Ningxin Chen
email: ningxin.chen@upmc.fr

Danièle Valdes

Christelle Marlin

Pierre Ribstein

Fabrice Alliot

Emmanuel Aubry

Hélène Blanchoud

Transfer and degradation of the common pesticide atrazine through the unsaturated zone of the Chalk aquifer (Northern France)

Keywords: Chalk groundwater, Atrazine, Unsaturated zone, Clay-with-flints

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

Introduction

Chalk aquifer provides a large quantity of drinking water in Europe [START_REF] Lapworth | Persistent and emerging micro-organic contaminants in Chalk groundwater of England and France[END_REF]. The groundwater extracted from the Chalk aquifer of the Paris Basin supplies 70% of the drinking water consumed in the north of France [START_REF] Lallahem | Structure et modélisation hydrodynamique des eaux souterraines : application à l'aquifère crayeux de la bordure nord du Bassin de Paris[END_REF]. In the UK, the Chalk aquifer provides almost 60% of the total withdrawal groundwater [START_REF] Azeez | Hydrology of the Chalk Aquifer in East Yorkshire from Spring Recession Analysis[END_REF]. It is also a substantial water resource to sustain and exchange with river flows [START_REF] Grapes | Dynamics of riveraquifer interactions along a chalk stream: the River Lambourn, UK[END_REF][START_REF] Sear | Groundwater dominated rivers[END_REF]. Water quality of the Chalk aquifer is an important issue. Beginning in the 1970s, the impact of intensive agriculture on groundwater has been demonstrated [START_REF] Headworth | Contamination of Groundwaters from Diffuse Sources arising from Farming Activities[END_REF][START_REF] Johnson | Penetration of herbicides to groundwater in an unconfined chalk aquifer following normal soil applications[END_REF]. Numerous studies have shown degradation of groundwater quality of the Chalk aquifer due to the use of fertilizers and pesticides [START_REF] Baran | Agricultural diffuse pollution in a chalk aquifer (Trois Fontaines, France): Influence of pesticide properties and hydrodynamic constraints[END_REF][START_REF] Brouyère | Migration of contaminants through the unsaturated zone overlying the Hesbaye chalky aquifer in Belgium: a field investigation[END_REF]. To protect groundwater, it's crucial to better understand the transfer of pesticides from the soil to the water table, and particularly how pesticides move through the unsaturated zone (UZ), which has not been fully understood because of limited access. The Saint-Martin-le-Noeud underground quarry provides access to the UZ of the Chalk aquifer with groundwater at different depths [START_REF] Barhoum | Spatial heterogeneity of high-resolution Chalk groundwater geochemistry Underground quarry at Saint Martin-le-Noeud, France[END_REF]. [START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF] studied the transfer of water and contaminants through the matrix in the UZ of the quarry but the transfer of the pesticides to the water table remains unclear.

Pesticides -Atrazine

The World Health Organization defines pesticides as chemical organic compounds that are used to kill pests, fungi and unwanted plants (weeds). The European Drinking Water Directive sets the maximum limit of 0.1 μg/L for each individual pesticide in drinking water and 0.5 μg/L for total pesticides, i.e., the sum of all the substances detected in a sample. Among pesticide substances, atrazine is one of the most widely applied herbicides. It is classified as a priority substance due to its toxicity to the environment (Official Journal of the European Communities, 2001). [START_REF] Gammon | A risk assessment of atrazine use in California: human health and ecological aspects[END_REF] showed that life-time exposure to atrazine may cause cancer. [START_REF] Araújo | Habitat fragmentation caused by contaminants: Atrazine as a chemical barrier isolating fish populations[END_REF] demonstrated that the presence of atrazine can affect the spatial distribution of fish in freshwater by forming a chemical barrier isolating fish populations. Exposure to atrazine can cause breaks in erythrocytes of Carrasius auratus, demonstrating the genotoxic potential of atrazine on fish [START_REF] Cavas | In vivo genotoxicity evaluation of atrazine and atrazinebased herbicide on fish Carassius auratus using the micronucleus test and the comet assay[END_REF]. In 2003, the application of atrazine was banned in the European Union, although it is still widely used in the United States [START_REF] Mahler | Similarities and differences in occurrence and temporal fluctuations in glyphosate and atrazine in small midwestern streams USA during the 2013 growing season[END_REF]. It is still one of the most frequently detected pesticides in freshwater [START_REF] Vryzas | Determination and aquatic risk assessment of pesticide residues in riparian drainage canals in northeastern Greece[END_REF].

Degradation is the process that transforms a substance into by-products.

The mechanisms of degradation are physical, chemical and biological [START_REF] Coats | Pesticide Degradation Mechanisms and Environmental Activation, in: Pesticide Transformation Products[END_REF]. The main degradation products are deethylatrazine (DEA), deisopropylatrazine (DIA) and hydroxyatrazine (HA). DEA is the most frequently detected degradation product of atrazine [START_REF] Cahoon | Chapter 6 -forensic water quality investigations: Identifying pollution sources and polluters[END_REF]. Biological processes are responsible for metabolizing atrazine to DEA in soil by microorganisms under aerobic conditions [START_REF] Solomon | Atrazine biodegradation efficiency, metabolite detection, and trzD gene expression by enrichment bacterial cultures from agricultural soil[END_REF]. The half-life (the time it takes for half of the pesticide to be degraded) of atrazine varies depending on soil type, soil pH and the atrazine quantity applied. [START_REF] Accinelli | Atrazine and metolachlor degradation in subsoils[END_REF] showed that the average half-life of atrazine in non-sterile soils is 49 days under aerobic conditions and 407 days under anaerobic conditions. The degradation of atrazine mainly occurs in soil, but it may also occur in the UZ with the presence of micro-organisms. [START_REF] Johnson | Potential for isoproturon, atrazine and mecoprop to be degraded within a chalk aquifer system[END_REF] found no potential degradation in the groundwater over a 200-day incubation. [START_REF] Issa | Degradation of atrazine and isoproturon in the unsaturated zone: a study from Southern England[END_REF] showed that the degradation of atrazine might occur in the UZ at a lower rate than in soil if micro-organisms are present.

Chalk aquifer

Chalk is a marine sedimentary rock. It is a carbonate rock, made up of more than 90% calcite and a variable proportion of dolomite [START_REF] Fabricius | Chalk: composition, diagenesis and physical properties[END_REF].

The Chalk aquifer has been considered a double/triple porosity aquifer made of matrix, fractures and conduits (in the most karstic areas). It is partly covered by a layer of clay-with-flints [START_REF] Laignel | Reconstruction of the Upper Cretaceous chalks removed by dissolution during the Cenozoic in the western Paris Basin[END_REF]. The clay-with-flints is the residual deposit formed from the dissolution, decalcification and cryoturbation of bedrock strata of the Chalk Group [START_REF] Jukes-Browne | The Clay-with-Flints; its Origin and Distribution[END_REF]. The thickness of the layer of clay-with-flints may reach several meters. [START_REF] Valdes | Infiltration processes in karstic chalk investigated through a spatial analysis of the geochemical properties of the groundwater: The effect of the superficial layer of clay-with-flints[END_REF] showed that the presence of clay-with-flints on the surface may have an impact on infiltration processes of water and solutes in the Chalk aquifer: it may store water on the surface and also create preferential pathways.

The conduits are developed in some areas of the Chalk aquifer, e.g., in Northern France. For the areas without conduits, the water transfer process may occur through the matrix and fractures. However, most studies considered that the main transfer process mainly occurs through the matrix [START_REF] Cooper | Soil controls on recharge to aquifers[END_REF][START_REF] Mathias | The significance of flow in the matrix of the Chalk unsaturated zone[END_REF]. The water transfer through fractures may be active occasionally depending on different conditions such as rainfall intensity and soil moisture [START_REF] Haria | Mechanisms of groundwater recharge and pesticide penetration to a chalk aquifer in southern England[END_REF][START_REF] Ireson | Controls on preferential recharge to Chalk aquifers[END_REF]. [START_REF] Van Den Daele | Unsaturated flow and solute transport through the Chalk: Tracer test and dual permeability modelling[END_REF] showed that the solute exchange between the matrix porewater and the porewater in the fractures is driven by two possible processes: advection and diffusion. For an organic component such as atrazine, the sorption, i.e,.

adsorption -desorption, is a major process controlling their fate [START_REF] Chefetz | Sorptiondesorption behavior of triazine and phenylurea herbicides in Kishon river sediments[END_REF]. Adsorption of an organic component on the Chalk delays the transport time, leading to a storage of the component in the Chalk UZ.

