J Kammer

P.-M Flaud

A Chazeaubeny

Raluca Ciuraru

K Le Ménach

E Geneste

H Budzinski

J M Bonnefond

Eric Lamaud

E Perraudin

K Le Menach

Eric Villenave
email: eric.villenave@u-bordeaux.fr

Biogenic volatile organic compounds (BVOCs) reactivity related to new particle formation (NPF) over the Landes forest

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

Introduction

Forest ecosystems are key components of our environment, representing around 30% of the Earth land surface (United Nations food and agricultural organization, 2015). Also, forests represent a large source of Volatile Organic Compounds (VOCs). It is widely recognized that 90% of VOCs are emitted by biogenic sources, isoprene and monoterpenes being the most emitted biogenic VOCs (BVOCs) in the atmosphere [START_REF] Guenther | A global model of natural volatile organic compound emissions[END_REF][START_REF] Sindelarova | Global dataset of biogenic VOC emissions calculated by the MEGAN model over the last 30 years[END_REF]. Once oxidized in the atmosphere, some of their products have sufficiently low volatility to partition between the gas and the particle phases. Through this process, forests act as a source of secondary organic aerosol (SOA), which play a crucial role in air quality and climate change [START_REF] Hallquist | The formation, properties and impact of secondary organic aerosol: current and emerging issues[END_REF]IPCC, 2013;[START_REF] Kazil | Aerosol nucleation and its role for clouds and Earth's radiative forcing in the aerosol-climate model ECHAM5-HAM[END_REF][START_REF] Pope | Health effects of fine particulate air pollution: lines that connect[END_REF].

New Particle Formation (NPF) is a process that consists of the secondary formation of atmospheric particles [START_REF] Dal Maso | Formation and growth rates of ultrafine atmospheric particles: a review of observations[END_REF]. Currently, NPF is estimated to contribute around half of the global Cloud Condensation Nuclei (CCN) at a global scale [START_REF] Merikanto | Impact of nucleation on global CCN[END_REF]. NPF was observed in a large number of locations worldwide [START_REF] Kulmala | Formation and growth rates of ultrafine atmospheric particles: a review of observations[END_REF]. However, processes governing NPF remain quite uncertain [START_REF] Andreae | The aerosol nucleation puzzle[END_REF][START_REF] Kulmala | Direct Observations of Atmospheric Aerosol Nucleation[END_REF][START_REF] Nallathamby | Particle nucleation in a forested environment[END_REF]. Until recently, it was accepted that NPF could not occur without the involvement of sulphuric acid in the first step, i.e. nucleation [START_REF] Andreae | The aerosol nucleation puzzle[END_REF][START_REF] Kulmala | How particles nucleate and grow[END_REF][START_REF] Kulmala | Direct Observations of Atmospheric Aerosol Nucleation[END_REF][START_REF] Nallathamby | Particle nucleation in a forested environment[END_REF]. In the atmosphere, gas phase sulphuric acid is mainly generated from SO2 reaction with OH radicals in the presence of water. It may also be produced in the gas phase by oxidation of SO2 with H2O2 or by reaction with Criegee intermediates [START_REF] Sipilä | Reactivity of stabilized Criegee intermediates (sCIs) from isoprene and monoterpene ozonolysis toward SO2; and organic acids[END_REF]. Due to its very low vapour pressure, sulphuric acid may easily form clusters with other molecules, i.e. water or oxygenated molecules [START_REF] Elm | Molecular interaction of pinic acid with sulfuric acid -exploring the thermodynamic landscape of cluster growth[END_REF][START_REF] Kulmala | How particles nucleate and grow[END_REF][START_REF] Neitola | Total sulfate vs. sulfuric acid monomer concenterations in nucleation studies[END_REF][START_REF] Praplan | Elemental composition and clustering behaviour of α-pinene oxidation products for different oxidation conditions[END_REF][START_REF] Riccobono | Oxidation products of biogenic emissions contribute to nucleation of atmospheric particles[END_REF]. Then, if this cluster is stable, it can grow through the condensation of extremely low volatility organic compounds (ELVOCs), produced from autoxidation (Ehn et al., 2014;[START_REF] Kulmala | Direct Observations of Atmospheric Aerosol Nucleation[END_REF][START_REF] Praplan | Elemental composition and clustering behaviour of α-pinene oxidation products for different oxidation conditions[END_REF][START_REF] Riccobono | Oxidation products of biogenic emissions contribute to nucleation of atmospheric particles[END_REF]. Recently, it was demonstrated from laboratory studies that NPF can be caused solely by α-pinene ozonolysis [START_REF] Kirkby | Ion-induced nucleation of pure biogenic particles[END_REF][START_REF] Tröstl | The role of low-volatility organic compounds in initial particle growth in the atmosphere[END_REF]. It was highlighted from the CLOUD chamber experiments that highly oxidized molecules (like HOMs) can be rapidly formed, and induce NPF, following autoxidation mechanisms [START_REF] Ehn | A large source of low-volatility secondary organic aerosol[END_REF][START_REF] Jokinen | Rapid autoxidation forms highly oxidized RO2 radicals in the atmosphere[END_REF]. Direct evidence of such implication of HOMs in the real atmosphere was highlighted very recently in the free troposphere, where one of the main driver of NPF could be anthropogenic VOCs [START_REF] Bianchi | New particle formation in the free troposphere: A question of chemistry and timing[END_REF]. More, [START_REF] Rose | Observations of biogenic ion-induced cluster formation in the atmosphere[END_REF] observed that biogenic ions can induce stable cluster formation. But identification of biogenic NPF still needs more investigations in real atmosphere, especially at pristine locations. As a consequence, new measurements in ecosystems poorly or not yet investigated are needed.

In this context, the goal of the present study was to reinforce our knowledge on the relation between BVOCs and NPF. For that purpose, two field measurement campaigns took place in the summers of 2014 and 2015 in the Landes forest, in the south west of France. A first paper by our group recently evidenced frequent nocturnal NPF in 2015, together with high monoterpene mixing ratios [START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF]. This new paper first presents a comparison between 2014 and 2015 field campaigns. Then, the focus is made on the main emitted BVOCs at the measurement site, their reactivity and their oxidation products both in the gas and the particulate phases. Finally, considering the recent findings, the link between in canopy BVOC reactivity and NPF is discussed.

Methodology

Site description

As part of the LANDEX-episode zero project, two intensive field campaigns were conducted, in the French Landes forest. The first campaign started from the 5 th of July until the 18 th of July 2014, and the second started from the 5 th to the 23 rd of July 2015. [START_REF] Moreaux | Paired comparison of water, energy and carbon exchanges over two young maritime pine stands (Pinus pinaster Ait.): effects of thinning and weeding in the early stage of tree growth[END_REF][START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF]. Due to the proximity of the Atlantic Ocean, the local climate is oceanic, and winds are consequently frequently originating from west-north west.

Field measurements

Meteorological parameter measurements have been performed on a mast during each campaign, at 15 m above ground level (a.g.l.). Air temperature and relative humidity (HMP 155, VAISALA HUMICAP), wind speed and direction (Windsonic 1, Campbell scientific), global solar radiation (CMP22 and CNR4, Campbell scientific) and rainfall was continuously measured at a half hourly scale. To complete these meteorological measurements, air mass backward trajectories have been computed using the NOAA HYSPLIT model [START_REF] Stein | NOAA's HYSPLIT atmospheric transport and dispersion modeling system[END_REF]. Daily backward trajectories were calculated for each day of the campaign using the GDAS (Global Data Assimilation System) meteorological dataset, indicating the origin of the analysed air mass at Bilos. Besides, eddy covariance fluxes of momentum, heat, water vapour and CO2 were measured at 15 m a.g.l., in order to evaluate the physiological state of the ecosystem. The details of the eddy covariance methods have been fully described elsewhere [START_REF] Aubinet | Estimates of the annual net carbon and water exchange of forests: The EUROFLUX methodology[END_REF][START_REF] Burba | A brief practical guide to eddy covariance flux measurements: principles and workflow examples for scientific and industrial applications[END_REF].

A mobile laboratory truck (the "Barracuda" facility), located in the centre of the area, was used for physico-chemical measurements with air tubing devoted to aerosol and gas sampling being located at 4 m a.g.l., corresponding to the canopy level. A 5 m Teflon tubing (4.5 mm inner diameter) was used for the gas phase measurements, whereas a 4 m long conductive silicone tubing (4.5 mm inner diameter) was used to sample particles. Both gas and particle sampling inlets were located right next (about 20 cm) to ensure the collection of the same air masses. Preliminary tests were performed by measuring the same air with and without tubing. As no difference was observed, it is assumed that no significant wall losses of terpenes occurred in tubing. During the campaign, ozone and nitrogen oxides (NOx = NO + NO2) mixing ratios were monitored through a UV absorption analyzer (APOA 370, HORIBA, detection limit (DL) = 0.5 ppb) and a chemiluminescence analyzer (APNA 370, HORIBA, DL = 0.5 ppb), respectively. Sulphur dioxide (SO2) measurements were performed only during the 2015 field campaign using a UV fluorescence monitor (API 100 E, Teledyne, DL = 0.4 ppb). All the three monitors (O3, NOx and SO2) have been calibrated before the campaigns, and leak checks were frequently performed using activated charcoal air filters. A scanning mobility particle sizer (SMPS, TSI model 3080) was used to characterize particle number size distribution every 10 minutes. It combines a differential mobility analyser (DMA, TSI model 3081) and an optical counter (CPC, TSI model 3772). Aerosol flow and sheath flow were set respectively to 0.5 L min -1 and 5 L min -1 to ensure a 1:10 ratio in the DMA. Under these flow conditions, particle number size distribution and concentration were measured for particles having an electric mobility diameter between 10.9 nm and 487 nm. Particle wall losses were calculated following the methodology described in [START_REF] Baron | Aerosol measurement: principles, techniques, and applications[END_REF].

