Sarah Fourour

Véronique Tocqueville

Frédéric Paboeuf

Gérald Lediguerher

Nadège Morin

Isabelle Kempf

Corinne Marois-Créhan

Coraline Mattei

Julien Dupont

Henri Wortham

Etienne Quivet
email: etienne.quivet@univ-amu.fr

Pathogenicity study of Mycoplasma hyorhinis and M. flocculare in specific-pathogen-free pigs pre-infected with M. hyopneumoniae

Keywords: Atmosphere, Surface coating, Degradation, Heterogeneous, Ozone, Pesticides

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

Introduction

Numerous studies have reported the presence of pesticides in the atmosphere in concentrations ranging from a few picograms per cubic meter to hundreds of nanograms per cubic meter [START_REF] Coscollà | Occurrence of currently used pesticides in ambient air of Centre Region (France)[END_REF][START_REF] Estellano | Assessing levels and seasonal variations of current-use pesticides (CUPs) in the Tuscan atmosphere, Italy, using polyurethane foam disks (PUF) passive air samplers[END_REF][START_REF] Lopez | Risk assessment of airborne pesticides in a Mediterranean region of Spain[END_REF][START_REF] Désert | Spatial and temporal distribution of current-use pesticides in ambient air of Provence-Alpes-Côte-d'Azur Region and Corsica, France[END_REF][START_REF] Villiot | Temporal and seasonal variation of atmospheric concentrations of currently used pesticides in Champagne in the centre of Reims from 2012 to 2015[END_REF]. At the same time, many adverse effects of pesticides on human health [START_REF] Clementi | Pesticides and fertility: An epidemiological study in Northeast Italy and review of the literature[END_REF][START_REF] Hatcher | Parkinson's disease and pesticides: a toxicological perspective[END_REF][START_REF] Amy | Organophosphate Pesticide Exposure and Attention in Young Mexican-American Children: The CHAMACOS Study[END_REF]Inserm 2013;[START_REF] Parrón | Environmental exposure to pesticides and cancer risk in multiple human organ systems[END_REF][START_REF] Kim | Exposure to pesticides and the associated human health effects[END_REF]) and on the environment [START_REF] Santadino | Glyphosate Sublethal Effects on the Population Dynamics of the Earthworm Eisenia fetida (Savigny, 1826)[END_REF][START_REF] Carvalho | Pesticides, environment, and food safety[END_REF]) have been evidenced. In particular, harmful health effects associated with pesticide exposure by inhalation of ambient air have been highlighted [START_REF] Lee | Community exposures to airborne agricultural pesticides in California: ranking of inhalation risks[END_REF][START_REF] Li | Occurrence, seasonal variation and inhalation exposure of atmospheric organophosphate and pyrethroid pesticides in an urban community in South China[END_REF][START_REF] Lopez | Risk assessment of airborne pesticides in a Mediterranean region of Spain[END_REF]. Therefore, it is important to improve our understanding of the atmospheric fate of pesticide.

In the atmosphere, pesticides are distributed between aqueous, gaseous, and particulate phases according to their physicochemical properties. Nevertheless, they are generally poorly present in the aqueous phase due to the low water solubility of most of them. Numerous pesticides are semi-volatile compounds, and a fraction of them is adsorbed on the atmospheric particles surface [START_REF] Sauret | Study of the effects of environmental parameters on the gas/particle partitioning of current-use pesticides in urban air[END_REF]. Pesticides can react with atmospheric oxidants such as ozone, hydroxyl (OH) and nitrate (NO3) radicals. Their atmospheric reactivity in the gas phase has been widely studied and the modeling software AOPWIN TM (Atmospheric Oxidation Program for Microsoft Windows, Software, [START_REF] Meylan | Computer estimation of the Atmospheric gas-phase reaction rate of organic compounds with hydroxyl radicals and ozone[END_REF] allows to estimate their rate constants with OH radicals and, to a lesser extent with ozone. For the particle phase, a few studies have investigated the heterogeneous degradation of pesticides with ozone (Mattei et al, 2018 and references therein), OH radicals (Socorro et al. 2016 and references therein), and NO3 radicals (Mattei 2019b, submitted). The influences of relative humidity and particle type on the pesticide reactivity have already been demonstrated [START_REF] Mattei | Heterogeneous atmospheric degradation of pesticides by ozone: Influence of relative humidity and particle type[END_REF](Mattei et al. , 2019a)). However, the influence of the amount of pesticide on the surface on the degradation kinetics was poorly investigated.

Before studying heterogeneous reactivity, authors calculate or estimate the particle surface coating level in the organic compounds under study. Assuming the formation of a uniform monolayer on the particle surface, these calculations are usually expressed in percentage of a monolayer. For pesticides, the total surface coating (i.e., the percentage of the particle surface covered by pesticide molecules, considering all the compounds adsorbed on the surface) varies greatly from a study to another, from 0.04% (Pflieger et al. 2009) to 69% (Al [START_REF] Rashidi | Heterogeneous Ozonolysis of Folpet and Dimethomorph: A Kinetic and Mechanistic Study[END_REF].

To the best of our knowledge, only one study focusing on the heterogeneous ozonolysis of chlorpyrifos (insecticide) adsorbed on sand particles investigated and demonstrated the influence of the particle coating level on pesticide degradation [START_REF] El Masri | Influence of the coating level on the heterogeneous ozonolysis kinetics and product yields of chlorpyrifos ethyl adsorbed on sand particles[END_REF]. The cited work shows that the increase of surface coating level from 6% to 60% of a monolayer slows the heterogeneous degradation kinetics down. If very few data are available for pesticides, further information on the influence of the surface coating level was published on other semi-volatile organic compounds such as Polycyclic Aromatic Hydrocarbons (PAH) adsorbed on fused silica plates [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF], on nonactivated silica gel [START_REF] Alebic-Juretic | Heterogeneous Polycyclic Aromatic Hydrocarbon Degradation with Ozone on Silica Gel Carrier[END_REF], on soot particles [START_REF] Pöschl | Interaction of Ozone and Water Vapor with Spark Discharge Soot Aerosol Particles Coated with Benzo[a]pyrene: O3 and H2O Adsorption, Benzo[a]pyrene Degradation, and Atmospheric Implications[END_REF], and on graphite and silica particles [START_REF] Perraudin | Kinetic Study of the Reactions of Ozone with Polycyclic Aromatic Hydrocarbons Adsorbed on Atmospheric Model Particles[END_REF][START_REF] Miet | Heterogeneous reactions of ozone with pyrene, 1-hydroxypyrene and 1-nitropyrene adsorbed on particles[END_REF]. For PAH, the reaction rates seem to be also influenced by the surface coating level.

The aim of this study is to investigate the influence of the surface coating level (0.3 to 15% of a monolayer) on the heterogeneous ozonolysis of four pesticides (cyprodinil, deltamethrin, permethrin, and pendimethalin) adsorbed on two types of model mineral particles (hydrophobic and hydrophilic silicas). These four pesticides were selected based on their presence in the atmosphere up to a dozen of ng m -3 [START_REF] Désert | Spatial and temporal distribution of current-use pesticides in ambient air of Provence-Alpes-Côte-d'Azur Region and Corsica, France[END_REF], on their toxicity (APVMA, 2017), and on their distribution between the gas and particle phases (AOPWIN TM , [START_REF] Meylan | Computer estimation of the Atmospheric gas-phase reaction rate of organic compounds with hydroxyl radicals and ozone[END_REF]. Moreover, they have been the subject of numerous studies of reactivity towards ozone [START_REF] Socorro | Heterogeneous reactions of ozone with commonly used pesticides adsorbed on silica particles[END_REF][START_REF] Mattei | Heterogeneous atmospheric degradation of pesticides by ozone: Influence of relative humidity and particle type[END_REF]) and OH radicals [START_REF] Socorro | The persistence of pesticides in atmospheric particulate phase: An emerging air quality issue[END_REF][START_REF] Mattei | Heterogeneous degradation of pesticides by OH radicals in the atmosphere: Influence of humidity and particle type on the kinetics[END_REF]. The two particle types were chosen as models for mineral atmospheric particles. They have different surface properties but a very close composition and morphology. For this work, particles coated with a various amount of pesticides were exposed in realistic conditions of temperature, relative humidity, and gaseous ozone concentration.

Materials and methods

Chemicals

Cyprodinil (purity 99.8%), deltamethrin (99.7%), pendimethalin (98.8%), and permethrin (98.3%) were purchased from Sigma-Aldrich (PESTANAL®, analytical standard) and were used as received. The chemical structures of the pesticides under study are depicted in Supplementary Information (SI) Fig. SI1 and their physicochemical properties are given in SI Table SI1.

Particles

Atmospheric mineral aerosols were mimicked using two commercial silica particles: AEROSIL R812 (Degussa) hereafter referred to as "hydrophobic silica", and AEROSIL 255 (Evonik Industries) referred to as "hydrophilic silica". Composition: hydrophobic and hydrophilic silicas (>99.8 wt%) contain primarily SiO2. Particle size: the average primary particle size of hydrophobic and hydrophilic silica particles ranges from 5 nm to 50 nm (Evonik, 2015). However, most of them can be arranged as agglomerates with an aggregate size coarsely measured mainly around 5 μm, sometimes up to 25 μm. Field observations show that pesticides are distributed in the fine (0.1-1 μm), ultrafine (0.03-0.1 μm), and coarse (1-10 μm) particle size fraction, and no pesticides were detected in the size fraction >10 μm [START_REF] Coscollà | Particle size distributions of currently used pesticides in ambient air of an agricultural Mediterranean area[END_REF][START_REF] Coscollà | Particle size distributions of currently used pesticides in a rural atmosphere of France[END_REF][START_REF] Xu | Size distribution and seasonal variations of particle-associated organochlorine pesticides in Jinan, China[END_REF]. Specific surface area (SSA; BET method): hydrophobic and hydrophilic silica particles have a specific surface area of (260 ± 30) m 2 g -1 and (255 ± 25) m 2 g -1 , respectively (Evonik, 2015). Surface chemistry and hygroscopicity: surfaces of hydrophobic particles are mainly covered by siloxanes whereas hydrophilic silica particles are mainly covered by silanols groups. Silanol groups have a hydrophilic nature but the siloxane groups are chemically inert and hydrophobic.

Particles coating

Hydrophobic and hydrophilic silica particles were independently coated with pesticides according to a liquid/solid adsorption. In an amber Pyrex bulb of 500 cm 3 , 600 mg of particles were mixed with a pesticide solution in dichloromethane (each of the 4 pesticides at a concentration of 20 mg L -1 in dichloromethane for HPLC, ≥99.8%, Sigma-Aldrich) of a variable volume according to the coating level required and 40 mL of dichloromethane. After a 5-min ultrasound treatment, dichloromethane was evaporated by a rotary evaporator (Rotavapor R-114, Büchi) at 40°C and 850 mbar. For a given volume of solution, this process allows a reproducible coating of the pesticides on the particle's surface [START_REF] Socorro | Heterogeneous reactions of ozone with commonly used pesticides adsorbed on silica particles[END_REF].

