Lena Barascou
email: lena.barascou@inrae.fr

Jean-Luc Brunet

Luc Belzunces

Axel Decourtye

Mickael Henry

Fourrier

Yves Le Conte

Cedric Alaux
email: cedric.alaux@inrae.fr

Pesticide risk assessment in honeybees: toward the use of behavioral and reproductive performances as assessment endpoints

Keywords: Apis mellifera, toxicology, hazard assessment, sublethal endpoints, regulatory test methods

 There is a gap between new evidence of pesticide toxicity in honeybees and regulatory toxicological bioassays  Current endpoints should be complemented with behavioral and reproductive endpoints  We review such endpoints and discuss their possible use in pesticide risk assessment  There is a need to translate toxicological research studies into regulatory test methods

Introduction

Plant protection products, also referred to as phytopharmaceutical products or pesticides, consist of active substances (with safeners or synergists) intended to protect plants against harmful organisms (e.g. insects, fungi) or prevent the growth of undesired plants (herbicides). Pesticides must be evaluated and approved by regulatory authorities before registration. Besides providing data on the physical and chemical properties, the mode of action and the efficacy, pesticide registration applications must include data on the product's fate in the environment, as well as its toxicity for humans and non-target organisms. Regulatory authorities can then provide an assessment of the risk presented by the pesticide and determine to what extent it is safe for human and environmental health.

As part of the overall risk assessment procedure, OECD (Organization for Economic Co-operation and Development) and EPPO (European and Mediterranean Plant Protection Organization) test guidelines require toxicological data on honeybees (Apis mellifera). Until very recently, honeybees were the only insect pollinator used for pesticide risk assessment and were considered a surrogate species for non-Apis bees (EPPO, 2010a(EPPO, , 2010b)). Likely because they are relatively easy to rear, their biology has been wellstudied, and they are one of the most important pollinators worldwide [START_REF] Ghazoul | Buzziness as usual? Questioning the global pollination crisis[END_REF][START_REF] Klein | Importance of pollinators in changing landscapes for world crops[END_REF]. Honey bees forage within large areas around their hives and are thus likely to be exposed to several pesticides either directly (collecting nectar, pollen and water) or indirectly (sharing food with nestmates: larvae and adult worker bees, queen and drones).

Over the last decades, progress in research has been made in evaluating the risk that pesticides pose to honeybees by assessing the toxicological effects. Extensive empirical data have therefore accumulated on pesticide effects, which have been compiled in several reviews (see for instance [START_REF] Alkassab | Sublethal exposure to neonicotinoids and related side effects on insect pollinators: honeybees, bumblebees, and solitary bees[END_REF][START_REF] Belzunces | Neural effects of insecticides in the honey bee[END_REF][START_REF] Blacquière | Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment[END_REF][START_REF] Cullen | Fungicides, herbicides and bees: A systematic review of existing research and methods[END_REF][START_REF] Desneux | The sublethal effects of pesticides on beneficial arthropods[END_REF][START_REF] Johnson | Honey bee toxicology[END_REF][START_REF] Poquet | Modulation of pesticide response in honeybees[END_REF]. Toxic effects vary depending on the dose/concentration, modes of action and exposure routes. More importantly, a new range of endpoints (biological parameters used to evaluate the toxicity of a chemical) have been used to identify sublethal effects (e.g. impairment of cognitive capacities, foraging activity, communication, and reproductive performances) [START_REF] Decourtye | Learning performances of honeybees (Apis mellifera L) are differentially affected by imidacloprid according to the season: Effects of imidacloprid on learning performances of honeybees[END_REF]Kairo et al., 2017b;[START_REF] Karahan | Sublethal imidacloprid effects on honey bee flower choices when foraging[END_REF]Zhang et al., 2020b). In addition, small doses or concentrations that were believed to be sublethal in laboratory bioassays proved to be lethal in field conditions (e.g. [START_REF] Henry | A common pesticide decreases foraging success and survival in honey bees[END_REF].

These recent findings raise concerns about risk assessment procedures for pesticide registration, which mainly focus on lethal effects. First, while sublethal effects do not directly cause the death of individual bees, they may impair individual and colony performance, as well as their pollination services [START_REF] Meikle | Sublethal effects of imidacloprid on honey bee colony growth and activity at three sites in the U[END_REF][START_REF] Prado | Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees[END_REF][START_REF] Tison | Effects of sublethal doses of thiacloprid and its formulation Calypso® on the learning and memory performance of honey bees[END_REF][START_REF] Woodcock | Country-specific effects of neonicotinoid pesticides on honey bees and wild bees[END_REF]. Second, exposures to pesticides, previously identified as nonlethal in lab assays, caused high mortality in natural conditions [START_REF] Henry | A common pesticide decreases foraging success and survival in honey bees[END_REF]. Therefore, current test procedures do not appear sufficient to protect honey bees. This growing gap between new evidence of pesticide toxicity (e.g. sublethal effects) in honeybees and the conservative toxicological bioassays needed for pesticide approval contribute to the controversy between stakeholders, policymakers, environmentalists and scientists [START_REF] Durant | Ignorance loops: how non-knowledge about bee-toxic agrochemicals is iteratively produced[END_REF][START_REF] Sgolastra | Bees and pesticide regulation: Lessons from the neonicotinoid experience[END_REF][START_REF] Storck | Towards a better pesticide policy for the European Union[END_REF][START_REF] Thompson | The relevance of sublethal effects in honey bee testing for pesticide risk assessment[END_REF]. In order to fill this gap and better assess the threat of pesticides to honeybees, we emphasize the need to complement current endpoints (essentially based on LD50 -dose at which 50% of the individuals die) with sublethal endpoints. We focus on behavioral and reproductive endpoints, which have received increasing interest due to their ecological relevance and the technical advances made in their recording. Furthermore, pesticides can have low-dose effects on these endpoints.

We review such endpoints and discuss which ones could be considered for pesticide risk assessments based on several criteria, such as the possibility of standardization, their monitoring simplicity and their ecological relevance. Finally, we highlight the future challenges that need to be addressed to better integrate and use these endpoints in pesticide risk assessment.

The rise of behavioral and reproductive endpoints in response to the risks associated with low exposure to pesticide

In the current regulatory framework for honeybees, the effects of pesticides are assessed by standard regulatory tests, in a tiered approach [START_REF] Efsa | EFSA Guidance Document on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees)[END_REF]EPPO, 2010aEPPO, , 2010b)). At a low tier, laboratory tests on active substances or formulated products are used on individual bees that are representative of different life stages (larval and adult). This is the first mandatory step that includes an acute toxicity test after oral or contact exposure in adults (OECD, 1998a(OECD, , 1998b) ) and larvae (OECD, 2013), and a chronic toxicity test on adults over 10 days (OECD, 2017). For acute toxicity tests, the LD50 is determined. Then, if a risk is identified as a result of this first step, supplementary tests are required at a higher tier (semifield and field tests). For instance, several key parameters of honeybee colony state may be measured, besides bee mortality, like foraging activity and honey and brood production (EPPO 2010a(EPPO , 2010b)).

However, particular attention should be paid to sublethal effects to better identify risk, and avoid situations where a pesticide might not undergo tests at a higher tier because of its supposedly low toxicity under current laboratory tests. We therefore need individual-level endpoints that are more reliable and relevant.

The primary endpoint of laboratory tests is the LD50, which suggests that the dose-response for a given substance follows either a proportional or a threshold dose-response relationship. However, there are several lines of evidence for multiphasic relationships. For instance, [START_REF] Suchail | Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera[END_REF] found that mortality rates in response to exposure to imidacloprid (neonicotinoid insecticide) increased for low doses, decreased for medium doses, and then increased in a dose-dependent manner for higher doses.

This suggests that significant effects can occur at doses lower than the ones used in mortality bioassays (LD50 tests) or under the NOEL (No Observed Effect Level). Such effects may occur in environments contaminated by low doses of pesticide residues and are usually sublethal with adverse effects on development [START_REF] Dai | Effects of sublethal concentrations of bifenthrin and deltamethrin on fecundity, growth, and development of the honeybee Apis mellifera ligustica[END_REF], metabolism (Decourtye et al., 2004a), immunity [START_REF] Aufauvre | Transcriptome analyses of the honeybee response to Nosema ceranae and insecticides[END_REF], behavior [START_REF] Yang | Abnormal foraging behavior induced by sublethal dosage of imidacloprid in the honey bee (Hymenoptera: apidae)[END_REF], and reproduction [START_REF] Kairo | Drone exposure to the systemic insecticide fipronil indirectly impairs queen reproductive potential[END_REF]. This may be translated into potential risk to colonies. For instance, the immunosuppression caused by clothianidin has been shown to promote the replication of the deformed wing virus [START_REF] Di Prisco | Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees[END_REF], a virus that has been frequently linked to colony losses [START_REF] Grozinger | Bee viruses: Eecology, pathogenicity, and impacts[END_REF].