The estimated particle loss rates were estimated to be 34.7% for the smallest particles (with a 10.9 nm diameter) analysed. The particle number and size distribution data were corrected to account for the calculated wall losses. Then, a proton transfer reaction -time of flightmass spectrometry (PTR-TOF-MS, Kore Technology) was used to study the composition of the gaseous phase, especially the evolution of BVOCs and their related oxidation products.

This instrument is based on the proton transfer reaction from H3O + to the analytes, a soft ionisation which prevents from strong fragmentation. Basically, the air mass composition could thus be analyzed by recording the temporal evolution of the MH + ions. During the campaign, the PTR-TOF-MS was operated at 600 V drift voltage and 1.3 mbar reactor pressure, ensuring an E/N ratio around 130 Td (1 Townsend = 10 -17 V cm -2 ), with E being the electric field strength in the reactor and N the gas number density. The PTR-TOF-MS was tuned to work at this E/N ratio as it is a good compromise to prevent from high fragmentation in the mass spectrometer and ambient relative humidity effect [START_REF] Pang | Biogenic volatile organic compound analyses by PTR-TOF-MS: Calibration, humidity effect and reduced electric field dependency[END_REF][START_REF] Tani | Measurement of monoterpenes and related compounds by proton transfer reaction-mass spectrometry (PTR-MS)[END_REF]. PTR-TOF-MS blanks and calibrations were daily performed using the vapour pressure (at 0°C and diluted in N2 flow) of a pure α-pinene solution, regulated by a mass flow controller (Millipore, Coastal Instruments). Typical day to day sensitivity variations were about 10 %.

The variation of the sensitivity may be sometimes larger, when the PTR-TOF-MS parameters (for example, the voltage applied to extraction lenses) were optimized during the campaign.

For non-calibrated compounds (other than α-pinene), calibration coefficients determined for α-pinene were used to consider the day-to-day sensitivity variations of the instrument. But as we could not consider that these calibration coefficients provide a real estimation of the concentration of these other compounds, then each signal was normalized by its maximum count number.

As monoterpenes C10H16 present many isomers and as the PTR-TOF-MS was only able to measure their total concentrations, an online gas chromatograph coupled to a flame ionization detector (GC-FID, airmoVOC, Chromatotec) was used to discriminate their speciation. The GC-FID sampled air at 0.11 L min -1 , and concentrated VOCs on a cold air trap composed of adsorbent Tenax TA (20-35 mesh, Chrompack). The desorbed VOCs to analyse were then separated using a Chrompack Sil 8CB low bleed capillary column, and finally detected with the FID. A complete description of the GC-FID can be found elsewhere [START_REF] Staudt | Monoterpene and sesquiterpene emissions from <i>Quercus coccifera</i> exhibit interacting responses to light and temperature[END_REF]. In 2015, the GC-FID was only available during the last five days of the campaign.

The chemical composition of particles collected during the 2015 field campaign was investigated using high performance liquid chromatography coupled to mass spectrometry.

Briefly, a high volume sampler (DA-80, Digitel, with an air flow set to 30 m 3 h -1 ) was used to collect PM2.5 (particles with an aerodynamical diameter smaller than 2.5 µm) onto quartz fiber filters (Ø = 150 mm). Different laboratory tests showed that ultrasonic extraction provides the highest and more reproducible extraction yields. If there are still some uncertainties related to this technique, it is widely used to measure pinic acid and pinonic acid in aerosol samples [START_REF] Anttila | Determination of organic acids in aerosol particles from a coniferous forest by liquid chromatography-mass spectrometry[END_REF][START_REF] Feltracco | Photo-oxidation products of α-pinene in coarse, fine and ultrafine aerosol: A new high sensitive HPLC-MS/MS method[END_REF][START_REF] Kourtchev | Molecular composition of biogenic secondary organic aerosols using ultrahigh-resolution mass spectrometry: comparing laboratory and field studies[END_REF][START_REF] Nozière | The Molecular Identification of Organic Compounds in the Atmosphere: State of the Art and Challenges[END_REF]. As a result, particles were extracted two times by placing a 47 mm diameter disc in 3 mL of a mixed acetonitrile/water solution (70/30) under ultrasonication for 15 min. Then, each extract was centrifugated and the supernatent was transfered in a 15 mL vial and concentrated to about 400 µL under a gentle pure nitrogen flow (99.995 % purity, Linde Gas SA) at 40 °C.

Samples were weighted using gravimetry (precision: 10 -4 g), then stored at -18°C until analysis. LC-HRMS analyses were carried out using an Agilent 1290 HPLC system coupled to an Agilent 6540 QToF mass spectrometer equipped with an Agilent Jet Stream electrospray ionization source (ESI) operating in the negative mode. The chromatographic separation was performed on a Zorbax Eclipse XDB-C18 (2.1×150 mm; 3.5 μm) regulated at 30°C. Gradient LC elution was performed at a flow rate of 0.5 mL min -1 using 0.2% formic acid in purified water as mobile phase A and 0.2% formic acid in acetonitrile as mobile phase B. Sample injection volume was set at 5 µL. In this study, pinic acid and pinonic acid were quantified using authentic standards of the two targeted compounds (pinic acid 98 % and cispinonic acid 98 %, Sigma-Aldrich) as external standards. More details about LC-QTOF-MS parameters can be found in the supplementary information (Table S1).

3 Results and discussion a,d). During this last campaign, the air temperature frequently exceeded 30°C during the day and was hardly ever below 15°C at night, which was not the case in July 2014. Such high temperature values were already reported to induce a thermic stress on trees [START_REF] Laothawornkitkul | Biogenic volatile organic compounds in the Earth system[END_REF]. One particularity of the site was that relative humidity increased to reach its maxima (i. which is very high for a forest stand [START_REF] Gu | Direct and indirect effects of atmospheric conditions and soil moisture on surface energy partitioning revealed by a prolonged drought at a temperate forest site[END_REF]. Such high Bowen ratios were not observed during the 2014 field campaign, with values only around 0.5 [START_REF] Kammer | Ozone production in a maritime pine forest in water-stressed conditions[END_REF].

Meteorological conditions during 2014 and 2015 field campaigns

Thus, a thermic stress (due to temperatures over 30°C) coupled to a hydric stress occurred during the 2015 campaign. The presence of such a stress situation may increase BVOC emissions [START_REF] Kesselmeier | Biogenic Volatile Organic Compounds (VOC): An Overview on Emission[END_REF][START_REF] Laothawornkitkul | Biogenic volatile organic compounds in the Earth system[END_REF][START_REF] Loreto | On the monoterpene emission under heat stress and on the increased thermotolerance of leaves of Quercus ilex L. fumigated with selected monoterpenes[END_REF][START_REF] Peñuelas | BVOCs and global change[END_REF]

General conditions of the campaigns

A previous study reported by our group [START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF] showed that NPF has been frequently observed during the 2015 field campaign, mostly during the night, although a few events (only 2) were also recorded during daytime. NPF events were defined as an increase of particle concentration in the nucleation mode followed by a subsequent growth during at least 2 hours [START_REF] Dal Maso | Formation and growth rates of ultrafine atmospheric particles: a review of observations[END_REF]. In 2014, only one NPF event was observed, occurring during nighttime. This leads to a NPF frequency of occurrence (i.e. the number of nights where NPF events were observed divided by the total number of nights during the field campaign) of about 8% during the 2014 field campaign. In 2015, 12 NPF events were reported, reaching a high frequency of occurrence of around 55% [START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF].

NPF thus occurred more frequently in a summer influenced by a hydric stress (according to the high Bowen ratio values measured during the 2015 campaign). This observation may further suggest that the presence of a hydric stress is indirectly inducing more NPF events because of stronger monoterpene emissions. Nevertheless, more studies about the effect of hydric stress on NPF will be needed to confirm such hypothesis. All the observed NPF events are listed in Table S2. A more complete analysis of NPF events (growth rates, nucleation rates, etc.) can be found in [START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF].

Analyses of wind directions and air mass back trajectories have shown that air mass origin did not affect NPF. During both campaigns, NPF were always recorded along (i) high relative humidity (i.e. RH ≥ 75%), (ii) temperatures higher than 16°C and (iii)

Monoterpene mixing ratios

The sum of monoterpene as well as ozone mixing ratios are represented in Figure 1 for both field campaigns. A strong diurnal cycle was observed in July 2014 as well as in July 2015 for both ozone and monoterpenes. A typical ozone diurnal cycle was observed, with higher values in the afternoon (Figure 1). The ozone cycle was mirrored by the monoterpenes cycle, with higher monoterpene mixing ratios values during the night, as previously reported in the Landes forest [START_REF] Simon | The Landes experiment: Monoterpenes emitted from the maritime pine[END_REF]. Monoterpenes were dominated from far by α-and βpinene, accounting for around 90% of total monoterpenes, in accordance with previous studies [START_REF] Riba | Diurnal variation in the concentration of αand βpinene in the Landes forest (France)[END_REF][START_REF] Simon | The Landes experiment: Monoterpenes emitted from the maritime pine[END_REF]. Maritime pines are well-known to be strong temperature driven BVOC emitters [START_REF] Kesselmeier | Biogenic Volatile Organic Compounds (VOC): An Overview on Emission[END_REF][START_REF] Lathiere | Impact of climate variability and land use changes on global biogenic volatile organic compound emissions[END_REF].