The percentage of particle surface coated with pesticides was calculated considering the following assumptions: the particles surface coating is uniform and below a monolayer. The pesticide molecule is considered as a sphere and its radius (ri, cm) is calculated by Equation (1):

r i = 3 4 3 A m N V × × × π (1)
where Vm is the molar volume (cm 3 mol -1 ) of pesticide and NA is the Avogadro number. The molar volume is calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994-2014 ACD/Labs).

where n is the amount of adsorbed pesticide (mole):

Si = 4 × π × ri 2 × NA × n (2)
The percentage of the coated aerosol surface Ti (%) by the adsorbed pesticide i is calculated as follow:

Ti = × 100 (3)
where S1 is the silica particle surface (m 2 g -1 ).

The total surface coated (including all compounds under study) was calculated by adding individual percentages of the aerosol coated surface:

T = ΣTi (4)
As a result, the percentage of the particle surface coated with total pesticide was between 0.3% and 15% of the monolayer according to the different experiments for hydrophobic and hydrophilic silicas, which correspond to pesticide loadings between 0.166 mg g -1 and 8.45 mg g -1 .

Ozonolysis experiments

The experimental setup was previously detailed in [START_REF] Socorro | Heterogeneous reactions of ozone with commonly used pesticides adsorbed on silica particles[END_REF] (Fig. 1).

Fig. 1: Experimental setup to study the heterogeneous ozonolysis of pesticides Briefly, a 500 cm 3 amber Pyrex bulb containing about 500 mg of dried particles coated with the mixture of pesticides was fixed to a modified rotary evaporator and placed in a thermostated water bath (25 ± 1 °C). Ozone was generated by flowing purified air through an ozone generator (UVP, LLC Upland, UK). Particles coated with pesticides were exposed to an air flux containing an ozone mixing ratio of 410 ppb (i.e., 1.03•10 13 molecules cm -3 )

constantly measured with a photometric ozone analyzer (O3 41M, Environnement S.A). The relative humidity (RH) was set at 55% ± 2%. The humidity level was reached by mixing dry and wet gaseous fluxes of purified air. Relative humidity was measured with a humidity probe (Hydrolog NT, Rotronic, USA, uncertainty 2% RH) and the total of all air fluxes in the reaction flask was 500 mL min -1 . Experiments lasted for 26 h.

Extraction and pesticides quantification

During ozone exposure, 30 mg aliquots of particles were regularly sampled in order to quantify the remaining adsorbed pesticides on their surface. Each 30 mg aliquot of particles was individually introduced in a 33 mL stainless steel cell with an internal standard solution (Triphenyl phosphate, 99.9%, Sigma-Aldrich) and pesticides were extracted by accelerated solvent extraction (ASE 350, Dionex) with dichloromethane. Then, the extracts were concentrated under a nitrogen flow with a concentration workstation (TurboVap II, Biotage).

The obtained solutions were analyzed with gas chromatography coupled to tandem mass spectrometry (GC/MS-MS), with a Trace GC Ultra (Thermo Scientific) coupled to a TSQ Quantum™ Triple Quadrupole (Thermo Scientific) using electron impact ionization (70 eV).

More details about ASE extraction, concentration, and GC/MS-MS analysis are available in [START_REF] Socorro | Heterogeneous reactions of ozone with commonly used pesticides adsorbed on silica particles[END_REF].

Determination of second-order rate constants and half-lives toward ozone

Considering that ozone was continuously drifting in the reactor and that it was, therefore, present in excess, a pseudo-first-order kinetic constant was assumed:

( ) ( ) = -( (! " ) # + (! " ) # + %& (! " ) # ) × ' (5)
where

(! " ) # (s -1
) is the pseudo-first-order rate constant of the reaction between ozone and the pesticides,

(! " ) # (s -1
) is the first-order desorption rate constant, %& (! " ) # (s -1 ) is the pseudo-first-order hydrolysis rate constant, ' (s) is the time of ozone exposure, and

( ) ( )
is the pesticide concentration normalized to the initial pesticide concentration.

The pseudo-first-order reaction rate constants for the heterogeneous ozonolysis of the particlephase pesticides were determined by analyzing their corresponding temporal profiles in a time frame from 0 to 26 h, for 410 ppb ozone mixing ratio (i.e., 1.03 • 10 13 molecules cm -3 ).

( (! " ) # + %& (! " ) #
) was determined experimentally by exposing the pesticides adsorbed on the surface of particles to a flow of humidified air, using the same experimental conditions as for the ozonolysis experiments but in absence of ozone. In order to determine the second-order rate constants

(! " ) ##
(cm 3 molecule -1 s -1 ), the experimental pseudo-first-order reaction rate constants

(! " ) #
were plotted as a function of the ozone concentrations. To simulate the kinetic mechanisms of the heterogeneous reactivity, the second-order rate constants

(! " ) ##
were calculated assuming only one ozone mixing ratio, i.e., 410 ppb, using a Langmuir-Rideal model, as done in previous studies concerning pesticide degradation by ozone and hydroxyl radicals [START_REF] Mattei | Heterogeneous atmospheric degradation of pesticides by ozone: Influence of relative humidity and particle type[END_REF](Mattei et al., , 2019a)). Indeed, the linearity of the first order kinetic constants versus the ozone concentration (with ozone mixing ratios ranging from 215 ppb to 917 ppb) was already demonstrated in [START_REF] Socorro | Heterogeneous reactions of ozone with commonly used pesticides adsorbed on silica particles[END_REF]. The second-order rate constant was calculated as follows:

(! " ) ## = (! " ) # / ) *(+, ) (6)
where ) *(+, ) is the constant ozone mixing ratio of 410 ppb used in the experiments, which is representative of a highly polluted atmosphere (Finlayson-Pitts and Pitts 2009).

Results and discussion

Degradation kinetics

In order to study the influence of pesticide surface coating level on the heterogeneous ozonolysis kinetics, four pesticides (cyprodinil, deltamethrin, permethrin, and pendimethalin)

were adsorbed together on mineral model particles (hydrophobic and hydrophilic silicas) at five levels of surface coating (T = 0.3%, 1%, 1.5% (only hydrophilic silica), 2.8% (only hydrophobic silica), 10%, and 15% of a monolayer) and exposed to 410 ppb of ozone. As an example, Fig. 2 represents the degradation kinetics for permethrin adsorbed on hydrophilic silica at different surface coating levels. Those data are not corrected by the loss of permethrin induced by hydrolysis and desorption. After correcting the raw data, pseudo-first-order and then second-order degradation rate constants were calculated according to section 2.6

(Determination of second-order rate constants and half-lives toward ozone). Pesticides were exposed together to ozone and the corrections with the hydrolysis and desorption kinetic rate constants were applied for all of them. The results show that the four pesticides were degraded by ozone and their second-order kinetic rate constants ranged from (1.1 ± 0.9) • 10 -19 cm 3 molecules -1 s -1 (permethrin, T = 15%, hydrophilic silica) to (1.2 ± 0.1) • 10 -18 cm 3 molecules -1 s -1 (deltamethrin, T = 1%, hydrophilic silica).

Fig. 2: Raw data of the degradation kinetics for permethrin on hydrophilic silica exposed to 410 ppb of ozone adsorbed at five surface coating levels

Influence of the surface coating for pesticides adsorbed on hydrophobic silica

Fig. 3 presents the second-order kinetic rate constants for the ozonolysis of the four pesticides under study adsorbed on hydrophobic silica as a function of the surface coating level that ranged from 0.3% to 15% of a monolayer.

Fig. 3: Second-order kinetic rate constants for the ozonolysis of four pesticides adsorbed on hydrophobic silica at 5 surface coating levels from 0.3% to 15% of a monolayer.

When they were adsorbed on hydrophobic silica, all four pesticides were degraded by ozone.

However, two different types of behavior toward the surface coating level were observed according to the pesticide nature.

Firstly, deltamethrin, permethrin, and pendimethalin had a parallel behavior; second-order kinetic rate constants were approximatly stable given the uncertainties until a given surface coating level, and above this level, their degradation kinetics decreased with increasing surface coating level. Second-order kinetic rate constants obtained for deltamethrin and permethrin, two pyrethroid insecticides, adsorbed on hydrophobic silica were similar, i.e., (5.4 ± 1.8) • 10 -19 cm 3 molecules -1 s -1 and (4.7 ± 1.2) • 10 -19 cm 3 molecules -1 s -1 from 0.3% to 10% of surface coating, respectively. No statistically significant degradation was observed at 15% surface coating. The kinetic behavior of pendimethalin was close to those of permethrin and deltamethrin with constant kinetic rates from 0.3% to 1% of surface coating (about (1.4 ± 0.8)

• 10 -19 cm 3 molecules -1 s -1 ) and not statistically significant (i.e., negligible degradation rate constants) from 2.8% to 15% surface coating.

Secondly, the behavior of cyprodinil was different from those of deltamethrin, permethrin, and pendimethalin. With increasing coating level, second-order kinetic rate constants increased and then decreased. The second-order kinetic rate constant for cyprodinil at 0.3% of surface coating ((5.0 ± 1.3) • 10 -19 cm 3 molecules -1 s -1 ) was slower than those obtained from 1% to 10% (about (8.7 ± 1.4) • 10 -19 cm 3 molecules -1 s -1 ). Cyprodinil degradation rate was then the slowest at 15% of surface coating ((3.2 ± 0.8) • 10 -19 cm 3 molecules -1 s -1 ).

Influence of the surface coating for pesticides adsorbed on hydrophilic silica

When they were adsorbed on hydrophilic silica, all four pesticides were degraded by ozone.

However, results concerning cyprodinil were associated with high uncertainties (about 10 times higher than for the other pesticides). Therefore, results for cyprodinil are not included in the discussion.

Fig. 4 presents the second-order kinetic rate constants for the ozonolysis of deltamethrin, pendimethalin, and permethrin adsorbed on hydrophilic silica as a function of the surface coating level from 0.3% to 15% of a monolayer.

Fig. 4: Second-order kinetic rate constants for the ozonolysis of deltamethrin, pendimethalin, and permethrin adsorbed on hydrophilic silica at 5 surface coating levels from 0.3% to 15% of a monolayer.

The three pesticides (deltamethrin, permethrin, and pendimethalin) followed the same trend given the uncertainties, i.e., steady degradation rates at low surface coating level (from 0.3% to 1.5%) and a gradual decrease of the degradation at higher surface coating level (≥ 1.5%).

The second-order degradation rate constant of pendimethalin was stable at 0.3%, 1%, and 1.5% of surface coating (about (9.7 ± 1.2) • 10 -18 cm 3 molecules -1 s -1 ), 2 times slower at 10% of surface coating ((3.8 ± 1.5) • 10 -19 cm 3 molecules -1 s -1 ), and 9 times slower at 15% of surface coating ((1.1 ± 1.1) • 10 -19 cm 3 molecules -1 s -1 ). The same trend was observed for permethrin and deltamethrin. The second-order degradation rate constants for permethrin and deltamethrin were about (1.0 ± 0.1) • 10 -18 cm 3 molecules -1 s -1 and about (1.1 ± 0.1) • 10 -18 cm 3 molecules -1 s -1 from 0.3% to 1.5% of surface coating, (5.5 ± 1.2) • 10 -19 cm 3 molecules -1 s -1 and (8.4 ± 1.8) • 10 -19 cm 3 molecules -1 s -1 at 10% of surface coating, and (1.1 ± 0.9) • 10 -19 cm 3 molecules -1 s -1 and (3.0 ± 1.0) • 10 -19 cm 3 molecules -1 s -1 ) at 15% of surface coating, respectively, which represent a degradation 9 and 3 times slower between the highest and lowest rate constant values.

