Paula Pérez-Rodríguez

Anne-Desirée Schmitt

Sophie Gangloff

Jérémy Masbou

Gwenaël Imfeld
email: imfeld@unistra.fr

Plants affect the dissipation and leaching of anilide pesticides in soil mesocosms: insights from compound-specific isotope analysis (CSIA)

Keywords: Anilide pesticides, Biodegradation, Carbon isotope fractionation, Leaching, Mesocosms, Rhizosphere, Soil mesocosms

The soil-plant system can act as a prevailing zone of pesticide dissipation in agroecosystems, which may influence pesticide leaching following rainfall events. Here we examined the contribution of leaching, dissipation and degradation of widely used anilide pesticides in planted and unplanted mesocosms with contrasting vineyard and forest soils. The mesocosms were spiked at 25 mg/kg with acetochlor, alachlor, Smetolachlor, butachlor and metalaxyl, and followed-up for 75 days. Successive rainfalls were applied on days 45, 60 and 75 to collect pesticide leachates. Bulk soil and rhizosphere samples were collected to evaluate pesticide dissipation. Up to 14% of initially spiked pesticides was exported by leaching. The first rainfall accounted for 69 to 99% of the total pesticide leaching. Pesticide dissipation in soil mesocosms on day 75 ranged from 58 to 99% and was larger in planted than in unplanted mesocosms.

Compound-specific stable isotope analysis (CSIA) of the pesticides showed that biodegradation in both the vineyard and forest soil mesocosms occurred mainly in the soil solution that was leached following rainfall events rather than in the bulk soil.

Changes in carbon isotope values (Δδ 13 C up to 13.9 ± 0.5‰) of acetochlor, alachlor, Smetolachlor and butachlor in leachates were more pronounced in planted than in unplanted mesocosms, indicating predominant pesticide degradation in the planted mesocosms. Pesticide biodegradation was favoured in the soil solution of soil-plant systems, independently of the soil type. Leaching of the soil solution, pesticide ageing and biodegradation in the bulk soil and the rhizosphere were the main processes of pesticide dissipation. Overall, this study emphasizes the variability of leaching and degradation of anilide pesticides in agricultural soils, and proposes a framework using CSIA to examine the contribution of dissipation processes in soil-plant systems.

Introduction

Anilide pesticides include widely used herbicides to control broad-leaf weeds and annual grasses in crops and fungicides commonly used in vineyards (EUROSTAT, 2020). Anilide pesticides such as the herbicides acetochlor, alachlor, S-metolachlor, butachlor and the fungicide metalaxyl can be exported off-site from agricultural fields during rainfall events, representing a risk for ground and surface waters. Off-site export of pesticides from agricultural settings largely depends on their dissipation in the soil, including pesticide loss by leaching following a rainfall event, as well as plant uptake, and ageing and degradation in the soil. Although agricultural plots are generally vegetated, leaching and biodegradation of anilide pesticides in the soil-plant system are generally studied independently.

Interactions between plants, soil and microorganisms can enhance pesticide availability and dissipation [START_REF] Chaudhry | Utilising the synergy between plants and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment[END_REF], making the soil-plant system an environmental hotspot for pollutant transformation [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. The soil-plant system includes the rhizosphere, which is characterized by diverse and intense microbial activity, respiration processes and nutrient uptake, mainly controlled by the water circulation [START_REF] Bardgett | Belowground biodiversity and ecosystem functioning[END_REF][START_REF] Hinsinger | Rhizosphere: biophysics, biogeochemistry and ecological relevance[END_REF][START_REF] Hinsinger | Rhizosphere: a new frontier for soil biogeochemistry[END_REF][START_REF] Mommer | Root-root interactions: towards a rhizosphere framework[END_REF][START_REF] Zhang | The unseen rhizosphere root-soil-microbe interactions for crop production[END_REF]. While organic contaminants are hardly adsorbed to organic matter and soil minerals, the root exudates from plants favour their desorption and availability, being directly accessed by macro-and microorganisms and plants [START_REF] Huang | Influences of artificial root exudate components on the behaviors of BDE-28 and BDE-47 in soils: desorption, availability, and biodegradation[END_REF]. Plant rhizosphere can enhance pesticide dissipation by sustaining specific microbial communities capable of pesticide transformation [START_REF] Diez | Pesticide dissipation and microbial community changes in a biopurification system: influence of the rhizosphere[END_REF], resulting in shorter pesticide half-lives in the rhizosphere compared to the bulk soil [START_REF] Hand | Evaluation of the rhizosphere contribution to the environmental fate of the herbicide prometryn[END_REF]. However, pesticide dissipation in the soil-plant system remains poorly understood as it depends mainly on the contribution of both degradative and non-degradative processes, which are themselves related to pesticide physicochemical properties and bioavailability [START_REF] Chaudhry | Utilising the synergy between plants and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment[END_REF][START_REF] Ren | Sorption, transport and biodegradationan insight into bioavailability of persistent organic pollutants in soil[END_REF][START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF].

Sorption, dilution and off-site transport can act simultaneously with degradation to dissipate pesticides in agricultural soil and making the dissipation difficult to disentangle using a mass balance approach relying on concentration analysis only.