Although emissions are higher during the day, the larger dilution of emissions in the surface layer and the higher oxidant levels conduct to lower BVOC mixing ratios during light hours.

More explanation about the diurnal cycle and the effect of the change in the boundary layer height on monoterpene mixing ratios can be found in [START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF]. In 2015,

Monoterpene/O3 reactivity

Time series of ozone and monoterpene mixing ratios (Figure 1) suggest that their respective diurnal cycles were closely anti-correlated. The monoterpene mixing ratios increased every night, as reported on Figure 1, when it is likely that no ozone was produced (because of its dependence on solar radiation), nor ozone advection from higher layers of the atmosphere as their kinetic rate constants are quite low [START_REF] Atkinson | Gas-phase tropospheric chemistry of biogenic volatile organic compounds: a review[END_REF]. A rough estimation, assuming pseudo-first order reactions between ozone and monoterpenes, led to a maximal contribution to the ozone decrease being around 50 %. Thus, there is also a possibility for other reactive BVOCs to be co-emitted with monoterpenes and react very fast with ozone (like β-caryophyllene, for example). Further modelling of ozone deposition and chemical reactions between ozone and BVOCs will be needed for a detailed estimation.

As monoterpene emissions come from the diffusion of resin stored in pools, the ratio between α-and β-pinene in maritime pine emissions is assumed to be constant [START_REF] Simon | The Landes experiment: Monoterpenes emitted from the maritime pine[END_REF]. Hence, changes in the α/β-pinene ratio can mostly be assigned to differences in oxidation processes, as the reactivity of α-and β-pinene with the main atmospheric oxidants differs (see Table 1). The daily evolution of the α/β-pinene ratio may provide an interesting tool to evaluate the importance of each oxidation processes. When the OH-initiated monoterpene photooxidation is the dominant process, β-pinene mixing ratio should decrease faster than that of α-pinene, because of its larger reactivity with OH (Table 1). In the morning, the α/β-pinene ratio measured at Bilos was increasing, meaning that photooxidation was increasingly dominated by OH radicals (Figure 2-b). In the afternoon, the α/β-pinene ratio was found to drop, during both 2014 and 2015 field campaigns. Such decrease reflects that the dominating oxidation process was changing, as the reaction rate constants of α-pinene with ozone and NO3 are known to be larger than for β-pinene (Table 1). It is recognized that NO3 reactivity is mostly significant during nighttime, as nitrate radicals are photolysed by solar radiations [START_REF] Seinfeld | Atmospheric Chemistry and Physics: From Air Pollution to Climate Change[END_REF]. As a result, the drop of the α/β-pinene ratio highlighted the dominant role of ozone oxidation processes in the afternoon.

The IUPAC recommended values [START_REF] Atkinson | Evaluated kinetic and photochemical data for atmospheric chemistry: Volume II: gas phase reactions of organic species[END_REF]

BVOC oxidation products

As the PTR-TOF-MS has a good mass resolution, and considering the high mixing ratios of both α-and β-pinene at the Bilos site, several detected ions were able to be attributed to biogenic oxidation products. The identification of the following ions was proposed based on the proximity of the peak with the theoretical mass, and considering the characteristics of the Bilos site (very rural, high monoterpene mixing ratios, etc.). However, a contribution of a compound with a m/z very closed to the proposed compound could not be fully excluded.

d) c)

First, m/z 139.11 was proposed to be attributed to nopinone (C9H14O) and m/z 169.12 to pinonaldehyde (C10H16O2), as both compounds were frequently reported at rural field sites [START_REF] Bourtsoukidis | From emissions to ambient mixing ratios: online seasonal field measurements of volatile organic compounds over a Norway spruce-dominated forest in central Germany[END_REF][START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF][START_REF] Park | Biogenic volatile organic compound emissions during BEARPEX 2009 measured by eddy covariance and flux-gradient similarity methods[END_REF][START_REF] Park | Eddy covariance emission and deposition flux measurements using proton transfer reaction -time of flight -mass spectrometry (PTR-TOF-MS): comparison with PTR-MS measured vertical gradients and fluxes[END_REF][START_REF] Rantala | Continuous flux measurements of VOCs using PTR-MS-reliability and feasibility of disjunct-eddy-covariance, surface-layer-gradient, and surface-layerprofile methods[END_REF][START_REF] Ruuskanen | Eddy covariance VOC emission and deposition fluxes above grassland using PTR-TOF[END_REF]. Relevant signals were also observed at m/z 135.11, m/z 151.10 and m/z 155.13, but their attribution was more difficult. The m/z 135.11 protonated ion corresponds to compounds of the molecular formula C10H14. Among several potential hydrocarbons, the m/z 135.11 ion may correspond to p-cymene, which has already been reported by [START_REF] Gratien | Surprising formation of p-Cymene in the oxidation of α-pinene in air by the atmospheric oxidants OH, O3 , and NO3[END_REF] as a secondary product of α-pinene photo-oxidation. Even if p-cymene has not yet been reported among monoterpene emissions from maritime pines, some works have already pointed out its emission from different plant species [START_REF] Bäck | Chemodiversity of a Scots pine stand and implications for terpene air concentrations[END_REF]. P-cymene could thus have been be emitted by understorey species (for which BVOC emissions remain unknown or poorly investigated), or result from α-pinene photo-oxidation. P-cymene levels were found to be highly correlated with pinonaldehyde and nopinone (R 2 = 0.8), suggesting the secondary origin of this compound.

The molecular formula [C10H14O]H + was proposed to be the major contributor of m/z 151.10 ion. This ion is recognized as a monoterpene oxidation product, and some studies attributed this mass to a significant pinonaldehyde fragment [START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF][START_REF] Holzinger | Emission, oxidation, and secondary organic aerosol formation of volatile organic compounds as observed at Chebogue Point, Nova Scotia: VOC emission and SOA formation[END_REF][START_REF] Park | Biogenic volatile organic compound emissions during BEARPEX 2009 measured by eddy covariance and flux-gradient similarity methods[END_REF][START_REF] Rantala | Continuous flux measurements of VOCs using PTR-MS-reliability and feasibility of disjunct-eddy-covariance, surface-layer-gradient, and surface-layerprofile methods[END_REF]. Note that i) other compounds such as myrtenal or verbenone, that may have primary sources, could also be important contributors to the m/z 151.10 signal [START_REF] Kim | Emissions and ambient distributions of Biogenic Volatile Organic Compounds (BVOC) in a ponderosa pine ecosystem: interpretation of PTR-MS mass spectra[END_REF] and ii) OH initiated oxidation of nopinone was also reported to yield products with m/z 151.10. Finally, the PTR-TOF-MS signal recorded at m/z 155.13 has been identified as C10H18O compounds. Several isomers can potentially contribute to this signal. Notably, primary OVOCS such as linalool or 1,8-cineol have already been reported at m/z 155.13 elsewhere [START_REF] Bourtsoukidis | From emissions to ambient mixing ratios: online seasonal field measurements of volatile organic compounds over a Norway spruce-dominated forest in central Germany[END_REF][START_REF] Park | Eddy covariance emission and deposition flux measurements using proton transfer reaction -time of flight -mass spectrometry (PTR-TOF-MS): comparison with PTR-MS measured vertical gradients and fluxes[END_REF][START_REF] Rantala | Continuous flux measurements of VOCs using PTR-MS-reliability and feasibility of disjunct-eddy-covariance, surface-layer-gradient, and surface-layerprofile methods[END_REF]. However, a contribution of secondary oxygenated compounds cannot be excluded. The presence of all such monoterpene oxidation products confirms that monoterpenes were oxidized at the Bilos site. Especially, pinonaldehyde and nopinone are known to be exclusively formed from the oxidation (by ozone or OH radical) of α-and βpinene, respectively, supporting the occurrence and the importance of the oxidation of monoterpenes in the Landes forest. Indeed, OH can be formed as a secondary product of the ozonolysis of monoterpenes. Any observed OH reactivity during nighttime would thus be an indication of a primary monoterpene ozonolysis process. It was finally assumed that the increase of nopinone and pinonaldehyde during the night was arising from monoterpene ozonolysis.

The temporal evolution of the compounds of interest measured during the 2015 field campaign is represented on Figure 3. In this section, we make the choice to focus on the 2015 data as i) oxidation product concentration values were frequently around the DL of the instrument in 2014 and ii) there was only one NPF event in 2014. This is consistent with lower monoterpene mixing ratios observed during this first campaign. As oxidation products were not quantified in this study, their corresponding signals were normalized to their maximum to allow the comparison of their temporal evolution profiles and diurnal dynamics.

Interestingly, all the oxidation products globally followed the same diurnal cycle, similar to that observed for monoterpenes (Figure 3). Their levels started to increase with the sunset, then decreased when the first rays of the sunlight appeared (Figure 3). The decrease of the monoterpene oxidation product could be explained by i) a higher dilution due to the increase of the surface layer height as soon as the first rays break the stable nocturnal boundary layer and ii) potential photolysis, especially in the case of pinonaldehyde and nopinone, as both are carbonyl compounds prone for photolysis. Although oxidation products are quite volatile and thus are not expected to directly contribute significantly to NPF, they highlight a strong reactivity between ozone and monoterpenes, also yielding (at night) to potential very low volatile oxidation products that may contribute to nocturnal NPF at Bilos.