Additionally, comparing effects observed in laboratory tests to field observations raised concerns about the underestimation of pesticide toxicity at sublethal doses, as illustrated by studies of aquatic populations of macroinvertebrates [START_REF] Rasmussen | Stream habitat structure influences macroinvertebrate response to pesticides[END_REF][START_REF] Schäfer | Thresholds for the Effects of Pesticides on Invertebrate Communities and Leaf Breakdown in Stream Ecosystems[END_REF]) (e.g. for Daphnia magna, the level of exposure to pesticides was found to be 10 to 100 times more harmful than that predicted by the EU's "first tier" risk assessment). In honeybees, these concerns were verified in field tests by recording the homing success of bees exposed to a dose of thiamethoxam assumed to be sublethal based on regulatory tests (nearly four times lower than the LD50). This sublethal dose caused significant failure in homing flight which could potentially decrease colony survival [START_REF] Henry | Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees[END_REF][START_REF] Henry | A common pesticide decreases foraging success and survival in honey bees[END_REF]. This discrepancy between the laboratory and the field could be explained by the differences between the safe, non-challenging conditions of the laboratory assays and the environmentally challenging conditions of the field.

The underestimation of risks related to low doses and the striking mismatch between laboratory and field tests highlight the need to complement the current regulatory evaluation of pesticides, essential for performing a preliminary screening of toxic effects, with sublethal and more ecologically-relevant tests.

In this context, the measurement of behavioral and reproductive performances in ecotoxicity tests has become increasingly popular. Notably, a keen interest in behavioral effects has recently been tracked in the US EPA ECOTOX database with records of 17 324 and 13 809 measurements for aquatic and terrestrial organisms, respectively (Ågerstrand et al., 2020). As explained by Ågerstrand et al. (2020), the relevance of behavioral endpoints is driven by several advantages, which we believe are also valid for reproductive endpoints. They improve the ecological relevance of pesticide risk assessment because they add ecological realism to the test conditions and help connect organismal response to populations, which are generally the ultimate protection goal [START_REF] Rudén | Assessing the relevance of ecotoxicological studies for regulatory decision making: eelevance assessment of ecotoxicological studies[END_REF]. These individual-level endpoints therefore should help predict effects on higher levels of biological organization (i.e. colonies for honeybees). In addition, pesticides can have low-dose effects on these endpoints, which can thus provide a more effective assessment of risks associated with low contamination levels of the environment as compared to the current mortality endpoints. Finally, regarding behavioral measurements, the emergence of new tracking technologies has increased the rate of acquisition and amount of in situ monitoring data [START_REF] Kays | Terrestrial animal tracking as an eye on life and planet[END_REF]. More importantly, such technology enables us to improve the robustness and precision of measurements and thus decrease the variability of ecotoxicological data, which can be relatively high when obtained through basic observations. The improved reproducibility of toxicity tests is notably essential for the regulatory assessments of pesticides.

Overview of behavioral and reproductive performances as assessment endpoints in honeybees

The investigation of pesticide effects at sublethal doses or concentrations in honeybees began in earnest in the 2000s (Fig. 1A). Since then, behavioral and reproductive endpoints have been more frequently included in toxicological research studies. The goal here is not to provide a systematic review but rather an overview of the main individual-based behavioral and reproductive endpoints by describing (i) their biological interests, (ii) the measurement methodologies and (iii) the main observed effects. For that purpose, we focused on the most studied endpoints (used in more than five research studies) that were grouped into the seven following categories: grooming, locomotion, learning and memory, foraging, homing flight, waggle dance and reproduction (Fig. 1B).

Grooming

The ability of worker bees to clean themselves (self-grooming) and their nestmates (allo-grooming) contributes to the prevention of the spread and multiplication of parasites and pathogens within the colony and is therefore related to colony health [START_REF] Evans | Socialized medicine: Individual and communal disease barriers in honey bees[END_REF]. Bees notably groom themselves or another bee in order to remove parasites from their body with their legs and mandibles.

Since grooming mainly involves motor functions, neurotoxic pesticides have been hypothesized to alter the performance of these behaviors. Grooming behavior has traditionally been evaluated by marking and tracking bees in observation hives or petri dishes. Only two studies investigated the influence of pesticides on allo-grooming; both reported a lack of effect after exposure to imidacloprid, bioinsecticides (essential oil and geraniol; [START_REF] Santos | Apis mellifera (Insecta: Hymenoptera) in the target of neonicotinoids: A one-way ticket? Bioinsecticides can be an alternative[END_REF], thiacloprid and tau-fluvalinate [START_REF] Retschnig | Effects, but no interactions, of ubiquitous pesticide and parasite stressors on honey bee (Apis mellifera) lifespan and behaviour in a colony environment: Stressor effects in honey bee colonies[END_REF]. Conversely, self-grooming performance appeared more sensitive to intoxication. While de [START_REF] De Mattos | Effects of synthetic acaricides on honey bee grooming behavior against the parasitic Varroa destructor mite[END_REF] found a reduction in the duration of self-grooming after exposure to acaricides (coumaphos, amitraz and tau-fluvalinate), bees intoxicated with pesticides, such as neonicotinoids (imidacloprid, thiamethoxam, clothianidin, dinotefuran) [START_REF] Williamson | Exposure to neonicotinoids influences the motor function of adult worker honeybees[END_REF], acetylcholinesterase inhibitors [START_REF] Williamson | Exposure to acetylcholinesterase inhibitors alters the physiology and motor function of honeybees[END_REF], permethrin [START_REF] Cox | Effects of permethrin on the behavior of individually tagged honey bees, Apis mellifera L. (hymenoptera: apidae)[END_REF] and oxalic acid (Schneider et al., 2012), showed an increased propensity to perform self-grooming. However, two toxicological studies found no effect of pyrethroids (including tau-fluvalinate and permethrin) [START_REF] Oliver | Pyrethroids and nectar toxins have subtle effects on the motor function, grooming and wing fanning behaviour of honeybees (Apis mellifera)[END_REF], imidacloprid or bioinsecticides on self-grooming [START_REF] Santos | Apis mellifera (Insecta: Hymenoptera) in the target of neonicotinoids: A one-way ticket? Bioinsecticides can be an alternative[END_REF].

Locomotion

Insecticides, such as pyrethroids and neonicotinoids, which target the voltage-sensitive sodium channels or nicotinic acetylcholine receptors, respectively, may directly impair neuronal transmission, muscle activation [START_REF] Hirata | Studies on the mode of action of neurotoxic insecticides[END_REF], and therefore locomotor activity (walking, flying). Moreover, some pesticides, like fungicides, may reduce energy production [START_REF] Degrandi-Hoffman | Effects of oral exposure to fungicides on honey bee nutrition and virus levels[END_REF] and therefore affect locomotion, especially flight capacity, as flying is one of the most intense and energy-demanding physiological processes in insects [START_REF] Dudley | The Biomechanics of insect flight: From, Function, Evolution[END_REF].

Walking locomotion experiments typically monitor the walking pattern of individual bees in standardized open field arenas and may reveal in-hive worker impairments during ongoing tasks in the colonies. Movement pattern is most often monitored by means of automated video tracking systems coupled with an ad-hoc image processing software that detects on a continuous basis the position of bees. Locomotion activity is typically indicated by the total covered distance or conversely by the length of time of immobility [START_REF] Hesselbach | Effects of the novel pesticide flupyradifurone (Sivanto) on honeybee taste and cognition[END_REF], also termed prostration [START_REF] Kadala | Honey bees long-lasting locomotor deficits after exposure to the diamide chlorantraniliprole are accompanied by brain and muscular calcium channels alterations[END_REF] or lethargy [START_REF] Williams | Comparative effects of technical-grade and formulated chlorantraniliprole to the survivorship and locomotor activity of the honey bee, Apis mellifera (L.)[END_REF], during a fixed period (typically between 3 to 15 min), and more recently, by circadian rhythms [START_REF] Delkash-Roudsari | Assessment of lethal and sublethal effects of imidacloprid, ethion, and glyphosate on aversive conditioning, motility, and lifespan in honey bees (Apis mellifera L.)[END_REF]. More subtle metrics may document abnormal trembling or convulsive movements of legs and wings [START_REF] Aliouane | Subchronic exposure of honeybees to sublethal doses of pesticides: effects on behavior[END_REF], as well as the loss of the righting reflex [START_REF] Oliver | Pyrethroids and nectar toxins have subtle effects on the motor function, grooming and wing fanning behaviour of honeybees (Apis mellifera)[END_REF]. Experiments may be either conducted in complete darkness using infrared recording systems [START_REF] Teeters | Using video-tracking to assess sublethal effects of pesticides on honey bees (Apis mellifera L.)[END_REF], or in the presence of a nearby light stimuli to explore the effect of exposure to pesticides on phototaxis [START_REF] Charreton | A locomotor deficit induced by sublethal doses of pyrethroid and neonicotinoid insecticides in the honeybee Apis mellifera[END_REF][START_REF] El Hassani | Effects of sublethal doses of acetamiprid and thiamethoxam on the behavior of the honeybee (Apis mellifera)[END_REF][START_REF] Ghazoul | Buzziness as usual? Questioning the global pollination crisis[END_REF]Tosi and Nieh, 2017). Additionally, when placed vertically, the arena experiment may reveal a potential interaction with geotaxis [START_REF] El Hassani | Effects of sublethal doses of acetamiprid and thiamethoxam on the behavior of the honeybee (Apis mellifera)[END_REF][START_REF] Ghazoul | Buzziness as usual? Questioning the global pollination crisis[END_REF]. Finally, more advanced versions of the experiments may introduce food or conspecifics into the arena to further implement food attraction or social interactions as a part of the walking pattern [START_REF] Ingram | Evaluating sub-lethal effects of orchardapplied pyrethroids using video-tracking software to quantify honey bee behaviors[END_REF][START_REF] Teeters | Using video-tracking to assess sublethal effects of pesticides on honey bees (Apis mellifera L.)[END_REF].

