Samir M Ahmad

Ana S Mestre
email: asmestre@fc.ul.pt

Nuno R Neng

Maria Conchi

Ovin Concepcion

Ana Ania

José Carvalho

Nogueira
email: nogueira@fc.ul.pt

Conchi O Ania

Ana P Carvalho

José M F Nogueira

M F Nogueira

Carbon-Based Sorbent Coatings for the Determination of Pharmaceutical Compounds by Bar Adsorptive Microextraction

Keywords: Carbon hydrochars, Activated Carbons, Sorbent coating, Bar adsorptive microextraction (BAµE), HPLC-DAD, Pharmaceutical compounds, Environmental water matrices

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

including surface water, sea water, tap water and wastewater. Polar and nonpolar pharmaceuticals were selectedsulfamethoxazole, triclosan, carbamazepine, diclofenac, mefenamic acid, 17α-ethinylestradiol, 17β-estradiol, estrone, gemfibrozil and clofibric acidas model compounds to cover distinct therapeutic classes. Despite having a less-developed porosity, data showed that "in-house" prepared nanoporous hydrochars obtained from carbohydrates at low temperature (e.g., 180 ºC) in the presence of an eutectic salt mixture compete with the best commercial activated carbons for this particular application. The combination of a micro and mesopore network with a rich oxygen-based surface chemistry yielding an acidic nature allowed these hydrochars to present the best overall recoveries (between 20.9 to 82.4 %) for the simultaneous determination of the ten target PhCs with very distinct chemical properties, by high performance liquid chromatography-diode array detection (HPLC-DAD).

Introduction

Pharmaceuticals and personal care products (PPCPs) encompass many different substances, such as drugs used in human and veterinary medicine, fragrances, sunscreen agents, and cosmetics ingredients. More than 3000 PPCPs are currently marketed and new molecules enter the market yearly and they are ubiquitously detected in treated wastewater [START_REF] Arpin-Pont | Occurrence of PPCPs in the marine environment: a review[END_REF]. Moreover, pharmaceuticals compounds (PhCs) are generally biologically active compounds that are often water soluble, therefore can be found in wastewaters and easily end up in natural waters, including ground water, river water, sea water and surface waters [START_REF] Caracciolo | Pharmaceuticals in the environment: Biodegradation and effects on natural microbial communities. A review[END_REF][START_REF] Mnif | Effect of endocrine disruptor pesticides: a review[END_REF][START_REF] Li | Occurrence, sources, and fate of pharmaceuticals in aquatic environment and soil[END_REF][START_REF] Zhao | Porous Aromatic Framework Modified Electrospun Fiber Membrane as a Highly Efficient and Reusable Adsorbent for Pharmaceuticals and Personal Care Products Removal[END_REF], usually at the trace level, ranging from μg L -1 to ng L -1 [START_REF] Richardson | Water Analysis: Emerging Contaminants and Current Issues[END_REF]. PhCs include multiple medicines classes, namely lipid regulators, antibacterials/antifungals, β-adrenergic blocking agents, analgesics, tranquilizers, hormones, and antiseptics, among many others [START_REF] Jiang | Occurrence, transportation, monitoring and treatment of emerging micro-pollutants in waste water -A review from global views[END_REF]. The concern for the possible ecotoxicological effects of these compounds is reflected on decisions of the European Parliament in the field of water policy. In 2013, three widely used PhCs, including the hormonal preparations 17α-ethinylestradiol and 17β-estradiol, as well as the painkiller diclofenac, were listed in the watch list of substances to be monitored in the European Union (EU) to support the determination of appropriate measures to address the risk posed by them (Directive 2013/39/EU) [START_REF]Council of 12 August 2013 amending Directives 2000/60/EC and 2008/105/EC as regards priority substances in the field of water policy[END_REF]. The first revision of this list in 2015 maintained these three pharmaceuticals (Directive 2015/495/EU) [START_REF]Commission Implementing Decision (EU) 2015/495 of 20 March 2015 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council, Off[END_REF] but in the most recent revision diclofenac, along with three other substances, was removed from the watch list since the commission concluded that sufficient high-quality monitoring data was already available (Directive 2018/840/EU) [START_REF]Commission Implementing Decision (EU) 2018/840 of 5 June 2018 establishing a watch list of substances for Union-wide monitoring in the field of water[END_REF]. For these reasons, there is a need for innovative analytical approaches that allow for their 5/40 quantification in environmental water matrices. Furthermore, considering the EU decision on water policy, the development of a simple methodology that allows the simultaneous analysis of the most representative emerging PhCs in water matrices continues to be a hot research topic. As water bodies contain trace amounts of a large range of PhCs with distinct physicochemical properties, the challenge relies on the development of quantification techniques comprising both polar and non-polar compounds.

Many analytical approaches have been proposed to analyze trace levels of

PhCs in environmental water matrices; these always include a sample enrichment step prior to gas or liquid chromatography [START_REF] Arpin-Pont | Occurrence of PPCPs in the marine environment: a review[END_REF][START_REF]Council of 12 August 2013 amending Directives 2000/60/EC and 2008/105/EC as regards priority substances in the field of water policy[END_REF][START_REF]Commission Implementing Decision (EU) 2015/495 of 20 March 2015 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council, Off[END_REF][START_REF]Commission Implementing Decision (EU) 2018/840 of 5 June 2018 establishing a watch list of substances for Union-wide monitoring in the field of water[END_REF]. In the last decades, the development of miniaturized passive sorption-based techniques for sample enrichment has increased, covering e.g., solid phase microextraction (SPME), stir bar sorptive extraction (SBSE) [START_REF] Nogueira | Stir-bar sorptive extraction -15 years making sample preparation more environment friendly[END_REF][START_REF] Nogueira | Novel sorption-based methodologies for static microextraction analysis: A review on SBSE and related techniques[END_REF][START_REF] Wang | In situ hydrothermally grown TiO2@C core-shell nanowire coating for highly sensitive solid phase microextraction of polycyclic aromatic hydrocarbons[END_REF], and bar adsorptive microextraction (BAµE) as most representative for trace analysis of polar to nonpolar analytes in aqueous media [START_REF] Neng | Development of a bar adsorptive microextraction-large-volume injection-gas chromatography-mass spectrometric method for pharmaceuticals and personal care products in environmental water matrices[END_REF][START_REF] Almeida | Determination of trace levels of parabens in real matrices by bar adsorptive microextraction using selective sorbent phases[END_REF][START_REF] Oliveira | Characterization of Pharmaceuticals and Personal Care products in hospital effluent and waste water influent/effluent by direct-injection LC-MS-MS[END_REF]. BAµE is based on the floating sampling technology enrichment process, allowing for the possibility of selecting the most convenient sorbent coating upon the target PhCs involved [START_REF] Neng | Development of a bar adsorptive microextraction-large-volume injection-gas chromatography-mass spectrometric method for pharmaceuticals and personal care products in environmental water matrices[END_REF][START_REF] Ahmad | Bar adsorptive microextraction (BAμE) coated with mixed sorbent phases-Enhanced selectivity for the determination of non-steroidal anti-inflammatory drugs in real matrices in combination with capillary electrophoresis[END_REF][START_REF] Almeida | Determination of steroid sex hormones in real matrices by bar adsorptive microextraction (BAμE)[END_REF].

In this regard, and recognizing the relevance of activated carbons as

Experimental

Reagents

Glucose, G (> 99 %), fructose, F (> 99 %) and sucrose, S (> 99 %) were obtained from AnalaR NORMAPUR (Leuven, Belgium were purchased from Panreac (Barcelona, Spain). The acrylic acid (AA, ≥ 99 %) was purchased from Merck (Hohenbrunn, Germany). All chemicals were used without further purification. Ultra-pure water was obtained from the Milli-Q water purification systems (Merck Millipore, Darmstadt, Germany).

