Léa Lansade
email: lea.lansade@inrae.fr

Julie Lemarchand

Fabrice Reigner

Cécile Arnould

Aline Bertin

Automatic brushes induce positive emotions and foster positive social interactions in group-housed horses

Keywords: Equus caballus, positive affect, facial expression, emotional indicators, welfare, environmental enrichment

In mammals, positive tactile contact is recognized as an effective tool for triggering positive affective states. In this study, we investigated the benefits of providing automatic rotative brushes for group-housed horses. Our three aims were: i. to determine whether horses used automatic brushes and if so how they used them; ii. to investigate whether the presence of these brushes induced positive social behaviours (allogrooming), or aggressiveness, as can be to conduct self-grooming, and also in all likelihood, an indirect benefit by facilitating positive social interactions. This study encourages the installation of automatic brushes in places where horses are kept to improve their welfare and provide comfort.

observed when there is competition for a desired resource; iii to provide a preliminary explanation of the role of the positive facial expression displayed by some horses while being groomed.

Two brushes were installed in a large stable with free access to a paddock in which 40 horses were housed 7h30/day. For four days, video-cameras placed above the brushes continuously filmed the horses. First, analysis of the video footage demonstrated that brushes were used by almost 90% of the horses, mainly on areas that are not easily accessible to another horse during allogrooming, such as the head. Secondly, it revealed that among the horses that used the brush, 25.7% expressed positive social behaviour (allogrooming) at the same time, while none expressed aggressive behaviours. The brush thus seems to act as a catalyst for affiliative behaviours rather than competition for a desired resource. Thirdly, we observed their facial expression, especially a positive one, described when the horse are groomed (neck moderately raised, eyes open or half-closed, upper lip extended, ears turned backwards almost in line with the nose). More than half of the horses displayed this while using the brushes. Interestingly, when a horse expressed this face, it had a six times greater chance to subsequently start allogrooming with a conspecific. Several possible explanations are discussed, including a social function. Further research is needed to validate other criteria to help qualify whether or not this positive facial expression is a signal of intentional communication. From a practical point of view, the results show that the brushes provide both a direct benefit to the horses enabling them

Introduction

In mammals, positive tactile contact is recognized as an effective tool for triggering positive affective states [START_REF] Burgdorf | The neurobiology of positive emotions[END_REF]. These positive effects of tactile contacts can be evaluated through changes in behaviour such as the expression of specific vocalizations, specific facial expressions, contact seeking, allogrooming (grooming between conspecifics) or a relaxed state in various species such as rodents [START_REF] Burgdorf | The neurobiology of positive emotions[END_REF][START_REF] Finlayson | Facial Indicators of Positive Emotions in Rats[END_REF], sheep [START_REF] Reefmann | Behavioural and physiological assessment of positive and negative emotion in sheep[END_REF][START_REF] Westerath | Are special feed and being brushed judged as positive by calves?[END_REF][START_REF] Tamioso | Behavior and body surface temperature as welfare indicators in selected sheep regularly brushed by a familiar observer[END_REF], cattle [START_REF] Schmied | Stroking of different body regions by a human: Effects on behaviour and heart rate of dairy cows[END_REF][START_REF] Westerath | Are special feed and being brushed judged as positive by calves?[END_REF], heifers [START_REF] Bertenshaw | Exploring heifers' perception of 'positive' treatment through their motivation to pursue a retreated human[END_REF], dogs [START_REF] Rehn | Dogs' endocrine and behavioural responses at reunion are affected by how the human initiates contact[END_REF] or horses [START_REF] Feh | Grooming at a preferred site reduces heart rate in horses[END_REF][START_REF] Mcbride | A preliminary study on the effect of massage to reduce stress in the horse[END_REF][START_REF] Mullard | Development of an ethogram to describe facial expressions in ridden horses (FEReq)[END_REF]. Positive tactile contact also has physiological correlates in horses. It induces changes in parameters such as heart rate [START_REF] Feh | Grooming at a preferred site reduces heart rate in horses[END_REF] or hormone levels, such as the basal oxytocin level [START_REF] Lansade | Facial expression and oxytocin as possible markers of positive emotions in horses[END_REF], for a review [START_REF] Vandierendonck | Coping in groups of domestic horses -Review from a social and neurobiological perspective[END_REF].

With the aim of improving welfare by promoting positive affective states, different ways of providing tactile contact have been studied in horses: allowing allogrooming with partners [START_REF] Snorrason | Social relationships in a group of horses without a mature stallion[END_REF], a handler scratching them with their hands [START_REF] Feh | Grooming at a preferred site reduces heart rate in horses[END_REF][START_REF] Lansade | Facial expression and oxytocin as possible markers of positive emotions in horses[END_REF], or by making different devices available, such as fixed brushes [START_REF] Lansade | Behavioral and Transcriptomic Fingerprints of an Enriched Environment in Horses (Equus caballus)[END_REF]. On cattle farms, automatically rotating brushes are also often provided and animals use them extensively [START_REF] Devries | Short communication: Usage of mechanical brushes by lactating dairy cows[END_REF][START_REF] Moncada | Use of a mechanical brush by dairy cows with chorioptic mange[END_REF] with positive results reported, such as a reduction in time spent inactive [START_REF] Velasquez-Munoz | Effect of a mechanical grooming brush on the behavior and health of recently weaned heifer calves[END_REF] or an increase in self-grooming [START_REF] Horvath | Characterizing grooming behavior patterns and the influence of brush access on the behavior of group-housed dairy calves[END_REF]. To date, the influence of an automatic brush on the behaviour of group-housed horses has not been studied. Thus, the present study on horses aimed to investigate this. Moreover, we recently showed in horses that positive tactile contacts trigger a positive affective state associated with a specific facial expression (called "positive facial expression" hereafter). Indeed, when scratched by humans, horses express a facial expressions with the following characteristics: neck moderately raised, eyes opened or half closed, upper lip extended and either immobile or twitching and ears turned backwards almost in line with the nose [START_REF] Lansade | Facial expression and oxytocin as possible markers of positive emotions in horses[END_REF][START_REF] Trösch | Horses feel emotions when they watch positive and negative horse-human interactions in a video and transpose what they saw to real life[END_REF]. Facial expressions may have a double function: expressing passive emotion and/or an intention to engage in specific activities with the receiver (Waller et al., 2017;[START_REF] Camerlink | Facial expression as a potential measure of both intent and emotion[END_REF]. The communication of intention is thought to regulate social interactions within groups, such as lipsmacks in chimpanzees that enable allogrooming to be coordinated and prolonged, and facilitate social cooperation. According to [START_REF] Townsend | Exorcising Grice's ghost: an empirical approach to studying intentional communication in animals[END_REF], to determine whether facial expressions are intentional at least two criteria should be used to evaluate them: the signal is only produced in the presence of the recipient (audience effect) and it is contingent on the behaviour of the recipient, which means that the behaviour follows the facial expression and -de facto-ends it (audience behaviour). The intentional use of facial expressions in social communication remains highly debated [START_REF] Waller | Rethinking primate facial expression: A predictive framework[END_REF] and to date has not been explored in horses.

This study had three aims. The first was to determine whether or not horses used automatic brushes, on what parts of their body, at what frequency, and how they triggered them. The second aim was to investigate whether the presence of these brushes induced positive social behaviours assessed by the expression of allogrooming behavior, or instead negative behaviours such as threats and aggressiveness, as can be observed when there is competition for a desired resource. The third aim was to provide a preliminary description of the role of facial expressions in horses, by determining whether the positive facial expression described when horses are groomed by humans was also expressed when they used the brush, and whether it played a social role or not. To this end, we investigated whether there was an audience effect when the positive face was expressed (i.e. if a conspecific was nearby when the signal was produced) and audience behaviour in response (with a contingent signal to the recipient's behaviour). We expected that the positive facial expression would be expressed while brushing but preferentially when another horse was nearby and that this facial expression would promote allogrooming.