Pesticide study in the Chalk aquifer

For the past 30 years, most studies on pesticides have focused on soil components [START_REF] Gutierrez | Long-term transfer of diffuse pollution at catchment scale: Respective roles of soil, and the unsaturated and saturated zones (Brévilles, France)[END_REF] because it is widely considered that less than 1% of pesticides pass through soil components [START_REF] Flury | Transport of Anions and Herbicides in a Loamy and a Sandy Field Soil[END_REF]. Some studies observing pesticides (especially atrazine) in the groundwater show that the concentration can be higher than 0.1 μg L -1 , and some even higher than 0.5 μg L -1 [START_REF] Vonberg | 20 years of long-term atrazine monitoring in a shallow aquifer in western Germany[END_REF][START_REF] Lapworth | Source and persistence of pesticides in a semi-confined chalk aquifer of southeast England[END_REF]. [START_REF] Thurman | A reconnaissance study of herbicides and their metabolites in surface water of the midwestern United States using immunoassay and gas chromatography/mass spectrometry[END_REF] observed the river-groundwater interaction with the exchange of pesticides and showed the importance of studying pesticides in groundwater because it may also impact surface water. Few studies took an interest in the transfer of atrazine in the UZ of the Chalk aquifer. [START_REF] Baran | Agricultural diffuse pollution in a chalk aquifer (Trois Fontaines, France): Influence of pesticide properties and hydrodynamic constraints[END_REF] studied the infiltration processes of water with atrazine from 1989 to 2006, showing that the pesticide concentrations remained stable during periods of low-flow and increased episodically after rapid infiltration. [START_REF] Chilton | Pesticide fate and behaviour in the UK Chalk aquifer, and implications for groundwater quality[END_REF] studied the main factors controlling the fate of pesticides in order to establish a model predicting pesticide behavior in the groundwater. However, these studies examined water samples from wells whose characteristics result from all the possible transfer processes in the Chalk aquifer, in both the unsaturated and saturated zones, in which it is difficult to focus on the transfer only through the UZ. [START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF] showed that the transfer of atrazine in the UZ of the Chalk aquifer occurs mainly through the matrix with a velocity estimated between 0.7 and 2.1 m year -1 for the water percolating in the Saint-Martin-le-Noeud quarry (Northern France).

Objective of the study

The aim of this study is to characterize the processes of the transfer and degradation of atrazine in the UZ of the Chalk aquifer. The study was conducted in the Saint-Martin-le-Noeud underground quarry in the Paris Basin (Northern France). [START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF] have analyzed the ceiling percolation water in the same quarry. They have shown that the percolation water resulted mainly from the transfer through matrix. The quarry gives also a direct access to groundwater. Water table outcrops in the quarry, forming 16 lakes with different characteristics of UZ. The present study focus on the 16 underground lakes (water table). This study followed the spatio-temporal variation of the concentration of atrazine and its main degradation product DEA in groundwater has been followed. Spatio-temporal variations of the pesticides and nitrate are analyzed to characterize transfer processes in the unsaturated zone. Compared to nitrate variation, the variation of atrazine and DEA highlights the transfer process of pesticides including sorption effect.

Materials and methods

Site settings

Located in Saint-Martin-le-Noeud, 70 km north of Paris (Fig. 1), the underground quarry is 1200 m long and 150 m wide at an altitude of 165 m a.s.l. The coordinates of the quarry is 49.4 • N and 2.05 • E. The quarry was exploited from the 12th to the 18th century for chalk as a construction material, using room-and-pillar method. The quarry has been studied since 2012 [START_REF] Barhoum | Spatial heterogeneity of high-resolution Chalk groundwater geochemistry Underground quarry at Saint Martin-le-Noeud, France[END_REF][START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF].

The climate data used from January 2016 to April 2018 are recorded at the Beauvais station managed by Météo-France. The station is located 7 km from the quarry and is nearly at the same altitude. The average annual temperature is 11 • C (minimum temperature in January and maximum temperature in July).

The annual rainfall amount is 670 mm (values from Météo-France for the 1981-2010 period). An agricultural zone overlies the quarry. The landowner over the quarry provided information on agricultural practices beginning in 1974: cultivated fields contain corn, wheat and rapeseed. He claims that atrazine was used from 1977 to 2003.

Geology and hydrogeology

The quarry has been excavated in the Upper Cretaceous chalk layer, within the limit of Coniacian and Turonian. The excavation followed the dip of Chalk layers. The dip is about 12 • NNE. The Bray anticline is located at the western side of the quarry (Fig. 1). The Chalk aquifer, where the quarry is located, is considered to be an aquifer with two porosity types: matrix and fractures, since no karstic conduit was discovered. The quarry is partly covered by a layer of the clay-with-flints. The total porosity of the Chalk aquifer was estimated at 40% [START_REF] Lafrance | Étude des effets de leau sur les phnomnes de rupture et de deformation affectant les carrières souterraines de craie[END_REF].

Effective rainfall infiltrates through the unsaturated zone (chalk with or without clay-with-flints) before arriving into the quarry, where the water table outcrops at deeper parts. The water table forms 16 underground isolated lakes in the quarry (Fig. 2). The infiltrated water flows into the lakes by two pathways: percolating water dropping from the quarry ceiling and water circulating through the pillars and walls of the lake. The water of the lakes (corresponding to groundwater) is therefore a mixture of water coming from ceiling percolation and water passing through walls and pillars.

Characterization of the unsaturated zone of the study sites

The UZ thickness (from the soil surface to the quarry ceiling) varies spatially from 18 to 30 m. The thickness of the layer of clay-with-flints, measured with an electromagnetic ground conductivity geophysical instrument (EM31), varies from 30 cm to more than 5 m [START_REF] Barhoum | Spatial heterogeneity of high-resolution Chalk groundwater geochemistry Underground quarry at Saint Martin-le-Noeud, France[END_REF]. Method EM31 measures the electrical conductivity of soil. Soil with clay-with-flints has higher electrical conductivity than soil without clay-with-flints. However, the relation between thickness of clay-with-flints and electrical conductivity of soil is not a linear relation. The exact thickness of clay-with-flints was measured for 3 sites: 30 cm for PED (lowest electrical conductivity, 20 mS m -1 ); 2 m for BLE (electrical conductivity medial, 47.5 mS m -1 ); more than 3 m for STA (highest electrical conductivity, 72 mS m -1 ).

According to [START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF], the 16 sites (Fig. 2) can be divided into three groups according to the UZ thickness and thickness of the clay-with-flints layer.

-Group 1 (G1): CAS, CHA, BLE, PIL, JAR and LIT. The sites have the highest UZ thickness (around 30 m) with a thin layer of clay-with-flints (electrical conductivity between 30 and 60 mS m -1 ).

-Group 2 (G2): NAT, TSA, STA, LUC, VOU, NIC and PI2. The sites have a high UZ thickness (around 28 m) with a thick layer of clay-with-flints (electrical conductivity higher than 70 mS m -1 ).

-Group 3 (G3): SQU, CAR and PED. The sites have the lowest UZ thickness (around 20 m) with a thin layer of clay-with-flints electrical conductivity less than 30 mS m -1 ).

Data and methods

Sampling and measurements

Groundwater was sampled in 16 underground lakes (water table outcrops and porewater percolated through the UZ in quarry ceiling). From 2016 to 2018, 14 campaigns were conducted: 2016-04-12, 2016-06-06, 2016-06-23, 2016-09-05, 2016-10-20, 2017-01-09, 2017-02-27, 2017-05-02, 2017-06-26, 2017-09-05, 2017-10-23, 2018-01-16, 2018-04-10, 2018-05-16. The campaigns are divided into three groups. Table 1 presents the measurements and analysis done during each campaign.

For all 16 sites, the lake level and percolating volume were measured. For BLE, STA and PED, daily data of percolating flow rate and lake level were recorded from 2016-01-01 to 2018-05-16. Daily time series of the percolating flow rate were recorded using rain gauges. Daily time series of the lake level were recorded using CTD-Diver loggers.

The percolating flow was calculated as the ratio of the collected water depth (collected percolation volume divided by beaker area) between two field campaigns. In case of overflow, the percolating flow was underestimated. For each site, the lake level was standardized considering the lowest level as 0. Therefore, the standardized lake level of each campaign is the difference between the lake level measured and the lowest level.

