Pauline Bellot

François Brischoux

Hélène Budzinski

Sophie M Dupont

Clémentine Fritsch

Sydney F Hope

Bruno Michaud

Marie Pallud

Charline Parenteau

Louise Prouteau

Steffi Rocchi

Frédéric Angelier

Chronic exposure to tebuconazole alters thyroid hormones and plumage quality in house sparrows (Passer domesticus)

Keywords: passerine birds, agroecosystem, fungicides, sublethal effects, thyroid hormones, body condition, sperm quality

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

It is estimated that, worldwide, only fourteen crops make up the majority of the human diet.

However, all of these crops are susceptible to diseases while growing in the field or after the harvest [START_REF] Strange | Plant Disease : A Threat to Global Food Security[END_REF], and fungal diseases represent a major risk not only to crop yield [START_REF] Fisher | Emerging fungal threats to animal, plant and ecosystem health[END_REF]), but also to food safety [START_REF] Strange | Plant Disease : A Threat to Global Food Security[END_REF]. Many pesticides, and more specifically fungicides, are massively used in agriculture to circumvent these problems and to reduce the economic costs and health issues of fungal pests. For example, fungicides accounted for more than 40% of total pesticide sales from 2011 to 2018 in the European Union [START_REF] Zubrod | Fungicides : An Overlooked Pesticide Class?[END_REF]. Fungicides can even represent up to 90% of the pesticides that are used for certain crops (e.g., orchards, vineyards, [START_REF] Zubrod | Fungicides : An Overlooked Pesticide Class?[END_REF].

Triazoles are a large family of systemic, broad-spectrum fungicides used in agriculture since the 1970s-80s (Ribas e [START_REF] Ribas E Ribas | Is the emergence of fungal resistance to medical triazoles related to their use in the agroecosystems? A mini review[END_REF]. They are designed to interfere with lanosterol 14αdemethylase (CYP51), an enzyme of the cytochrome P450 monooxygenase (CYP) superfamily [START_REF] Becher | Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens[END_REF]. In fungi, CYP51 plays a key role in the biosynthesis of ergosterol, a sterol located in fungal membranes and involved in membrane permeability and fluidity [START_REF] Daum | Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae[END_REF]. One of the main problems associated with agricultural triazoles is their potential effect on non-target species, through non-target physiological mechanisms (e.g., [START_REF] Fernández-Vizcaíno | Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge[END_REF][START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF][START_REF] Bellot | Experimental Exposure to Tebuconazole Affects Metabolism and Body Condition in a Passerine Bird, the House Sparrow (Passer domesticus)[END_REF]. In general, the effects of fungicides, and more particularly triazoles, are understudied in wildlife. Indeed, most studies focus on acute ecotoxicity tests and with model species commonly used in toxicology (e.g., zebrafish, earthworm, Binev et al. 2005;[START_REF] Sancho | Short-term exposure to sublethal tebuconazole induces physiological impairment in male zebrafish (Danio rerio)[END_REF][START_REF] Andreu-Sánchez | Acute toxicity and bioconcentration of fungicide tebuconazole in zebrafish (Danio rerio)[END_REF][START_REF] Raby | Acute Effects of Binary Mixtures of Imidacloprid and Tebuconazole on 4 Freshwater Invertebrates[END_REF][START_REF] Rico | Lethal and sub-lethal effects of five pesticides used in rice farming on the earthworm Eisenia fetida[END_REF]. Although these studies elucidate the biological targets of triazoles and their modes of action, they are not necessarily representative of the effects of triazoles at concentrations found in the environment. In addition, model species are often laboratory-raised species, and thus, are not always representative of the ecosystems where triazoles are used. For wildlife, acute exposures to high concentrations of triazoles are scarce and correspond to sporadic events, such as accidental contamination (e.g., [START_REF] Capel | Accidental input of pesticides into the Rhine River[END_REF]. Therefore, to better understand the sublethal effects that triazoles may have on non-target species, it is necessary to study the impact of long-term exposure of triazoles on wildlife, and especially vertebrate sentinel species of agroecosystems. Among vertebrates, birds are particularly relevant to study this question because many bird species exploit or live near agricultural areas and are potentially exposed to triazoles through multiple routes of exposure [START_REF] Robinson | The diet of seed-eating birds on lowland farmland[END_REF][START_REF] Lopez-Antia | Risk assessment of pesticide seed treatment for farmland birds using refined field data[END_REF][START_REF] Fernández-Vizcaíno | Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge[END_REF], 2022;[START_REF] Ortiz-Santaliestra | Egg overspray with herbicides and fungicides reduces survival of red-legged partridge chicks[END_REF][START_REF] Angelier | High contamination of a sentinel species by azoles in vineyards : A study of common blackbirds (Turdus merula) in multiple habitats in western France[END_REF]). This contamination is likely associated with chronic exposure to low concentrations of triazoles, for example in water, soil, air and seeds (e.g., [START_REF] Kahle | Azole Fungicides : Occurrence and Fate in Wastewater and Surface Waters[END_REF][START_REF] Rokbani | Heterogeneous oxidation of two triazole pesticides (diniconazole and tebuconazole) by OH-radicals and ozone[END_REF][START_REF] Pelosi | The contributions of enchytraeids and earthworms to the soil mineralization process in soils with fungicide[END_REF][START_REF] Lopez-Antia | Risk assessment of pesticide seed treatment for farmland birds using refined field data[END_REF], 2021). In this context, specific bird species (e.g., non-migratory species) can be relevant sentinel species of agroecosystems because they rely on this specific environment to complete their annual life cycle. As such, they should be chronically exposed to specific triazoles that are used for agricultural purposes and, thus, represent a powerful model to better understand the impact of environmental concentrations of triazoles on key organismal traits. Experimental studies have shown that chronic or subchronic exposure to triazoles can induce reprotoxic effects in birds [START_REF] Grote | epoxiconazole causes changes in testicular histology and sperm production in the japanese quail (Coturnix coturnix japonica)[END_REF][START_REF] Lopez-Antia | Brood size is reduced by half in birds feeding on flutriafol-treated seeds below the recommended application rate[END_REF]). These effects may arise from an interaction of triazoles with non-fungal sterol synthesis [START_REF] Zarn | Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase[END_REF]. Thus, triazoles can act as major endocrine disruptors by modulating the expression and activity of enzymes such as cytochrome P450 aromatase [START_REF] Goetz | Disruption of Testosterone Homeostasis as a Mode of Action for the Reproductive Toxicity of Triazole Fungicides in the Male Rat[END_REF][START_REF] Taxvig | Endocrine-Disrupting Activities In Vivo of the Fungicides Tebuconazole and Epoxiconazole[END_REF][START_REF] Jacobsen | Persistent developmental toxicity in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides[END_REF][START_REF] Warrilow | Azole Affinity of Sterol 14α-Demethylase (CYP51) Enzymes from Candida albicans and Homo sapiens[END_REF][START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF] with potential implications for multiple metabolic pathways, and also for reproduction [START_REF] Poulsen | Tebuconazole disrupts steroidogenesis in Xenopus laevis[END_REF][START_REF] Huang | Endocrine disruption by azole fungicides in fish : A review of the evidence[END_REF]. Indeed, pesticides, and possibly triazoles can have a direct impact on reproduction by causing testicular abnormalities that affect spermatogenesis and the mobility and quality of germ cells (carbamates and neonicotinoids [START_REF] Tokumoto | Effects of Exposure to Clothianidin on the Reproductive System of Male Quails[END_REF][START_REF] Mohanty | Thyroid disrupting pesticides impair the hypothalamic-pituitarytesticular axis of a wildlife bird, Amandava amandava[END_REF]Humann-Guilleminot et al. 2019;triazoles: Machado-Neves et al. 2018). For example, triazoles could alter the fertility of individuals, notably through a decrease in sperm quantity and quality (e.g., [START_REF] Taxvig | Endocrine-Disrupting Activities In Vivo of the Fungicides Tebuconazole and Epoxiconazole[END_REF][START_REF] Grote | epoxiconazole causes changes in testicular histology and sperm production in the japanese quail (Coturnix coturnix japonica)[END_REF][START_REF] Pereira | Sperm quality of rats exposed to difenoconazole using classical parameters and surface-enhanced Raman scattering: classification performance by machine learning methods[END_REF].

More recently, triazoles have also been suspected to affect key endocrine mechanisms, such as the Hypothalamic-Pituitary-Thyroid (HPT) axis and the resulting regulation of thyroid hormones (THs). These hormones also play a key role in gonadal development and the regulation of seasonal reproduction in mammals and birds [START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF][START_REF] Mohanty | Thyroid disrupting pesticides impair the hypothalamic-pituitarytesticular axis of a wildlife bird, Amandava amandava[END_REF]. The first evidence highlighting the effect of triazoles on thyroid disruption comes from the study of aquatic models (e.g., zebrafish, [START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF][START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF]). However, the potential effects of azole fungicides on thyroid functioning have been overlooked in non-aquatic vertebrates. In adults birds, the HPT axis is also a key physiological system, which is, for example, involved in (i) seasonal processes, notably the moulting process that allows birds to renew their plumage, and (ii) metabolism and thermoregulation (reviewed by [START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF].

An increase in THs, such as triiodothyronine (T3) and thyroxine (T4), triggers the initiation of moulting [START_REF] Groscolas | The endocrine control of reproduction and molt in male and female emperor (Aptenodytes forsteri) and adelie (Pygoscelis adeliae) penguins[END_REF][START_REF] Groscolas | How to Molt While Fasting in the Cold : The Metabolic and Hormonal Adaptations of Emperor and King Penguins[END_REF][START_REF] Jenni-Eiermann | Temporal uncoupling of thyroid hormones in Red Knots : T3 peaks in cold weather, T4 during moult[END_REF], as experimentally shown in the white-crowned sparrow (Zonotrichia leuchophrys gambelii) [START_REF] Pérez | Effects of thyroid hormone manipulation on pre-nuptial molt, luteinizing hormone and testicular growth in male white-crowned sparrows (Zonotrichia leuchophrys gambelii)[END_REF]. Similarly, TH levels have been linked to metabolism in multiple bird species and increased TH levels are associated with increased maintenance costs (basal metabolic rate, [START_REF] Chastel | Pre-breeding energy requirements : Thyroid hormone, metabolism and the timing of reproduction in house sparrows Passer domesticus[END_REF][START_REF] Elliott | Thyroid hormones correlate with resting metabolic rate, not daily energy expenditure, in two charadriiform seabirds[END_REF].