Flying locomotion is more difficult to document. It requires more sophisticated apparatus like flight mills, whereby the tested bees are tethered to a balanced arm and allowed to fly circularly around an axis [START_REF] Tong | Combined nutritional stress and a new systemic pesticide (flupyradifurone, Sivanto®) reduce bee survival, food consumption, flight success, and thermoregulation[END_REF]Tosi et al., 2017). Alternatively, bees may be harnessed to a fixed support and exposed to air flow and optic flow stimuli in order to elicit flight [START_REF] Liao | Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin[END_REF]. Flight experiments typically document flight duration until exhaustion, total covered distance, as well as average flight velocity.

Most of the studies reported significant locomotion deficits in bees exposed to pesticides, particularly pyrethroids and neonicotinoids. Walking experiments typically reported increased immobility [START_REF] Aliouane | Subchronic exposure of honeybees to sublethal doses of pesticides: effects on behavior[END_REF][START_REF] El Hassani | Effects of sublethal doses of fipronil on the behavior of the honeybee (Apis mellifera)[END_REF], righting difficulties [START_REF] Williamson | Exposure to neonicotinoids influences the motor function of adult worker honeybees[END_REF] and shorter walking distances [START_REF] Charreton | A locomotor deficit induced by sublethal doses of pyrethroid and neonicotinoid insecticides in the honeybee Apis mellifera[END_REF][START_REF] Ingram | Evaluating sub-lethal effects of orchardapplied pyrethroids using video-tracking software to quantify honey bee behaviors[END_REF]. However, common patterns are difficult to identify across studies due to the broad variety of experimental designs and peculiarities related with bee age [START_REF] Charreton | A locomotor deficit induced by sublethal doses of pyrethroid and neonicotinoid insecticides in the honeybee Apis mellifera[END_REF], topical vs. oral exposure [START_REF] El Hassani | Effects of sublethal doses of acetamiprid and thiamethoxam on the behavior of the honeybee (Apis mellifera)[END_REF][START_REF] Ghazoul | Buzziness as usual? Questioning the global pollination crisis[END_REF], exposure duration [START_REF] Williams | Comparative effects of technical-grade and formulated chlorantraniliprole to the survivorship and locomotor activity of the honey bee, Apis mellifera (L.)[END_REF] or timescale after exposure [START_REF] Kadala | Honey bees long-lasting locomotor deficits after exposure to the diamide chlorantraniliprole are accompanied by brain and muscular calcium channels alterations[END_REF]. Similarly, if a reduction in flying capacities was generally reported by experiments with flight mills [START_REF] Liao | Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin[END_REF][START_REF] Tong | Combined nutritional stress and a new systemic pesticide (flupyradifurone, Sivanto®) reduce bee survival, food consumption, flight success, and thermoregulation[END_REF]Tosi et al., 2017), depending on the type of exposure, opposite effects could also be induced on specific flight traits [START_REF] Tong | Combined nutritional stress and a new systemic pesticide (flupyradifurone, Sivanto®) reduce bee survival, food consumption, flight success, and thermoregulation[END_REF].

Learning and memory

One commonality of nearly all studies investigating pesticide effects on learning and memory in honeybees is the use of a Pavlovian conditioning protocol, the olfactory conditioning of the "Proboscis Extension Response" (PER). Other paradigms for testing learning and memory (e.g., habituation, visual learning, aversive learning) are available, but have been less used in toxicological studies [START_REF] Armengaud | Effects of imidacloprid on the neuralprocesses of memory in honey bees[END_REF][START_REF] Guez | Contrasting effects of imidacloprid on habituation in 7-and 8-day-old honeybees (Apis mellifera)[END_REF][START_REF] Lambin | Imidacloprid-induced facilitation of the Proboscis Extension Reflex habituation in the honeybee[END_REF][START_REF] Muth | A neonicotinoid pesticide impairs foraging, but not learning, in freeflying bumblebees[END_REF][START_REF] Urlacher | Measurements of Chlorpyrifos Levels in Forager Bees and Comparison with Levels that Disrupt Honey Bee Odor-Mediated Learning Under Laboratory Conditions[END_REF]. The olfactory PER conditioning protocol is notably useful for excluding confounding variables and experimental noise in learning assays. It enables one to reproduce, under laboratory conditions, the conditioning process occurring on the flower [START_REF] Menzel | Learning and memory in honeybees: from behavior to neural substrates[END_REF]. Whereas bees initially exhibit PER (unconditioned response) to antennal contact with sucrose (reward), they learn to initiate PER in response to an odor alone (conditioned response) when this contact was previously paired with this odor [START_REF] Giurfa | Invertebrate learning and memory: fifty years of olfactory conditioning of the proboscis extension response in honeybees[END_REF]. Although [START_REF] Muth | A neonicotinoid pesticide impairs foraging, but not learning, in freeflying bumblebees[END_REF] proposed an original method of PER conditioning in free-flying bees, PER experiments are commonly carried out on restrained individuals in the laboratory. The PER protocol has proven very useful in addressing many aspects of bee learning, such as extinction learning, and stimulus generalization and discrimination [START_REF] Giurfa | Invertebrate learning and memory: fifty years of olfactory conditioning of the proboscis extension response in honeybees[END_REF].

Multiple authors have investigated the effects of pesticides with the PER assay with varying parameters (e.g. pesticide concentration, honeybee age, season, subspecies, and trial number, inter-trial interval and performance measures). Underlying these toxicological studies is that sublethal doses of neurotoxic pesticide affect the learning and memory of bees [START_REF] Taylor | Impairment of a classical conditioned response of the honey bee (Apis mellifera L.) By sublethal doses of synthetic pyrethroid insecticides[END_REF], thereby reducing individual foraging efficiency. This explains why, besides some studies on fungicide or insect-growth regulators, PER assays were mostly used to test insecticides with neurotoxic activity, [START_REF] Abramson | The effect of insecticides considered harmless to honey bees (Apis mellifera): proboscis conditioning studies by using the insect growth regulators tebufenozide and diflubenzuron[END_REF]Decourtye et al., 2004b). As of now, nearly thirty pesticides have been tested with this bioassay; the majority of studies being performed with the active ingredient (rarely the 'ready to use' formulation of the farmer, except for [START_REF] Abramson | The effect of pymetrozine (Plenum WG-50®) on proboscis extension conditioning in honey bees (Apis mellifera: Hybrid var. Buckfast)[END_REF]Tison et al., 2017b)). By building on this strong background, [START_REF] Siviter | Quantifying the impact of pesticides on learning and memory in bees[END_REF] conducted a meta-analysis to quantify pesticide effects on learning and memory. They showed that both neonicotinoid and non-neonicotinoid pesticides cause significant decreases in learning and memory capacity at field-realistic exposure levels and under both chronic and acute exposure. The impacts at the adult stage of larval exposure remain relatively unexplored [START_REF] Papach | Larval exposure to thiamethoxam and American foulbrood: effects on mortality and cognition in the honey bee Apis mellifera[END_REF][START_REF] Yang | Impaired Olfactory Associative Behavior of Honeybee Workers Due to Contamination of Imidacloprid in the Larval Stage[END_REF]. However, [START_REF] Siviter | Quantifying the impact of pesticides on learning and memory in bees[END_REF] suggested that learning performances of bees could be more sensitive to pesticides when exposed during larval development.

Foraging activity

Foraging consists of repeated visits to different flowers for collecting pollen and nectar in an average radius of 1.5 to 5 km (up to 10 km if the floral resources are scarce) [START_REF] Beekman | Long-range foraging by the honey-bee, Apis mellifera[END_REF][START_REF] Steffan-Dewenter | Honeybee foraging in differentially structured landscapes[END_REF]. Typically performed by older bees foraging involves many complex cognitive functions such as learning (e.g. visual and olfactory stimuli of flowers, landscape characteristics), orientation and navigation [START_REF] Dyer | Honey bee orientation: a backup system for cloudy days[END_REF][START_REF] Menzel | Memory dynamics in the honeybee[END_REF], and is also energetically costly. Because foragers are usually the first bees to encounter pesticides while visiting flowers and can bring back pesticide-contaminated pollen and nectar to the hive, foraging is considered as the starting point of colony intoxication. For these reasons (frequent risk of exposure, behaviorally challenging tasks), the influence of pesticides on foraging has long been investigated.