Nighttime reactivity of BVOC oxidation products

In order to go further in the understanding of the role of oxidation products observed in the forest, it was decided to particularly focus on a short period, on m/z 139.11 and m/z 169.12 signals, corresponding to nopinone and pinonaldehyde respectively (Figure 4). This is of particular interest as they are directly related to α-pinene (for pinonaldehyde) and β-pinene (for nopinone) oxidation. To the best of our knowledge, no direct comparison of diurnal behaviours of both nopinone and pinonaldehyde from online field measurements has ever been reported. The Figure 4 presents the temporal evolution of nopinone, pinonaldehyde, total monoterpenes and ozone mixing ratios during two nights (12/13 and 13/14 July 2015), representative of the entire campaign. It can be observed that during both nights, pinonaldehyde signal first increased faster than that of nopinone, reaching its maximum within a few hours and then decreased throughout the night. At the end of the night, a new increase was observed for pinonaldehyde mixing ratios, which finally returned to its daily level in the morning. The nocturnal dynamics of nopinone was observed to be simpler.

Nopinone level started to increase at the sunset and decreased in the morning as turbulence reappeared. Such a strong difference between the nocturnal profiles of these two compounds probably results from their different physico-chemical properties and related reactivity. The formation yields of pinonaldehyde and nopinone from ozonolysis of α-and βpinene have been reported to be highly dependent on the physico-chemical conditions [START_REF] Lee | Gas-phase products and secondary aerosol yields from the ozonolysis of ten different terpenes[END_REF]. More, the range of formation yields for both compounds are quite similar and very large (Table 2). If formation yields did not directly allow elucidating the faster increase of pinonaldehyde, the higher reaction rate constant of α-pinene ozonolysis (compared to βpinene) may at least partially explain such trend.

Table 2: Formation yields of nopinone and pinonaldehyde from β-and α-pinene ozonolysis, reaction rate constants with the main atmospheric oxidants OH, O3 and NO3 and saturation vapour pressures (Ps in Torr). The second difference between the two nighttime product time profiles was the decrease of pinonaldehyde during the night, not (or less) observable in the case of nopinone, highlighting a larger sink for pinonaldehyde than for nopinone (Figure 4), assumed to be due to its faster reactions with atmospheric oxidants (Table 2). The reaction of pinonaldehyde with ozone is known to be very slow, and may not be responsible of such a decrease. NO3-initiated reaction might be an important removal process for pinonaldehyde but would have required larger concentrations of nitrate radicals that those expected at the very low NOx levels measured during the campaign (Figures S3.a and S4.a). In addition, NO3 reaction rate constants with α-and β-pinene are at least 100 times faster than nopinone and pinonaldehyde (Table 1). As a result, any NO3 radicals present at Bilos would first react with monoterpenes, not allowing to explain the decrease of pinonaldehyde.

Compounds

Many studies have reported the secondary formation of OH radicals following the monoterpene ozonolysis, with yields up to 1 [START_REF] Rickard | OH Yields in the Gas-Phase Reactions of Ozone with Alkenes[END_REF][START_REF] Aschmann | OH radical formation from the gas-phase reactions of O3 with a series of terpenes[END_REF][START_REF] Fick | OH radical formation from the gasphase reactions of O3 with a series of terpenes[END_REF][START_REF] Atkinson | Gas-phase tropospheric chemistry of biogenic volatile organic compounds: a review[END_REF][START_REF] Forester | Hydroxyl radical yields from reactions of terpene mixtures with ozone: Hydroxyl radical yields from reactions of terpene mixtures with ozone[END_REF]. Moreover, [START_REF] Faloona | Nightime observations of anomalous high levels of hydroxyl radicals above a deciduous forest canopy[END_REF] reported unexpected high OH concentrations at a deciduous forest site during nighttime. Therefore, it is reasonable to hypothesize that the nighttime pinonaldehyde decrease was due to its reaction with OH (this reaction being 5 times faster than that of nopinone) (Table 2). Obviously, future measurement of OH radicals at the Bilos site will be required to deeply answer to this question.

Another hypothesis can be put forward to tentatively explain the different profiles of pinonaldehyde and nopinone: their potential condensation on the particle phase. Indeed, the saturation vapour pressures of the two compounds differ by one order of magnitude, pinonaldehyde being 10 times less volatile than nopinone at ambient temperature (Table 3).

But [START_REF] Kavouras | Formation and gas/particle partitioning of monoterpenes photo-oxidation products over forests[END_REF] have shown that both compounds are usually mostly present in the gas phase. As a result, gas/particle partitioning might only weakly impact their concentration in the gas phase. Lastly, heterogeneous reactions (as for example, reactions with amines) could not be excluded from this discussion, and may play an important role in the fate of both products [START_REF] Duporté | Nitrogen-containing low volatile compounds from pinonaldehydedimethylamine reaction in the atmosphere: a laboratory and field study[END_REF], but the investigation of such reactions was beyond the scope of the present study and might be considered in the further measurement campaigns at Bilos.

Contribution of BVOC oxidation products to NPF

This work raises the question of the link between the in-canopy reactivity of monoterpenes and the NPF events observed at night. In a recent paper [START_REF] Kammer | Observation of nighttime new particle formation over the French Landes forest[END_REF], we reported that NPF always occurred i) when the condensation sink was low, ii) after the nocturnal boundary layer was taking hold and iii) for the nights where monoterpene mixing ratios were high, suggesting their contribution to nocturnal NPF. In the section 3.5, we relate that the mixing ratios of monoterpene oxidation products increased at night. The Figure 4 presents two consecutive nights (12/13 and 13/14 July 2015) where NPF occurred, as a typical example of what was observed during the campaign. At night, the sum of monoterpene mixing ratios started to increase about 1-2 hours before NPF took place, at the same time as nopinone and pinonaldehyde. This time might corresponded to a switch from condensation to nucleation, resulting from the drop of the condensation sink and the higher abundance of low volatile intermediates. To go further, a better knowledge of nanometer particle formation and size-resolved chemical composition of aerosols will be necessary. Figure 4 shows that monoterpenes and their oxidation products levels increased simultaneously, which means that the main drivers that are triggering NPF are probably physical. However, the time lag between the simultaneous increase and the beginning ot the NPF event may be due to i) the inability of the SMPS to catch the first steps of NPF and ii) the required time for condensable vapors to reach their saturation limit. This last hypothesis suggests a fast oxidation process, able to generate very low volatile gases in a short time. Auto-oxidation process has been

shown to be able to produce highly oxidized multifunctional organic compounds (HOMs) very fast, which can then promote particle nucleation and growth [START_REF] Jokinen | Rapid autoxidation forms highly oxidized RO2 radicals in the atmosphere[END_REF]Kirkby et al., 2016;[START_REF] Tröstl | The role of low-volatility organic compounds in initial particle growth in the atmosphere[END_REF]. Hence, simultaneously to nopinone and pinonaldehyde generations, HOMs may have been produced by fast auto-oxidation processes, participating in fine to nocturnal NPF [START_REF] Jokinen | Rapid autoxidation forms highly oxidized RO2 radicals in the atmosphere[END_REF]. In addition, OH radicals were very probably produced from the ozonolysis of monoterpenes during the night. This is supported by the time evolution of pinonaldehyde, which decreased in the middle of the night, probably due to its reaction with OH. Thus, secondary reactions involving OVOCs might also be involved in NPF. The lag time described above might also be the time required for these secondary reactions to take place. But, even if the reactions between OH and OVOCs could be important for NPF at Bilos, the main source of OH during nighttime was arising from the primary ozonolysis of monoterpenes, highlighting the importance this process as an initial step. To go further, the contribution of each oxidation pathways (i.e. ozone and OH) to NPF will need more investigations. Considering that most of NPF events occurred during nighttime and the very low SO2 mixing ratios observed at Bilos, it was not expected that sulfuric acid was involved, emphasizing again the role of BVOCs. The recent studies about NPF from pure BVOC ozonolysis, and the high monoterpene mixing ratios at our site let us hypothesise that ozonolysis of monoterpenes -and potentially other BVOCs -may be one of the main drivers of nocturnal NPF at the Bilos site [START_REF] Kirkby | Ion-induced nucleation of pure biogenic particles[END_REF][START_REF] Tröstl | The role of low-volatility organic compounds in initial particle growth in the atmosphere[END_REF].

Molecular tracers of BVOC oxidation in the particle phase

To support our hypothesis about the contribution of BVOCs to the nocturnal NPF, SOA tracers from α-and β-pinene oxidation were investigated in the PM2.5 particulate fraction collected during the 2015 field campaign. Even if the chemical composition of PM2.5 and newly formed particle could be different, it is well-recognized that pinic and pinonic acids are good tracers for SOA formed from α-and β-pinene ozonolysis [START_REF] Claeys | Terpenylic acid and related compounds from the oxidation of α-pinene: Implications for new particle formation and growth above forests[END_REF][START_REF] Faiola | Chemical characterization of biogenic SOA generated from plant emissions under baseline and stressed conditions: inter-and intra-species variability for six coniferous species[END_REF][START_REF] Hallquist | The formation, properties and impact of secondary organic aerosol: current and emerging issues[END_REF][START_REF] Jaoui | Identification and quantification of aerosol polar oxygenated compounds bearing carboxylic or hydroxyl groups. 2. Organic tracer compounds from monoterpenes[END_REF][START_REF] Kristensen | Denuder/filter sampling of organic acids and organosulfates at urban and boreal forest sites: Gas/particle distribution and possible sampling artifacts[END_REF][START_REF] Nozière | Atmospheric chemistry in stereo: A new look at secondary organic aerosols from isoprene[END_REF][START_REF] Yasmeen | Mass spectrometric characterization of isomeric terpenoic acids from the oxidation of α-pinene, β-pinene, d-limonene, and delta-3-carene in fine forest aerosol[END_REF]. Thus, the presence of such markers in PM2.5 would support our hypothsis. During July 2015, pinic and pinonic acids were detected and quantified in all samples. Both products were found in large quantities in the particles collected at Bilos, in every 24 hours filters. Mean concentration of pinic acid was 18.3 ng m -3 , and reached the maximum value of 33.8 ng m -3 (Table 3). These values are in the same order of magnitude as those previously reported in studies conducted in other rural forested areas around the world. For example, the concentration of pinic acid measured in a boreal forest (Hyytiälä, Finland) during summertime was 7.2 ng m -3 , and reached a maximal concentration of 31 ng m -3 . Pinic acid concentrations reported at other sites (Table 3) were reported to be beween 4 and 8 ng m -3 , slightly lower than those measured at Bilos. This is consistent with the high monoterpene mixing ratios presented above. The mean concentrations of pinonic acid during the campaign was 10.2 ng m -3 , also close to the reported values in Table 3. The high concentrations of these markers arising from α-and β-pinene oxidation measured in this work support the high reactivity between monoterpenes and ozone, which is an important oxidation pathway for monoterpenes during nighttime at the Bilos site. This result attests that oxidation products of pinenes are well transferred to the particle phase, resulting from either gas/particle transfer during NPF or condensation. Further measurements of the size-resolved chemical composition of aerosols will be necessary to differentiate these processes.