Chemical analysis

Lake water from the 16 sites was sampled for nitrate, atrazine and DEA analysis. The nitrate concentration was analyzed by ion chromatography (Dionex ISC 3000). The accuracy is 1 mg L -1 with 5% uncertainty. The ionic balance error was checked with other major ions (HCO -

3 , K + , N a + , Ca 2+ , M g 2+ , Cl -; SO 2- 4
). Atrazine, DEA, DIA and HA were analyzed using an on-line method with liquid chromatography-tandem mass spectrometry (Agilent Technology)

with filtered samples (by 0.2 μm filters) [START_REF] Blanchoud | Submitted: Rapid on-line solid phase extraction LC MS/MS analysis for atrazine, its by-products, simazine and S metolachlor in groundwater samples[END_REF]. The quantification limit of this method is 20 ng L -1 with 20% uncertainty.

Atrazine degradation rate

Atrazine degradation rate can be expressed by DAR. DAR, introduced by [START_REF] Adams | Formation and transport of deethylatrazine in the soil and vadose zone[END_REF], is the ratio of the concentration of DEA, the main degradation product of atrazine to the concentration of atrazine, the primary molecule.

The equation to calculate DAR is: DAR = DEA / Atrazine. This parameter is used as an indicator of atrazine degradation processes. A higher DAR value means greater degradation of atrazine.

Results

Spatial variation

Figure 3 presents UZ thickness, the electrical conductivity of soil (indicator of the thickness of clay-with-flints), hydrodynamics (total percolation and standardized lake level), quality (concentration of nitrate, atrazine and DEA) and DAR of the lake for the 16 sites. In Fig. 3, the six campaigns in group A (April 2016 to September 2017, Table 1) were used for the boxplots, and all ten campaigns in groups A and B (Table 1) were used to calculate the total percolation

between April 2016 and September 2017. Within 1200 m, high spatial variation was observed in the hydrodynamics and the quality of the groundwater.

Among the 16 sites, the total percolation varied from 180 cm to more than 1500 cm during the same period covering nearly 1.5 years. The amplitude of lake level varied from 11 to 134 cm. LUC and VOU (G2) behaved very differently from the 14 other sites. These two sites had the highest total percolation (more than 1500 cm) but the smallest lake level amplitude (11 cm for LUC and 17 cm for VOU). Most of the sites had a lake level amplitude between 40 and 80 cm with a total percolation less than 1000 cm.

High spatial variation of nitrate concentration was observed. The medians of nitrate concentrations of the 16 sites varied between 23 ± 1 and 93 ± 4 mg for the Chalk aquifer in the UK.

N O - 3 L -1 .
High spatial variation of atrazine and DEA concentration was observed.

The concentration of DIA and HA is lower than the limit of quantification, so these two degradation products of atrazine are not presented in this study. The High spatial variation of DAR was observed. The DAR medians for the 16 sites varied between 2.9 and 10.3, with the highest DAR value observed for the lakes in the quarry at 41.9. These DAR values partly match what other studies had found, e.g., maximum DAR around 3 for the groundwater of the Luxembourg's Sandstone aquifer [START_REF] Farlin | Estimating pesticide attenuation from water dating and the ratio of metabolite to parent compound[END_REF]. The high DAR values may mean more degradation of atrazine in the soil, or also result from the different transfer processes in DEA and atrazine.

Spatial variations were clearly observed between the three groups. For the sites with thin layers of clay-with-flints (G1 and G3) the lake level amplitude was between 46 and 134 cm. The G1 sites, with 30-m UZ thickness, had a low concentration in nitrate (less than 40 mg N O - 3 L -1 , atrazine (less than 150 ng L -1 ) and DEA (less than 450 ng L -1 ). The G3 sites with a UZ thickness less than 20 m showed higher concentrations in nitrate (medians up to 93 ± 4 mg

N O - 3 L -1
), atrazine (medians up to 202 ± 40 ng L -1 ) and DEA (medians up to 1127 ± 225 ng L -1 ). The difference of nitrate, atrazine and DEA concentrations between G1 and G3 could be due to the different UZ thicknesses of the sites.

For G2, the sites have a thick layer of clay-with-flints and an UZ thickness around 28 m (about the same depth as for G1, thicker than G3). Water had a DAR higher than the G1 and G3 sites. However, high spatial variation of the quality of the groundwater was observed within G2. LUC and VOU were particularly different from the 14 other sites. These two sites had the highest concentrations of atrazine and DEA and the highest DAR, and also nitrate concentration. The hydrodynamic features and water quality at LUC and VOU could indicate that the transfer processes are different for these two sites.

Temporal variation

The boxplots in Fig. 3 show a temporal variability of the hydrodynamics and quality of the lakes between the campaigns. The temporal variability differed between the sites and between the parameters. For example, the nitrate boxplots showed small variability between the campaigns for most of the sites. But for DAR, the temporal variability between the campaigns varied greatly between the sites: most of the sites showed very little variability while DAR at VOU varied between 5 and 30.

To understand the temporal variation in nitrate, atrazine, DEA and DAR, the data are presented over time (Fig. 4). Four sites are presented: two sites with a thin layer of clay-with-flints (BLE in G1 and PED in G3) and two sites under a thick layer of clay-with-flints (STA and VOU in G2). Figure 4 presents the time series of the monthly rainfall amount, percolating flow rate, standardized lake level, nitrate, atrazine, DEA and DAR for the four sites (BLE, STA, VOU and PED) between 1 January 2016 and 16 May 2018.

The annual rainfall recorded at the Beauvais station was 750 mm for 2016 and 600 mm for 2017. The distribution of the rainfall varied from one year to the other. An intense rainfall event occurred in May 2016 with a monthly rainfall more than 170 mm. January 2018 was also a wet period with monthly rainfall greater than 100 mm. The temporal variation of the lake level and the percolating flow were, therefore, different between the two years. The lake level at VOU (Fig. 4) presented little temporal variation, which is different from what was observed at the three other sites. BLE, STA and PED show a high temporal variation in lake level (e.g., lake level amplitude was 100 cm for BLE).

Temporal variation was observed for the lake level and the percolating flow rate at BLE, STA and PED: in 2016, the percolation stopped from November to December 2016, and percolation stopped earlier in 2017 from July to August.

The highest percolating flow rate in 2016 was more than twice as high as in

2017. The lake level variation presented the same behavior as the percolating flow rate. The maximum lake level variation in 2016 was also more than twice as high as in 2017. The temporal lake level and percolating flow rate variation between 2016 and 2017 may result from the temporal variation of rainfall.

The nitrate concentration shows a low variation for BLE, STA and PED, unlike hydrodynamics. Atrazine and DEA, on the other hand, show high temporal variation and little temporal variation for DAR. The differences between the temporal variation of nitrate and the temporal variation of atrazine and DEA mean that the transfer processes differ for the two molecules. For BLE, STA and PED, the temporal variation seems to be synchronized between atrazine and DEA but not synchronized with the lake level: the highest concentration of atrazine and DEA is delayed compared to the highest lake level and the lowest concentration of atrazine and DEA was observed at the same time as the lowest lake level (January 2018).

The temporal variation of the lake level and the percolating flow at VOU differed from BLE, STA and PED. The VOU lake level showed a little variation. The percolating flow of VOU was uninterrupted but the intensity of the percolation varied over time. The fact that the temporal variation was observed for the percolating flow but not for the VOU lake level indicates that the VOU lake may be recharged continuously by other infiltrated water (water passing through walls and pillars).

High temporal variation was observed in atrazine, DEA and DAR for VOU.

However, the temporal variations of atrazine differed from that of DEA. The et al., 2007;[START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF] but this cannot exclude the presence of transfer through fractures.

Degradation processes

In this study, most of the G1 and G3 sites had a DAR value around 3.5 with a very low temporal variation despite the high temporal variation observed in the concentration of atrazine and DEA. [START_REF] Farlin | Estimating pesticide attenuation from water dating and the ratio of metabolite to parent compound[END_REF] also found DAR values around 3 for the groundwater of the Chalk aquifer. Several sites of G2, covered by a thick layer of clay-with-flints, had DAR values higher than 6. [START_REF] Martins | Sorption and desorption of atrazine on soils: The effect of different soil fractions[END_REF] observed that atrazine could be easily adsorbed by clay-with-flints. Considering that the clay-with-flints may be responsible for the storage of water and atrazine on the surface, atrazine at the G2 sites may stay longer on the surface, leading to more degradation. Degradation of atrazine is a biological process, normally under aerobic condition. The degradation of atrazine is considered occurring mainly in the soil with nearly no degradation in the UZ (anaerobic condition) [START_REF] Johnson | Potential for isoproturon, atrazine and mecoprop to be degraded within a chalk aquifer system[END_REF]. Groundwater in the quarry is under aerobic condition. Therefore, degradation of atrazine may also occur in the quarry if micro-organisms are present [START_REF] Issa | Degradation of atrazine and isoproturon in the unsaturated zone: a study from Southern England[END_REF][START_REF] Solomon | Atrazine biodegradation efficiency, metabolite detection, and trzD gene expression by enrichment bacterial cultures from agricultural soil[END_REF]. The different DAR values in the groundwater related to the different thicknesses of the clay-with-flints layer indicate that the clay-with-flints on the surface may favor atrazine degradation processes with potential atrazine degradation in the groundwater if micro-organisms are present.