Compound-specific stable isotope analysis (CSIA) may provide a concentrationindependent evaluation of pesticide degradation in the environment [START_REF] Elsner | Compound-specific isotope analysis (CSIA) of micropollutants in the environment -current developments and future challenges[END_REF] by evaluating changes in the stable isotope ratios (e.g., 13 C/ 12 C, and 15 N/ 14 N) within the molecule. Dilution processes such as transport or sorption, generally do not alter stable isotope ratios. However, pesticide molecules with light and heavy isotopes are degraded at slightly different rates, resulting in a kinetic isotope effect quantifiable by CSIA [START_REF] Meite | Impact of rainfall patterns and frequency on the export of pesticides and heavy-metals from agricultural soils[END_REF][START_REF] Elsayed | Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands[END_REF][START_REF] Melsbach | Dual-element isotope analysis of desphenylchloridazon to investigate its environmental fate in a systematic field study: a long-term lysimeter experiment[END_REF]Schürner et al., 2016;[START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF]. CSIA allowed to evaluate the bioavailability of pyrethroids in soil [START_REF] Xu | A new approach to estimate bioavailability of pyrethroids in soil by compound-specific stable isotope analysis[END_REF], and the degradation of γ-hexachlorocyclohexane [START_REF] Qian | Assessment of hexachlorcyclohexane biodegradation in contaminated soil by compound-specific stable isotope analysis[END_REF] and polybrominated diphenyl ethers [START_REF] Wang | Assessing microbial degradation degree and bioavailability of BDE-153 in natural wetland soils: implication by compound-specific stable isotope analysis[END_REF]. Using CSIA, we recently demonstrated that biodegradation and sorption mainly drove dissipation of S-metolachlor and butachlor, respectively, in contrasted agricultural soils [START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF]. However, this study also brought up new questions. How do soil characteristics affect leaching, dissipation and biodegradation of anilide pesticides following rainfall events? What is the role of plants and the rhizosphere in the dissipation and biodegradation of anilide pesticides?

In the present study, we evaluated and compared the contribution of processes responsible for dissipation of anilide pesticides, including acetochlor (ACE), alachlor (ALA), S-metolachlor (SM), butachlor (BUT) and metalaxyl (MTY), in planted and unplanted soil mesocosms exposed to successive rainfall events. These five anilide pesticides were selected since they represent a major pesticide class used worldwide, although their applicability and toxicity largely vary. The herbicides acetochlor and alachlor are not allowed in agricultural crops in the EU, while S-metolachlor is mostused in the EU and South America, and butachlor in Asia [START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF].

Metalaxyl is used as a fungicide to control air-and soil-borne diseases in crops and vineyards (PPDB, 2019). While sharing the same general chemical structure (i.e., acyl derivatives of aniline), the anilide pesticides selected in this study display a broad spectrum of physico-chemical properties, which may drive their dissipation in soil-plant systems (see Supporting Information SI-A). Their dissipation in soil-plant systems have been found to be quicker in the rhizosphere than in the bulk soil [START_REF] Bai | Alterations of microbial populations and composition in the rhizosphere and bulk soil as affected by residual acetochlor[END_REF][START_REF] Elsayed | Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands[END_REF], although they are usually studied individually. Two contrasted forest and vineyard soils were used to evaluate the effect of soil characteristics on pesticide dissipation processes using CSIA. We hypothesized that pesticide biodegradation would be larger in the planted mesocosms and in the soil solution that may leach after a rainfall event. In contrast, non-degradative processes were expected to predominate in non-planted mesocosms and in the bulk soil.

Materials and Methods

Chemicals

Anilide standards of acetochlor (ACE), alachlor (ALA), S-metolachlor (SM), butachlor (BUT) and metalaxyl (MTY) (PESTANAL, purities: 98%, 98%, 98%,96.9% and 99.6%, respectively), metolachlor-d 11 (analytical grade purity: >97%), dichloromethane (DCM), acetonitrile (ACN) (HPLC grade, purity: >99.9%), and anhydrous magnesium sulfate (reagent grade: >97%) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of the pesticide mixture for soil spiking and standards for analysis were prepared as 1 g/L in DCM and ACN, respectively.

Aliquots were stored at -20°C. Primary-secondary amine (PSA) was supplied by Supelco (Bellefonte, PA, USA).

Experimental soils

A vineyard and a forest soil with contrasted physicochemical characteristics were collected, respectively, on November 23 and 24, 2017, from two wellcharacterized sites. The vineyard soil was collected from a plot in the experimental catchment of Rouffach France,47° 57' 43.0'' N,7° 17' 27.3'' E,altitude: 200 m asl) [START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF][START_REF] Duplay | Copper, zinc, lead and cadmium bioavailability and retention in vineyard soils (Rouffach, France): the impact of cultural practices[END_REF]. The forest soil was collected from the Strengbach experimental catchment (Haut-Rhin, France, 48° 12' 57.9'' N, 7° 11' 52.5'' E, altitude: 850 m asl) [START_REF] Gangloff | Factors controlling the chemical composition of colloidal and dissolved fractions in soil solutions and the mobility of trace elements in soils[END_REF].

Sixty kg of topsoil were collected by random sub-sampling at each site with a distilled water-and ethanol-cleaned shovel. Sub-samples of vineyard topsoil (0-10 cm, Hor. A) were collected at ten locations across vine inter-rows from a 1486 m 2 plot managed under conventional wine-growing practices. Sub-samples of forested topsoil (0-10 cm, mostly organic horizon) were collected from eight different random locations of a spruces plot (1500 m 2 ). Soil samples were transported in coolers into the laboratory, dried at 30°C with an air-forced oven and sieved using a 2-mm mesh.

After complete homogenization by mixing in quarters up to four times, an aliquot of each dried soil was weighed and dried at 105 °C until constant weight was achieved to quantify the initial soil humidity. Soil physicochemical characteristics were measured following ISO methods and procedures, as detailed in SI-B.

Mesocosms setup

Two series of mesocosm experiments with pesticide-spiked soil, planted and unplanted, were set up in triplicate for each soil type (Figure 1). An additional two series of planted and unplanted mesocosms amended with sterile-filtered water without pesticides was run in parallel as nonspiked 'blank' experiments (data not shown). In total, twelve spiked mesocosm experiments and twelve nonspiked 'blank' experiment were followed in parallel.