In this study, we experimentally tested the impact of environmental concentration of tebuconazole on key traits of a sentinel bird species of agroecosystems, the house sparrow (Passer domesticus). Tebuconazole is the most widely used triazole fungicides in the world and it is found in multiple agroecosystems, especially in air [START_REF] Désert | Spatial and temporal distribution of current-use pesticides in ambient air of Provence-Alpes-Côte-d'Azur Region and Corsica, France[END_REF], soil [START_REF] Kalogridi | Part II: temporal and spatial distribution of multiclass pesticide residues in lake sediments of northern Greece: application of an optimized MAE-LC-MS/MS pretreatment and analytical method[END_REF], and water [START_REF] Kahle | Azole Fungicides : Occurrence and Fate in Wastewater and Surface Waters[END_REF]. To date, only a few studies have evaluated the impact of tebuconazole on terrestrial non-target vertebrate species: partridges [START_REF] Fernández-Vizcaíno | Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge[END_REF][START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF]), bats (Machado-Neves et al. 2018), and amphibians [START_REF] Poulsen | Tebuconazole disrupts steroidogenesis in Xenopus laevis[END_REF][START_REF] Bernabò | Tissues injury and pathological changes in Hyla intermedia juveniles after chronic larval exposure to tebuconazole[END_REF]. However, none of these experimental studies have tested the impact of tebuconazole on the levels of TH and related physiological traits. Thus, the aim of this study was to experimentally test the impact of exposure to tebuconazole on the HPT axis in wild-caught house sparrows that were kept in captivity under controlled conditions. We also examined the impact of tebuconazole on plumage quality and body condition, two phenotypic traits that are closely related to the HPT axis, as well as on sperm quality.

To do this, we randomly chose half of our wild-caught captive house sparrows to be exposed to tebuconazole through their drinking water ('exposed sparrows'), while the other sparrows were not exposed ('control sparrows'). We then examined the impact of this experimental treatment on the (1) levels of THs (T3 and T4), (2) plumage quality, using the morphometric characteristics of feathers, (3) body condition (body mass corrected for body size: body condition index (BCI)), and (4) the morphology of the sperm. Plumage quality, body condition and sperm quality are closely related to individual performance and fitness. Indeed, feathers enable birds to perform vital functions such as flight, signalling and thermoregulation [START_REF] Swaddle | Plumage Condition Affects Flight Performance in Common Starlings : Implications for Developmental Homeostasis, Abrasion and Moult[END_REF][START_REF] Dawson | Rate of moult affects feather quality : A mechanism linking current reproductive effort to future survival[END_REF][START_REF] Jenni | The Biology of Moult in Birds[END_REF]. Similarly, the BCI is a commonly used proxy of the physiological state of an individual, and disruption of metabolism usually translates into an abnormal BCI. Finally, sperm morphology may be an indicator of sperm quality and reproductive success [START_REF] Helfenstein | Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus[END_REF]. We predicted that exposure to tebuconazole would (1) disrupt the HPT axis and result in reduced TH levels (T3 and T4), as previously shown in laboratory aquatic model species [START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF]); (2) affect moult, with a detrimental impact on plumage quality (smaller and less dense feathers in exposed sparrows); (3) result in an increased BCI, because tebuconazole should lower metabolism [START_REF] Bellot | Experimental Exposure to Tebuconazole Affects Metabolism and Body Condition in a Passerine Bird, the House Sparrow (Passer domesticus)[END_REF], and thus maintenance costs, through its impact on TH levels (reduced TH levels, prediction 1), and (4), reduce sperm quality by altering sperm morphology.

Material and Method

Study species

This study was conducted on captive adult's house sparrows (Passer domesticus, Linnaeus, 1758). House sparrows are an appropriate sentinel species for assessing the contamination of agroecosystems because they are sedentary, strongly linked to agricultural environments (feeding, nesting, and breeding), and can be impacted by agricultural practices and landscapes [START_REF] Swaileh | Monitoring urban heavy metal pollution using the House Sparrow (Passer domesticus)[END_REF]Humann-Guilleminot et al. 2019). A total of 39 sparrows were used in this experiment (n females = 20 and n males = 19) that were captured in Western France in 2019 (Secondigné-sur-Belle). Birds were acclimated to captivity for two weeks before the start of the experiment. All sparrows were maintained in captivity in 4 similar outdoor aviaries (length: 3 m, width: 2 m, height: 2 m). The birds were maintained in oudoor aviaries to keep them under natural seasonal temperature cycles. Birds were split in the aviaries by treatment type and sex: control birds were split into two aviaries for a total of 9 females and 9 males. Exposed birds were split into the two other aviaries for a total of 11 females and 10 males. As females and males were kept in separate aviaries for the duration of the experiment, no eggs were laid. These sample sizes differ slightly between the two treatments, as some of the birds did not adapt well to captivity and were released.

Each aviary was equipped with artificial shelter boxes (i.e., one box per bird). The sparrows were fed ad libitum with a mixture of commercial seeds, millet sprays, grit, and salt/mineral blocks. In all aviaries, water was dispensed ad libitum through a drinking trough and was changed on a weekly basis.

Experimental design

Individuals were exposed to tebuconazole through their drinking water a source of exposure that is thus far less studied in comparison to the ingestion of treated seeds (e.g., [START_REF] Lopez-Antia | Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole[END_REF][START_REF] Lopez-Antia | Brood size is reduced by half in birds feeding on flutriafol-treated seeds below the recommended application rate[END_REF][START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF][START_REF] Fernández-Vizcaíno | Bird exposure to fungicides through the consumption of treated seeds : A study of wild red-legged partridges in central Spain[END_REF]). In the two control aviaries, control individuals had access to regular tap drinking water without tebuconazole. In the two exposed aviaries, tebuconazole (Sigma-Aldrich, CAS. No: 107534-96-3, purity: ≥ 98.0 %) was diluted with tap water using a precision balance (± 0.0001 g) to reach a concentration of 550 µg.L-1.As in the control group, the water was changed once per week.

Based and showed that the individuals from the control group were free of contamination and that the individuals from the exposed group had an average plasma concentration of 59.7 pg.g -1

tebuconazole. Moreover, we chose this water concentration of tebuconazole because we have previously shown that it results in an environmentally realistic plasma contamination [START_REF] Angelier | High contamination of a sentinel species by azoles in vineyards : A study of common blackbirds (Turdus merula) in multiple habitats in western France[END_REF]. Exposure to tebuconazole started on 6 November 2019 and lasted until 29 June 2020 (i.e., until the end of sampling, see below, Fig 1). We specifically decided to expose individuals to tebuconazole during seven months to test the impact of a long-term exposure to environmental doses of tebuconazole on TH and key physiological parameters. Although the duration of exposure to tebuconazole is rather long in our experiment, such a long-term chronic exposure can be found in some agroecosystems that rely on multiple treatments of fungicides (e.g., vineyards where tebuconazole is used from April to August), and also in mixed landscapes with multiple crops that used triazoles during different periods (e.g., cereal crops in autumn and winter, orchards, vegetables, vineyards in spring and summer). In order to verify that there was no major difference in drinking behaviour due to treatment, we analysed the hydration state (plasma osmolarity, Vapro2 osmometer, Elitech group, France) of several control and exposed sparrows (for individuals with sufficient plasma left, n = 9 for control sparrows and n = 9 for exposed sparrows). This parameter has been shown to vary according to sparrow drinking behaviour [START_REF] Brischoux | Short-term dehydration influences baseline but not stress-induced corticosterone levels in the house sparrow (Passer domesticus)[END_REF]. We found no difference in plasma osmolarity (F(1,16) = 0.64, p = 0.436), suggesting that there is no major difference in drinking behaviour between control and exposed sparrows. Tebuconazole concentrations of exposed and control drinking water were assayed by an independent accredited analytical laboratory (QUALYSE laboratory, La Rochelle, France). Tebuconazole was undetectable in control water while tebuconazole concentrations were 550 µg.L -1 in the water of exposed aviaries (by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)).

Feather morphometry

We used a standardized protocol to study plumage quality of exposed and control sparrows. In

April 2020, we plucked the two inner rectrices (central tail feathers) of all sparrows, Fig 1.

These plucked feathers had grown before birds were captured and before the experimental exposure to tebuconazole (i.e., in August/September 2019 during the annual post-nuptial moult)

and they, therefore, did not allow us to test the impact of tebuconazole exposure on plumage quality. However, feather plucking stimulates the growth of new feathers, and all inner rectrices had regrown within a month in most of individuals (n = 30). Then, in May 2020, after 6 months of exposure to tebuconazole, we plucked these two inner rectrices that had regrown while birds were either exposed to tebuconazole or not in order to test the impact of tebuconazole exposure on plumage quality, Fig 1 . Five morphometric parameters were then assessed on the plucked tail feathers of control and exposed house sparrows (initial and regrown). The morphometric parameters of both rectrices of each individual were averaged for each measurement timepoint.

Only individuals with data for both measurement times (initial: measure 1, regrown: measure 2, Fig 1 ) were considered for statistical analysis (n = 30 sparrows, with exposed and control individuals equally distributed). The first three morphometric parameters were measured using a high-resolution balance (feather mass, ± 0.01 mg) and digital caliper (total feather length and diameter of the calamus ± 0.01 mm). Standardized pictures of the feather were also taken, and feather area was calculated using a dedicated image software (ImageJ, v1.53). Lastly, the density of feathers was calculated by dividing the mass of the feathers by their area.