Material and Method

Welfare statement

This study was non-invasive: we filmed the animals in their normal living condition without particular intervention. No permission from the Animal Ethics Committee was needed.

Animals, housing and characteristics of the brushes

The study was conducted on 40 female Welsh ponies, with an average age of 7.56+0.42 (mean+sem) years old and height at withers of 1m to 1.20m, reared at the Animal Physiology Experimental Unit PAO, INRAE (DOI: 10.15454/1.5573896321728955E12). The herd was established four weeks before the beginning of the observations. From then on, they lived in a group at pasture and every day from 08:00 to 15:30 they were brought into a large straw-covered stabling area (11x35m) with free access to an adjoining paddock (10x35m). Hay and water were available ad libitum in the stabling area.

Two brushes were present in the middle of the stabling area 10m apart. The brushes were those usually used for goats and which start rotating when touched by an animal (Mini rotating brush for goats and calves, MSB DeLaval). Each brush was attached to a mobile axis, enabling it to move from a vertical to a horizontal position and thus reach different parts of the pony's body (Fig. 1). The top of the brush was at a height of 120cm, corresponding approximately to the pony's' withers. The dimensions of each brush were 72x30 cm and the rotation speed was 25.5 rotations/min.

Behavioural observations

After 4 weeks of familiarization to the presence of the brushes in the stabling area, a camera was fixed above each brush to film the horses using it. Four periods from 08:00 to 15:30 were recorded, two in week 5 (14, 15 May) and two in week 6 (22, 23 May), for a total of 30 hours of observation. The film footage was subsequently viewed by an experienced observer (JL) to record the following behaviours which were divided into three categories according to the different aims.

To characterize the use of the automatic brushes (aim 1), we recorded: the number of sequences when a horse activated the brush, and identified the animal; the time the brush was used for each sequence (i.e. the time the brush was in contact with the horse); the part of the body that activated the brush (head, forehand or hindquarters, Fig. 3) or if it was already activated when the horse arrived. The percentage of time spent brushing each of these areas (time on one area of the body/ total time of use*100) was also calculated.

Then, we determined what type of behaviour was performed during the brushing (aim 2). For each sequence, we recorded whether at the same time as the horse used the brush it demonstrated an affiliative behaviour such as allogrooming (the horse is top to tail with a conspecific and both mutually nibbling different parts of each other's bodies) or an aggressive behaviour such as threatening to bite and biting (ears are backward, neck and head are stretched toward the opponent and accompanied by a quick movement toward it, without the teeth touching the opponent -biting is similar, but the teeth touch the opponent), threatening to kick and kicking (the ears are backward, the croup is toward the opponent and the horse raises one or both hind limbs or kicks out the hind limbs -kicking is similar, but the limbs touch the opponent), chasing or attacking. For each sequence, this was recorded as a binary response:

Yes/No.
For each sequence, we also evaluated the facial expressions of the horses while they used the brush (aim 3): a positive facial expression (first described during grooming by a person, see [START_REF] Lansade | Facial expression and oxytocin as possible markers of positive emotions in horses[END_REF], a negative facial expression, or neither of them (see Fig. 2 for a description of each expression). The negative facial expression differs from the threatening behaviors as the former is not explicitly directed toward a conspecific (the head and neck are not stretched toward a specific opponent and it is not accompanied by a quick movement toward it; the horse does not raise or kick out its hind limbs). When the four zones used to describe the facial expressions were not entirely visible (neck, ears, eyes and lips) the sequence was excluded from the analyses. Only one of these facial expressions was ever observed in any one sequence of brush use, never both of them. Finally we noted whether the positive facial expressions were expressed when there was another horse within 2m (Yes/No) and whether it was followed by allogrooming that de facto ended the facial expression (Yes/No). Initially, we had planned to do this kind of analysis for the negative facial expression to determine whether it was also followed by the aggressive behaviours, but since aggressive behaviours were not expressed, this was not possible.

Statistical Analysis

Two-proportion z tests using the Monte Carlo Method (5000 simulations, [START_REF] Fleiss | Statistical Methods for Rates and Proportions[END_REF][START_REF] Vose | Risk Analysis -A Quantitative Guide[END_REF] were conducted to compare the proportion of affiliative behaviours vs aggressive behaviours; to compare the proportion of positive vs negative facial expressions; and to test whether positive facial expressions were associated with affiliative behaviours (allogrooming).

For the latter, we compared the proportion of allogrooming after a positive facial expression vs that without a positive facial expression.

We observed 139 sequences during which a horse used the brush. Of the 139 times the brushes were used, it was possible to determine 132 times when allogrooming took place or not, 111 times when there was a positive facial expression or not and 131 times when there was a negative facial expression or not. These numbers differed since it was not always possible to validate all the criteria presented in Fig. 2. For instance, sometimes it was not possible to see all the criteria required to validate the presence of a positive facial expression, because for example the lips were not visible on the video, and therefore their position was unclear. In that case, a positive facial expression could not be validated and we considered the data as missing.

The two variables 'allogrooming' and 'positive facial expression' could be matched for 105 sessions of brush use. The statistical analyses presented in the results section were conducted on these respective numbers of sequences.

Results

Descriptive analysis of the use of the automatic brushes

Over the four 08:00 to 15:30 periods, 35 of the 40 horses used a brush (i.e. 87.5%) and the brushes were activated 139 times. The mean + sem for each horse was 3.97+2.93 times, with a minimum of once and a maximum of 11 times per horse. The mean + sem duration of use was 55.28+ 4.125 s. Of the 139 times the brush was activated, the majority were triggered by the horse's head, followed by the forehand and then the hindquarters. In a quarter of the cases the brush was already activated when the horse arrived (Fig. 3a). The horses mainly scratched their heads, followed by their hindquarters and then their forehand (Fig. 3b).

Aggressive and affiliative behaviours resulting from the use of the brushes

There were no cases of aggressive behaviour when horses used the brush. By contrast, affiliative behaviour (allogrooming) was expressed in 7.57% of the sequences, by nine different horsesone horse allogroomed twice (comparison between the proportion of aggressive behaviour and affiliative behaviour: 0/139 vs 10/132 sequences, two-proportion z test using the Monte Carlo Method, diff=0.076, p= 0.001).

Facial expressions observed while the horse used the brush and the social context in which they appeared

There were only 3.82% of sequences with a negative facial expression (5/131 sequences, from 5 different horses), but 39.94% with a positive facial expression (41/111 sequences, from 22 different horses, two-proportion z test using the Monte Carlo Method, diff=0.33, p<0.0001, Fig. 4).

Regarding the 'audience effect' hypothesis, there was always at least one other horse present less than 2 m from the horse being brushed with the exception of one case (and in that case, the horse did not produce a specific facial expression), thus it was not possible to test this hypothesis.

Regarding the 'audience behaviour' hypothesis, among the 105 sequences for which the variables "allogrooming" and "positive facial expression" matched, horses displayed a facial expression in 37 sequences and did not in 68 sequences. Interestingly, when the horse expressed a positive facial expression during a sequence there was 18.91% chance that an allogrooming event with another horse would take place immediately after it (7/37 sequences, for 7 different horses). This was 6.4 times less if the horse had not previously made a positive facial expression (2.94%: 2/68 sequences, for 2 different horses). These proportions are significantly different (two-proportion z test using the Monte Carlo Method, diff=-0.16, p=0.008, Fig. 5). The 9 (7+2) sequences of allogrooming involved nine different horses being brushed. We can also notice that allogrooming was never observed after a horse had expressed a negative facial expression (n=5).

