# Urban mixtures of non-essential trace metal elements affect the behaviour and stress levels of a passerine bird in a laboratory experiment

Clément Parnet, Sylvie Massemin, Anne Boos, Pascale Ronot, Frédéric Angelier, Charline Parenteau, Julien Courtecuisse, Navina D Liebermann-Lilie, Josefa Bleu

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Clément Parnet, Sylvie Massemin, Anne Boos, Pascale Ronot, Frédéric Angelier, et al.. Urban mixtures of non-essential trace metal elements affect the behaviour and stress levels of a passerine bird in a laboratory experiment. Ecotoxicology and Environmental Safety, 2025, 302, pp.118647. 10.1016/j.ecoenv.2025.118647. hal-05166045

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# Urban mixtures of non-essential trace metal elements affect the behaviour and stress levels of a passerine bird in a laboratory experiment

Clément Parnet<sup>a, *, ID</sup>, Sylvie Massemin<sup>a</sup>, Anne Boos<sup>a</sup>, Pascale Ronot<sup>a</sup>, Frédéric Angelier<sup>b</sup>, Charline Parenteau<sup>b</sup>, Julien Courteuisse<sup>a</sup>, Navina D. Liebermann-Lilie<sup>c</sup>, Josefa Bleu<sup>a ID</sup>

## ARTICLE INFO

Edited by Dr Yong Liang

Keywords:

Antagonism effect

Food

Heterophil/lymphocyte

Taeniopygia castanotis

Trace metal elements

## ABSTRACT

Trace Metal Elements (TME) are known for their toxicity, which is higher for non-essential TME (nTME) than for essential TME (eTME). However, little is known about the toxicity of eTME and nTME mixtures at concentrations relevant to the urban environment. Since birds are commonly used for biomonitoring, we investigated the behavioural and physiological responses of a passerine model - the zebra finch (*Taeniopygia castanotis*) formerly known as *Taeniopygia guttata*) - to TME mixtures in a laboratory setting. Eighty males were divided into 3 exposure groups and 1 control group. Each group was then divided into *ad libitum* and intermittent feeding groups to better represent challenging natural conditions. The birds were exposed to TME at urban relevant levels via drinking water for 2 months. Three mixtures were tested: one containing only nTME (cadmium, arsenic, lead), one containing the same nTME plus eTME (zinc, copper, nickel), and one containing the same nTME and double the amount of eTME. Our results showed that the more contaminated the birds were with nTME, the less time they spent at the feeders and the more stressed they were. The birds showed an aversion to the mixtures containing TME, particularly eTME. This suggests that birds are able to detect TME in water. Birds exposed to eTME accumulated more copper and less cadmium than the birds exposed only to nTME, highlighting the antagonism between the two types of TME. This study demonstrates that nTME can directly impact the behaviour and stress levels of birds at urban relevant doses.

## 1. Introduction

Trace metal elements and metalloids (TME) occur naturally in small quantities in ecosystems (Gouzy and Ducos, 2008). The natural sources of TME are erosion, volcanism and geochemical background (Gouzy and Ducos, 2008). However, anthropogenic activities such as mining, industry, transport, and heating are the main sources of TME emissions and environmental pollution (Charlesworth et al., 2011). TME can have different physiological effects depending on their essentiality: Essential TME (eTME) such as zinc (Zn) or copper (Cu) are necessary for the metabolism of organisms at low doses but become toxic if these doses are exceeded (Mertz, 1993). Conversely, non-essential TME (nTME) such as lead (Pb) or cadmium (Cd) have no identified physiological role (Mertz, 1993) and are toxic for organisms, even at low doses.

Exposure to TME has been associated with several adverse effects in animal populations, especially non-essential TME (e.g. muscle

weakness, anaemia, death (Liu, 2003)). Among wild species, birds are well studied as they are considered as good sentinels of their environment (García-Fernández, 2014), mainly because their individual health and degree of contamination are considered reliable proxies of the quality of their habitat. For instance, urbanisation has been shown to be associated with reduced reproductive success in great tits (Parus major) and blue tits (Cyanistes caeruleus) (Chatelain et al., 2021a). This effect of urbanization on breeding success was correlated with higher levels of Cd, As, Pb and Cu contamination. Similarly, urban-related contaminations in Cd and Pb were correlated with an increase in stress hormone levels (corticosterone) in the feathers of common blackbird (Turdus merula) (Meillère et al., 2016). These studies highlight the putative link between TME contamination and physiological stress that can be found in urban environments. Wild birds are widely used to monitor environmental pollution by TME (Dmowski, 1999), but there is only few experimental studies measuring the direct toxicity of urban relevant

<footnote><sup>a</sup> Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg F-67000, France</footnote>

<footnote><sup>b</sup> Centre d'Etudes Biologiques de Chizé, CNRS-Université de La Rochelle, UMR-7372, Villiers-en-Bois, France</footnote>

<footnote><sup>c</sup> Department of Animal Behaviour - Bielefeld University, Bielefeld, Germany</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: parnet.clement@gmail.com (C. Parnet).</footnote>
TME mixtures on birds (Chatelain et al., 2016a, 2016b, 2016c, 2018; Dauwe et al., 2002, 2006; Saulnier, 2022; Saulnier et al., 2020).

In a mixture, the toxicity of the contaminants can add up (Beyer et al., 2014). Different TME interact with each other and influence the overall toxicity of the mixture: for example, two-by-two tests on the nematode (Caenorhabditis elegans) have shown different interactions of TME (Wah Chu et al., 2002). This work showed simple additive effects of mercury (Hg) and Pb or nickel (Ni) and Cd on nematodes mortality (Wah Chu et al., 2002). It also highlighted antagonism of Zn with Cd and synergistic effects of Cu with Zn, Cd, Pb, Ni, Hg, chromium (Cr), cobalt (Co), manganese (Mn) and aluminium (Al) on the mortality of nematodes (Wah Chu et al., 2002). These antagonistic and synergistic effects are called 'cocktail effects' (Beyer et al., 2014). However, the biological effects of these interactions are still poorly understood and seem to dramatically vary depending on the considered TME and the number of TME interacting. For example, previous studies highlighted antagonism between Cu and Cd (Levander, 1978) whereas some have shown Cu and Cd as synergistic (Wah Chu et al., 2002; Driessnack et al., 2016), while several studies have reported antagonism of Zn with Pb and Cd (Wah Chu et al., 2002; Levander, 1978; Prasanthi et al., 2010). It has been hypothesised that the physiological role of TME might explain the cocktail effect occurring between TME in a mixture (Wani et al., 2021; Stankevičiūtė et al., 2018). For example, the role of Zn in physiology as an eTME could play a role in its antagonism when co-exposed in mixtures with nTME such as Cd or Pb. Although mixtures are more environmentally relevant than pure TME, few studies have investigated the impact of such mixtures at urban-relevant doses.