To be able to record individual-based foraging activity, experiments are generally performed in a flight tunnel and with a feeder on which bees can forage. The frequency of visits to a feeder by forager bees (marked with a color dot on the thorax), as well as the time period between visits, can then be determined. Exposure to pesticides can be carried out in different ways, either by providing colonies with a pesticide-contaminated sugar solution [START_REF] Ramirez-Romero | Effects of Cry1Ab protoxin, deltamethrin and imidacloprid on the foraging activity and the learning performancesof the honeybee Apis mellifera, a comparative approach[END_REF][START_REF] Yang | Abnormal foraging behavior induced by sublethal dosage of imidacloprid in the honey bee (Hymenoptera: apidae)[END_REF] or individually by submitting the bees to an acute treatment (topical or oral route) [START_REF] Guez | Methyl parathion modifies foraging behaviour in honeybees (Apis mellifera)[END_REF][START_REF] Karahan | Sublethal imidacloprid effects on honey bee flower choices when foraging[END_REF]. A decrease in visit frequency and an increase in time period between visits were generally observed upon exposure to pesticides [START_REF] Karahan | Sublethal imidacloprid effects on honey bee flower choices when foraging[END_REF][START_REF] Ramirez-Romero | Effects of Cry1Ab protoxin, deltamethrin and imidacloprid on the foraging activity and the learning performancesof the honeybee Apis mellifera, a comparative approach[END_REF][START_REF] Yang | Abnormal foraging behavior induced by sublethal dosage of imidacloprid in the honey bee (Hymenoptera: apidae)[END_REF]. However, depending on the dose, opposite effects could be observed. For instance, doses of 10 and 50 ng of methyl parathion induced a decrease and an increase in the frequency of visits to the feeder, respectively [START_REF] Guez | Methyl parathion modifies foraging behaviour in honeybees (Apis mellifera)[END_REF]. Only recently, the emergence of new technologies (Radio Frequency Identification -RFID, optic bee counters) has enabled more detailed analysis of foraging activity and a move from non-challenging foraging activity in a flight tunnel, under controlled conditions, to field foraging activity [START_REF] Decourtye | Honeybee tracking with microchips: a new methodology to measure the effects of pesticides[END_REF][START_REF] Streit | Automatic life-long monitoring of individual insect behaviour now possible[END_REF]. By recording, on a continuous basis, the activity of individual bees at the hive entrance (time of each exit and entrance of tagged bees), these technologies allow access to different individual foraging traits, such as the number and duration of trips (daily and total) or the age at the onset of foraging. Apart from an early study, which demonstrated a short-term reduction in foraging activity and longer foraging trips between the hive and a feeder after exposure to clothianidin and imidacloprid (Schneider et al., 2012), no clear effect was observed on the daily number and duration of trips in the field when bees were exposed, to fieldrealistic doses of insecticides (Colin et al., 2019b;[START_REF] Hesselbach | Chronic exposure to the pesticide flupyradifurone can lead to premature onset of foraging in honeybees Apis mellifera[END_REF]. However, a significant premature onset of foraging was often reported (Colin et al., 2019b;[START_REF] Hesselbach | Chronic exposure to the pesticide flupyradifurone can lead to premature onset of foraging in honeybees Apis mellifera[END_REF][START_REF] Shi | Sublethal acetamiprid doses negatively affect the lifespans and foraging behaviors of honey bee (Apis mellifera L.) workers[END_REF]. Precocious foraging was then accompanied by a reduction in lifespan and total foraging activity.

The onset of foraging was delayed in bees exposed to mixtures of pesticides at very low doses (from 100 to 10 000 times lower than their LD50 values) [START_REF] Prado | Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees[END_REF]; such bees exhibited a lowered daily foraging activity. Scaling up to a higher tier of study, [START_REF] Henry | Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees[END_REF] investigated the foraging activity and mortality of bees originating from colonies placed near fields of rape oilseed treated with thiamethoxam. Although no modification in foraging traits was reported, an increased mortality rate, likely due to failure in homing flights, was observed. Finally, foraging efficiency was analyzed by [START_REF] Prado | Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees[END_REF] by measuring the amount of nectar and pollen collected by bees (exposed to pesticide at emergence): no effect of exposure to pesticides was found on nectar foraging but individual foragers returned to the colonies with a lower amount of pollen. [START_REF] Morfin | Sublethal exposure to clothianidin during the larval stage causes long-term impairment of hygienic and foraging behaviour of honey bees[END_REF] also observed the impairment of pollen foraging in bees exposed to clothianidin at the larval stage. This higher sensitivity of pollenforaging bees, as opposed to nectar foraging, has also previously been reported for bumblebees following exposure to pesticides [START_REF] Feltham | Field realistic doses of pesticide imidacloprid reduce bumblebee pollen foraging efficiency[END_REF][START_REF] Gill | Combined pesticide exposure severely affects individual-and colony-level traits in bees[END_REF], and therefore seems to be a hallmark of stressed bees [START_REF] Bordier | Stress decreases pollen foraging performance in honeybees[END_REF][START_REF] Lach | Parasitized honey bees are less likely to forage and carry less pollen[END_REF].

Homing flight

Over the last thirty years, sublethal doses of pesticides have been suspected to affect orientation and homing flight of foragers. This concern originated with the depopulation of hives observed by beekeepers near fields sowed with seed-dressing-treated crops. Orientation performance of foragers exposed to low doses of an insecticide were initially investigated using a complex maze device [START_REF] Decourtye | Sublethal effects of fipronil on the ability of honeybees (Apis mellifera L.) to orientate in a complex maze[END_REF]. This device relies on associative learning between a visual mark to navigate in the maze and a reward of sugar solution [START_REF] Zhang | Honeybee memory: navigation by associative grouping and recall of visual stimuli[END_REF]. Indeed, a bee exposed to a pesticide during foraging trips may incorrectly learn and/or memorize visual patterns for navigation, causing disorientation and bee loss due to homing failure [START_REF] Desneux | The sublethal effects of pesticides on beneficial arthropods[END_REF]. These experiments are based on the use of limited cues whereas navigation in natural conditions relies on a combination of several guidance mechanisms and cognitive functions (see above). More realistic experimental situations were designed and showed an impact of insecticides on the homing performances of foragers by recording the flight activity between a feeder and the colony located 8 m away in semi-field conditions [START_REF] Vandame | Alteration of the homing-flight in the honey bee Apis mellifera L. exposed to sublethal dose of deltamethrin[END_REF] or 500 m away in field conditions [START_REF] Bortolotti | Effects of sublethal imidacloprid doses on the homing rate and foraging activity of honey bees[END_REF].

More recently, [START_REF] Matsumoto | Reduction in homing flights in the honey bee Apis mellifera after a sublethal dose of neonicotinoid insecticides[END_REF] showed an increase in the proportion of homing failure of foragers, released 500 m away from the colony, when exposed to sublethal doses of neonicotinoid or pyrethroid insecticides. However, the number of individuals monitored simultaneously and the time span of the observations may present experimental difficulties in these studies using marked bees (paint or colored, numbered tags) [START_REF] Decourtye | Honeybee tracking with microchips: a new methodology to measure the effects of pesticides[END_REF][START_REF] Pahl | Large ccale homing in honeybees[END_REF]. The emergence of automatic tracking technology helped to solve these problems and study the effects of pesticides on free-ranging foragers in field conditions. Using RFID technology with continuous recording (48 hours), [START_REF] Henry | A common pesticide decreases foraging success and survival in honey bees[END_REF] showed that foragers released 1 km away from their colony returned to the hive at a significantly lower rate when exposed to a sublethal dose of thiamethoxam. Such effects were confirmed with colonies placed near rape oilseed crop originating from seed-dressing treated with Cruiser®-containing thiamethoxam [START_REF] Henry | Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees[END_REF]. Foragers originating from these colonies and tracked with RFID microchips disappeared at a faster rate with increasing field exposure (e.g. increased mortality rate of 10% due to non-returning bees per 15 ha of treated crop in a radius of 1 km around the colony) and this excess mortality increased over time. The homing flight failure might be explained by impairments to flight capacity (e.g. motor functions) (Tosi et al., 2017) and cognitive functions, such as the retrieval of previously learned information (e.g. visual cues) [START_REF] Fischer | Neonicotinoids interfere with specific components of navigation in honeybees[END_REF][START_REF] Tison | Honey bees' behavior is impaired by chronic exposure to the neonicotinoid thiacloprid in the field[END_REF]. These assumptions are further supported by the fact that negative effects on homing performances were more pronounced at lower temperatures (below 28°C) and in environments with a greater density of landmarks (e.g. hedges, forest borders) [START_REF] Henry | Pesticide risk assessment in free-ranging bees is weather and landscape dependent[END_REF]. Finally, the health status of colonies and especially varroa infestation levels may also impact the homing performances of foragers. Experimental data and predictive modelling show a three times greater effect of thiamethoxam on foragers originating from colonies infested by 5 varroa mites per 100 bees as compared to foragers from varroa-free colonies [START_REF] Monchanin | Hazard of a neonicotinoid insecticide on the homing flight of the honeybee depends on climatic conditions and Varroa infestation[END_REF].

Waggle dance

When returning from a food rich area, successful foragers share information about its distance and direction through body movements to recruit naive bees [START_REF] Dyer | The biology of the dance language[END_REF]. The dancing bee notably performs repeated waggle dances (loops with a shape of a '8') if the food source is located at more than 50 meters away. In the waggle dance, composed of a waggle and return phase, the axis of the '8' represents the direction of the food source relative to the sun position, and the number of waggle runs inform receivers of its distance. Waggle dance performance therefore requires detecting and integrating into the central nervous system several environmental parameters linked to spatial information, which are then encoded into body movements. Pesticides, and particularly insecticides, that target the nervous system, would have an expected impact on forager ability to communicate food location.

