domain: sde chim

text:
Yara Arbid

Mohamad Sleiman

Claire Richard

Photochemical interactions between pesticides and plant volatiles

Keywords: Chlorothalonil, imidacloprid, thyme's leaves, photoreduction, photooxidation, photonitration

Among the numerous studies devoted to the photodegradation of pesticides, very scarce are those investigating the effect of plant volatiles. Yet, pesticides can be in contact with plant volatiles after having been spread on crops or when they are transported in surface water, making interactions between the two kind of chemicals possible. The objectives of the present study were to investigate the reactions occuring on plants. We selected thyme as a plant because it is used in green roofs and two pesticides : the fungicide chlorothalonil for its very oxidant excited state and the insecticide imidacloprid for its ability to release the radical NO 2 under irradiation. Pesticides were irradiated with simulated solar light first in a solvent ensuring a high solubility of pesticides and plant volatiles, and then directly on thyme's leaves. Analyses were conducted by headspace gas chromatography-mass spectrometry (HS-GC-MS), GC-MS and liquid chromatography-high resolution mass spectrometry (LC-HRMS). In acetonitrile, chlorothalonil photosensitized the degradation of thymol, -pinene, 3-carene and linalool with high quantum yields ranging from 0.35 to 0.04, and was photoreduced, while thymol underwent oxidation, chlorination and dimerization. On thyme's leave, chlorothalonil was photoreduced again and products arising from oxidation and dimerization of thymol were detected. Imidacloprid photooxidized and photonitrated thymol in acetonitrile, converting it into chemicals of particular concern. Some of these chemicals were also found when imidacloprid was irradiated dispersed on thyme's leaves. These results show that photochemical reactions between pesticides and the plants secondary metabolites 2 can take place in solution as on plants. These findings demonstrate the importance to increase our knowledge on these complex scenarios that concern all the environmental compartments.

Introduction

Pesticides undergo degradation processes in surface waters [START_REF] Pehkonen | The Degradation of Organophosphorus Pesticides in Natural Waters: A Critical Review[END_REF], on the surface of soil and of leaves [START_REF] Katagi | Photodegradation of Pesticides on Plant and Soil Surfaces[END_REF] yielding breakdown chemicals. The potential effects of these chemicals on wildlife and human health have attracted numerous studies aiming for identification and to improve knowledge on their physico-chemical, biological and toxicological properties (Martínez Vidal et al., 2019[START_REF] De Souza | Occurrence, impacts and general aspects of pesticides in surface water: A review[END_REF].

Pesticides are generally photodegraded when they absorb solar light [START_REF] Burrows | Reaction pathways and mechanisms of photodegradation of pesticides[END_REF][START_REF] Marussi | Direct photodegradation of 36 organic micropollutants under simulated solar radiation: Comparison with free-water surface constructed wetland and influence of chemical structure[END_REF] or when they are irradiated in the presence of natural chromophoric species acting as sensitizers (natural organic matter) or photoinducers (nitrate ions, ferric species…) [START_REF]The role of indirect photochemical degradation in the environmental fate of pesticides: a review[END_REF][START_REF] Ossola | Singlet Oxygen Quantum Yields in Environmental Waters[END_REF][START_REF] Marussi | Direct photodegradation of 36 organic micropollutants under simulated solar radiation: Comparison with free-water surface constructed wetland and influence of chemical structure[END_REF][START_REF] Brand | Bolte Degradation Photoinduced by Fe(III): Method of Alkylphenol Ethoxylates Removal in Water[END_REF]. Photochemical reactions between two pesticides can also take place. In our group, we have demonstrated that mixing mesotrione and nicosulfuron [START_REF] Ter Halle | The effect of mixing two herbicides mesotrione and nicosulfuron on their photochemical reactivity on cuticular wax film[END_REF] or chlorothalonil and cycloxydim [START_REF] Monadjemi | Photoreduction of chlorothalonil fungicide on plant leaf models[END_REF] enhances the rate of photodegradation of each of them. Thiophanate-methyl was also shown to favor the photodegradation of methyl-bifenazate [START_REF] Hamdache | Unravelling the reactivity of bifenazate in water and on vegetables: Kinetics and byproducts[END_REF]. These results were explained by the capacity of chlorothalonil and the primary photoproduct of thiophanate-methyl to generate oxidant triplet excited states and singlet oxygen capable of oxidizing other pesticides. In addition, reactions can be drastically affected by the medium. This was illustrated in the case of the insecticide methyl-bifenazate, the photodegradation of which was measured to be 70-fold faster on green pepper skin than on a film of paraffinic wax [START_REF] Hamdache | Unravelling the reactivity of bifenazate in water and on vegetables: Kinetics and byproducts[END_REF]. This behaviour was rationalized by the presence of water from pepper. These results bring evidence that the fate of pesticides can be affected by the presence of other chemicals present in the medium apart from the well-known sensitizers or photoinducers.

Volatile compounds emitted by plants are the subject of intense research because of their implication in atmospheric chemistry, air pollution and defense of plants against pathogens [START_REF] Atkinson | Atmospheric Degradation of Volatile Organic Compounds[END_REF][START_REF] Mellouki | Atmospheric Chemistry of Oxygenated Volatile Organic Compounds: Impacts on Air Quality and Climate[END_REF][START_REF] Wannaz | Air pollutants effect on monoterpenes composition and foliar chemical parameters in Schinus areira L[END_REF][START_REF] Hammerbacher | Roles of plant volatiles in defence against microbial pathogens and microbial exploitation of volatiles[END_REF]. However, to the best of our knowledge, scenarios in which they could interact with pesticides were rarely considered in the literature. These potential reactions concern all the environmental compartments. When a pesticide is dispersed on the crop leaves just after been applied, it is surrounded by plant's volatiles facilitating reactions between each group of chemicals at the solid gas interface. Moreover, pesticides and plant volatiles can be transported to surface water after their solubilization in the run off water and can interact in the aquatic compartment. Plant volatiles correspond to a large group of chemicals : mono, di and triterpenes/terpenoids, phenols [START_REF] Dudareva | Plant Volatiles: Recent Advances and Future Perspectives[END_REF][START_REF] Abbas | Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering[END_REF][START_REF] Mochizuki | Effect of growth temperature on monoterpene emission rates of Acer palmatum[END_REF].

They bear functionalities such as H-atoms, double bonds and aromatic rings giving them possibilities to be involved in a lot of photochemical reactions.

Here, we investigated the photochemical reactivity of two pesticides : the fungicide chlorothalonil (CT) and the insecticide imidacloprid (IMD) in the presence of the main thyme volatiles : thymol (T), linalool, 3-carene, -pinene. These terpenes and thymol are common to other plant species [START_REF] Salehi | Thymol, thyme, and other plant sources: Health and potential uses[END_REF]. Both pesticides are susceptible to have an effect on the plant volatiles : CT shows photooxidant properties via its triplet excited state or the formation of singlet oxygen ( 1 O 2 ) (Ter Halle 2010) while IMD releases NO and NO 2 under irradiation making it a potential nitrosating/nitrating agent [START_REF] Palma | New Route to Toxic Nitro and Nitroso Products upon Irradiation of Micropollutant Mixtures Containing Imidacloprid: Role of NO x and Effect of Natural Organic Matter[END_REF]. Out of nitrogen oxides, NO and NO 2 are considered toxic [START_REF] Skalska | Trends in NOx abatement : a review[END_REF]. Experiments were first conducted in solution to evidence the reactions. Acetonitrile was chosen for its ability to ensure a high solubility of all the studied chemicals. Then, pesticides were directly irradiated on leaves of thyme to approach the real conditions closely. ). Acetonitrile (ACN) was purchased from Carlo Erba Reagents (HPLC Plus Gradient grade-ACS-Reag).

Thyme (Thymus Vulgaris) was freely grown under natural sunlight in a garden for seven years in Beaumont (France) and regularly watered. In the case of IMD, we undertook a comparative set of experiments on a thyme plant (Thymus Vulgaris) bought in a garden center.

Samples preparation.

All stock solutions were prepared in ACN and kept in the dark at 4°C before use. Final solutions were prepared by diluting stock solutions with ACN. For the experiments on thyme leaves, twigs were cut using scissors. Each twig contained 30 leaves (average area of each leave of 0.03 cm 2 ) for a total area of 1.8 cm 2 considering the two faces.

Twigs were dipped in IMD or CT solution (7.5×10 -4 M) for 30 s, left to dry freely for an hour, and then put in hermetically sealed headspace (HS) vials to be either used for control experiments in the dark or irradiated. Dark controls in the absence of pesticides were conducted with twigs directly put in HS vials. The surface concentration of IMD and CT on twigs was evaluated as follows : twigs covered by CT were immersed and agitated in 2 mL of acetonitrile for 2 min while those covered by IMD in 5 mL of acetonitrile. Solutions were then analyzed by high performance liquid chromatography coupled to a photodiode-array detector(HPLC-DAD) to determine the CT's and IMD's concentrations.