In the laboratory, several avian models have been used to test urban relevant mixtures. For example, it has been shown in the common pigeon (Columba livia) that Zn co-exposure can reduce both the mass loss induced by Pb and the overall Pb concentration of Pb in the blood (Chatelain et al., 2016b). In the same way, Zn and Pb co-exposure can also reduce the negative effects of Pb exposure on maternal transfer of immune components (Chatelain et al., 2016a). Zn has also been shown to reduce the negative effects of urban Pb exposure on the bacterial community in the feathers (Chatelain et al., 2016c). These results suggest a compensatory effect between Zn and Pb. Another well studied avian model in toxicology is the zebra finch (Taeniopygia castanotis) formerly known as *Taeniopygia guttata*) and a few studies have investigated the effects of Pb exposure at urban relevant levels on zebra finches (Dauwe et al., 2002, 2006). This model was also exposed to an urban relevant mixture of several TME (Co, Cr, Ni, Cu, Zn, As, Pb, Cd) with adverse effects on telomere length, a proxy of cellular ageing (Saulnier et al., 2020). However, these experiments are often conducted in ideal and non-realistic conditions that cannot be found in the wild (i.e., low energy expenditure and *ad libitum* feeding), questioning their robustness under non-laboratory conditions.

One of the major challenges for organisms in the wild is energy intake and in particular foraging that allows them to sustain their annual energetic needs. The foraging behaviour of birds have been shown to be affected by proximity to TME polluted sites and indirectly by their pollution (Brotons et al., 1998). For example, the lack of carotenoid rich diet can reduce the antioxidant and therefore the TME defence capacity of wild great tits in an urbanization context (Eeva et al., 2005). In addition, as the maintenance cost of organisms increases with detoxification processes (Hoffmann, 1997), the energetic needs of contaminated birds should increase, impacting their feeding behaviour and food intake (Alcaraz, 2005). For these reasons, the effects of TME contamination on the foraging behaviour and its link with the body condition of birds should be investigated.

Several protocols aimed at replicating the stochasticity of food availability in the wild and testing its effect on stress and energy trade-offs in controlled environment exist in the literature on zebra finches. One protocol consists in providing access to food 4 h per day, and has been shown to have strong effects on birds' metabolism and reproduction (Mishra et al., 2020; Prabhat et al., 2020). Marasco et al. (2015).

proposed another protocol to mimic a stochastic access to food rather than an energetic stress by randomly restricting access to food for 4.9 h per day on 4 random days per week (Marasco et al., 2015). This study highlighted that the food-restricted birds had higher basal corticosterone levels without having any effect on the birds' body mass (Marasco et al., 2015). This protocol enables to experimentally investigate TME toxicity in a control environment with more environmentally relevant rearing conditions.

This study aims to investigate the potential buffering effects of eTME on the toxicity of nTME in a laboratory bird species: the zebra finch. We tested the following hypotheses: 1) nTME are primarily responsible for the adverse effects of urban mixtures. 2) eTME can mitigate the detrimental effects of nTME. 3) More challenging rearing conditions for animals exacerbate the detrimental effects of nTME. By exposing zebra finches to urban-relevant and realistic mixtures of eTME (Zn, Cu, Ni) and nTME (Cd, Pb, As), we investigated the contamination levels of the birds and the adverse effects of such contamination on behaviour and physiological proxies of stress. We based the composition of our TME mixtures on previous studies (Chatelain et al., 2016a; Dauwe et al., 2006; Snoeijns et al., 2005) in order to replicate the exposure experienced by great tits in a medium-sized European city, as determined by their faecal TME levels (Saulnier, 2022; Saulnier et al., 2020). Since the TME content of feathers reflects the contamination of the birds (Saulnier et al., 2020; Dauwe et al., 2004; Goede et al., 1986), we used it as a proxy for individual levels of contamination. Since physiological stress is intrinsically linked with metabolic rate variation (Jimeno et al., 2023, 2018) and glucocorticoid secretion (Angelier et al., 2013; Cockrem, 2007; Landys et al., 2006; Romero et al., 2009), we monitored feather corticosterone levels, which provide a temporal integration of this hormonal signal of stress (Bortolotti et al., 2008; Jenni-Eiermann et al., 2015). We also measured physiological stress using the heterophils / lymphocytes (H/L) ratio, which is a more general proxy for chronic stress (Davis et al., 2008; Goessling et al., 2015). The total time spent eating and the mass of each bird were monitored to track feeding behaviour. Finally, this study also investigated the potential interactive effects of non-ideal laboratory conditions on our behavioural and physiological variables of interests by implementing stochastic food access and TME contamination.

Based on our hypotheses, we therefore expected the following results: 1) A higher concentration of nTME should result in a higher H/L ratio and corticosterone levels, as well as reducing the mass and increasing the feeding time of birds; 2) these adverse effects of nTME should be less pronounced in birds that are exposed to both nTME and eTME than in those that are only exposed to nTME; 3) birds with random access to food should exhibit higher H/L ratios and corticosterone levels, which should exacerbate the detrimental effects of the TME mixture on their physiological stress, body mass loss and behaviour.

## 2. Material and methods

In this experiment, we compared the TME feather levels, H/L ratios and corticosterone levels of adult male zebra finches before and after 65 days of exposure to a TME mixture containing a variable quantity of eTME and a fixed quantity of nTME. Throughout the experiment, we also monitored how much of the mixture the birds drank and how long they spent at their feeder.

### 2.1. Animal model and housing conditions

The zebra finch *Taeniopygia castanotis* previously referred as *Taeniopygia guttata* (del Hoyo and Collar, 2016) is a small passerine bird of the Estrildidae family that originates from Australia. It is a well-established avian model organism for studies in the fields of neuroscience, behavioural ecology and physiology (Griffith et al., 2010; Mello, 2014) and more recently ecotoxicology (Jaspers, 2015). All zebra finches used in this experiment came from the domesticated stock population of the Department of Animal Behaviour at Bielefeld
University (Germany). For each bird the breeding history and pedigree was controlled and known.

The experiment was carried out from February to April 2022 and approved by the French national ethics committee (project APAFIS 26929). Eighty male zebra finches, aged between 2 and 3 years, were randomly assigned to 16 cages of 150 × 70 x 100 cm³, each containing 5 individuals. In average, the birds had a body mass of 13.62 g before the experiment. Body mass is not statistically different between the groups (Supplementary Table S5, ANOVA tests, p-value > 0.05). Similarly, absence of significant differences in individual size (tarsus length) between cages was verified (ANOVA tests, p-value > 0.05). Cages were placed in 2 rooms with a 14-h day/ 10-h night cycle, at a fixed temperature of 23°C and a relative humidity of 30 %. Each cage was provided with a feeder containing a seed mixture Oiseaux Exotique Super® from Delinature®- Ets Hassenforder (Réguisheim, France) composed of 62 % foxtail millet, 14 % proso millet, 10 % canary grass, 5 % red panis, 4 % red millet, 3 % japanese millet and 2 % niger seeds.