To decipher potential effects of pesticides, several components of the waggle dance can be measured, such as the number of loops, the angle of the dance, the duration of the waggle run and the frequency of the vibrations produced by the bees [START_REF] Dyer | The biology of the dance language[END_REF]. The simplest methods for studying the waggle dance are visual observation or video recording of a colony in an observation hive. Using such techniques, a precursor study by [START_REF] Schricker | The Effect of Sublethal Doses of Parathion on Honeybee Behaviour. I. Oral Administration and the Communication Dance[END_REF] reported a pesticide effect on the waggle dance, with changes in the dance angle in a stepwise fashion (instead of linearly) in bees exposed to parathion. Then, three studies, investigating the toxic effect of imidacloprid on the waggle dance, found a reduced number of dance circuits [START_REF] Eiri | A nicotinic acetylcholine receptor agonist affects honey bee sucrose responsiveness and decreases waggle dancing[END_REF] and decreased precision of directional information (i.e. larger variance in the angle of the waggle phase) (Kirchner, 1999;Zhang et al., 2020a). A similar impairment of dance communication was found upon exposure to deltamethrin (Zhang et al., 2020b). A more automatic recording of waggle dance, based on the measurements of electrical fields, was developed by [START_REF] Greggers | Reception and learning of electric fields in bees[END_REF]. They found that dancing bees emit low-(movements of the abdomen, 16Hz) and high-frequency (buzzing of the wings, 230 Hz) electrical fields, which allowed them to determine the number of waggle runs [START_REF] Greggers | Reception and learning of electric fields in bees[END_REF]. Using this technique, [START_REF] Tison | Honey bees' behavior is impaired by chronic exposure to the neonicotinoid thiacloprid in the field[END_REF] found a significant reduction in the number of waggle dances per hour in bees intoxicated by the neonicotinoid thiacloprid as compared to control bees.

Queen and drone fertility

As early as the 1960s, it was shown that exposure to chemicals can directly alter sexual behavior, mating success, sex ratio and fertility, notably through a decrease in spermatozoa viability and number. The pioneering study of Carson found that the organochlorine insecticide DDT (Dichlorodiphenyltrichloroethane) causes reproductive injuries in birds, leading to a decline in their populations [START_REF]Silent spring[END_REF]. Evidence for pesticide-induced reproductive toxicity in honeybees emerged 40 years later and was especially intriguing, given that the reproductive castes (queen and drones) are generally exposed to much lower concentrations of pesticides than worker bees. The queen and drones normally feed on food processed and supplied by workers, which can lead to exposure to concentrations below the limit of detection of analytical methods [START_REF] Kairo | Drone exposure to the systemic insecticide fipronil indirectly impairs queen reproductive potential[END_REF] that represents between 0.001 and 0.016 % of the original pesticide concentration found in the diet consumed by workers bees [START_REF] Böhme | From field to food-will pesticide-contaminated pollen diet lead to a contamination of royal jelly?[END_REF]. However, they may be exposed during their development to higher levels of pesticides contained in the wax [START_REF] Chauzat | Pesticide residues in beeswax samples collected from honey bee colonies (Apis mellifera L.) in France[END_REF][START_REF] Mullin | High levels of miticides and agrochemicals in north american apiaries: implications for honey bee health[END_REF]).

An impairment of queen fertility could translate into either a temporary or permanent absence of egg laying, or the laying of unfertilized eggs (drones), leading to poor colony development or ultimately colony failure [START_REF] Büchler | Standard methods for rearing and selection of Apis mellifera queens[END_REF][START_REF] Pettis | Fluvalinate treatment of queen and worker honey bees (Apis mellifera L) and effects on subsequent mortality, queen acceptance and supersedure[END_REF][START_REF] Rangel | The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth[END_REF][START_REF] Vanengelsdorp | Idiopathic brood disease syndrome and queen events as precursors of colony mortality in migratory beekeeping operations in the eastern United States[END_REF].

Queen fertility after pesticide exposure has been investigated at both the colony and individual level.

Colony-level examination usually consists of spiking the colony food supplement with pesticide and then analyzing queen egg-laying rate, brood area, hive population and food stocks (Colin et al., 2019a;[START_REF] Meikle | Sublethal effects of imidacloprid on honey bee colony growth and activity at three sites in the U[END_REF][START_REF] Odemer | Chronic exposure to a neonicotinoid pesticide and a synthetic pyrethroid in full-sized honey bee colonies[END_REF][START_REF] Woodcock | Country-specific effects of neonicotinoid pesticides on honey bees and wild bees[END_REF]. Using this procedure, colonies that were chronically exposed to the neonicotinoids, imidacloprid, thiamethoxam and clothianidin, demonstrated a significant decrease in queen egg-laying followed by reduced brood production [START_REF] Sandrock | Impact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure[END_REF][START_REF] Wu-Smart | Sub-lethal effects of dietary neonicotinoid insecticide exposure on honey bee queen fecundity and colony development[END_REF]. Egg-laying reduction was notably associated with lower queen mobility (Wu-Smart and Spivak, 2016). Finally, colony foraging on rapeseed grown from seeds coated with thiamethoxam was followed by an increase in drone brood production [START_REF] Henry | Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees[END_REF]. Such an effect could reflect either a higher investment of colonies in reproduction or an impairment of egg fertilization, the latter suggesting damage to spermatozoa in queens. These impairments caused by pesticides did not trigger colony collapse, even if a greater number of supersedures were reported [START_REF] Bendahou | Biological and biochemical effects of chronic exposure to very low levels of dietary cypermethrin (Cymbush) on honeybee colonies (Hymenoptera: Apidae)[END_REF][START_REF] Sandrock | Impact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure[END_REF]. A major drawback of colony-level experiments is separating the direct and indirect effects of the pesticide on the observed reduction in egg-laying. The pesticide may be acting directly on the queen or may be decreasing worker foraging performance, resulting in reduced colony resources essential to brood production.

An alternative or complementary approach is to determine the effects of pesticides directly on queen physiology and fertility (body weight, the structure and the development of ovaries, the quantity and viability of spermatozoa stored in queen spermatheca). Negative impacts on ovary weight and fertility impairments were observed in young queens exposed during their development to coumaphos, fluvalinate and neonicotinoids via contaminated food [START_REF] Haarmann | Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations[END_REF][START_REF] Pettis | Effects of coumaphos on queen rearing in the honey bee, Apis mellifera[END_REF][START_REF] Williams | Neonicotinoid pesticides severely affect honey bee queens[END_REF]. Exposure to pesticides at the larval stage through contaminated wax also resulted in reduced queen egg-laying rate and attractiveness to workers [START_REF] Walsh | Queen honey bee (Apis mellifera) pheromone and reproductive behavior are affected by pesticide exposure during development[END_REF].

Regarding drones, the count and the viability rate of spermatozoa are two major biological parameters of fertility from which the quantity of spermatozoa available for queen fertilization can be deduced.

Sperm count is determined using a microscopic cell count chambers or by flux cytometry analyses. The reproductive toxicity of pesticides in drones has also been investigated during the larval and adult stage.

By supplying colonies with food contaminated with neonicotinoids during drone development [START_REF] Ciereszko | Sperm parameters of honeybee drones exposed to imidacloprid[END_REF][START_REF] Straub | Neonicotinoid insecticides can serve as inadvertent insect contraceptives[END_REF] or exposing drones during their development to beewax previously sprayed with miticides or agrochemicals [START_REF] Fisher | Exposure to pesticides during development negatively affects honey bee (Apis mellifera) drone sperm viability[END_REF], a consistent decrease in sperm viability and motility was found. To determine the reproductive toxicity of environmental concentration of the insecticide fipronil on adult drones, experiments were performed in semi-field conditions under insect-proof tunnels or in laboratory conditions. In semi-fields conditions, colonies foraged on a pesticide-contaminated feeder, whereas in laboratory conditions, drones were reared with worker bees in cages provided with pesticide-contaminated pollen. For all exposure methods, fipronil did not affect drone survival, maturity rates (number of drones that provided sperm after stimulation) and semen volumes, but significantly altered drone fertility by lowering spermatozoa concentration and viability, which reduced the amount of living sperm available for reproduction [START_REF] Kairo | Drone exposure to the systemic insecticide fipronil indirectly impairs queen reproductive potential[END_REF](Kairo et al., , 2017b)).

Relevance of behavioral and reproductive performance as assessment endpoints

The goal of this section is to provide general insights into the reliability and relevance of the different behavioral and reproductive endpoints in regards to the risk assessment procedure, i.e. to determine to what extent each of them is appropriate for chemical regulations. For that purpose, we primarily relied on criteria cited for test method validation in a regulatory hazard assessment context: simplicity, sensitivity, robustness, reproducibility (OECD, 2005). Simplicity implies that the endpoint method is relatively easy, and time-and cost-effective, for use in a regulatory context. Sensitivity indicates whether the method used to assess the endpoint can accurately detect dose-dependent effects. Robustness refers to the stability of responses to minor variations in experimental procedures and reproducibility to the ability to duplicate the results across laboratories/studies. We grouped both robustness and reproducibility criteria into one: the standardization level. The more toxicological studies have used a

given method, the better we can evaluate its sensitivity and standardization level. Finally, we discussed the ecological relevance of endpoints, which indicates whether effects measured in individual bees can be related to consequences for colonies in field conditions.