Irradiation. Solar UV radiations reaching the earth's surface contain UVA (315-400 nm) and a part of UVB (295-315 nm). The absorption spectrum of CT and IMD overlapping the radiations reaching the earth's surface within the wavelength range 295-350 nm (Figure SI-1A), we used the following irradiation devices to simulate solar light. The first one (device 1) was used to calculate the quantum yield of CT photodegradation in the presence of thyme's volatiles. Solutions containing CT (10 -4 M) + volatiles (10 -3 M) were put in a cuvette and irradiated at 313 nm (UVB) in parallel beam using a high pressure mercury lamp equipped with an Oriel monochromator. A radiometer QE65000 from Ocean optics was used to measure the photon flux I 0 : (3.0±0.2)×10 14 photons cm -2 s -1 at 313 nm. Samples were taken at chosen irradiation times and directly analyzed by HPLC-DAD to follow the degradation profile of CT. To monitor the disappearance of CT or IMD (10 -4 M) in ACN alone or in the presence of volatiles (10 -3 M) and detect new photoproducts we needed to use a device delivering a more intense photon flux. Solutions (20 ml) were placed in a cylindrical Pyrex reactor (1.4 cm internal diameter) sealed with an air-tight silicon cap surrounded by 3 polychromatic tubes simulating UVB (Philips, TL20W/01-RS, λ max = 313 nm) installed inside a custom-made cylindrical irradiation device (device 2). Aliquots were sampled at chosen irradiation times and directly analyzed by HPLC-DAD to monitor the degradation profiles of CT or IMD and UHPLC coupled to high resolution mass spectrometry (LC-HRMS) and gas chromatography coupled to mass spectrometry (GC-MS) to identify photoproducts. The third device (device 3) was a solar simulator CPS Atlas equipped with a xenon arc lamp, a filter cutting off wavelengths shorter than 290 nm and a cooling system maintaining the bottom of the device at 10°C. The global irradiance in the range of 300-800 nm was set at 500 W m -². It represents a medium value at 40°N latitude in summer. This device was used to irradiate the HS vials containing thyme twigs alone or covered with IMD or CT. In the case of IMD, 2 h of irradiation were enough to detect significant amounts of nitro derivatives of thymol. For CT, due to the difficulty to demonstrate the interaction between CT and thyme's volatile, we extended the irradiation until 6 h. Vials were then analyzed directly by Headspace gas chromatography coupled to mass spectrometry (HS-GC-MS) or chemicals contained in and on twigs were extracted by ACN as previously explained and analyzed by LC-HRMS or GC-MS. Irradiation experiments in solution were duplicated and on twigs duplicated or triplicated.

Analyses. UV-Vis absorption spectra of volatiles, CT, IMD, TQ, and TQH 2 were recorded using a Varian Cary 3 spectrophotometer. CT and IMD degradation profiles were tracked by HPLC-DAD using a NEXERA XR HPLC-DAD apparatus. The analytical column was a Phenomenex-kinetex C 18 (100 mm×2.1 mm, 2.6 μm particle size) for CT experiments and EC 150/4.6 NUCLEODUR C 18 endcapped column for IMD analyses. The eluent was a mixture of ACN and water and the flow rate was set at 0.2 and 1 mL min -1 for CT and IMD, respectively. The HPLC conditions for CT were as follows: the elution gradient started with 30% ACN maintained for 2 min and then linearly increased to reach 80% after 13 min; while that for IMD started with 30% ACN which was maintained for 10 min. Photoproducts were analyzed and quantified using HRMS on an Orbitrap Q-Exactive (Thermoscientific) coupled to an ultra-high performance liquid chromatography (UHPLC) instrument Ultimate 3000 RSLC (Thermoscientific). The column was a Kinetec EVO C 18 (100×2.1 mm), particle size of 1.7 μm (Phenomenex). The elution gradient started with 15% acetonitrile maintained for 5 min, then the percentage of acetonitrile was linearly increased to reach 55% after 5 min. The flow was set at 0.45 mL min -1 . Analyses were made in positive (ESI + /ESI -) electrospray mode. Identification of the major constituents was carried out using the Xcalibur software and when necessary by matching retention times with those of authentic standards. The m/z of proposed structures differed by less than 5 ppm from the theoretical mass. Standard solutions of TQ were analyzed immediatly after have been prepared. The limit of detection LOD in the case of TQ was estimated to 10 -6 M by analyses of authentic solutions.

Photoproducts were also analyzed using an Agilent 6890 GC coupled to an Agilent 5973 MS.

The separation was carried out using a HP-5 µs column (25 m×0.25 mm×0.25 μm) operating initially at 80°C over 1 min, followed by a 10°C min -1 ramp to reach 200°C and then a 30⁰ C min -1 ramp to reach 260°C maintained for 1 min (to calculate the degradation of terpenes) in the case of CT experiments, while the analysis was operated initially at 70°C followed by a 10°C min -1 ramp to reach 160°C for 5 min and then a 30°C min -1 ramp to reach 250°C maintained for 2 min in the case of IMD. The flow rate was 1 mL.min -1 with an injection volume of 1 µL in both cases. T and some of its photoproducts were quantified after derivatization with N,O-Bis (trimethylsilyl)trifluoroacetamide (BSTFA). The derivatizing solution (2 mL) was prepared by mixing 60 µL of BSTFA with 1.4 mL sample. Separation was achieved as previously described for CT experiments.

To determine the volatile metabolites produced by the plant, HS-GC-MS (Shimadzu HS-20 coupled with QP2010SE) was used. Twigs of 0.2 g alone or after dipping in CT solution were transferred into a 20 mL headspace glass vial, then left in the dark or irradiated and incubated

for 5 min at 80⁰ C. The analytical column (Mega 5-MS 30 m×0.25 mm) was operated initially at 50°C for 1 min, followed by an 8°C min -1 ramp to reach 170°C and held for 4 min, and then a 15°C min -1 ramp to reach 275°C maintained for 1 min. The mass spectrometer source was heated to 200°C, and signals were detected between mass to charge ratios (m/z) of 50 and 350.

Identification of the major constituents was carried out using the NIST 17 database and when necessary by matching retention times and ion fragments with those of authentic standards.

For calibration, a stock solution of T was prepared in ethanol at a concentration of 1 g L -1 .

Quantification of T was performed by means of 5-point calibration curve for which the concentrations of standards ranged from 2-100 mg L -1 . All standard solutions were stored in dark at 2-8°C in amber glass vials with Teflon-lined caps. They were found to be stable under short-term (48 h) storage conditions. A linear response was obtained with a correlation coefficient (R 2 ) of 0.997. Selectivity was evaluated by analyzing a set of blank samples and monitoring the absence of interferences with S/N ratios higher than 3. No significant interfering peaks were observed in the ion chromatogram time window monitored for T. The precision of the method was determined by calculating the relative standard deviation (RSD) of six replicate measurements of diluted T solution (10 mg L -1 ). All RSDs were acceptable and less than 7% indicating a good precision. The limit of detection LOD was calculated from the standard deviation (σ) of multiple measurements (n=7) of diluted T solutions within a factor of 5 of the background signal. LOD was set equal to 3σ which resulted in a value of 13 pg. The Limit of quantification (LOQ) was defined as 10σ or 43 pg. In all cases, LOD and LOQ were significantly below the levels determined in the thyme extracts (0.07 to 2.7 mg).

Quantum yields of CT photodegradation in the presence of terpenes in acetonitrile. The quantum yield of CT photodegradation (Φ CT ) was determined using Eq 1:

Φ CT =Δc/(Ia×Δt) Eq 1
where Δc/Δt was the rate of CT consumption expressed in M s -1 and Ia was the rate of photon absorption by CT at 313 nm expressed in Einstein L -1 s -1 . Ia was obtained using Eq 2:

Ia= Io×(1-10 -ε×ℓ×[CT]o )×1000/ℓ Eq 2
where ε= 1760 M -1 cm -1 , [CT] o = 10 -4 M, and ℓ= 1 cm.

Laser flash photolysis. Laser flash photolysis experiments were carried out using an Applied Photophysics LKS.60 apparatus equipped with a Nd 3+ :YAG laser Quanta-Ray GCR-130 used in a right-angle geometry with respect to the monitoring light beam. Samples were irradiated using the fourth harmonic (266 nm, 9 ns pulse duration) in a quartz cuvette. 3 CT* was generated by irradiating CT (5×10 -5 M in aerated ACN) and its decay monitored at 330 nm.