## 2.2. Experimental procedure

After a one-month habituation period, the cages were divided into 8 experimental groups of 10 individuals each in 2 different rooms and cages. The groups consisted of 2 cross treatments: TME mixtures exposure and the random food access. There were 4 different groups for the TME treatment (control, nTME alone, nTME + low eTME and nTME + high eTME) and 2 different groups for the food treatment (ad libitum and random) resulting in 8 experimental groups (Table 1). Exposure to environmentally relevant levels of TME was derived from Saulnier et al., 2020. The control condition “Ctrl” was given bottled mineral water Montcalm® (Auzat, France). Exposure to the TME mixtures was carried out via drinking water using the same bottled mineral water Montcalm® of low mineral content. TME were added in water as metal chlorides salts with a purity of 98–99 % from Alfa Aesar (Haverhill, Massachusetts, USA), except for arsenic, which was added as a sodium arsenite (NaAsO₂ – 0.1 N) solution from Alfa Aesar (Haverhill, Massachusetts, USA). The “nTME alone” condition contained 5 mg.L⁻¹ of As, 10 mg.L⁻¹ of Pb and 1 mg.L⁻¹ of Cd. The “nTME + low eTME” condition contained 5 mg.L⁻¹ of As, 10 mg.L⁻¹ of Pb, 1 mg.L⁻¹ of Cd and 100 mg.L⁻¹ of Zn, 24 mg.L⁻¹ of Cu, 0.5 mg.L⁻¹ of Ni. The “nTME + high eTME” condition contained 5 mg.L⁻¹ of As, 10 mg.L⁻¹ of Pb, 1 mg.L⁻¹ of Cd and 200 mg. L⁻¹ of Zn, 48 mg.L⁻¹ of Cu, 1 mg.L⁻¹ of Ni (see Table 1). Each mixture was available for the birds in polyethylene plastic bottle. The stability of each solution was measured by performing ICP-MS measurement in the solution 3 days after dilution in plastic polyethylene bottle and showed variation of less than 5 % of aimed concentration.

The manipulation of the temporal availability of food was carried out using a protocol from the literature (Marasco et al., 2015). The ad libitum condition consisted of continuous access to food for the birds while the “random” condition consisted of restricted access to food (access to food

for the 5 first or last 5 h of the day, randomly distributed over 4 days per week, see Marasco et al. (2015).

The initial sample size was 80 individuals per sampling time but due to natural deaths (no visible signs of sickness) of individuals, the final sample size (Table 1) was 68 individuals (10 Control + ad libitum; 8 Control + random; 9 nTME alone + ad libitum; 6 nTME alone + random; 9 nTME + low eTME + ad libitum; 7 nTME + low eTME + random; 10 nTME + high eTME + ad libitum; 9 nTME + high eTME + random). These accidental deaths were balanced between treatments and were unrelated to the experimental protocol (Log rank tests, all p > 0.05).

Birds were sampled before the start of the exposure on day 0 and at the end of the experiment 65 days later. At both times, 100 µL of blood was collected using a heparinised capillary tube, of which 5 µL was used to make blood smears on microscope glass slides, and the remainder was stored at –20°C for further analysis. The blood smears were then placed for 10 min in a 100 % ethanol bath from CARLO ERBA Reagents (Val-de-Reuil, France) for fixation. At the same time, we collected two feathers from each side of the bird: the two 4th primary remiges and the two most external rectrices. Collecting flight feathers enables us to identify each feather precisely (for re-plucking the same one after growth) and ensure that we have enough material for chemical analysis, as flight feathers are bigger than covering feathers. These 4 feathers were also collected at the beginning of the acclimatisation period and at mid-term to allow them to regrow and accumulate TME and corticosterone during their growth. Individuals’ tarsus length was measured at the beginning of the experiment (± 0.1 mm) and birds were weighted at each sampling time (± 0.01 g).

## 2.3. Exposure and contamination analyses

Since the amount of water drank by each bird reflected its exposure to the TME mixture, the amount of water consumed for each cage was measured every 2–3 days when the bottles were replaced. The amount of water consumed was divided by the time spent and the number of birds in each cage during this time period in order to obtain the mean daily water consumption per bird. Since this method did not account for individual variability in water consumption (and uptake of the mixtures), we used feathers to monitor individual contamination. Feathers are known to accumulate TME during the time they are perfused with blood, i.e. during their growth (Dauwe et al., 2004; Goede et al., 1986). This growth period lasts from 21 to 26 days (Zann, 1996a) and the amount of TME accumulated in the feather during this period depends on the amount of TME circulating in the blood during the same period. The concentration of TME was measured with the following protocol: each 4th primary remige was first washed with ultrapure water before being digested overnight at 40°C in nitric acid bath HPLC grade from Merck (Darmstadt, Germany). The remaining solution was diluted with ultrapure water to a final volume of 5 mL before being analysed using ICP-MS Agilent 7700 from Agilent (Santa Clara, California, United States) to

<table><caption>**Table 1**
Experimental groups combining mixture and food treatment.</caption><thead><tr><th rowspan="2">Group name</th><th colspan="6">Mixture treatment TME composition (mg.L<sup>-1</sup>)</th><th rowspan="2">Food treatment</th><th rowspan="2">Number of birds (start of the experiment)</th><th rowspan="2">Number of birds (end of the experiment)</th></tr><tr><th>As</th><th>Pb</th><th>Cd</th><th>Zn</th><th>Cu</th><th>Ni</th></tr></thead><tbody><tr><td>Ctrl + ad libitum</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Ad libitum</td><td>10</td><td>10</td></tr><tr><td>Ctrl + random</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>Random</td><td>10</td><td>8</td></tr><tr><td>nTME alone + ad libitum</td><td>5</td><td>10</td><td>1</td><td>0</td><td>0</td><td>0</td><td>Ad libitum</td><td>10</td><td>9</td></tr><tr><td>nTME alone + random</td><td>5</td><td>10</td><td>1</td><td>0</td><td>0</td><td>0</td><td>Random</td><td>10</td><td>6</td></tr><tr><td>nTME + low eTME + ad libitum</td><td>5</td><td>10</td><td>1</td><td>100</td><td>24</td><td>0.5</td><td>Ad libitum</td><td>10</td><td>9</td></tr><tr><td>nTME + low eTME + random</td><td>5</td><td>10</td><td>1</td><td>100</td><td>24</td><td>0.5</td><td>Random</td><td>10</td><td>7</td></tr><tr><td>nTME + high eTME + ad libitum</td><td>5</td><td>10</td><td>1</td><td>200</td><td>48</td><td>1</td><td>Ad libitum</td><td>10</td><td>10</td></tr><tr><td>nTME + high eTME + random</td><td>5</td><td>10</td><td>1</td><td>200</td><td>48</td><td>1</td><td>Random</td><td>10</td><td>9</td></tr></tbody></table>
measure Cu, Ni, As, Pb, Cd content and ICP-AES 720ES from Varian (Santa Clara, California, United States) to measure Zn content. For both ICP methods, calibration was performed using a standard curve generated from a multi-element standard at 1 g.L⁻¹ from CPI International (Santa Rosa, California, United States). The analytical limit of detection (LOD) for each analysis was determined as 3 times the baseline of the signal curve. Values for several elements were left-censored, i.e. below their LOD. In this case, values were replaced by their LOD divided by the squared root of 2. This is a robust alternative for left-censored data (Adjei and Stevens, 2022) and has already been used in similar study (Saulnier et al., 2020).