In the present study, the concentrations of markers in the particle phase were in the same range as the reported values (or around twice as high), whereas monoterpene mixing ratios were at least one order of magnitude higher (Table 3). Such differences in the ratio between particulate SOA tracers concentrations and gaseous monoterpene mixing ratios suggest that different physico-chemical processes may occur at our site.

To further investigate the link between in-canopy monoterpene reactivity and nocturnal NPF, night and day filter sampling should be performed. [START_REF] Mochizuki | Long-term measurement of terpenoid flux above a Larix kaempferi forest using a relaxed eddy accumulation method[END_REF] observed strong differences in the concentration of night and day particulate SOA tracers. In the case of nocturnal NPF identified in this study, such information could help to unravell the detailed processes. the This will be considered in a next field campaign implemented at Bilos. Note that a large panel of other biogenic SOA tracers were detected in the particles collected. The detailed chemical composition of atmospheric particles collected during the campaign will be the subject of a future publication.

Table 3: Comparison of pinic and pinonic acid concentrations measured at the Bilos site in profiles, suggesting a possible secondary source of OH radicals from monoterpene ozonolysis during the night. Further work will be necessary to adress this issue. The particle phase analysis allowed to identify and quantify pinic and pinonic acids, confirming the oxidation of monoterpenes observed at Bilos as well as the contribution of these precursors to SOA. Considering the differences between 2014 and 2015 field campaigns in the frequency of occurrence of NPF, the monoterpene mixing ratios, and the observed monoterpene in-canopy reactivity, monoterpene ozonolysis is proposed to very likely contribute to nocturnal NPF at our site. Measurements of size-resolved aerosol composition will be required to determine more specifically to which extent monoterpene oxidation products are involved in nucleation and/or aerosol growth in the Landes pine tree forest.

The high NPF frequency of occurence observed in 2015 may be associated to the direct consequence of the hydric/thermic stress, which promotes strong BVOC emissions.

Considering that the number of hydric stress periods will continue to increase due to the global warming, it can be hypothesized that NPF events may be more and more important in southwestern France. More studies will thus be required to confirm the effect of hydric stress on NPF.

  Meteorological parameters are crucial as they may greatly influence BVOC emissions and the oxidative capacity of the atmosphere. The Bilos site is for instance frequently subject to periods of large hydric stress during summer. Detailed meteorological conditions during 2014 and 2015 field campaigns can be found in Figures S1 and S2. For each campaign, the conditions were very different. The 2015 field campaign was very hot, with few rain events (see Figures S1.a, d and Figures S2.

  e. 100%) almost every night, in both July 2014 and 2015. No rainfall was observed from the middle of June 2015 (meteorological parameters outside the campaign range are available from the ICOS network) and only few mm of rain was recorded between the 5 th and the 22 nd July in 2015. As a result, a very weak water vapour flux was observed in July 2015, whereas the sensible heat flux was very high (Figure S2.b), especially comparing to 2014 fluxes (Figure S1.b). The Bowen ratio (between sensible heat fluxes and water vapour fluxes) is a powerful tool to evaluate the hydric stress level. During drought, the water vapour fluxes decrease whereas the heat fluxes increase (by compensation to conserve the energy balance), making the Bowen ratio particularly elevated. Indeed, the 2015 campaign was characterized by Bowen ratio values about 10,

  monoterpene mixing ratios frequently (i.e. almost half of the nights) reached around 15 ppb, whereas they were always lower than 10 ppb in 2014, except the night of July 16 th to 17 th (Figure 1). The highest values were reached the 21 st of July 2015, where the sum of monoterpene mixing ratios exceeded 35 ppb (Figure 1.b). The very high mixing ratios of monoterpenes observed in 2015 were assumed to be the result of the meteorological conditions, and more specifically the thermic/hydric stress. The high frequency of occurrence of NPF in 2015 occurred for the highest monoterpene mixing ratios, emphasizing the role of monoterpenes at night at Bilos.

(

  because of the nocturnal stratified boundary layer). The anti-correlation between ozone and monoterpenes can be observed on Figure 2.a. Interestingly, when ozone was close to 0, monoterpene mixing ratios values were at least of 5 ppb during the corresponding nights (Figure 2.a). Reaction between ozone and monoterpenes in the early night could thus be expected. Such reactions could only partially explain the ozone drop to levels close to zero,

Figure 1 :

 1 Figure 1: Time series of the sum of monoterpene mixing ratios (in ppb, green line) and ozone mixing ratios (in ppb, red line) for a) 2014 and b) 2015 field campaigns.

  Figure 2.b shows that the α/β-pinene ratio mean diurnal cycle was more pronounced in 2015. Solar radiation, taken as a simple proxy for OH concentration, and ozone mixing ratios, were higher in 2015 than in 2014 (Figures 2.c and 2.d). Higher OH concentration or ozone mixing ratios led to a more pronounced increase or drop in the α/β-pinene ratio values, respectively. The Figure 2.b illustrates that the mean diurnal cycle of the α/β-pinene ratio logically followed the solar radiation and ozone diurnal profiles. The analysis of the mean diurnal evolution of the α/β-pinene ratio also supports the hypothesis of the importance of ozone reactions with monoterpenes in the afternoon/evening. To check this hypothesis, the presence (or absence) of monoterpene oxidation products in the gas phase was investigated.

Figure 2 :

 2 Figure 2: a) Anti-correlation between ozone and monoterpene mixing ratios b) mean diurnal profile of the α/β-pinene ratio c) mean diurnal profile of the ozone mixing ratios d) mean diurnal profile of global radiation, during each campaign. The error bars represent the standard errors of each variable. For each graphics, light blue squares represent 2014 dataset, and dark blue crosses represent 2015 dataset. (Universal time = local time -2h)

Figure 3 :

 3 Figure 3: Half hourly evolution of m/z 135.11 (C10H14), m/z 139.11 (C9H14O, nopinone), m/z 151.10 (C10H14O), m/z 155.13 (C10H18O), m/z 169.12 (C10H16O2, pinonaldehyde) and their sum. Y axes are unit less as signals (number of counts) have been normalized to their maximum. Missing data are due to calibrations and blanks of the instrument. The blue shaded areas denote the nighttime periods (defined as the time when global radiation was below 50 W m -2 ).

a

  [START_REF] Lee | Gas-phase products and secondary aerosol yields from the ozonolysis of ten different terpenes[END_REF] and references therein b[START_REF] Calogirou | Gas-phase terpene oxidation products: a review[END_REF] c recommended IUPAC values[START_REF] Atkinson | Evaluated kinetic and photochemical data for atmospheric chemistry: Volume II: gas phase reactions of organic species[END_REF] d[START_REF] Hallquist | The formation, properties and impact of secondary organic aerosol: current and emerging issues[END_REF]

Figure 4 :

 4 Figure 4: Size distribution of particles between 10 nm and 478.3 nm, during two typical NPF events observed at Bilos in July 2015. On lower panels: the half hourly evolution of m/z 139.21 (C9H14O, nopinone) and m/z 169.24 (C10H16O2, pinonaldehyde) normalized signals, total monoterpene and ozone mixing ratios are represented. Intensities of oxidation product signals (number of counts) have been normalized to their maximum. Blue shaded areas denote the nighttime periods (defined as the time when global radiation was below 50 W.m -2 ).

  The nearest urban area is Bordeaux and surroundings, at 40 km northeast, and the Atlantic Ocean is 23 km west of the site. The measurement area is a maritime pine (Pinus pinaster Aiton) stand sowed in 2004, with an understorey mostly

	composed of gorse (Ulex europaeus L.), grass (Molinia caerula (L.) Moench) and heather
	(Calluna vulgaris (L.) Hull). Tree height was around 7 m in 2014 and 8 m in 2015, over a
	sandy hydromorphic podzol. A more complete description of Bilos site can be found
	elsewhere

The measurement site, located at Bilos (44°29'39.69"N, 0°57'21.75"W, and 37 m above sea level) in the northern part of the Landes forest, is a measurement site of the European Integrated Carbon Observation System (ICOS).