The reactivity of 3 CT* with thyme's volatiles was measured within the concentration range 10 -4 -2×10 -3 M. In the case of IMD, no transient species were detected.

Photoproducts toxicity. The toxicity of photoproducts formed during the irradiations was estimated using the freely available ECOSAR (ECOlogical Structure-Activity Relationship) computer program (v 2.0). It predicts the toxicity of a molecule using a structure-activity approach. The acute (LC50, the concentration that is lethal to half of fish after 96 h of exposure to a certain molecule) and chronic toxicities (ChV) of CT, IMD, and formed photoproducts on fish in fresh waters as well as logKow were obtained. The program (v 2.0) is available from the website: epa.gov/tsca-screening-tools/ecological-structure-activityrelationships-ecosar-predictive-model. were the main detected compounds. T is a phenolic compound while linalool, 3-carene and pinene are terpenes (Scheme 1). The amount of extractable T from thyme's twigs by ACN was measured by GC-MS. Based on several replicates using twigs containing 30 leaves (weight 0.2 g), it could be estimated to range between 0.35 and 2.7 mg per g of wet twigs.

After that, we analyzed the non-volatile chemicals. For this, two vials containing two twigs each were left in the dark for 4h, extracted in 2 ml of ACN and analyzed by UHPLC-HR-MS and GC-MS. Several peaks directly connected to T were found (Table 1). By UHPLC-HR- for thymolhydroquinone). We therefore concluded that TO was the benzyl alcohol derivative ( and7). By GC-MS, TO, TQ and several isomers of TO-TO -2H were also detected.

MS

The second objective was to investigate the effect of irradiation on the formation of these compounds. For this, two vials containing two twigs each were irradiated in the solar simulator for 4h and extracted using ACN. The same compounds were detected and the effect of irradiation on their formation can be seen in Table 2. The peak areas were between 15 and 100-fold higher after irradiation than in the dark showing the drastic effect of light. 1A). In contrast, the irradiation of CT (10 -4 M) in the presence of each of these volatiles (10 -3 M) led to the fast disappearance of CT (Figure 1A)

Interactions between CT and thyme's volatiles

and the thyme's volatiles (Figure 1B). As shown in Figure 1A, the CT photodegradation increased in the order T > -pinene > 3-carene > linalool. In accordance, the quantum yield of CT photodegradation ( CT ) was equal to 0.35, 0.20, 0.15, 0.04 for T, -pinene, 3-carene and linalool, respectively against < 0.001 for CT alone. Concerning the thyme's volatiles loss, T was the fastest to disappear, followed by -pinene, 3-carene, and linalool (Figure 1B).

To confirm the ability of thyme's volatiles to react with 3 CT* and measure the bimolecular rate constant of the reaction (k r ), we undertook laser flash photolysis experiments. CT was irradiated in the presence of each of these chemicals added at a concentration comprised between 310 -4 and 2×10 -3 M while the decay of 3 CT* was monitored at 330 nm (Figure 2). Alone, in air-saturated ACN, 3 CT* disappeared by an apparent first order kinetic with k = 1.610 6 s -1 corresponding to the sum

k d + k O2 [O 2 ],
where k d is the rate constant of deactivation by collision with the solvent molecules, k O2 the rate constant of reaction with oxygen and [O 2 ] the concentration of oxygen in the solution. In the presence of T, 3 CT* disappeared faster than alone showing that a reaction between 3 CT* and T took place (Figure 2A). This reaction might be an energy transfer or an electron/H atom transfer reaction. The energy level of 3 CT* was previously estimated to lay at 275 kJ mol -1 based on phosphorescence measurements at 77 K in pentane [START_REF] Porras | Humic substances enhance chlorothalonil phototransformation via photoreduction and energy transfer[END_REF]. This is lower than the energy level reported for phenol  314-334 kJ.mol -1 [START_REF] Becker | Theoritical and Interpretation of fluorescence and phosphorescence[END_REF] making the energy transfer from 3 CT* to T unlikely. Moreover, given the ability of 3 CT* to oxidize phenols [START_REF] Monadjemi | Photoreduction of chlorothalonil fungicide on plant leaf models[END_REF], an electron or H atom transfer from T to 3 CT* with formation of the reduced CT radical (CT -. /CTH . ) and the phenoxyl radical (T -H

. ) (Scheme 2) looks more probable .

Scheme 2 : Reaction of 3 CT* with thymol

The shape of the time-resolved absorption spectrum of Figure 3A revealed the formation of a secondary species that could be therefore either the reduced CT radical or T -H . . Its significant absorption measured at 330 nm matches more with the reduced CT radical [START_REF] Bouchama | Richard Photoreactivity of chlorothalonil in aqueous solution[END_REF]) than with T -H . that shows maxima at 390 and 410 nm [START_REF] Venu | Oxidation Reactions of Thymol: A Pulse Radiolysis and Theoretical Study[END_REF]. Due to the overlapping of the two species absorption, a numerical analysis of the differential equations was necessary to determine the k r value. The best fit of the experimental data was obtained by taking k r = 7.010 9 M -1 s -1 ,  CT* / phenoxyl = 2.0, and a first order decay rate constant of 510 5 s -1 for the reduced CT radical in air-saturated medium (Monadjemi et al., 2014). Individual evolution of these species are shown in the inset of Figure 2A.

In the case of -pinene, 3-carene and linalool, the absorbance at 330 nm completely decayed within 1 s following the flash (Figure 2B). This result demonstrated the reaction of 3 CT* with terpenes. However, with these compounds, the formation of the reduced CT radical was not observed (Figure 2B). Values of k r could be deduced from the linear plot of k, the obserbed first order rate constant, vs [terpene] (inset of Figure 2B). Assuming k

= k d + k O2 [O 2 ] +k r [terpene]
, one got k r = 2.610 9 , 2.810 9 and 3.910 9 M -1 s -1 for -pinene, 3carene, and linalool, respectively. The oxidation of terpenes by 3 CT* was likely due to the presence of labile H atoms in their structures and the non-observation of the 330 nm radical could be tentatively explained by an in-cage reaction between the reduced CT radical and the oxidized terpene radical leading directly to photoproducts (Scheme 3 in the case of 3-carene).

If one admits that the relative importance of process a that yielded photoproducts and process b in which CT was regenerated depended on the structure of the terpene, then one can also explain why  CT varied from 0.20 for -pinene to 0.04 for linalool whereas the k r values laid in a very narrow range for these two compounds. Finally, we found that all four thyme's volatiles readily reacted with 3 CT*. The higher reactivity of T compared to that of -pinene, 3-carene, and linalool is in line with the presence of an easily abstractable phenolic H atom. and was named TCl (Table 1). By GC-MS, we detected TCl again, CT -Cl and CT -2Cl . The formation of dechlorinated CT confirmed that photoreduction took place. CT -Cl was also detected upon the irradiation of CT+3-carene, but no other photoproducts were found by GC-MS and no photoproducts at all were detected with -pinene and linalool.

Based on these data and on the known CT photoreactivity, the reaction pathways shown in Scheme 4 can be proposed to explain the reaction between CT and T. In neat acetonitrile, 3 CT* is generated with a quantum yield close to unity and trapped by O 2 (k O2 = (1±0.1)10 9 M -1 s -1 ) to yield 1 O 2 with a quantum yield equal to 0.85±0.06 [START_REF] Bouchama | Richard Photoreactivity of chlorothalonil in aqueous solution[END_REF]. T will therefore compete with O 2 for the reaction with 3 CT*. Taking k r = 7.010 9 M -1 s -1 and [O 2 ] = 1.910 -3 M in air-saturated acetonitrile solution at 24°C [START_REF] Murov | Handbook of Photochemistry Second Edition[END_REF], one can estimate that T (10 -3 M) could trap 79% of 3 CT* while 21% of O 2 . In our experimental conditions, T was thus mainly photooxidized through a direct reaction with 3 CT*, however, given the known good reactivity of singlet oxygen with phenolic compounds [START_REF] Wilkinson | Rate constants for the decay and reactions of the lowest electronically excites singlet state of molecular oxygen in solution[END_REF], a part of the loss of T may be attributable to the reaction T + 1 O 2 . Once formed, T -H . should be further oxidized into TO, TQ and yield dimeric species. The detection of TCl demonstrates that CT can also photoinduce the chlorination of other chemicals. A possible explanation is the release of chlorine atoms from CT -. followed by their recombination with T -H . (Scheme 4). Such a reaction also rationalizes the formation of dechlorinated CT. The values of k r for terpenes were 1.8 to 2.7-fold lower than for T. Therefore, the percentage of 3 CT* trapped by terpenes at 10 -3 M should be consequently lower than for T and the amount of singlet oxygen generated higher. The photooxygenation of terpenes bearing double bonds such as -pinene, linalool and 3-carene is therefore likely in our conditions,

3 CT * h 1 CT * CT k d CT O 2 k O2 T k r O 2 + CT + 1 O 2 T -H T oxidation products O 2 CT + HO 2 CT -Cl + Cl
given their reactivity with singlet oxygen [START_REF] Chiron | Photochemical hydroperoxidation of terpenes I. Synthesis and characterization of α-pinene, β-pinene and limonene hydroperoxides[END_REF].