Atrazine transfer process in the Chalk aquifer UZ

Chen et al. ( 2019) showed that the transfer process of atrazine is different from the transfer process of nitrate and estimated the transfer velocity of nitrate and atrazine in the Chalk UZ about 1 m year -1 . This shows that the transfer of the contaminants, nitrate and pesticide, is mainly driven by piston flow. With this hypothesis, the difference of the concentration of nitrate, atrazine and DEA between the sites of G1 and G3 may be due to the difference of the sites' depths.

Deeper the groundwater is, less contaminant is presented. [START_REF] Baran | Agricultural diffuse pollution in a chalk aquifer (Trois Fontaines, France): Influence of pesticide properties and hydrodynamic constraints[END_REF] observed that pesticides can be transferred through both matrix and fractures in UZ of the Chalk aquifer. A number of studies [START_REF] Keim | Convergent Flow in Unsaturated Fractured Chalk[END_REF][START_REF] Smith | Water Movement in the Unsaturated Zone of High and Low Permeability Strata by Measuring Natural Tritium[END_REF] suggested that transfer of water and contaminants through fractures can account for 30% of the total recharge and, in certain conditions, transfer through fractures may dominate during the recharge period. [START_REF] Foster | Mechanisms of Groundwater Pollution by Pesticides[END_REF] showed that, unlike nitrate, pesticides are more likely transferred through fractures. In this study, nitrate shows a small temporal variation, independent of water tables dynamic. This may lead to a conclusion that the transfer of nitrate occurs mainly through matrix by piston flow. Atrazine and DEA, on the contrary, show a high temporal variation, which seems to be linked to water tables dynamic. Therefore, the transfer of atrazine and DEA is different from the transfer of nitrate: other than matrix, it may occur mainly through fractures. In a recent study, [START_REF] Chen | Water, nitrate and atrazine transfer through the unsaturated zone of the Chalk aquifer in northern France[END_REF], who studied percolation water in the same quarry, considered percolation as water mainly passing through matrix with no atrazine and no DEA (concentration lower than Quantification limit, 20 ng L -1 ) found in the percolation water of the G1 sites (water before 1977).

In the present study, lake water at the G1 sites showed higher concentrations than percolation water (Fig. 3) in atrazine (medians varied between 67 ± 13.4

and 149 ± 14.8 ng L -1 ) and in DEA (medians varied between 257 ± 51.4 and 514 ± 102.8 ng L -1 ). Therefore G1 lakes include some water infiltrated between 1977 and 2003, younger than percolation water. The lakes are recharged by a transfer quicker than the transfer through matrix. Consequently, a significant proportion of the recharge is likely transfer through fractures.

Since atrazine and DEA are organic components, the transfer of these substances in the Chalk aquifer UZ may be influenced by the sorption process [START_REF] Wefer-Roehl | Sorption of organic contaminants in a fractured chalk formation[END_REF]. [START_REF] Ezra | Retardation of organobromides in a fractured chalk aquitard[END_REF] showed that the transfer of organic components in the Chalk UZ can be delayed due to adsorption. Nitrate, a soluble molecule, is less influenced by sorption, compared to the pesticides. In the present study, temporal variations were observed in atrazine and DEA but not in nitrate. These results indicate that the transfer processes are different between nitrate and pesticides. Moreover, the maximum pesticide concentrations in the water table occurred a few weeks later than the maximum of the lake level (Fig. 4). This could be explained by the sorption processes (adsorption and desorption). Atrazine and DEA adsorbed in the UZ are likely desorbed after the arrival of water recharging the water table. There is a 4-month delay due to the time for desorption.

Hysteresis (water level -pesticide concentration)

To better understand the relationships between lake level and the concentration of atrazine and DEA over time, Two types of hysteresis were observed (Fig. 5a): i) no clear hysteresis for VOU, perhaps because the lake level does not present temporal variation; ii) counterclockwise hysteresis clearly observed in atrazine and DEA for BLE, STA and PED. Atrazine and DEA showed similar hysteresis, meaning that the variation of atrazine and the variation of DEA are synchronized. A sketch of the counterclockwise hysteresis is presented into four steps (Fig. 5b).

(1) Start of the recharge: the concentrations of atrazine and DEA remain stable as the lake level starts to rise: not enough time for desorption.

(2) Recharge continues: concentrations of atrazine and DEA start to increase as the lake level continues to rise: there is enough time for the desorption.

(3) Decrease of recharge: concentrations of atrazine and DEA increase to the maximum value as the lake level starts to drop: desorption continues.

(4) End of recharge: concentrations of atrazine and DEA decrease to the minimum value as the lake level drops to the minimum: there is no more desorption. All desorbed pesticides have been leached out and the concentration decreases to the lowest value.

The sites with temporal variation observed in lake level showed synchronized transfers of atrazine and DEA. The transfer of the pesticides is delayed by the adsorption and desorption processes.

Water table beneath clay-with-flints formation

The LUC and VOU sites had a behavior different from the other 14 sites.

Both presented little temporal variation in terms of lake level but the highest concentrations of atrazine and DEA. [START_REF] Valdes | Infiltration processes in karstic chalk investigated through a spatial analysis of the geochemical properties of the groundwater: The effect of the superficial layer of clay-with-flints[END_REF] Unlike other sites, VOU lake atrazine and DEA concentrations have different temporal variations (Fig. 4). The VOU temporal variation of atrazine is similar to other sites, so the transfer of atrazine is similar for all the sites, independent of the thickness of the layer of clay-with-flints. However, the DEA of LUC and VOU time series differ from the 14 other sites and no relation was observed between the lake level and DEA for LUC and VOU. These results may be explained by the different sorption capacities between atrazine and DEA. Sorption capacity depends on materials and the physico-chemical properties of the organic component [START_REF] Coquet | Sorption of Pesticides Atrazine, Isoproturon, and Metamitron in the Vadose Zone[END_REF]. [START_REF] Bottoni | Leaching indices of some major triazine metabolites[END_REF] showed that atrazine has lower mobility than DEA in soil with a Koc (Soil Organic Carbon-Water Partitioning Coefficient) of 111 mL g -1 for atrazine and a Koc of 38 mL g -1 for DEA (higher Koc indicates higher capacity of being adsorbed by soil). [START_REF] Brouwer | Adsorption of transformation products of atrazine by soil[END_REF] and [START_REF] Mersie | Adsorption and Desorption of Atrazine, Deethylatrazine, Deisopropylatrazine, and Hydroxyatrazine on Levy Wetland Soil[END_REF] showed that atrazine is more adsorbed than DEA on soils, meaning that atrazine is more easily to be adsorbed by the Chalk aquifer than DEA, and also more difficult to desorb than DEA.

Since we assumed that the transfer of atrazine and DEA for LUC and VOU occurs mainly through preferential pathways, the transfer of DEA may occur through fractures with very little adsorption in the Chalk aquifer. Atrazine is more easily adsorbed, so its transfer is delayed by sorption. The preferential pathways may result in different transfer processes of atrazine and DEA with the transfer of DEA quicker because DEA desorbs more easily.

Conclusion

This study shows very high concentrations of atrazine and its degradation product, DEA, in groundwater, which is the output of the UZ under an agricultural area. The transfer velocities from soil surface towards the water table of atrazine and DEA are estimated between 0.45 and 2.21 m/year. Spatial varia-tions were observed in the groundwater concentration of nitrate, atrazine and DEA, and also in DAR in the groundwater, which may be related to the properties of the UZ of the Chalk aquifer (thickness of clay-with-flints and depth).

Temporal variations were observed in the concentration of atrazine and DEA.

The spatio-temporal variation of atrazine, DEA and DAR shows that:

-The transfer of atrazine and DEA occurs through both matrix and fractures, and the transfer is delayed by the sorption in the UZ.

-Clay-with-flints may favor the degradation of atrazine by creating a "perched water table", leading to atrazine staying near the surface for a long time.

-Clay-with-flints can create preferential pathways, through which the transfers of atrazine and DEA are quicker. The transfers of atrazine and DEA differ in their preferential pathways because DEA is more easily to be desorbed.