  As a consequence, and taking into account the nocturnal nature of NPF, it is highly probable that sulphuric acid was not responsible for nocturnal NPF at Bilos, conversely to classical daytime NPF. Ozone mixing ratios presented a typical diurnal cycle with maximal values reached during the afternoon, and frequently dropped under the analyser limit (i.e. 0.5 ppb) in the middle of the night. The nights of July 15 th and 16 th in 2014 and 12 th to 15 th , 17 th , 18 th and 20 th in 2015 are perfect examples of this ozone drop (Figures

	S3.c and S4.c). The meteorological conditions observed during these nights indicate that
	these ozone drops occurred under stratified atmosphere conditions (low wind speed and high
	1/u*).
	NPF were very likely influenced by local emissions, limiting the impact of transported
	compounds.
	NO, NO2, SO2 (only for 2015 campaign) and ozone mixing ratios are represented on Figures
	S3 and S4. NOx mixing ratios were very low during both campaigns, confirming the very rural

1/u* (where u* is the friction velocity) over 2.5 s m -1 , indicating vertical stratification (this parameter was usually below 2 s m -1 during the day where turbulence occurred, see the meteorological parameters in Figures S1 and S2 during NPF events). It shows that NPF started once the stable nocturnal boundary layer was taking hold. As suggested by the air mass analysis, nocturnal feature of the Bilos site. The mixing ratios of SO2, which is recognized as a key species for daytime nucleation, was always around the DL value of the instrument (only available in 2015, Figure S4.b).

Table 1 :

 1 Chemical kinetic rate constants of reactions of α-and β-pinene with the main

	atmospheric oxidants (OH, O3 and NO3)
		Rate constant (at 298K, in cm 3 molecule -1 s -1 )
		OH	O3	NO3
	α-pinene	(5.3±0.2) × 10 -11	(9.0±0.2) × 10 -17	(6.2±0.1) × 10 -12
	β-pinene	(7.6±0.1) × 10 -11	(1.9±0.3) × 10 -17	(2.5±0.1) × 10 -12

07/072019 07/092019 07/112019 07/13/2019 07/15/2019 07/17/2019 07/19/2019 07/21/2019 07/23/2019 07/072019 07/092019 07/112019 07/13/2019 07/15/2019 07/17/2019 07/19/2019 07/21/2019 07/23/2019

Acknowledgements

This work was supported by the French Environment and Energy Management Agency (ADEME), the CNRS-INSU LEFE-CHAT research program, the NEEDS action, and the PEPS IdEx Bordeaux. The authors want to acknowledge the Bilos ICOS team (Christophe Chipeaux, Sébastien Lafont and Denis Loustau) for meteorological data and site availability and Michael Staudt (CEFE-CNRS) for the loan of the GC-FID during the campaign. We also thank Didier Garrigou (ISPA-INRA) for his precious help during the field campaigns, especially in the installation and the maintenance of instruments.

Conclusions

Two intensive field measurement campaigns were conducted in the French Landes forest, at Bilos, in the summers of 2014 and 2015. Frequent nocturnal NPF events were observed during periods of strong thermic and/or hydric stress (dry and hot summer). In parallel, monoterpene mixing ratios (dominated by α-and β-pinene) reached very high levels almost every night, frequently over 20 ppb in summer 2015. A strong anti-correlation between ozone and monoterpene mixing ratios was highlighted, suggesting a high chemical reactivity. This hypothesis was reinforced by the mean diurnal variation of the ratio between α-and β-pinene mixing ratios. Products from the oxidation of α-and β-pinene, namely pinonaldehyde and nopinone, were measured in the gas phase. The diurnal profiles of these two oxidation products is consistent with the high reactivity between α-and β-pinene mostly with ozone in the evening. Furthermore, pinonaldehyde and nopinone presented different nocturnal

Competing interests:

The authors declare no competing of interest.


Kernéis

Domenech De Cellès

Mira Jp

Variable impact of an antimicrobial stewardship programme in three intensive care units: time-series analysis of 2012-2017 surveillance data

Keywords: antimicrobial stewardship, carbapenems, Pseudomonas aeruginosa, timeseries modeling, intensive care unit

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

Introduction

Multidisciplinary antimicrobial stewardship (AMS) programmes have proven effective to decrease mortality, to improve quality of antibiotic prescriptions and to reduce unnecessary antibiotic use [START_REF] Davey | Interventions to improve antibiotic prescribing practices for hospital inpatients[END_REF][START_REF] Schuts | Current evidence on hospital antimicrobial stewardship objectives: a systematic review and meta-analysis[END_REF]. Implementation of such programmes is therefore strongly encouraged in all healthcare settings [START_REF] Barlam | Implementing an Antibiotic Stewardship Program: Guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America[END_REF][START_REF] Pulcini | Antibiotic stewardship: update and perspectives[END_REF]. However, when planning implementation of an AMS programme at a hospital scale, prioritizing strategies may be challenging, especially in resource-limited settings. Both restrictive and enabling approaches have been used, and their impact is variable [START_REF] Davey | Interventions to improve antibiotic prescribing practices for hospital inpatients[END_REF][START_REF] Tamma | What Is the More Effective Antibiotic Stewardship Intervention: Preprescription Authorization or Postprescription Review With Feedback?[END_REF][START_REF] Mehta | Comparison of prior authorization and prospective audit with feedback for antimicrobial stewardship[END_REF][START_REF] Delory | Impact of a program combining pre-authorization requirement and post-prescription review of carbapenems: an interrupted time-series analysis[END_REF]. In a recent prospective crossover trial [START_REF] Tamma | What Is the More Effective Antibiotic Stewardship Intervention: Preprescription Authorization or Postprescription Review With Feedback?[END_REF], a restrictive approach consisting in requirement of pre-prescription approval (PPA), was compared to a post-prescription review with feedback (PPRF) approach. PPRF was more efficient to reduce antibiotic use, suggesting that prioritizing such intervention would have the most significant impact [START_REF] Tamma | What Is the More Effective Antibiotic Stewardship Intervention: Preprescription Authorization or Postprescription Review With Feedback?[END_REF]. Conversely, other authors reported an increase of cefepime, piperacillin/tazobactam and vancomycin use when PPA was replaced to PPRF in a prospective non comparative study [START_REF] Mehta | Comparison of prior authorization and prospective audit with feedback for antimicrobial stewardship[END_REF].

PPRF requires important human resources that hamper simultaneous implementation in all medical wards of a health-care setting. In most published studies, programmes were either implemented at a hospital scale with substantial dedicated human and financial resources, or restricted to specific departments [START_REF] Chung | Antimicrobial stewardship: A review of prospective audit and feedback systems and an objective evaluation of outcomes[END_REF]. There is currently limited evidence on the impact of PPRF at a hospital scale in real-life conditions. In the French context, AMS programmes are primarily supervised by infectious diseases clinicians in addition to their routine clinical tasks, with limited dedicated time to AMS activities [START_REF] Pulcini | Human resources estimates and funding for antibiotic stewardship teams are urgently needed[END_REF]. Following national and international recommendations, we implemented in our 1500-bed university hospital a stewardship programme based on PPA and PPRF targeting carbapenem prescriptions. The goal of this study was to assess and compare, in real-life conditions, the impact of PPA and PPRF on antibiotic use and resistance of Pseudomonas aeruginosa in three intensive care units (ICU) within a same healthcare setting.

Methods

Setting, participants and intervention

This retrospective observational study was conducted between 2012 and 2017 in the Hôpitaux Universitaires Paris Centre (HUPC), a 1500-bed university hospital, Paris, France. Patients from three distinct intensive care units were included: ICU1 (24 beds) including patients with severe medical conditions (e.g. severe community-acquired sepsis, out-of-hospital cardiac arrest, etc.), ICU2 (10 beds) post-operative patients after gastrointestinal surgery and ICU3 (16 beds) post-operative patients following thoracic surgery. All ICU patients who had received at least one dose of carbapenem (meropenem or imipenem) over the study period were included. The study was conducted in accordance with STROBE-AMS guidelines [START_REF] Tacconelli | STROBE-AMS: recommendations to optimise reporting of epidemiological studies on antimicrobial resistance and informing improvement in antimicrobial stewardship[END_REF].

PPA alone, then PPA + PPRF were successively implemented in 2012 and 2016 in all medical, surgical and intensive care units of the hospital for carbapenem prescriptions. The programme was supervised as part of routine work by the multidisciplinary team available on site, composed of one pharmacist in charge of antibiotics deliveries and of three Infectious Diseases (ID) physicians dedicated to giving daily individual bedside advice for patients with infectious diseases. No additional resources were allocated to implement the AMS programme. Procedures for PPA and PPRF were as reported elsewhere [START_REF] Tamma | What Is the More Effective Antibiotic Stewardship Intervention: Preprescription Authorization or Postprescription Review With Feedback?[END_REF][START_REF] Canouï | Assessment of appropriate carbapenem prescribing in stewardship programmes[END_REF]. With PPA the prescriber had to seek input from the pharmacists prior to administration of carbapenems. For each carbapenem prescription, the role of the pharmacist was to check whether the indication was concordant with the local guideline, and if not, to discuss alternatives with the clinician in charge. In the second period (PPA+PPRF), in addition to PPA, pharmacists identified new carbapenem prescriptions on a daily basis and sent the list of patients to the ID physicians of the AMS team. In the ensuing 48-72 hours, ID physicians visited the patients, reviewed the clinical charts and interviewed the prescribing physicians during a bedside visit. Based on available diagnostic tests and clinical course, the AMS team advised the clinician with recommendations for stopping, discontinuing, or adjusting therapy. Both ID physicians and pharmacists followed a local guideline derived from national recommendations on treatment durations and promotion of non-carbapenems alternatives [START_REF] Canouï | Assessment of appropriate carbapenem prescribing in stewardship programmes[END_REF][START_REF] Bretonnière | Strategies to reduce curative antibiotic therapy in intensive care units (adult and paediatric)[END_REF][START_REF] Wintenberger | Proposal for shorter antibiotic therapies[END_REF]. The guideline also recommended preferential use of meropenem rather than imipenem based on literature data suggesting better tolerance and greater in vitro activity against Gram-negative pathogens [START_REF] Mohr | Update on the Efficacy and Tolerability of Meropenem in the Treatment of Serious Bacterial Infections[END_REF].