Thyroid hormones

After 6 months of exposure, on 26 and 27 May, blood samples (100-150µL) were successfully collected from all but one individual by puncture of the brachial vein to assess the effect of the treatment on total triiodothyronine (T3) and total thyroxine (T4) of the house sparrows (n = 37,

Fig 1)

. All blood samples were centrifuged, and red blood cells and plasma were separated and stored at -20 °C until the assay. Plasma concentrations of total T3 and T4 were determined using radioimmunology at the Centre d'Études Biologiques de Chizé (CEBC) as previously described by [START_REF] Chastel | Pre-breeding energy requirements : Thyroid hormone, metabolism and the timing of reproduction in house sparrows Passer domesticus[END_REF]. The lower limits of detection for T3 and T4 were 0.08 ng.mL - 1 and 0.602 ng.mL -1 respectively. For each hormone, all samples were run in duplicates in a single assay. The intra-assay CVs were 9.49% and 11.36% for T3 and T4, respectively.

Individual condition

To assess the effect of the treatment on the body condition of the sparrows, body mass was

Sperm morphology

In order to test the effect of the experimental treatment on the sperm morphology of the sparrows, we took a sperm sample during the breeding season (10 June 2020, Fig 1). These sperm samples were obtained by faecal and abdominal massage described in detail by [START_REF] Girndt | Method matters : Experimental evidence for shorter avian sperm in faecal compared to abdominal massage samples[END_REF]. This method allowed us to have a successful collection for 17 males including 8 males from the control group and 9 males from the exposure group. Each sperm sample was collected directly into a 0.5 ml microtube containing 200 μl of 5% formalin. At the laboratory, 10 µl of each sample was put on a slide to perform microscopic observation of sperm cells at 400x magnification on a bright field setting. For each individual, 100 photos of distinct intact sperm were taken (based on the method of [START_REF] Girndt | Method matters : Experimental evidence for shorter avian sperm in faecal compared to abdominal massage samples[END_REF]2019). The different components of the sperm (i.e., head, including acrosome, flagellum, including midpiece) were measured by a single observer from digital images using ImageJ software (v1.53). Total sperm length was calculated as the sum of head and flagellum measurements [START_REF] Girndt | Method matters : Experimental evidence for shorter avian sperm in faecal compared to abdominal massage samples[END_REF]2019).

Statistical analysis

All statistical analyses were carried out in Rstudio (R version 4.2.1). To test the effect of the experimental treatment (i.e., exposed to tebuconazole or control) on the levels of TH, feather morphometry, body condition (BCI), and sperm length, linear mixed models (LMM) and linear models (LM) were used. All models were selected using the backward stepwise selection method by eliminating non-significant terms (p > 0.100). After conducting LMMs, specific comparisons were conducted using contrast procedures with the Satterthwaite approximation to describe the differences that were found between different groups of birds. The contrasts were made on the selected minimal model. We checked that all models met the assumptions of normality and homoscedasticity of residuals.

First, the plasma levels of T3 and T4 were analysed using linear models (LM) with two explanatory variables (treatment: control or exposed; sex: female or male) and their interaction.

A log-transformed function was applied to the T4 thyroid hormone data to improve the distribution and homogeneity of the model residuals.

Second, because the morphometric parameters of the feathers were correlated, a principal component analysis (PCA) was performed. The first principal component (PC1) of the PCA explained 57.4% of the variation. PC1 was significantly and positively correlated with total feather length, (r = 0.92, p < 0.001), feather mass (r = 0.91, p < 0.001), feather area (r = 0.80, p < 0.001), diameter of the calamus (r = 0.60, p < 0.001), and feather density (r = 0.42, p < 0.001).

The second principal component (PC2) explained 28.9% of the variation.

Results

Thyroid Hormones (T3 and T4)

The "Treatment" factor did not significantly influence the T3 levels of sparrows (Table 1), whereas there was a significant effect of the "Sex" factor (Table 1), with higher T3 concentrations in females than in males (Fig 2 .A). The treatment had a significant effect on the levels of T4 (Table 1). Individuals exposed to tebuconazole had significantly lower T4 levels than controls (Fig 2 .B). T4 levels were not influenced by sex (Table 1). The "Treatment x Sex" interaction had no significant effect on T3 or T4 levels (Table 1), demonstrating that the influence of the experimental treatment on TH did not differ significantly between sexes (Fig

2.A.B).

Feather morphometrics

PC1 and PC2 scores significantly varied according to the "Time" factor (Table 2). Specifically, the regrown feathers were overall smaller and less dense relative to the initial feathers (Fig 3).

Although there was no significant effect of either the "Sex" or "Treatment" factors on PC1 and PC2 scores (Table 2), there was a significant effect of multiple interactions on both PC1 and PC2 scores (Table 2), demonstrating that the influence of the treatment on PC1 and PC2 was complex and differed between sexes and groups.

Regarding PC1 scores, the "Time x Sex" and "Time x Treatment x Sex" interactions were significant (Table 2), demonstrating that the influence of the experimental treatment on this parameter differed significantly depending on the time of measurement (i.e., before or after feather regrowth) and the sex of the individuals (Fig Regarding PC2 scores, the "Treatment x Sex", and "Time x Treatment" interactions were significant (Table 2), demonstrating that the influence of the experimental treatment on this parameter differed significantly depending on the time of measurement (i.e., before or after feather regrowth) and the sex of the individuals (Fig Specifically, feathers of exposed males were denser than those of control males before exposure to tebuconazole (contrast: t(48.5) = -1.94, p = 0.058) but this difference was not significant for the regrown feathers, i.e., after exposure (contrast: t(48.5) = -1.37, p = 0.178, Fig 3 .D).

Body Condition Index

Although the factors "Sex", "Time", and "Treatment" did not significantly influence the BCI of individuals, the "Time x Treatment", and "Time x Treatment x Sex" interactions had significant effects on the BCI of the individuals (Table 3). This showed that the influence of the experimental treatment on the BCI differs significantly according to the sex of the individual Table 3: Effects of the timing of mass measurement (M1, M2 or M3), treatment (control or exposed to tebuconazole), sex (female or male), and their interactions on the BCI of sparrows.

Individual identity was added as a random factor. Significant terms are indicated by the following codes: '***' < 0.001 '**' < 0.010 '*' < 0.050 '.' < 0.100. All models were selected using the backward stepwise selection method by eliminating non-significant terms (p > 0.100). The abbreviations used are as follows: Degrees of freedom in numerator (NumDF), degrees of freedom in denominator (DenDF), the Chi-square statistic (khi²-value), the p-value associated (Pr (> khi²)).

Sperm morphology

Sperm head length, flagellum length, and total length were not significantly affected by treatment (control or exposed, Table 4). Sperm head length averaged (mean ± SE): 14.85 µm ± 0.033 for controls and 14.68 µm ± 0.027 for exposed sparrows. The length of the sperm flagellum was on average 86.95 µm ± 0.15 for the controls and 87.09 ± 0.12 for the exposed sparrows. Finally, the total length of the spermatozoa was 101.80 µm ± 0.16 for the controls and 101.77 µm ± 0.12 for the exposed sparrows.

Discussion

In this study, we showed for the first time in wild-caught birds that chronic exposure to a widely used triazole fungicide (tebuconazole) could significantly reduce the levels of T4. However, we did not find any effect of such exposure to tebuconazole on the levels of T3. In female sparrows, exposure to tebuconazole also affected the structure of the feathers that grew during the experiment, with larger but less dense feathers for exposed individuals compared to controls.

In addition, body condition did not vary considerably throughout the experiment, except for the control females that experienced a significant increase in their body condition towards the end of the experiment. Finally, sperm morphology was not affected by the treatment. Overall, we demonstrated sublethal effects of tebuconazole on key physiological parameters, such as the HPT axis and one other related physiological trait (plumage quality) in vertebrates that are commonly found in agroecosystems.

Thyroid hormones

Although T3 levels were not significantly affected by the chronic exposure to tebuconazole, our experimental treatment was associated with a significant decrease in circulating T4 levels.

In agreement with our results, previous studies have shown that an exposure to tebuconazole could induce an alteration of the HPT axis, and notably a decrease in T4 levels in laboratory model species [START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF][START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF]. Indeed, zebrafish showed a significant decrease in T4 levels when exposed to tebuconazole concentrations of 200 and 500 µg.L -1 [START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF].

Similar effects were also found in zebrafish larvae, with a significant decrease in T4 levels when larvae were exposed to a tebuconazole concentration of 4 mg.L -1 [START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF], although this effect was not found at lower concentrations (1 and 2 mg.L -1 ). Furthermore, and interestingly, [START_REF] Bernabò | Effects of long-term exposure to two fungicides, pyrimethanil and tebuconazole, on survival and life history traits of Italian tree frog (Hyla intermedia)[END_REF] found an effect of tebuconazole on the success and speed of metamorphosis in tadpoles, suggesting a disrupting effect of tebuconazole on the HPT axis.

Here, we showed for the first time that such dysregulation of the HPT axis may also occur in wild-caught terrestrial vertebrates. Importantly, the activity of the thyroid gland is under the control of the pituitary, which governs the biosynthesis and secretion of the thyroid stimulating hormone (TSH, encoded by tshα and tshβ, Fig 5, [START_REF] Hall | Thyroid-stimulating Hormone Response to Synthetic Thyrotrophin-releasing Hormone in Man[END_REF][START_REF] Harris | The Physiological Role of Thyrotropin-Releasing Hormone in the Regulation of Thyroid-Stimulating Hormone and Prolactin Secretion in the Rat[END_REF][START_REF] Capen | Correlation of mechanistic data and histopathology in the evaluation of selected toxic endpoints of the endocrine system[END_REF][START_REF] Stoker | Evaluation of ammonium perchlorate in the endocrine disruptor screening and testing program's male pubertal protocol : Ability to detect effects on thyroid endpoints[END_REF]. When TH levels become insufficient, the levels of TSH increase in order to stimulate the secretion of THs by the thyroid and restore homeostasis. Some studies have shown that exposure to azoles (difenoconazole, hexaconazole and tebuconazole, [START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF][START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF]) cause a significant increase in the transcription of genes related to TSH production and secretion (i.e., tshβ), which could result from a stimulation of the HPT axis in response to decreased T4 levels [START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF][START_REF] Pirahanchi | Physiology, Thyroid Stimulating Hormone[END_REF]). To test this hypothesis, it would be interesting now to measure the transcriptomic activity of tshβ to know if it is inversely proportional to the decrease in T4 observed in the sparrows exposed to tebuconazole or not.