Similar reactions can take place in water or in media enriched in water because 3 CT* was also detected in water/acetonitrile (90 :10, v/v) with a quantum yield of formation also close to unity [START_REF] Bouchama | Richard Photoreactivity of chlorothalonil in aqueous solution[END_REF]. However, changing the solvent is expected to induce some changes in the pathway ratios. For instance, the percentage of 3 CT* trapped by O 2 will decrease significantly from acetonitrile to water because the O 2 concentration is reduced by a factor of about 7 (from 1.9×10 -3 M to 2.7×10 -4 M in air-saturated solutions at 24°C). This may favor the direct reaction between 3 CT* and the two volatiles T and linalool for which the water solubility is good and thus the CT photoreduction. In contrast, for 3-carene and pinene that show a very low water solubility, CT photoreduction should be insignificant and 3 CT* could mainly lead to singlet oxygen. However, the lifetime of singlet oxygen is shorter in water (4 s, [START_REF] Rodgers | Lifetime of ( 1 O 2 ) in Liquid Water As Determined by Time-Resolved Infrared Luminescence Measurements[END_REF] than in acetonitrile (61-68 s, [START_REF] Hurst | Lifetime of Singlet Oxygen in Solution Directly Determined by Laser Spectroscopy[END_REF] which will affect negatively the rate of terpenes photooxidation. Last, the higher polarity of water compared to acetonitrile may increase k r , although it is already quite high in acetonitrile.

Irradiation of CT on thyme's leaves

We started the experiments on thyme's leaves by estimating the amount of CT deposited on two twigs after their dipping in a solution of CT (7.5×10 -4 M). In the 2 ml of ACN used for the extraction, the CT concentration was of 3.4×10 -5 M. Based on a total leaves area of 3.6 cm 2 , we finally got a CT concentration of 51 g per cm 2 , which is in the range of the surfacic concentrations calculated for typical CT application rates of 0.5-1 kg ha -1 .

Then, CT was irradiated for 6 h in the solar simulator on thyme's leaves or deposited on a glass dish chosen as an inert support and ACN extracts analysed by GC-MS (Figure 4).

While no CT photodegradation was observed on dishes, an almost full loss of CT on thyme's leaves was measured. CT -Cl and CT -2Cl were formed (Inset of Figure 3) confirming the photoreduction of CT when applied on thyme's leaves. T that was present in high amounts in the thyme's twigs was the candidate to induce this photoreduction, even though its involvement in the reaction cannot firmly demonstrated.

Moreover, the GC-MS area of T peak increased similarly after 4 h of irradiation for thyme's twigs irradiated in the absence of CT and for thyme's twigs irradiated in the presence of CT from (2.80.4)×10 6 to (2.30.9)×10 8 (Table 2). The presence of CT did not affect the nature of T byproducts (TO, TQ, TQO, T-TO -2H and T-TO -2H ) and their peak area measured after 4 h of irradiation and in the dark (Table 2). The only evidence of an interaction between CT and T came from the analyses of the gazeous phase of irradiated vials by HS-GC-MS.

Indeed, in this case, a drastic loss of T was observed . The apparent discrepancy between the HS-GC-MS analyses conducted directly on the volatiles present in the vials and the GC-MS analyses that required extraction of chemicals by ACN can be explained as follows. CT deposited on leaves consumed T present in the gazeous phase, and the weak volatility of T limited any reequilibration through remission from leaves allowing to detect the T loss by HS-GC-MS. However, this T consumption was not measurable after extraction by ACN because extraction allowed the recovering of the great part of T present in the leaves while the loss of T that was related to the initial concentration of CT on the twigs laid below the measurement uncertaincies. Indeed, we estimated that 5.7×10 -7 mole of T could be extracted while 6.7×10 -8 mole of CT were present on twigs. As one mole of CT was expected to oxidize one mole of T at its best, the amount of T consumed was less than 12% of T extracted, thus negligible.

To conclude, we demonstrated that in solution photochemical reactions between CT and thyme's volatiles, in particular T, took place with photoreduction of CT and photooxidation/photochloration of T. On thyme's leaves, photoreduction of CT was also observed. The involvement of T in this reaction was likely based on the data obtained in solution, but could not be firmly confirmed.

Interactions between IMD and thyme's volatiles

Irradiation of IMD and T in acetonitrile solution

After 30 min of irradiation alone in device 2, IMD (10 -4 M) disappeared by 6.5×10 -5 M (65%). Three photoproducts were identified (Table 3). In the presence of T (10 -3 M), IMD disappeared by 3.0×10 -5 M (30%) after 30 min of irradiation (Table 4), i. e. less than alone. On the other hand, the T loss was of 7% after 2 h, yielding a consistent consumption of around 1.7×10 -5 M after 30 min (Table 4). IMD -NO2+H , IMD -HNO and IMD-IMD -N2O3 were detected again but in much smaller amounts than in the absence of T (Table 4). In addition, we detected along TO, a peak at m/z = 194. NO 2 bond and led to the formation of the radical IMD -NO2 that is further converted into IMD - NO2+H after the abstraction of an H atom [START_REF] Palma | New Route to Toxic Nitro and Nitroso Products upon Irradiation of Micropollutant Mixtures Containing Imidacloprid: Role of NO x and Effect of Natural Organic Matter[END_REF]. The formation of TNO 2 could be explained by the oxidation of T by NO 2 into the corresponding phenoxyl radical followed by the addition of NO 2 onto it [START_REF] Marussi | Direct photodegradation of 36 organic micropollutants under simulated solar radiation: Comparison with free-water surface constructed wetland and influence of chemical structure[END_REF]. Similarly, the nitration of TO into TONO 2 should involve the oxidation of TO by NO 2 followed by the addition of NO 2 on the corresponding phenoxyl radical. The lower rate of IMD loss in the presence of T was in favor of a competition between IMD and T for the reaction with NO 2 and suggests that NO 2 reacted with IMD, for example, by H atom abstraction.

Irradiation of IMD on thyme's leaves

As for CT, we estimated the amount of IMD deposited on two twigs after their dipping in a solution of IMD (7.5×10 -4 M). In the 5 ml of ACN used for the extraction, we measured a IMD concentration of 3.9×10 -5 M. Based on a total leaves area of 3.6 cm 2 , we finally got a IMD concentration of 142 g per cm 2 that is enough to observe reactions.

Vials containing twigs covered with IMD were irradiated in the solar simulator for 2 h, and in parallel other vials were left in the dark for the control. The ACN extracts were then analyzed by UHPLC-HR-MS. In the samples left in the dark, we detected TO, TNO 2 , TONO 2 and TO-TNO 2 (Table 4), confirming that the NO 2 release from IMD could also take place in the dark. As shown in Table 4, upon irradiation of IMD on twigs, TNO 2 and TO-TNO 2 where detected in amounts equivalent to those observed in the dark . However, for TONO 2 a 50folds enhancement was measured. The presence of IMD little increased the formation of TO compared to thyme irradiated in the absence of IMD. Another set of experiments was undertaken on the same variant of thyme just bought in a garden center and thus much younger and much less exposed to solar light. In this case also, nitration reactions were observed under irradiation but they yielded TNO 2 and not TONO 2 as for the older thyme.

Accordingly, the young thyme contained much less TO than the older one.

Therefore, we conclude that IMD generated nitrocompounds when it was in contact with T, with an increase of the phenomenon under light exposure. The reaction involved the radical NO 2 that was released in the medium mainly photochemically but also spontaneously.

T had also an effect of the fate of IMD : by quenching NO 2 , it increased its lifetime.