The multi-sites with different UZ properties is a simple system (output of the UZ), which makes it possible to study how the UZ properties (UZ thickness and thickness of clay-with-flints) impact the transfer and degradation processes of pesticides. It is possible to apply this study to other studies on a larger scale, e.g., the study of the transfer of pesticides in the Chalk aquifer of the Paris Basin. 2016-04-12, 2016-06-23, 2016-10-20, 2017-01-09, 2017-02-27, 2017-09-05, 2017-10-23, 2018-01-16, 2018-04-10, 2018-05-16. b, Sketch of the hysteresis observed between lake level variation and pesticide concentration.

  The highest nitrate concentration observed was 95 ± 4 mg N O - 3 L -1 . The concentrations observed in the quarry were at the upper end of what other studies had found. For example, El Gaouzi et al. (2013) observed a nitrate concentration between 34 and 49 mg N O - 3 L -1 in the Paris Basin. Sorensen et al. (2015) found nitrate concentrations varied between 30 and 60 mg N O - 3 L -1

  medians of the atrazine concentrations at the 16 sites varied between 55±11 and 202 ± 40 ng L -1 . The medians of the DEA concentrations of the 16 sites varied between 269±53 and 1727±345 ng L -1 . The highest atrazine concentration was 501 ± 100 ng L -1 and the highest DEA concentration was 3941 ± 788 ng L -1 . These atrazine and DEA concentrations were much higher than any published values in other studies. For example,[START_REF] Baran | Agricultural diffuse pollution in a chalk aquifer (Trois Fontaines, France): Influence of pesticide properties and hydrodynamic constraints[END_REF] recorded that, from 2000 to 2006, for the Paris Basin, groundwater atrazine varied between 50 and 100 ng L -1 and DEA varied between 100 and 200 ng L -1 .[START_REF] Hakoun | Factors controlling spatial and temporal patterns of multiple pesticide compounds in groundwater (Hesbaye chalk aquifer, Belgium)[END_REF] observed the maximum concentrations of atrazine and DEA less than 200 ng L -1 concentration of atrazine and DEA for the Chalk aquifer in Belgium.

  highest concentration of atrazine was observed in January 2017 and the lowest concentration of atrazine was observed in January 2018, with the highest concentration of DEA observed in April 2016. The differences between the temporal variation of atrazine and DEA indicate that the transfer processes of atrazine and of DEA are different for VOU. The DAR value at VOU showed a high temporal variation between 3 and 30. The highest DAR value was observed in January 2018 when the concentration of atrazine was at the lowest level.4. DiscussionSince atrazine was used for the treatment of crops from 1977 to 2003, the high concentrations observed in atrazine and DEA indicate that the groundwater in the quarry has a residence time of one to several decades. Assuming a transfer within the UZ by piston flow, the apparent residence time between 14 and 40 years can be calculated(2017 -2003 = 14 years and 2017 -1997 = 40 years).With an UZ thickness between 18 and 31 m, the transfer velocity of atrazine and DEA for the groundwater in the quarry can be estimated between 0.45 and 2.21 m/year. The apparent velocities match the matrix velocities (Van den Daele

  Fig.5 was plotted to present the hysteresis between the water level and atrazine and DEA concentrations for four lakes (BLE, STA, VOU and PED). Data from April 2016 to May 2018 were used.

  suggested that the layer of clay-with-flints may have two roles in the infiltration process: it can store water near the surface and it can also accelerate the infiltration by creating preferential pathways beneath it. The high concentrations of atrazine and DEA observed in the LUC and VOU lakes may result from the quick transfer through preferential pathways under the clay-with-flints. Below the layer of clay-withflints, a perched water table may form near the surface between the Chalk and the clay-with-flints due to the contrast of hydraulic conductivity. The stored water then infiltrates by the preferential pathways, recharging LUC and VOU lakes continuously, explaining the low variation of lake levels and continuous percolating flow of these two sites. The presence of preferential pathways may allow micro-organisms entering the UZ, leading to the degradation of atrazine in groundwater. LUC and VOU have higher DAR than other sites and this may be because of higher degradation rate in soil or because of the degradation of atrazine in the lakes with the presence of micro-organisms.LUC and VOU also had the highest temporal variation of DAR. The high temporal variation of DAR values may result from the differences observed be-tween the atrazine concentration and the DEA concentration time series. This difference between the temporal variation of atrazine concentration and DEA concentration is due to the different transfer processes in atrazine and DEA.

Figure 1 :

 1 Figure 1: Location of the study site with the zoom of the geology of the study site.

Figure 2 :

 2 Figure 2: Schematic cross section of the Saint-Martin-le-Noeud quarry with the presentation of the 16 sites in the quarry.

Figure 3 :

 3 Figure 3: UZ thickness and electrical conductivity of the 16 sites. The total percolation between April 2016 and September 2017 and the boxplot of lake level variation, of concentration of nitrate, atrazine and DEA, and of DAR of the lake of the 16 sites. Campaigns used for the box are: 2016-04-12, 2016-06-23, 2016-10-20, 2017-01-09, 2017-02-27, 2017-09-05. The box range between the 25th and 75th percentiles, whiskers correspond to minimum and maximum, horizontal bars indicate the median. The 16 sites are divided into three groups: G1, G2 and G3.

Figure 4 :

 4 Figure 4: Time series of rainfall, lake level variation, percolating flow, nitrate, atrazine, DEA and DAR from 2016-01-01 to 2018-06-31 for BLE, STA, VOU and PED. Time series of percolating flow is presented with reversed y-axis. Arrows of the percolating flow of VOU indicate that the values are underestimated.

  29

Figure 5 :

 5 Figure 5: Hysteresis between lake level and pesticide concentration. a, hysteresis between atrazine, DEA and lake level variation for BLE, STA, Vou and PED. Campaigns used are:

Table 1 :

 1 The measurements and analyses made for each campaign.

	Groups	Campaigns	Sites	Measurements	Analyses
		2016-04-12, 2016-06-23,
				percolating flow rate,	nitrate, atrazine,
	A	2016-10-20, 2017-01-09,	16 sites
				lake level	DEA
		2017-02-27, 2017-09-05
		2016-06-06, 2016-09-05,		percolating flow rate,
	B		16 sites		N.A.
		2017-05-02, 2017-06-26		lake level
		2017-10-23, 2018-01-16,	BLE, STA,	percolating flow rate,	nitrate, atrazine,
	C
		2018-04-10, 2018-05-16	VOU, PED	lake level	DEA

Acknowledgments

The authors thank the Region Ile-de-France and the EC2CO project for financial support and the IPSL federation for additional financial support. This work was performed within the framework of the CRAIE observation site, part of the KARST observatory network (www.sokarst.org) initiated by INSU/CNRS, which aims to strengthen knowledge-sharing and to promote cross-disciplinary research on karst systems. Thanks to the Conservatoire d'espaces naturels de Picardie for providing the access to the quarry.


Taylor Leroy
email: taylor-lucrece.leroy@laposte.net

Elisabeth Monnet
email: elisabeth.monnet@univ-fcomte.fr

Stéphane Guerzider
email: stephane.guerzider@hotmail.fr

Pascale Jacoulet
email: p1jacoulet@chu-besancon.fr

Pierre-Emmanuel Falcoz
email: pierre-emmanuel.falcoz@chru-strasbourg.fr

Marie Gainet-Brun

Jean Lahourcade
email: j.lahourcade@chi-hautecomte.fr

Faraj Alfreijat
email: faraj.alfreijat@hnfc.fr

Hamadi Almotlak
email: halmotlak@chu-besancon.fr

Bernardino De Bari
email: bdebari@chu-besancon.fr

Olivier Adotevi
email: er.adotevi@univ-fcomte.fr

Didier Pernet
email: dpernet@chu-besancon.fr

Jean-Charles Polio
email: dr.jean-charles.polio@laposte.net

Maxime Desmarets
email: maxime.desmarets@univ-fcomte.fr

M.D Anne-Sophie Woronoff
email: asworonoff@chu-besancon.fr

Virginie Westeel
email: virginie.westeel@univ-fcomte.fr

Sophie Woronoff

Let us not underestimate the long-term risk of SPLC after surgical resection of NSCLC

Keywords: non-small cell lung cancer, surgery, second primary lung cancer, postoperative radiotherapy, follow-up

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

Introduction

Lung cancer is the most common cause of cancer death worldwide [START_REF]Fact Sheets by Cancer[END_REF] . Non-small cell lung carcinoma (NSCLC) accounts for around 85% of lung cancer cases. The standard treatment for patients with stage I and II NSCLC is surgery based, with or without postoperative chemotherapy depending on the pathological T and N status. Treatment for stage III NSCLC consists either of concurrent chemoradiation, or surgery with perioperative chemotherapy with or without thoracic radiotherapy [START_REF]NCCN Guidelines® Clinical Practice Guidelines in Oncology[END_REF] . After surgery, the 5-year survival rates range between 23 and 83%, depending on stage [START_REF] Goldstraw | The IASLC Lung Cancer Staging Project: Proposals for Revision of the TNM Stage Groupings in the Forthcoming, eighth ed. TNM Classification for Lung Cancer[END_REF] .