Discussion

This study first demonstrates that automatic rotating brushes were used by a large proportion of horses living in a group. It also revealed that when horses used these brushes more positive social behaviours (allogrooming) than aggressive behaviours were expressed. Finally, if a horse expressed a positive facial expression during a sequence, it had a six times greater chance to subsequently start allogrooming with a conspecific than if it did not.

Description of brush use

Like cows (e.g. [START_REF] Devries | Short communication: Usage of mechanical brushes by lactating dairy cows[END_REF][START_REF] Horvath | Characterizing grooming behavior patterns and the influence of brush access on the behavior of group-housed dairy calves[END_REF][START_REF] Velasquez-Munoz | Effect of a mechanical grooming brush on the behavior and health of recently weaned heifer calves[END_REF][START_REF] Moncada | Use of a mechanical brush by dairy cows with chorioptic mange[END_REF], horses spontaneously use automatic brushes, as 87% of the horses in this study used them during the four 7h30 observation periods. The duration of each use was generally short being less than a minute, but this corresponds to the durations described in other species, for instance, bout duration in calves was 18 s [START_REF] Zobel | Calves Use an Automated Brush and a Hanging Rope When Pair-Housed[END_REF]. The number of visits to the brushes (4 visits per animal over 30h, i.e. 3.2 visits / 24h) is also on the same scale as that observed in adult cows : 4.5 visits / 24h in [START_REF] Mandel | The effect of food location, heat load, and intrusive medical procedures on brushing activity in dairy cows[END_REF] or 7.7 visits / 24h in [START_REF] Devries | Short communication: Usage of mechanical brushes by lactating dairy cows[END_REF]. This figure is also similar to that described in Islandic horses for allogrooming sequences [START_REF] Snorrason | Social relationships in a group of horses without a mature stallion[END_REF].

Regarding the areas brushed the most, we might have expected to find the base of the neck or the withers, which according to a study on Camargue horses corresponds to the area of the body that horses mutually scratch during allogrooming [START_REF] Feh | Grooming at a preferred site reduces heart rate in horses[END_REF]. However, this was not the case in our study, as the horses spent more time scratching their head and hindquarters than the base of the neck which was nevertheless an area easily accessible to the automatic brush. In particular, the head is not an area that horses mutually groom probably because it is not so easy for them, but it was the area brushed the most by the automatic brush.

The brushes thus appear to supplement the allogrooming horses carry out. That could be particularly true in spring and summer, when potential itching due to flies might explain why horses use the brushes more on areas not typically associated with allogrooming (the present observations were made in May). The time spent using the brush could also reflect a tendency to play with them, although this hypothesis cannot be verified.

To activate the brush, horses mainly used their head and their forehand. However, it should be noted that in a quarter of the cases they used brushes that had already been activated by another horse. It is possible that this was based on a process of social facilitation ("when the behaviour of a conspecific changes the motivation of the observer, resulting in the tendency of individual animals to do what other individuals are doing") or local enhancement ("when the behaviour of the demonstrator results in an increase in the salience of a particular stimulus, and the observer's motivation to investigate that stimulus may be increased"),processes previously described in the horse [START_REF] Rørvang | Social Learning in Horses-Fact or Fiction[END_REF]. The horses could thus have used the brush because a conspecific had just previously used it. This raises the question of the role these brushes could play in social cohesion.

Induction of affiliative behaviours

The most striking result of this study is that the brushes induced more affiliative than aggressive behaviours. This is noteworthy as we could have expected the contrary regarding a limited resource with only two brushes for 40 horses. Indeed, there could have been a form of competition to access this resource, with aggressive behaviours of a threatening or biting type, as can be observed regarding a vital and rare source, such as at a watering place [START_REF] Rutberg | Dominance, aggression frequencies and modes of aggressive competition in feral pony mares[END_REF]. No such behaviour was observed. On the contrary, we were surprised to note that while horses were being brushed by the automatic brush they simultaneously started allogrooming. The brush thus seems to act as a catalyst for affiliative behaviour. To confirm this, in a future study it would be interesting to count the total number of these affiliative behaviours in the group of horses that can access the brushes compared to groups of horses that cannot. In any case, our finding suggests that the presence of brushes directly improved horse welfare by providing them with comfort, as evidenced by the presence of positive facial expression in almost half of the brushing sequences, but also indirectly, by fostering positive social relations between the horses, which is essential to maintain social cohesion (review:

VanDierendonck and Spruijt, 2012).

Social context of expressing the positive facial expression

Finally, this study provides initial leads into understanding the social role of horses facial expressions, and in particular the positive facial expression observed when horses are groomed [START_REF] Lansade | Facial expression and oxytocin as possible markers of positive emotions in horses[END_REF]. First, we aimed to investigate whether there was an audience effect (if a conspecific was nearby when the facial expression was produced). However, this effect could not be tested because in all sequences except one, there was a horse less than 2m from the brushed horse. Second, our aim was to investigate whether there was audience behaviour in response. Results show that if a horse expressed this face during the brushing sequence (half of the horses were observed doing this), it had a six times greater chance to subsequently start allogrooming with a conspecific than if it had not expressed that face. There are at least three possible explanations of this result.

The first corresponds to the audience behaviour hypothesis: the positive facial expression could promote allogrooming and could be used intentionally to communicate. However, to confirm this explanation further studies are necessary to identify supplementary criteria. Among the criteria put forward by [START_REF] Townsend | Exorcising Grice's ghost: an empirical approach to studying intentional communication in animals[END_REF], there is the "Manipulation of the attentional state of the recipient". To investigate this, we could determine whether "attention-getting behaviours were directed towards a recipient" before the facial expression was expressed [START_REF] Liebal | Use of gesture sequences in chimpanzees[END_REF]. We could also check whether there was an exchange of gazes between the signaler and the recipient, given that in horses, gaze alternations to manipulate the attention of the recipient have already been described in another context [START_REF] Malavasi | Evidence of heterospecific referential communication from domestic horses (Equus caballus) to humans[END_REF]. In our study, there were too many horses in front of the brushed horse to be able to distinguish clearly this type of behaviour. Moreover, as it is known that horses preferentially display allogrooming with certain privileged partners (review: VanDierendonck and Spruijt, 2012), it could also be interesting to determine the composition of the audience, and investigate whether this potential signal was only produced in the presence of certain recipients.

A second explanation is related to a phenomenon of emotional contagion. In a previous experiment, we showed that horses tended to engage in allogrooming toward an experimenter simply by watching video footage of another horse expressing this positive facial expression while it was scratched by a handler [START_REF] Trösch | Horses feel emotions when they watch positive and negative horse-human interactions in a video and transpose what they saw to real life[END_REF]. This behaviour was interpreted as a phenomenon of positive emotional contagion. To discriminate between intentional communication and emotional contagion, we could try to determine whether the positive facial expression of horses using the brush specifically promotes allogrooming between these particular horses and privileged partners, or also allogrooming between two third-party horses (who are not using the brushes).

Finally, a third explanation is that the animals who displayed the positive facial expression could be those which appreciated being scratched by the brush the most, and which were the most motivated to be groomed whether with a brush or by a conspecific. In this case they could also be the most motivated to subsequently start allogrooming with another horse.