Antibiotic use

Carbapenem use was estimated from aggregate data of imipenem and meropenem pharmacy deliveries, and expressed as Defined Daily Doses (DDD) per 1000 occupied bed-days (BD).

DDD were defined according to ATC/DDD index 2014 [START_REF]WHO Collaborating Centre for Drug Statistics Methodology[END_REF]. Imipenem and meropenem have the same DDD of 2 grams per 24 hours. However, individual dosing may vary widely between patients, mainly depending on the severity of clinical presentation (typically 1.5g to 3g/24 hours for imipenem and 3 to 6 g/24h for meropenem, the highest doses being used in the most critical patients [START_REF] Giannella | Effect of combination therapy containing a high-dose carbapenem on mortality in patients with carbapenem-resistant Klebsiella pneumoniae bloodstream infection[END_REF]). As previously described, using non-standard doses of antibiotics can affect calculation of DDD [START_REF] Barlam | Implementing an Antibiotic Stewardship Program: Guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America[END_REF]. For instance, the highest recommended dose would account for 1.5 DDD for imipenem (3g/24 hours) and 2 DDD for meropenem (6g/24 hours), therefore biasing comparisons between molecules. To account for these discrepancies, we randomly selected ten prescriptions of imipenem and meropenem per year and per intensive care unit (n=360 prescriptions) and collected data on individual doses actually prescribed. For each unit, we then calculated a correction coefficient, defined as the mean daily dose of meropenem divided by the mean daily dose of imipenem. Corrected meropenem consumptions were calculated by dividing raw consumptions by the correction coefficient in each unit.

Antimicrobial resistance

Identification of microorganisms used MALDI-TOF and susceptibility testing was performed by disc diffusion methods according to EUCAST guidelines. An isolate was considered non susceptible when tested and interpreted as resistant (R) or with intermediate susceptibility (I) in accordance with the French and European clinical breakpoint criteria (EUCAST) [START_REF]Surveillance of antimicrobial resistance in Europe -Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net)[END_REF]. Data were expressed as percentage of resistance (PR), defined as the percentage of non-susceptible (I+R) isolates, out of all isolates with antimicrobial susceptibility testing. Duplicates from the same patient were eliminated taking only the first by date of sample collection and isolate source, and defined as the presence of similar susceptibility patterns for all antibiotics from the standard list defined by the EUCAST for the species. We first studied the global resistance to carbapenems, defined as resistance to either imipenem, meropenem, or both. Trends of individual resistance to one of the two molecules (imipenem or meropenem) were secondly assessed and are presented in the supplementary material. The incidence of extendedspectrum β-lactamase-producing Enterobacteriacae (ESBL-E) from all isolates was assessed per year and per 1000 BD.

Data analysis

Two periods were analyzed: 2012-2015 (PPA alone) and 2016-2017 (both PPA and PPRF).

Analysis of aggregated data was performed through a controlled interrupted time-series method. The time-series was divided into a pre and post intervention segment. We estimated the three-monthly consumption of carbapenems with a multiple linear regression model

= + × + × ( ) , - + ( + ! ) × + × ( )
, -

In this model, t is a continuous variable indicating the time in trimester since 2012. β0 is the intercept which estimates the baseline consumption of carbapenems, β1 is the change in consumption during the pre-intervention period, β2 is the change of slope between the postintervention and the pre-intervention period, and γ the parameter associated to the incidence of ESBL. Since local epidemiology may affect the use of carbapenems, all models were adjusted on the annual incidence of ESBL-E.

We estimated the three-monthly percentage of resistance (PR) of P. aeruginosa to carbapenems with a Poisson loglinear regression model:

log ( % & ' (' ) = ) + × + log( % & (' ) , - + ( + ! ) × + log( % & (' ) , -
Statistical analysis and modeling were performed using the R software (3.3.2) [START_REF] Core | R: A language and environment for statistical computing[END_REF].

Anonymized routine surveillance data were stored on a secured database regularly declared to the national competent authorities (Commission Nationale Informatique et Libertés). For this type of observational retrospective study, formal consent and permission of an institutional review board are not required.

Results

Antibiotic use

Over There was however a switch between imipenem and meropenem: while overall consumption of imipenem steadily decreased, meropenem increased over the PPA period in all three units (tables S1 and S3, supplementary material).

Within each ICU, carbapenem use showed different patterns. In ICU 1, carbapenem use was stable over the PPA period, along with a progressive replacement of imipenem by meropenem (Figure 1). S4).

Discussion

In this retrospective study based on surveillance data, we report heterogeneous impact of an AMS programme within three ICUs of the same hospital. Carbapenem use did not change over the PPA period in all three ICUs, and significantly decreased in only one ICU after implementation of PPRF. A switch from imipenem to meropenem was observed in the three ICUs during the PPA period. Resistance rates showed similar patterns and were stable over the study period within the three units.

As previously reported, PPA alone had a limited impact on the overall carbapenem use in our setting, apart from a switch from imipenem to meropenem use. This switch during the study period was probably related to adherence to local guidelines, actively promoted by the pharmacists and ID physicians of the AMS team [START_REF] Mohr | Update on the Efficacy and Tolerability of Meropenem in the Treatment of Serious Bacterial Infections[END_REF]. In the trial by Tamma et al., antibiotic consumptions remained steady during PPA periods in both arms [START_REF] Tamma | What Is the More Effective Antibiotic Stewardship Intervention: Preprescription Authorization or Postprescription Review With Feedback?[END_REF]. A Cochrane review and meta-analysis confirmed that restrictive strategies were less effective than enabling interventions with feedback to enhance compliance to antibiotic strategies [START_REF] Davey | Interventions to improve antibiotic prescribing practices for hospital inpatients[END_REF]. Moreover, several studies revealed unintended consequences of PPA: negative professional culture through breakdown in trust and communication [START_REF] Baysari | Audit and feedback of antibiotic use: utilising electronic prescription data[END_REF][START_REF] Linkin | Effect of communication errors during calls to an antimicrobial stewardship program[END_REF] and delay in time to first antibiotic dose [START_REF] Strom | Unintended Effects of a Computerized Physician Order Entry Nearly Hard-Stop Alert to Prevent a Drug Interaction: A Randomized Controlled Trial[END_REF] . In practice, there are many barriers for implementation of PPA in a healthcare setting. PPA requires someone to be available to answer requests in real time, 24/7. Antibiotic approval practices may also differ between stewardship practitioners depending on their background, level of experience [START_REF] Canouï | Assessment of appropriate carbapenem prescribing in stewardship programmes[END_REF] . In many health-care settings, pharmacists are dedicated to this task. However, even if extensively trained on antibiotic stewardship, most pharmacists feel to have limited capacity to influence antibiotic decision-making, mainly due to the perception of antibiotic use as a medical responsibility [START_REF] Broom | What role do pharmacists play in mediating antibiotic use in hospitals? A qualitative study: Table 1[END_REF] . This is particularly true in ICUs where patients are critically unwell, at high risk for carriage of multidrug-resistant bacteria, and where physicians are highly experienced in prescribing antibiotics. All these limitations raise concerns on effectiveness of PPA in practice and even more of its medico-economic impact.

PPRF showed encouraging positive effects [START_REF] Tamma | What Is the More Effective Antibiotic Stewardship Intervention: Preprescription Authorization or Postprescription Review With Feedback?[END_REF][START_REF] Delory | Impact of a program combining pre-authorization requirement and post-prescription review of carbapenems: an interrupted time-series analysis[END_REF][START_REF] Abbara | Impact of a multimodal strategy combining a new standard of care and restriction of carbapenems, fluoroquinolones and cephalosporins on antibiotic consumption and resistance of Pseudomonas aeruginosa in a French intensive care unit[END_REF] , leading to its positioning as a cornerstone of AMS programmes [START_REF] Pulcini | Factors associated with adherence to infectious diseases advice in two intensive care units[END_REF] . In our study, PPRF showed a significant impact on carbapenem consumption in one out of three ICU units. In comparison, Abbara et al. reported in a single ICU a decrease in broad spectrum antibiotics use -among which carbapenemsand resistance of P. aeruginosa after a strategy combining: i) systematic PPRF of all antibiotics, ii) restriction of broad spectrum antibiotics through local guidelines. While our AMS programme targeted only carbapenems in the whole hospital, their AMS programme targeted all broad-spectrum antibiotics in a single ICU. Like PPA, PPRF is a resourceconsuming strategy since members of the AMS team must be available on site to review prescriptions within 24-48 hours. Thus, focusing the AMS programme on a single ICU could partly explain their different results. Second, effectiveness of the strategy highly depends on adherence of the caring physicians to recommendations of the AMS team [START_REF] Pulcini | Factors associated with adherence to infectious diseases advice in two intensive care units[END_REF][START_REF] Evelyn | Why Don't They Listen? Adherence to Recommendations of Infectious Disease Consultations[END_REF]. Factors that drive adherence to ID advices are not fully understood. Several reports underlined that the strongest drivers were stewards and prescribers' behavior and beliefs, rather than patients' characteristics [START_REF] Pulcini | Factors associated with adherence to infectious diseases advice in two intensive care units[END_REF][START_REF] Evelyn | Why Don't They Listen? Adherence to Recommendations of Infectious Disease Consultations[END_REF][START_REF] Perozziello | Experiences and perspectives of implementing antimicrobial stewardship in five French hospitals: a qualitative study[END_REF]. Recent qualitative work investigated that issue, putting forward factors such as local culture and practices, physicians' personal experiences, involvement of supra-level bodies like hospital authorities, physician's interest and willingness to conform to guidelines, competition between physicians and AMS team's qualifications and tasks, lack of interaction between the AMS team and senior doctors while care delivery is very hierarchically structured [START_REF] Perozziello | Experiences and perspectives of implementing antimicrobial stewardship in five French hospitals: a qualitative study[END_REF][START_REF] Charani | Implementation of antibiotic stewardship in different settings -results of an international survey[END_REF][START_REF] Rzewuska | Prioritizing research areas for antibiotic stewardship programmes in hospitals: a behavioural perspective consensus paper[END_REF]. Because these factors are very heterogeneous, it is difficult to anticipate in which setting PPRF is likely to have the highest impact.