## 2.4. Feather corticosterone assay

Feather were used to measure the avian hormone corticosterone, which reflects an average value of corticosterone in the blood circulation during the 21–26 days growth period of the feather (Zann, 1996a). Feather corticosterone assays were conducted at the Centre d'Etudes Biologique de Chizé (Villiers-en-Bois, France). Corticosterone was extracted from the rectrice feathers by following a modified protocol described in Bortolotti et al. (2008). The calamus of the feathers was cut, and the remaining part of the feathers was cut in two pieces and placed in a test tube with 2 mL of 80 % methanol from Hipersolv VWR International (Fontenay-sous-Bois, France). The samples were placed in a sonicating water bath for 30 min, then 2 min in a vortex, followed by an overnight incubation at 50°C. The methanol was then separated from feather material and evaporated at 60°C under a nitrogen flow. The dried extract was reconstituted with PBS buffer used in the radioimmunoassay (RIA). Then, 100 µL of extract were incubated overnight at 4°C with 4000 cpm of the appropriate H3-steroid from Perkin Elmer (Waltham, Massachusetts, US) and polyclonal sheep antiserum from Merck (Darmstadt, Germany). The bound fraction was then separated from free fraction by addition of dextran-coated charcoal and activity was counted on a tri-carb 2810 TR scintillation counter from Perkin Elmer (Waltham, Massachusetts, US). Feather extracts, spiked with corticosterone, were serially diluted in the assay buffer and their displacement curves were parallel to the standard curve. Inter- and intra-assay variations were respectively 16.71 % and 11.74 %. Corticosterone lowest detectable concentrations in feather extract was 56.3 pg. mL⁻¹. According to the manufacturer, cross-reactions of corticosterone antiserum were as follows: 11-dehydrocoelicosterone (0.67 %), deoxycorticosterone (1.5 %), 18-hydroxy-deoxycorticosterone (<0.01 %), cortisone (<0.01 %), cortisol (<0.01 %), progesterone (0.004 %), aldosterone (0.2 %).

## 2.5. Heterophils/lymphocytes (H/L) ratio

To measure basal chronic stress level of birds, we monitored the H/L ratio in blood throughout the experiment. Ethanol-fixed blood smears were stained with a May - Grünwald - Giemsa stain bath of 1:10 v/v Slow Giemsa stain diluted with phosphate-buffered saline all from Sigma-Aldrich®- Merck (Darmstadt, Germany) at pH 7.2 for 40 min. The smears were then washed and dried on absorbent paper. The smears were read under a light microscope Axioplan 2 Zeiss (Oberkochen, Germany) at 1000x magnification using Nikon x100 oil immersion objectives (Tokyo, Japan). The number of heterophils and lymphocytes was determined for 100 leukocytes per slide. Only one experimenter performed the counts after checking for self-repeatability (N = 20, Intraclass Correlation Coefficient of 97.2 %).

## 2.6. Feeding behaviour monitoring

In order to monitor the feeding behaviour of birds, each bird was equipped with a Radio Frequency Identification (RFID) ring from Eccel Technology (Leicester, United Kingdom). The feeder was equipped with a RFID antenna and a reader that monitored which individual was at the

feeder and when, allowing us to measure the time spent at the feeder and the feeding habits. The TITAN 4004 OEM board, from TECTUS Technology (Bergisch Gladbach, Germany), was employed for its compatibility with the EM4102 PIT TAG protocol of the rings. Data acquisition was managed by an open-source application, rfid.app (https://gitlab.in2p3.fr/rfid.app, on 13/12/2024). This application recorded sensor-generated events on a computer from Raspberry Pi (Cambridge, United Kingdom), storing data with timestamps in a dedicated database. A custom antenna design was developed specifically for integration with a 2GR "Unika model 433" bird feeder from 2GR (Correggio, Italy). This antenna was constructed using 3D printed components and 0.23 mm enameled copper wire, achieving an enhanced maximum reading range of approximately 50 mm (https://rfid.app/pages.in2p3.fr/rfid.app/wiki/latest/Hardware/Antenna_Feeder/, visited on 13/12/2024). The RFID data were processed to remove records corresponding to nighttime activity, times when there was no food available and erroneous RFID reads. We then calculated the total time (in seconds) spent at the feeder by each individual over the 65-day period. The RFID monitoring was validated by comparing video recordings of eating events with RFID detection and time at the feeder. We obtained a good correlation between the time at the feeder filmed and using RFID (n = 18, p < 0.001, Spearman correlation coefficient = 0.86) and a good correlation with eating events and time at the feeder (n = 24, p < 0.001, Spearman correlation coefficient = 0.94) (See supplements for more information on the validation of the RFID method). Based on this validation, we consider time spent at the feeder to be a reliable indicator of eating time. However, this parameter should be interpreted as feeding behaviour and not as an indicator of food intake.

## 2.7. Statistical analyses

Data were analysed using R 4.3.0 (Core Team, 2024). All statistical tests with a p-value below 0.05 were considered significant. The lack of effect of the treatments on survival was assessed using a Cox risk analysis (R packages survminter 0.4.9 (Kassambara et al., 2021) and survival 3.6–4 (Therneau et al., 2000)).

We tested the effects of mixture and food treatments and their interaction on daily water consumption using linear mixed models (package lme4 1.1–35.5 (Bates et al., 2015) and lmerTest 3.1–3 (Kuznetsova et al., 2017)). Then, to describe individual contamination patterns for each TME, we tested: (i) the effects of mixture treatments, time (day 0 and day 65) and their interactions, (ii) the effects of food treatments, time and their interactions, on TME feather concentration using Scheirer-Ray-Hare tests (package rcompanion 2.4.3 (Mangiafico, 2024)). Dunn post-hoc pairwise comparisons were performed for significant interactions (package FSA 0.9.5 (Ogle et al., 2023)).