In any case, our result shows that the brush could act as a catalyst for positive emotions and affiliation behaviours within the group.

Conclusions

This study shows that 87.5% of the horses observed used the brushes. The automatic brushes provide both a direct benefit to the horses enabling them to conduct self-grooming, and also in all likelihood, an indirect benefit by facilitating positive social interactions (allogrooming).

They do not trigger aggressiveness, as can be sometimes observed when there is competition for a desired resource. The brush thus would appear to act as a catalyst for affiliative behaviours.

These results promote the installation of automatic brushes in places where horses are kept to improve their welfare and provide comfort. Our results also show that more than half of the horse expressed a positive facial expression while using the brush. When they displayed this facial expression, they had a six times greater chance to subsequently start allogrooming with a conspecific than if they did not. For a clearer interpretation of this result further research is needed to validate other criteria to qualify whether or not this positive face is a signal of intentional communication.

Figures captions

Figure 1: Automatic brush being used by a horse in the study

Figure 2 .

 2 Figure 2. Behaviour and facial expressions recorded while the horse used the brush

Figure 4 .Figure 5 .

 45 Figure 4. Affiliative or aggressive behaviours and facial expressions observed while the horse

Acknowledgements

We thank the staff at the Animal Physiology Experimental Unit PAO, INRA (DOI: 10.15454/1.5573896321728955E12) for their contribution to this experiment. We would like to thank Estel Blasi for drawing the horse images and Sue Edrich from the translation agency Interconnect for correcting the English manuscript.

Highlights

Declaration of Competing Interest

The authors report no declarations of interest.


Frédéric Mercier

Magaly Angénieux

Nathalie Kondjoyan

Saïd Abou

El Karam

Patrick Blinet

Angélique Travel

Eric Royer

Elisabeth Baéza

Ronan Cariou

Jérémy Ratel
email: jeremy.ratel@inrae.fr

Saïd Abouelkaram

Elisabeth Baéza-Campone

Catherine Jondreville

Erwan Engel

Identification by volatolomics of hydrocarbon, oxygenated, sulfur and aromatic markers of livestock exposure to α-hexabromocyclododecane

Keywords: Volatolomics, α-HBCDD, Headspace-SPME, GC-MS, Livestock, Food safety

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

INTRODUCTION

Hexabromocyclododecane (HBCDD) is a brominated flame retardant that has been a targeted substance in the Stockholm Convention since May 2013. HBCDD is classified as a substance of very high concern (SVHC) on the REACH candidate list due to its persistent bioaccumulative and toxic (PBT) properties. For non-occupationally exposed persons, dietary exposure is a major route of total HBCDD intake [START_REF] Covaci | Hexabromocyclododecanes (HBCDs) in the Environment and Humans: A Review[END_REF]. Food contaminations by HBCDD are especially problematic because of sometimes high levels, so that the ingestion of a single contaminated animal-derived food can significantly increase consumer exposure to it [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF]. Based on direct HBCDD detection in food, the current analytical methods for the surveillance of HBCDD contamination are efficient and sensitive but are hindered by cumbersome implementation related in particular to the ubiquitous occurrence of HBCDD. These routine monitoring techniques thus do not allow rapid cost-effective largescale methods, which seem essential for strengthening food safety with respect to this contaminant [START_REF] Meurillon | How to secure the meat chain against toxicants? Innovative Food Science & Emerging Technologies[END_REF].

To overcome these limitations, alternative approaches based on omics have emerged to detect contaminations. They are inspired by research showing the usefulness of the rapid costeffective analysis of expired volatile organic compound (VOC) markers in clinical diagnosis [START_REF] Hakim | Volatile organic compounds of lung cancer and possible biochemical pathways[END_REF]. [START_REF] Berge | Use of Volatile Compound Metabolic Signatures in Poultry Liver to Back-Trace Dietary Exposure to Rapidly Metabolized Xenobiotics[END_REF] proved the concept that the signature of the liver metabolome in small compounds, the volatolome, was modified for chickens in response to exposure in their diet to different xenobiotics including brominated flame retardants such as polybrominated diphenyl ethers. Liver is a key target for the determination of markers of exposure to contaminants because of its major function in protecting the organism from potentially toxic chemical insults. Bouhlel et al. (2018) took this concept and identified some VOCs that were impacted in the liver volatolomes of chickens exposed to three types of micropollutants ̶ a pesticide, an environmental contaminant and a coccidiostatic agent. As HBCDD impacts the metabolism of exposed organisms with clear indications of toxicological effects in the liver [START_REF] Cantón | Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats[END_REF][START_REF] Germer | Subacute effects of the brominated flame retardants hexabromocyclododecane and tetrabromobisphenol A on hepatic cytochrome P450 levels in rats[END_REF], liver volatolomics could be useful for identifying markers of HBCDD exposure and back-tracing HBCDD food contamination. Ratel et al. used volatolomics to reveal exposure of laying hens to γ-HBCDD [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] and reported a list of VOCs in livers impacted by γ-HBCDD exposure. The study of [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] showed that at least four additional issues needed to be addressed: (i) this first study performed on HBCDD focused on the γ-isomer, whereas the α-HBCDD isomer is probably more relevant because it predominates in the environment, in animal tissues and in animal-derived food [START_REF] Marvin | Hexabromocyclododecane: Current Understanding of Chemistry, Environmental Fate and Toxicology and Implications for Global Management[END_REF][START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF][START_REF] Koch | Review of hexabromocyclododecane (HBCD) with a focus on legislation and recent publications concerning toxicokinetics and-dynamics[END_REF][START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF], (ii) this first list of VOC candidate markers needed filtering to screen for the most robust one, (iii) the proof of concept had to be confirmed on more realistic HBCDD levels of feed contamination, and (iv) the scope had to be extended to other farm animals.

Based on control/test experiments, the present study aimed first to confirm the utility demonstrated by [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] of liver volatolomics in demonstrating the exposure of laying hens to HBCDD, by considering the α-HBCDD isomer. In addition, realistic exposure levels of laying hens were implemented and the potential of egg volatolomics was explored.

Egg is widely consumed and the literature has reported high HBCDD levels in chicken eggs, sometimes above the mg kg -1 LW level (Hiebl and Vetter, 2017;[START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF]. According to its lipid rate, egg yolk may contain many volatile compounds and it is therefore very relevant for considering the implementation of non-invasive volatolomics methods for food safety surveillance. Liver volatolomes were also investigated for two other major monogastric farm animals, namely broilers and pigs. The potential markers discovered are discussed especially in the light of the candidates found in the two main studies carried out from liver volatolomics of livestock exposed or not to micropollutants: the study of [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF], based on the exposure of laying hens to γ-HBCDD, and that of Bouhlel et al. (2018), based on the exposure of broilers to different micropollutants.