Carbon materials synthesis and characterization

The hydrochars were prepared by hydrothermal carbonization of carbohydrates: 15 cm 3 of 1.5 mol L -1 glucose or sucrose solutions introduced in an autoclave and heated at 190 ºC during 5 h [START_REF] Mestre | High performance microspherical activated carbons for methane storage and landfill gas or biogas upgrade[END_REF][START_REF] Mestre | Sustainable activated carbons prepared from a sucrose-derived hydrochar: Remarkable adsorbents for pharmaceutical compounds[END_REF]. These samples are labeled as HG or HS according with the carbohydrate precursor (glucose and sucrose, respectively). Modified sucrose-derived hydrochars were prepared by a similar protocol but adding acrylic acid (e.g., 7, 14 or 27 wt.%) to the starting sucrose aqueous solution [START_REF] Duarte | Styrene oxidation catalyzed by copper(II) C-scorpionates in homogenous medium and immobilized on sucrose derived hydrochars[END_REF][START_REF] Demir-Cakan | Carboxylate-rich carbonaceous materials via one-step hydrothermal carbonization of glucose in the presence of acrylic acid[END_REF]. The modified hydrochars were labeled as HSAAx, where x stands for the amount of acrylic acid. The sucrose-derived hydrochar was chemically activated as indicated elsewhere [START_REF] Mestre | High performance microspherical activated carbons for methane storage and landfill gas or biogas upgrade[END_REF][START_REF] Mestre | Sustainable activated carbons prepared from a sucrose-derived hydrochar: Remarkable adsorbents for pharmaceutical compounds[END_REF]. Briefly, superactivated carbons HS/H800 and HS/C800 were prepared by activation of An activated carbon prepared by steam activation of expanded corkboard granules at semi-industrial scale following the procedure reported in ref. [START_REF] Mestre | Activated carbons prepared from industrial pre-treated cork: Sustainable adsorbents for pharmaceutical compounds removal[END_REF] was also used (Cork/S800). Commercial powdered activated carbon (PAC) materials CN1 from Cabot-Norit (Com/CN1, supplied by Salmon & Cia. (Lisbon, Portugal)) and R from Riedel-de-Haën (Com/R, Seelze, Germany) were tested for comparison purposes. Further details on the synthesis and characterization of some of these carbons were previously reported (see cited references), and it is presented on table 1 for comparison purposes. a Synthesis and characterization reported in [START_REF] Mestre | Activated carbons prepared from industrial pre-treated cork: Sustainable adsorbents for pharmaceutical compounds removal[END_REF] b Synthesis and characterization reported in [START_REF] Mestre | High performance microspherical activated carbons for methane storage and landfill gas or biogas upgrade[END_REF][START_REF] Mestre | Sustainable activated carbons prepared from a sucrose-derived hydrochar: Remarkable adsorbents for pharmaceutical compounds[END_REF] c Synthesis reported in [START_REF] Mestre | Sustainable activated carbons prepared from a sucrose-derived hydrochar: Remarkable adsorbents for pharmaceutical compounds[END_REF][START_REF] Duarte | Styrene oxidation catalyzed by copper(II) C-scorpionates in homogenous medium and immobilized on sucrose derived hydrochars[END_REF] and characterization reported in [START_REF] Duarte | Styrene oxidation catalyzed by copper(II) C-scorpionates in homogenous medium and immobilized on sucrose derived hydrochars[END_REF] d Synthesis and characterization reported in [START_REF] Duarte | Styrene oxidation catalyzed by copper(II) C-scorpionates in homogenous medium and immobilized on sucrose derived hydrochars[END_REF]

A BET  1000 m 2 /g Micro+mesopores Similar V micro but V meso of Com/CN1

Characterization of the carbon materials

The textural properties of the porous hydrochars and commercial PACs were characterized by N 2 adsorption isotherms at -196 ºC in an automatic volumetric apparatus from Micromeritics (ASAP 2010). Before N 2 adsorption-11/40 desorption the samples (60 -100 mg) were outgassed at 120 ºC overnight (ca. 17 h) under vacuum (pressure < 10 -2 Pa). The apparent surface area, A BET , was estimated from the N 2 adsorption data in the p/p 0 range of the BET plot that assures: positive interception on the ordinate of the BET plot (C > 0) and n ads (1p/p 0 ) continuously increases with p/p 0 [START_REF] Rouquerol | Is the bet equation applicable to microporous adsorbents?[END_REF][START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF]. The total pore volume, V total , was quantified with the Gurvich rule [29], the micropore volume, V micro , was calculated with the α s method taking as reference the isotherm reported by Rodríguez-Reinoso et al. [30]. ACRESCENTAR ultra e super… The mesopore volume, V meso , corresponds to the difference V total -V micro .

The surface chemistry properties of the synthesized porous hydrochars and commercial PAC were assessed through the determination of the pH at the point of zero charge, pH PZC , according to the reverse mass titration method [START_REF] Noh | Estimation of the point of zero charge of simple oxides by mass titration[END_REF] using a Symphony SP70P pH meter. Diffuse reflectance infrared Fourier transform (DRIFT) spectra of the porous hydrochars were recorded in a Nicolet Magma-IR560 spectrometer provided with a high sensitivity mercury cadmium telluride detector (MCT-A) that operates at cryogenic temperature. Each spectrum was obtained using the powdered samples with no KBr addition and corresponds to the accumulation of 256 scans, recording with a spectral resolution of 2 cm -1 in the mid-IR (4000 -650 cm -1 ) spectral range. The thermal analysis (Setaram Labsys) was carried out on 15 mg of sample under a nitrogen flow rate of 50 cm 3 min -1 up to a final temperature of 900 ºC (heating rate of 15 ºC min -1 ). The ash content of the glucose-derived porous char was determined in the same equipment after exposure of the sample at 600 ºC in air (50 cm 3 min -1 ) for 3 h (constant mass).

12/40

The morphology of the porous hydrochars was evaluated by Scanning Electron Microscopy (SEM) performed at a Zeiss Supra 55 VP apparatus with 5 kV as accelerating voltage and using the powdered samples coated with iridium.

Standard preparation and water samples

Stock solutions of individual analytes (1,000.0 mg L -1 ) used for the working standard mixture were prepared in MeOH and stored at 4 ºC and renewed every month. Working standard mixtures of 1.0 mg L -1 were daily prepared in MeOH and used for spiking sample assays. For instrumental calibration, standard mixtures were prepared in MeOH by appropriate dilution from stock solutions.

The water samples were obtained in the metropolitan area of Lisbon (Portugal). The sea water samples were collected in the coastal area near Estoril and Costa da Caparica, the lake water from an artificial lake (Campo Grande) and the tap water samples from the public water supply system of Lisbon and Almada. The estuarine water samples were collected at Ponta do Mato, a Tagus river estuary beach. The wastewater treatment plant (WWTP) samples were obtained from Alcântara WWTP after primary decantation and filtration. All samples were collected in clean amber glass bottles and filtered with paper filters (125 mm of diameter, 10-13 µm of pore size, Cat No 1001 125, Whatman; Amersham, U.K.) and kept refrigerated at -20 ºC until being used.

BAµE-µLD methodology

The BAµE devices (7.5 mm in length and 3 mm in diameter) were in-house prepared as indicated in previous works [START_REF] Neng | Adsorptive micro-extraction techniques--novel analytical tools for trace levels of polar solutes in aqueous media[END_REF][START_REF] Almeida | Improvements on bar adsorptive microextraction (BAmuE) technique--application for the determination of insecticide repellents in environmental water matrices[END_REF]. After being produced, the 13/40 devices were stored at room temperature in closed glass flasks. The BAµE devices were cleaned with MeOH and ultra-pure water before use. Considering that microextraction bars are relatively inexpensive and easy to prepare, and aiming to avoid any possible contamination, they were discarded after each use.

The BAµE-μLD procedure was performed by placing a 25 mL aliquot of the water samples in a 25 mL sampling glass flask, followed by the introduction of a BAµE device, previously coated with npHS, and a conventional Teflon magnetic stirring bar. The assays were performed in a multipoint agitation plate (Variomag H+P Labortechnik Multipoint 15, Oberschleissheim, Germany) at room temperature using 1,000 rpm for 16 h (pH 2.0). After microextraction, the devices were removed from the samples with clean tweezers, dried for a few seconds using a lint-free tissue and placed into glass vial inserts containing 100 μL of ACN, ensuring their total immersion prior to ultrasonic treatment (Branson 3510, Zurich, Switzerland) at room temperature for 60 min. After μLD, the devices were removed, 100 μL of ultra-pure water was added, the vials vortexed for 10 s, and closed, followed by HPLC-DAD analysis. The standard addition method (SAM) was applied in real samples assays using four concentration levels ranging from 8.0 µg L -1 to 104.0 µg L -1 for all PhCs under study. Blank assays were also performed using the same procedure but without spiking ("zero-point" assays). The assays on real matrices were performed in triplicate.

Instrumental set-up

HPLC-DAD analysis were carried out on a benchtop Agilent 1100 series LC chromatographic system (Agilent Technologies, Waldbronn, Germany) equipped with a vacuum degasser (G1322A), autosampler (G1313A), thermostated column compartment (G1316A), quaternary pump (G1311A) and a diode array detector (G1315B). The data acquisition and system control were the flow rate was set at 0.6 mL min -1 . For identification purposes, standard addition was used, by spiking the samples with pure standards, as well as by comparing the relative retention time and peak purity with the UV/vis spectral reference data. For quantification purposes, calibration curves using the external standard methodology were performed. Instrument linearity was calculated by injecting ten standard solutions having concentration from 10.0 or 30.0 µg L -1 (depending on the compound) to 5,000.0 µg L -1 , where it was possible obtain determination coefficients (r 2 ) higher than 0.99 for the target compounds. For recovery calculations, peak areas obtained from each assay were compared with the peak areas of standard controls used for spiking. Peak areas were obtained by integration of each target compounds corresponding 15/40 peak using the mentioned software. The sensitivity of the instrumental system was checked through the limits of detection (LODs) and quantification (LOQs) calculated with signal-to-noise (S/N) of 3/1 and 10/1, where it ranged from 1.0 µg L -1 to 10.0 µg L -1 and between 5.0 µg L -1 to 27.0 µg L -1 for all the PhCs under study, respectively. The instrumental precision was evaluated by consecutively injecting a standard mixture (n = 6, 1.0 mg L -1 ), resulting in relative standard deviations (RSD) lower than 2.9 %.

Results and discussion

Carbon material selectivity as sorbent coating

To maximize the microextraction efficiency by the BAµE-µLD process, thirteen carbon materials were tested as enrichment phase for the recovery of PhCs using carbamazepine, 17α-ethinylestradiol, 17β-estradiol, diclofenac and triclosan as model compounds. These carbon materials were selected to cover a large range of textural features (from non-porous to highly porous samples with A BET up to 2400 m 2 /g) and surface properties (acidic, neutral or basic, with pH PZC ranging from 2 to 11), as it can be observed in tables 1 and 2. Additional information on the physicochemical properties of the studied carbons is summarized in the Supporting Information (topic S1). The evaluation assays were made in triplicate using ultra-pure water samples spiked at the 16.0 µg L -1 level.