Discussion on the influence of the surface coating on the degradation kinetics

Aggregate shielding effect

On both hydrophilic and hydrophobic silicas, the degradation kinetics were generally slowed down by an increase of the surface coating level [START_REF] El Masri | Influence of the coating level on the heterogeneous ozonolysis kinetics and product yields of chlorpyrifos ethyl adsorbed on sand particles[END_REF]. Indeed, when the surface coating increases, the probability of pesticides forming aggregates also increases. This

can induce an 'aggregate shielding' effect, making a fraction of the pesticide molecules less accessible to oxidation by ozone. The shielding effect was observed in this study as a decrease of the kinetic constants with increasing surface coating level, and even with the total inhibition of the reactivity of some of the pesticides at the highest surface coating level tested.

To our knowledge, only a few studies investigated the influence of the surface coating level of organic compounds on their heterogeneous degradation by ozone. One study only focused on the degradation of a pesticide (chlorpyrifos) [START_REF] El Masri | Influence of the coating level on the heterogeneous ozonolysis kinetics and product yields of chlorpyrifos ethyl adsorbed on sand particles[END_REF]. Other studies took an interest on the heterogeneous degradation of PAH and especially of benzo[a]pyrene (BaP) by ozone [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF][START_REF] Alebic-Juretic | Heterogeneous Polycyclic Aromatic Hydrocarbon Degradation with Ozone on Silica Gel Carrier[END_REF][START_REF] Pöschl | Interaction of Ozone and Water Vapor with Spark Discharge Soot Aerosol Particles Coated with Benzo[a]pyrene: O3 and H2O Adsorption, Benzo[a]pyrene Degradation, and Atmospheric Implications[END_REF][START_REF] Perraudin | Kinetic Study of the Reactions of Ozone with Polycyclic Aromatic Hydrocarbons Adsorbed on Atmospheric Model Particles[END_REF][START_REF] Miet | Heterogeneous reactions of ozone with pyrene, 1-hydroxypyrene and 1-nitropyrene adsorbed on particles[END_REF]).

The heterogeneous ozonolysis of chlorpyrifos adsorbed on sand particles has been shown to be strongly influenced by the surface coating level of the pesticide [START_REF] El Masri | Influence of the coating level on the heterogeneous ozonolysis kinetics and product yields of chlorpyrifos ethyl adsorbed on sand particles[END_REF].

This was possibly due to the use of high pesticide concentrations adsorbed on particles ranging from 10 µg g -1 to 100 µg g -1 which correspond to a surface coating level of 6% to 60% of a monolayer. Degradation rate constants were found to be 70 times slower at 60% of surface coating level than at 6%. This decrease in kinetic constants followed an exponential trend, explained by the authors as a shielding effect induced by the increasing coating level.

Our results also show a decrease of the kinetic constants, however, coating levels employed in our study were lower (from 0.3% to 15%) than theirs (from 6% to 60%). In the present work the decrease in kinetic constants was observed only above a given surface coating level (1% to 3%) whereas El Masri et al. ( 2016) observed the decrease of the kinetic constants over the entire surface coating level range from 6% to 60%. This allows us to show that, with increasing surface coating level, degradation rates are at first constant, as observed in our study, and then, they start to decrease, as observed in our study and by El Masri et al. ( 2016).

It could be expected that the 'aggregate shielding' effect would decrease as the compound is degraded. However, this would only be the case if the degradation products would desorb to the gas phase. Yet, it was previously shown that the heterogeneous ozonolysis of deltamethrin and permethrin leads to degradation products including some that remain in the particle phase [START_REF] Socorro | The persistence of pesticides in atmospheric particulate phase: An emerging air quality issue[END_REF]. Also, the study of the degradation of chlorpyrifos [START_REF] El Masri | Influence of the coating level on the heterogeneous ozonolysis kinetics and product yields of chlorpyrifos ethyl adsorbed on sand particles[END_REF] also focuses on one of its degradation products: chlorpyrifos oxon. This degradation product is not reacting with ozone, and it remains in the particle phase, acting as a barrier to ozone diffusion. In this way, even as pesticides are degraded by ozone, the 'aggregate shielding' effect can remain.

The heterogeneous ozonolysis of PAH on diverse surfaces (silica gel, silica particles, fused silica plates, graphite, and soot particles) was also shown to be influenced by the surface coating level, with kinetic constants slowed down by the increase of the surface coating [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF][START_REF] Alebic-Juretic | Heterogeneous Polycyclic Aromatic Hydrocarbon Degradation with Ozone on Silica Gel Carrier[END_REF][START_REF] Pöschl | Interaction of Ozone and Water Vapor with Spark Discharge Soot Aerosol Particles Coated with Benzo[a]pyrene: O3 and H2O Adsorption, Benzo[a]pyrene Degradation, and Atmospheric Implications[END_REF][START_REF] Perraudin | Kinetic Study of the Reactions of Ozone with Polycyclic Aromatic Hydrocarbons Adsorbed on Atmospheric Model Particles[END_REF][START_REF] Miet | Heterogeneous reactions of ozone with pyrene, 1-hydroxypyrene and 1-nitropyrene adsorbed on particles[END_REF]. Unfortunately, most of those studies only considered the variation in the degradation on the degradation kinetics between the sub-monolayer and super-monolayer coating and therefore underline the difference in reactivity for PAH in the bulk phase and the dispersed phase due to shielding effect. Nevertheless, [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF] pointed out that the kinetic constants for the ozonolysis of PAH adsorbed on fused silica plates is proportional to the ozone concentration for low surface coating level (one-third of a monolayer) but that this cannot be extended for higher surface coatings (aggregates or bulk phase). Again, this indicates that under a given surface coating level, no aggregates are formed, and so kinetic constants are independent of the surface coating level and that above this surface coating level, the formation of aggregates induces a shielding effect affecting the degradation kinetics.

Influence of particle nature

Generally, literature is consistent with the fact that the increase of the surface coating level induces a decrease in the degradation kinetics constants, as observed in this study. However, some studies mentioned a shielding effect appearing above the level of the monolayer coating [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF][START_REF] Alebic-Juretic | Heterogeneous Polycyclic Aromatic Hydrocarbon Degradation with Ozone on Silica Gel Carrier[END_REF][START_REF] Pöschl | Interaction of Ozone and Water Vapor with Spark Discharge Soot Aerosol Particles Coated with Benzo[a]pyrene: O3 and H2O Adsorption, Benzo[a]pyrene Degradation, and Atmospheric Implications[END_REF][START_REF] Perraudin | Kinetic Study of the Reactions of Ozone with Polycyclic Aromatic Hydrocarbons Adsorbed on Atmospheric Model Particles[END_REF][START_REF] Miet | Heterogeneous reactions of ozone with pyrene, 1-hydroxypyrene and 1-nitropyrene adsorbed on particles[END_REF]. In this study, as well as in the study by [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF], the shielding effect was observed far below a monolayer. Aggregate formation is therefore possible for a low concentration of adsorbed compounds (i.e., 1-1.5% of a monolayer). Figures 3 and4 show that the surface coating level for which second-order kinetic rate constants start to decrease seems to be dependent on the particle type (it is lower on hydrophilic silica than on hydrophobic silica). This probably shows that the nature of the interaction between organic compound and particle is crucial in the formation of aggregates. Indeed, if there is a good affinity, the organic compound is likely to adsorb homogeneously on the surface, but in case of poor affinity, organic compounds are likely to adsorb on a few sites and then to form aggregates around the prior-adsorbed compounds. Therefore, the nature of the particle and of the adsorbed compound also plays a role in the effect of the surface coating level.

In the present work, carried out with silica particles, the kinetic profiles do not show any plateau, thereby suggesting that the shielding effect is only partial (it only significantly slows down the degradation kinetics but does not entirely hinder degradation). But some studies concerning the heterogeneous degradation of PAH by oxidants such as ozone, NO2 and hydroxyl radicals reported the presence of a plateau in the degradation time trends (Esteve et al. 2004(Esteve et al. , 2006;;[START_REF] Perraudin | Kinetic Study of the Reactions of Ozone with Polycyclic Aromatic Hydrocarbons Adsorbed on Atmospheric Model Particles[END_REF][START_REF] Miet | Heterogeneous reactions of ozone with pyrene, 1-hydroxypyrene and 1-nitropyrene adsorbed on particles[END_REF]. This means that, some molecules are not accessible to the oxidant, because they are hidden by other PAH or by particle phase degradation products. In those studies, this plateau phenomenon was observed on the surface of organic particles (soot, diesel particles) but not on the surface of silica particles, possibly because of a different disposition of PAH molecules according to the nature of the surface.

Thus, because of different surface repartition of molecules, the particle type can influence the degradation kinetics.

Lastly, our previous study [START_REF] Mattei | Heterogeneous atmospheric degradation of pesticides by ozone: Influence of relative humidity and particle type[END_REF] showed a faster ozonolysis of deltamethrin, permethrin, and pendimethalin when adsorbed on hydrophilic silica than hydrophobic silica at a surface coating of about 3% of a monolayer in the same experimental conditions as in this study, explained by a greater affinity of ozone for hydrophilic silica than hydrophobic silica.

This observation is also valid for low surface coating (≤ 10%), as can be seen in Figure 3 and 4. However, at 15% surface coating, kinetic constants were comparable whatever the particle surface was. This shows that for a high surface coating level, the particle nature does not have an influence on the degradation kinetics because the 'aggregate shielding' effect dominates over the particle nature effect.

Atmospheric implications and conclusion

The influence of surface coating between 0.3% and 15% of a monolayer on the heterogeneous ozonolysis kinetics of four commonly used pesticides (cyprodinil, deltamethrin, permethrin, and pendimethalin) was investigated. Degradation rate constants were slowed down when the surface coating level increased above 1% to 3% of a monolayer, by a factor of maximum 10, due to shielding effect. This effect is due to the formation of aggregates of the organic compound on the particle surface, which makes the reaction between the oxidant and the molecules included in the aggregates more difficult or impossible. The surface coating level for which this shielding effect appears depends on the surface type, probably because of the variability of affinity between the organic compound and the surface.

As already mentioned for relative humidity and particle type [START_REF] Mattei | Heterogeneous atmospheric degradation of pesticides by ozone: Influence of relative humidity and particle type[END_REF], these results

highlight the need to be consistent in surface coating when doing experimental determination of kinetic rate constants in order to be able to compare results from different laboratories and different studies.

Moreover, this work was conducted regarding the degradation of pesticides by ozone, but it could be likely that the shielding effect observed could also affect the degradation kinetic rates of pesticides with other atmospheric oxidants such as hydroxyl and nitrate radicals.

This study shows that pesticide degradation was slowed down for weak surface coating level (i.e., weak pesticide loading) without taking into account the presence of other SVOC.