Estimation of the photoproducts toxicities

Using the ECOSAR computer program (version 2.0), we estimated the potential acute (LC 50 ) and chronic toxicity (ChV) of the starting chemicals and their degradation products (Table 5). CT is classified as a moderate toxic chemical. Its photoreduction by T and terpenes in CT -Cl and CT -2Cl decreases the acute and chronic toxicity on fish from 6.98 to 74.40 mg L -1 and 0.81 to 7.57 mg L -1 , respectively and also the bioaccumulation, but these compounds are still classified as moderatly toxic compounds. On the other hand, TO is less toxic on fish than T with LC 50 of 20.90 against 1.86 mg L -1 and ChV of 2.08 against 0.21 mg L -1 and less bioaccumulated. In contrast, TQ, T-TO -2H , TO-TO -2H and TCl are more toxic than T with LC 50  1.12 mg L -1 and ChV  0.16. TQ, T-TO -2H and TCl are classified as highly toxic. TCl was not detected in the experiments on thyme's leaves, this might be due to a high value of K ow and a diffusion of this compounds inside the cuticle preventing its extraction by ACN. In the case of IMD, T inhibited the pesticide degradation rate and any new chemical deriving from IMD was formed. Other good H-donor chemicals such as phenolic compounds contained in plants may have the same effect and therefore increase the lifetime of IMD and lengthen its insecticidal effect. T and TO underwent nitration. TONO 2 is moderatly toxic as T with higher LC 50 but lower ChV, while TNO 2 is classified as highly toxic with LC 50 = 1.39 mg L -1 and CHV = 0.15. Therefore, chemicals that are more toxic than the starting compound can be formed during these reactions.

Conclusion :

In this work, we investigated the photochemical reactions between pesticides and plant's volatiles in solution and leaf surfaces and we demonstrated that interactions take place in both media. The interactions modified the fate and the transformation pathways of the chemicals and depended on the chemicals structure and the matrix. This was illustrated by the examples of the fungicide chlorothalonil and the insecticide imidacloprid chosen as pesticides and thymol, -pinene, 3-carene and linalool as plant's volatiles. Studying these reactions is relevant for several reasons. First, they can occur in the real conditions on plants and in surface waters and affecting the fate of the chemicals, these reactions can also alter the phytosanitary treatments shortening the lifetime of the pesticide for example, and lead to repeated and/or increased treatments. These are therefore important factors to consider in the design of pesticides. Secondly, the interactions can potentially generate toxic by-products leading to the pollution of water bodies and of air. The air pollution may be observed in particular in the cities where green roofs are installed. Indeed, their growth in many urban zones in the world could be accompagnied by increased possibilities of reactions between plant's volatiles and other chemicals such as the pesticides used to protect the roof plants which might finally lead to an enhanced formation of by-products. For all these reasons, it seems necessary to get a better insight into these overlooked reactions often referred to as « cocktail effect ».

Results 1 .

 1 Characterization of the thyme's constituents This first objective was to determine what are the main volatile compounds in thyme's twigs, to estimate the amount of T in thyme's twig and to identify and quantify the chemicals related to T. Volatile compounds released from thyme's twigs after two hours in the dark in the HS vials were analyzed by HS-GC-MS (Figure SI-2). T, linalool, 3-carene and -pinene

  , one got a peak at m/z = 165.0901 in ES -corresponding to [M+O-H + ] -and named TO. By MS-MS, TO gave two main fragments at 149.0593 and 135.0443 corresponding to the loss of O and CH 2 O, respectively (Figure SI-3). The oxidation of T can take place on the isopropyl group, on the methyl group or on the ring. No loss of H 2 O was observed ruling out an oxidation of the isopropylic carbon atoms. The oxidation of the ring would lead to thymolhydroquinone. However, TO was not assigned to thymolhydroquinone because the two compounds had different retention times in HPLC (3.3 min for TO against 4.5 min for thymolhydroquinone), and different maximum of absorption (278 nm for TO against 290 nm

2. 1 .

 1 Irradiation of CT and thyme's volatiles in acetonitrile solution Neither T, 3-carene, linalool and -pinene nor CT underwent significant photodegradation when irradiated alone in ACN solution. Thyme's volatiles do not absorb the radiations emitted by the tubes (Figure SI-1B), while CT absorbs the radiations (Figure SI-1A) but yields a triplet excited state 3 CT* efficiently deactivated by oxygen (7.810 8 M -1 s -1 , Monadjemi et al., 2011) (Figure

  photoproductsCT + 3-carene

  Scheme 4: Mechanism of CT photolysis in the presence of T

  The main one gave a peak at m/z = 211.0742/213.0710 in ES + (Figure SI-9) and corresponded to [M-NO 2 +H+H + ] + , it was assigned to the imino derivative IMD -NO2+H . Two other minor peaks were detected at m/z = 223.0385/225.0356 in ES -(Figure SI-10) corresponding to [M-NO-H-H + ] -and at m/z = 432.0744/434.0715/436.0689 (Figure SI-11) corresponding to [2M-N 2 O 3 -H + ] -. They were named IMD -HNO and IMD-IMD -N2O3 , respectively.

  photoproducts formation, in particular that of IMD -HNO and IMD-IMD -N2O3 while undergoing

  2H+H + ] + and was assigned to the quinonic derivative TQ (Table1). Its maximum of absorption at 250 nm was fully in line with this assignement. The other peak at m/z = 179.0704 in ES -corresponding to [M+2O-2H-H + ] -was attributed to the quinonic derivative of TO, TQO (FigureSI-5). The two peaks showing m/z = 313.1806 and 329.1754 in ES -were assigned to dimers, T-TO -2H and TO-TO -2H , respectively

Table 1). The peak detected at m/z = 165.0901 in ES + (Figure SI-4) corresponded to [M+O-

Acknowledgments :

This research was supported by a PhD fellowship from the University Clemront-Auvergne/French Ministry of Higher Education and Research. The authors would like to thank Martin Leremboure (Engineer CNRS) and Frédéric Emmenegger (Tech CNRS) for UHPLC-MS analyses, and Guillaume Voyard (Engineer CNRS) for HPLC-DAD analyses.


Tayonara S Lima

Maria Fátima

S Silva

Xirley P Nunes

Andrea V Colombo

Helinando P Oliveira

Patrícia L Goto

Henrique L Piva

Antonio C Tedesco

Marigilson P Siqueira-Moura
email: marigilson.moura@univasf.edu.br

Muriel Blanzat

Prof Pontes Marigilson

Siqueira De

Ph.D Moura

Cineole-containing nanoemulsion: development

Keywords: Development, stability, and antibacterial activity, stability, and antibacterial activity 1, 8-cineole, Spontaneous emulsification, Nanoemulsion, Stability, Antibacterial activity

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.

Introduction

In nature, bioactive oils based on terpene (isoprene) and terpenoid compounds are synthesized by aromatic plants as secondary metabolites, commonly known as essential oils (EO) or volatile oils. Monoterpenes are formed by two isoprene units (10 carbon atoms) and constitute more than 80 % of EO composition [START_REF] Asbahani | Essential oils: From extraction to encapsulation[END_REF][START_REF] Bakkali | Biological effects of essential oils -A review[END_REF][START_REF] Bilia | Essential Oils Loaded in Nanosystems: A Developing Strategy for a Successful Therapeutic Approach[END_REF][START_REF] Matos | Essential oils in nanostructured systems: Challenges in preparation and analytical methods[END_REF][START_REF] Slamenova | Cytotoxic, anti-carcinogenic and antioxidant properties of the most frequent plant volatiles[END_REF].

According to each plant source, EO comprise widely varied monoterpenes, sesquiterpenes, and phenylpropenes combinations. There is a terpenic oxide among such monoterpenes, namely 1,8-cineole (C10H18O; M.W. 154.25 g mol -1 ), which is a cyclic ether found as the main constituent in EO obtained from eucalyptus species. This monoterpene is a limpid and colorless oily liquid with a strong camphor-like odor and also called 1,8-epoxy-p-menthane, eucalyptol, and cajeputol [START_REF] Adak | Nanoemulsion of eucalyptus oil: An alternative to synthetic pesticides against two major storage insects (Sitophilus oryzae (L.) and Tribolium castaneum (Herbst)) of rice[END_REF][START_REF] Ali | Essential oils used in aromatherapy: A systemic review[END_REF][START_REF] De | Constituents of aromatic plants: eucalyptol[END_REF][START_REF] Salehi | Insights into Eucalyptus genus chemical constituents, biological activities and health-promoting effects[END_REF][START_REF] Slamenova | Cytotoxic, anti-carcinogenic and antioxidant properties of the most frequent plant volatiles[END_REF].

Owing to its bactericidal and fungicidal activities as well as flavor and fragrances properties, 1,8-cineole has been used by food, cosmetic, and pharmaceutical industries.

However, despite all these applications, the use of 1,8-cineole or EO containing this terpene is limited due to its limited thermal stability, substantial volatility, and low water solubility [START_REF] Adak | Nanoemulsion of eucalyptus oil: An alternative to synthetic pesticides against two major storage insects (Sitophilus oryzae (L.) and Tribolium castaneum (Herbst)) of rice[END_REF][START_REF] Hammoud | New findings on the incorporation of essential oil components into liposomes composed of lipoid S100 and cholesterol[END_REF]Prakash et al., 2018a;Prakash et al., 2018b).