As presented in figure 2a, the recovery efficiency of the carbon materials for the five selected PhCs is strongly dependent on both the type of material and the target pharmaceutical compound. From the set of activated carbons tested (samples Com/CN1, Com/R, Cork/S800, HS/C800 and HS/H800), the commercial sample Com/CN1 with a BET area higher than 1000 m 2 /g, a micro+mesopore structure and slightly acidic character (i.e., pH PZC of 5.1) outperformed the steam activated cork carbon (sample Cork/S800) with lower surface area and basic character, as well as the activated sucrose-derived hydrochars (samples HS/H800 and HS/C800) with a better-developed micropore structure and acidic nature. The non-porous hydrochars (figure 2b) failed to recover diclofenac and, while the samples HG and HS were able to recover ca. 40 % of the remaining PhCs, the acrylic acid modified hydrochars (HSAAx%) failed the recovery of 17α-ethinylestradiol and 17β-estradiol. On the contrary, the acidic nanoporous hydrochars (figure 2c) displaying micro-/mesopore networks and moderate surface areas (Tables 1 and2) allowed to recover the five target PhCs. Furthermore, the bar prepared using sample npHS as coating allowed to obtain recoveries comparable to the best commercial activated carbon for all the PhCs (ranging between 42-78%), with the exception of carbamazepine.

Sample A BET (m 2 g -1
)

V total a (cm 3 g -1
)

V meso b (cm 3 g -1 ) α s Method pH PZC V α total (cm 3 g -1
)

V α ultra (cm 3 g -1
)

V α super (cm 3 g -1

) 18/40 To gather a deeper understanding of the contribution of the adsorption and desorption processes in the overall recovery of the lowest performing materials, three of them were selected for a complementary assay. Samples HS/H800, HG and HSAA27% were tested for the recovery of the five PhCs using higher initial concentrations, thereby facilitating the quantification after the concentration step (figure S3, SI). In the case of sample HS/H800 (superactivated carbon), the low recoveries can be attributed to the irreversible adsorption of the targets inside the well-developed microporous structure; on the contrary, for the non-porous hydrochars, most of the molecules remain in the water matrix, thus the less efficient recovery of polar PhCs seems to be related with their low affinity towards the carbon coating phase. Under these experimental conditions, recoveries of 100% were obtained for triclosan -the most nonpolar target-with these non-porous hydrochars, whereas the recovery of carbamazepine -the most polar compound of the set-would be hindered by the irreversible retention on the acidic surface of such carbon coatings.

npHG
Interestingly, the relatively high recoveries of herein prepared nanoporous hydrochars characterized by lower surface areas than commercial activated carbons (tables 1 and 2) points out to the paramount importance of combining micro-mesoporosity and appropriate surface chemistry. The porous hydrochars are composed by agglomerates of interconnected units in the nanometric scale (20 -50 nm) that originate a pore system in the mesoscale (figure 3). This could be important to assure an efficient and fast adsorption and desorption of the target compounds during the enrichment step. Figure 4 illustrates the influence of the properties of the above mentioned thirteen carbon materials on their performance as enrichment phases. The four graphics present the correlation between selected carbon properties (pH PZC vs A BET , V total , % V micro and % V meso ) and the percentage of removal attained for the set of five PhCs (carbamazepine, 17α-ethinylestradiol, 17β-estradiol, diclofenac and triclosan). The number of occurrences corresponding to removals in the first quartile is systematically higher at low pH PZC values associated with percentages of micropores between 15 % and 50 % and consequently to the presence of a micro+mesopore network.

Summarising, these set of data points out that the selection of an adequate carbon coating for BAμE-μLD/HPLC-DAD to assure high recoveries should consider sorbents combining a well-developed pore structure in the full micro to mesopore range, along with acidic surface groups (i.e. phenol and carboxylic acid, see DRIFT spectra discussion in Supporting Information).

In light of these preliminary results for the nanoporous hydrochars with the set of five targets, these carbon materials were further tested as enrichment phase for the recovery of the 10 PhCs and benchmarked vs two commercial PACs (figure 5). As seen, under the experimental conditions tested (ca. pH 5.5) none of the carbon materials were able to recover clofibric acid, although Com/R presented the lowest performance for the majority of PhCs, with the exceptions were sulfamethoxazole and carbamazepine. Such low recoveries may be partially since this carbon was the only material displaying a slightly positively charged surface under our experimental conditions (i.e., pH solution < pH PZC 6.5). Despite a less developed pore network, the nanoporous hydrochars compare favourably with the commercial activated carbons, except in the case of sulfamethoxazole and carbamazepine. This might be attributed to their negatively charged surface charge (i.e., pH solution > pH PZC 2.7 -3.1). For ; Equilibrium: 16 h (1000 rpm) pH 5.5; μLD: ACN (100 µL) 60 min.

After choosing the best performing carbon materials through BAμE for the determination of the target PhCs from aqueous media, the µLD parameters, as well as the equilibrium time, stirring rate, ionic strength, matrix polarity, and sample pH were also evaluated. All the results the optimization, with exception of the latter which will be discussed in the following, can be consulted in SI.

The chemical characteristics of the water matrix were also assessed, in particular pH, ionic strength and polarity. Figure 6 shows the recovery yields with sample CN1, the solution pH strongly influenced the recovery of all the target analytes. This was somewhat expected since it is well known that this parameter affects the ionic or neutral forms of the PhCs in solution [START_REF] Neng | Development of a bar adsorptive microextraction-large-volume injection-gas chromatography-mass spectrometric method for pharmaceuticals and personal care products in environmental water matrices[END_REF], as well as the surface charge of the carbon materials. Under strong acidic pH, clofibric acid -analyte with the lowest pK a value-was recovered with yields between 70 to 90 % for all tested sorbent phases. This points out that the determination of clofibric acid by BAµE using carbon coatings as enrichment phases, is only possible when this semipolar acidic compound is in the protonated form. Our previous results using cork-derived activated carbons as enrichment materials also identified the critical role of pH in the quantification of clofibric acid, with the best results attained at acidic pH [START_REF] Neng | Cork-based activated carbons as supported adsorbent materials for trace level analysis of ibuprofen and clofibric acid in environmental and biological matrices[END_REF]. Table S1 (SI) showed that regardless the solution pH there is an overall increase in the recovery yields with the increase in the log D values. In general, at solution pH 2.0, the selected PhCs present higher octanol solubility (favoring both adsorption and desorption), thus contributing to higher recovery yields.

When the solution pH is higher than or similar to the pK a of a given PhCs, its recovery gets close to zero, justifying the greater reduction in the recovery yields in compounds with lower pK a values. It can so be concluded that the lower recoveries are related with the deprotonation of the target compounds, thus with the higher stability in the water matrix and consequently lower affinity towards the porous solids.

Validation of the BAμE(HC-S)-μLD/HPLC-DAD methodology

By using the optimized experimental conditions for each carbon-based sorbents (Com/CN1, npHF and npHS), it was possible to attain average recoveries between 20.3 and 90.9 % for all target compounds in ultra-pure water spiked at the 16.0 µg L -1 level. Comparatively, better results were obtained using npHS as carbon coating with most of the PhCs, thus the validation assays of the BAμE(npHS)-μLD/HPLC-DAD methodology were carried out using this sorbent phase (table S1, SI).

The sensitivity of the methodology was checked through the LODs (0.5 -1.5 µg L -1 ) and LOQs (1.5 -5.0 µg L -1 ), calculated with signal to noise ratios S/N of 3/1 and 10/1, respectively. The methodology was also evaluated through intraday and interday repeatability assays, calculating the corresponding relative standard deviations (RSD) [START_REF] Comtois-Marotte | Analysis of emerging contaminants in water and solid samples using high resolution mass spectrometry with a Q Exactive orbital ion trap and estrogenic activity with YES-assay[END_REF]. Interday repeatability assays were carried out as six replicates a day in three consecutive days and intraday repeatability assays consisted in six replicates performed in the same day (using three spiking levels). Good precisions were achieved for the intraday repeatability assays, with RSD ≤ 8.4 % (diclofenac), and for interday repeatability, was ≤ 12.3 % (mefenamic acid), under optimized experimental conditions. Assays were also performed in ultra-pure water having concentrations ranging from 5.0 to 104.0 µg L -1 , where convenient linearity was obtained, with r 2 higher than 0.99 for all the target PhCs under study. Table S2 (SI) summarizes the LODs, LOQs, as well as intraday and interday precision levels achieved by the proposed methodology, under optimized experimental conditions.