MATERIALS AND METHODS

Animal feeding

Compositions of experimental feeds for the three farm animals were detailed in Table S1. Contaminated diets were obtained by replacing 5 g of clean soy oil in the control diet by 5 g of soy oil spiked at appropriate levels with α-HBCDD. The synthesis of α-HBCDD has been described by [START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF]. Briefly, technical HBCDD, containing 1, 5, and 93% of α-, β-, and γ-HBCDD, respectively, was enriched in α-isomer by thermal rearrangement (172 ± 0.4 °C, 6 h), according to a method adapted from that described by [START_REF] Szabo | Toxicokinetics of the flame retardant hexabromocyclododecane alpha: effect of dose, timing, route, repeated exposure, and metabolism[END_REF]. Purification was performed on neutral silica gel and magnesium silicate manually packed solid phase extraction (SPE) columns and also by preferential precipitation at -20 °C, using dichloromethane and n-hexane. The purity of resulting crystals has been estimated at 99.3% α-HBCDD, η-HBCDD being the only identified impurity. Crystals of α-HBCDD were dissolved in acetone used to spike soy oil at the targeted concentration. All spiked feeds were prepared at the same time in the INRAE PEAT feed mixing unit (Nouzilly, France) by using dilutions of a single 50 µg g -1 contaminated oil prepared by LABERCA (Nantes, France). However, for pigs, the volume of contaminated feeds needed for all the experiment was too high to be produced in the INRA PEAT facilities. To overcome this issue, the contaminated feeds for pigs have been concentrated 10 times more than the expected level, then contaminated feed was brought each day of the experiment in the pig trough in a 1:10 ratio (w/w) mixed with uncontaminated feed.

For the three farm animals, a contaminated diet with the target concentration of 50 ng α-HBCDD g -1 was prepared. This concentration was expected to enable the animal-derived products to reach several hundreds of ng of HBCDD g -1 LW, as previously reported in heavily contaminated samples shown by French monitoring plans [START_REF] Jondreville | Accumulation of αhexabromocyclododecane (α-HBCDD) in tissues of fast-and slow-growing broilers (Gallus domesticus)[END_REF]. For laying hens and pigs, a second lower dose of 5 ng g -1 feed was tested to obtain animal-derived products with more realistic α-HBCDD contamination levels. For broilers, the high occupancy of the INRAE facilities did not allow testing two exposure doses with individual animal cages.

Animal testing

Animal experiments have been designed to study the exposure to α-HBCDD in feed of laying hens, slow-growing broiler chickens and growing pigs. These experiments were conducted in compliance with Directive 2010/63/EU in France and approved by the relevant ethics committee. Animal experiments were conducted in appropriate facilities with cages allowing feed ingestion of individual animals to be monitored. Laying hens, broilers and pigs were fed with non-contaminated feeds (control groups) or with α-HBCDD contaminated feeds (exposed groups) for 18, 12 and 16 weeks, respectively. The main features of the three experiments conducted in laying hens, broilers and pigs are summarized in Table 1, with the detailed conditions of each animal experiment which are given in Table S2. Information about body weight and feed ingested of animals during animal testing is given in Table S3.

Slaughtering and sampling

At the end of the exposure period, all animals were sacrificed and the weight of each warm carcass was recorded at slaughter (Table S3). Poultry and pigs were killed after a 12-hour and a 18-hour fast, respectively, by electronarcosis followed by exsanguination (electric stunner for poultry, Ducatillon, Cysoing, France; Morphee M4 electric stunner for pigs, Lelong & Cie, Savigny, France). "Control samples" and "exposed samples" correspond to the samples collected from control and exposed animal carcasses, respectively. For eggs, the eggs laid before slaughter and on the day of slaughter were collected. The egg laid before slaughter was dedicated to HBCDD analysis. It was weighed and removed from its shell before separating and storing at -20°C the white and yolk. The egg laid on the day of slaughter was dedicated to volatolomics analysis. It was collected, immersed in liquid nitrogen and stored at -80 °C. For fat and muscle samples dedicated to HBCDD quantification, abdominal fat and muscles from one thigh (without skin and bone) for poultry and samples of back fat and semi-membranous muscle for pigs were collected, weighed and stored at -20°C. For liver samples dedicated to HBCDD quantification and volatolomics, the whole liver for poultry and a sample prepared from the left lateral and right medial lobes for pigs were collected, weighed, immersed in liquid nitrogen, wrapped in aluminum foil, vacuum-packed, and stored at -80 °C. No visible sign of pathology was detected during the autopsy carried out during the cutting of the postslaughter animals. Data about weight and lipid content of collected samples are given in Table S3.

HBCDD quantification

HBCDD isomers (α, β and γ) were analysed in feed and animal samples according to a method covered by the scope of the ISO/IEC 17025:2005 accreditation of the LABERCA laboratory, and described by [START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF]. Briefly, all glassware and Na2SO4 were baked prior to use at 400 °C for 4 h or at 650 °C for 6 h, respectively. Feed was dried in an oven at 80 °C. Other matrices, animal tissues and egg yolk, were lyophilized. The sample size ranges used for HBCDD quantification was given for each matrix in Table S4.

Lipids were extracted by pressurized liquid extraction (PLE) over three successive static cycles (100 bar, 120 °C) (SpeedExtractor, Büchi, Switzerland) with a toluene/acetone mixture (70:30, v/v), evaporated to dryness, and weighed. Purification steps were conducted on a SPE column manually packed with Na2SO4, neutral, and acidic (H2SO4) silica gel using hexane and dichloromethane, followed by partitioning between n-hexane and 1 N NaOH. Final extracts were reconstituted into a mixture of methanol/water 80:20 (v/v). HBCDD isomers were analyzed by LC-ESI(-)-MS/MS (6410, Agilent Technologies). Separation was achieved on a Hypersil Gold column (100 mm × 2.1 mm, 1.9 μm) (Thermo Scientific, San Jose, CA, USA) fitted to a 1260 series HPLC pump. The mobile phase was constituted of 20 mM ammonium acetate (A) and a mixture of acetonitrile/methanol 1:1 (v/v) (B) in isocratic conditions (A/B 30:70, v/v). The transitions monitored through the triple-quadrupole mass filter corresponded to [M -H] -→ [Br] -. Each analytical series comprised a procedural blank and a quality control sample. Quantification was achieved according to the isotopic dilution principle (13C-labeled isomers as internal standards and 2H18-β-HCBDD as external standard). Limits of quantification (LOQ) were determined individually for each sample and isomer, based on a signal-to-noise ratio of 3. A limit of reporting (LoR) higher than each LOQ was established by matrix, species and isomer (considering procedural contamination as well), as presented in Table 2.

Volatolome analysis

All of each egg yolk and liver were ground in liquid nitrogen for 1 and 3 min, respectively, into a fine homogeneous powder using a home-made stainless steel ball mill. A 1.2 g aliquot of powder was placed in a 20mL-glass vial (Supelco, Sigma-Aldrich, St. Louis, MO), sealed under a nitrogen flow with magnetic caps with PTFE/silicone septa (Supelco, Sigma-Aldrich), and stored at -80 °C. The volatolome of powdered liver and egg yolk samples was analyzed by headspace-solid-phase microextraction (HS-SPME) coupled to gas chromatography -mass spectrometry (GC-MS) according to [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF]. Briefly, the following steps were carried out with an automated sampler (AOC-5000 Shimadzu, Japan): (i) preheating of the sample for 10 min in the agitator (500 rpm), (ii) SPME trapping (75 µm carboxen/polydimethylsiloxane, 23-gauge needle, Supelco) of the volatile organic compounds (VOCs) for 30 min. For the liver samples, the extraction temperature was set at 40 °C, as recommended by [START_REF] Bouhlel | Solid-phase microextraction set-up for the analysis of liver volatolome to detect livestock exposure to micropollutants[END_REF] for better extraction of liver VOCs with a narrower analytical variability and improved sensitivity. For egg yolk samples, this temperature had to be increased to 60 °C to boost extraction rates. After extraction, thermal desorption was performed at 250 °C for 2 min in the GC inlet. Further VOC analysis was performed by GC/MS-full scan (GC2010; QP2010+, Shimadzu). VOCs were injected in splitless mode into a DB-5MS capillary column (60 m × 0.32 mm × 1 µm; Agilent J&W) with Helium as carrier gas at a flow rate of 1 ml.min -1 . Oven temperature was held at 40 °C for 5 min, ramped to 230 °C at 3 °C.min -1 , and held at 230 °C for 10 min. The temperature of the transfer line between GC and MS was set at 230 °C. Temperature was fixed at 180 °C in the MS source and 150 °C in the MS quadrupole. Electron impact energy was set at 70 eV, and data was collected in the range m/z 33-250 at 10 scans per second.