However, they can also participate to the shielding effect as mentioned for PAH [START_REF] Wu | Fluorescence spectroscopic study of reactions between gaseous ozone and surface-adsorbed polycyclic aromatic hydrocarbons[END_REF][START_REF] Alebic-Juretic | Heterogeneous Polycyclic Aromatic Hydrocarbon Degradation with Ozone on Silica Gel Carrier[END_REF][START_REF] Pöschl | Interaction of Ozone and Water Vapor with Spark Discharge Soot Aerosol Particles Coated with Benzo[a]pyrene: O3 and H2O Adsorption, Benzo[a]pyrene Degradation, and Atmospheric Implications[END_REF][START_REF] Perraudin | Kinetic Study of the Reactions of Ozone with Polycyclic Aromatic Hydrocarbons Adsorbed on Atmospheric Model Particles[END_REF][START_REF] Miet | Heterogeneous reactions of ozone with pyrene, 1-hydroxypyrene and 1-nitropyrene adsorbed on particles[END_REF]). In fact, the highest the surface coating in SVOC is, the slowest their degradation will be, influencing, as a result, the ability of those compounds to spread in the environment and impact air quality.

Finally, few field studies measured atmospheric concentrations with respect to particle surface sizes, making it impossible to calculate the real surface coating in SVOC. This shows the need for more field measurements to better describe the real surface coating of pesticides on atmospheric particles.

Acknowledgments

21

This work has been carried out thanks to the support of the COPP'R project "Modelling of atmospheric contamination by plant protection products at the regional scale" funded by the PRIMEQUAL -AGRIQA « Agriculture et qualité de l'air » program. C. Mattei received a doctoral grant from the French Environment and Energy Management Agency (ADEME) and the Region Provence-Alpes-Côte d'Azur.


Anquez Benoit

Perret Franck

Oldrich Navratil

Frederic Cherqui

Philippe Namour
email: philippe.namour@inra.fr

Innovative low-cost and open source turbidity senor for monitoring urban water management

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

1

  Université Lumière -Lyon 2 (Bâtiment Europe), 5, avenue Pierre Mendès-France, 69676 Bron cedex, France 2 Univ Lyon, INSA-LYON, Université Claude Bernard Lyon 1, DEEP, F-69621, F-69622, Villeurbanne, France 3 INRAE Lyon-Grenoble Auvergne-Rhône-Alpes, UR RiverLy 5, rue de la Doua, CS 20244, 69625 Villeurbanne Cedex, France Material & Methods Material & Methods Field tests in Chassieu town (69) Lyon East Field tests in Chassieu town (69) Lyon East Lab Tests (in sunlight) Lab Tests (in sunlight)  Require sensor upgrade & lock-in amplifier • 5 min standby scheduled between two measurements • Data transfer via LoRaWAN protocol • OpenDataEau platform for sharing data  Request a ruggedized and cheap encapsulation Solar panel Top lid Sensor cap Data logger housing Probe housing Encapsulation made of PVC pipe parts: • Easy to find, cheap and robust material, • Electronic components remain accessible, • Do not attract attention in the field. Mobile lab sun-testing unit Setup downstream of the other measuring devices, to avoid any interferences Storm-water runoffs from the Chassieu's industrial zone Sunlight Impact (Voltage change over time) Remote monitoring system built for a total cost ≈ 200 € HT Data transfer via LoRaWAN protocol to OpenDataEau platform for sharing data Suitable for any low-LED powered by a square wave signal (0-3.3 V) to subtract the solar IR (front & back) • Solar panel power supply for stand-alone probe • Control of the turbidity sensor power supply (lockin detection) Electrical setup of the data logger: 1. NanoTimer SparkFun TPL5110, 2. Real Time Clock DS3231, 3. Arduino MKR Wan 1310 PCB 4. LoRa antenna & 5. Sensor connections 6. LiPO Battetry & 6'. LiPo rider card start of probe in water (


Keran Li

Pierre-Yves Dugas

Elodie Bourgeat-Lami

Muriel Lansalot

Polymer-encapsulated γ-Fe 2 O 3 nanoparticles prepared via RAFT-mediated emulsion polymerization

Keywords: Iron oxide, RAFT, emulsion polymerization, magnetic, morphology, cryo-TEM

Composite organic/inorganic latexes encapsulating iron oxide (IO) nanoparticles of maghemite (γ-Fe 2 O 3 ) were successfully synthesized by surfactant-free reversible addition-fragmentation chain transfer (RAFT)-mediated emulsion polymerization. γ-Fe 2 O 3 was first dispersed in an aqueous solution containing a statistical copolymer constituted of acrylic acid (AA) and n-butyl acrylate (BA) units, prepared by RAFT polymerization (so-called macroRAFT agent). Taking benefit from the affinity of carboxylic acid groups for iron oxide, the P(AA 10 -co-BA 10 ) macroRAFT agent was adsorbed onto the surface of IO nanoparticles leading to the formation of macroRAFT/IO clusters. The interaction between the macroRAFT agent and the IO surface was investigated by the study of the adsorption isotherms, indicating that the amount of adsorbed macroRAFT agent increased with increasing macroRAFT concentration. However, a high fraction of the macroRAFT chains (up to 47%) remained in the aqueous phase. The clusters were then engaged in batch emulsion polymerization of styrene or of methyl methacrylate (MMA)/BA mixtures. IO encapsulation was however unsuccessful, and a phase separation between the polymer and the IO clusters was observed. In contrast, semi-batch emulsion polymerization of MMA/BA (90/10 wt ratio) led to effective encapsulation. Morphology studies suggest that the formation of stable latexes containing large IO clusters mainly depends on the concentration of the macroRAFT agent and the pH. Under optimized conditions, a latex with superparamagnetic properties (M s = 16.2 emu g -1 ) encapsulating almost all the initial IO nanoparticles was successfully produced.

INTRODUCTION

The synthesis of magnetic polymer particles has been focusing the attention of the scientific community for more than two decades. In most cases, these hybrid colloids incorporate magnetic iron oxide (IO) nanoparticles such as magnetite (Fe 3 O 4 ) or maghemite (γ-Fe 2 O 3 ), and find a wide range of applications due to their superparamagnetic properties. They can be used as solid support for purification, extraction and concentration of biomolecules in the biomedical field or for oil recovery for environmental purpose, as contrast agents in magnetic resonance imaging, as mediator in hyperthermia, or as carrier for guided drug delivery [START_REF] Lu | Magnetic Nanoparticles: Synthesis, Protection, Functionalization, and Application[END_REF][START_REF] Jeong | Superparamagnetic Colloids: Controlled Synthesis and Niche Applications[END_REF][START_REF] Boyer | The design and utility of polymer-stabilized iron-oxide nanoparticles for nanomedicine applications[END_REF][START_REF] Philippova | Magnetic polymer beads: Recent trends and developments in synthetic design and applications[END_REF][START_REF] Pavía-Sanders | Robust Magnetic/Polymer Hybrid Nanoparticles Designed for Crude Oil Entrapment and Recovery in Aqueous Environments[END_REF][START_REF] Kalia | Magnetic polymer nanocomposites for environmental and biomedical applications[END_REF]. To efficiently play their role, these particles should meet certain criteria including an homogeneous distribution and a good encapsulation of the IO to avoid any leakage of the magnetic material, associated with a narrow particle size distribution to ensure a uniform response to an external magnetic field. Depending of the targeted utilization, the particle size must be finely tuned and appropriate surface functionalities should be considered. Particles in the submicrometer range are particularly attractive due to their low sedimentation rate and their large specific surface area for molecules immobilization.

A substantial amount of work has already been dedicated to the preparation of IO/polymer particles by free radical emulsion [START_REF] Bourgeat-Lami | Organic/Inorganic Composite Latexes: The Marriage of Emulsion Polymerization and Inorganic Chemistry[END_REF], miniemulsion [START_REF] Hu | Organic-inorganic nanocomposites synthesized via miniemulsion polymerization[END_REF][START_REF] Weiss | Miniemulsion Polymerization as a Means to Encapsulate Organic and Inorganic Materials[END_REF][START_REF] Qi | Recent advances in the preparation of hybrid nanoparticles in miniemulsions[END_REF][START_REF] Asua | Challenges for Industrialization of Miniemulsion Polymerization[END_REF][START_REF] Flesch | Poly(ethylene glycol) surface coated magnetic particles[END_REF], dispersion or precipitation [START_REF] Rahman | Organic-Inorganic Hybrid Magnetic Latex[END_REF] polymerization. However, to the best of our knowledge, only one of these numerous studies takes advantage of the great possibilities offered by the reversible-deactivation radical polymerization (RDRP) techniques, and more specifically in this case, by the reversible addition-fragmentation chain transfer (RAFT) process. Indeed, Chakraborty et al. [START_REF] Chakraborty | Synthesis of Magnetic Polystyrene Nanoparticles Using Amphiphilic Ionic Liquid Stabilized RAFT Mediated Miniemulsion Polymerization[END_REF] have recently reported a RAFT-mediated miniemulsion polymerization approach involving IO nanoparticles.

Using an oil soluble initiator and amphiphilic ionic liquids as surfactants, styrene polymerization was performed in the presence of an organosoluble trithiocarbonate RAFT agent carrying carboxylic acid groups, which are well-known for their affinity for the IO surface. Fine tuning of the molar ratio of the initiator and the RAFT agent and varying the final conversion led to the production of relatively stable magnetic polystyrene (PS) particles incorporating up to 27 wt% of IO (with respect to PS).

A few years ago, a surfactant-free and RAFT-based emulsion polymerization approach was proposed by Nguyen et al. for the direct encapsulation of inorganic particles in water [START_REF] Nguyen | Pigment Encapsulation by Emulsion Polymerization Using Macro-RAFT Copolymers[END_REF]. The strategy employed living amphipathic random copolymers of acrylic acid (AA) and n-butyl acrylate (BA) adsorbed on zirconia or alumina-coated TiO 2 pigments, to encourage the emulsion polymerization to occur at the particle surface. The random nature of the copolymer prevented the macroRAFT copolymers from self-assembling into micelles, minimizing likewise the unwanted formation of new particles via micellar nucleation, while the incorporation of hydrophobic units in the polymer chains increased the affinity of the hydrophobic monomers for the particle' s environment, promoting their encapsulation. Ali et al. [START_REF] Ali | Polymer Encapsulated Gibbsite Nanoparticles: Efficient Preparation of Anisotropic Composite Latex Particles by RAFT-Based Starved Feed Emulsion Polymerization[END_REF] confirmed the versatility of Nguyen's strategy by extending it to the encapsulation of gibbsite platelets using similar P(AA-co-BA) macroRAFT agents.

Following the same approach, Zgheib et al. [START_REF] Zgheib | Cerium oxide encapsulation by emulsion polymerization using hydrophilic macroRAFT agents[END_REF] reported the successful encapsulation of cerium oxide (CeO 2 ) nanoparticles still using P(AA-co-BA) macroRAFT agents.