Performance comparison with other microextraction techniques

A comparison of the performance of the BAμE(npHS)-μLD/HPLC-DAD methodology and other established static-based microextraction techniques is shown in table 3 [START_REF] Balakrishnan | Determination of sulfonamide 39/40 antibiotics in wastewater: A comparison of solid phase microextraction and solid phase extraction methods[END_REF][START_REF] Salami | Microextraction in packed sorbent for analysis of sulfonamides in poultry litter wastewater samples by liquid chromatography and spectrophotometric detection[END_REF][START_REF] Gilart | Novel coatings for stir bar sorptive extraction to determine pharmaceuticals and personal care products in environmental waters by liquid chromatography and tandem mass spectrometry[END_REF][START_REF] Al-Hadithi | A solid bar microextraction method for the liquid chromatographic determination of trace diclofenac, ibuprofen and carbamazepine in river water[END_REF][START_REF] Duy | Using a novel sol-gel stir bar sorptive extraction method for the analysis of steroid hormones in water by laser diode thermal desorption/atmospheric chemical ionization tandem mass spectrometry[END_REF][START_REF] Antoniou | Analysis of Selected Pharmaceutical Compounds and Endocrine Disruptors in Municipal 40/40 Wastewater Using Solid-Phase Microextraction and Gas Chromatography[END_REF][START_REF] Pintado-Herrera | Environmentally friendly analysis of emerging contaminants by pressurized hot water extraction-stir bar sorptive extraction-derivatization and gas chromatography-mass spectrometry[END_REF][START_REF] Silva | Advances in stir bar sorptive extraction for the determination of acidic pharmaceuticals in environmental water matrices Comparison between polyurethane and polydimethylsiloxane polymeric phases[END_REF][START_REF] Giordano | Ibuprofen, carbamazepine and β-estradiol determination using thin-film microextraction and gas chromatography-mass spectrometry[END_REF]. As it can be observed, the developed methodology using BAμE devices coated with sucrose-based hydrochar presents similar or better recovery yields than other miniaturized enrichment techniques, e.g., SBSE coated with PDMS, PA or EG [START_REF] Gilart | Novel coatings for stir bar sorptive extraction to determine pharmaceuticals and personal care products in environmental waters by liquid chromatography and tandem mass spectrometry[END_REF] or SBME coated with C8 polymer [START_REF] Al-Hadithi | A solid bar microextraction method for the liquid chromatographic determination of trace diclofenac, ibuprofen and carbamazepine in river water[END_REF] for the extraction of carbamazepine; TFME coated with PDMS [START_REF] Giordano | Ibuprofen, carbamazepine and β-estradiol determination using thin-film microextraction and gas chromatography-mass spectrometry[END_REF] for the extraction of 17--estradiol; SBSE coated with PDMS [START_REF] Pintado-Herrera | Environmentally friendly analysis of emerging contaminants by pressurized hot water extraction-stir bar sorptive extraction-derivatization and gas chromatography-mass spectrometry[END_REF] for the extraction of clofibric acid; SBSE coated with PU [START_REF] Silva | Advances in stir bar sorptive extraction for the determination of acidic pharmaceuticals in environmental water matrices Comparison between polyurethane and polydimethylsiloxane polymeric phases[END_REF] for the extraction of diclofenac, gemfibrozil or mefenamic acid. Additionally, the BAμE(npHS)-μLD/HPLC-DAD methodology sensitivity is similar when compared to analogue instrumental systems [START_REF] Salami | Microextraction in packed sorbent for analysis of sulfonamides in poultry litter wastewater samples by liquid chromatography and spectrophotometric detection[END_REF][START_REF] Al-Hadithi | A solid bar microextraction method for the liquid chromatographic determination of trace diclofenac, ibuprofen and carbamazepine in river water[END_REF][START_REF] Silva | Advances in stir bar sorptive extraction for the determination of acidic pharmaceuticals in environmental water matrices Comparison between polyurethane and polydimethylsiloxane polymeric phases[END_REF] and/or some mass spectrometry or tandem mass spectrometry instrumental systems [START_REF] Duy | Using a novel sol-gel stir bar sorptive extraction method for the analysis of steroid hormones in water by laser diode thermal desorption/atmospheric chemical ionization tandem mass spectrometry[END_REF][START_REF] Giordano | Ibuprofen, carbamazepine and β-estradiol determination using thin-film microextraction and gas chromatography-mass spectrometry[END_REF]. On the other hand, instrumental apparatus that employ mass spectrometry or tandem mass spectrometry expectedly presented lower LODs than the proposed technique [START_REF] Gilart | Novel coatings for stir bar sorptive extraction to determine pharmaceuticals and personal care products in environmental waters by liquid chromatography and tandem mass spectrometry[END_REF][START_REF] Antoniou | Analysis of Selected Pharmaceutical Compounds and Endocrine Disruptors in Municipal 40/40 Wastewater Using Solid-Phase Microextraction and Gas Chromatography[END_REF][START_REF] Giordano | Ibuprofen, carbamazepine and β-estradiol determination using thin-film microextraction and gas chromatography-mass spectrometry[END_REF][START_REF] Pintado-Herrera | Environmentally friendly analysis of emerging contaminants by 41/40 pressurized hot water extraction-stir bar sorptive extraction-derivatization and gas chromatography-mass spectrometry[END_REF].

PPCP

Concluding remarks

Thirteen carbon sorbents prepared from different precursors and methodologiescommercial activated carbons, hydrochars and nanoporous hydrochars, and superactivated carbonswere tested as sorbent coatings for bar adsorptive microextraction followed by microliquid desorption and high performance liquid chromatography-diode array detection (BAµE(npHS)-µLD/HPLC-DAD methodology) for the simultaneous determination of traces of

  adsorbents of micropollutants, it is interesting to evaluate the potential of different types of carbon materials -e.g., carbohydrate-derived hydrochars and their activated counterparts-, as coating phases for a selective extraction of a wide range of PhCs. Hydrochars can be obtained by the hydrothermal carbonization of a carbohydrate precursor under mild synthesis conditions using water as solvent. As opposed to activated carbons prepared from conventional activation routes, hydrochars are essentially non-porous solids characterized by 6/40 displaying a surface chemistry rich in oxygen groups. The addition of eutectic salt mixtures during the hydrothermal carbonization allows to obtain nanoporous hydrochars with hierarchical porosity [20] and a rich amphoteric surface chemistry. The combination of these features will prove to play a crucial role in their high performance toward the quantification of PhCs with distinct physicochemical properties. This work aims to evaluate the performance of BAµE devices coated with various carbon materials to simultaneously monitor traces of pharmaceutical compounds (i.e. sulfamethoxazole, triclosan, carbamazepine, diclofenac, mefenamic acid, 17α-ethinylestradiol, 17β-estradiol, estrone, gemfibrozil and clofibric acid) in deionized water and in environmental water matrices. The BAµE was used as the first step in the quantification and identification of the PhCs, followed by microliquid desorption (μLD) and then high performance liquid chromatography with diode array detection (HPLC-DAD). The performance of the distinct carbon materials covering activated carbons, nanoporous carbohydrate-based hydrochars and superactivated hydrochars has been discussed in the light of their porous and chemical features. The validation and application of the optimized methodology (including the influence of polarity, back-extraction time and solvent, stirring rate) for real water matrices is fully discussed, as well as the comparison with other microextraction-based techniques.

Figure 1 -

 1 Figure 1 -Chemical structures, pK a and log K O/W values of the analysed PhCs [21].

  HS/H800 V micro is almost the double of HS/C800 pH PZC  4

  performed by the software LC3D ChemStation (version Rev.A.10.02[1757], Agilent Technologies). Analyses were performed on a Kinetex hexyl phenyl column, 150.0 × 4.6 mm, 2.6 µm particle size (Phenomenex, Torrence, U.S.A.). The samples were analysed using a gradient mobile phase consisting of 2.5 % acetic acid/ultra-pure water (v/v, solvent A) and ACN (solvent B). The employed elution gradient was as follows: 0 min -80/20 (solvent A/B); 24 min -20/80 (solvent A/B); 27 min -20/80 (solvent A/B); 30 min -80/20 (solvent A/B); 35 min -80/20 (solvent A/B). All solvents were previously filtered (125 mm in diameter, 10-13 µm in pore size, 1001 125, Whatman, U.K.) to remove suspended particles, if any. The detector was set at 280 nm and the column temperature at 20 ºC. The injection volume was 40 µL with a draw speed of 200 µL min -1 and

Figure 2 - 1 ;

 21 Figure 2 -Effect of sorbent selectivity on the recovery yields of the five mentioned PhCs using commercial and lab-made activated carbons (a), lab-made hydrochars (b) and lab-made nanoporous hydrochars (c) as sorbent coating obtained by BAμE-μLD/HPLC-DAD. BAµE coating: 1.0-3.0 mg; Spiking level: 16.0 µg L -1 ; Equilibrium: 16 h (1000 rpm) pH 5.5; μLD: ACN (100 µL) 60 min.

Figure 3 -

 3 Figure 3 -SEM images of the nanoporous hydrochars.

Figure 4 -

 4 Figure 4 -Influence of carbon materials properties in the removal percentages of five target PhCs: pH at the point of zero charge (pH PZC ) versus BET area (a), total pore volume (b), percentage of micropore volume (c), and percentage of mesopore volume (d). Circles represent the percentage of removal (in quartiles) adjusted for the removal range of each PhC, Q1 corresponding to top 25 % removal and Q4 to bottom 25 % removal, and circle sizes are directly proportional to the percentage of occurrence.

  , the recoveries ranged between 40 %(17-β-estradiol) and 80 % (17-α-ethinyl estradiol and estrone), with the nanoporous hydrocarbons prepared from sucrose and fructose competing closely with the best performing commercial carbon (sample Com/CN1) for the majority of the target compounds. Considering all this, further optimization assays were carried out on microextraction bars prepared using samples npHS, npHF and Com/CN1as carbon coatings.