Contrary to our prediction, we did not find any effect of tebuconazole on the levels of T3. [START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF] showed that exposing zebrafish to tebuconazole caused a significant decrease in T3 levels (dose: 200 and 500 µg.L -1 ) but [START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF] reported in another study that exposure to tebuconazole resulted in a significant increase in T3 levels in zebrafish larvae (4 mg.L -1 ).

Interestingly, [START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF], found similar results to our study. Indeed, after exposing zebrafish larvae to difenoconazole (another triazole), a significant decrease in T4 levels was observed while no significant effect was observed on T3 levels. Altogether, these results suggest that the effect of tebuconazole on T3 levels may depend on the dose of tebuconazole, lifehistory stages, and/or species.

At the mechanistic level, the decrease in T4 levels may be enhanced by an increased activation of type II deiodase (encoded by dio2, Fig 5 .A). Type II deiodase is involved in the catalytic conversion of T4 to its more active form T3 [START_REF] Bianco | Deiodinases : Implications of the local control of thyroid hormone action[END_REF], so its activation could lead to a decrease in the stock of T4 (as suggested by Pandey & Mohanty, 2015, Fig 5.A). Supporting this idea, [START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF] found that difenoconazole can cause an increase in dio2 transcription. In agreement with the results of [START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF], we did not observe any change in the levels of T3, although an increase in the conversion of T4 to T3 by type II deiodase should logically lead to an increase in T3 levels. However, the levels of T3 also depend on the activity of TH-degrading enzymes, such as type III deiodase (dio3, [START_REF] Bianco | Deiodinases : Implications of the local control of thyroid hormone action[END_REF], which activates the conversion of T3 into inactive forms (diiodothyronine, T2, [START_REF] Dentice | Local impact of thyroid hormone inactivation[END_REF]. Therefore, tebuconazole could affect the activation of these enzymes (dio2 and dio3)

and result in decreased T4 levels, but stable T3 levels [START_REF] Dentice | Local impact of thyroid hormone inactivation[END_REF][START_REF] Pandey | The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupt the pituitary-thyroid axis of a wildlife bird[END_REF]. Alternatively, tebuconazole could also affect T4 transcription. If the transcriptomic activity of T4 is decreased but the levels of T3 are maintained, it is possible that exposed individuals show an increased biosynthesis of T3 independently of T4 in order to compensate for the decrease in T4 levels (Fig 5 .B). Under normal circumstances, about 80% of T3 is formed from T4, while only 20% is produced directly by the thyroid [START_REF] Pirahanchi | Physiology, Thyroid Stimulating Hormone[END_REF]. Thus, as suggested by [START_REF] Pandey | The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupt the pituitary-thyroid axis of a wildlife bird[END_REF], the induction of a T4-independent T3 biosynthetic pathway could explain why T4 levels decrease but T3 levels are maintained (Fig

5.B).

Therefore, there is no clear consensus on how tebuconazole affects the thyroid system. Our study shows that tebuconazole seems to disrupt the HPT axis in wild terrestrial vertebrates, as previously found in aquatic models [START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF][START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF][START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF]). However, its mode of action could be complex and may involve several metabolic pathways (Fig. 5, see also [START_REF] Kjaerstad | Effects of azole fungicides on the function of sex and thyroid hormones[END_REF][START_REF] Yang | Effects of pubertal exposure to thiazole-Zn on thyroid function and development in female rats[END_REF][START_REF] Yu | Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole[END_REF][START_REF] Liang | Exposure to difenoconazole causes changes of thyroid hormone and gene expression levels in zebrafish larvae[END_REF][START_REF] Li | Parental exposure to tebuconazole causes thyroid endocrine disruption in zebrafish and developmental toxicity in offspring[END_REF][START_REF] Zhang | Amphibian (Rana nigromaculata) exposed to cyproconazole : Changes in growth index, behavioral endpoints, antioxidant biomarkers, thyroid and gonad development[END_REF]). To further understand the impact of tebuconazole on the HPT axis, it would be relevant to use a transcriptomic approach to identify the biological targets responsible for this disruption of the thyroid axis.

Feather quality

After exposure to tebuconazole, exposed individuals had larger but less dense feathers than the controls, but this effect was only found in females. To our knowledge, our study is the first to provide evidence for an effect of triazoles on plumage quality. Although we could not demonstrate that these effects were linked to a disruption of the HPT axis, this is plausible because the relationship between TH and the moulting process is well established [START_REF] Daum | Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae[END_REF][START_REF] Wilson | The Annual Cycle of Thyroid Activity in White-Crowned Sparrows of Eastern Washington[END_REF][START_REF] Groscolas | How to Molt While Fasting in the Cold : The Metabolic and Hormonal Adaptations of Emperor and King Penguins[END_REF][START_REF] Cherel | Plasma thyroid hormone pattern in king penguin chicks : A semialtricial bird with an extended posthatching developmental period[END_REF][START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF]. Previous studies have shown that moulting can be induced by the administration of exogenous TH, while the removal of the thyroid gland leads to the inhibition of moult or regrowth of artificially plucked feathers [START_REF] Voitkevich | The feathers and plumage of birds. The feathers and plumage of birds[END_REF][START_REF] Hahn | Adjustments of the prebasic molt schedule in birds[END_REF][START_REF] Kuenzel | Neurobiology of molt in avian species[END_REF][START_REF] Pérez | Effects of thyroid hormone manipulation on pre-nuptial molt, luteinizing hormone and testicular growth in male white-crowned sparrows (Zonotrichia leuchophrys gambelii)[END_REF].

We found that tebuconazole caused a decrease in circulating T4 levels in exposed sparrows and several studies have demonstrated that T4 plays a key role in the moulting process of birds [START_REF] Groscolas | How to Molt While Fasting in the Cold : The Metabolic and Hormonal Adaptations of Emperor and King Penguins[END_REF][START_REF] Cherel | Plasma thyroid hormone pattern in king penguin chicks : A semialtricial bird with an extended posthatching developmental period[END_REF][START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF]. For example, [START_REF] Groscolas | The endocrine control of reproduction and molt in male and female emperor (Aptenodytes forsteri) and adelie (Pygoscelis adeliae) penguins[END_REF], showed a positive correlation between peak plasma T4 and the onset of moult in birds, suggesting a more important role of T4 than T3 in this process [START_REF] Groscolas | The endocrine control of reproduction and molt in male and female emperor (Aptenodytes forsteri) and adelie (Pygoscelis adeliae) penguins[END_REF]. These findings are supported by several studies in multiple bird species [START_REF] Decuypere | Physiological basis of induced moulting and tissue regeneration in fowls[END_REF][START_REF] Cherel | Fasting in king penguin. II. Hormonal and metabolic changes during molt[END_REF][START_REF] Cherel | Plasma thyroid hormone pattern in king penguin chicks : A semialtricial bird with an extended posthatching developmental period[END_REF][START_REF] Jenni-Eiermann | Temporal uncoupling of thyroid hormones in Red Knots : T3 peaks in cold weather, T4 during moult[END_REF][START_REF] Vézina | Hormonal correlates and thermoregulatory consequences of molting on metabolic rate in a northerly wintering shorebird[END_REF].

According to these studies and the results that we obtained on T4, we can hypothesise that the interference of tebuconazole with the HPT axis may have caused a change in the quality of the feathers that regrew during the phase of exposure to tebuconazole. However, it is important to note that we are not aware of any study that has linked circulating TH levels with feather quality because most studies focused on the onset or the rate of molting rather than on the quality of the feathers produced (e.g., [START_REF] Decuypere | Physiological basis of induced moulting and tissue regeneration in fowls[END_REF][START_REF] Cherel | Plasma thyroid hormone pattern in king penguin chicks : A semialtricial bird with an extended posthatching developmental period[END_REF][START_REF] Pérez | Effects of thyroid hormone manipulation on pre-nuptial molt, luteinizing hormone and testicular growth in male white-crowned sparrows (Zonotrichia leuchophrys gambelii)[END_REF]). In addition,

we found an effect of tebuconazole on feather quality mainly in females, while the T4 levels of both males and females were affected by tebuconazole. This suggests that the observed effects on feather quality could also be related to the effect of tebuconazole on other mechanisms. For example, feather quality and moult processes have been also linked to levels of glucocorticoids (corticosterone in birds) and testosterone [START_REF] Nolan | Testosterone and Avian Life Histories : Effects of Experimentally Elevated Testosterone on Prebasic Molt and Survival in Male Dark-Eyed Juncos[END_REF][START_REF] Stoehr | The effects of elevated testosterone on plumage hue in male House Finches[END_REF][START_REF] Desrochers | Exogenous and endogenous corticosterone alter feather quality[END_REF][START_REF] Siefferman | Effect of Prenatal and Natal Administration of Testosterone on Production of Structurally Based Plumage Coloration[END_REF][START_REF] Jenni-Eiermann | Corticosterone : Effects on feather quality and deposition into feathers[END_REF]. Previous studies have found that triazoles can also affect the synthesis of these two steroids in vertebrates through their action on cytochrome P450 enzymes [START_REF] Goetz | Disruption of Testosterone Homeostasis as a Mode of Action for the Reproductive Toxicity of Triazole Fungicides in the Male Rat[END_REF][START_REF] Taxvig | Endocrine-Disrupting Activities In Vivo of the Fungicides Tebuconazole and Epoxiconazole[END_REF][START_REF] Yang | Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats[END_REF][START_REF] Draskau | In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole[END_REF]. Therefore, disruption of sex hormones, such as testosterone, could potentially explain the sex-dependent effect of tebuconazole on feather quality. Further studies of these endocrine axes are therefore required to better understand the impact of triazoles on molt processes and feather quality.

The regrown feathers of the exposed females were overall larger and less dense relative to control females. This structural modification of feathers can potentially have implications for individual performance (i.e., survival and reproduction) through an impact on flight quality, the ability of individuals to thermoregulate, or the ability to express secondary sexual signals through plumage [START_REF] Swaddle | Plumage Condition Affects Flight Performance in Common Starlings : Implications for Developmental Homeostasis, Abrasion and Moult[END_REF][START_REF] Dawson | Rate of moult affects feather quality : A mechanism linking current reproductive effort to future survival[END_REF][START_REF] Jenni | The Biology of Moult in Birds[END_REF]. Future studies should now examine the impact of tebuconazole on these feather-related traits.