The macroRAFT-mediated emulsion polymerization strategy offers a high degree of versatility with respect to the hydrophobic/hydrophilic composition and chemical nature of the macroRAFT agent, and was recently extended to the encapsulation of many kinds of inorganic particles or fillers [START_REF] Cenacchi-Pereira | Encapsulation with the Use of Controlled Radical Polymerization[END_REF][START_REF] Zetterlund | Controlled/Living Radical Polymerization in Dispersed Systems: An Update[END_REF][START_REF] Bourgeat-Lami | Synthesis of Nanocapsules and Polymer/Inorganic Nanoparticles Through Controlled Radical Polymerization At and Near Interfaces in Heterogeneous Media[END_REF] such as carbon nanotubes [START_REF] Zhong | A versatile encapsulation method of noncovalently modified carbon nanotubes by RAFT polymerization[END_REF][START_REF] Nguyen | Polymer coating of carboxylic acid functionalized multiwalled carbon nanotubes via reversible addition-fragmentation chain transfer mediated emulsion polymerization[END_REF],

and silica [START_REF] Bourgeat-Lami | Synthesis of polymer/silica hybrid latexes by surfactant-free RAFT-mediated emulsion polymerization Macromolecules[END_REF]. Similar strategies were also reported for various metals (Zn, Mo), metal oxides (BaTiO 3 , TiO 2 , Al 2 O 3 , CuO, ZrO 2 ) [START_REF] Daigle | A Simple Method for Forming Hybrid Core-Shell Nanoparticles Suspended in Water[END_REF], and quantum dots [START_REF] Das | Copolymer nanosphere encapsulated CdS quantum dots prepared by RAFT copolymerization: synthesis, characterization and mechanism of formation[END_REF][START_REF] Das | Synthesis of single-core and multiple-core core-shell nanoparticles by RAFT emulsion polymerization: Lead sulfide-copolymer nanocomposites[END_REF], using however sodium dodecyl sulfate as molecular surfactant. To the best of our knowledge, iron oxide encapsulation has however never been reported.

In this study, IO nanoparticles were encapsulated via RAFT-mediated emulsion polymerization. As mentioned above, carboxylic acid groups can readily interact with the surface of IO. Therefore, P(AA-co-BA) macroRAFT agents were first synthesized before being adsorbed onto IO nanoparticles. Their adsorption induced the agglomeration of the nanoparticles, and the aggregates could not be reverted to individual particles upon sonication, leading to the formation of finite sized clusters of IO in a similar way as reported earlier by Zgheib et al. [START_REF] Zgheib | Cerium oxide encapsulation by emulsion polymerization using hydrophilic macroRAFT agents[END_REF] for the encapsulation of CeO 2 nanoparticles using similar macroRAFT agents. However, compared with CeO 2 , the shape of the clusters was different. The interaction between the macroRAFT agent and the IO nanoparticles was investigated by the study of adsorption isotherms, and the dispersion state of the clusters was carefully examined. The clusters were then used as seeds for the emulsion polymerization of either styrene or a monomer mixture of BA and methyl methacrylate (MMA) to form a polymeric shell around the clusters.

Both batch and semi-batch processes were evaluated, and in the latter case the impact of macroRAFT concentration, IO concentration and pH was studied.

EXPERIMENTAL SECTION

Materials

4,4'-azobis(4-cyanopentanoic acid) (ACPA) (Fluka, 98 %), ethyl acetate (Aldrich, anhydrous 99.8 %), acrylic acid (AA, Acros, 99.5 %), n-butyl acrylate (BA, 99%, Acros Organics), methyl methacrylate (MMA, 99%, Aldrich), styrene (St, 99%, Aldrich), 1,3,5-trioxane (Acros, 99.5 %) and sodium hydroxide (NaOH 0.1N, standard, Acros Organics) were used as received. Water was deionized before use (Purelab Classic UV, Elga LabWater). Iron oxide nanoparticles of maghemite (γ-Fe 2 O 3 ) were synthesized using the coprecipitation method adapted from Massart et al. [START_REF] Massart | Preparation of Aqueous Magnetic Liquids in Alkaline and Acidic Media[END_REF] and detailed elsewhere [START_REF] Li | Surfactant-free emulsion polymerization stabilized by ultrasmall superparamagnetic iron oxide particles using acrylic acid or methacrylic acid as auxiliary comonomers[END_REF]. 4-Cyano-4-thiothiopropylsulfanyl pentanoic acid (CTPPA) was obtained by reaction of ACPA with bis(propylsulfanylthiocarbonyl) disulfide according to the literature [START_REF] Boursier | Controlled radical polymerization of styrene in miniemulsion mediated by PEO-based trithiocarbonate macromolecular RAFT agents[END_REF].

Methods

Synthesis of the macroRAFT agent.

The poly(acrylic acid-co-n-butyl acrylate) (P(AA 10 -co-BA 10 )) macroRAFT agent containing a trithiocarbonate chain end was synthesized by copolymerization of AA and BA (50/50 mol%) in 1,4-dioxane at 80 °C using CTPPA as a control agent and ACPA as an initiator [START_REF] Zgheib | Cerium oxide encapsulation by emulsion polymerization using hydrophilic macroRAFT agents[END_REF]. 0.2 mmol of chain transfer agent (CTPPA) was introduced in a round-bottom glass flask and 0.078 mmol of 1,3,5-trioxane, 6 mmol of AA, 6 mmol of BA and 0.02 mmol of ACPA were added. The mixture was dissolved in 4 mL of 1,4-dioxane and the flask was purged with nitrogen for 30 minutes and sealed. The glass flask was then heated to 80 °C to start the polymerization. The reaction was conducted for 5h. Samples were taken during polymerization to determine conversion as a function of time and molar mass evolution with conversion. Finally, the obtained polymer was purified by precipitation in diethyl ether and characterized by size exclusion chromatography (SEC-THF) after methylation: P(AA 10 -co-BA 10 ), M n = 2040 g mol -1 , Ð = 1.2.

Adsorption of P(AA 10 -co-BA 10 ) macroRAFT agent onto IO nanoparticles.

The adsorption of the P(AA 10 -co-BA 10 ) macroRAFT agent onto IO was investigated using the depletion method as reported by Zgheib et al. for the adsorption of similar macroRAFT agents onto CeO 2 [START_REF] Zgheib | Cerium oxide encapsulation by emulsion polymerization using hydrophilic macroRAFT agents[END_REF]. Briefly, different solutions of the macroRAFT copolymer (pH = 6.0) were first prepared with concentrations ranging from 4 to 20 g L -1 , and mixed with equal amounts of the IO dispersion (20 g L -1 and pH = 2.2) to cover a range of concentrations from 2 to 10 g L -1 (i.e. from 1 to 5 mmol L -1 ) while maintaining a fixed IO concentration of 10 g L -1 . In each case, the macroRAFT solution was added to the IO dispersion drop by drop, which should favor the formation of large aggregates [START_REF] Qi | Influence of the Formulation Process in Electrostatic Assembly of Nanoparticles and Macromolecules in Aqueous Solution: The Mixing Pathway[END_REF]. After additional stirring for 1h, the mixture was sonicated for 3 min. The purpose of sonication was to disperse the coarse aggregates formed soon after the addition of the macroRAFT agent solution. It was found that 3 min were necessary to get clusters with the narrowest size distribution (Figure S3, Supporting Information).

The dispersions were then ultracentrifuged at 80 000 rpm (Allegra TM 64R Centrifuge) for 2 h to isolate the supernatant, and the macroRAFT agent concentration determined by UV analysis on the basis of a pre-established calibration curve using the specific absorption of the C=S bond of the trithiocarbonate function with a maximum at 308 nm. The adsorbed amount of macroRAFT was then calculated by difference between the initial and equilibrium concentrations according to:

Adsorbed P(AA 10 -𝑐𝑜-BA 10 ) (µmol m -2 ) = (! ! !! ! )! ! × 100 (1)
where C 0 (mmol L -1 ) is the initial macroRAFT agent concentration, C e (mmol L -1 ) is its equilibrium concentration in the supernatant, V (L) is the volume of solution and m (g) is the mass of iron oxide.

Emulsion polymerization in the presence of the macroRAFT agent/IO clusters.

Batch emulsion polymerization reactions were performed in a 50 mL three-necked round bottom flask equipped with a condenser. As a representative example (Latex 3, Table 1), 10 mL of the cluster dispersion of IO (10 g L -1 ) coated with P(AA 10 -co-BA 10 ) (10 g L -1 ) was placed in the flask and 0.0042 g of ACPA ([macroRAFT agent]/[ACPA]

= 3) was added, followed by the addition of 1.6 g of St. This suspension was deoxygenated by purging with nitrogen for 30 min. Then, the flask was immersed in an oil bath and heated to 80 °C to start the polymerization. The same procedure was used for semi-batch experiments except for the monomer addition. 1.6 g of a deoxygenated mixture of MMA and BA (80/20, wt/wt) were fed in at a rate of 0.4 g h -1 using a Dosimat autotitrator. The reaction was left 6 hours in course, 4 hours under monomer feed and 2 additional hours to complete the polymerization. Polymer conversion was determined by gravimetric analysis.

Determination of the magnetic fraction (MF) of the particles, of the IO content in the magnetic fraction (IO MF ) and of the fraction of magnetically-separated IO (MS IO ).

To determine the fraction of particles that could be effectively captured by a magnet (hereafter referred to as the magnetic fraction, MF, in %), magnetic separation was applied to the final latex. Typically, a strong magnet (ferrite) was applied to the outside wall of a vial containing 1 mL of the composite latex suspension for 1 min.

The magnet was then held in position and the liquid was poured out of the vial. The magnet was then removed, and the residual solid weighed after evaporation of water at 100 °C in an oven. MF was defined as:

𝑀𝐹 % = ! ! ! ! ×100 (2)
Where m A is the total mass of solid in the latex and m B is the mass of separated magnetic particles.

The IO content in the magnetic fraction (IO MF , %) was then determined by thermogravimetric analyses (TGA), and used to calculate the fraction of magnetically-separated IO, MS IO (%), which corresponds to the percentage of IO present in the magnetic fraction with respect to the initial amount of IO and defined as:

𝑀𝑆 !" % = ! !" !"#$%$&"' ! !" ×100 (3)
Where m IO is the mass of IO used initially in the emulsion polymerization reaction, and m IOseparated is the mass of IO in the magnetic particles separated by the magnet:

m IOseparated = !" !"
!"" ×𝑚 ! (4)

Characterization techniques

Fourier Transform InfraRed (FTIR) spectroscopy. FTIR spectra were recorded from KBr pellets at room temperature using a Nicolet iS50 FTIR spectrometer from Thermo Scientific equipped with a deuterated triglycine sulfate (DTGS)

detector. Background and sample were acquired using 32 scans at a spectral resolution of 4 cm -1 from 4000 to 400 cm -1 . Spectral data were obtained using OMNIC Software from Thermo Scientific.

Size Exclusion Chromatography (SEC).

The molar masses of the macroRAFT agent were determined by Size Exclusion Chromatography in THF (THF-SEC). SEC measurements were carried out at 40 °C with a flow rate of 1 mL min -1 using toluene as a flow rate marker. Before analyses, carboxylic acid groups of the polymer were methylated in a THF/H 2 O (90/10 v/v%) mixture using tri(methylsilyl)diazomethane as methylation agent [START_REF] Hashimoto | New Methods and Reagents in Organic Synthesis. A Simple Efficient Preparation of Methyl Esters with Trimethylsilyldiazomethane (TMSCHN2) and Its Application to Gas Chromatographic Analysis of Fatty Acids[END_REF], to prevent interactions between acid groups and the stationary phase. Samples were filtered on a 0.45 µm pore size membrane and analyzed at 3 mg

Dynamic Light Scattering (DLS).