Data treatment

All calculations and statistical analyses applied to animal testing and HBCDD quantification data have been previously detailed [START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF][START_REF] Jondreville | Accumulation of αhexabromocyclododecane (α-HBCDD) in tissues of fast-and slow-growing broilers (Gallus domesticus)[END_REF].

RESULTS

HBCDD in feed

HBCDD concentrations determined in feed and animal tissues are summarized in Table 2.

Control feeds used for the three animals tested were HBCDD-free according to limits of reporting. For contaminated feed intended to contain 50 ng α-HBCDD g -1 spiked feed, compliant concentrations of 40, 38 and 31.8 ng α-HBCDD g -1 feed were measured for laying hens, broilers and pigs, respectively. The concentration of α-HBCDD in contaminated feeds was 20-36% lower than expected. For the second lot of contaminated feed intended to contain 5 ng α-HBCDD g -1 spiked feed for laying hens and pigs, concentrations of 3.62 ng and 3.42 ng α-HBCDD g -1 feed were measured, respectively. The intended 1:10 ratios between the two α-HBCDD levels (5 and 50 ng α-HBCDD g -1 ) in spiked feed were moderately well kept following the preparation of contaminated feed, with a 1:9.0 ratio for laying hens and 1:9.3 for pigs. The difference observed in feed between the expected and measured concentrations is probably related to the uncertainty in the weight of the produced crystals of α-HBCDD used for spiking oils. But no degradation product of α-HBCDD has been identified in feed and only α-HBCDD measured values were considered for further calculations. No β-HBCDD was detected in any spiked feed. Some γ-HBCDD was quantified at levels of 0.18 and 0.14 ng g -1 fw in the high-and low-level feeds, respectively, representing 0.45 and 3.9% of the total HBCDD.

HBCDD in animal samples

The results of HBCDD quantification in animal samples obtained at the end of the exposure period are presented in Table 2. The results of tissue distribution and transfer to eggs of ingested α-HBCDD have been detailed in laying hens and in broilers by [START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF] and [START_REF] Jondreville | Accumulation of αhexabromocyclododecane (α-HBCDD) in tissues of fast-and slow-growing broilers (Gallus domesticus)[END_REF], respectively. The exposed animals, at any level, performed as well as the control animals in terms of body weight, growth rate, feed efficiency, and laying rate, and the weight and lipid content of their tissues were not significantly different [START_REF] Dominguez-Romero | Tissue Distribution and Transfer to Eggs of Ingested α-Hexabromocyclododecane (α-HBCDD) in Laying Hens (Gallus domesticus)[END_REF], Jondreville et al., 2017). While the HBCDD levels in ground liver samples from control laying hens should have been almost zero, traces of αand γ-HBCDD isomers have been detected, with 2.2 ± 1.8 ng and 5.0 ± 3.3 ng g -1 lw for αand γ-isomer, respectively (Table 2). The γ-HBCDD content was very similar to that from exposed hens (2.6 ± 1.7 ng and 5.6 ± 4.1 ng γ-HBCDD g -1 lw for the exposure "dose 5" and "dose 50", respectively; Table 2), which suggested a systematic contamination in all ground liver samples. The profile in HBCDD isomers determined in ground control liver samples was fairly constant. Dominated by the γ-isomer (29%, 5%, 66% of α-, βand γ-HBCDD, respectively), it matched that of a technical mixture. HBCDD is known as a ubiquitous environmental contaminant commonly found in industrial and domestic polymeric materials. The analysis of the polystyrene box used during the liver grinding (Laberca analysis ID 14.1842.3) has revealed a similar profile in HBCDD isomers (26%, 14%, 60% of α-, βand γ-HBCDD, respectively). HBCDD traces detected in ground control liver samples could thus be ascribed to the polystyrene box. The analyses of HBCDD quantification pointed out that the feeds given to control animals were α/β/γ-HBCDD-free and that the liver grinding step was the source of the contamination revealed in control ground liver samples.

Accordingly, the volatolome of the liver from control hens can then be used to reveal α-HBCDD-exposure markers by comparison with the volatolome of liver from exposed animals deliberately fed with α-HBCDD-contaminated feeds. In the experiments conducted on pigs and laying hens, the α-HBCDD concentration measured in tissues was proportional to the level of diet exposure of the pigs and laying hens.

Change in volatolomes in response to α-HBCDD exposure

To confirm the promising interest of liver volatolomics [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] and to explore the potential of egg volatolomics for revealing livestock exposure to HBCDD, changes in volatolomes were investigated in SPME-GC-MS signals obtained from livers and eggs of control and α-HBCDD exposed animals. Analyses of liver volatolomes found 98, 105, and 134 VOCs for laying hens, broilers and pigs, respectively, and 51 VOCs were detected in egg yolk volatolomes (Table S5).

Laying hens

The results of liver volatolomics presented in Table 3 show that 8 VOCs were impacted in the livers of laying hens for the two exposure levels. Figure S1 presents the first map of PCA plotted on these candidate markers. The hepatic disturbance was thus visible in the volatolome even for realistic low doses of α-HBCDD. Levels of HBCDD in feed in our study (5 and 50 ng α-HBCDD g -1 feed) were reduced by a factor of 20 and 200 compared to the levels of HBCDD used in Ratel et al. (2017) (0.1 and 10 μg γ-HBCDD g -1 feed). Table 3 includes mainly hydrocarbon, oxygenated (alcohols, ketones, acids), aromatic and sulfur compounds. These results are consistent with the list of candidate markers published by [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] in response to γ-HBCDD exposure, the authors highlighting in the livers of laying hens mainly hydrocarbon compounds (alkanes and branched alkanes), oxygenated compounds (alcohols, aldehydes, ketones) and aromatic compounds. Hydrocarbons. Heptane (increased in exposed animals) and 2,2,4-trimethylpentane (decreased in exposed animals) were found as significant markers of α-HBCDD exposure. Several hydrocarbons were previously found as markers in the livers of laying hens after a γ-HBCDD exposure, with levels in these compounds also increased or decreased in exposed animals according to the compound considered [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF]. The changes in the hydrocarbon content of livers in response to α-HBCDD exposure may result from an imbalance between their production, mainly due to unsaturated fatty acid peroxidation by reactive oxygen species (ROS), and their hydroxylation, by detoxifying enzymes resulting in the production of alcohols [START_REF] Hakim | Volatile organic compounds of lung cancer and possible biochemical pathways[END_REF]. Alcohols. Table 3 includes 2-butanol. In response to the exposure to toxic xenobiotics, the level of alcohols in livers could result from an equilibrium between anabolism (e.g. hydroxylation of hydrocarbons) and catabolism (e.g. activation of CYP-450) reactions.