Figure 5 -

 5 Figure 5 -Effect of sorbent selectivity on the PhCs recovery yields with different carbon based materials as sorbent coating obtained by BAμE-μLD/HPLC-DAD. BAµE coating: 1.0-3.0 mg; Spiking level: 16.0 µg L -1

  BAμE devices at different solution pH between 2 and 10 for the three selected carbon coatings. With the exception of carbamazepine recovery

Figure 6 - 1 ;

 61 Figure 6 -Effect of matrix pH on the PhCs recovery yields using npHS (a), npHF (b) and

Figure 7 -

 7 Figure 7 -Chromatograms obtained from assays performed on an ultrapure water sample spiked at the 16.0 µg L -1 level (a) and a non-spiked WWTP sample (b), performed and by BAµE(npHS)-µLD/HPLC-DAD, under optimized experimental conditions.

Table 1 -

 1 Summary of the main textural and surface properties of the carbon materials used as coatings (apparent surface area -A BET , micropore volume -V micro (pores with widths < 2 nm), mesopore volume -V meso (pores with widths between 2 and 50 nm), pH at the point of zero charge -pH PZC ).

	Type	Carbon material	Textural properties	Surface properties
	Activated	Commercial
	carbons	(Com/CN1 and
		Com/R)

Table 2 -

 2 Textural properties of the porous hydrochars and commercial activated carbons, and pH PZC .

	a Evaluated at p/p 0 = 0.975 in the N 2 adsorption isotherms at -196 ºC

b Difference between V total and V α total

PhCs in aqueous samples.

Acknowledgments

The authors wish to thank Fundação para a Ciência e a Tecnologia (FCT, Portugal) for funding Centro de Química e Bioquímica (UID/MULTI/00612/2019) and Centro de Química Estrutural (Pluriannual funding 2020-2023). SMA thanks FCT for the PhD grant (SFRH/BD/107892/2015), ASM acknowledges FCT for the financial support to the EMBRACE project (CEECIND/01371/2017) and NRN acknowledges FCT for the financial support through the contract established from DL 57/2016. The authors also wish to thank Salmon & Cia (Portugal) for offering the commercial Norit carbon material.

Application to environmental water matrices

To evaluate the applicability of BAμE(npHS)-μLD/HPLC-DAD methodology to real matrices, assays were performed in sea, lake, estuarine, tap and WWTP water samples, through the SAM. This approach is usually the best strategy for quantification purposes to determine the levels of analytes under study and to reduce possible matrix interferences in real samples [START_REF] Neng | Development of a bar adsorptive microextraction-large-volume injection-gas chromatography-mass spectrometric method for pharmaceuticals and personal care products in environmental water matrices[END_REF][START_REF] Almeida | Determination of trace levels of parabens in real matrices by bar adsorptive microextraction using selective sorbent phases[END_REF][START_REF] Ahmad | Application of bar adsorptive microextraction (BAμE) for anti-doping control screening of anabolic steroids in urine matrices[END_REF]. In a first step, the water matrices were spiked with four working standards to produce the corresponding spiking levels (8.0 µg L -1 to 104.0 µg L -1 ) for the ten PhCs under study. "Zero-point" assays were also performed without spiking to ensure maximum control of the analytical methodology. Good linear correlations were achieved, with r 2 higher than 0.99. The chromatograms of neat standard mix at the 8.0 μg L -1 level for the ten PhCs (figure 7a) and WWTP sample without spiking obtained by BAμE(npHS)-μLD/HPLC-DAD, under optimized experimental conditions (figure 7b), exemplify the results obtained proving high sensitivity at the trace level. The proposed methodology allowed to quantify two of the analytes under study in the WWTP sample (carbamazepine: 4.01 ± 0.46 μg L -1 ; diclofenac: 1.99 ± 0.21 μg L -1 ).

ten PhCs from distinct therapeutic classes in synthetic and environmental water matrices.

The lab-made nanoporous hydrochars proved to compete with commercial activated carbon adsorbents for the enrichment of the PhCs, due to a hierarchical pore structure in the full micro-mesopore range combined with a rich surface chemistry composed of acid O-groups that favour both the adsorption and desorption of the target compounds, thus contributing to a high performance at the trace level. The method also demonstrated to be an environmentally friendly approach, easy to implement, sensitive, robust and requiring low sample volume. In short, the proposed analytical methodology seems to be a very effective microextraction-based alternative to fulfill the EU Decision 2015/495, as well as the USEPA guidelines for PhCs monitoring, particularly if combined with tandem mass spectrometry systems, which may provide even better detection limits.

Conflicts of Interest

The authors declare no conflict of interest.


Olivier Mascaro
email: olivier.mascaro@gmail.com

Marie Aguirre

Mélanie Brun

Auriane Couderc

Hugo Mercier

Pauline Le Floch

Aurélie Giraud

Nonverbal Rhetoric: Two-to Four-year-old Children Select Relevant Evidence When Trying to Influence Others

Keywords:

to all the families and toddlers that participated in

Nonverbal Rhetoric: Two-to Four-year-old Children Select Relevant Evidence When Trying to Influence Others Psychologists have long noted how early some reason-giving and reason-evaluating capacities develop and the crucial cognitive role that these abilities play in development.

Piaget claimed that "logical reasoning is an argument which we have with ourselves, and which reproduces internally the features of a real argument" (Piaget, 1928, p. 204; see also [START_REF] Doise | The social development of the intellect[END_REF][START_REF] Perret-Clermont | Social Interaction and Cognitive Development in Children[END_REF][START_REF] Vygotsky | Mind in society: The development of higher psychological processes[END_REF]. More recent theories have stressed the evolutionary importance of argumentative and, more generally, reason-giving and reason-evaluating skills, suggesting that these skills should develop early [START_REF] Mercier | Why do humans reason? Arguments for an argumentative theory[END_REF], 2017;[START_REF] Tomasello | A natural history of human thinking[END_REF]. Yet, to this date, we do not know how early reasongiving develops. To address this developmental issue, we test the existence and nature of reason-giving during toddlerhood, using a non-verbal paradigm.

Recent experiments suggest that preschoolers' reason-giving already shows two signatures of argumentative skills: (i) a capacity to recognize when producing reasons is needed and (ii) some ability to select appropriate reasons. In one experiment, separate pairs of 3-and 5-year-olds played a game together [START_REF] Köymen | Reasoning during joint decisionmaking by preschool peers[END_REF]. In this game, some moves were more conventional than others. Both age groups were more likely to provide reasons when making unconventional than conventional moves. This finding shows that preschoolers are already attuned to whether a situation warrants providing reasons. Another experiment suggested that preschoolers possess some capacity to select reasons that are effective and convince others. In [START_REF] Köymen | Preschoolers use common ground in their justificatory reasoning with peers[END_REF], 3-and 5-year-olds were able to adjust their reason-giving to the state of knowledge of their interlocutor. Children were provided with a good reason to perform an unconventional action, either as pairs or individually, and they were then asked to perform the unconventional action, always as pairs. When the children were paired with another child who had heard the reason at the same time as them, they were less likely to use this reason in their interaction, compared to when they were paired with another child who had not heard the reason at the same time as them. Preschoolers could thus use common knowledge between themselves and their interlocutor, or lack thereof, to adjust their reason-giving strategies (see also [START_REF] Mammen | The reasons young children give to peers when explaining their judgments of moral and conventional rules[END_REF].

In sum, preschoolers are sensitive to whether reasons are needed and they provide their interlocutors with relevant reasons when attempting to influence them. The goal of this article is to investigate whether younger children's nonverbal communicative behaviors already show these two signatures of reason-giving skills. As we argue presently, many of the basic skills required to produce reasons are likely to develop early during infancy and toddlerhood.

Recognizing When Reasons Are Needed

Reason-giving is most appropriate when we believe our audience has ignored, doubted, or disagreed with what we have communicated-or when we deem our audience likely to do so. Two-year-olds already have some sensitivity to such disagreements. For instance, they contradict adults who name objects incorrectly, often by using negation [START_REF] Choi | The semantic development of negation: a cross-linguistic longitudinal study[END_REF][START_REF] Hummer | On the origins of denial negation[END_REF][START_REF] Kim | Development of the concept of truth-functional negation[END_REF][START_REF] Koenig | Infants' understanding of false labeling events: The referential roles of words and the speakers who use them[END_REF][START_REF] Pea | Origins of verbal logic: Spontaneous denials by two-and three-year olds[END_REF][START_REF] Rakoczy | Done wrong or said wrong? Young children understand the normative directions of fit of different speech acts[END_REF]. Toddlers and preschoolers also react to third-party assessments of propositions. They prefer to learn what an informant communicates if other people assent rather than dissent to this testimony [START_REF] Fusaro | Children assess informant reliability using bystanders' non-verbal cues[END_REF][START_REF] Lee | Little liars: Development of verbal deception in children[END_REF]. Moreover, toddlers and children recognize that someone might explicitly deny the truth of a proposition. This ability is most evident in their capacity to process truth-functional negations [START_REF] Austin | Young children's understanding of denial[END_REF][START_REF] Feiman | What do you mean, no? Toddlers' comprehension of logical "no" and "not[END_REF][START_REF] Hill | Inferential reasoning by exclusion in children (Homo sapiens)[END_REF][START_REF] Mascaro | Epistemology for beginners: two-to five-year-old children's representation of falsity[END_REF][START_REF] Mascaro | The moral, epistemic, and mindreading components of children's vigilance towards deception[END_REF]. In short, a sensitivity to explicit disagreements about the truth of a proposition is already present during toddlerhood. As a result, explicit markers of disagreement such as denials are likely to help children identify when they need reasons to influence others, at least from toddlerhood onwards.