4.1. Test endpoints with low standardization level and uncertain ecological relevance: grooming, waggle dance

The observation of bee behaviors has traditionally been conducted either directly by the observer or by means of video recording. If both methods are relatively easy to perform and do not require expensive materials, complexity in behavioral recording can arise from the targeted behavior itself. For instance, some behaviors may occur at a relatively low rate, making observations quite time-consuming to obtain enough sampling events, while others might be quite complex and require trained observers to record the full behavioral pattern. The waggle dance falls into both categories, which might explain why almost all toxicological studies focusing on the waggle dance tested only one dose or concentration. It is therefore currently not possible to determine whether pesticides can cause dose-dependent effects on the waggle dance. Similarly, few studies with grooming behavior as an endpoint used several doses or concentrations of pesticides. To our knowledge, only Williamson et al. (2014a;2013) exposed bees to several sublethal doses of pesticides and reported effects on grooming in a dose-dependent manner.

Regarding the robustness and reproducibility of methods, the waggle dance seems promising as revealed by three studies performed by different laboratories, which found similar effects of imidacloprid on waggle dance performance [START_REF] Eiri | A nicotinic acetylcholine receptor agonist affects honey bee sucrose responsiveness and decreases waggle dancing[END_REF]Kirchner, 1999;Zhang et al., 2020b). However, some inconsistencies across studies testing the same substance were found for grooming behaviors. For instance, bees exposed to low doses of tau-fluvalinate showed either a decrease in grooming [START_REF] De Mattos | Effects of synthetic acaricides on honey bee grooming behavior against the parasitic Varroa destructor mite[END_REF] or a lack of effect [START_REF] Oliver | Pyrethroids and nectar toxins have subtle effects on the motor function, grooming and wing fanning behaviour of honeybees (Apis mellifera)[END_REF]. Such a discrepancy might be due to variability in experimental conditions (duration of exposure, dose, size of bee groups…) rather than to the recording of the behavior itself, which is rather straightforward.

The main shortcoming for the use of these endpoints in pesticide risk assessment might be the limited extrapolation of effects on the consequences for the colonies. Grooming is essential to colony hygiene, but to what extent a decrease or increase in grooming performance of individual bees will affect colony development and survival, is largely unknown. Regarding the waggle dance, several studies have attempted to quantify the benefits of spatial information on colony foraging success. [START_REF] Schürch | Dancing Bees Improve Colony Foraging Success as Long-Term Benefits Outweigh Short-Term Costs[END_REF] found, via agent-based simulations, that spatial information was beneficial in the long-term in almost all ecological conditions, but it remains to be empirically validated. In reality, in experimental studies, the benefits were often null or occurred only in very specific habitats. For instance, benefits were observed in Asian tropical habitats but not in temperate habitats [START_REF] Dornhaus | Why do honey bees dance?[END_REF], in the winter or in urban areas [START_REF] Sherman | Honeybee colonies achieve fitness through dancing[END_REF], and then, only in habitats with high floral species richness and dense floral patches [START_REF] Donaldson-Matasci | How habitat affects the benefits of communication in collectively foraging honey bees[END_REF]. Overall, these studies suggest that pesticide-induced disoriented dances might not have a clear impact on foraging success of the colony.

Test endpoints with high standardization level but lower ecological relevance:

learning/memory, locomotion

The high interest in PER conditioning for evaluating pesticide effects on learning and memory can be explained by its simplicity. The method benefits from 60 years of methodological and conceptual development [START_REF] Giurfa | Invertebrate learning and memory: fifty years of olfactory conditioning of the proboscis extension response in honeybees[END_REF], is cheap, does not require specific technical expertise, and provides fast results for a large number of individuals. Most importantly, after reviewing the dozens of toxicological studies that used PER conditioning and taking into account possible technical bias across laboratories, [START_REF] Siviter | Quantifying the impact of pesticides on learning and memory in bees[END_REF] found that size effects on both learning and memory were robust between studies. Therefore, PER conditioning seems to provide reliable data for a wide range of doses and application procedures, which makes it a serious endpoint candidate for chemical regulation policy.

The main weakness of PER conditioning is its ecological relevance. Drawing general conclusions about the impacts in field conditions of poor learning performances measured in the laboratory is challenging [START_REF] Decourtye | Assessing the sublethal effects of pesticides on the honey bee[END_REF][START_REF] Thompson | Behavioural Effects of Pesticides in Bees -Their Potential for Use in Risk Assessment[END_REF]. Since cognitive abilities can directly affect functional behaviors (see [START_REF] Morand-Ferron | Studying the evolutionary ecology of cognition in the wild: a review of practical and conceptual challenges: Evolutionary ecology of cognition in the wild[END_REF] for a review), an impairment of cognitive capacities is expected to affect honeybee foraging performance. However, while reversal learning performance is associated with foraging experience (cumulated foraging time) [START_REF] Cabirol | Relationship between brain plasticity, learning and foraging performance in honey bees[END_REF], it remains unclear whether and how bee cognitive abilities contribute to individual foraging success and efficiency, and ultimately to colony performance. In bumblebees no difference in the rate of food collection and the number of foraging trips per day was found between fast and slow learners [START_REF] Evans | Fast learning in free-foraging bumble bees is negatively correlated with lifetime resource collection[END_REF]. There is a lack of studies using exposure methods that attempt to mimic field realistic scenarios. Therefore, the extrapolation of PER conditioning results to individual and colony foraging success is a key challenge that needs to be addressed in regard to its selection as an assessment endpoint.

Methods for analyzing potential locomotor disabilities upon exposure to pesticides also proved to be sensitive since several studies were able to detect dose-dependent effects on bee locomotion (e.g.

significant differences between exposures differing by 2 to 10-fold in dose) [START_REF] Ingram | Evaluating sub-lethal effects of orchardapplied pyrethroids using video-tracking software to quantify honey bee behaviors[END_REF]Tosi and Nieh, 2017;[START_REF] Williamson | Exposure to neonicotinoids influences the motor function of adult worker honeybees[END_REF]. The robustness and reproducibility of methods is more difficult to assess due to a relatively low number of research studies. However, the recording of bee activity in an arena is rather straightforward: it simply requires a camera and data that can be analyzed in a semi-automatic way [START_REF] Kadala | Honey bees long-lasting locomotor deficits after exposure to the diamide chlorantraniliprole are accompanied by brain and muscular calcium channels alterations[END_REF]. The standardization of endpoints measured with the flight mill is also promising. This latter method was developed in the 1940s and is increasingly used in a broad range of scientific areas, such as invasion biology, population dynamics, and pest management, with over 400 publications recorded [START_REF] Naranjo | Assessing insect flight behavior in the laboratory: a primer on flight mill methodology and what can be learned[END_REF]. In addition, there is a free online protocol for building a standard and affordable flight mill that can record the flight activity of up to 16 insects simultaneously [START_REF] Attisano | A Simple Flight Mill for the Study of Tethered Flight in Insects[END_REF]. Tests using flight mills might need qualified staff but the recording and analysis can also be done in an automatic manner with sensors and customized scripts. The use of standard flight mills in honeybee toxicological research thus has the potential to provide robust data. However, as for PER conditioning, the ecological relevance of bee activity recorded in indoor conditions remains to be determined. Impairment of locomotion ability, due to alteration of either energetic metabolism [START_REF] Liao | Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin[END_REF] or brain and muscle excitability [START_REF] Kadala | Honey bees long-lasting locomotor deficits after exposure to the diamide chlorantraniliprole are accompanied by brain and muscular calcium channels alterations[END_REF], may affect the performance of foragers or in-hive bees (like nurse bees). For instance, flight mills have been largely used for better understanding migration in insects, and flight studies have been shown to support field observations [START_REF] Minter | The tethered flight technique as a tool for studying life-history strategies associated with migration in insects: Tethered flight for studying insect movement[END_REF]. The challenge though is to define how locomotion patterns obtained in lab-controlled conditions can be used to predict honeybee performance in the field and to what extent it could affect colonies.

4.3. Test endpoints with promising standardisation levels and/or ecological relevance: foraging, homing flight, queen and drone fertility

The evaluation of pesticide toxicity on foraging activity and homing flights has recently been boosted by the emergence of automatic tracking technologies. The major drawbacks of these bioassays are the cost of equipment and the need for specific technical expertise. However, the toxicity assays are usually performed in more field-realistic conditions than other behavioral endpoints, and both foraging activity and homing flights integrate a wide range of biological functions (learning/memory, orientation, locomotion, energetic metabolism…) with which pesticides with different modes of action may interfere. This may explain why these endpoints are receiving growing interest despite the relative complexity of the measurements. A great majority of these studies have shown consistency in the results obtained (e.g. decrease in the number of visits to the feeder, precocious onset of foraging and mortality, and lowered homing success). It is still difficult to assess the robustness and reproducibility of methods used for recording foraging activity, because studies were mostly performed on different pesticides.