Body condition index

Contrary to our prediction, we did not find any major effects of tebuconazole on the body condition of individuals. Most individuals maintained their body condition throughout the experiment, independently of the treatment. The only significant effect was an increase in BCI for control females towards the end of the experiment (M3), whereas this increase was not observed in exposed females or control and exposed males. This puzzling result may be an artifact, although our sample size is quite robust for such an experimental study. Indeed, most previous studies have not reported any significant effect of exposure to triazoles on the body condition of birds [START_REF] Lopez-Antia | Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole[END_REF], 2018;[START_REF] Pandey | The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupt the pituitary-thyroid axis of a wildlife bird[END_REF][START_REF] Fernández-Vizcaíno | Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge[END_REF]). However, it could also be linked to a biological process: control females may have accumulated body reserves at the end of the experiment in preparation for breeding.

Indeed, females usually increase their body mass a few days/weeks before laying their eggs (e.g., [START_REF] Kvalnes | Correlates of egg size variation in a population of house sparrow Passer domesticus[END_REF][START_REF] Hennin | Pre-breeding energetic management in a mixed-strategy breeder[END_REF]) while this phenomenon is not detectable in males.

Tebuconazole exposure may have delayed or even suppressed the initiation of reproduction in females by interfering with seasonal endocrine processes, explaining then this absence of body weight gain in exposed females. Accordingly, studies have reported that triazoles may impair reproduction in birds (e.g., Fernandez-Vizcaino et al 2020;[START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF]. Moreover, triazoles are also known to interfere with cytochrome P450 enzymes, which are associated with the regulation of sex hormones (e.g., CYP19, which is involved in the conversion of androgens to estrogens, [START_REF] Poulsen | Tebuconazole disrupts steroidogenesis in Xenopus laevis[END_REF][START_REF] Saxena | Modelling inhibition of avian aromatase by azole pesticides[END_REF]. Since estrogens (such as estradiol) are known to control feeding behaviour, fat deposition and body condition in vertebrates, it is possible that deregulation of the steroid axis during the reproductive period could result in a sex-dependent effect of treatment on body condition. Interestingly, [START_REF] Pandey | The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupt the pituitary-thyroid axis of a wildlife bird[END_REF] found a significant decrease in the mass of exposed birds in response to fungicides, but only during the breeding phase, suggesting that the effects of fungicides on the body condition of individuals may be more apparent during the energetically demanding reproductive stage [START_REF] Pandey | The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupt the pituitary-thyroid axis of a wildlife bird[END_REF]. We do not know the exact age of our adult sparrows as they were caught in the wild, but it would also be interesting to see if age could influence the response to tebuconazole exposure. We must, however, remain cautious because exposed birds and controls unexpectedly differed in body condition at the beginning of the experiment, making it difficult to draw strong conclusions. Moreover, the sparrows had access to food ad libitum in our experiment and captivity limits mobility and therefore energy expenditure. These ideal nutritional conditions may have masked a potential impact of tebuconazole on metabolism and/or food consumption, and individuals may have been able to maintain their BCI despite the tebuconazole treatment. Finally, it would be interesting to further test how tebuconazole may affect body condition. For example, it is possible that tebuconazole has an impact on food intake, water consumption, metabolism, or/and activity, all of which influence body condition.

However, [START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF] showed no effect of tebuconazole on food intake in redlegged partridges suggesting that there is no effect on palatability or hunger. Future studies should therefore examine the impact of tebuconazole on body mass under realistic food situations and test whether exposure to tebuconazole affects food consumption, activity and reproduction.

Sperm morphology

Chronic exposure to tebuconazole prior to the breeding season did not result in any significant effect on sperm length in exposed sparrows. This result is contrary to our prediction as tebuconazole is known to interfere with steroid hormones and catalase and to have a negative impact on the reproduction of individuals [START_REF] Poulsen | Tebuconazole disrupts steroidogenesis in Xenopus laevis[END_REF][START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF][START_REF] Huang | Endocrine disruption by azole fungicides in fish : A review of the evidence[END_REF]). In addition, some studies have shown that pesticides can affect male germ cells, such as causing a decrease in the quantity and density of these cells, a reduction in testicular mass and an interruption of spermatogenesis [START_REF] Tokumoto | Effects of Exposure to Clothianidin on the Reproductive System of Male Quails[END_REF][START_REF] Mohanty | Thyroid disrupting pesticides impair the hypothalamic-pituitarytesticular axis of a wildlife bird, Amandava amandava[END_REF]Humann-Guilleminot et al. 2019). Machado-Neves et al. ( 2018) also showed that bats treated with 1 ml.L -1 of tebuconazole for 30 days had morphometric changes in their testes and epididymis suggesting a potential effect of tebuconazole on the gonadal axis. Importantly, it would be interesting to complement our measurements with an assessment of sperm motility, sperm count, abnormalities, and oxidative damage, because fungicides may affect these variables despite no effect on sperm morphology. Therefore, further studies would be needed to understand how tebuconazole impacts fertility and reproductive success in wildlife.

Conclusion

Overall, our results showed physiological effects of tebuconazole on a sentinel species of agroecosystems, the house sparrow. These effects include a significant decrease in T4 levels, and an alteration of feather structure, which altogether suggests a dysregulation of thyroid homeostasis by tebuconazole. Therefore, our study highlights the importance of assessing the potential risks of chronic exposure to tebuconazole for wild vertebrates living in agroecosystems.

  taken three times during the experiment (n = 38, Fig 1): once at the start of the experiment (M1), a second time after 5 months (M2), and a third time after 6 months of experimentation (M3, Fig 1). BCI was calculated by extracting the residuals from the linear regression of individual body mass as a function of tarsus length (F(1,112) = 44.28, p < 0.001). Control and exposed sparrows did not differ in body size (tarsus, F(1,36) < 0.01, p = 0.932).

Fig 1 .

 1 Fig 1. Timeline describing the different steps of the experiments. The five dots highlight a key event during the experiment. The two red borders highlight the period of exposure of the experimental individuals to tebuconazole.

Fig 1 )

 1 Fig 1), and their interactions to test the effect of tebuconazole on feather morphometry (PC1 or PC2). The identity of individuals was added as a random effect in the models because individuals were sampled twice (measures 1 and 2).

Fig 2 .

 2 Fig 2. Plasma concentrations of T3 (2.A) and T4 (2.B) in ng.mL -1 according to treatment (control or exposed to tebuconazole) and sex (female or male).Control and exposed individuals are represented in black and gray, respectively. Females and males are represented by circles and triangles, respectively. The concentration of T3 did not differ significantly between exposed and control sparrows ('ns', p > 0.100), whereas the concentration of T4 was significantly lower for exposed sparrows compared to control sparrows ('*' , p < 0.050). Means ± SE are presented.

Table 1 :

 1 Effect of treatment (control or exposed to tebuconazole) and sex (female or male) on the levels of T3 and T4 of house sparrows. A log-transformed function was applied to the T4 data in statistical models. Significant terms are indicated by the following codes: '***' p < 0.001 '**' p < 0.010 '*' p < 0.050 '.' p < 0.100. All models were selected using the backward stepwise selection method by eliminating nonsignificant terms (p > 0.100). The abbreviations used are as follows: Degrees of freedom in numerator (NumDF), degrees of freedom in denominator (DenDF), Fisher's statistics (F-value), the p-value associated (Pr (> F)).

  3.A.B). Specifically, the regrown feathers of exposed females were significantly larger than those of control females (measure 2, contrast: t(41.2) = -2.72, p = 0.009, Fig 3.A), whereas this difference was not significant for the initial feathers (measure 1, contrast: t(41.2) = -1.00, p = 0.322, Fig 3.A). For males, there was no difference in feather size between exposed and control individuals at any time (measure 2, contrast: t (41.2) = 0.64, p = 0.524 and measure 1, contrast: t(41.2) = -0.68, p = 0.500, Fig 3.B).

  3.C.D).Interestingly the regrown feathers of exposed females (measure 2) were significantly less dense than those of control females (contrast: t(48.5) = 2.34, p = 0.024), whereas this difference was not significant for the initial feathers (measure 1, contrast: t(48.5) = -0.60, p = 0.549, Fig 3.C). The feathers of exposed males were overall denser than those of the control males (contrast: t(26.0) = -2.08, p = 0.048, Fig 3.D).

Fig 3 .

 3 Fig 3. PC1 scores (feather size, 3.A and 3.B) and PC2 scores (feather density, 3.C and 3.D) as a function of the time of measurement (initial feathers corresponding to measurement 1 and regrown feathers corresponding to measurement 2). Figures 3.A and 3.C show the results obtained for females and figures 3.B and 3.D show the results obtained for males. Control and exposed individuals are represented in black and gray, respectively. Significant terms are indicated by the following codes: '*' p < 0.050, '.' p < 0.100 and 'ns' p > 0.150. The means ± SE are presented.

  and the time of the measurement (M1, M2 or M3, Fig 4). Specifically, the BCI of control females varied over time, where they had a higher BCI at time M3 compared to time M1 (contrast: t(68.0)= -4.45, p < 0.001, Fig 4.A). However, the BCI of exposed females did not vary over time (contrasts: all p > 0.345 Fig 4.A). Moreover, exposed females had a greater BCI than control females at time M1, i.e., before exposure to tebuconazole (contrast: t(59.1) = -2.31, p = 0.024) whereas this difference became marginal at time M2, i.e., after 5 months of exposure (contrast: t(59.1) = -1.97, p = 0.054) and non-significant at time M3, i.e., after 6 months of exposure (contrast: t(59.1) = 1.20, p = 0.235, Fig 4.A). In control and exposed males, there was no significant variation in BCI among times M1, M2 and M3 (contrasts: all p > 0.089, Fig 4.B).

Fig 4 .