Selecting Efficient Means of Influencing Others

To influence others, one needs more than recognize when they disagree (or might disagree) with one's claims. One also needs to select behaviors that can efficiently influence others, an ability that is likely to improve with the development of representations of mental states (also known as mentalizing or theory of mind, [START_REF] Premack | Does the chimpanzee have a theory of mind?[END_REF]. Indeed, children's use of others' mental states when making influence attempts improves throughout childhood when practicing deception [START_REF] Lee | Little liars: Development of verbal deception in children[END_REF][START_REF] Mascaro | Gullible's travel: How honest and trustful children become vigilant communicators[END_REF][START_REF] Mascaro | Optimistic expectations about communication explain children's difficulties in hiding, lying, and mistrusting liars[END_REF], and honest persuasion [START_REF] Bartsch | Children's use of mental state information in selecting persuasive arguments[END_REF][START_REF] Bartsch | Children's attention to others' beliefs during persuasion: Improvised and selected arguments to puppets and people[END_REF]. Furthermore, the amount and quality of children's attempts in persuasion tasks correlates positively with their capacity to predict explicitly agents' behavior on the basis of their beliefs (Bartsch, London & Campbell, 2007, Study 2;[START_REF] Kołodziejczyk | Children's understandings of characters' beliefs in persuasive arguments: links with gender and theory of mind[END_REF][START_REF] Lonigro | Theory of mind, affective empathy, and persuasive strategies in school-aged children[END_REF][START_REF] Peterson | Nimble negotiators: How theory of mind (ToM) interconnects with persuasion skills in children with and without ToM delay[END_REF][START_REF] Slaughter | I can talk you into it: Theory of mind and persuasion behavior in young children[END_REF]. In short, available evidence suggests that the development of mentalizing abilities constrains the set of persuasion strategies that are available to children. This hypothesis makes a straightforward developmental prediction: the first reason-giving behaviors to emerge are likely to be those that involve precociously developing mentalizing abilities.

One of the most primary forms of mentalizing abilities is the capacity to recognize informational access (e.g., [START_REF] Martin | What cognitive representations support primate theory of mind?[END_REF][START_REF] Wellman | The child's theory of mind[END_REF]. Infants and toddlers have some sensitivity to the factors that influence what others know. They recognize that communicative actions can inform others [START_REF] Liszkowski | 12-and 18-month-olds point to provide information for others[END_REF][START_REF] Martin | Understanding the abstract role of speech in communication at 12months[END_REF][START_REF] Song | Can an agent's false belief be corrected by an appropriate communication? Psychological reasoning in 18-monthold infants[END_REF][START_REF] Vouloumanos | Do 6-month-olds understand that speech can communicate[END_REF][START_REF] Vouloumanos | Twelve-month-old infants recognize that speech can communicate unobservable intentions[END_REF]; for a review see [START_REF] Harris | Infants understand how testimony works[END_REF]. Furthermore, infants and toddlers recognize that visual perception provides people with information about their surroundings [START_REF] Luo | Do 12.5-month-old infants consider what objects others can see when interpreting their actions?[END_REF][START_REF] Luo | Recognizing the role of perception in action at 6 months[END_REF][START_REF] Moll | Level 1 perspective-taking at 24 months of age[END_REF][START_REF] Sodian | Now I see it but you don't: 14-month-olds can represent another person's visual perspective[END_REF][START_REF] Southgate | Action anticipation through attribution of false belief by 2-year-olds[END_REF]. For example, in [START_REF] Moll | Level 1 perspective-taking at 24 months of age[END_REF], toddlers interacted with an adult searching for an object. One candidate object was hidden from the adult but was visible to the participant. The other candidate object was visible to both the participant and the adult.

When asked to help the adult find the object that she was looking for, 24-month-old toddlers gave the adult the object that was hidden from her. These results suggest that toddlers have some sensitivity to what other people can see. Thus, infants and toddlers recognize some of the conditions enabling informational access in others. This capacity is likely to support the development of the first reason-giving behaviors geared towards influencing others.

In sum, two basic capacities required to produce reasons efficiently are present well before children can recruit them in a verbal context: a sensitivity to disagreements and expectations about the kind of experiences (such as seeing) that can inform others. Here, we investigate whether these two abilities support the early development of reason-giving in toddlers. No experimental study has specifically tested reason-giving at such an early age, in part because previous studies relied on measures of verbal production. To be suitable for very young children, our study relies on nonverbal behaviors, a possible precursor of verbal reason-giving.

Operationalization Principle

In our study, we tested whether toddlers adjust their nonverbal behaviors to the context when trying to provide support for their claims. We manipulated whether the exact same action (rotating a box) was relevant by varying the presence of explicit disagreement.

An experimenter asked toddlers to indicate the location of a box containing a toy. Participants could provide their answer by pointing at the correct box or by grasping it. They could also manipulate the position of the boxes by rotating them. In the Window condition, a box containing a toy was opaque except for a transparent window that faced the participants but was hidden from the experimenter. Thus, participants could provide additional evidence about the toy's location by rotating the box and making the toy visible to the experimenter through the transparent window. The Fully Transparent condition used the same procedure as the Window condition except that the box containing the toy (as well as the other boxes) was entirely transparent, making the toy visible to the experimenter at all times. Thus, in this condition, participants could not provide additional evidence about the location of the toy by rotating the box.

During the baseline phase, there was no reason for participants to assume that the experimenter disagreed with them. In contrast, during the test phase, the experimenter contradicted participants after they indicated where the toy was located. We hypothesized that during the test, the participants would rotate the box more often when this action was relevant to provide evidence for their claims (i.e., in the Window condition). We chose to test 2-to 4-year-old participants because we wanted to (i) assess reason-giving at the earliest age possible, and (ii) evaluate possible developmental effects. Since we used denials in our experimental setup, we did not test one-year-olds because, at that age, the understanding of truth-functional negation is not well consolidated [START_REF] Austin | Young children's understanding of denial[END_REF][START_REF] Feiman | What do you mean, no? Toddlers' comprehension of logical "no" and "not[END_REF].

Material and Methods

Participants

Two groups of 2-to 4-year-old children participated in this study (Window condition: n = 35, M age = 35.51 months, range = 23-48 months, 14 girls; Fully Transparent condition: n = 36, M age = 35.67 months days, range = 23-48 months, 13 girls). Eleven additional participants were excluded (M age = 31.27 months, range = 24-41 months, 6 girls) because they did not understand the task, i.e., they did not answer correctly by pointing towards or reaching for the location of the dog in at least three out of the four baseline trials (5), because of technical failure (1), or because participants stopped interacting with the experimenter after being contradicted (5). Our rejection rate (11 participants out of 82, 13%) is similar to what has been observed in comparable studies (e.g., [START_REF] Moll | Level 1 perspective-taking at 24 months of age[END_REF][START_REF] Moll | How does it look? Level 2 perspective-taking at 36 months of age[END_REF]. However, it should be noted that our sample is representative only of the children kept in the analysis, and who were thus able to understand the task and willing to engage with the experimenter. The mean age of the excluded participants tended to be lower than the mean age of the participants in the Window condition (t(44) = 1.78, p = .082, d = .69) and in the Fully Transparent condition (t(45) = 1.78, p =.071, d = .72), presumably because the youngest of our participants were more likely to fail to understand the task.

The research reported in this manuscript followed the guidelines of the Declaration of Helsinki and it was approved by the local board of daycare centers involved in the project.

The written informed consent of participants' caregivers was obtained prior to the study.

Institutional Review Board approval from ethics committees for bio-medical research was not sought because this project involves purely behavioral measures, and it falls out of the scope of biomedical research (Title: Non-verbal rhetoric; Institution: Institute for Cognitive Sciences Marc Jeannerod). The participants were recruited from urban daycare centers and preschools in a large French city. They came from mixed socioeconomic backgrounds and were native French speakers. They were randomly assigned to one of the two conditions (Window vs. Fully Transparent).

We aimed at achieving the largest possible sample size, given our recruitment opportunities. Therefore, we tested all the available participants within our target age range in the schools that we contacted. To assess the resulting power to detect a difference between participants' number of rotations of the box across conditions (Window vs. Fully Transparent) with a Fisher Exact test, we ran a compromise power analysis with G-Power 3.1 [START_REF] Faul | G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences[END_REF]. This analysis postulated a low probability to rotate the box during the trials of the Fully Transparent condition (p r = .1) and a large effect size (α = .05, β = .05, OR = 6.71, Chen, Cohen & Chen, 2010). It revealed that our sample size yielded an implied power equal to .90.

Materials and Procedure

Participants were tested in a quiet room at their daycare center. The main experimenter was aware of the study's hypotheses (as in many comparable experiments, e.g., [START_REF] Moll | Level 1 perspective-taking at 24 months of age[END_REF][START_REF] Warneken | Altruistic helping in human infants and young chimpanzees[END_REF]. A daycare employee familiar to the participants, and unaware of the hypotheses of the study, acted as a confederate and was present in the room during the entire experiment. Both the experimenter and the confederate were instructed not to influence participants' choices during the warm-up and test phases. One camera (temporal resolution = 25 frames/second) recorded participants' behaviors. Toddlers sat in front of the experimenter, across a child-size table. The experimenter said: "Look, I brought little boxes to play with." She placed four plastic boxes (8 x 6 x 5 cm) on a rectangular cardboard tray (60 x 27 cm) positioned in the center of the table, approximately 30 cm from the participants. Three of the boxes were empty; a small plastic dog was glued inside the fourth box. Boxes were closed with lids, and they were locked so that participants could not open them easily.