Each soil mesocosms consisted of a rhizotron device, made of polypropylene plastic bottles with a nylon filter at the bottom (0.45 µm) and filled with 600 g of homogenized and dried vineyard or forest soil. Mesocosms were spiked with aqueous stock solutions containing ACE, ALA, SM, BUT and MTY. Soil bulk densities were 2.0 and 0.7 g/cm 3 for vineyard and forest soils, respectively. Pesticide spiking in the aqueous solution was adjusted to 50% (W/W) of soil water content to ensure that both soil types reached water holding capacity (see SI-C for details). To homogenize water content and pesticide spiking (SI-D), sterile artificial rainwater (SI-E) was introduced by capillarity from the bottom of the mesocosms, while 100 mL of aqueous pesticide solution was added with a pipette at the top of the mesocosms. Final pesticide concentrations of each pesticide in the spiked soil mesocosms was 25 mg/kg, corresponding to severe environmental concentrations [START_REF] Meite | Impact of rainfall patterns and frequency on the export of pesticides and heavy-metals from agricultural soils[END_REF][START_REF] Lefrancq | High frequency monitoring of pesticides in runoff water to improve understanding of their transport and environmental impacts; 28242219[END_REF].

The mesocosms were placed randomly in a climate chamber (FDM LFC900SX PRO, Italy) and incubated for 75 days under controlled conditions of temperature (20 °C), humidity (70% RH) and photoperiodic lighting (16 h light, 8 h dark) with fluorescent LED lamps at 170 µmol photons/m 2 .

Mesocosm operations

Each of the planted mesocosms were seeded with 320-340 Italian ryegrass (Lolium multiflorum sp.) at 1-2 seeds per cm² [START_REF] Bourceret | Mapping the centimeter-scale spatial variability of PAHs and microbial populations in the rhizosphere of two plants[END_REF]. Ryegrass was chosen since it is an herbaceous fast-growing species widely used to cover vineyard soils [START_REF] Fernández-Calviño | Cu immobilization and Lolium perenne development in an acid vineyard soil amended with crushed mussel shell[END_REF]. Mesocosms without pesticides were incubated as parallel duplicate control mesocosms to determine the necessary incubation time to reach the vegetation maturity stage.

The soil water content of the mesocosms was controlled by weighing daily each mesocosm and adjusted if necessary. A soil water content of 50% (W/W) was maintained across the experiment by spraying MilliQ ® water on the top of the mesocosms.

The preparation of artificial rainfall and the rainfall simulator system are described in SI-E. Artificial rainfall events consisted of a total volume of 300 mL, corresponding to a rainfall depth of 15 mm, applied on the mesocosms with a duration of 12 min at 62 mm/h. This rainfall event pattern represents a medium duration event with intermediate intensity in a temperate climate [START_REF] Meite | Impact of rainfall patterns and frequency on the export of pesticides and heavy-metals from agricultural soils[END_REF]. A first artificial rainfall (R1) was applied when plants in control nonspiked mesocosms reached the elongation stage (i.e., 45 days after seeding). The second and third artificial rainfalls (R2, R3) were applied 15 (day 60) and 30 days after R1 (day 75), respectively.

Mesocosms sampling

The sampling scheme and sample preparation procedures are detailed in SI-F.

One day after pesticide spiking (day 1), triplicate soil sub-samples were collected randomly from each mesocosm. Then, they were homogenized and pooled separately per experimental condition. Leachate samples were collected after each artificial rainfall on days 45, 60 and 75, denoted as L45, L60 and L75, respectively. Leachates were sampled separately from each mesocosm. Half of the leachate volume was immediately filtered using a 0.45 µm glass fibre filter for pesticide analysis. The other half was filtered using a 0.45 µm cellulose acetate filter for pH, DOC, DIC, major anions and cations and minor nutrient analysis. For each condition, one mesocosm (day 45) and duplicate mesocosms (day 75) were sacrificed to collect bulk soil, rhizosphere, plant aerial parts and roots.

Pesticide quantification and CSIA

Pesticides were extracted from soil and rhizosphere samples using a solvent extraction and cleanup procedure adapted from [START_REF] Ivdra | Validation of user-and environmentally friendly extraction and clean-up methods for compound-specific stable carbon isotope analysis of organochlorine pesticides and their metabolites in soils[END_REF]. Pesticides were extracted from leachates following a protocol adapted from US EPA Method 525.2 and described by [START_REF] Elsayed | Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands[END_REF]. Briefly, a SPE (solid phase extraction) procedure was followed, using SolEx C18 cartridges (1 g, Dionex®, CA, USA) on an AutoTrace 280 SPE system (Dionex®, CA, USA). Concentrations of ACE, ALA, SM, BUT and MTY from soils and leachates were measured using GC/MS as described in SI-G and SI-H.

The carbon isotope compositions (δ 13 C) of ACE, ALA, SM, BUT and MTY and nitrogen (δ 15 N) of SM were analysed using a GC-C-IRMS system consisting of a TRACE™ Ultra Gas Chromatograph (ThermoFisher Scientific) coupled via a GC IsoLink/Conflow IV interface to an isotope ratio mass spectrometer (IRMS DeltaV Plus, ThermoFisher Scientific) configured as described in Masbou et al. (2018) and detailed in the SI-I.

Every injection was checked to ensure that it remained within the linearity ranges (SI-I). A set of in-house BTEX (for C-CSIA), IAEA-600 caffeine (for N-CSIA), and pesticide standards with known isotopic composition, determined using an elemental analyser-isotopic ratio mass spectrometer (Flash EA IsoLinkTMCN IRMS, Thermo Fisher Scientific, Bremen, Germany) was measured at least every ten injections to control analytical reproducibility and accuracy of measurements.

Extraction and measurement methods used for pesticide CSIA from soil and leachate samples did not cause significant isotope fractionation (Δδ 13 C≤1‰ and Δδ 15 N≤0.5‰) (SI-I, J and K). Analytical uncertainty of 0.5 ‰ and 0.7 ‰ was attributed to each measurement based on ±1 SE of, respectively, mean δ 13 C and δ 15 N values from triplicate measurements for each sample, and the long-term reproducibility measurements.

Data analysis

Water budget, nutrient and pesticide leaching

Water mass budget [START_REF] Thornthwaite | The water balance[END_REF] was calculated as follows:

(1)

where P corresponds to precipitation and SWC to soil water content or the water mass per mass of dry soil. ETP and L correspond to potential evapotranspiration from planted mesocosms and to leachates, respectively.

The mass water budget for each compartment was used to estimate the pesticide exports following each rainfall event.