 4 Fig 4. Body condition index of individuals (BCI) as a function of the timing of mass measurement (M1, M2 or M3), treatment (control or exposed to tebuconazole) and the sex of individuals (female 4A or male 4B). Control and exposed individuals are represented in black and gray, respectively. Significant terms are indicated by the following codes: '*' p < 0.050, '.' p < 0.100 and 'ns' p > 0.150. Means ± SE are presented.

Fig 5 .

 5 Fig 5. Schematic representation of the different scenarios proposed for the mode of action of tebuconazole on the house sparrow endocrine system. (A) By induction of type II deiodase activity resulting in increased conversion of T4 to T3 to ensure production of active TH at the expense of a decrease in T4 stock. (B) By inhibition of T4 transcription or release leading to compensation of T3 production by a T4 independent biosynthetic pathway. These examples are not exhaustive and hypotheses (A) and (B) are non-mutually exclusive. The flash symbol represents potential biological targets of tebuconazole in the negative feedback loop of thyroid hormones.

Table 2 : Effects of time of measurement on feather morphometric parameters (initial or after regrowth), treatment (control or exposed to tebuconazole), sex (female or male), as well as their interactions on PC1 scores (feather size) and PC2 scores (feather density), respectively.

 2

	Individual identity was added as a random factor. Significant terms are indicated by the
	following codes: '***' p < 0.001 '**' < 0.010 '*' p < 0.050 '.' p < 0.100. All models were selected
	using the backward stepwise selection method by eliminating non-significant terms (p > 0.100).
	The abbreviations used are as follows: Degrees of freedom in numerator (NumDF), degrees
	of freedom in denominator (DenDF), the Chi-square statistic (khi²-value), the p-value
	associated (Pr (> khi²)).
	Response variables	Explanatory factors	NumDF	Den	khi²-	Pr (> khi²)
				DF	value
	PC1 (~ Length)	Time	1	26	35.49
						< 0.001 ***
		Treatment	1	26	2.50	0.114
		Sex	1	26	< 0.001	0.976
		Time : Treatment	1	26	0.13	0.718
		Time : Sex	1	26	6.82	0.009 **
		Treatment : Sex	1	26	2.09	0.148
		Time : Treatment : Sex	1	26	4.70	0.030 *
	PC2 (~ Density)	Time	1	28	43.43	< 0.001 ***
		Treatment	1	26	0.39	0.534
		Sex	1	26	2.53	0.112
		Time : Treatment	1	28	5.01	0.025 *
		Time : Sex	1	27	1.74	0.187
		Treatment : Sex	1	26	5.11	0.024 *
		Time : Treatment : Sex	1	26	1.72	0.190

Table 4 : Effects of treatment (control or exposed to tebuconazole) on the length of the different sperm components (head, flagellum and total length).

 4 Individual identity was added as a random factor. Significant terms are indicated by the following codes: '***' < 0.001 '**' < 0.010 '*' < 0.050 '.' < 0.100. The abbreviations used are as follows: Degrees of freedom in numerator (NumDF), degrees of freedom in denominator (DenDF), Fisher's statistics (F-value), the p-value associated (Pr (> F)).

	Response	Explanatory factors NumDF	DenDF	F-value	Pr (> F)
	variables
	Head length	Treatment	1	14.4	1.68	0.215
	Flagellum length	Treatment	1	15.0	0.15	0.700
	Total length	Treatment	1	15.0	0.04	0.844

Acknowledgments and Funding

We thank E. Beaugeard, E. Daniaud, B. Mohring, P. Pardon, L. Lièvre and N. Imbert for their help during the course of this study. This research has been funded by the Agence Nationale de la Recherche (ANR project VITIBIRD ANR-20-CE34-0008 to F.A.), by the Région Nouvelle-Aquitaine (MULTISTRESS project), by the Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES project BiodiTox #2019/1/031 to F.B.), by the Centre National de la Recherche Scientifique and by MITI-PEPSAN (mission for transversal and interdisciplinary initiatives to C.F.). LIENS laboratory of the University of La Rochelle for their technical support. The platform PLATINE (https://www.ubordeaux.fr/Recherche/Plateformes/Sciences-de-l-environnement/Plateforme-de-chimieanalytique-organique-environnementale-PLATINE) of the University of Bordeaux and UMR EPOC is acknowledged for technical and analytical support.

Statements & Declarations

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Credit author statement


Pauline Bellot

François Brischoux

Clémentine Fritsch

Aurélie Goutte

Fabrice Alliot

Steffi Rocchi

Frédéric Angelier

Evidence of environmental transfer of tebuconazole to the eggs in the house sparrow (Passer domesticus): An experimental study

Keywords: Triazole fungicides, agroecosystems, sentinel species, egg-laying, maternal transfer

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Since the 1960s, the intensification of agricultural practices has been identified as a major cause of decline for birds, especially farmland species [START_REF] Donald | Agricultural intensification and the collapse of Europe's farmland bird populations[END_REF][START_REF] Stanton | Analysis of trends and agricultural drivers of farmland bird declines in North America : A review[END_REF]). In addition to habitat loss and fragmentation, the massive use of pesticides has also been suggested to be involved in these population declines (e.g., reviewed by [START_REF] Stanton | Analysis of trends and agricultural drivers of farmland bird declines in North America : A review[END_REF]. Indeed, about 2 million tonnes of pesticides are used each year worldwide to protect crops from potential diseases and pests [START_REF] Ali | Environmental and Health Effects of Pesticide Residues[END_REF] and they are commonly detected in wildlife, including birds [START_REF] De Souza | Occurrence, impacts and general aspects of pesticides in surface water : A review[END_REF][START_REF] Montaigu | Identifying agricultural pesticides that may pose a risk for birds[END_REF]. This contamination can result either from a direct exposure of tissues to the pesticides (i.e., inhalation or trans-cutaneous absorption, [START_REF] Vyas | Dermal insecticide residues from birds inhabiting an orchard[END_REF] or from an oral exposure through the consumption of water, seeds, or any other contaminated food [START_REF] Lopez-Antia | Risk assessment of pesticide seed treatment for farmland birds using refined field data[END_REF][START_REF] Fernández-Vizcaíno | Bird exposure to fungicides through the consumption of treated seeds : A study of wild red-legged partridges in central Spain[END_REF]. Due to these multiple routes of exposure, pesticides are detectable in the blood, organs, feathers, and faeces of birds [START_REF] Van Drooge | Organochlorine residue levels in livers of birds of prey from Spain : Inter-species comparison in relation with diet and migratory patterns[END_REF][START_REF] Abbasi | Use of feathers to assess polychlorinated biphenyl and organochlorine pesticide exposure in top predatory bird species of Pakistan[END_REF][START_REF] Mateo | Risk assessment of lead poisoning and pesticide exposure in the declining population of red-breasted goose (Branta ruficollis) wintering in Eastern Europe[END_REF][START_REF] Rial-Berriel | Micro QuEChERS-based method for the simultaneous biomonitoring in whole blood of 360 toxicologically relevant pollutants for wildlife[END_REF][START_REF] Esther | Noninvasively Collected Fecal Samples as Indicators of Multiple Pesticide Exposure in Wild Birds[END_REF] indicating a contamination of the organisms.

Since, many farmland birds spend a significant part of their life cycle in agroecosystems, they may be chronically exposed to pesticides [START_REF] Bro | Quantification of potential exposure of gray partridge (Perdix perdix) to pesticide active substances in farmlands[END_REF]. Such a chronic exposure to pesticides can affect reproduction (e.g., [START_REF] Lopez-Antia | Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole[END_REF]) and potentially the development of the progeny [START_REF] Garcês | Teratological effects of pesticides in vertebrates : A review[END_REF][START_REF] Ortiz-Santaliestra | Egg overspray with herbicides and fungicides reduces survival of red-legged partridge chicks[END_REF], especially if the embryo is exposed to pesticides. Indeed, a few field studies have reported the presence of pesticides in eggs, demonstrating a potential exposure for the embryos [START_REF] Bro | Quantification of potential exposure of gray partridge (Perdix perdix) to pesticide active substances in farmlands[END_REF][START_REF] Bro | Residues of plant protection products in grey partridge eggs in French cereal ecosystems[END_REF][START_REF] Corcellas | Pyrethroid insecticides in wild bird eggs from a World Heritage Listed Park : A case study in Doñana National Park (Spain)[END_REF][START_REF] Venugopal | Levels and distribution pattern of organochlorine pesticide residues in eggs of 22 terrestrial birds from Tamil Nadu, India[END_REF]. Although some pesticides seem to be able to penetrate the eggshell and chorionic membrane [START_REF] Bro | Quantification of potential exposure of gray partridge (Perdix perdix) to pesticide active substances in farmlands[END_REF], they can also be transferred from the mother to the egg during vitellogenesis (e.g., persistent organic pollutants, [START_REF] Verreault | Maternal transfer of organohalogen contaminants and metabolites to eggs of Arctic-breeding glaucous gulls[END_REF][START_REF] Kitulagodage | Adverse effects of fipronil on avian reproduction and development : Maternal transfer of fipronil to eggs in zebra finch Taeniopygia guttata and in ovo exposure in chickens Gallus domesticus[END_REF][START_REF] Ruuskanen | Effects of parental exposure to glyphosate-based herbicides on embryonic development and oxidative status : A long-term experiment in a bird model[END_REF][START_REF] Jouanneau | A Bad Start in Life? Maternal Transfer of Legacy and Emerging Poly-and Perfluoroalkyl Substances to Eggs in an Arctic Seabird[END_REF]).