However, a dose-dependent effect of sublethal doses of acetamiprid was found on the onset of foraging [START_REF] Shi | Sublethal acetamiprid doses negatively affect the lifespans and foraging behaviors of honey bee (Apis mellifera L.) workers[END_REF], indicating that the recording of foraging activity with tracking technologies is a sensitive method.

Homing flight tests were identified as a useful method to assess the effects of sublethal doses of pesticides on honey bees in field conditions [START_REF] Efsa | EFSA Guidance Document on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees)[END_REF][START_REF] Efsa | Scientific Opinion on the science behind the development of a risk assessment of Plant Protection Products on bees (Apis mellifera, Bombus spp. and solitary bees)[END_REF]. The method has been tested since 2015 with a dozen laboratories in different environments [START_REF] Fourrier | The homing flight method to assess the effect of sublethal doses of plant protection products on the honey bee in field conditions: results of the ring tests and proposal of an OECD TG[END_REF]. During five years of ring test, the majority of laboratories performed the test successfully. In addition, this method has proved to be sensitive because dose-effect responses have been found [START_REF] Monchanin | Hazard of a neonicotinoid insecticide on the homing flight of the honeybee depends on climatic conditions and Varroa infestation[END_REF]. As a whole, the results show that the method is well standardized. The homing flight test, which was proposed to OECD standard protocols for pesticide risk assessment, is currently under evaluation. Even if it has been validated for only thiamethoxam, there is growing evidence for low-dose effects of others insecticides, fungicides and herbicides, on honeybee navigation, foraging activities and flight performances (e.g. [START_REF] Balbuena | Effects of sublethal doses of glyphosate on honeybee navigation[END_REF][START_REF] Bortolotti | Effects of sublethal imidacloprid doses on the homing rate and foraging activity of honey bees[END_REF][START_REF] Liao | Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin[END_REF][START_REF] Matsumoto | Reduction in homing flights in the honey bee Apis mellifera after a sublethal dose of neonicotinoid insecticides[END_REF][START_REF] Prado | Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees[END_REF][START_REF] Vandame | Alteration of the homing-flight in the honey bee Apis mellifera L. exposed to sublethal dose of deltamethrin[END_REF]. Such endpoints have a high ecological relevance because of the obvious link between the loss of foragers and/or decreased foraging activity and colony development. The rate of forager loss and activity decline that could lead to a critical reduction of the colony size remains an open question. However, population dynamics models might help to fill this gap. For instance, effects on homing failure and foraging performances can be converted into death rate and foraging parameters (resource collection, age at the onset of foraging…), respectively, and implemented into models to investigate the consequences on colony development and survival.

The field of reproductive toxicity in honeybees has been growing recently. Multiple studies are now available that investigated the reproductive toxicity of the same compound and have revealed consistent results. For instance, both [START_REF] Haarmann | Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations[END_REF] and [START_REF] Pettis | Effects of coumaphos on queen rearing in the honey bee, Apis mellifera[END_REF] showed that exposure to coumaphos during development negatively affects queen weight at emergence. Moreover a series of studies using both Tier 1 (laboratory test) and Tier 2 (semi-field test) approaches to analyze the reproductive toxicity of fipronil found similar results. By first showing that drones reared under laboratory and semi-field conditions had similar semen quality [START_REF] Ben Abdelkader | Semen quality of honey bee drones maintained from emergence to sexual maturity under laboratory, semi-field and field conditions[END_REF], they found consistent toxic effects of chronic exposure to fipronil across different times of the year and among years, i.e. higher spermatozoa mortality and lower spermatozoa production (Kairo et al., 2017b(Kairo et al., , 2017a[START_REF] Kairo | Drone exposure to the systemic insecticide fipronil indirectly impairs queen reproductive potential[END_REF]. This could serve as a basis for the development of standardized methods for testing the reproductive toxicity of pesticides. If the methods using reproduction as assessment endpoints provide robust results, their sensitivity is less known. Besides Wu-Smart and Spivak (2016) who described a dose-dependent effect of imidacloprid on queen egg-laying rate, most studies tested a single dose or concentration of pesticide. This is likely due to the complex and time-consuming protocols. A clear strength of these reproductive endpoints in pesticide risk assessment is their great ecological relevance:

impairment of queen and drone fertility can be readily extrapolated to consequences at the colony level.

A decrease in queen egg-laying would generally translate into lower brood production and queen replacement by workers (supersedure) [START_REF] Hendriksma | Stimulating nautral supersedure of honeybee queens, Apis mellifera[END_REF][START_REF] Sandrock | Impact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure[END_REF]; the latter being risky for the colony fate if it fails. In addition, smaller queens tend to mate with a lower number of drones and to have a lower sperm count in the spermatheca [START_REF] Delaney | The physical, insemination, and reproductive quality of honey bee queens (Apis mellifera L.)[END_REF]. Reproductive endpoints are especially interesting given that the risk associated with poor fertility has been quantified in the literature. For instance, it was found via artificial insemination that a sperm count lower than 4 million results in premature queen supersedures [START_REF] Harizanis | The quality of insemination of queen honey bees mated under commercial conditions[END_REF]Woyke, 1989c).

Additionally, queens that are considered by beekeepers as failing (e.g. poor brood production) or that need to be changed have a lower sperm viability (median value of 55%) than healthy queens (92%) [START_REF] Pettis | Colony Failure Linked to Low Sperm Viability in Honey Bee (Apis mellifera) Queens and an Exploration of Potential Causative Factors[END_REF]. Such observations indicate that environmental exposure to fipronil represents a high risk for honeybee colonies. Indeed, [START_REF] Kairo | Drone exposure to the systemic insecticide fipronil indirectly impairs queen reproductive potential[END_REF] found that queens inseminated with semen from drones exposed to an environmentally-relevant concentration of fipronil had 62% sperm viability and 3.14 million spermatozoa on average (vs 75% sperm viability and 4.62 million spermatozoa for control queens). They would thus fall into the category of failing queens. robustness and ecological relevance) (Fig. 2). The test on foraging activity at a syrup feeder scored, on average, the best for all of the criteria. In particular, it was considered easier to implement and more robust than the homing flight test. The homing flight test was well noted for sensitivity, reproducibility of results and the best rated for ecological relevance. The test measuring the effects on social communication (waggle dance) received the lowest score (Fig. 2). While it was not possible to have the variability of the participants' responses, the results of this participative session reflect a reality observed by the participants, namely that there are no behavioral tests that respond perfectly to all of the proposed criteria, and that it is rather a trade-off between the different criteria that must be found.

Challenges for better integrating behavioral and reproductive performances in pesticide risk assessment

Translation of toxicological research studies into regulatory test methods

Research studies performed by academics and testing methods used by governments, industries and independent laboratories generally share the same goal, which is identifying and determining potential hazards of pesticides. However, research studies are often hypothesis-driven or exploratory with relatively complex protocols, as opposed to testing methods that rely on simpler and highly standardized protocols. As a consequence, few research studies share the same methodological designs for the measurement of a given endpoint and the robustness and reproducibility of measurements are difficult to assess. Moving from an exploratory to a standard method therefore requires defining standard measurements (such as the LD50 in mortality tests), sharing full details of protocols, building a broader database and developing comparative studies. A larger number of tested doses/concentrations and replicates will notably help determine whether the method is reliable enough for chemical regulation.

Regarding behavioral endpoints, the standardization and analysis of measurements currently benefits from the emergence of new technologies. Labor-intensive and time-consuming recordings of behaviors are now faster and easier thanks to computational tools (e.g. machine learning-based system) and electronic tagging of animals. In addition, such tools contribute to removing or reducing opportunities for human observation error. For instance, optic bee counters and RFID proved to be very efficient in automatically tracking the foraging and homing flights of hundreds of honeybees in toxicological studies. Most importantly they drastically reduced the measurement complexity of these highly ecologically-relevant endpoints, making them more accessible to regulatory test methods. Computer vision algorithms have also been developed to track and phenotype the communication and in-hive behaviors of honeybees. For example, by tracking thousands of bees the BeesBook system allows for the automatic detection and decoding of communication dances [START_REF] Wario | Automatic methods for long-term tracking and the detection and decoding of communication dances in honeybees[END_REF]. Similarly, the Bee Behavioral Annotation System [START_REF] Blut | Automated computer-based detection of encounter behaviours in groups of honeybees[END_REF] and the technology developed by [START_REF] Gernat | Automated monitoring of behavior reveals bursty interaction patterns and rapid spreading dynamics in honeybee social networks[END_REF] enable the monitoring of honeybee worker social interactions (trophallaxis, antennation). The next step will be to test the efficiency and sensitivity of computer vision algorithms in detecting toxicological effects on bees, as recently performed by [START_REF] Siefert | Chronic within-hive video recordings detect altered nursing behaviour and retarded larval development of neonicotinoid treated honey bees[END_REF] for investigating the influence of neonicotinoids on nursing behavior. The improved phenotyping of behaviors that are, by definition, complex, dynamic and noisy will facilitate the potential integration of behavioral endpoints in pesticide risk assessment.