The particle size (hydrodynamic diameter, D h ) was measured by DLS using the Zetasizer NanoZS instrument from Malvern. The data were collected at 173° using the fully automatic mode of the Zetasizer system, and depending on the size distribution, either the monomodal cumulant analysis or the CONTIN analysis was performed. The broadness of the distribution was given by a dimensionless number called poly value determined from the autocorrelation function using the second-order method of cumulant analysis.

Cryogenic-transmission electron microscopy (cryo-TEM).

PMMA is known to be particularly sensitive to electron damage. Therefore, in order to prevent degradation of the latex particles due to radiation damage at room temperature and allow reliable determination of particle morphology, all samples were characterized by cryo-TEM.

The diluted samples were dropped onto 300 Mesh holey carbon films (Quantifoil R2/1) and quench-frozen in liquid ethane using a cryo-plunge workstation (made at LPS Orsay). The specimens were then mounted on a precooled Gatan 626 specimen holder, transferred in the microscope (Phillips CM120) and observed at an accelerating voltage of 120 kV (Centre Technologique des Microstructures (CTµ), platform of the Université Claude Bernard Lyon 1, Villeurbanne, France).

Magnetic measurements.

A vibrating sample magnetometer (VSM, BHV-55) was used to measure the magnetization (r) as a function of the magnetic field intensity (H) at room temperature. Magnetization of both IO nanoparticles and magnetic particles was measured using the Weiss extraction method [START_REF] Joumaa | Surface modification of iron oxide nanoparticles by a phosphate-based macromonomer and further encapsulation into submicrometer polystyrene particles by miniemulsion polymerization[END_REF], decreasing the magnetic field from 21 to 0 kOe. This dynamic method makes use of the variation of flux induced in a coil when moving the sample in the field. Measurements were performed on dried samples. Specific magnetization (M in emu g -1 ) of a given sample was defined as:

𝑀 = 4300×𝜎×𝛼 𝑚 ( 5
)
where 4300 is the apparatus constant, m the mass of the sample (g), δ the raw electric signal value (a.u.), and α the correction of the magnetic image for strong magnetic fields. M S , the specific saturation magnetization was obtained by extrapolation of M for strong magnetic fields (1/H → 0).

Thermogravimetric analyses (TGA).

The IO content in the magnetic fraction of the particles was determined by TGA on a TA instruments Q5000 IR. Typically, 10 mg of the dried sample were accurately weighed and heated from 25 to 800 °C at a rate of 10 °C min -1 under an oxygen atmosphere.

RESULTS AND DISCUSSION

The aqueous dispersion of iron oxide nanoparticles used in this study was synthesized by the precipitation of ferrous and ferric iron in an alkaline environment, followed by oxidation with Fe(NO 3 ) 3 and peptization using HNO 3 [START_REF] Li | Surfactant-free emulsion polymerization stabilized by ultrasmall superparamagnetic iron oxide particles using acrylic acid or methacrylic acid as auxiliary comonomers[END_REF]. The resulting IO nanoparticles spontaneously redispersed in water to produce a colloidal sol stable at a pH around 2.2. The surface of the nanoparticles was positively charged (ζ = + 50 mV at pH = 2.2) with an isoelectric point of 7.4. The DLS analysis indicated a hydrodynamic diameter of 25 nm (poly = 0.25). In addition, the specific surface area determined by BET was 166 m 2 g -1 . These values will be used in the rest of the study.

As mentioned in the Introduction, magnetic particles should fulfill some requirements to ensure an effective magnetic separation, including the encapsulation of a high amount of IO into submicronic particles. To meet these criteria, iron oxide clusters of finite size (typically larger than 100 nm) were first prepared before being used as seeds in emulsion polymerization. To favor the formation of such clusters and then the polymerization from their surface, the adsorption of P(AA 10 -co-BA 10 )

macroRAFT agents onto IO was first investigated in details.

/iron oxide clusters

The amount of P(AA 10 -co-BA 10 ) adsorbed onto the IO nanoparticles was determined by the depletion method using different solutions of the macroRAFT agent mixed with equal amounts of the IO dispersion to cover copolymer concentrations ranging from 2 to 10 g L -1 (i.e. from 1 to 5 mmol L -1 ) while maintaining a fixed IO concentration of 10 g L -1 . It is worth noting that the pH of the mixture was impacted by the macroRAFT concentration: the pH steadily increased from 3.2 to 4.7 when the macroRAFT concentration increased from 2 to 5 g L -1 , to finally level off at 6 for higher macroRAFT amounts (6 to 10 g L -1 ). The adsorption isotherm of the P(AA 10 -co-BA 10 ) macroRAFT agent onto the IO nanoparticles is shown in Figure 1a. It plots the adsorbed amount per unit area (µmol m -2 ) as a function of the equilibrium (residual) concentration in solution, C eq . The amount of adsorbed macroRAFT agent increased from 0.5 to 1.5 µmol m -2 with increasing the concentration of macroRAFT agent in the suspension until a plateau was reached.

Indeed, the polymer chains progressively saturate the IO surface as the macroRAFT agent concentration increases. The proportion of adsorbed macroRAFT agent therefore decreased from 80% to 53% with increasing the initial macroRAFT agent concentration, indicating that an increasing part of the macroRAFT chains remained in the aqueous phase (Figure 1b). The amount of adsorbed macroRAFT was also quantified by TGA for P(AA 10 -co-BA 10 ) concentrations of 1, 3 and 5 mmol L -1 (see Figure S1a in the Supporting Information), and the weight fraction of macroRAFT increased accordingly with the initial concentration (13, 27 and 30 wt%, respectively). The measured weight loss of the three samples was in good agreement with the amount determined by the depletion method using UV analysis (Figure S1b).

As mentioned above, macroRAFT adsorption on the IO nanoparticles led to their aggregation into finite size clusters. To gain further insight into the formation of the clusters, the size and zeta potential of the IO/macroRAFT clusters were measured as a function of the macroRAFT concentration (from 2 to 10 g L -1 , corresponding to 1.0 and 5.0 mmol L -1 ) for an IO concentration in the mixture of 10 g L -1 (Figure 2). Again, the macroRAFT solution (pH = 6) was added to the IO dispersion and then sonicated for 3 min. For the lowest concentrations (< 2.5 mmol L -1 ) the IO precipitate could not be efficiently redispersed and DLS measurements were not reliable in that case as particle agglomerates could be observed by the naked eye. The size of the clusters then decreased from 1121 to 122 nm (with a poly value of 0.21) with increasing macroRAFT concentration from 2.5 to 5 mmol L -1 (Figure 2a). The observed instability at low macroRAFT concentration (≤ 2.5 mmol L -1 ) is due to the low pH value of the suspension (from 3.2 to 4.7 as the macroRAFT concentration increases).

Indeed, considering the pKa values reported for PAA (pKa (PAA) ≈ 6 [START_REF] Plamper | Synthesis, Characterization and Behavior in Aqueous Solution of Star-Shaped Poly(acrylic acid)[END_REF]), the macroRAFT copolymer is likely protonated at low pH values, which would lead to a poor stabilization efficiency. An additional experiment was performed where the pH was increased to 6, and indeed the size of the clusters was significantly reduced (down to around 120 nm). The zeta potential of the pristine IO nanoparticles was + 50 mV and decreased from + 0.8 mV for the lowest macroRAFT content (2 g L -1 , pH = 3.2) to around -60 mV for the highest macroRAFT content (10 g L -1 , pH = 6.0) (Figure 2b). Therefore, the stability of macroRAFT/IO clusters increased with increasing macroRAFT concentration. Zeta potential results are in agreement with the DLS results.

In addition, FTIR studies show that the macroRAFT copolymer is adsorbing through complexation between the carboxylate groups and the Fe atoms. This causes destabilization and therefore aggregation into finite sized clusters. From a more detailed investigation of the FTIR spectra (see Figure S4 and Table S1 in the Supporting Information), we hypothesize that the macroRAFT copolymer adsorbs as loops oriented flat on the surface of IO but with tails that extend into the solution in order to provide colloidal stability to the formed clusters.

Finally, the IO/macroRAFT clusters produced from the mixing of the 20 g L -1 IO dispersion and the 20 g L -1 P(AA 10 -co-BA 10 ) solution (i.e. the point at 10 g L -1 or 5 mmol L -1 in Figure 2b) were observed by cryo-TEM (Figure 3). The clusters exhibit a non-spherical, irregular shape with a fractal geometry and a rather broad size distribution in agreement with their high poly values determined by DLS. A closer look at the images indicates that the clusters are slightly anisotropic with their longer dimensions roughly comprised between 70 and 130 nm, which is also in agreement with DLS (122 nm). Note that a similar fractal behavior was reported during the complexation of iron oxide nanoparticles with poly(trimethylammonium ethylacrylate)-b-poly (acrylamide) diblock copolymers [START_REF] Berret | Controlled Clustering of Superparamagnetic Nanoparticles Using Block Copolymers: Design of New Contrast Agents for Magnetic Resonance Imaging[END_REF][START_REF] Berret | Stable oxide nanoparticle clusters obtained by complexation[END_REF]. The fractal geometry of the aggregates suggests a diffusion-limited aggregation mechanism [START_REF] Lin | Universality in colloid aggregation[END_REF]. This first series of experiments showed that the adsorption of P(AA 10 -co-BA 10 ) onto the IO nanoparticles was effective for macroRAFT concentrations comprised between 6 and 10 g L -1 (corresponding to a pH value of 6 for the final dispersion) leading to the formation of clusters with a size close to 120 nm, which were however not easily separated by a magnet. Therefore, many parameters were then investigated to form stable dispersions of macroRAFT/IO clusters with larger sizes: the mixing pathway (direct versus drop by drop), the homogenization step (time and type of device), the pH (during or after mixing), the IO concentration (10 to 60 g L -1 ) or the macroRAFT agent concentration (10 to 40 g L -1 ). All these methods showed that controlling at the same time the size and the dispersity of the clusters was difficult to achieve. On the one hand, stable suspensions could be obtained but with relatively small IO clusters (≤ 200 nm) and reasonable dispersity (poly values ≤ 0.2), which could not be efficiently separated by applying an external magnetic field. On the other hand, big IO clusters could be produced (≥ 200 nm) but with large particle size distributions (poly values ≥ 0.3). In the range of experimental conditions studies, the formation of relatively big clusters (D h ≥ 200 nm) with reasonable dispersities (poly values ≤ 0.2) was however not successful.

Consequently, the idea was to form large clusters during the emulsion polymerization step. Indeed, a partial (and limited) loss of cluster stability could result in further aggregation of IO clusters. The IO/macroRAFT clusters being more stable at pH 8 than pH 6 (information drawn from the many routes investigated to increase the cluster size), emulsion polymerizations was thus first performed at pH 6 in a batch process, using different hydrophobic monomers (either styrene or MMA/BA mixtures). The next experiments were performed in semi-batch with MMA/BA mixture (90/10 in weight). Taking into account the adsorption studies (Figure 2b), the amount of macroRAFT was gradually decreased to induce controlled cluster aggregation. On the other hand, the concentration of IO was increased to produce high IO content magnetic composite particles. The pH was also increased to 8 in some cases to improve the cluster stability. All these routes are presented in the following section.