To compute an index of individual contamination taking into account all TME we performed a principal component analysis (PCA) with the scaled concentrations of Zn, Cu, Ni, Pb, As and Cd in feathers (packages factoextra 1.0.7 (Kassambara and Mundt, 2020) and FactoMineR 2.11 (Lê et al., 2008)). We kept the first 2 dimensions (PC1 and PC2) from the eigenvalue selection. Since the use of PCA dimensions remove time from our models, we performed Scheirer-Ray-Hare tests and Dunn post-hoc pairwise comparison on PC1 and PC2 to see how the time variable and treatments explained the dimensions. PC1 and PC2 were then used as explanatory variables in the other models.

Linear mixed models were used to analyse the effects of treatments on: corticosterone level in feathers, H/L ratio, total time spent at the feeder and body mass. Corticosterone concentrations and H/L ratios were log-transformed to fit model assumptions. We tested both the effect of the mixture treatment as a categorical variable and the effect of individual contamination using TME concentration levels (PC1 and PC2) on each parameter in two separate models. In the first set of models, we also included: food treatments, time and their interactions. In the second set of models, we included only food treatments and their interactions (the effect of time being explained by PC1, see results). For the model
explaining body mass we also included tarsus length as a covariate. For each model, we checked conformity with underlying assumption using R package DHARMa 0.4.6 (Hartig et al., 2024). For linear mixed models, we included individual ID as a random intercept for corticosterone, H/L, mass and cage ID as a random intercept for water consumption and time at the feeder. For each analysis we selected the best model by comparing the AIC of all possible models (function dredge, package MuMln 1.48.4 (Barton, 2024)). When several models had comparable AIC (difference of AIC < 2), we chose the most parsimonious model. We performed ANOVA III tests and Sidak post-hoc tests (package emmeans 1.10.4 (Lenth et al., 2024)) for categorical variables. When interactions were significant, simple slopes analyses (package interactions 1.2.0 (Long, 2024)) were performed. Dataset and R scripts access information are available in the Data and resource availability section.

## 3. Results

### Exposure

Daily water consumption is a proxy for monitoring the exposure of birds to the mixture treatments. The mixture treatment affected individual mean daily water consumption (Fig. 1). Birds exposed to TME drank significantly less than control birds (Fig. 1) i.e., 13.07 mL per day in average (see supplementary Table S1 for mean values). The difference was greater for birds exposed to eTME than for those exposed to nTME alone, compared with the control group. Birds exposed to eTME significantly drank less (In average 2.51 and 3.65 per day) than birds exposed to TME alone, but there was no difference between low and high eTME (Fig. 1). No difference in daily water consumption was detected according to the food treatment (variable not retained in the best model).

### Contamination

Individual contamination was measured as the concentration of TME in feathers (Supplementary Table S2). Ni, Cd and As were the only elements with values below the LOD with 23.5 %, 20.6 % and 4.4 % of the total values respectively (Supplementary Table S2). A significant

increase in concentration was found to be related to exposure time, depending on the mixture treatment, for the concentrations of Cu, As, Cd and Pb in feathers (Supplementary Table S3 A). The concentration of Cu increased significantly between T0 and T65 in the “nTME+ low eTME” and “nTME+ high eTME” groups (Fig. 2A). There was no significant increase in the other groups not exposed to Cu (“Ctrl”, “nTME alone”, Fig. 2A). The concentration of As and Pb increased significantly in all groups exposed to nTME (Figs. 2B and 2D). The pattern was different for Cd. The concentration of Cd only increased significantly in the “nTME alone” when no significant time effect was detected in the “nTME+ low eTME” and “nTME+ high eTME” groups (Fig. 2C). Finally, the concentration of Ni only significantly increased with time (6.9 % increase between Day 0 and 65), independently of the mixture treatment, and the concentration of Zn did not change significantly during the experiment (Supplementary Table S3 A). No difference in the contamination levels in any TME was associated with the food treatment (Supplementary Table S3 B).

We performed a PCA with the concentrations of As, Cd, Cu, Ni, Pb and Zn in feathers (Fig. 3). The first 2 dimensions explained 53.6 % of the variance (respectively 29.7 % for the first dimension PC1 and 23.9 % for the second dimension PC2). PC1 was significantly explained by Pb (46.2 %) and As (39.8 %) and PC2 by Zn (47.7 %) and Cu (37.1 %). Therefore, PC1 can be considered as the nTME contamination gradient and PC2 as the eTME contamination gradient. The effect of time, mixture treatment and their interaction were significant for PC1 but not for PC2 (Supplementary Table S4). Moreover, no association with the food treatment was found for either dimension (Supplementary Table S4).

## 3.2. Feather corticosterone levels and H/L ratios

Mean H/L ratios and corticosterone levels are available in the supplementary Table S5. Both feather corticosterone concentration and blood H/L ratios increased significantly with time independently of mixture treatment (Table 2 Model A and model B and supplementary Table S6). The interaction between time and food treatment was significant for H/L ratio, showing a significant increase in the H/L ratios between day 0 and day 65 only in the group exposed to random food access (Supplementary Table S7). Both markers were not associated with the mixture treatments. In terms of individual contamination, there was a significant positive correlation between the concentration of corticosterone in feathers and PC1 (Table 3 Model E and supplementary Table S7). There was also a significant interaction between PC1 and food treatment for the H/L ratio (Table 3 Model F), with a significant increase of the H/L ratio with increasing PC1 value only in the group with random access to food (Fig. 4). Both PC2 and mixture treatment groups did not show any significant effect on any of the 2 markers. When looking at link between mass and H/L ratio, no significant effect of the mass on H/L ratio variation through time has been highlighted (N = 68 birds, n = 136 measurements, R² = 0.016, *Est* = 0.069, s.e = 0.068, t-value = 1.025, *p*-value = 0.309). Similarly, there was no significant effect of the mass variation on feather corticosterone level variation through time (N = 68 birds, n = 136 measurements, R² = 0.013, *Est* = -1.394, s.e = 1.522, t-value = -0.916, *p*-value = 0.363).