After surgery, most international guidelines recommend regular follow-up visits and chest computed tomography (CT), but the optimal duration and frequency of follow-up remain unclear. Several studies have shown that lung cancer survivors remain at high risk of developing a second primary (metachronous) lung cancer (SPLC), even more than 5 years after treatment [START_REF] Walsh | Is follow-up of lung cancer patients after resection medically indicated and cost-effective?[END_REF][START_REF] Martini | Factors influencing ten-year survival in resected stages I to IIIA non-small cell lung cancer[END_REF][START_REF] Westeel | Relevance of an intensive postoperative follow-up after surgery for non-small cell lung cancer[END_REF][START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF][START_REF] Rice | The risk of second primary tumors after resection of stage I nonsmall cell lung cancer[END_REF][START_REF] Kinsey | Predicting the Mortality Benefit of CT Screening for Second Lung Cancer in a High-Risk Population[END_REF] . However, studies investigating the risk of SPLC from resected NSCLC series reported widely varying estimates of this risk, with very few of them taking into account that the patients operated on for NSCLC are at particularly high competing risk of death, due to recurrence of the initial disease and to comorbidities.

Therefore, the primary study objective was to estimate the long-term incidence function of SPLC in a population-based cohort of patients operated on for Stage I to III NSCLC while taking account of the competing risks of death during follow-up. The secondary objective was to identify patient and tumor characteristics, as well as perioperative treatments associated with an increased risk of SPLC (while factoring in competing risks of death).

Material and methods

Study population

Patients were identified using the cancer registry for the Doubs and Belfort administrative districts of France. All incident cases of NSCLC diagnosed between January 1, 2002, and December 31, 2015, were retrieved. Of those, all patients aged 18 years or older with a completely resected pathological Stage I to III NSCLC were included. Staging was determined according to the 6 th edition of the Union for International Cancer Control (UICC) TNM classification for cases resected before 2009, and according to the 7 th edition thereafter [START_REF] Sobin | TNM Classification of Malignant Tumours[END_REF][START_REF] Sobin | TNM Classification of Malignant Tumours[END_REF] . Complete resection was defined as microscopically free surgical margins, and either systematic mediastinal lymph node dissection or selective nodal sampling. Patients were also eligible if they had incomplete resection due to carcinoma in situ in the resection margins or metastatic tumors due to pulmonary nodules in another ipsilateral lobe according to the 6 th edition of the UICC-TNM classification. The exclusion criteria were: death within 30 days of surgery, synchronous lung cancer or a history of prior cancer within the previous 5 years (except for basal cell carcinoma of the skin or carcinoma in situ of the uterine cervix). A consensus approach on follow-up had been adopted for the Doubs and Belfort region. The follow-up comprised clinic visits and chest CT every 6 months for 2 years, then every year between years 3 and 5, and lastly chest X-ray or chest CT yearly beyond 5 years.

Data sources and variables studied

The data, routinely collected by the registry and from medical records, were as follows: date of diagnosis, topography, histology, date of surgery, pathological classification of the primary tumor (pT) and regional lymph nodes (pN), and perioperative treatment (chemotherapy; thoracic radiotherapy). Histology was dichotomized into adenocarcinoma and nonadenocarcinoma (Appendix 1). Pathology reports were retrospectively examined to determine whether the resection was complete. Smoking history was retrospectively collected. Patients were classified as never-smokers (if they had smoked less than 10 cigarettes in their lifetime), former smokers (who had quit >1 year before diagnosis) or current smokers, and smoking was quantified in pack-years.

A distinction was made between recurrence and SPLC using the Martini and Melamed criteria [START_REF] Martini | Multiple primary lung cancers[END_REF] . According to this definition, a new, distinct pulmonary malignancy was considered a SPLC if it fulfilled any of the following three criteria: 1) histologic type different from that of the index tumor; 2) same histologic type as that of the index tumor but diagnosed at least 2 years later; or 3) same histologic type as that of the index tumor, diagnosed within 2 years, but located in a different lobe or lung, with no involvement of lymphatics common to both, and no evidence of extrapulmonary metastasis [START_REF] Martini | Multiple primary lung cancers[END_REF] . Vital status, date of death, and last available follow-up date were extracted from the medical records and registry database. An active search for vital status at December 31, 2017 was carried out.

Statistical analysis

Quantitative variables were expressed by median and range, and qualitative variables by number and percentage. The event of primary interest was the occurrence of SPLC. As many patients may die before experiencing the event of interest due to poor NSCLC prognosis or comorbidities, death from any cause was considered as a competing risk. Consequently, we used a competing-risks approach [START_REF] Fine | Gray RJA proportional hazards model for the subdistribution of a competing risk[END_REF] . The time-to-event was calculated from the date of surgical resection. Patients who experienced no event during follow-up were censored at the time of last contact.

We employed the cumulative incidence function (CIF) method to estimate the "net" probability of SPLC over time among survivors of the first lung cancer, i.e., the incidence of this event in the cohort before the occurrence of death, the competing event. Firstly, annualized risks of SPLC, corresponding to the cumulative incidence over 12-month intervals, were estimated along with the corresponding 95% confidence intervals (95% CI). For this estimation, patients who were still at risk were recalculated at the beginning of each follow-up year. CIF curves were estimated from surgery to the last observed time point, and were generated for the whole cohort according to the following variables: gender, age at initial surgery, smoking status and number of pack-years, pT and pN, histology, and perioperative treatment (chemotherapy and/or radiotherapy) of initial lung cancer. The CIF curves were compared using Gray's test.

Then the Fine-Gray model was used to analyze the prognostic effect of each variable on the sub-distribution hazard function of SPLC [START_REF] Austin | Introduction to the Analysis of Survival Data in the Presence of Competing Risks[END_REF] , that is, the relative change in the instantaneous rate of the event occurrence in patients who were event-free or experienced a competing event. Significance was assessed with the Wald test, and all variables with a p value of 0.2 or less were retained for multivariate analysis. Both unadjusted and adjusted sub-distribution relative hazards were estimated along with the corresponding 95% CI [START_REF] Lau | Competing Risk Regression Models for Epidemiologic Data[END_REF] . In the final multivariate model, the proportional hazard assumption was verified for all significant covariates using the Schoenfeld residuals method. A time-by-covariate interaction term was added to the model when the assumption was not met [START_REF] Latouche | A competing risks analysis should report results on all cause-specific hazards and cumulative incidence functions[END_REF] . All statistical tests were 2-sided, with a significance level at 0.05. SAS Version 9.4 (SAS Institute, Inc., Cary, NC, USA) was used to perform all statistical analyses.

Results

A total of 556 patients with completely resected pathological Stage I to III NSCLC were identified (Figure 1). We excluded 16 patients who died within 30 days of surgery, 2 patients with synchronous lung cancer, and 16 patients with a history of prior cancer within the previous 5 years. Patient characteristics at baseline are summarized in Table 1. In the 522 eligible patients, median age was 64 years (range: 38-86), 73.6% of patients were males, and 15.1% never-smokers. In ever-smokers, the median number of pack-years was 40 (1-160 2). The risk of SPLC persisted over time (Figure 3). In eight of the 84 cases, SPLC occurred within 2 years of surgery, and all had a pathological type different from that of the index tumor. The pathological type was unknown in 7 (43%) of the 16 SPLC diagnosed during year 3, in 7 (37%) of the 19 diagnosed during year 4, and in 5 (38%) of the 13 diagnosed during year 5. It was different from that of the index tumor in 4 (25%), 3 (16%) and 6 (46%), respectively. In the first 5 years after surgery, the annual risk peaked at year 4 (5.8%, 95% CI: 3.6-8.6%). The risk persisted beyond year 9, with 7 SPLC cases among the 92 patients alive at the beginning of year 10 after surgery of the index lung cancer. Among these patients, the risk of SPLC during the following 5 years reached 14.3% (95% CI: 9.6-27.0%).