In order to prevent terpenes and phenylpropanoid derivatives volatilization or oxidation, as well as their degradation by light and for improving the chemical stability, the encapsulation of these compounds into nanostructured systems has been commonly used.

Several nanocarriers have been well designed to entrap EO or their main compounds in this context. Among these carriers, the choice covers polymeric nanocapsules or nanoparticles, liposomes, phytosomes, nanoemulsions, microemulsions, solid lipid nanoparticles, and nanostructured lipid carriers [START_REF] Asbahani | Essential oils: From extraction to encapsulation[END_REF][START_REF] Bilia | Essential Oils Loaded in Nanosystems: A Developing Strategy for a Successful Therapeutic Approach[END_REF][START_REF] Harwansh | Nanoemulsion: Promising nanocarrier system for delivery of herbal bioactives[END_REF][START_REF] Matos | Essential oils in nanostructured systems: Challenges in preparation and analytical methods[END_REF]Prakash et al. 2018a;Prakash et al., 2018b;[START_REF] Zhao | Development of pHsensitive self-nanoemulsifying drug delivery systems for acid-labile lipophilic drugs[END_REF].

Oil-in-water nanoemulsion is the most often-used colloidal lipid carrier to incorporate EO. According to recent review conducted by [START_REF] Matos | Essential oils in nanostructured systems: Challenges in preparation and analytical methods[END_REF], almost half of all nanostructured systems containing EO was represented by nanoemulsified dispersions.

Nanoemulsions (NE) are ultrafine dispersions constituted of two immiscible liquids (organic and aqueous phases) stabilized by surfactant(s). Despite being considered nonequilibrium systems (thermodynamically unstable), nanoemulsions are metastable or kinetically stable colloidal dispersions [START_REF] Anton | Nano-emulsions and Micro-emulsions: Clarifications of the Critical Differences[END_REF][START_REF] Gupta | Nanoemulsions: formation, properties and applications[END_REF][START_REF] Helgeson | Colloidal behavior of nanoemulsions: Interactions, structure, and rheology[END_REF][START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Singh | Nanoemulsion: Concepts, development and applications in drug delivery[END_REF][START_REF] Solans | Nano-emulsions: Formation by low-energy methods[END_REF].

In general, nanoemulsions are produced using low or high-energy methods. High-energy emulsification approaches, i.e., work-based emulsification methods, usually require sophisticated and expensive devices, namely high-pressure homogenizers, ultrasound generators, and microfluidizers. On the other hand, low-energy methods or thermodynamic methods are easy production processes based on simple stirring and changing some formulation parameters, e.g., surfactant concentration and type, oil-water ratio, temperature, etc. Usually low-energy emulsification methods have been categorized as phase inversion composition, phase inversion temperature, emulsion inversion point, and spontaneous emulsification [START_REF] Anton | The universality of low-energy nano-emulsification[END_REF][START_REF] Gupta | Nanoemulsions: formation, properties and applications[END_REF][START_REF] Helgeson | Colloidal behavior of nanoemulsions: Interactions, structure, and rheology[END_REF][START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Safaya | Nanoemulsions: A review on low energy formulation methods, characterization, applications and optimization technique[END_REF][START_REF] Singh | Nanoemulsion: Concepts, development and applications in drug delivery[END_REF][START_REF] Solans | Nano-emulsions: Formation by low-energy methods[END_REF].

The main reason for incorporating EO or their major constituents into the lipid core of nanoemulsion is directly related to bioactive molecules improving their stability and subsequent enhanced bioefficacy [START_REF] Asbahani | Essential oils: From extraction to encapsulation[END_REF][START_REF] Bilia | Essential Oils Loaded in Nanosystems: A Developing Strategy for a Successful Therapeutic Approach[END_REF][START_REF] Harwansh | Nanoemulsion: Promising nanocarrier system for delivery of herbal bioactives[END_REF][START_REF] Matos | Essential oils in nanostructured systems: Challenges in preparation and analytical methods[END_REF]. Also, EO-based nanoemulsion formulations have received increasing attention as effective nanocarriers improving antimicrobial activity of bioactive components (Prakash et al., 2018a;Prakash et al., 2018b). Nevertheless, available information about preparation, stability, and biological activities of nanoemulsions containing isolated bioactive compounds from EO is still scarce, requiring more investigations. Therefore, this study aimed to develop cineole-containing nanoemulsion formulation using an isothermal low-energy emulsification method and assess its accelerated stability and antibacterial activity.

Materials and methods

Materials

1,8-Cineole (Eucalyptol 99 %, d = 0.921 g mL -1 at 25 °C), sorbitan monooleate (SMO), polyoxyethylen-20 sorbitan monolaurate (SML-20), polyoxyethylen-20 sorbitan monooleate (SMO-20), sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Medium-chain triglycerides (MCT, d = 0.94 g mL -1 at 20 °C) oil was kindly supplied from Brasquim (Porto Alegre-RS, Brazil).

Nanoemulsion preparation

Nanoemulsions (NE) were prepared using a method based on a spontaneous emulsification procedure described by [START_REF] Saberi | Stabilization of vitamin E-enriched mini-emulsions: Influence of organic and aqueous phase compositions[END_REF] with slight modifications.

Briefly, spontaneous emulsification was carried out by addition of an organic phase consisting of carrier oil (MCT), 1,8-cineole, and surfactant(s), to an aqueous phase (phosphate buffer solution pH 7.4) under magnetic stirring (200 rpm) and ambient temperature kept constant throughout process. Initially, different ratios between phases and surfactant mixtures were assessed to find more suitable formulations resulting in final composition (wt. %) of the five nanoemulsions reported in Table 1. Thus, both carrier oilto-cineole and surfactant-to-oil weight ratios were kept constant at 1.0 and 2.0, respectively. The proportion of surfactant:oil:water was set at 10:10:80 (wt. %), respectively, and for F1, F3, and F5 formulations the surfactant combinations were maintained at HLB 12 (Table 1). All formulations were prepared three times (n=15) and stored at 4 °C (±2). Unloaded NE (F5 formulation) was obtained from organic phase composed only of carrier oil (MCT) and surfactants using the same spontaneous emulsification process.

Droplet size measurement

The hydrodynamic diameter of droplets and polydispersity index (PdI) of the NE formulations were determined by photon correlation spectroscopy (PCS) at 25 °C and a scattering angle of 173° (Zetasizer ® Nano ZS, Malvern PCS Instruments, UK). All samples were diluted (1/100) with ultra-purified water. The reported values are average ±SEM of three different batches of each colloidal dispersion.

Zeta potential measurement

Zeta (ζ) potential of NE was measured by electrophoretic mobility using a Zetasizer ® Nano ZS apparatus (Malvern PCS Instruments, UK). The Smoluchowski model was used to estimate ζ potential from electrophoretic mobility. The analyses were conducted at 25 °C, and the samples were appropriately diluted (1/100) with ultra-purified water. Values reported are average ±SEM of three different batches of each colloidal dispersion.

Forced stability study

Stability study was performed using the analytical centrifugation method with photometric detection at 865 nm (LUMiSizer ® 611, LUM GmbH, Germany). The samples of NE formulations were incorporated into polycarbonate cell and equilibrated to 25 °C before analyses. Measurements were carried out at 3,618 rpm for 4.5 h resulting in accelerated migration of the droplets and integration of transmission profiles within a selected region of the holding cell allowed to get information about dispersion stability.

The analyses were carried out using specific cuvettes with a radius position of 129.5 mm and based on results, it was possible to predict the shelf life of the NE samples from following equation (1) [START_REF] Goto | Application of aluminum chloride phthalocyanine-loaded solid lipid nanoparticles for photodynamic inactivation of melanoma cells[END_REF]:

Shelf life (seconds) = Measurement time x Relative Centrifugal Force (1)

Incorporation efficiency

1,8-cineole was determined using a UV spectrophotometric method described by [START_REF] Yin | Optimization and characterization of 1,8-cineole/hydroxypropyl-β-cyclodextrin inclusion complex and study of its release kinetics[END_REF], with slight modifications. Calibration curves were constructed for 1,8-cineole in n-hexane at different concentrations (w/v) and their absorbances detected at 250 nm using an Even UV/VIS spectrophotometer (Ionlab, Paraná, Brazil). Total monoterpene amount in the F1 formulation was assessed with the addition of an aliquot of the formulation in n-hexane (1:3, v/v). This mixture was put in an ultrasonic bath at 30 °C for 30 min, and then centrifuged at 4,000 rpm for 20 min. Next, the amount of 1,8-cineole was determined in the upper phase using UV spectrophotometric method as mentioned above. Free 1,8-cineole amount was determined by measuring the non-incorporated monoterpene present in a clear ultrafiltrate obtained through separation of aqueous phase using an ultrafiltration/ultracentrifugation procedure (Microcon Ultracel YM-100, Millipore, Ireland) at 10,583 x g for 60 min at 4 °C (novatecnica, Microcentrifuge NT 805, Piracicaba-SP, Brazil). The same procedure used to determine 1,8-cineole in NE formulation was applied to quantify free monoterpene fraction in the dispersion medium (aqueous phase). All analyses were performed in triplicate and the incorporation efficiency (IE %) of 1,8-cineole in F1 formulation was calculated from following equation

(2) [START_REF] Rodrigues | Preparation and characterization of nanoemulsion containing a natural naphthoquinone[END_REF]:

IE (%) = (TCin -FCin/ThCin) x 100 (2)
Where, TCin is the total 1,8-cineole amount; FCin, free 1,8-cineole amount; ThCin, theoretical 1,8-cineole amount.