## 3.3. Feeding behaviour and body mass

The mean time spent at the feeder is available in the supplementary Table S1. Individual total amount of time spent at the feeder was independent of the food treatment, with a marginal effect of the mixture treatments (Table 2, model C). Only the “nTME + low eTME” group had a higher time spent at the feeder than the “Ctrl” group (Fig. 5). In terms of individual contamination, there was no significant effect of PC2 on total individual time at the feeder but there was a positive effect of PC1 (Table 3, model G and supplementary Table S7), showing that the more contaminated the birds were, the more time they spent at the feeder. Yet,

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Effect of the mixture treatment on mean individual daily water consumption. N = 16 cages, n = 432 measurements, R² Marginal = 0.708, R² Conditional = 0.747, Anova III, df = 8, F-value = 62.62, p-value < 0.001. Similar letters showing no-significant differences between groups at the alpha risk of 5 % (Sidak multiple pairwise comparisons test). “Ctrl” = control condition given mineral water only. “nTME alone” = condition exposed to 5 mg. L⁻¹ of As, 10 mg.L⁻¹ of Pb and ¹ mg.L⁻¹ of Cd. “nTME+ low eTME” = condition exposed to 5 mg.L⁻¹ of As, 10 mg.L⁻¹ of Pb, 1 mg.L⁻¹ of Cd and ¹⁰⁰ mg.L⁻¹ of Zn, 24 mg.L⁻¹ of Cu, 0.5 mg.L⁻¹ of Ni. “nTME+ high eTME” condition exposed to 5 mg.L⁻¹ of As, 10 mg.L⁻¹ of Pb, 1 mg.L⁻¹ of Cd and 200 mg.L⁻¹ of Zn, 48 mg.L⁻¹ of Cu, 1 mg.L⁻¹ of Ni.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Effect of treatment groups on the concentration of TME in feather in mg/kg (dry matter) before exposure (Day 0) and after 65 days of exposure (Day 65). A) Copper concentrations; B) Arsenic concentrations; C) Cadmium concentrations; D) Lead concentrations. Similar letters represent non-significant differences between groups at the alpha risk of 5 % (Dunn multiple pairwise comparison tests).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Principal Component Analysis of TME feather concentrations. N = 68 individuals, n = 136 feathers. Contribution to dimension 1 (PC1): Pb 46.2 %, As 39.8 %, Cu 11.8 %, Cd 1.0 %, Zn 0.8 %, Ni 0.3 %. Contribution to dimension 2 (PC2): Zn 47.7 %, Cu 37.1 %, As 9.6 %, Ni 2.3 %, Pb 2.0 %, Cd 1.3 %.</figcaption></figure>

those birds were not significantly heavier (Table 3, and see the mean body mass value per group in the supplementary Table S5). Body mass was only explained by the null model (Table 2, model D), meaning that neither treatment nor time was associated with variation in this variable. However, lower body mass was significantly correlated with a higher PC1 (Table 3, model H and supplementary Table S5) suggesting

that lighter individuals were more contaminated than heavier individuals.

## 4. Discussion

This study aimed to determine whether urban relevant TME mixtures have an effect on the behaviour and the physiology of zebra finches and whether these effects can be modulated by the presence of eTME in the mixtures or by food availability. Overall, we found evidence that, birds exposed to TME mixtures avoided the mixture by drinking less, although they still accumulated Cu, Pb, As and Cd. This avoidance is accentuated when the treatment contains eTME. Birds exposed to eTME accumulated less Cd than birds exposed to nTME alone. The mixture treatment affected the H/L ratio and the corticosterone level of the birds and marginally affected the feeding behaviour of the birds. Indeed, the level of nTME contamination was positively correlated with the H/L ratio and the corticosterone levels in birds, a relationship that was exacerbated by food restriction. nTME levels were also positively correlated with the time spent at the feeder without inducing body mass changes.

### 4.1. Water avoidance

Contrary to our initial expectations, birds showed a strong avoidance of the TME mixture particularly for mixtures containing eTME. Although zebra finches are native to semi-arid regions of Australia they show “flexibility rather than specialisation” (Zann, 1996b) regarding water access. The lowest mean consumption (2.5 mL/day) detected was in the range of the regular zebra finches water consumption of 3 mL/day (Zann, 1996b), indicating that this avoidance should not have induced water deprivation. We can therefore rule out effects linked to dehydration on our physiological markers, e.g. on corticosterone (Brischoux
<table><caption>**Table 2**
ANOVA Type III results on Linear Mixed Models outputs testing the effects of food and mixture treatments and time.</caption><thead><tr><th colspan="10">Variable of the best selected model</th></tr><tr><th>Model</th><th>Explained</th><th>Transformation of explained</th><th>Explanatory</th><th>n</th><th>R<sup>2</sup><sub>Marginal</sub></th><th>R<sup>2</sup><sub>Conditional</sub></th><th>df (Num)</th><th>F-value</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="2">A</td><td rowspan="2">Corticosterone in feathers</td><td rowspan="2">Log</td><td>Time</td><td rowspan="2">136</td><td rowspan="2">0.098</td><td rowspan="2">0.098</td><td>1</td><td>14.73</td><td>&lt; 0.001</td></tr><tr><td>Food</td><td>1</td><td>0.40</td><td>0.532</td></tr><tr><td rowspan="2">B</td><td rowspan="2">H/L ratio</td><td rowspan="2">Log</td><td>Time</td><td rowspan="2">136</td><td rowspan="2">0.112</td><td rowspan="2">0.444</td><td>1</td><td>18.42</td><td>&lt; 0.001</td></tr><tr><td>Food: Time</td><td>1</td><td>10.75</td><td>0.002</td></tr><tr><td>C</td><td>Total time spent at the feeder</td><td></td><td>Mixture</td><td>54</td><td>0.280</td><td>0.427</td><td>3</td><td>4.10</td><td>0.052</td></tr><tr><td>D</td><td>Body mass</td><td>No covariates selected</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<table><caption>**Table 3**
ANOVA Type III results on Linear Mixed Models outputs testing the effects of the individual index of contamination (PC1 and PC2) and the food treatment.</caption><thead><tr><th colspan="10">Variable of the best selected model</th></tr><tr><th>Model</th><th>Explained</th><th>Transformation of explained</th><th>Explanatory</th><th>n</th><th>R<sup>2</sup><sub>Marginal</sub></th><th>R<sup>2</sup><sub>Conditional</sub></th><th>df (Num)</th><th>F-value</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="2">E</td><td rowspan="2">Corticosterone in feathers</td><td rowspan="2">Log</td><td>PC1</td><td rowspan="2">136</td><td rowspan="2">0.051</td><td rowspan="2">0.051</td><td>1</td><td>7.26</td><td>0.008</td></tr><tr><td>PC1</td><td>1</td><td>25.04</td><td>&lt; 0.001</td></tr><tr><td rowspan="2">F</td><td rowspan="2">H/L ratio</td><td rowspan="2">Log</td><td>Food</td><td rowspan="2">136</td><td rowspan="2">0.141</td><td rowspan="2">0.537</td><td>1</td><td>0.94</td><td>0.427</td></tr><tr><td>PC1: Food</td><td>1</td><td>9.21</td><td>0.003</td></tr><tr><td>G</td><td>Total time spent at the feeder</td><td></td><td>PC1</td><td>54</td><td>0.103</td><td>0.350</td><td>1</td><td>4.93</td><td>0.032</td></tr><tr><td rowspan="2">H</td><td rowspan="2">Body mass</td><td rowspan="2"></td><td>PC1</td><td rowspan="2">136</td><td rowspan="2">0.149</td><td rowspan="2">0.570</td><td>1</td><td>4.15</td><td>0.045</td></tr><tr><td>Tarsus length</td><td>1</td><td>14.18</td><td>&lt; 0.001</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Effects of PC1 and food treatment on individual heterophils/lymphocytes ratios (log-transformed). N = 68 birds, n = 136 measurements. When food is random: Est = 0.439, Standard error = 0.080, T-value = 5.48, p-value < 0.001. When food is ad libitum: Est = 0.107, S.E = 0.074, T-value = 1.45, p-value = 0.151.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Effect of mixture treatment on the time spent at the feeder. N = 14 cages, n = 54 individual measured. Similar letters showing no-significant differences between groups given by Sidak multiple pairwise comparisons test at the alpha risk of 5%.</figcaption></figure>

contamination.