Pesticide loads (µg) in leachates were estimated from individual pesticide concentrations (in µg/L) in leachates (L45, L60 and L75), and the leached volume ( , in L) per mesocosm at each sampling time t:

(2)

The leachate export, i.e., the proportion of pesticide mass leached from a mesocosm with respect to the initial amount of spiked pesticides, and the first flux contribution, i.e., the contribution of the first leachate (L45) to the total pesticide export by leaching, were calculated according to Eq. 3 and 4, respectively:

(3)

(4)
where is the total mass of pesticide exported during the experiment, is the mass spiked of each pesticide, and is the pesticide mass in L45.

Quantification of pesticide dissipation

Pesticide dissipation in each mesocosm, accounting for the difference between the initial amount of the spiked pesticides and the losses by leaching, degradation, plant uptake and ageing in the soil and rhizosphere, was estimated using Eq. 5:

(5) where and represent pesticide mass [µg] at the initial sampling time (t1) and time t (45, 75) in soil mesocosms, respectively.

Dissipation kinetics

Pesticide data in each condition were fitted to an exponential decay single firstorder (SFO) model according to NAFTA guidelines (USEPA). Dissipation rates (k)

were estimated when the model provided a significant fit (R 2 > 0.6 and p < 0.05) from n ≥ 3 points (SI-N).

Carbon stable isotope notation and calculation

and values were normalized by the Vienna Pee Dee Belemnite (VPDB) standard for carbon and by the international AIEA600 air standard for nitrogen as follows:

(1)

where is expressed in per thousand ( ) and refers to the ratio of heavy ( ) to light ( ) isotopes of the element in the analysed samples and the international standards. Samples were injected in triplicate, and values are reported as the associated arithmetic mean. Each measurement was checked to remain within the linearity ranges for C and N. A set of in-house BTEX (for C), caffeine (AIEA 600, for N) and pesticide (for C and N) standards with known isotopic composition (determined by EA-IRMS) was measured at least every ten injections to control the measurement quality. Reference and compositions of BTEX and pesticide standards were determined at our isotope facility using an elemental analyser-isotopic ratio mass spectrometer (Flash EA IsoLinkTMCN IRMS, Thermo Fisher Scientific, Bremen, Germany).

The average isotope value of the residual nondegraded fraction of herbicides varies according to the Rayleigh equation:

(7)
where f is the residual substrate fraction, δ 13 C t the corresponding isotope values at time t and δ 13 C 0 is the initial isotope value for carbon of each compound at the beginning of the experiment (f = 1).

The isotopic fractionation factor ε relates changes of isotope ratios to the extent of transformation. The ε c factor was determined based on carbon isotope data obtained across the experiment (days 1 -75) using a linear regression according to:

(8)

where δ 13 C 0 and δ 13 C t are carbon isotope ratios for each compound studied, and C 0 and C t are concentrations at time 0 (day 1) and t, respectively. Values of carbon isotopic fractionation ɛ c for the pesticides were determined if (i) isotope fractionation during the incubation period was significant (Δδ 13 C>2‰, Álvarez-Zaldívar et al., 2008), (ii) the linear regression of the Rayleigh plot was significant (R 2 > 0.6, P < 0.05), and (iii) n ≥ 3 sampling times were available.

Quantification of pesticide biodegradation

The extent of biodegradation (B C ) of all compounds studied at time t in planted and unplanted mesocosms was estimated from δ 13 C values obtained in the present study using the inverse form of the Rayleigh equation [START_REF] Elsner | Insight into methyl tert-butyl ether (MTBE) stable isotope fractionation from abiotic reference experiments[END_REF]. Isotopic fractionation factors (ɛ c ) of anilide pesticides retrieved from [START_REF] Elsayed | Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands[END_REF] for ACE (-3.5±0.5) and ALA (-2.0±0.3), Masbou et al., (2018aMasbou et al., ( , 2018b) ) for BUT (-3.7±0.7) and MTY (-2.0±1.3 in F and -0.9±0.5 in V soil), respectively and Álvarez-Zaldívar et al.

(2018) for SM (-1.5±0.5) were used to estimate B C,t (CSIA) , since the experiments were carried out in biotic mesocosms with the same or similar soils:

(9)

Results and discussion

Water balance, plant growth and soil characteristics

Water leaching accounted for 75±3% ( ±σ) and 82±2% of the initial rainfall in planted and unplanted vineyard mesocosms, and for 34±4% (planted and unplanted) in forest mesocosms (SI-L).

Plant growth started in spiked planted microcosms from day 45 onwards, which correlated with the first rainfall. At day 75, plant biomass in spiked and nonspiked planted mesocosms was, respectively, 0.1 and 26.8 g/kg soil (dry aerial part). The length of plant aerial parts ranged from 3-5 and 15-20 cm, respectively, while roots ranged over a length of 1-4 and 12 cm respectively, with a density of 1.1•10 -3 -5.5•10 -4 and 2.2•10 -2 -6.7•10 -3 g (dry weight)/cm 3 soil, respectively, emphasising the suppressing effect of pesticides on plant growth in spiked mesocosms.

Plants contributed to decrease pH in vineyard soil due to nutrient uptake and leaching of basic nutrients. Leachate pH values from vineyard mesocosms were slightly lower in planted mesocosms (7.2-7.5) than in unplanted mesocosms (7.6-7.8), although soil pH values remained constant over the experiment (planted: 7.8-8.0, unplanted: 8.0-8.3) (Table 1). In contrast, the forest soil pH (initial pH = 3.4) increased in both planted (up to 4.0) and unplanted (up to 3.8) mesocosms during the experiment, whereas the pH of leachates varied from 3.4 to 3.6 in both types of mesocosms. Acid root exudates during plant growth may displace major cations such as K or Mg in the soil solution, which likely increased the pH of the acidic forest soil [START_REF] Canarini | Root exudation of primary metabolites: Mechanisms and their roles in plant responses to environmental stimuli[END_REF] (SI-B).

