ECHANGE project to V.L. (2021–2023) and Ifremer (ICEfish project). J.D. PhD grant was founded by "Université de Bretagne Occidental" and by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program "Investissements d'Avenir" embedded in France 2030.

# CRediT authorship contribution statement

**Jimmy Devergne:** Writing – original draft, Methodology, Formal analysis, Data curation. **Arianna Servili:** Writing – review & editing, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Sylvain Jodet:** Writing – review & editing, Formal analysis, Data curation. **Titouan Brandicourt:** Formal analysis, Data curation. **Christophe Lebigre:** Writing – review & editing, Validation, Methodology, Formal analysis. **Sophie Collet:** Methodology, Data curation. **Olivier Mouchel:** Writing – review & editing, Data curation. **Marie Lou Fleury:** Methodology, Data curation. **Sabine Roussel:** Writing – review & editing, Validation. **Véronique Loizeau:** Writing – review & editing, Project administration, Methodology, Funding acquisition, Formal analysis, 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.

# Acknowledgments

Special thanks to Marie-Madeleine Le Gall and all the members of the PHYTNESS unit (Ifremer-LEMAR) for their assistance in the experimental work and fish maintenance.

# Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.aquatox.2025.107528.

# Data availability

The data presented in this study are available in the supplementary materials.

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