The CIF curves for SPLC according to perioperative index cancer treatment are shown in Figure 4. Fourteen of the 44 patients who received radiotherapy developed a SPLC. SPLC risks were significantly higher among patients who received postoperative radiotherapy than among those who received surgery or perioperative chemotherapy only (5, 10 After adjusting for time of diagnosis, age, smoking (pack-years) and pN, the initial perioperative cancer treatment was the main factor associated with SPLC hazard. Compared to surgery alone, postoperative radiotherapy significantly increased SPLC hazard (2.79 [1.41-5.52], p=0.003), whereas perioperative chemotherapy did not (1.32 [0.77-2.26], p=0.31). SPLC hazard tended to be higher among patients aged 70 and older compared to their younger counterparts (1.57 [0.98-2.51]). All variables included in the final model met the proportional hypothesis assumption, except for age, for which the time interaction term was significantly associated with the risk of SPLC (p=0.01). When the interaction term was included in the model, increased age became significantly associated with a higher risk of SPLC up to year 4 after surgery (2.51 [1.39-4.54]). Relative hazard estimations for the other covariates were not modified (data not shown).

Discussion

In this exhaustive population series of 522 resected Stage I to III NSCLC patients with a long follow-up (median 59 months, maximum of more than 15 years), our results showed a high risk of SPLC (84 patients, 16.1%). Cumulative incidence, which took account of competing risks of death, was 20.2% at 10 years and 25.2% at 14 years. SPLC cases were observed throughout follow-up, up to year 14 after initial surgery. In a multivariate analysis that took competing risks of death into account, patients who received postoperative radiotherapy (with or without chemotherapy) had a significantly higher risk of developing SPLC than did patients who had surgery with or without chemotherapy (hazard ratio = 2.79; 95% CI: 1.41-

5.52).

Second primary lung cancer

The majority of the mostly retrospective series have reported SPLC rates after surgery for Stage I to III NSCLC of up to 8.6% [START_REF] Walsh | Is follow-up of lung cancer patients after resection medically indicated and cost-effective?[END_REF][START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF][START_REF] Rice | The risk of second primary tumors after resection of stage I nonsmall cell lung cancer[END_REF][START_REF] Thakur | Risk of Second Lung Cancer in Patients with Previously Treated Lung Cancer: Analysis of Surveillance, Epidemiology, and End Results (SEER) Data[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF][START_REF] Boyle | Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis[END_REF][START_REF] Lou | Patterns of recurrence and second primary lung cancer in earlystage lung cancer survivors followed with routine computed tomography surveillance[END_REF][START_REF] Pairolero | Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease[END_REF] . Only the single-center trial of 124 resected Stage IA NSCLC by Lamont et al. has reported a 15.3% SPLC rate close to ours [START_REF] Lamont | Systematic Postoperative Radiologic Follow-up in Patients With Non-Small Cell Lung Cancer for Detecting Second Primary Lung Cancer in Stage IA[END_REF] . In studies with median follow-up durations similar to this study, the highest SPLC rates reported were around 8% [START_REF] Walsh | Is follow-up of lung cancer patients after resection medically indicated and cost-effective?[END_REF][START_REF] Thakur | Risk of Second Lung Cancer in Patients with Previously Treated Lung Cancer: Analysis of Surveillance, Epidemiology, and End Results (SEER) Data[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] . In a Surveillance Epidemiology and End Results (SEER) cohort including 20032 patients who survived 5 years after an initial diagnosis of lung cancer, the median 10year risk of SPLC was 8.36% [START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF] .

One of the difficulties in estimating the risk of SPLC revolves around the definition used to distinguish new primaries from lung recurrences. Most studies have applied the same definition as Martini and Melamed, which considers distinct pulmonary malignancies with the same histologic results as being SPLC if they are diagnosed at least two years after the index primary lung cancer [START_REF] Westeel | Relevance of an intensive postoperative follow-up after surgery for non-small cell lung cancer[END_REF][START_REF] Thakur | Risk of Second Lung Cancer in Patients with Previously Treated Lung Cancer: Analysis of Surveillance, Epidemiology, and End Results (SEER) Data[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF][START_REF] Boyle | Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis[END_REF][START_REF] Lou | Patterns of recurrence and second primary lung cancer in earlystage lung cancer survivors followed with routine computed tomography surveillance[END_REF] . Others have defined SPLC as a new, distinct lung malignancy diagnosed more than 5 years after the primary tumor, using a stricter definition than the Martini and Melamed criteria [START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF][START_REF] Rice | The risk of second primary tumors after resection of stage I nonsmall cell lung cancer[END_REF][START_REF] Pairolero | Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease[END_REF] . Comparing radiographic and metabolic appearance, growth patterns, comprehensive histologic assessments, genomic hybridization and biomarker patterns has been proposed to differentiate separate primary lung cancers from metastatic foci in the eighth TNM classification, yet even this is considered to be associated with a substantial rate of misclassification [START_REF] Detterbeck | The IASLC Lung Cancer Staging Project: Background Data and Proposed Criteria to Distinguish Separate Primary Lung Cancers from Metastatic Foci in Patients with Two Lung Tumors in the Forthcoming Eighth Edition of the TNM Classification for Lung Cancer[END_REF] . In our study, all the eight cases of SPLC diagnosed within the two first years of surgery had a histological type different from that of the index tumor, suggesting that these tumors were indeed SPLC. Although the risk of recurrence decreases after two years, misclassifications cannot be eliminated as 13 (27%) of the 48 SPLC had the same histologic type as the index primary lung cancer. Some differences in patient characteristics are associated with a higher risk of SPLC, and these should also be considered when attempting to understand the risk of SPLC. After surgery for Stage III NSCLC, the risk of recurrence is higher than the risk of SPLC [START_REF] Lou | Differences in Patterns of Recurrence in Early-Stage Versus Locally Advanced Non-Small Cell Lung Cancer[END_REF] . Even in series excluding resected Stage III NSCLC, the SPLC rate has not exceeded 8.6% [START_REF] Rice | The risk of second primary tumors after resection of stage I nonsmall cell lung cancer[END_REF][START_REF] Lou | Patterns of recurrence and second primary lung cancer in earlystage lung cancer survivors followed with routine computed tomography surveillance[END_REF] . Age cannot be the reason for the difference either, since our median age was comparable to that of the published literature [START_REF] Rice | The risk of second primary tumors after resection of stage I nonsmall cell lung cancer[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] . The risk of SPLC may also be linked to smoking history [START_REF] Boyle | Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis[END_REF] . The 15% non-smoker rate in our study was comparable to the rate of non-smokers in other trials [START_REF] Rice | The risk of second primary tumors after resection of stage I nonsmall cell lung cancer[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] . A quantification of smoking in pack-years is often lacking in the literature. When available, the number of pack-years was, as in our study, retrospectively collected and therefore at risk of information bias. None of the trials, ours included, has provided information on smoking cessation after surgery. Most of the series published are older, ranging from 1972 to 2012, and taken together they cover more time before the year 2000 than after. Progress over time in clinical and pathological diagnosis may likewise have influenced the 16.1% SPLC rate observed in this series, as may regional surveillance practices with regular follow-up visits and chest CT.

Finally, the risk of SPLC is strongly influenced by the number of patients at risk over the study period. Therefore, such an analysis should take into account the actual number of pa-tients at risk over time. Only few studies have considered competing risks, mostly death from comorbidity or recurrence of the index lung cancer [START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] . In the study by Ripley et al., which only included surgically treated patients and used the same definition for SPLC as in the present study, the CIF of SPLC at 10 years was around 20%, exactly the same as that observed here [START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] . Our results were based on a recent series and used appropriate methods to adjust for competing risks, and they clearly show that the risk of SPLC may be even higher than we usually think and should not be underestimated.

Annual SPLC risks increased after the first two years. In the study by Lou et al., the annual SPLC rate increased from 3 to 6 per 100 person-years between years 1 and 5 after surgery [START_REF] Lou | Differences in Patterns of Recurrence in Early-Stage Versus Locally Advanced Non-Small Cell Lung Cancer[END_REF] .

These results are consistent with those observed in our study, and reflect the definition used to distinguish SPLC from lung recurrences [START_REF] Martini | Multiple primary lung cancers[END_REF] . Concordant results were reported in a populationbased cohort from the Public Health England National Cancer Registration and Analysis Service, showing an increased incidence of subsequent lung cancer for at least a decade from the first diagnosis [START_REF] Barclay | Incidence of second and higher order smoking-related primary cancers following lung cancer: a population-based cohort study[END_REF] . The risk of SPLC persists after 5 years, and even after 10 years, with the SPLC rate being 5.6% in year 14 after surgery. As observed in the literature, the risk of SPLC after lung cancer surgery did not plateau [START_REF] Thakur | Risk of Second Lung Cancer in Patients with Previously Treated Lung Cancer: Analysis of Surveillance, Epidemiology, and End Results (SEER) Data[END_REF][START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] , proving the need for follow-up of these patients until death.