Emulsion polymerization using macroRAFT/IO clusters as seeds

Batch emulsion polymerization

The first encapsulation experiments were performed in batch at pH = 6 using either a mixture of MMA and BA (90/10 wt % or 80/20 wt %) or pure styrene (Table 1). The preparation of the clusters was identical to that previously described for the adsorption studies (i.e. drop by drop addition of P(AA 10 -co-BA 10 ) macroRAFT to the IO dispersion, followed by 1h of stirring and 3 min of sonication). P(AA 10 -co-BA 10 ) and IO concentrations (both 10 g L -1 ) were those leading to stable clusters of ca. 120 nm (Figure 2a). The final conversion was slightly higher for the 90/10 MMA/BA mixture (96 %) than for the 80/20 mixture (94%) or pure St (92 %). In all cases, the latex was stable with a hydrodynamic diameter close to 130 nm for the MMA/BA series, and a bit larger (D h = 160 nm) for PSt (Table 1). It seems however that the IO particles were not encapsulated (Figure 4). Indeed, cryo-TEM images of the three latexes show the presence of aggregated clusters of IO likely located on the surface of the polymer particles and forming a separate phase, reflecting a poor affinity between IO and the polymer phase. Moreover, the interaction between IO and the polymer particles seems lower in Latex 3 (PSt) than in Latex 1 or 2. This is likely because St is more hydrophobic than MMA and BA. Therefore, St has a poorer affinity for the macroRAFT-coated IO particles than MMA or BA, and so do the resulting polymers.

In addition, a population of pure polymer particles can also be clearly identified on the micrographs. The presence of this population of secondary nucleated particles may be related to the amount of free macroRAFT in water. Indeed, the adsorption isotherm in Figure 1b shows that only 53 % of the macroRAFT agent is adsorbed on the IO surface under the conditions used in the emulsion polymerizations (i.e.

[IO] =

[macroRAFT] = 10 g L -1 , Table 1), while the remaining 47 % stays in the aqueous phase. As a result, chain extension of these free P(AA 10 -co-BA 10 ) chains in water with BA/MMA or St leads to amphiphilic block copolymers which are able to self-assemble to form polymer particles devoid of IO according to the polymerization-induced self-assembly process [START_REF] Zetterlund | Controlled/Living Radical Polymerization in Dispersed Systems: An Update[END_REF][START_REF] Canning | A Critical Appraisal of RAFT-Mediated Polymerization-Induced Self-Assembly[END_REF][START_REF] Lansalot | Polymerization-Induced Self-Assembly: the Contribution of Controlled Radical Polymerization to the Formation of Self-Stabilized Polymer Particles of Various Morphologies[END_REF]. Several hypotheses can be put forward to explain the failure of IO encapsulation. First, the presence of monomer droplets may promote macroRAFT desorption from the IO surface. The adsorbed amount would be then lower than the minimum amount necessary for successful encapsulation. This may be accentuated by the formation of the secondary nucleated particles, which may also compete for macroRAFT adsorption to the detriment of encapsulation. Second, the monomer accumulated in the polymer particles may act as a plasticizing agent, increasing polymer chain mobility and allowing the IO clusters to migrate to the polymer/water interface in a thermodynamically controlled process.

Note that a similar phase separation phenomenon was already reported in the literature using a related macroRAFT strategy in batch [START_REF] Zgheib | Cerium oxide encapsulation by emulsion polymerization using hydrophilic macroRAFT agents[END_REF][START_REF] Zhong | A versatile encapsulation method of noncovalently modified carbon nanotubes by RAFT polymerization[END_REF][START_REF] Bourgeat-Lami | Synthesis of polymer/silica hybrid latexes by surfactant-free RAFT-mediated emulsion polymerization Macromolecules[END_REF].

Semi-batch emulsion polymerization

The previous results showed the limits of the batch process to ensure IO encapsulation.

Therefore, the next series of experiments were performed in semi-batch. Indeed, the process used for the encapsulation of inorganic particles in the presence of macroRAFT agents, either batch or semi-batch, has been shown to influence the overall mechanism of encapsulation [START_REF] Cenacchi-Pereira | Encapsulation with the Use of Controlled Radical Polymerization[END_REF][START_REF] Zetterlund | Controlled/Living Radical Polymerization in Dispersed Systems: An Update[END_REF][START_REF] Bourgeat-Lami | Synthesis of Nanocapsules and Polymer/Inorganic Nanoparticles Through Controlled Radical Polymerization At and Near Interfaces in Heterogeneous Media[END_REF]. As mentioned in the previous section, monomer droplets are present in a batch process and the macroRAFT agents may thus partition between the different phases. In addition, the monomer may act as a plasticizer, decreasing the glass transition temperature of the copolymer shell, promoting polymer chain mobility, which may push the inorganic particles towards the polymer/water interface. All or part of these drawbacks should be avoided, at least minimized, in a semi-batch process.

The detailed experimental procedure is presented in the experimental section. The protocol followed for the formation of the macroRAFT/IO clusters was the same as for the batch process, except that after stirring for 1h, the clusters were prepared either by sonication (when the pH was 6) or by sonication followed by a pH increase to 8.

The encapsulation experiments were in all cases performed with a semi-continuous feed of MMA/BA (90/10 wt/wt) (Table 2). In the first series of experiments (Latex 4, 5 and 6), the IO concentration was kept at 10 g L -1 , while the concentration of macroRAFT agent was decreased from 10 to 6 g L -1 . These concentrations correspond to the conditions used in the adsorption experiments shown in Figure 2, leading to a pH value of 6 for the IO/macroRAFT dispersion. As expected, the IO cluster size showed a slight increase from 121 nm to 146 nm with a decrease of macroRAFT agent concentration from 10 to 6 g L -1 . The latexes obtained after emulsion polymerization were stable. For the three experiments, DLS results showed a slight increase in the particle size, which could be consistent with a successful encapsulation of the starting clusters. Indeed, the corresponding cryo-TEM images (Figure 5) confirmed that IO was exclusively located in the latex particles, with however an uneven distribution among the loaded particles probably correlated with the rather broad distribution in size of the initial clusters. Cryo-TEM also shows a good agreement with the DLS results. Yet, many free latex particles and small magnetic composites were observed in the three samples. Interestingly, the composite particles have an irregular shape that may follow the shape of the original IO nanoclusters, which suggests that the clusters have been acting as seeds for the emulsion polymerization. When the macroRAFT concentration decreased (from Latex 4 to 6), the fraction of pure polymer particles decreased. Indeed, the proportion of free macroRAFT (determined from the adsorption isotherm in Figure 1b) was 47% in Latex 4, which was higher than for Latex 5 (36%) and Latex 6 (33%) and, as mentioned above, the presence of free macroRAFT promotes secondary nucleation.

Because the total amount of monomer was kept the same (1.6 g) in all the experiments, the thickness of the polymer shell logically increased when the fraction of secondary-nucleated particles decreased. The fraction of separated magnetic particles (MF), the IO amount in the magnetic fraction (IO MF ) and the fraction of magnetically-separated IO (MS IO ) were then determined to confirm TEM observations. The MF increased from 13% to 36% with decreasing the macroRAFT concentration. In other words, the fraction of the pure polymer particles and of the composite ones that were not magnetic enough to be separated by the magnet decreased. In addition, TGA showed that the IO content in the magnetic fraction, IO MF , decreased from 19% to 15%, which was in agreement with the increase of shell thickness. Nevertheless, as mentioned above, the magnetic fraction, MF, increased when the macroRAFT concentration decreased, indicating that more IO nanoparticles were encapsulated, or at least present in aggregates big enough to allow their magnetic separation. Accordingly, MS IO increased from 35 % to 75 % with decreasing macroRAFT concentration from 10 to 6 g L -1 .

In order to further increase the amount of IO in the final latex, the concentration of both IO and P(AA 10 -co-BA 10 ) was increased to 20 g L -1 , 30 g L -1 and 40 g L -1 (Latex 7, 8 and 9). The IO cluster size of the three samples before polymerization was almost the same, around 135 nm (in accordance with the results of the preliminary experiments, data not shown). This series of latexes showed results similar to those obtained for lower IO and macroRAFT contents (Latex 4, 10 g L -1 of both IO and macroRAFT). Indeed, secondary nucleation occurred due to the presence of free macroRAFT as could be expected from the previous adsorption studies (Figure 1b), as ca. 50% of the macroRAFT should remain in water. Again, the particles showed distorted shapes, which may be similar to those of the original clusters. The IO particles were not uniformly distributed among the composite particles, which could be related to the size inhomogeneity of the original clusters (Figure 6). Indeed, some contained a lot of IO while others contained only one or two IO nanoparticles. In this group of experiments, MF increased from 23% to 44% in good agreement with the increase of the initial IO content (from 20 to 40 g L -1 ). IO MF was nevertheless similar for the 3 latexes (ca. 20%), and so was MS IO (38 ± 2%). The distribution of IO inside the polymer particles looked similar in the three cases. 2).

Right: same samples at higher magnification.

To decrease the secondary nucleation while keeping the uniformity of IO clusters, the concentration of macroRAFT agent was first decreased to 30 and 25 g L -1 (Latex 10 and 11, respectively) keeping a constant IO concentration of 40 g L -1 . The size of the clusters only slightly increased compared to Latex 9 (140 and 142 nm versus 139 nm).

Accordingly, the size of the final particles slightly increased from 146 to 152 nm (Latex 9, 10 and 11 in Table 2). Cryo-TEM images clearly show that the amount of free particles decreases gradually with decreasing the concentration of macroRAFT agent (Figures 6 and7). 2 for detailed experimental conditions). Right: same samples at higher magnification.

Accordingly, MF increased from 44% to 85%, accompanied by a slight increase of IO MF (20 to 25%). TGA confirmed that MS IO also increased from 40% to 95%. In order to increase the size of the IO clusters and consequently that of the final composite particles, the concentration of macroRAFT agent was further decreased to 20 g L -1 (Latex 12). However, the clusters were not stable. It seems that a minimal amount of macroRAFT (for a given concentration of IO) is necessary to maintain the

stability. This is in agreement with the DLS results shown in Figure 2a. Nevertheless, decreasing the concentration of macroRAFT agent, within a certain range, allows the synthesis of bigger magnetic composite particles.

As the use of less macroRAFT agent clearly impacts the stability of the final latex, the influence of the initial IO/macroRAFT cluster stability was then investigated by varying the pH of the IO/macroRAFT mixture. Indeed, right after the preparation of the clusters, the pH was raised to 8. Whereas no stable clusters were obtained for Latex 12, for the same concentration of both IO and macroRAFT, the additional step of pH increase allowed the formation of stable clusters (Latex 13, Table 2), with a diameter of 91 nm. This improvement can reasonably be associated with the contribution of more carboxylate groups to the stability of the clusters. After polymerization, Latex 13 was stable with a diameter of 135 nm, higher than that of the clusters, which clearly indicates partial aggregation during the polymerization. The pH increase from 6 to 8 was thus able to provide stable clusters but the amount of macroRAFT was however likely not high enough to provide stability to the growing particles. Some particles thus aggregated to reduce the total surface area. This experiment thus indicates that the stability of the latex depends on both the concentration of the macroRAFT agent and the pH. Cryo-TEM image of this latex showed that encapsulated particles were smaller than those from Latex 11 for instance, and that many particles only incorporated a few IO nanoparticles (Figure 8).

Therefore, some composite particles (with low IO amount) could not be separated magnetically. The fraction of separated magnetic particles was 72% with still a IO MF value close to 25 %. The fraction of magnetically-separated IO, MS IO , was 73 %.