Several alcohols were included in the list of [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF], and primary and secondary alcohols have been proposed as candidate markers for VOC-based clinical diagnoses [START_REF] Hakim | Volatile organic compounds of lung cancer and possible biochemical pathways[END_REF]. 2-Butanol can be thus considered as particularly useful for revealing exposure of laying hens to α-HBCDD. Ketones. Like for alcohols, these compounds are at the crossroads of cell metabolism elicited in response to exposure to toxic xenobiotics. The levels of 2,5octanedione could be modified because of an impact of the toxic exposure on the lipid metabolism with a high oxidation rate of fatty acids or on the protein metabolism with amino acid metabolism-induced ketone formation [START_REF] Hakim | Volatile organic compounds of lung cancer and possible biochemical pathways[END_REF]. Sulfur compounds. Two compounds (sulfur dioxide, dimethylsulfone) were identified as markers at the highest HBCDD dose. Among the candidate markers not classified according to their chemical structure ("others" class) by [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF], we can note two sulfur-containing compounds (thiazole and thiadiazole). Bouhlel et al. (2018) also highlighted dimethylsulfone and carbon disulfide among the most important liver VOC contributors to the separation of control chickens and exposed chickens to micropollutants such as pesticides or polychlorobiphenyls.

In the review of Shubert et al. ( 2004) on the medical diagnostic potential of endogenous VOCs, the authors report a possible relation between impairment of liver function, which could be initiated by exposure to α-HBCCD given its toxicological effects reported in the liver [START_REF] Cantón | Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats[END_REF][START_REF] Germer | Subacute effects of the brominated flame retardants hexabromocyclododecane and tetrabromobisphenol A on hepatic cytochrome P450 levels in rats[END_REF], and level of sulfur-containing compounds.

The generation of these compounds may be connected to an incomplete metabolism of methionine in the transamination pathway. Aromatic compounds. Table 3 shows 3 aromatic compounds derived from alkylbenzenes among candidate markers: styrene, o-xylene and α-methylstyrene. Alkylbenzenes were already highlighted by [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] when animals were exposed to γ-HBCDD. In their study, the levels of all alkylbenzene candidate markers were higher in exposed laying hens, like in our study. o-Xylene and α-methylstyrene were also among the VOCs identified by [START_REF] Bouhlel | Solid-phase microextraction set-up for the analysis of liver volatolome to detect livestock exposure to micropollutants[END_REF] as the major contributors to the separation of control and exposed chickens based on liver volatolomics. Although it is largely agreed that alkylbenzenes have an exogenous origin [START_REF] Hakim | Volatile organic compounds of lung cancer and possible biochemical pathways[END_REF], their level in the liver could be impacted by cellular and enzymatic defense mechanisms elicited to eliminate hazardous compounds such as α-HBCDD. It is of note that we found styrene, o-xylene, αmethylstyrene and one unknown VOC (RI 1028) as candidate markers in the livers of laying hens for both α-HBCDD doses. However, the abundance of these VOCs and the α-HBCDD concentration in liver were not significantly correlated according to the R-Pearson test (r Pearson indices between -0.13 and -0.46; p values between 0.26 and 0.75).

Regarding the egg volatolome, only 2-butanol was affected by the α-HBCDD exposure (Table 3). This VOC was also identified as a marker in the livers of laying hens exposed to the higher dose of α-HBCDD. 2-Butanol is a secondary alcohol in one of the VOC families reported as likely to be impacted by a chemical risk exposure [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF]. Given the high utility of eggs in any non-invasive volatolomics-based control, it would be interesting to deepen our knowledge of the potential impact of liver disorders on egg composition. [START_REF] Saraswati | Increased egg production of Japanese quail (Cortunix japonica) by improving liver function through turmeric powder supplementation[END_REF] showed that liver functions modified yolk precursor synthesis and depositions in the developing follicles. It would also be of interest to improve egg volatolome analysis by implementing recent advances in sample preparation and volatile fraction collection [START_REF] Majchrzak | Sample preparation and recent trends in volatolomics for diagnosing gastrointestinal diseases[END_REF].

Broilers

The exposure to α-HBCDD generates a detectable metabolic disturbance in the liver volatolome of broilers. The first map of PCA plotted on the candidate markers allows the separation of case/control groups to be visualized (Figure S1). With levels higher in control animals, the candidate markers listed in Table 4 are hydrocarbon (2-methylbutane) or aromatic (phenol, 2-phenoxyethanol, methoxybenzene and styrene) compounds. These chemical families were already highlighted in the liver volatolome of laying hens exposed to α-HBCDD (Table 3) and to γ-HBCDD [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF]. Concerning aromatic compounds, styrene was already identified as affected in liver of laying hens for the two exposure doses of α-HBCDD (Table 3). Styrene levels were detected significantly higher in control hens than in exposed hens. These results make it promising candidate marker. Phenol was already highlighted in the livers of laying hens as a candidate marker of γ-HBCDD exposure by [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF], with levels also higher in the livers of controls. To explain changes in liver phenol content in response to γ-HBCDD exposure, the authors hypothesize an imbalance between production and degradation of phenol related to (i) the double involvement of CYP-450 enzymes in the detoxication process of toxic xenobiotics and in microsomal hydroxylation of phenol in liver, and (ii) the hepatic degradation of tyrosine and tryptophan amino acids responsible for phenol generation, which could be increased in the case of liver function impairment. Our result thus confirms that phenol is a promising candidate marker to reveal differences in liver metabolism after a diet exposure to HBCDD.

Pigs

Table 5 shows that 1 and 9 VOCs were significantly impacted when animals were exposed to 5 and 50 ng α-HBCDD g -1 feed, respectively. This result suggests a dose effect and possibly a higher metabolic response threshold in the case of this animal. Figure S1 presents the first map of PCA plotted on the candidate markers revealed for the exposure at 50 ng α-HBCDD g -1 feed. The candidate markers, levels of which were all higher in exposed animals, were mainly oxygenated compounds (alcohols, ketones, lactones) and one sulfur-containing compound (carbon disulfide). Among alcohols, we found the primary alcohol 3-methyl-1butanol. This compound was reported as a hepatic candidate marker for exposure of laying hens to γ-HBCDD [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF], with levels also higher in exposed animals. Carbon disulfide was reported in the liver volatolome of chickens by [START_REF] Bouhlel | Solid-phase microextraction set-up for the analysis of liver volatolome to detect livestock exposure to micropollutants[END_REF] as a major contributor to the discrimination between control chickens and chickens exposed to pesticide.

Further work is needed to identify the unknown VOC (RI 1132), which is a candidate marker for pigs and broilers for the higher α-HBCDD level.

CONCLUSION

The present paper confirms that liver volatolome is relevant to highlight metabolic disturbances induced by the exposure of animals to a chemical contamination with α-HBCDD. Based on realistic α-HBCDD exposures, our work supports the study of [START_REF] Ratel | Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD)[END_REF] by detecting, by liver volatolomics, the α-HBCDD exposure of laying hens. It shows the effectiveness of this approach in the case of two other farm animals, namely broilers and pigs. But given the numbers of animals involved in this proof-of-concept study, further work is needed to assess the robustness of the markers identified before considering to use them for food safety surveillance. The chemical families of most candidate markers are consistent with the two main cellular reactions put forward as affecting the anabolism and catabolism of VOCs studied as marker of pathologies as cancers [START_REF] Hakim | Volatile organic compounds of lung cancer and possible biochemical pathways[END_REF]. First, oxidative stress could be induced, with synthesis of reactive oxygen species that leak from the mitochondria or from peroxidated polyunsaturated fatty acids in the cell membranes. Second, detoxifying enzymes like cytochrome P-450 enzymes could be induced with catalysis of the oxidation of organic substances. Additional case/control experiments need to be investigated to go further in assessing the robustness of the candidate markers, understanding of the cellular reaction mechanisms which affect the VOC production and ascertaining the relationship between level of risky exposure and volatolomic response. In this purpose, repeating in vivo experiments with higher numbers of animals, especially for volatolomic studies on pig livers and poultry eggs, or implementing in vitro experiments with hepatocyte culture cells are two very useful opportunities. The recent advances in sensors for non-invasive and early detection of VOCs [START_REF] Li | Functional metal-organic frameworks as effective sensors of gases and volatile compounds[END_REF] should be followed since they may lead rapidly to the design of routine sensors for easy, rapid detection of volatolomics markers useful for food chemical safety surveillance. Level found higher in liver from "control" animals compared to "exposed animals" te marker with its standard deviation (in bracket).