Among pesticides, the family of triazole fungicides is of primary concern because of its ubiquitous use in agroecosystems [START_REF] Ribas E Ribas | Is the emergence of fungal resistance to medical triazoles related to their use in the agroecosystems? A mini review[END_REF]. For example, tebuconazole is the most widely used triazole fungicide in the world and it is detected in the majority of environmental compartments, such as air [START_REF] Désert | Spatial and temporal distribution of current-use pesticides in ambient air of Provence-Alpes-Côte-d'Azur Region and Corsica, France[END_REF], soil [START_REF] Kalogridi | Part II: temporal and spatial distribution of multiclass pesticide residues in lake sediments of northern Greece: application of an optimized MAE-LC-MS/MS pretreatment and analytical method[END_REF], and water [START_REF] Kahle | Azole Fungicides : Occurrence and Fate in Wastewater and Surface Waters[END_REF]). Importantly, it has been convincingly demonstrated that tebuconazole is the most frequently ingested pesticide by farmland bird species [START_REF] Lopez-Antia | Risk assessment of pesticide seed treatment for farmland birds using refined field data[END_REF][START_REF] Fernández-Vizcaíno | Bird exposure to fungicides through the consumption of treated seeds : A study of wild red-legged partridges in central Spain[END_REF]. In addition, this fungicide is highly suspected to affect reproductive output [START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF], to disrupt physiological systems [START_REF] Bellot | Experimental Exposure to Tebuconazole Affects Metabolism and Body Condition in a Passerine Bird, the House Sparrow (Passer domesticus)[END_REF], and to act as an endocrine disruptor during development [START_REF] Robinson | Triazole induced concentration-related gene signatures in rat whole embryo culture[END_REF][START_REF] Li | Endocrine disrupting effects of tebuconazole on different life stages of zebrafish (Danio rerio)[END_REF], including in birds [START_REF] Fernández-Vizcaíno | Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge[END_REF]. However, and surprisingly, very little data is currently available regarding the potential contamination of eggs by triazoles in agroecosystems.

A recent avian study showed that tebuconazole is rapidly absorbed, distributed to the different tissues (liver, brain, kidneys, and muscles), metabolised, and then excreted [START_REF] Gross | Uptake, Metabolism, and Elimination of Fungicides from Coated Wheat Seeds in Japanese Quail (Coturnix japonica)[END_REF].

Because of the short half-life of tebuconazole in tissues [START_REF] Konwick | Bioaccumulation and biotransformation of chiral triazole fungicides in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Gross | Uptake, Metabolism, and Elimination of Fungicides from Coated Wheat Seeds in Japanese Quail (Coturnix japonica)[END_REF], the bioaccumulation and biomagnification risk of tebuconazole, and therefore the possibility of transfer of tebuconazole to the egg, are estimated to be low. However, some authors suggest that contamination of eggs could indeed occur because some contaminants would not be retained egg by the mother and might therefore be easily transferred to the egg during vitellogenesis [START_REF] Kadokami | Maternal transfer of organochlorine pesticides, polychlorinated dibenzo-pdioxins, dibenzofurans, and coplanar polychlorinated biphenyls in frogs to their eggs[END_REF][START_REF] Verreault | Maternal transfer of organohalogen contaminants and metabolites to eggs of Arctic-breeding glaucous gulls[END_REF]. A past report from the Food and Agriculture Organization (FAO) suggests that tebuconazole may be transferred to eggs because radiolabeled tebuconazole was found in multiple tissues, including eggs, after oral administration in laying hens (Ecker and Webe, 1991, Unpublished;Beedle and Ying, 2002, Unpublished). To our knowledge, data are however really scarce and no published and available study has experimentally evaluated whether tebuconazole can be transferred from the mother to the egg in birds.

In this study, we experimentally tested whether parental exposure to tebuconazole could result in egg contamination by this fungicide in a sentinel bird species of agroecosystems, the house sparrow (Passer domesticus). Captive sparrows were chronically exposed to tebuconazole through drinking water (exposed group) or not (control group) during the pre-breeding and breeding season as tebuconazole is used during these periods in several agroecosystems (vineyards, orchards, vegetable farming). During this experiment we opportunistically collected abandoned eggs to test whether the eggs of exposed females contained tebuconazole.

MATERIALS & METHOD

Study species and experimental design

This study was conducted on captive adult house sparrows. A total of 32 adult sparrows were used in this experiment (Nfemales = 16 and Nmales = 16). All sparrows were maintained in captivity in 6 similar outdoor aviaries (length: 4 m, width: 3 m, height: 3 m). Each aviary was equipped with artificial shelter boxes (i.e., one box per bird) and a shrub. The sparrows were fed ad libitum with a mixture of commercial seeds, millet on the stack, sand and salt/minerals blocks.

In all aviaries, water was dispensed through drinking trough and changed every week. Of the 6 aviaries, three were exposed to tebuconazole (exposed group) and the three others were not exposed to tebuconazole (control group). The sex ratio was balanced in each aviary and the birds were divided by treatment type as follows (Aviary A: 6 controls; Aviary B: 6 exposed; Aviary C: 4 controls; Aviary D: 6 exposed; Aviary E: 6 controls; Aviary F: 4 exposed). Exposed individuals were treated with tebuconazole through their drinking water. In the three exposed aviaries, tebuconazole (Sigma-Aldrich, CAS. No: 107534-96-3, purity: ≥ 98.0 %) was diluted with tap water using a precision balance (±0.0001 g) to reach a concentration of 550 µg.L -1 .

The NOAEL (no observed adverse effect level) of tebuconazole in birds is estimated to be 5.8 mg/kg bw/day (Colinus virginianus, http://sitem.herts.ac.uk/aeru/ppdb/en/Reports/610.htm), while the exposure by our treatment is estimated to be much lower (i.e., 0.165 mg/kg bw/day), because a bird drinks approximately 30 percent of its body weight per day [START_REF] Bartholomew | The water economy of land birds[END_REF]. This exposure concentration was also chosen because it has been shown to result in realistic plasma contamination (i.e., similar to the plasma contamination of wild birds living in vineyards, [START_REF] Bellot | Experimental Exposure to Tebuconazole Affects Metabolism and Body Condition in a Passerine Bird, the House Sparrow (Passer domesticus)[END_REF]. In three control aviaries, control individuals had access to regular tap drinking water without tebuconazole. The concentration of tebuconazole in the drinking water of the exposed and control groups was analysed to confirm an exposure concentration of 550 µg.L -1 in the exposed individuals and a tebuconazole-free water for the control individuals (by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS, Qualyse laboratory, La Rochelle, France)). Exposure to tebuconazole began in December 2020 until July 2021 (7 months). This exposure period was chosen because tebuconazole can be applied in winter but also during the spring-summer period. For example, it can be used on vines from 7 to 8 leaves until bunch closure against mildew or powdery mildew corresponding to the sparrow breeding period. During this breeding period (May to July), sparrows formed pairs and laid eggs, some of which were abandoned by the parents without being incubated. These eggs were collected opportunistically and stored at -20°C until chemical analysis. A total of 24 eggs from 18 clutches were recovered (11 eggs from 8 clutches for the 'exposed parents' group and 13 eggs from 10 clutches for the 'control parents' group). In a few cases, 2 or 3 eggs from the same clutch were collected (3 eggs: 2 clutches; 2 eggs: 2 clutches, 1 egg: 14 clutches). This opportunistic collection of abandoned eggs was chosen for ethical reasons, and it is unlikely to lead to any bias because control and exposed sparrows showed similar rates of egg abandonment. The concentration of tebuconazole was determined in exposed and control eggs (albumen and yolk, without shell). The limit of quantification (LOQ) was 0.23 ng.g -1 dry weight and the limit of detection (LOD) was 0.075 ng.g -1 dry weight. Pesticides were determined by LC-MS/MS in Paris, France. The protocol for the determination of pesticides is detailed in the supplementary material.

Statistical analysis

All statistical analyses were performed in Rstudio (R version 4.0.3). When several eggs from the same clutch were collected, the concentrations of tebuconazole of all the eggs were averaged to avoid pseudo-replication (N= 8 clutches for the exposed group; N= 10 clutches for the control group). In order to test the effect of the parental exposure to tebuconazole (exposed vs. control group) on the quantification or not of tebuconazole in the collected clutches, a GLM (Generalized Linear Model) was performed with a binomial distribution and a logit link function (dependent binary variable: contaminated or uncontaminated clutch; dependent factor: exposed or control). In addition, to test the effect of the experimental treatment (exposed vs. control) on the concentration of tebuconazole found in the collected clutches, a non-parametric test was used with the NADA package (Peto-Prentice test, [START_REF] Shoari | Toward improved analysis of concentration data : Embracing nondetects[END_REF]. This test allowed us to consider censored values, i.e., below the limit of quantification (N = 8). The censored values were replaced by LOQ / 2 (i.e., 0.115 ng.g -1 ).