## 4.2. Urban relevant contamination

In this study, we aimed to achieve contamination levels typical of the contamination of urban-living birds by referring to urban great tits faecal concentration of TME (Saulnier et al., 2020). Using existing literature on urban-living passerine birds (blue tits and great tits in several cities in Poland (Chatelain et al., 2021a, 2021b) and in Antwerp, Belgium (Dauwe et al., 2000) and great tits in Strasbourg, France (Saulnier et al., 2023), house wrens (Troglodytes aedon) and American robins (Turdus migratorus) in Jersey city, New Jersey, USA (Hofer et al., 2010) and the house sparrow (Passer domesticus) in the West band (Swaileh et al., 2006)), we calculated ranges of TME concentrations found in feathers: 55–890 mg.kg⁻¹ of Zn, 4.9–41 mg.kg⁻¹ of Cu, 0.4–62 mg.kg⁻¹ of Pb, 0.007–3.8 mg.kg⁻¹ of Cd and between 0.2 and 8 mg.kg⁻¹ of As (Supplementary Table S8). The zebra finches involved in our study displayed contamination levels in feathers that were within

et al., 2020). A lower water consumption of TME-treated birds compared to the control group had already been shown (Saulnier, 2022) without highlighting any detrimental effect of such low water consumption on other variables. Since other taxa were proven to be able to detect TME either via taste (drosophila (Li et al., 2023; Xiao et al., 2022) and humans (Lawless et al., 2005); Pirkwieser et al., 2021; Reddy et al., 2010) or smell (humans (Lawless et al., 2005); Pirkwieser et al., 2021; Reddy et al., 2010) and avoid TME (earthworms (Chatelain, 2023)). This suggests that either zebra finches have a similar ability to detect and avoid TME such as Cd, As or Pb in the drinking water and especially Zn, Cu or Ni, or that they have a neophobia to taste or smell (Krause et al., 2018). Further studies are needed to investigate the cognitive processes and pathways underlying this avoidance behaviour. From a more practical point of view, this result means that the contamination of the birds may be different from what we would expect in each group. We have addressed this issue through individual monitoring of feather
the calculated ranges for passerine birds for Zn, Cu, Cd and Pb. However, they displayed As levels up to 2 times higher than the maximum value found in blue tits (Chatelain et al., 2021a). Although the level of As in the mixtures was lower than other TME like Pb, it seems that its accumulation ability in the feather is higher. Similar trends have been showed in tree sparrow (Passer montanus) and light-vented bulbul (Pycnonotus sinensis) with a higher concentration of As in feather than muscle or stomach (Yang et al., 2018). In magpie (Pica pica), feathers and liver displayed similar As concentrations (Yang et al., 2018) but as these birds were living in a mining area, they displayed As feather content up to 2 times higher than the zebra finches involved in our study. Overall, it seems that the contamination level of zebra finches in our study is at an intermediate level between the urban and mining environments for As and coherent with an urban environment for the other TME.

### 4.3. nTME contamination and adverse effects

Birds exposed to nTME accumulated them in their feathers, with higher concentrations measured after exposure to mixtures. This corroborates previous work suggesting that feathers can be used as a proxy for avian contamination (Dauwe et al., 2002; Saulnier et al., 2020; Abdullah et al., 2015), although the direct correlation between exposure and feather content depends on the TME considered (Dmowski, 1999). According to the PCA results, the overall nTME contamination gradient was mostly explained by variation in Pb and As feather concentrations. This is in accordance with a previous study showing a strong relationship between exposure level and feather level for Pb and As, but not for Cd (Dauwe et al., 2004). However, in our experiment, many of the post-exposure Cd values were very low, limiting our ability to measure changes in Cd levels.

We first hypothesized that nTME should be responsible for the adverse effects on the zebra finches. Accordingly, the exposure to nTME induced a physiological stress for birds as exposure to Pb and As was positively correlated with the H/L ratio and corticosterone concentrations. However, in common pigeons, there is no effect of Pb contamination on plasmatic corticosterone levels (Chatelain et al., 2018). The result we observed could be explained by the fact that we exposed the birds to several nTME at the same time which could result in a higher toxicity.

We also measured an average value of corticosterone in the feather, which integrates both baseline and temporary surges in corticosterone levels (i.e., stress response) following acute stress, such as handling (Bortolotti et al., 2008). Since corticosterone is involved in energy balance (Landys et al., 2006), and has been shown to be intrinsically related with fasting and feeding behaviour (Astheimer et al., 1992), changes in corticosterone levels following exposure to TME are expected to be related with changes in feeding behaviour, and more specifically with a more intense foraging behaviour. This is what we observed, as the time spent at the feeder was positively correlated to the exposure to Pb and As without changes in individual body mass. This can be interpreted as a slower feeding process, either on the activity itself (seeds sorting or husking...) or as a lower absorption or increased metabolism. For example, young common terns (Sterna hirundo) show a loss of feeding efficiency (slower feeding rate, increased dropping rate) impacting the energetic level (lower weight gain) when exposed to Pb (Gochfeid et al., 1988). However, and in contrast to our study, Pb exposure also affected individual body mass. This difference can be explained by the fact that we only studied adult birds, which may be less sensitive to Pb than growing individuals. Another reported effect of corticosterone is that it can induce changes in body composition, with loss of muscle and gain of fat tissue without affecting food intake or drinking frequency in dark-eyed juncos (Junco hyemalis) (Gray et al., 1990). Similarly chronic stress has been shown to reduce the accuracy and speed of movements (Metz et al., 2005). It is then possible that the increase in time spent at the feeder is a behavioural consequence of the increase in chronic stress

caused by contamination. To disentangle these effects, a study investigating both the time spent eating and the amount of food intake is needed. Regarding the eTME contamination, Cu was the only eTME to accumulate in feathers when birds were exposed to mixtures containing eTME. This corroborates previous results showing accumulation of Cu in blood (Saulnier et al., 2020) and feather (Saulnier, 2022).

Therefore, as stated in our first hypothesis, the adverse effects of urban relevant TME mixture on feeding behaviour and individual stress can be attributed to nTME contamination.