In order to produce larger particles, the concentration of macroRAFT agent was decreased to 10 g L -1 (Latex 14). The size of this stable latex (178 nm) was almost twice as much as that of the initial IO cluster (94 nm). Again, this clearly indicates that the IO clusters partially aggregated during the polymerization. Cryo-TEM image of the latex also confirmed the presence of larger magnetic composite nanoparticles (Figure 8). 95 % of the latex particles could be separated magnetically, with a IO MF value of 28%, and MS IO reaching 99%. Nevertheless, when the concentration of macroRAFT agent was further decreased to 8 g L -1 (Latex 15), the macroRAFT agent was not able to maintain the stability of the particles during the polymerization, even if the clusters were stable at the beginning (102 nm). 2 for detailed experimental conditions). Right : same samples at higher magnification.

Finally, the magnetic properties of the latex exhibiting the most promising features, i.e.

Latex 14 (Table 2), were investigated. When a magnet was applied for 1 min, almost all the magnetic composite nanoparticles could be separated (MF = 95 %). The magnetic properties of the separated composite nanoparticles from Latex 14 were then investigated using a vibrating sample magnetometer. Figure 9 shows the magnetization curve of the latex. The magnetic particles possess superparamagnetic properties as no remanence was observed when the magnetic field was removed. The

CONCLUSIONS

Composite organic/inorganic latexes encapsulating IO nanoparticles were successfully synthesized by surfactant-free RAFT-mediated emulsion polymerization using P(AA 10 -co-BA 10 ) macroRAFT copolymers to both stabilize the initial iron oxide dispersion and to facilitate the growth of polymer onto the inorganic surface. Taking benefit from the affinity of carboxylic acid groups for transition metal oxides, P(AA 10 -co-BA 10 ) macroRAFT copolymers carrying a trithiocarbonate chain end were adsorbed onto the surface of IO leading to the formation of macroRAFT/IO clusters.

From the adsorption isotherm, the mass of macroRAFT agent adsorbed on the IO surface increased with increasing macroRAFT concentration. However, a high amount of the macroRAFT chains (up to 47%) remained in the aqueous phase. Batch emulsion polymerization of St or of MMA/BA mixtures resulted in unsuccessful encapsulation, leading to a phase separation between the polymer and the IO cluster.

During batch emulsion polymerization, the polymer chains grew not only on the IO clusters surface but also in water. In contrast, semi-batch emulsion polymerization of MMA/BA (90/10 wt ratio) led to effective encapsulation, the polymer shell following the irregular contours of the original clusters. Morphology studies suggest that the formation of stable latexes containing large IO clusters mainly depends on the pH and the concentration of the macroRAFT agent. The concentration of the latter must be high enough to ensure the cluster formation and their stability, while providing free macroRAFT agents in the continuous phase that will participate to the stabilization of the growing composite particles. In addition, we showed that working at pH 8 rather than 6 allowed reducing the amount of macroRAFT agent while forming smaller clusters that however underwent partial aggregation during the polymerization, which in fine led to bigger particles. In the best case, a stable latex (178 nm) encapsulating almost all the initial IO nanoparticles (99%) with 95% of the particles being magnetic was produced. This composite latex exhibited superparamagnetic properties (M s = 16.2 emu g -1 ).

1 H

 1 NMR spectroscopy. The individual molar conversions of AA and BA during the macroRAFT agent synthesis were determined by1 H NMR spectroscopy in DMSO-d 6 at room temperature (Bruker DRX 300), by the relative integration of the protons of the internal reference (1,3,5-trioxane) and of the vinylic protons of the monomers.

mL - 1 .

 1 Separation was carried out on three columns from Malvern Instruments [T6000 M General Mixed Org (300 × 8 mm)]. The device (Viscotek TDA305) was equipped with a refractive index (RI) detector (λ = 670 nm). The number-average molar mass (M n ) and dispersity (Đ = M w /M n , with M w : weight-average molar mass) were derived from the RI signal by a calibration curve based on polystyrene standards (PS from Polymer Laboratories). pH measurements. pH values were measured on a Mettler Toledo SevenEasy pH meter with an InLab Routine Pro combination electrode, calibrated with 4, 7, and 10 pH buffer solutions.

3. 1 .

 1 P(AA 10 -co-BA 10 ) adsorption onto IO and formation of P(AA 10 -co-BA 10 )

Figure 1 .

 1 Figure 1. a) Isotherm for P(AA 10 -co-BA 10 ) adsorption onto the IO surface and b) evolution of the proportion of adsorbed macroRAFT as a function of initial macroRAFT concentration. IO concentration in the final mixture: 10 g L -1 .

Figure 2 .

 2 Figure 2. a) Hydrodynamic diameter and b) zeta potential of the IO/macroRAFT clusters as a function of the macroRAFT concentration. [IO] mixture = 10 g L -1 .

Figure 3 .

 3 Figure 3. Cryo-TEM images of IO/macroRAFT clusters obtained when mixing the IO dispersion with the P(AA 10 -co-BA 10 ) solution at pH = 6 ([IO] mixture = 10 g L -1 and [P(AA 10 -co-BA 10 )] mixture = 10 g L -1 or 5 mmol L -1 ). For comparison purpose, the white circle shows the size determined by DLS (D h = 122 nm).

Figure 4 .

 4 Figure 4. Cryo-TEM images of the latex particles obtained after batch emulsion polymerization of hydrophobic monomer(s) in the presence of IO clusters (10 g L -1 ) coated with P(AA 10 -co-BA 10 ) macroRAFT agent (10 g L -1 ). Latex 1: MMA/BA 80/20 (wt/wt), Latex 2: MMA/BA 90/10 (wt/wt) and Latex 3: pure St. The inset shows a magnified portion of the image.

  8 g; Monomer: 1.6 g (feed rate = 0.4 g h -1 ); [macroRAFT]/[ACPA] = 3; reaction time = 6.0 h; T = 80 °C. b pH of IO/macroRAFT mixture. c Final conversion determined by gravimetric analysis. d Initial (before monomer addition) and final diameters and poly values (D h,i , poly i and D h,f , poly f respectively) measured by DLS. e Magnetic fraction of the particles determined using equation (2). f IO content of the magnetic fraction as determined by TGA. g Fraction of magnetically-separated IO calculated using equation (3).

Figure 5 .

 5 Figure 5. Left: cryo-TEM images of the polymer-IO composite particles obtained after emulsion polymerization of MMA/BA (90/10) in the presence of IO clusters coated with P(AA 10 -co-BA 10 ) macroRAFT agent (pH = 6) prepared using various concentrations of macroRAFT (Latex 4 to 6, See Table2for detailed experimental conditions). Right: same samples at higher magnification.

Figure 6 .

 6 Figure 6. Left: cryo-TEM images of the polymer-IO composite particles obtained after emulsion polymerization of MMA/BA (90/10 in weight) in the presence of IO clusters coated with P(AA 10 -co-BA 10 ) macroRAFT agent prepared using varying IO and macroRAFT concentrations (pH = 6) for a fixed 1:1 ratio (Latex 7 to 9, Table2).

Latex 9 Figure 7 .

 97 Figure 7. Left : cryo-TEM images of the polymer-IO composite particles obtained after emulsion polymerization of MMA/BA (90/10) in the presence of IO clusters coated with P(AA 10 -co-BA 10 ) macroRAFT agent (pH = 6) prepared using various concentrations of macroRAFT (Latex 10 and 11, SeeTable 2 for detailed

  The three last experiments (i.e. Latex 13 to 15) clearly show the crucial role played by the initial concentration of the macroRAFT agent, which should ensure a good balance between the amount of macroRAFT adsorbed onto the IO clusters and the amount of free polymer chains present in the continuous phase. Indeed, as previously mentioned in the literature[START_REF] Nguyen | Pigment Encapsulation by Emulsion Polymerization Using Macro-RAFT Copolymers[END_REF] it appears that a certain quantity of free macroRAFT is necessary to maintain the stability of the growing particles while their surface area increases during the polymerization. However, this concentration should remain low enough to prevent secondary nucleation. Based on the adsorption isotherm presented in Figure1, the concentration of free macroRAFT decreases from Latex 13 to Latex 15, where this concentration is actually too low to ensure the stability.

Figure 8 .

 8 Figure 8. Left : cryo-TEM images of the polymer-IO composite particles obtained after emulsion polymerization of MMA/BA (90/10) in the presence of IO clusters coated with P(AA 10 -co-BA 10 ) macroRAFT agent (pH = 8) prepared using various concentrations of macroRAFT (Latex 13 and 14, SeeTable 2 for detailed

  magnetization M s was 16.2 emu g -1 . A comparison with bulk IO indicates that the amount of IO in this latex was 27 wt %, which was in good agreement with the TGA data, 28 wt % (FigureS2in the Supporting Information).

Figure 9 .

 9 Figure 9. Magnetization versus applied magnetic field curve of the magnetic fraction of Latex 14 recovered after exposing the hybrid suspension to a magnet for 1 min. The magnetization is given per gram of composite particles.

Table 1 .

 1 Batch emulsion polymerizations performed in the presence of P(AA 10 -co-BA 10 )/IO clusters.

	Latex a P(AA 10 -co-BA 10 ) (g L -1 )	IO (g L -1 )	Monomer	Conv. c (%)	D h,i /D h,f (nm)	d	poly i /poly f	d
	1	10	10	MMA/BA b (80/20)	94	129/131 0.20/0.13
	2	10	10	MMA/BA b (90/10)	96	123/134 0.22/0.11
	3	10	10	St	92	132/161 0.21/0.23

a Monomer: 1.6 g / Water: 9.8 g; [macroRAFT]/[ACPA] = 3; pH = 6; reaction time = 6 h; T = 80 °C. b

Weight ratio. c Final conversion determined by gravimetric analysis. d Initial (before monomer addition) and final diameters and poly values (D h,i , poly i and D h,f , poly f respectively) measured by DLS.

Table 2 .

 2 Semi-batch emulsion polymerizations of MMA/BA (90/10 in weight) performed in the presence of IO coated with P(AA 10 -co-BA 10 ).

	Latex a	IO (g L -1 )	macroRAFT (g L -1 )	pH b Conv. c (%)	D h,i /D h,f (nm)	d	poly i /poly f	d	MF e (%)	IO MF (%)	f	MS IO (%)	g
	4	10	10	6	81	121/125 0.20/0.17		13	19		35
	5	10	8	6	82	132/151 0.21/0.12		18	18		44
	6	10	6	6	77	146/157 0.17/0.12		36	15		75
	7	20	20	6	87	135/137 0.20/0.17		23	20		36
	8	30	30	6	86	135/142 0.20/0.21		33	21		38
	9	40	40	6	89	139/146 0.17/0.16		44	20		40
	10	40	30	6	86	140/149 0.17/0.15		62	22		62
	11	40	25	6	85	142/152 0.19/0.16		85	25		95
	12	40	20	6				Unstable clusters
	13	40	20	8	76	91/135		0.19/0.19		72	24		73
	14	40	10	8	71	94/178		0.17/0.21		95	28		99
	15	40	8	8	/	102/unstable 0.20/unstable

a Water: 9.

ACKNOWLEDGMENTS

Financial support from the Chinese Scholarship Council is gratefully acknowledged.