Characteristics associated with the risk of SPLC

In multivariate analysis adjusted for age, smoking status, period of diagnosis and pN, thoracic radiotherapy after surgery for the index cancer was the only variable significantly associated with SPLC risk (sdRH 2.79; 95% CI: 1.41-5.52). The two periods of diagnosis, 2002-2009 and 2010-2015, were chosen to take into account the change in the TNM classification in 2010, from the 6 th to the 7 th edition. Because only 10 of the 44 patients who received postoperative radiotherapy had no chemotherapy, the interaction between postoperative radiotherapy and chemotherapy was not tested, meaning that the heightened incidence could have been due to an additive or synergistic effect of radiotherapy and chemotherapy.

There are only a few series in which the correlation between thoracic radiotherapy after surgery and the risk of SPLC have been analyzed. In the SEER database of 20032 Stage I to IV NSCLC patients who survived 5 years after an initial lung cancer, the proportion of patients who received radiotherapy was 20.6% in both the censored and SPLC groups [START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF] . However, in that series, all NSCLC stages were eligible, and both surgery and thoracic radiotherapy were given to 1435 patients (11%). The treatment variable is reported not to influence the risk of SPLC, but no information on the specific risk of postoperative radiotherapy is available. Patients treated with chemotherapy and radiotherapy for Stage III NSCLC probably have a higher competing risk of recurrence than a risk of SPLC. Of the 1484 patients included in the series by Duke University Medical Center, postoperative radiotherapy did not influence the risk of SPLC, but only 85 patients received such treatment [START_REF] Boyle | Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis[END_REF] . The postoperative radiotherapy (PORT) meta-analysis of 14 trials and 2343 patients evaluating surgery versus surgery plus radiotherapy showed an 18% relative increase in the risk of death after radiotherapy in patients with completely resected NSCLC [START_REF] Burdett | Postoperative radiotherapy for non-small cell lung cancer[END_REF] . The SPLC rate was not reported. Nevertheless, postoperative radiotherapy may be useful for Stage III N2 NSCLC [START_REF] Douillard | Impact of Postoperative Radiation Therapy on Survival in Patients With Complete Resection and Stage I, II, or IIIA Non-Small-Cell Lung Cancer Treated With Adjuvant Chemotherapy: The Adjuvant Navelbine International Trialist Association (ANITA) Randomized Trial[END_REF] , a question which should be answered by the LungART randomized trial [START_REF] Faivre-Finn | Lung ART: Phase III study comparing postoperative conformal radiotherapy to no post-operative radiotherapy in patients with completely resected non-small cell lung cancer and mediastinal N2 involvement[END_REF] . It ought to be noted that, despite its potential interest, we did not perform a pattern of relapse analysis to verify that the SPLC we observed in radiated patients occurred inside the radiation fields, and so we cannot conclude that radiotherapy was directly responsible for the increased incidence of SPLC in our patients. A confusion bias cannot be completely ruled out, as patients who are delivered postoperative radiotherapy were those at high risk of recurrence. However, these patients are more likely to develop an early recurrence and all the SPLC diagnosed within the first two postoperative years had a histologic type different from that of the index tumor. Still, considering the con-troversial data in the literature on the impact of postoperative radiotherapy on lung cancer patients, and the results of the PORT meta-analysis [START_REF] Burdett | Postoperative radiotherapy for non-small cell lung cancer[END_REF] , the significant association that we showed between an increased risk of SPLC and radiotherapy should be particularly borne in mind when postoperative radiation is being discussed.

Of the other variables, only smoking history expressed as pack-years was reported to be associated with an increased risk of SPLC on multivariate analysis in the Duke University series of 1484 patients cited above [START_REF] Boyle | Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis[END_REF] . In the SEER database, high-risk individuals were defined according to deciles based on age, histology, and disease extent [START_REF] Han | Risk Stratification for Second Primary Lung Cancer[END_REF] . In our study, there was a trend toward a correlation between increasing age and the risk of SPLC (p=0.06). In the study by Boyle et al., on Cox multivariate analysis per 10 pack-years, smoking was significantly associated with the risk of developing SPLC (hazard ratio 1.08; 95% CI 1.01-1.16; p=0.03) [START_REF] Boyle | Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis[END_REF] . In contrast, we found that pack-year exposure was not significantly associated with SPLC hazard (p=0.21) on Fine-Gray multivariate modeling. As in our study, CIF curves of SPLC did not differ according to smoking status (p=0.16) in the study by Ripley et al. [START_REF] Ripley | Second Primary Lung Cancers: Smokers Versus Nonsmokers After Resection of Stage I Lung Adenocarcinoma[END_REF] .

To conclude, this study using appropriate statistical methods to take account of competing risks showed that after complete resection for NSCLC the CIF of SPLC was high (around 20% at 10 years and 25% at 14 years) and did not plateau. Patients who received postoperative thoracic radiotherapy were at higher risk of SPLC. These data confirm the need for lifelong follow-up of patients who undergo surgery for NSCLC, with the objective of screening for SPLC.

Figures legends

Figure 1: Flow chart of study patients. NSCLC: non-small cell lung cancer; SPLC: second primary lung cancer Figure 2: Cumulative incidence function of SPLC considering the competing risk of death during follow-up (N=522 patients with surgical resection for NSCLC). SPLC: second primary lung cancer; NSCLC: non-small cell lung cancer Figure 3: Annual risk of SPLC after surgery with competing risk of death factored in. SPLC: second primary lung cancer; Deaths during the year; Patients at risk at the beginning of the year (patients at risk at the beginning of the previous year -number of deaths during the year -number of patients lost to follow-up) Figure 4: Cumulative incidence of SPLC by subgroup of first cancer treatment, with competing risk of death factored in. SPLC: second primary lung cancer

  ). Adenocarcinoma accounted for 52.3% of all cases, and Stage I or II 75.7%. A total of 233 patients (44.6%) received perioperative chemotherapy (preoperative 68 patients, postoperative 127 patients, both pre and postoperative 38 patients), and 44 patients (8.4%) postoperative thoracic radiotherapy (plus preoperative chemotherapy 8 patients, plus postoperative chemotherapy 24 patients, plus both pre and postoperative chemotherapy 2 patients). 5%) died without evidence of SPLC, and 258 (49.4%) were still alive, showing no evidence of SPLC at the end date. The median time from surgery to SPLC was 47 months (1-163). Of the 84 patients who developed a SPLC, 60 (71.4%) died during follow-up. The treatment for SPLC was surgery ± chemotherapy ± radiotherapy in 20 patients (23.8%), stereotactic radiotherapy in 6 patients (7.1%), radiotherapy ± chemotherapy in 13 patients (15.5%), chemotherapy in 32 patients (38.1%), and best supportive care in 12 patients only (14.3%).The treatment for SPLC was unknown in one patient.The CIF of SPLC during the follow-up period was 20.2% at 10 years (95% CI: 15.3-23.2),

	Median follow-up was 59 months (2-190). Eighty-four patients (16.1%) developed a SPLC,
	180 (34.and reached 25.2% at 14 years (95% CI: 19.4-31.3; Figure

  and 14-year risks [95% CI]: 25.8% [13.2-40.6], 40.7% [23.6-57.2] and 40.7% [23.6-57.2]; 9.3 [5.8-13.7], 15.5% [10.3-21.5] and 23.0% [14.9-32.1]; 12.8% [8.6-17.9], 18.4% [13.1-24.5] and 24.8% [16.1-34.6], respectively, p=0.003). Sub-distribution relative hazards (sdRHs) estimated from Fine-Gray univariate and multivariate models are presented in Table 2. On univariate analysis, SPLC hazard was significantly increased for pN2 (sdRH [95% CI] 1.84 [1.08-3.15], p=0.02) compared to pN0/1, and for postoperative radiotherapy (2.88 [1.54-5.38], p=0.004) compared to surgery alone. As they were associated with the risk of SPLC with a p value < 0.20, the variables time of diagnosis, age, pack-years, pN, and treatment of the index tumor were retained in multivariate modeling.

Acknowledgments

The authors thank all those who contributed to the inclusion of cancer patients in the registries, in particular pathologists, medical information staff in public and private healthcare in-stitutions, medical staff of health insurance companies, chest physicians and oncologists, as well as medical practitioners. The authors also extend their gratitude to the registry team for their help in data recording.

French cancer registries are supported by the French National Cancer Institute and Santé publique France, the French national public health agency, as well as by local institutions and the French League Against Cancer.