Window condition

In the Window condition, each box was entirely covered with opaque white paint except for a transparent window (4.5 x 4.5 cm) located on its front side. The experimenter placed the boxes so that their opaque sides faced her, while the transparent windows faced the toddlers. Participants could thus see the boxes' contents-including where the dog wasat all times. In contrast, it was not possible to see the boxes' content from the experimenter's point of view.

The experimenter explained: "Let's play a game. You have to find the little dog."

Then, during each baseline trial, the experimenter asked the prompt question: "Where is the dog?" We evaluated the participants' answer to the prompt question in the same way in the baseline, pre-test, and test trials. In all cases, participants could answer by pointing at or reaching for one of the boxes. For each trial, we considered that the participants selected accurately the box containing the toy when they pointed at it, reached for it, or rotated it.

When a child's answer to the prompt question was ambiguous or directed towards more than one box, the experimenter repeated the prompt question until the participant selected only one box. When participants pointed at or reached for an empty box in response to the prompt question, we assumed that they failed to select the correct box. During the baseline trials, when the participant failed to indicate the correct box, the experimenter pointed at the baited box and said: "Here is the dog."

In the baseline, pre-test and test trials, we also recorded whether participants answered the prompt question by rotating the box containing the dog so that its front part (i.e., the transparent window in the Window condition) faced the experimenter. To do so, participants had to rotate the box clockwise or counterclockwise around its vertical axis more than 90° from its position at the beginning of the experiment (thus making it possible for the experimenter to see the dog from her viewpoint in the Window condition). In a given trial, a participant was allowed to both point at and rotate the same box (no participant pointed at one box and rotated another one).

Once the child had pointed at or reached for the box containing the plastic dog, the experimenter proceeded to the next baseline trial. At the end of each baseline trial, the experimenter swapped the location of the box containing the dog with the location of one of the empty boxes. There were four baseline trials. For each trial, the experimenter placed the box containing the dog in a different position, using the following fixed order: right end of the row (trial 1), third position from the right end of the row (trial 2), left end of the row (trial 3), and second position from the right end of the row (trial 4). The left and right sides referenced here are from the participants' viewpoint. The experimenter displaced the box containing the dog prior to each of the baseline trials.

After the fourth baseline trial, the experimenter placed the box containing the dog in the second position from the right end of the row of boxes. She reached for her cell-phone in her pocket while saying "Oh, I have a phone call, I'll be right back" before leaving the room.

While she was away, the confederate took the box containing the dog and placed it at the left end of the row. After staying outside the room for approximately 15 seconds, the experimenter came back in the testing room. She sat across the table in front of the participant and said: "Let's continue the game!" and the pre-test trial began. The experimenter asked: "Where is the dog?" Once participants had pointed at, reached for, or rotated one of the boxes, the experiment proceeded to the test phase. The first test trial started by the experimenter contradicting participants by saying: "No… the dog is not there…" before asking the prompt question again: "Where is the dog?" Once the participant had pointed at, reached for, or rotated one of the boxes, the experimenter proceeded to the next test trial. Unlike in the baseline trials, during the pre-test and test trials, the experimenter did not indicate the real location of the dog to the participants when they failed to select the correct box. Each test trial started by the experimenter using a specific sentence to contradict the participants. During the second trial she contradicted participants by saying: "Is the dog there? No, I don't think so," and during the third trial, she contradicted participants by saying: "Well, it's not there!" During the test trials, the boxes were not manipulated by the experimenter or by the confederate. The experimenter simply contradicted the participant three times (once per test trial), each time after the child answered the question about the location of the dog ("Where is the dog?"). We used only three test trials because we were worried that our participants might stop responding after being contradicted too many times.

The fact that the boxes could be rotated was never demonstrated to the participants.

Fully Transparent condition

The Fully Transparent condition followed the same procedure as the Window condition, except that the plastic boxes used in the game were entirely transparent. Thus, both the participant and the experimenter could see the location of the plastic dog at all times.

Coding and analysis

For each baseline and test trial, we coded whether participants accurately indicated the location of the dog, by pointing at or by reaching for the baited box. For each participant, we computed the proportion of trials in which they correctly indicated the location of the dog, resulting in three accuracy ratios ranging from 0 to 1, one for the baseline trials, one for the pre-test trial, and one for the test trials. We also assessed whether participants rotated the box containing the dog during each baseline trial, during the pre-test trial, and during each test trial. Each participant scored 1 for each trial in which they rotated the box, for a total number of potential rotations per participant ranging from 0 to 4 during baseline trials and from 0 to 3 during test trials.

Data were coded offline. A primary coder aware of the study's hypotheses coded the entire dataset, and a secondary coder unaware of the study's hypotheses double-coded a subset of the data (n = 26). The inter-rater agreement was high for all our measures (average ρ = .93, range = .87-.99; agreement on whether the participants rotated the box or not during the test trials: κ = 1). We analyzed the data from the primary coder. All the statistical analyses reported in this paper are two-tailed. We performed Mann-Whitney U tests by compiling the null distribution of the rank-sum statistic using exact permutations [START_REF] Bergmann | Different outcomes of the Wilcoxon-Mann-Whitney test from different statistics packages[END_REF]. Statistics were computed using R 3.4.3 (R Core Team, 2018), with the following packages: lme4 (v1.1-19; Bates, Maechler, Bolker, & Walker, 2015), exactRankTests (v0.8-29;Hothorn & Hornik, 2017), and coin (v1.2-2, Hothorn, Hornik, van de Wiel & Zeileis, 2008).

Results

Preliminary analyzes revealed that Condition (Window vs. Fully Transparent) had no effect on participants' accuracy ratios during baseline trials (M = .96, Mdn = 1, SD = .09 vs. M = .96, Mdn = 1, SD = .09, U = 1275, Z = .27, r = .03, p = 1, Mann-Whitney U test), pre-test trials (M = 1, Mdn = 1, SD = 0 vs. M = 1, Mdn = 1, SD = 0) or during test trials (M = .89, Mdn = 1, SD = .18 vs. M = .88, Mdn = 1, SD = .17, U = 1283, Z = .33, r = .04, p = .76, Mann-Whitney U test). In short, there was no detectable difference in participants' ability to locate the dog across conditions. The participants' accuracy ratios were significantly lower during the test trials than during the baseline trials in both conditions (Window condition: W + = 105, W -= 15, Z = -2.56, r = .31, p = .01; Fully Transparent condition: W + = 118, W -= 18, Z = -2.59, r = .31, p = .01, Wilcoxon tests for matched pairs). Similarly, the participants' accuracy ratios were significantly lower during the test trials than during the pre-test trial in both conditions (Window condition: W + = 0, W -= 55, Z = -2.80, r = .33, p = .005; Fully Transparent condition: W + = 0, W -= 78, Z = -3.06, r = .36, p = .002, Wilcoxon tests for matched pairs). These results reveal that the participants reacted to being contradicted by the experimenter, and that they were sensitive to the negative evaluation of their answer by the experimenter.

Furthermore, Condition (Window vs. Fully Transparent) had no effect on participants' tendencies to rotate the boxes during the baseline trials (4 participants out of 35 rotated the box at least once in the Window condition, 1 participant out of 36 did so in the Fully Transparent condition, OR = 4.52, p = .199, Fisher Exact test) or during the pre-test trial (3 participants out of 35 rotated the box at least once in the Window condition, and 3 participants out of 36 did so in the Fully Transparent condition, OR = .69, p = 1, Fisher Exact test). Thus, when participants were not contradicted by the experimenter, their tendency to rotate the boxes was comparable-and very low-across conditions. Detailed descriptive statistics for the number of rotations per condition during the baseline, pre-test and test phase are reported in Table 1.

Descriptive Statistics for the Number of Rotations

Window condition

Fully Next, we analyzed whether participants rotated the box containing the dog during each trial of the test phase as a binary outcome, using Generalized Linear Mixed Models (GLMMs) with a binomial distribution (logit link). To explicitly take into account repeated measures, all mixed models included participant identity as a random intercept. We fit two different kinds of models: (1) the null models, featuring participant identity as the only predictor, and (2) the hypothesis-driven models, which included Condition as an additional factor, We used likelihood ratio tests (LRTs) to compare the overall fit of each of these models. For the estimates of the model parameters, the 95% confidence intervals were assessed by computing a likelihood profile and finding the appropriate cutoffs based on the LRT.

We built the hypothesis-driven model by including participant identity as a random We also performed additional analyses to assess the effect of a set of exploratory variables on the participants' tendency to rotate the box during the test phase, focusing on the effects of Gender, Number of rotations during the baseline phase, Number of rotations during the pre-test phase, Age, and interaction between Age and Condition (see the Supplemental Materials). These analyses revealed only an effect of the number of rotations during the baseline phase, indicating that the participants that rotated the target box more during the baseline phase also rotated it more during the test phase. These analyses also confirmed that the effect of Condition (Window vs. Fully Transparent) remained significant, even after taking into account the effect of all the exploratory variables.