Leaching of dissolved organic carbon (DOC) was 2-fold higher in the forest than in the vineyard mesocosms (Table 1), which can be related to the higher OM content in forest soil (SI-B). However, DOC concentrations in leachates from unplanted mesocosms were higher than leachates from planted mesocosms for both soil types, suggesting higher DOC retention in planted mesocosms during plant growth, and distinct carbon turnover in planted and unplanted mesocosms. Cation exchange capacity (CEC) was slightly higher in the vineyard than in the forest mesocosms (Table 1), reflecting larger cation uptake from the soil matrix, especially after pesticide leaching, which might contribute to the larger plant growth in the vineyard mesocosms.

Pesticide leaching

Pesticide leaching from the vineyard mesocosms was generally higher than from the forest mesocosms (Figure 2). The contribution of the first rainfall event to pesticide export by leaching ranged from 69 up to 99% (Table 2). The first rainfall event following pesticide application thus exported the largest fraction of pesticides, as previously observed with synthetic [START_REF] Hunsche | Mancozeb wash-off from apple seedlings by simulated rainfall as affected by drying time of fungicide deposit and rain characteristics[END_REF] and Cu-based fungicides [START_REF] Pérez-Rodríguez | Modeling losses of copper-based fungicide foliar sprays in wash-off under simulated rain[END_REF][START_REF] Pérez-Rodríguez | Rainfall-induced removal of copper-based spray residues from vines[END_REF]. The pesticide concentrations in leachates normalized per kg of mesocosm soil varied from 1•10 -3 mg/kg (ACE) up to 3.3 mg/kg (MTY) (Figure 2).

Pesticide leaching from the unplanted vineyard mesocosms was larger than leaching from the planted mesocosms, whereas leaching was similar in the planted and unplanted forest mesocosms. However, pesticide leaching from both soil types, ordered from highest to lowest, followed a MTY > SM ≈ BUT > ALA ≈ ACE pattern (Figure 2).

MTY leaching exports accounted for up to 12% in unplanted and 7% of initially spiked MTY in planted vineyard mesocosms, whereas it remained <3% in forest mesocosms (Table 2). The relatively high leaching of MTY in both vineyard and forest mesocosms reflects the high solubility (SI-A) and low sorption propensity of MTY [START_REF] Fernández-Calviño | Metalaxyl mobility in acid soils: evaluation using different methods[END_REF]. However, leaching exports were in the range of those established by the EU [START_REF] Efsa | Conclusion on the peer review of the pesticide risk assessment of the active substance metalaxyl-M[END_REF]. Lower leaching exports of MTY in the planted mesocosms (Table 2) suggests that plants enhanced MTY retention and/or biodegradation, possibly by sustaining rhizosphere processes associated with MTY transformation [START_REF] Baker | Degradation of metalaxyl-M in contrasting soils is influenced more by differences in physicochemical characteristics than in microbial community composition after reinoculation of sterilised soils[END_REF].

Leaching of SM from vineyard unplanted mesocosms (8% in total) was three times higher than leaching of ACE and ALA. However, leaching exports of SM, ACE and ALA were similar in planted mesocosms (3%), emphasizing the effect of plants on SM retention in vineyard mesocosms. SM and BUT leaching from forest mesocosms remained <1.4%, especially in unplanted mesocosms (Table 2). ALA leaching exports from vineyard and forest mesocosms remained <3% and <2%, respectively, across the experiment (Table 2). Leaching exports of ALA were similar to those in soils with higher SOM and acidic pH [START_REF] Heyer | Comparison of the leaching behaviour of alachlor and its metabolites under field and laboratory conditions[END_REF] and in Baltic soils with low organic matter [START_REF] Sakaliene | Sorption and predicted mobility of herbicides in Baltic soils[END_REF] (ranging from 0.6 to 1.5%) but were lower than those found in tropical soils (about 6%) from Brazil [START_REF] Giori | The role of sugarcane residues in the sorption and leaching of herbicides in two tropical soils[END_REF]. These differences underscore that soil properties affected ALA partitioning and its concentration in the soil solution. Similarly, ALA, ACE leaching in vineyard mesocosms was higher in unplanted (3%) than in planted vineyard mesocosms (1%), whereas it accounted for <1% in forest mesocosms.

Larger pesticide leaching in vineyard than in forest mesocosms stems from a combination of soil characteristics, including soil texture, hydraulic retention time (HRT), water holding capacity (WHC) and pesticide mobilization. The compaction of the clay-loam soil in vineyard mesocosms led to water ponding during rainfall events, as observed previously with the same soil [START_REF] Meite | Impact of rainfall patterns and frequency on the export of pesticides and heavy-metals from agricultural soils[END_REF]. Water ponding in vineyard mesocosms reduced the water infiltration rate while increasing the HRT and soil-water interactions compared to forest mesocosms. A longer HRT and soil-water interaction time favoured soil dispersion and thus pesticide remobilisation. This, together with a limited WHC (34 ± 3.5%, see SI-C), increased pesticide leaching from vineyard mesocosms. In contrast, and despite a sand-loam texture reducing the HRT of the forest soil, a higher organic matter content and WHC (48 ± 0.8%, see SI-C), as well as the type of clay, mainly 2:1 with larger size of interlayer spaces, limited pesticide leaching from forest mesocosms (SI-M).

Pesticide dissipation

Pesticides could be quantified in the bulk and rhizosphere soil samples, whereas low root and biomass development hampered pesticide quantification in the plants.

Pesticide dissipation up to day 45 ranged from 30 to 88% in the vineyard and forest mesocosms and was similar in planted and unplanted mesocosms (Figure 3). However, pesticide dissipation mostly increased in planted mesocosms from day 45 onwards, paricularly in forest mesocosms (Figure 3), reaching up to 99% dissipation on day 75, indicating that plant growth observed from day 45 onwards favoured pesticide dissipation (data not shown). Pesticides suppressed plant growth before day 45, which limited the influence of plants on pesticide dissipation up to day 45. The first rainfall on day 45 washed off 69 to 99% of the total amount of leached pesticide (Figure 3), which reduced the herbicide inhibition effect. Less pesticide in the soil mesocosms allowed plant growth from day 45 onwards with concomitant rhizosphere development.