### 4.4. Effect of eTME co-exposure

Then, we hypothesized that co-exposure to eTME with nTME should mitigate the adverse effects of nTME exposure on contamination level, behaviour and physiological stress. We found that eTME and nTME co-exposure affected Cd accumulation levels. When co-exposed with eTME, Cd did not accumulate significantly in feathers, meaning that the exposure to Cu, Zn and Ni probably prevented the accumulation of Cd in feathers. Birds exposed to the "nTME + High eTME" treatment, significantly accumulated Cu, revealing antagonistic effects of Cu on Cd. This result supports findings of antagonism of Cu and Cd when Cu is in excess (Gochfeid et al., 1988). However, this reduction in Cd accumulation did not translate into a reduction in the effects of the exposure to the mixture on physiological stress or feeding behaviour, suggesting that these effects of the mixture were certainly more related to As and Pb contamination than Cu and Cd contamination (see above). Indeed, we only tested one concentration of Cd and two eTME concentrations. Testing a wider range of concentrations would have helped highlighting the interactions between TME, although such approach is unpractical and difficult to conduct because of the number of birds and rooms required.

Contrary to our predictions, we did not find any effect of the eTME co-exposure on Zn feather concentration, even though Zn is considered to have high affinity for feather's keratin (Dmowski, 1999). Moreover, previous studies showed accumulation of Zn in blood and feather over time (Saulnier, 2022; Saulnier et al., 2020) using the exact same exposure procedure with similar concentration of Zn as in our "nTME + Low eTME" treatment. Our results do not reveal antagonism between Pb and Zn as we expected from literature (Chatelain et al., 2016a; Dauwe et al., 2002).

Therefore, we did not find a significant effect of eTME on behaviour or corticosterone and H/L ratio adverse outcome but we showed a reduction on Cd accumulation induced by eTME and specifically Cu.

### 4.5. Effect of random food access

Finally, we hypothesised that birds exposed to random food access (mimicking stochastic nature of food availability in natural conditions) would have higher levels of corticosterone level and H/L ratio and stronger adverse effect of nTME exposure on these markers of physiological stress. However, we did not find an increase in feather corticosterone levels associated to random food access. Feathers corticosterone level is thought to integrate both baseline and temporary surges in corticosterone levels (i.e. corticosterone stress response) over the period of feather growth (Jenni-Eiermann et al., 2015) but it has also been shown to be more tightly associated with acute stress response rather than baseline stress level (Bortolotti et al., 2008). This discrepancy could explain why random food access did not have any significant effect on feather corticosterone levels in our study, while the same food restriction protocol has been shown to affect baseline corticosterone levels in zebra finches in blood (Marasco et al., 2015).

The H/L ratio is a general proxy of physiological stress and it has previously been related to baseline circulating corticosterone levels (Davis et al., 2008). In accordance to our expectations, the random food access treatment was significantly associated to increased H/L ratio of birds without affecting their body mass (Marasco et al., 2015, 2018). In fact, the H/L ratio was the only marker affected by the food availability
treatment. Our results indicate that: in the case of *ad libitum* access to food, higher contamination level of Pb and As were associated to higher H/L ratio and feather corticosterone levels, while – in the case of random food access – the relation between Pb and As contamination level and H/L ratio was even steeper. This suggests that our food access treatment strengthened the adverse effect of nTME on this proxy of physiological stress. In addition, food access treatment had no effect on body mass, feeding behaviour or water consumption.

These results highlight the importance of ecologically relevant laboratories studies considering multiple stressors simultaneously (in this case TME contamination and random food access). Specifically, we showed that food availability (*ad libitum* vs. random access treatment) affected individual physiological stress level with no effect on body mass and that this treatment could exacerbate the negative effects of nTME on stress, even though these effects did not appear on other nTME related adverse effects.

## 5. Conclusion & perspectives

This work aimed to experimentally test the effects of TME mixture on a passerine bird in a controlled environment with urban relevant conditions. From our results, we can conclude that even at middle-sized cities relevant doses, nTME can affect the feeding behaviour and induce chronic stress on adult male birds. Although the toxic effect is sub-lethal, we cannot exclude potential long-term effects of TMEs on the birds. Indeed, this study does not provide insight on the pathways involved and the resistance and detoxification processes at stake. Antagonistic effect of Cu contamination on Cd contamination has been shown without effect on stress or behaviour. However, it is known that such TME contaminations can affect oxidative stress (Koivula et al., 2010) and telomere length (Saulnier et al., 2020) in laboratory experiments as well as in the wild (Stauffer et al., 2017). The effect of TME interactions should be further investigated notably at a molecular scale.

Since birds were non-breeding adults, they had more energy to allocate to detoxification and maintenance processes (Alcaraz, 2005) than birds investing in reproduction, which may explain their coping ability. Further studies focusing on energetically demanding life stages such as hatchlings, breeding females or embryonic stages are needed to better understand the overall effect of urban relevant TME contamination on birds' physiology and wild birds' populations.

Finally, studies using more environmentally relevant laboratory experiments are needed, especially for elucidating the role of nutrition in physiological responses to contamination, as we observed more important effect of the treatment when birds are under challenging conditions (reduced food availability).

### CRediT authorship contribution statement

**Josefa Bleu:** Writing – review & editing, Visualization, Validation, Project administration, Investigation, Funding acquisition, Conceptualization. **Navina D. Liebermann-Lilie:** Writing – review & editing, Resources. **Julien Courteuisse:** Writing – review & editing, Software, Resources, Methodology. **Charline Parenteau:** Writing – review & editing, Methodology, Investigation. **Frédéric Angelier:** Writing – review & editing, Validation, Methodology. **Pascale Ronot:** Writing – review & editing, Methodology, Investigation. **Anne Boos:** Writing – review & editing, Validation, Resources, Methodology. **Sylvie Massemin:** Writing – review & editing, Validation, Supervision, Investigation, Conceptualization. **Clément Parnet:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis, Data curation.

### Funding

This work was supported by the French National Agency of Research (Agence Nationale de la Recherche) project TRAMET ANR-21-

CE34-0015-01.

### Declaration of Competing Interest

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

### Acknowledgements

We thank D. Bock, A. Hranitzky, N. Spanier and H. Gachot-Neveu for their help with animal care; J. Deleau and J. Anizan for their help with RFID method pre-study; R. Colominas-Ciuró for his teaching on blood smears reading; We thank the "Service d'Analyses Biologiques du CEBC", especially Emmanuelle Grimaud for its technical help in conducting laboratory analyses; P. Korsten, C. Schradin and L. Makuya for their feedbacks.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2025.118647.

### Data availability

Data and R script are available on Zenodo repository at 10.5281/zenodo.14925359. rfid.app can be found on GitLab at https://gitlab.in2p3.fr/rfid.app.

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