The evaluation of pesticide reproductive toxicity requires the implementation of strong human and technical expertise, both in beekeeping and in the laboratory, which can be limiting for regulatory test methods. Thus, the next step would be to simplify measurement methods. The recent development of cytometry flux methods to determine spermatozoa number and viability in either queen spermatheca or drones has led to more accurate and robust quantification [START_REF] Pettis | Colony Failure Linked to Low Sperm Viability in Honey Bee (Apis mellifera) Queens and an Exploration of Potential Causative Factors[END_REF][START_REF] Tofilski | Flow cytometry evidence about sperm competition in honey bee (Apis mellifera)[END_REF][START_REF] Yániz | Sperm Quality Assessment in Honey Bee Drones[END_REF]. Such endpoints notably have the advantage of communicating actual or future queen quality. Identifying in queens and drones the most important route of exposure to pesticides would also help to narrow down tests to the most relevant life stages (larva, adult) and modes of exposure (contact, oral).

Improve the extrapolation from effects on individuals to colony response

To improve the ecological relevance of behavioral and reproductive endpoints, causative links between individual effects and colony response are needed. However, to confidently detect an effect at the colony level, large colony sample size has to be used. This might explain why none of the field studies developed to assess neonicotinoid effects had the power to detect an effect on the size and the fate of the colony based on the EFSA standard [START_REF] Franklin | Moving beyond honeybee-centric pesticide risk assessments to protect all pollinators[END_REF]. Efforts are being made to lower variability inherent to semi-field and field methodologies and thus reduce the Minimum Detectable Difference (MDD) [START_REF] Wang | Reduction of variability for the assessment of side effects of toxicants on honeybees and understanding drivers for colony development[END_REF]. The use of population dynamics models may help to integrate behavioral and reproductive endpoints into a risk assessment framework. The challenge of scaling up the consequences of adverse behavioral and reproductive effects from individuals to populations (or colonies) as a whole is known as the extrapolation problem [START_REF] Forbes | The extrapolation problem and how population modeling can help[END_REF]. Population dynamics models are promising tools in that respect. By population models, [START_REF] Forbes | The extrapolation problem and how population modeling can help[END_REF] refer to mechanistic models that relate individual level responses to changes in population size and structure.

Such models have been developed for honey bees [START_REF] Becher | BEEHAVE: a systems model of honeybee colony dynamics and foraging to explore multifactorial causes of colony failure[END_REF][START_REF] Khoury | Modelling food and population dynamics in honey bee colonies[END_REF][START_REF] Khoury | A quantitative model of honey bee colony population dynamics[END_REF][START_REF] Schmickl | HoPoMo: A model of honeybee intracolonial population dynamics and resource management[END_REF][START_REF] Torres | Modeling Honey Bee Populations[END_REF] and investigated as potential, useful tools to inform pesticide risk assessment because they allow for the joint implementation of mortality endpoints as well as a range of behavioral and reproductive endpoints. To date, most models have been used to assess the consequences of lethal pesticide effects at different individual developmental stages, including eggs, larvae, pupae, in-hive workers and ultimately foragers (e.g. [START_REF] Rumkee | Predicting Honeybee Colony Failure: Using the BEEHAVE Model to Simulate Colony Responses to Pesticides[END_REF][START_REF] Schmolke | Honey bee colony-level exposure and effects in realistic landscapes: An application of BEEHAVE simulating clothianidin residues in corn pollen[END_REF].

Model users may also tune those mortality parameters, jointly with other sublethal endpoints, to simulate the consequences of pesticide exposure from individual response to risk of colony collapse. Two sublethal parameters may be readily implemented in the context of pesticide risk assessment, namely homing failure and queen egg-laying rate. Homing failure can be readily combined with natural forager death rate to assess its global effect on the colony dynamics [START_REF] Bryden | Chronic sublethal stress causes bee colony failure[END_REF][START_REF] Henry | A common pesticide decreases foraging success and survival in honey bees[END_REF].

Likewise, reduced egg laying rate is easily introduced into population dynamics models by simply tuning down the daily egg production to simulate impaired queen reproductive activity [START_REF] Henry | Predictive systems models can help elucidate bee declines driven by multiple combined stressors[END_REF][START_REF] Rumkee | Predicting Honeybee Colony Failure: Using the BEEHAVE Model to Simulate Colony Responses to Pesticides[END_REF]. Models are also useful tools to reveal how combined sublethal stresses at the individual scale can act in concert to exacerbate global colony collapse risks [START_REF] Henry | Predictive systems models can help elucidate bee declines driven by multiple combined stressors[END_REF].

Still, additional sublethal parameters along with appropriate mathematical response functions need to be formally implemented into population models. Most importantly, their predictive power remains to be thoroughly tested with empirical data.

We do not suggest the use of an in silico approach as a substitute for empirical studies with colony level endpoints, but rather as a tool to narrow down the possible scenarios (e.g. level and duration of exposure) that might lead to the decline of several colony traits, such as brood and adult population size, honey production and colony collapse [START_REF] Henry | Predictive systems models can help elucidate bee declines driven by multiple combined stressors[END_REF]. A modelling approach should also give insight into the effect size and time delay before the impact of exposure to pesticide on colony traits. This is particularly important since a pesticide might be considered safe for honeybee colonies if field experiments are not sufficiently well designed, i.e. small-scale experiments and/or short-term monitoring. Therefore, once a risk has been identified at the individual level, population dynamics models may represent a first step screening process to design field experiments able to confidently detect potential changes at the colony level.

Conclusions

New endpoints need to be implemented at the screening level of pesticide risk assessment to consider the risk posed by sublethal doses and concentrations in honeybees. We showed here that several behavioral and reproductive endpoints are available thanks to the development of exploratory research studies in ecotoxicology. These endpoints are not all equally standardized and some are simpler than others. However, one of the most important criteria in the decision-making process of risk assessment relies on the ability of the endpoint to communicate information on the potential impact on the population. In this context, homing and reproductive performances have great potential. While measured at the individual level, both provide great insight into the risk at the colony level, since colony demography and dynamics, like for any population, are primarily governed by birth (fecundity) and death (survival) of individuals. However, for using behavioral and reproductive endpoints in pesticide regulation, further work is needed to develop standardized test protocols.

Around 95% of toxicological studies that used behavioral and reproductive endpoints to investigate the non-target effects of pesticides on honeybees have focused on insecticides (Fig. 1B). This is likely explained by the fact that insecticides are intended to affect insects and generally target the nervous system. In contrast, fungicides and herbicides are usually not considered as a threat to honeybees due to their high LD50 values [START_REF] Zioga | Plant protection product residues in plant pollen and nectar: a review of current knowledge[END_REF]. However, there is now growing evidence for lethal and sublethal effects induced by such pesticides [START_REF] Cullen | Fungicides, herbicides and bees: A systematic review of existing research and methods[END_REF]. Notably, fungicides may affect honeybee energetic metabolism [START_REF] Degrandi-Hoffman | Effects of oral exposure to fungicides on honey bee nutrition and virus levels[END_REF], which can translate into impaired behavioral performance [START_REF] Liao | Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin[END_REF][START_REF] Prado | Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees[END_REF]. In addition, in terms of tonnage, insecticide use is outweighed by fungicides and herbicides [START_REF] Cullen | Fungicides, herbicides and bees: A systematic review of existing research and methods[END_REF]. There is thus a clear need for filling the gap on the evaluation of fungicide and herbicide toxicity. In this context, behavioral and reproductive endpoints are of great interest, especially given that field-realistic exposures to fungicides and herbicides are generally below their LD50 values [START_REF] Ostiguy | Honey bee exposure to pesticides: a four-year nationwide study[END_REF][START_REF] Prado | Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees[END_REF].

Future research on fungicides and herbicides will notably have to screen a range of endpoints and investigate which ones are the most appropriate for use in regulatory risk assessment, as they might differ between pesticide type and family.

4. 4 .

 4 Relevance of behavioural endpoints from the point of view of stakeholdersThe relevance of some behavioral endpoints within the context of pesticide risk assessment was further evaluated in a participative session of a workshop organized by ITSAP-Institut de l'Abeille and ANSES in January 2020 with different European stakeholders: scientists, beekeeping representatives, regulators, contract laboratory representatives and manufacturers (23 participants). The representatives worked especially on the standardization levels of four behavioral endpoints and associated test methods (PER, waggle dance, foraging activity and homing flight) within the perspective of chemical regulation. The test methods were noted according to the 5 assessment criteria (simplicity, sensitivity, reproducibility,

Figure 1 .

 1 Figure 1. Bibliometric of honeybee toxicological research studies using individual-based behavioral and reproductive endpoints. (A) Cumulative number of publications per year on behavioral and reproductive endpoints. The Y-axis on the left refers to the number of publications per endpoints (colored lines) and the Y-axis on the right to total number of publications (thick black line).The X-axis refers to the years of publication. (B) Breakdown of pesticide types (left) and endpoints (right). The thickness of each line connecting a pesticide type to a given endpoint is proportional to the number of studies.

Figure 2 .

 2 Figure 2. Average scores (±s.e.) assigned by all of the stakeholders to each of the four test methods for the five assessment criteria considered. For each of the test methods, score ranges from 0 (low) to 5 (high); n= 23 participants.

Figure 1 1255

 1 Figure 1 1255

Acknowledgments

This project received funding from the European Horizon 2020 research and innovation programme under grant agreement no. 773921 (LB, YLC and CA).