Pesticide dissipation patterns were similar in the vineyard and forest mesocosms, and globally ordered from lowest to highest solubility in water, as follows: BUT > ACE > ALA ≈ SM > MTY (SI-A).

Total dissipation of BUT, including leaching, was similar, although slightly higher in vineyard (>98%, k > 0.17 ± 0.17 day -1 ) than in forest mesocosms (>96%, k = 0.09 ± 0.09 day -1 ) (Figure 3 and Table S5, SI-N). The BUT dissipation, even when the contribution of leaching was considered, was faster in the vineyard planted mesocosms than previous estimations using the same soil [START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF]. In contrast, dissipation in forested soil was like the dissipation in a paddy soil with similar SOM content studied by [START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF]. Fast BUT dissipation can be related to its high K oc and lower water solubility, and sorption was likely the main process affecting apparent BUT dissipation in mesocosm experiments [START_REF] Gevao | Bound pesticide residues in soils: a review[END_REF][START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF].

Dissipations of ACE and ALA was >98% and >90% in the vineyard and the forest mesocosms, respectively, and was similar in both planted and unplanted mesocosms (Figure 3 and Table S5, SI-N). Dissipation kinetics in the vineyard mesocosms are in agreement with typical half-lives (DT 50 )soil values (SI-A), as well as previous observations in paddy soils [START_REF] Yu | Dissipation dynamics and residue of four herbicides in paddy fields using HPLC-MS/MS and GC-MS[END_REF] and in multi-contaminated soils with similar physicochemical properties [START_REF] Jiang | The effect of antibiotics on the persistence of herbicides in soil under the combined pollution[END_REF]. Significantly lower halflives in the rhizosphere than in the bulk soil were found for ACE [START_REF] Bai | Alterations of microbial populations and composition in the rhizosphere and bulk soil as affected by residual acetochlor[END_REF], suggesting that degradation is more extensive in the planted mesocosms. DT 50 values for ALA inferred from vineyard mesocosms were similar to reference values, while the values for forest mesocosms were similar to those reported in field studies and higher than reference values (up to 21 days, University of Hertfordshire, 2019) (SI-A), also reflecting the higher affinity of ALA to forest SOM compared to ACE (ALA K OC > ACE K OC ). Lower ACE and ALA dissipation in forest than in vineyard mesocosms was consistent with slower dissipation rates (Table S5, SI-N) and with results of a simulated organic matrix biobed experiment with high organic matter content [START_REF] Chu | Enhanced dissipation of selected herbicides in a simulated organic matrix biobed: A system to control on-farm point-source pollution[END_REF].

Similarly, total dissipation of SM was >96% (Figure 3; Table S5, SI-N), slightly higher in planted than in unplanted mesocosms, and higher than in previous studies for the same soil [START_REF] Torabi | Dissipation of S-metolachlor and butachlor in agricultural soils and responses of bacterial communities: insights from compound-specific isotope and biomolecular analyses[END_REF]. Higher dissipation compared to previous studies reflects the contribution of SM leaching to total dissipation and suggests that plants enhanced SM dissipation. Dissipation rates were slower in the forest (Table S5, SI-N) than in the vineyard mesocosms, and corresponded to longer half-lives (33 to 51 d) than the dissipation rates observed in an acidic soil with similar physicochemical properties [START_REF] Gluhar | Dissipation of mecoprop-P, isoproturon, bentazon and S-metolachlor in heavy metal contaminated acidic and calcareous soil before and after EDTA-based remediation[END_REF].

Total MTY dissipation was larger than 97% and 58% in the vineyard and the forest mesocosms, respectively (Figure 3), and dissipation rates were faster in the vineyard (0.04-0.05 d -1 ) than in the forest (0.01 d -1 ) mesocosms (Table S5, SI-N).

Slower MTY dissipation in forest mesocosms is consistent with MTY half-lives in soils at pH lower than 7.5 [START_REF] Sukul | Influence of biotic and abiotic factors on dissipating metalaxyl in soil[END_REF] or more acidic [START_REF] Buerge | Enantioselective degradation of metalaxyl in soils: Chiral preference changes with soil pH[END_REF].

Pesticide dissipation in mesocosms resulted from leaching of the soil solution following a rainfall event, as well as pesticide ageing and biodegradation in the bulk soil and the rhizosphere. Overall, pesticides and soil physicochemical characteristics (SI-A) could be related to pesticide dissipation patterns in the mesocosms, with generally lower dissipation in the forest than in the vineyard mesocosms (Fig. 3). Higher organic matter content (5.3%) and, putatively, the type of clay (2:1 abundance) in forest soil enhanced pesticide sorption due to the availability of more sorption sites with larger sizes of interlayer spaces. Such conditions can limit pesticide availability, and thus pesticide biodegradation and export. In addition, lower pH of the forest soil canincrease pesticide half-lives [START_REF] Baker | Degradation of metalaxyl-M in contrasting soils is influenced more by differences in physicochemical characteristics than in microbial community composition after reinoculation of sterilised soils[END_REF] and affect microbial communities [START_REF] Fernández-Calviño | Growth response of the bacterial community to pH in soils differing in pH[END_REF][START_REF] Rousk | Investigating the mechanisms for the opposing pH relationships of fungal and bacterial growth in soil[END_REF], which in turn can influence pesticide biodegradation.

However, pesticide distribution and dissipation differed not only among pesticides but also between planted and unplanted mesocosms (Table 2 andFigure 3).