Electron microscopy analysis

NE formulation was analyzed using transmission electron microscopy (TEM) technique.

Aliquots ( 100 µL) of cineole nanoemulsion (F1 formulation) were put onto copper TEM grids and negatively stained with 1 % (w/v) sodium phosphotungstate solution; the excess sample was thoroughly removed by filter paper. After that, the samples were dried at room condition before analyses. Transmission electron micrographs of the samples were taken using a Microscope HT7700 Hitachi (Fukuoka, Japan) operated at accelerating voltage of 80 KV and emission 12 µA.

Antibacterial activity assay

The minimal bactericidal concentration of free 1,8-cineole and cineole nanoemulsion (F1 formulation) were determined by microdilution techniques in Mueller-Hinton broth (MHB) [START_REF] Clsi | Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically[END_REF]. Briefly, serial twofold dilutions of free 1,8-cineole and F1

formulation were carried out in MHB using 96-well microplates. Bacterial suspension (10 8 CFU mL -1 ) cultured in TSB at 37 °C for 24 h were inoculated onto wells (200 µL final volume). Microplates were incubated at 35 °C for 24 h. Bacterial growth control (MHB+ strain + DMSO 10 %), sterility control (MHB), and positive controls with gentamicin and chlorhexidine digluconate were performed simultaneously on assays. An aliquot of 10 µL of each well was inoculated on Mueller-Hinton Agar (MHA) at 37 °C for 24 h for colonies growth. Minimal bactericidal concentration was defined as the lowest 1,8-cineole or F1 concentration that did not allow visible growth on MHA agar.

Experiments were performed three times.

Statistical analysis

All experiments were performed three times and data are expressed as average value (±SEM). Statistical analysis was performed by One-way ANOVA followed by Tukey's pairwise comparisons using the Prism GraphPad Software. The statistical significance was set at p <0.05.

Results and discussion

In this study, cineole nanoemulsions were prepared using the spontaneous emulsification method as an isothermal approach applied to volatile and hydrophobic organic compounds. In order to prepare colloidal dispersions containing 1,8-cineole, as aqueous phase was used PBS (pH 7.4) while organic phase consisted of a mixture of cineole and MTC (1:1 w/w), and different types of surfactants.

For emulsions obtained herein, there was a variation of visual appearance among the fluid nanoemulsified dispersions from slightly turbid with bluish reflection to milky opaque dispersion reflecting directly the dispersed phase formed in each colloidal formulation.

As shown in Table 2, the main properties of nanoemulsified systems were dependent on both surfactant types and organic phase composition. The types of nonionic surfactants used in the formulations demonstrated a considerable influence over droplet average diameter and size distribution of the colloidal dispersions. Hence, the smallest droplets (<115 nm) and with the narrowest size distribution (PdI <0.2 and monomodal) were observed for F1 formulation (p <0.05), which was prepared containing SML-20 and SMO as pair of nonionic surfactants. In contrast, the largest droplets with broader size distribution were noticed from dispersion prepared with SMO-20 and SMO surfactants (F3 formulation). Likewise, for those formulations containing only SML-20 or SMO-20 as a surfactant, i.e., F2 and F4 formulations, respectively, the droplet average sizes were higher than 150 nm and the droplet size distribution was bimodal with PdI values above 0.2. Moreover, when the organic phase composition was altered to contain only MCT carrier oil, the droplet average diameter and size distribution were significantly larger than other NE formulations, as can be particularly observed comparing F1 and F5

formulations (p <0.05). Similarly, the organic phase composition also influenced both droplet size e PdI of NE formulations, e.g., F5 formulation, whose organic phase consisted entirely of MCT oil presented relatively large droplets (p <0.05). This result is in accord with earlier research developed by [START_REF] Saberi | Fabrication of vitamin E-enriched nanoemulsions: Factors affecting particle size using spontaneous emulsification[END_REF] in which the increasing MCT oil concentrations resulted in larger droplets from mini-emulsions. Such influence might be explained by the significant impact of the MCT carrier oil on surfactant spontaneous curvature and solubility affecting directly formation of small droplets [START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Saberi | Stabilization of vitamin E-enriched mini-emulsions: Influence of organic and aqueous phase compositions[END_REF].

The mechanism assigned to the formation of small-sized droplets using spontaneous emulsification is directly connected to the fast displacement of surfactant(s) from organic phase into aqueous phase. Afterward, both phases come into contact; this mixture results in the rapid diffusion of surfactant(s) from organic to aqueous phase producing turbulence at oil/water interface and consequent formation of fine droplets [START_REF] Anton | The universality of low-energy nano-emulsification[END_REF][START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Saberi | Fabrication of vitamin E-enriched nanoemulsions: Factors affecting particle size using spontaneous emulsification[END_REF]. It is commonly recognized that some factors can affect the formation and stability of nanodroplets designed from spontaneous emulsification approach. Among such factors are organic phase composition, type and concentration of surfactant(s) and/or cosurfactant(s), and conditions related to emulsifying process [START_REF] Helgeson | Colloidal behavior of nanoemulsions: Interactions, structure, and rheology[END_REF][START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF].

Zeta (ζ) potential reflects surface charge of droplets and it has been related to stability of colloidal formulations. High surface charge density may imply NE's long-term physical stability due to relevant repulsive electrostatic forces among the droplets preventing rapid aggregation and flotation of dispersed phase [START_REF] Mcclements | Natural emulsifiers -Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance[END_REF][START_REF] Singh | Nanoemulsion: Concepts, development and applications in drug delivery[END_REF]. All NE formulations presented significant negative values of zeta potential (< -20 mV, Table 2) which may be related to free ionized groups from components of organic phase. It is worth mentioning that additionally electrostatic stabilization of the dispersed phase, the nanoemulsion physical stability is also a result of van der Waals, hydrophobic, and steric interactions among droplets [START_REF] Gupta | Nanoemulsions: formation, properties and applications[END_REF][START_REF] Helgeson | Colloidal behavior of nanoemulsions: Interactions, structure, and rheology[END_REF][START_REF] Mcclements | Natural emulsifiers -Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance[END_REF][START_REF] Singh | Nanoemulsion: Concepts, development and applications in drug delivery[END_REF]. formulations [START_REF] Fernandes | Ibuprofen nanocrystals developed by 2 2 factorial design experiment: A new approach for poorly water-soluble drugs[END_REF]. For each NE sample, 255 transmission profiles were recorded in intervals of 65 s, and flotation velocities of oil droplets from undiluted samples of the NE were determined at 25 °C (Table 3).

Fig. 1 shows NIR transmission profiles obtained for F1 and F5 formulations while Fig. 2 shows transmission profiles for F2, F3, and F4 formulations. F1 and F5 formulations presented similar transmission profiles (Fig. 1) with the lowest clarification process, i.e., minimal phase separation due to the dispersed phase's flotation. These results were confirmed by flotation velocity obtained for F1 and F5; for these two formulations, the flotation velocities were the lowest among all samples as well as a narrower velocity distribution was also observed with only slight speed variation of droplet migration between D10 % and D90 % as shown in Table 3. Thus, we considered that F1 and F5