Table 5. Pigs -Volatile compounds in liver volatolome impacted by the exposure to a-HBCDD. Values are the mean of abundances (×10 4 ) of each candidate marker with its standard deviation (in bracket). Level found higher in liver from "control" animals compared to "exposed animals"

Candidate markers

  Peak areas of the VOCs were integrated from the SPME-GC-MS signals using a mass fragment selected for both being specific to the sought-after molecule and free of any coelution with a home-made automatic algorithm developed in Boulhel et al. (2017) under Matlab R2017 (The MathWorks, Natick, USA). VOCs were tentatively identified on the basis of both mass spectra, by comparison against the NIST 17 mass spectral library (version 2.3, build May 4 2017), and retention indices (RI), by comparison with published RI values and those of our internal database. For calculation of the experimental RI, an alkane standard solution (Supelco, Sigma-Aldrich) was analyzed by SPME-GC-MS at the end of the analytical campaign. Data were processed using Statistica (version 12, StatSoft) and R (version 2.1.4., http://www.R-project.org) software. Student's t-test and one-way ANOVA were performed on the abundances of the VOCs monitored in volatolomes for the comparisons of control vs. one or two groups of α-HBCDD contaminated animals.

a

  MS + RI, mass spectrum and RI agree with literature data; MS, mass spectrum agrees with literature spectrum b Mass fragment used for area determination c,d Retention indices on a DB5 capillary column from experimental run (c) or bibliographic data (d) [1][START_REF] Engel | Correction of the data generated by mass spectrometry analyses of biological tissues: Application to food authentication[END_REF] [2] [START_REF] Radulovic | Comparative study of the leaf volatiles of Arctostaphylos uva-ursi (L.) Spreng. and Vaccinium vitis-idaea L. (Ericaceae), Molecules[END_REF] [3] Cajka et al., 2007; [4] [START_REF] Aaslyng | Comparison of the aroma characteristics of acid-hydrolyzed and enzyme-hydrolyzed vegetable proteins produced from soy[END_REF] [5] [START_REF] Beaulieu | Identification of volatile compounds in cantaloupe at various developmental stages using solid phase microextraction[END_REF] e Diet contaminated with the target concentration of 5 or 50 ng α-

Table 3 .

 3 Laying hens -Volatile compounds in liver volatolome impacted by the exposure to a-HBCDD. Values are the mean of abundances (×10 4 ) of each candidate marker with its standard deviation (in bracket).

								Liver		Egg yolk
	Candidate markers	Tentatively identification a	m/z b	Exp. RI c	Ref. RI d	control	dose 50 e	dose 5 e	control dose 50 e	dose 5 e
	Hydrocarbons
		2,2,4-trimethylpentane	MS+RI		688	687 [1]	5.7 (9.1%)		1.4** (24.7%)
		heptane	MS+RI		700	700 13.7 (26.9%)		21.8* (5.3%)
	Alcohols
		2-butanol	MS+RI		602	603 [1] 13.4 (32.8%) 23.7* (3.9%)		42.5 (22.0%)	82.8* (1.1%)
	Ketones	MS+RI
		2,5-octanedione	MS+RI		985	985 [2]	2.0 (47.1%)		6.6* (26.1%)
	Acids
		acetic acid	MS+RI		575	602 [1] 512.8 (29.7%) 775.5* (2.6%)
	Aromatic compounds
		styrene	MS+RI		898	897 [1]	4.0 (11.2%) 2.7* (15.7%) 2.6* (16.4%)
		o-xylene	MS+RI		898	898 [3]	5.8 (1.6%) 3.7* (15.9%) 3.5* (26.6%)
		a-methylstyrene	MS+RI		994	994 [4]	1.1 (0.3%) 2.1* (19.6%) 4.1** (13.7%)
	Sulfur compounds
		sulfur dioxide	MS		<500		32.7 (7.2%) 20.9* (18.6%)
		dimethylsulfone	MS+RI		906	915 [1]	9.7 (6.1%) 38.3* (23.3%)
	Unknown
		unknown			1028		0.4 (31.3%) 1.7** (11.7%) 3.5* (26.2%)
		unknown			1141		0.3 (39.4%)		1.5** (4.7%)
	c,d	Retention indices on a DB5 capillary column from experimental run (c) or bibliographic data (d)
		[1] Engel and Ratel, 2007; [2] Xie et al., 2008; [3] Vasta et al., 2007; [4] Cajka et al., 2007.
	e	Diet contaminated with the target concentration of 5 or 50 ng α-HBCDD g -1
	*	p <0,05
	** p <0,01
		Level found higher in liver from "control" animals compared to "exposed animals"

a MS + RI, mass spectrum and RI agree with literature data; MS, mass spectrum agrees with literature spectrum b Mass fragment used for area determination

Table 4 .

 4 Broilers -Volatile compounds in liver volatolome impacted by the exposure to a-HBCDD. Values are the mean of abundances (×10 4 ) of each candidate marker with its standard deviation (in bracket). RI, mass spectrum and RI agree with literature data; MS, mass spectrum agrees with literature spectrum Retention indices on a DB5 capillary column from experimental run (c) or bibliographic data (d) [1][START_REF] Engel | Correction of the data generated by mass spectrometry analyses of biological tissues: Application to food authentication[END_REF] [2] Leffingwell and Alford, 2011; [3] Vasta et al., 2007; [4] de[START_REF] De Simon | Volatile compounds in acacia, chestnut, cherry, ash, and oak woods, with a view to their use in cooperage[END_REF]

	Candidate markers	identification a Tentatively	m/z b Exp. RI c Ref. RI d	control	dose 50 e
	Hydrocarbons
	2-methylbutane	MS	72	508	63.9 (31.2%)	28.2* (45.1%)
	Aromatic compounds
	styrene	MS+RI	104	898 897 [1] 13.7 (19.2%)	8.9* (32.2%)
	methoxybenzene	MS+RI	108	922 918 [2]	9.3 (29.5%)	3.2** (21.0%)
	phenol	MS+RI	94	976 983 [3] 92.4 (23.2%)	38.3** (54.3%)
	2-phenoxyethanol	MS+RI	94	1207 1220 [4] 270.7 (26.2%) 38.9** (100.9%)
	Unknown
	unknown		77	1132	10 (16.2%)	3.4** (35.2%)

a MS + b Mass fragment used for area determination c,d e Diet contaminated with the target concentration of 50 ng α-HBCDD g -1 * p <0,05 ** p <0,01

ACKNOWLEDGMENTS

The authors thank the French Ministry for Food, Agriculture and Fisheries for financial support from the Compte d'Affectation Spéciale Développement Agricole et Rural (CASDAR project 1256). They are also grateful to the technical staff of PEAT and BOA (INRAE Nouzilly) and of GIE VGS (IFIP Villefranche-de-Rouergue) for diet preparation, animal rearing and sample collection.

ns were not zero because of an accidental contamination during liver sample treatment (see paragraph 3.2. in Results section).