Our results also support the idea that the rhizosphere is a major hotspot of pesticide dissipation in planted systems [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. Pesticide biodegradation in the rhizosphere can be quick [START_REF] Kuzyakov | Rhizosphere size and shape: temporal dynamics and spatial stationarity[END_REF], and rhizosphere processes can influence a large volume of surrounding soil [START_REF] Dennis | Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities?[END_REF]. However, the presence of plants limited pesticide leaching in the vineyard and forest soils, which emphasizes the role of the rhizosphere on water and pesticide transfer in the plant-soil system [START_REF] Hinsinger | Rhizosphere geometry and heterogeneity arising from root-mediated physical and chemical processes[END_REF][START_REF] Hinsinger | Rhizosphere: biophysics, biogeochemistry and ecological relevance[END_REF]. Lower quantities of pesticides were found in the rhizosphere, and more generally the planted mesocosms, than in the bulk soil. The well-developed rhizosphere in vineyard mesocosms might have favoured the microbial biomass and specific microorganisms involved in pesticide degradation [START_REF] Bourceret | Mapping the centimeter-scale spatial variability of PAHs and microbial populations in the rhizosphere of two plants[END_REF]. In contrast, larger SOM and different clays structures in the forest mesocosms increased pesticide sorption and residence time [START_REF] Giori | The role of sugarcane residues in the sorption and leaching of herbicides in two tropical soils[END_REF], which limited pesticide leaching.

Overall, our results emphasise that plants and their rhizosphere influenced anilide pesticide dissipation, and putatively biodegradation, which is further discussed below using CSIA data.

Insights into anilide pesticide biodegradation in soil mesocosms by

CSIA

CSIA of pesticides was carried out in leachate and bulk soil samples, whereas pesticide amounts were insufficient for CSIA in the aerial parts of plants and rhizosphere soil samples.

In bulk soil, changes in δ 13 C values were less pronounced than in leachates and only occurred only at day 75 (Δδ 13 C up to 8.2 ± 0.8‰ for ACE in bulk soil of planted vineyard mescososms at day 75) (Figure 4). The absence of significant isotope fractionation of SM, BUT and MTY in the bulk soil before 75 days (Δδ 13 C ˂2‰, within the range of analytical uncertainty) suggests that sorption and leaching contributed more to SM, BUT and MTY dissipation in bulk soil than biodegradation. Based on physicochemical properties of the pesticides (SI-A) and mass balance (Figure 3), the contribution of sorption was higher for SM and BUT than for ACE and ALA, whereas leaching was the main MTY dissipation process, limiting the role of biodegradation of SM, BUT and MTY although dissipation apparently occurred.

In contrast, biodegradation dominated over other dissipation processes in the soil solution of both the vineyard and the forest mesocosms from day 45 onwards.

Significant isotope fractionation observed in leachates from the mesocosms indicated the occurrence of in situ degradation (Figure 4A). Carbon isotope composition (δ 13 C) of pesticides in leachates changed significantly following rainfall events on days 60 and 75, with changes of isotopic ratios (Δδ 13 C) up to 13.9 ±0.5‰ and 12.5 ±0.8‰ (ALA) in the planted vineyard and forest mesocosms, respectively (Figure 4A). Overall, isotopic fractionation of pesticides was significantly lower in both the planted and unplanted forest mesocosms than in vineyard mesocosms (p<0.05). Isotopic fractionation factors ε C that relate changes of isotope ratios to the extent of transformation [START_REF] Elsner | A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants[END_REF] were determined for ACE and ALA, using the isotopic fractionation factors cited previously, since good fitting with the Rayleigh model was observed (R 2 > 0.6, p<0.05; n<5). The values of ε C obtained for ACE and ALA with Eq. 8 ranged from -0.8 ± 0.4‰ (ACE in the planted forest mesocosms) up to -1.5 ± 0.6‰ (ALA in the vineyard mesocosms), which were slightly higher than the values of ε C obtained previously in [START_REF] Elsayed | Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands[END_REF] in a wetland mesocosms and significantly higher than the values of ε C obtained during abiotic hydrolysis by Masbou et al., (2018a). ε C values did not differ significantly between planted and unplanted vineyard and forest mesocosms (p<0.05). The extent of biodegradation in leachates from the vineyard and the forest mesocosms for ACE, ALA, and SM, estimated on day 75 based on isotopic data (Eq. 9), reached 93, 98 and 95 to 100%, respectively (Table 3).

Another important finding is that biodegradation was more pronounced in leachates from planted than from unplanted mesocosms (Table 3). Changes in δ 13 C values in leachates after 45 days were larger for ACE, ALA, SM and BUT in the vineyard planted mesocosms (Δδ 13 C up to 13.9 ± 0.5‰ for ALA at day 75) than in leachates from unplanted mesocosms and in the bulk soil (Figure 4). In leachates from the forest planted mesocosms, changes in δ 13 C values increased over time and were larger (Δδ 13 C up to 9.1 ± 0.4 for ACE) than in unplanted mesocosms (Δδ 13 C up to 4.4 ± 0.5‰ for ACE) on day 75 (Figure 4).

Overall, biodegradation was identified as the major process responsible for ACE, ALA and SM dissipation in the soil solution from both vineyard and forest mesocosms. Biodegradation occurred mainly in the soil solution where pesticides are mostly available to microorganisms [START_REF] Megharaj | Bioremediation approaches for organic pollutants: a critical perspective[END_REF]. Despite the lack of sterile experiments in this study, abiotic degradation of pesticides in the mesocosms are likely minor, especially under neutral/acidic conditions where the anilide pesticides were shown to be stable to hydrolysis (EXTOXNET, 2000;[START_REF] Zheng | Identification of UV photoproducts and hydrolysis products of butachlor by mass spectrometry[END_REF]Masbou et al., 2018a). Hence, we assumed that observed changes in the carbon isotopic composition of pesticide in the mesocosms reflected in situ biodegradation.

Nondegradative processes, including loss by leaching and sorption, also contributed to SM, BUT and MTY dissipation in the bulk soil of vineyard and forest mesocosms. ACE and ALA were biodegraded mainly in the leachates from the vineyard mesocosms, while biodegradation contributed to the dissipation of all pesticides except BUT in the leachates from the forest mesocosms (Table 3). This also highlights the potential of CSIA to differentiate dissipation processes in soil, by estimating the contribution of pesticide biodegradation versus pesticide sorption. Pesticide CSIA based on carbon alone may, however, not be sufficient to distinguish different pathways of transformation of pesticides. Multielement CSIA (ME-CSIA) of anilide pesticides (δ 2 H, δ 13 C, δ 15 N and δ 37 Cl) could be applied for this purpose.