Discussion

Between the ages of 2 and 3-or even earlier-toddlers have been shown to pay attention to disagreements [START_REF] Hummer | On the origins of denial negation[END_REF][START_REF] Koenig | Infants' understanding of false labeling events: The referential roles of words and the speakers who use them[END_REF], and they are sensitive to the kind of input-such as visual perception-that can provide others with information [START_REF] Moll | Level 1 perspective-taking at 24 months of age[END_REF][START_REF] Nurmsoo | Preschoolers' perspective taking in word learning: Do they blindly follow eye gaze?[END_REF]. The present experiment assessed whether young children could select relevant evidence when attempting to influence others. support their claims about the toy's location. Thus, a substantial subset of the participants established on their own the relevance of rotating the box as a way of supporting their claims.

Our study was not designed to assess capacities for reason-giving at the individual level. Therefore, it is difficult to make strong claims about the participants that did not rotate the box in the Windows condition. They might have either (i) not realized that they could rotate the box, or (ii) might have not dared to do it, or (iii) might have not realized that rotating the box was an appropriate way of providing support for their claims. Our data do not allow us to disentangle between these possibilities. Crucially, the fact that the participants' tendency to rotate the box was significantly higher in the Window condition than in the Fully Transparent condition indicates that by 2 years of age, at least a large subset of children is capable of selecting relevant evidence when providing support for a conclusion.

Three important features may explain why young children succeeded in engaging in reason-giving in our study. First, our participants had to provide support for something true.

By contrast, the capacity to persuade someone of something false appears to develop later, during the preschool years (e.g., [START_REF] Lee | Little liars: Development of verbal deception in children[END_REF][START_REF] Mascaro | Optimistic expectations about communication explain children's difficulties in hiding, lying, and mistrusting liars[END_REF][START_REF] Russell | The 'windows task'as a measure of strategic deception in preschoolers and autistic subjects[END_REF]. Thus, the fact that in our experiment, children were asked to give reasons in a benevolent and truthful manner might have contributed to their success.

Second, in our study non-verbal reason-giving consisted in making an object visible to support claims about its location. The informative value of direct visual perception is recognized early by infants and toddlers (e.g. [START_REF] Luo | Recognizing the role of perception in action at 6 months[END_REF][START_REF] Moll | Level 1 perspective-taking at 24 months of age[END_REF][START_REF] Nurmsoo | Preschoolers' perspective taking in word learning: Do they blindly follow eye gaze?[END_REF]. By the age of 2, children also have sufficient perspective taking abilities to place an object behind a screen in order to hide it from someone [START_REF] Flavell | Young children's knowledge about visual perception: Hiding objects from others[END_REF][START_REF] Mcguigan | The relation between hiding skill and judgment of eye direction in preschool children[END_REF][START_REF] Mascaro | Optimistic expectations about communication explain children's difficulties in hiding, lying, and mistrusting liars[END_REF]. Importantly, previous studies of perspective taking have not assessed children's ability to recruit the evidential weight of perception to provide support for a claim. Our study reveals that 2-and 3-year-olds can use their perspective taking abilities to manipulate an object to make it visible for an adult and that they do so when it is relevant to support claims about the object's location.

A third reason why many children succeeded in our task is that we made the disagreement between the experimenter and the participant explicit, building upon young children's capacity to contradict an adult who is mistaken [START_REF] Hummer | On the origins of denial negation[END_REF][START_REF] Koenig | Infants' understanding of false labeling events: The referential roles of words and the speakers who use them[END_REF]. Crucially, when contradicting someone, a child may simply deny this individual's claims without trying to select the most relevant evidence that may change the individual's mind. By contrast, in our study, the difference between children's behavior in the Window and in the Transparent conditions shows that children modulate their response to convey relevant information in order to provide support for their claims.

Previous studies suggest that children can evaluate others' reasons from 2 years of age [START_REF] Castelain | Evidence that 2 years old children are sensitive to argument strength[END_REF][START_REF] Castelain | The influence of power and reason on young Maya children's endorsement of testimony[END_REF][START_REF] Corriveau | Why does rain fall?": Children prefer to learn from an informant who uses noncircular explanations[END_REF][START_REF] Koenig | Beyond semantic accuracy: Preschoolers evaluate a speaker's reasons[END_REF][START_REF] Mercier | Early sensitivity to arguments: How preschoolers weight circular arguments[END_REF]Mercier, Sudo, Castelain, Bernard, & Matsui, in press). Yet, to evaluate reasons and to produce them to influence others are quite different tasks. Indeed, to evaluate a reason, one needs to assess the epistemic weight of a piece of information that is already presented to us as a reason. By contrast, to produce reasons efficiently, one needs to find a decent reason in a vast space of possible reasons [START_REF] Mercier | Looking for arguments[END_REF]. Moreover, finding a good reason means assessing the persuasive value of a piece of evidence from someone else's viewpoint. This process requires to take into account some of the audience's mental states (knowledge, beliefs) that may differ from one's own. Our study indicates that children adjust their reason-giving attempts to the informational access of their audience from 2 years of age onward.

Observational data suggest that children start providing what appears to be reasons nearly as soon as they can produce sentences, between the ages of 18 and 24 months [START_REF] Kuczynski | Development of children's noncompliance strategies from toddlerhood to age 5[END_REF][START_REF] Kuczynski | A developmental interpretation of young children's noncompliance[END_REF][START_REF] Perlman | If-then contingencies in children's sibling conflicts[END_REF]. By the age of three, children have been said to "generate and think about positive and negative reasons for pursuing different courses of action or for holding specific sets of beliefs" (Stein & Bernas, 1999, p. 97; see also [START_REF] Dunn | Development of justification in disputes with mother and sibling[END_REF].

Our data shed light on the cognitive underpinnings of these early reason-giving behaviors.

One route to discover what kind of behavior is likely to influence others is simply to rely on previous successes. Across repeated instances of social interactions, very young children may discover that by engaging in certain behaviors (e.g., smiling, crying, shouting, insisting, negating a contradictor's viewpoint), they are more likely to get others to comply with their requests or to agree with them (e.g., [START_REF] Weiss | Persuasive strategies used by preschool children[END_REF][START_REF] Bartsch | Young children's persuasion in everyday conversation: Tactics and attunement to others' mental states[END_REF]. This kind of fixed strategy is efficient up to a point. A more powerful way of influencing others is to rely on abstract principles that guide the selection of reasons, which one may call naïve rhetoric. A naïve rhetoric is likely to include some sensitivity to the role of evidence in persuading others. We find that the roots of naïve rhetoric are already present during toddlerhood. In our study, participants did not rely on a fixed, pre-established strategy.

If they had, they would have rotated the boxes equally across conditions (Window vs. Fully Transparent). Instead, toddlers were sensitive to the type of evidence that their interlocutor already had access to. When trying to provide support for their claims, toddlers were more likely to provide the experimenter with a piece of information when it was novel rather than when it was already known.

Observing flexible reason-giving in toddlers introduces a host of novel questions about the development of argumentative skills, and naïve rhetoric. Do toddlers assume that some sources of evidence (e.g., direct perception) are stronger than others (e.g., testimony)? Do they make assumptions about the way evidence from different sources are combined? Do they evaluate the well-formedness of their own reasons? Our results indicate that addressing these questions and therefore testing the preverbal roots of human reason-giving during infancy and toddlerhood is feasible.

  Figure1).

Figure 1 .

 1 Figure 1. Number of rotations of the box containing the dog during the test phase per

  Two-to four-year-old children were presented with an experimenter who asked them about the location of a toy placed in one of four boxes, and the experimenter disagreed with the information initially provided by the participant. In the Fully Transparent condition, all boxes were transparent, so the children did not have any obvious means of providing further support for their claims to the experimenter. In contrast, in the Window condition only the children, and not the experimenter, could see in which box the toy had been placed. Thus, the children had the opportunity to turn the box toward the experimenter as an additional means of supporting her claim about the toy's location.Children rarely turned the baited box when the boxes were fully transparent. In contrast, children were more likely to turn the box in the proper context: when the experimenter was disagreeing with them and when turning the box could reveal new and relevant information to the experimenter (i.e., in the Window condition). These results are compatible with two-non-mutually exclusive-interpretations of what children attempted to achieve by rotating the baited box in the Window condition. A first possibility is that the participants aimed to change the experimenter's belief about the location of the target toy by showing it to her. Second, it is possible that by rotating the baited box the children attempted to justify their answer by demonstrating to the experimenter that they had visual access to the location of the toy. Both of these interpretations suggest the presence of precursors of skills crucial for reason-giving: deciding when to use reason-giving and choosing reasons appropriately as a function of the context.Overall, our participants rotated the box at a low baseline rate. This pattern can be explained by the fact that (i) in the Fully Transparent condition, rotating the box was irrelevant, and (ii) the participants had to discover on their own that the boxes could be rotated. Remarkably, approximately half of the participants rotated the box at least once (16 participants out of 35, 46%) in the Windows condition, in which this action was relevant to

Table 1 .

 1 Descriptive Statistics for the Number of Rotations During the Baseline, Pre-test and Test Trials per Condition (Window vs. Fully Transparent).

	Transparent condition

centers that hosted the project (Crèche this study. This research was supported by grants from the "Agence Nationale pour la Recherche" (PragTICAL, ANR-14-ACHN-0020; VICCOM, ANR-16-TERC-0001-01, EUR FrontCog ANR-17-EURE-0017) to OM, and HM (respectively).