were the most stable NE formulations. Moreover, this result confirms that the inclusion of 1,8-cineole in the nanoemulsified system could not alter long-term formulation stability. Meanwhile, F2, F3, and F4 formulations showed a strong clarification behavior (Fig. 2) resulting in a significant creaming separation process and a wider distribution of droplet flotation velocity when compared with F1 and F5. These findings indicate that a substantial droplet aggregation might take place during the storage period for F2, F3, and F4 formulations resulting in more fast flotation due to larger oil droplets and/or their aggregates in the colloidal dispersion. In spite of different droplet sizes between F1 and F5 formulations, the packing of surfactant molecules adsorbed could result in a shielding coating in the oil droplets preventing aggregation of the dispersed phase and consequent improvement physical stability of these formulations. Therefore, such an outcome may be attributed to the surfactant monolayer's optimum curvature formed at oil/water interface of droplets in the F1 and F5 nanoemulsions [START_REF] Komaiko | Low-energy formation of edible nanoemulsions by spontaneous emulsification: Factors influencing particle size[END_REF][START_REF] Saberi | Fabrication of vitamin E-enriched nanoemulsions: Factors affecting particle size using spontaneous emulsification[END_REF]. The main destabilization mechanisms of nanoemulsified systems are related to close contact among droplets which may result in flocculation, Ostwald ripening, coalescence, and lastly phase separation. Thus, any aggregation process of the dispersed phase may trigger such instabilities and, consequently, the coarsening of the colloidal formulations [START_REF] Gupta | Nanoemulsions: formation, properties and applications[END_REF][START_REF] Helgeson | Colloidal behavior of nanoemulsions: Interactions, structure, and rheology[END_REF][START_REF] Singh | Nanoemulsion: Concepts, development and applications in drug delivery[END_REF]. In this study, it seems reasonable to assume that the adsorbed layer of SML-20 and SMO surfactants on the oil droplets increased the steric stabilization of F1 and F5 formulations avoiding oil droplet aggregation. It is also important to note that the addition of MCT carrier oil to formulation could prevent droplets' growth caused by instability primary mechanism of nanoemulsions known as Ostwald ripening, which is characterized as the mass transfer from smaller to the larger oil droplets [START_REF] Gupta | Nanoemulsions: formation, properties and applications[END_REF][START_REF] Helgeson | Colloidal behavior of nanoemulsions: Interactions, structure, and rheology[END_REF]. Furthermore, considering that there was no signal of instability for F1 and F5 formulations as well as this analysis at 3,618 rpm for 4.5 h corresponded to a predicted shelf life of 12 months, we could consider F1 and F5 NE as stable formulations for a such period of time.

Accordingly, in view of all results described above, concerning NE formulation having the smallest oil droplets and narrow size distribution along with improved physical stability, we selected F1 formulation for further studies. Moreover, as expected the assessment of the incorporation efficiency of 1,8-cineole in NE formulation (F1) confirmed high entrapment (86.3 % ±0.87) into dispersed phase mainly due to limited water solubility of this monoterpene. Transmission electron microscopy (TEM) has been considered a successful technique in order to analyze size and shape droplets, size distribution, and agglomeration tendency of the dispersed phase in nanoemulsified systems [START_REF] Falsafi | Morphology and microstructural analysis of bioactive-loaded micro/ nanocarriers via microscopy techniques[END_REF]. TEM analyses were carried out for F1 formulation and images are depicted in Fig. 3. As can be noted from photomicrographs, throughout F1 formulation non-aggregated oil droplets with irregular spherical shapes were observed. Furthermore, oil droplet sizes viewed by TEM technique were similar to those measured by dynamic light scattering analysis. All these findings confirm the presence of nanosized oil droplets with uniform size distribution in the F1 formulation and its enhanced physical stability as previously related. The potential activity of free 1,8-cineole and cineole NE (F1 formulation) against

Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pyogenes strains was assessed using the broth microdilution method. As depicted in Table 4, the bactericidal concentrations of nanoemulsion containing 1,8-cineole were 2-fold lower than those found for free 1,8-cineole. More specifically, S. aureus and E. faecalis were more sensitive to the cineole NE when compared to free monoterpene, i.e., nanoemulsified cineole was the most effective in killing these two bacteria. Meanwhile, cineole nanoemulsion was only slightly more active against S. pyogenes in comparison with free cineole (Table 4). Both gentamicin (1.66 mg mL -1 ) and chlorhexidine digluconate (1.2 mg mL -1 ) were found to be more active than pure cineole or cineole-NE against Grampositive bacteria.

Unlike these findings herein about free and nanoemulsified cineole against Gram-positive strains, [START_REF] Harkat-Madouri | Chemical composition, antibacterial and antioxidant activities of essential oil of Eucalyptus globulus from Algeria[END_REF] found that the essential oil of Eucalyptus globulus, in which the major compound was 1,8-cineole, was more active against Gram-negative strains. Despite this, it is claimed that EO are relatively less active against Gram-negative than Gram-positive microorganisms due to the uppermost layer of lipopolysaccharide present on Gram-negative bacteria, thus limiting the diffusional process of lipophilic molecules [START_REF] Harkat-Madouri | Chemical composition, antibacterial and antioxidant activities of essential oil of Eucalyptus globulus from Algeria[END_REF]Prakash et al., 2018a). Furthermore, the enhanced antibacterial activity from nanoemulsified cineole could mainly be related to the increased surface area from ultra-small sized droplets in the nanoemulsion formulation allowing passive transport through the outer cell membrane (Prakash et al., 2018a).

Table 4.

Minimal bactericidal concentrations of free cineole and cineole-containing nanoemulsion (F1 NE formulation) against Gram-positive bacteria.

Bacterial strains

Free cineole (mg mL -1 ) Cineole-NE (mg mL -1 )

Conclusions

In summary, we have designed nanoemulsions containing 1,8-cineole using low energy emulsification, which is commonly known as spontaneous emulsification method. The most stable nanoemulsion formulation (F1) consisted mainly of very small sized droplets with monomodal size distribution, and negative zeta potential. Stability study demonstrated that F1 formulation could be considered stable with lower droplet migration velocity. In addition, high incorporation efficiency of 1,8-cineole into F1 formulation was demonstrated and also the photomicrographs from TEM analysis confirmed that F1 formulation was formed by nanosized droplets and no aggregation tendency.

Antibacterial activity of cineole nanoemulsion against S. aureus and E. faecalis was enhanced (2-fold) compared to free 1,8-cineole. Hence nanoemulsified dispersions could be considered like a relevant lipid nanocarrier applied for inclusion of hydrophobic terpenic compounds. Moreover, these results encourage further studies about antibacterial activity of nanoemulsified cineole on preservation of food, cosmetic, and pharmaceutical products.

  photometric detection enabled physical stability assessment of the NE formulations. The

Fig. 1 .

 1 Fig. 1. Normalized NIR transmission profiles obtained with cineole-containing NE (F1)

Fig. 2 .

 2 Fig. 2. Normalized NIR transmission profiles obtained with F2, F3, and F4 NE

Fig. 3 .

 3 Fig. 3. TEM photomicrographs of cineole-containing NE (F1 formulation).

Table 1 .

 1

	Composition (wt. %) of NE formulations.
	NE	SML-20	SMO-20	SMO	MCT	Cin	PBS
	F 1	6.2	-	3.8	5.0	5.0	80
	F 2	10	-	-	5.0	5.0	80
	F 3	-	7.2	2.8	5.0	5.0	80
	F 4	-	10	-	5.0	5.0	80
	F 5	6.2	-	3.8	10	-	80
	SML-20: polyoxyethylen-20 sorbitan monolaurate; SMO-20: polyoxyethylen-20 sorbitan
	monooleate; SMO: sorbitan monooleate; MCT: medium-chain triglycerides; Cin: 1,8-
	cineole; PBS: 10 mM phosphate buffer solution pH 7.4.

Table 2 .

 2

	Droplet size (hydrodynamic diameter), polydispersity index (PdI), and ζ potential of NE
	formulations (average ±SEM, n = 3).
	NE	Size (nm)	PdI	ζ potential (mV)
	F1	111.5 (±0.79) a	0.182 (±0.007) b	-36.75 (±0.33)
	F2	203.4 (±6.68)	0.303 (±0.017)	-29.43 (±0.54)
	F3	212.8 (±8.67)	0.223 (±0.004)	-38.60 (±0.17)
	F4	159.3 (±1.28)	0.240 (±0.015)	-19.75 (±0.36) c
	F5	264.5 (±1.17)	0.321 (±0.014)	-42.02 (±0.42)
	a p <0.05 compared to F2, F3, F4, and F5 formulations concerning size.

b p <0.05 compared to F2, F3, F4, and F5 formulations concerning PdI.

Table 3 .

 3

	Distribution (D) of velocity (µm s -1 ) from droplet flotation of NE formulations measured
	at 25 °C using analytical photocentrifugation.
	NE	D10 %	D50 %	D90 %	Mean	velocity
		(µm s -1 )	(µm s -1 )	(µm s -1 )	(±S.D.)
	F1	1.280	1.619	2.290	1.634 (±0.377)
	F2	1.605	89.190	260.551	4.462 (±104.703)
	F3	1.510	3.106	199.099	3.140 (±82.126)
	F4	1.820	168.326	268.696	8.540 (±94.195)
	F5	1.291	1.641	2.279	1.645 (±0.364)

Acknowledgments

This study was supported by CAPES/COFECUB agreement (#Ph-C 859-15).