Here only few leachate samples satisfied the requirements for reliable δ 15 N measurements of SM in the vineyard mesocosms. Changes in the δ 15 N values for SM were similar in the planted (Δδ 15 N up to 2.8 ± 0.7‰ on day 75) and the unplanted (Δδ 15 N up to 2.9 ± 0.7‰ on day 75) mesocosms (SI-O). As an attempt to examine the degradation mechanisms of SM in the mesocosms, a dual-isotope plot of the changes in isotope ratios between N and C (Δδ 15 N vs. Δδ 13 C) was drawn to retrieve the Λ values from the slopes (SI-O). Distinct Λ values suggest different biodegradation mechanisms in the planted (Λ = Δδ 15 N/Δδ 13 C = 0.7 ± 0.6, 95% CI, n=4) and the unplanted (Λ = 1.8 ± 0.8, 95% CI, n=4) mesocosms. Interestingly, the Λ value retrieved from the planted mesocosm was like the Λ value previously observed during SM hydrolysis (Λ = 0.8 ± 0.7; Masbou et al., 2018a). Although these results emphasize the potential of ME-CSIA, identification of the SM degradation pathways based on transformation products and the associated C and N isotope fractionation is required to interpret the degradation mechanisms of SM in soil-plant systems. Currently and due to the above-mentioned limitations, the contribution of carbon to the molar mass of anilide pesticides makes carbon a reliable candidate to evaluate in the routine the occurrence of biodegradation using CSIA at typical low (sub-mg/L) concentrations of pesticides in soil-plant systems.

However, when the reaction involves the breakage of a bond containing chlorine as a single heteroatom in the pesticide molecule (e.g., the C-Cl bond for the biotic or abiotic hydrolysis of metolachlor), the expected strong Cl fractionation may be more informative than carbon to evaluate in situ degradation [START_REF] Ponsin | Compound-Specific Chlorine Isotope Analysis of the Herbicides Atrazine, Acetochlor, and Metolachlor[END_REF]. In addition, Cl-CSIA for anilide herbicides with GC-MS typically requires smaller amounts of compounds than for C-CSIA with GC-IRMS.

Biodegradation was generally more pronounced in leachates from planted than from unplanted mesocosms. Plants can enhance pesticide sorption on SOM or/and minerals within the rhizosphere, while root exudates favour pesticide desorption and availability, thereby increasing biodegradation [START_REF] Huang | Influences of artificial root exudate components on the behaviors of BDE-28 and BDE-47 in soils: desorption, availability, and biodegradation[END_REF][START_REF] Lefevre | Root exudate enhanced contaminant desorption: an abiotic contribution to the rhizosphere effect[END_REF][START_REF] Violante | Biogeochemical processes at soil-root interface[END_REF]. While microbial (co-)metabolism is a major degradation processes for anilide pesticides [START_REF] Elsayed | Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands[END_REF][START_REF] Imfeld | Toward integrative bacterial monitoring of metolachlor toxicity in groundwater[END_REF], the presence of a plant-root system in the soil can sustain rhizosphere microbial communities through root exudates, mainly consisting of labile organic matter [START_REF] Mommer | Root-root interactions: towards a rhizosphere framework[END_REF]. We hypothesized that the plant-root system altered the carbon/nitrogen balance in the rhizosphere and affected the microbial ability to use carbon [START_REF] Jones | Carbon flow in the rhizosphere: carbon trading at the soil-root interface[END_REF], and rhizosphere microbial communities were associated with anilide degradation. These processes appear to be decoupled from the bulk soil and the absence of plants may limit the microbial degradation functions of soil [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF], although our results suggest that biodegradation in bulk soil of planted mesocosms was more pronounced than in the unplanted mesocosms.

Conclusions

The leaching and dissipation of five widely used anilide pesticides in planted and unplanted vineyard and forest soil mesocosms were investigated and compared. Most importantly, this study shows the potential of CSIA to evaluate the occurrence and extent of biodegradation of pesticide mixtures in plant-soil systems.

Although physicochemical characteristics of the pesticides studied differed significantly, our approach showed significant pesticide biodegradation in the soil solution following rainfall events rather than in the bulk soil, which reflected their bioavailability of the pesticides. However, the contribution of leaching to the total dissipation of pesticide remained limited, and pesticide biodegradation depended mainly on the physicochemical characteristics of soil and pesticides.

In conclusion, this study proposes an approach to tease apart the dissipation processes of anilide pesticides in the soil-plant system. In the future, a similar approach may be adapted to various soil-plant systems and at different scales to examine realistic micropollutant dissipation scenarios accounting for in situ biodegradation and the role of rhizosphere.

  Analysis of dissipation kinetics, leachate export and CSIA of the pesticides allowed the evaluation of the contribution of degradative and non-degradative processes dissipation in soil and the leachates of planted and unplanted mesocosms and for two contrasted vineyard and forest soils. The results indicate that both degradative and nondegradative processes contributed to the apparent dissipation of BUT, SM, ALA, ACE and MTY in planted and unplanted mesocosms. The contribution of leaching, ageing in soil and biodegradation varied, depending mainly on the physicochemical characteristics of the pesticides, the SOM content and the occurrence of plants. High affinity of pesticide to SOM and clays may increase sorption, whereas pesticide degradation was enhanced by plants and their rhizosphere, which may increase pesticide bioavailability and desorption and eventually favour pesticide leaching.

Acknowledgements

Paula Pérez-Rodríguez was supported by a Postdoctoral fellowship from the Galicia Regional Government: "Consellería de cultura, educación e ordenación universitaria & GAIN, Xunta de Galicia, ED481B-2017/31", Spain. This study was funded by IDEX grant BioDisphère from the University of Strasbourg and by the French National Research Agency ANR through grant ANR-18-CE04-0004-01, project DECISIVE. The authors wish to acknowledge two anonymous reviewers for helpful comments and suggestions, as well as Amélie Aubert, Boris Droz, Colin Fourtet and Benoît Guyot for their help with analytical measurements.