RESULTS & DISCUSSION

Tebuconazole was quantified in all the clutches of exposed females (Figure 1) and the number of quantifiable samples was significantly higher for clutches from exposed females (exposed group) compared to those from control females (χ² = 14.7, df = 1, p < 0.001). Indeed, 80% of the clutches in the control group were below the LOQ while all clutches in the exposed group were above the LOQ. Overall, the concentration of tebuconazole in sparrow eggs was significantly affected by the experimental treatment with the levels of tebuconazole in the eggs of exposed parents being significantly higher than those in the eggs of control parents (χ² =17.8, df = 1, p < 0.001, Figure 1). In this study, we demonstrated for the first time that chronic exposure to tebuconazole in breeding sparrows results into egg contamination, demonstrating that tebuconazole can be transferred from the environment to the egg in birds. This result may appear surprising because of the rapid biotransformation potential of tebuconazole in tissues. Indeed, previous studies have investigated the toxicokinetic of tebuconazole in birds and they have shown that tebuconazole has a high clearance rate [START_REF] Konwick | Bioaccumulation and biotransformation of chiral triazole fungicides in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Gross | Uptake, Metabolism, and Elimination of Fungicides from Coated Wheat Seeds in Japanese Quail (Coturnix japonica)[END_REF]) and a moderate lipophilicity (EFSA, 2014). Accordingly, [START_REF] Gross | Uptake, Metabolism, and Elimination of Fungicides from Coated Wheat Seeds in Japanese Quail (Coturnix japonica)[END_REF] did not show any bioaccumulation potential of tebuconazole in the Japanese quail even after repeated oral contamination with low or high doses of tebuconazole (3.08 or 9.24 µg per day respectively) for 10 consecutive days and the possibility of maternal transfer of this compound to the egg was not favoured [START_REF] Lopez-Antia | Birds feeding on tebuconazole treated seeds have reduced breeding output[END_REF]). However, this fungicide is considered to be relatively lipophilic with an octanol/water partition coefficient (log Kow) of 3.7 (EFSA, 2014), and some studies suggest that maternal transfer may be favoured by low-Kow and/or less persistent compounds as these would be weakly recalcitrant in maternal tissues and therefore potentially more easily transferred into the egg [START_REF] Kadokami | Maternal transfer of organochlorine pesticides, polychlorinated dibenzo-pdioxins, dibenzofurans, and coplanar polychlorinated biphenyls in frogs to their eggs[END_REF][START_REF] Verreault | Maternal transfer of organohalogen contaminants and metabolites to eggs of Arctic-breeding glaucous gulls[END_REF]. A few studies have measured the levels of pesticides including azoles in eggs [START_REF] Gómez-Ramírez | An overview of existing raptor contaminant monitoring activities in Europe[END_REF][START_REF] Bro | Quantification of potential exposure of gray partridge (Perdix perdix) to pesticide active substances in farmlands[END_REF][START_REF] Bro | Residues of plant protection products in grey partridge eggs in French cereal ecosystems[END_REF][START_REF] Corcellas | Pyrethroid insecticides in wild bird eggs from a World Heritage Listed Park : A case study in Doñana National Park (Spain)[END_REF]. Interestingly, [START_REF] Bro | Residues of plant protection products in grey partridge eggs in French cereal ecosystems[END_REF] found relatively high concentrations of four triazoles in the eggs of wild grey partridges (e.g., 21 ng.g -1 cyproconazole, 13 ng.g -1 difenoconazole or tebuconazole < 10 ng.g -1 , on whole eggs by gas chromatography-tandem mass spectrometry). These concentrations can be 10 times higher than those found in our experiment. However, the route of contamination remained an open question and such high concentrations may result from direct transfer from the environment to the egg of these ground-nesting birds during spraying of pesticides [START_REF] Bro | Residues of plant protection products in grey partridge eggs in French cereal ecosystems[END_REF]. Because tebuconazole was found in the eggs of exposed sparrows, our study suggests that tebuconazole may be transferred from the environment into the egg through the ingestion of tebuconazole by the mother. Importantly, this demonstrates that the embryo can be exposed to tebuconazole when it is used in the environment (i.e., on agricultural land).

In our study, the contamination of the eggs by tebuconazole may have been amplified by the chronic exposure to this fungicide (i.e., several months before egg-laying). Indeed, continuous exposure to tebuconazole may have resulted in oxidative damage, and as a result in hepatotoxicity in exposed sparrows [START_REF] Ferreira | Assessment of oxidative stress in Rhamdia quelen exposed to agrichemicals[END_REF][START_REF] Yang | Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats[END_REF]. Such hepatotoxicity may have led to a lower ability of the liver to metabolize the parent compound, resulting in an accumulation of tebuconazole in the mother tissues. Similarly, a chronic exposure to tebuconazole may also lead to the saturation of phase I and II enzymes or xenobiotic transporters, and this may lead to a reduced biotransformation of tebuconazole in the mother tissues [START_REF] Drouillard | Quantifying maternal and dietary sources of 2,2′,4,4′,5,5′-hexachlorobiphenyl deposited in eggs of the ring dove (Streptopelia risoria)[END_REF]. Finally, it cannot be excluded that tebuconazole may interfere at different levels with the induction and/or repression of xenobiotic biotransformation pathways (e.g., [START_REF] Yang | Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats[END_REF], leading to accumulation of the parent substance or increased toxicity.

In this study, we exposed individuals chronically, including the breeding and egg-laying season.

Although this exposure mimics a realistic environmental scenario (farmland birds are repeatedly exposed to azole compounds for several months in agroecosystems, especially in vineyards, orchards or vegetable crops), it did not allow us to fully understand the kinetic of the maternal transfer of tebuconazole into the egg. More specifically, it is unclear whether tebuconazole is transmitted from mother to egg by maternal tissue or more directly by the intestinal route. Although the presence of tebuconazole in the plasma of exposed parents has been confirmed [START_REF] Bellot | Experimental Exposure to Tebuconazole Affects Metabolism and Body Condition in a Passerine Bird, the House Sparrow (Passer domesticus)[END_REF], it would be interesting to conduct a study with radiolabelled tebuconazole to follow its kinetics during the laying period. Furthermore, we were not able to test to what extent and for how long the maternal transfer of tebuconazole into the egg persists after exposure of the female to this triazole because females were continuously exposed to tebuconazole in our study. It would therefore be interesting to expose birds during the prebreeding only and to subsequently test whether tebuconazole or its metabolites can be found in maternal tissues and into eggs during the laying period. This would allow us to test whether the maternal transfer to the eggs is directly reversible after stopping the exposure or whether these effects persist until the complete elimination of tebuconazole in the maternal tissues or in the digestive system. Future studies should also measure the metabolites of tebuconazole in eggs to know the mixture of compounds to which the embryo is likely to be exposed. This is of primary importance because the presence of xenobiotics during embryonic development could cause deleterious effects on the developing chick [START_REF] Lopez-Antia | Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole[END_REF], 2015;[START_REF] Garcês | Teratological effects of pesticides in vertebrates : A review[END_REF][START_REF] Ortiz-Santaliestra | Egg overspray with herbicides and fungicides reduces survival of red-legged partridge chicks[END_REF][START_REF] Ruuskanen | Effects of parental exposure to glyphosate-based herbicides on embryonic development and oxidative status : A long-term experiment in a bird model[END_REF]. For example, Ortiz-Santaliestra et al. (2020) showed that experimental spraying of red-legged partridge eggs with tebuconazole at environmental concentrations resulted in a reduction in chick survival of around 26%, as well as physiological effects such as increased levels of alkaline phosphatase activity. These potential effects need to be further explored, particularly with regard to long-term effects that persist into adulthood.

Figure 1 :

 1 Figure 1: Tebuconazole concentration of sparrow eggs (ng.g -1 dry weight) according to the experimental treatment (control parents, N = 13 eggs or exposed parents, N = 11 eggs). The eggs of control or exposed parents are shown in light and dark blue, respectively.Tebuconazole concentrations were significantly different between the two groups ('***' < 0.001). Means ± SE are presented. The dotted line represents the analytical limit of quantification (LOQ = 0,23 ng.g -1 dry weight). Detected concentrations that were below the LOQ were graphically replaced by the LOQ / 2 (= 0.115 ng.g -1 dry weight).

Acknowledgements

We thank L. Lievre, E. Daniaud and B. Mohring, for their help during the course of this study. This research has been funded by the Agence Nationale de la Recherche (ANR project VITIBIRD ANR-20-CE34-0008 to F.A.), by the Région Nouvelle-Aquitaine (MULTISTRESS project to O.C.), by the Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES project BiodiTox #2019/1/031 to F.B.), by the Centre National de la Recherche Scientifique and by MITI-PEPSAN (mission for transversal and interdisciplinary initiatives to C.F.).

Ethical Statement

All applicable institutional and/or national guidelines for the care and use of animals were followed. This work was approved by the French authorities (COMETHEA ethic committee and Ministère de L'Enseignement Superieur, de la Recherche et de L'innovation) under permit #APAFIS#12918-2018010515574796.

SUPPLEMENTARY DATA

Dosage of pesticides

A total of 24 eggs were recovered opportunistically (N = 11 eggs from the exposed parent group and N = 13 eggs from the control parent group). Eggs (albumen and yolk, without eggshells) were lyophilized (alpha 1-4 D, Fisher, Illkirch, France) for 48h and ground with a mortar and a pestle to obtain a fine powder. Samples (0.5 g dry weight) were spiked with 50 ng of labelled internal standard (Tebuconazole D6, CIL Cluzeau, Sainte Foy la Grande, France) and stored overnight at 4°C. Accelerated solvent extraction (ASE 350; Thermo Fisher Scientific, Illkirch, France) was performed using methanol (VWR, Fontenay-sous-Bois) though 2 extractions (10 min, 70°C) and purging cycles (100s, solvent flush: 60% of cell volume). After concentration to approximately 1mL, using the EZ-2 (Biopharma Technologies) and then under a nitrogen stream at 40°C, the extracts were recovered in ultra-pure water (100 mL) with 0.2 g of Na2EDTA (Sigma, France). Solid Phase Extraction (SPE) was performed using HLB Chromabond (Macherey Nagel, France, 150 mg, 6 mL), conditioned with 5 mL of methanol and 5 mL of ultra-pure water. Extracts were eluted with 10 mL of methanol, concentrated (EZ-2 and nitrogen stream), recovered in 200 µL of ultra-pure water and methanol (50/50), rinsed twice with 70 µL of methanol, deposited on a filter tube (0.2 μm, nylon; VWR) and centrifuged (5000 rpm, 1 min). Analyses of tebuconazole was performed using an Agilent 1200 liquid chromatograph (LC) interfaced to a 6410B triple quadrupole mass spectrometer system (MS/MS) (Agilent Technologies) with an electrospray ionization (ESI) source in positive mode (N2: 350 °C; gas flow: 11 L/min; capillary: 4000 V). Five μL of extract were injected and separated using a Nucleoshell Biphenyl column (2 × 150 mm, 2.7 μm; Macherey Nagel) with a fritted disc (0.2 μm) heated at 40 °C at a flow of 0.65 mL/min. Two solvents were used as mobile phase, Acetonitrile (solvent A) and ultra-pure water (solvent B), both acidified with 0.1% of formic acid. The elution gradient was a transition from 98% to 0% of B in 9.5 min followed by 2 min of 100% of A and then a return to the initial conditions (98% of B) during 2 min for a total analysis time of 15 min.

The limit of quantification (LOQ) was calculated using the signal-to-noise (S/N) ratios, by extracting spiked (50 ng) matrix-matched egg samples (n = 3). LOQ was calculated as 10 times the ratio between spiked amount and S/N ratios. LOQ was 0.23 ng.g -1 of egg in dry weight. The recovery ratio was also evaluated in spiked egg samples, using the same extraction and quantification protocol as for the samples. The recovery ratio was 92 % ± 5.1 % (mean ± SD). During each analytical run, blanks and quantification controls were regularly used in order to crosscheck the quantification quality.