Our results on antibiotic resistance were deceiving, despite a marginal decrease of imipenem resistance during the PPA+PPRF period in a single ICU. The number of P.aeruginosa isolates in this ICU was low which could account for a high variability of results. Moreover, this was observed in only one ICU, while imipenem consumption had been previously decreasing for several years in all three ICUs. If the switch from imipenem to meropenem had an impact on P.aeruginosa resistance to imipenem, we would have assumed to see a sustainable and similar effect in the three ICUs, which we did not observe.

Several studies suggest a strong correlation between carbapenem use and the proportion of carbapenem-resistant P. aeruginosa isolates, particularly in settings where chromosomally encoded resistance mechanisms (loss of OprD porin, overproduction of the MexAB efflux system) are predominant, like in France. For instance, in a retrospective study in a French ICU [START_REF] Angebault | Emergence of imipenem resistant gram-negative bacilli in intestinal flora of intensive care patients[END_REF], imipenem resistance of P. aeruginosa strains was mainly due to chromosomally encoded resistance mechanisms, and the main risk factor for resistance was prior exposure to imipenem (Odds ratio = 6.8 [95% CI, 1.2 to 50.2] when patients received > 3 days of imipenem). The correlation between antibiotic use and emergence of resistance is not straightforward for other Gram-negative bacteria (i.e. resistance to 3GC and ESBL-Enterobacteriacae), explaining why we specifically chose to focus on the pairing of "P.

aeruginosa / carbapenems" to evaluate the impact of the programme.

Failure to show any impact of our strategies on carbapenems resistance could be due to a real lack of effect, to confounding factors, or a too short period of observation after implementation of PPRF (2 years). The effect of AMS programmes on antibiotic resistance, in particular of P. aeruginosa, remains unclear and inconsistent between studies [START_REF] Davey | Interventions to improve antibiotic prescribing practices for hospital inpatients[END_REF][START_REF] Baur | Effect of antibiotic stewardship on the incidence of infection and colonisation with antibioticresistant bacteria and Clostridium difficile infection: a systematic review and metaanalysis[END_REF][START_REF] Karanika | Systematic Review and Meta-analysis of Clinical and Economic Outcomes from the Implementation of Hospital-Based Antimicrobial Stewardship Programs[END_REF].

Actual evidence shows too much variance in microbial outcomes to reliably assess the effect of change in antibiotic use on gram-negative bacteria and to discriminate the impact of each strategy [START_REF] Karanika | Systematic Review and Meta-analysis of Clinical and Economic Outcomes from the Implementation of Hospital-Based Antimicrobial Stewardship Programs[END_REF].

This study has several limitations. As it is an uncontrolled study, PPA and PPRF occurred in two different periods, during which other changes could have impacted antibiotic prescribing practices. Because carbapenems are mainly used in the treatment of ESBL-E infections, we adjusted our models on the incidence of ESBL-E isolates from each unit. However, in all models, the parameter was not statistically significant, meaning that the ESBL-E incidence had no significant impact on carbapenem consumption over the study period. Still, other factors impacting bacterial resistance were not assessed, as fluoroquinolones' cross-over impact on carbapenem resistance, clinical characteristics of the patients, iatrogenic factors (use of invasive devices, mechanical ventilation, etc.) [START_REF] Falagas | Risk factors for the isolation of multi-drug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa: a systematic review of the literature[END_REF][START_REF] Boyer | Pseudomonas aeruginosa acquisition on an intensive care unit: relationship between antibiotic selective pressure and patients' environment[END_REF][START_REF] Mesaros | Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium[END_REF]. Another limitation is that we did not assess adherence to PPRF, while it is a major success factor in non-coercive strategies [START_REF] Broom | What role do pharmacists play in mediating antibiotic use in hospitals? A qualitative study: Table 1[END_REF][START_REF] Pulcini | Factors associated with adherence to infectious diseases advice in two intensive care units[END_REF]. Finally, there was a high variability over time in carbapenem consumption on a microscale level in each ICU. Because we reported antibiotic deliveries and not antibiotic use, this may be explained by stockpiling in the three units.

Until now, most published studies analyzed the effect of AMS strategies in a single setting, while we compared the effect of one strategy led by the same AMS team in three different settings from the same hospital [START_REF] Baur | Effect of antibiotic stewardship on the incidence of infection and colonisation with antibioticresistant bacteria and Clostridium difficile infection: a systematic review and metaanalysis[END_REF]. Analyzing impact at hospital level increases statistical power, but can also hide differential impacts within different medical units. Our study reports observational surveillance data, offering the opportunity to observe real-life effects of PPA and PPA+PPRF. In this pragmatic approach, our goal was to observe whether any trend would be observed after implementation of the programme, and to compare different settings.

This approach does not allow to formally relate a change in resistance to a change in antibiotic use. However, our data show that in real-life conditions, when implementing an AMS program, variable impact may be observed depending on the setting, within the same hospital. Taking that variability into account is important for deciding how to effectively split resources allocated to AMS programmes.

Conclusion:

In real life conditions, after implementation of an AMS programme based on systematic post-prescription review of all carbapenem prescriptions, we report a decrease of antibiotic use in only one out of three ICUs, with no clear impact on carbapenem resistance of P. aeruginosa. This suggests that within the same hospital, systematic post-prescription review by an antibiotic expert may have a heterogeneous impact from a unit to another. Due to financial and organizational constraints, such an approach should be prioritized in specific wards and predictors of successful implementation still need to be assessed. Black line: corrected carbapenem consumption (imipenem+meropenem).

Blue line: imipenem consumption.

Red line: corrected meropenem consumption.

Overall incidence of ESBL in all ICUs, from 2012 to 2017, per 1000 occupied bed-days :

  the 3 ICUs, raw consumption of carbapenems was 131 DDD/1000 BD in 2012 and 162 DDD/1000 BD in 2017. In the sample of 360 prescriptions, the mean individual dosage was 2.5g /24 hours (standard error (SE)=1.0) for imipenem and 5.2g /24 hours (SE=1.5) for meropenem, leading to a correction coefficient of 2. The interrupted time-series showed no change in overall corrected carbapenem use during the PPA period (β1=+0.1, 95% confidence interval (CI)= -13.1;+13.3), and no change of slope over the PPA+PPRF period (β2=-3.3, CI= -9.4;+2.8).

Figure 1 .

 1 Figure 1.

Figure 2 Figure 1 :

 21 Figure 2.

  and ICU 3, a switch from imipenem to meropenem also occurred during the PPA period, but we observed no impact of PPRF neither on overall consumption, nor imipenem and meropenem use.

	As shown in table 1, in all units, the annual incidence of ESBL-E was not statistically
	associated with carbapenem use.
	Antimicrobial resistance
	The overall PR of P. aeruginosa to carbapenems in the three ICUs was of 40% in 2012 and
	35% in 2017 (imipenem : 33% in 2012, 27% in 2017; meropenem : 23% in 2012, 24% in
	2017).

After implementation of PPRF, overall carbapenem consumption significantly dropped, related to a change of slope for both imipenem (β2=-7.57, CI = -12.69; -2.45, table

1

) and, to a lesser extent, meropenem use (β2=-11.39 CI = -25.23; +2.46, table

1

). In ICU 2

Global resistance of P. aeruginosa to carbapenems (imipenem and/or meropenem) was stable during the PPA period, with no significant change after implementation of PPRF over the three units (figure

2

, table 2). Similar patterns were observed in ICU 1 and ICU 2. In ICU 3, a significant decrease in global resistance was observed in the PPA+PPRF period, related to a decrease of imipenem resistance (β2=-0.19, CI=-0.4;-0.01, table

S2

), whereas meropenem resistance remained stable (β2=-0.06, CI=-0.26;+0.15, table

Figure 2: Proportion of resistance to imipenem and/or meropenem in Pseudomonas aeruginosa isolates over time This figure is online-only color a

  3.34, 3.34, 2.71, 2.71, 2.13, 2.11 ) in all ICUs, b) in ICU 1, c) in ICU 2, d) in ICU 3 Total number of isolates per year, from 2012 to 2017 : ICU 1 [34/27/23/30/20/19], ICU 2 [12/12/14/5/8/7], ICU 3 [15/11/24/21/8/7]

Acknowledgments: none.

Funding: none.

Declarations of interest:

MDdC received research funding from Pfizer for a project related to meningococcal epidemiology and independent from this work. Other co-authors declare no conflict of interest.

Tables

 Table 1.Parameters of the multiple linear regression models for corrected carbapenem use