Heterogeneous Photochemistry of Agrochemicals at the Leaf Surface

Aug 14, 2017 - The photoreactivity of plant activator benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), commonly named acibenzolar-S-me...
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Heterogeneous Photochemistry of Agrochemicals at the Leaf Surface: A Case Study of Plant Activator Acibenzolar‑S‑methyl M. Sleiman,* P. de Sainte Claire, and C. Richard Equipe Photochimie CNRS, UMR 6296, Institut de Chimie de Clermont-Ferrand (ICCF), 63178 Aubière, France Université Clermont Auvergne, CNRS, SIGMA Clermont, ICCF, F-63000 Clermont-Ferrand, France S Supporting Information *

ABSTRACT: The photoreactivity of plant activator benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), commonly named acibenzolar-S-methyl, was studied on the surfaces of glass, paraffinic wax films, and apple leaves. Experiments were carried out in a solar simulator using pure and formulated BTH (BION). Surface photoproducts were identified using liquid chromatography coupled with electrospray ionization and high-resolution Orbitrap mass spectrometry, while volatile photoproducts were characterized using an online thermal desorption system coupled to a gas chromatography−mass spectrometry (GC−MS) system. Pure BTH degraded quickly on wax surfaces with a half-life of 5.0 ± 0.5 h, whereas photolysis of formulated BTH was 7 times slower (t1/2 = 36 ± 14 h). On the other hand, formulated BTH was found to photolyze quickly on detached apple leaves with a half-life of 2.8 h ± 0.4 h. This drastic difference in photoreactivity was attributed to the nature and spreading of the BTH deposit, as influenced by the surfactant and surface characteristics. Abiotic stress of irradiated apple leaf was also shown to produce OH radicals which might contribute to the enhanced photodegradability. Eight surface photoproducts were identified, whereas GC−MS analyses revealed the formation of gaseous dimethyl disulfide and methanethiol. The yield of dimethyl disulfide ranged between 1.5% and 12%, and a significant fraction of dimethyl disulfide produced was found to be absorbed by the leaf. This is the first study to report on the formation of volatile chemicals and OH radicals during agrochemical photolysis on plant surfaces. The developed experimental approach can provide valuable insights into the heterogeneous photoreactivity of sprayed agrochemicals and could help improve dissipation models. KEYWORDS: benzothiadiazole, pesticides, apple, half-life, volatile, dimethyl disulfide

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INTRODUCTION With a projected increase in the world population from 7.4 billion in 2016 to 9.7 billion in 2050, the pressure to achieve high crop yields will be ever growing.1 Thus, the use of pesticides will likely continue to rise, despite concerns over environmental pollution and associated health risks. In recent years, technological advances in biochemical and molecular biological tools have improved our understanding of plant− pathogen interactions.2−4 This led to the discovery of promising alternatives, in particular a new generation of plant protection products, termed activators or synthetic elicitors, as illustrated in Table 1.5 Exogenous application of elicitors could trigger systemic acquired resistance (SAR)-like responses that mimic pathogen-induced resistance in plants. One of the most frequently used elicitors is benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), commonly named acibenzolar S-methyl, which was introduced by Syngenta.6,7 BTH is an analogue of salicylic acid that is naturally produced by plants.5 The product is commercialized in the United States as Actigard 50WG, in Europe as BION, and in other parts of the world as Blockade and Boost to control downey mildew on vegetables and to manage a range of fungal, bacterial, and viral diseases of crops such as tomato, cucumber, broccoli, tobacco, melon, apple, and pear trees.8−11 BTH and other synthetic elicitors are typically applied by foliar spray. Upon spraying on crops, the active ingredients remain at the surface of leaves as a solid deposit after water evaporation. If they absorb solar radiation, photolysis can take place and accelerate the © 2017 American Chemical Society

Table 1. Synthetic Plant Defense Elicitors, Commercialized (*) or Emerging

dissipation of active ingredients, reducing the efficiency of the treatment and increasing production costs due to the need for repeated applications. Thus, it is essential to study the photoreactivity of such chemicals under environmentally relevant conditions, as an important step to evaluate their Received: Revised: Accepted: Published: 7653

June 6, 2017 August 13, 2017 August 14, 2017 August 14, 2017 DOI: 10.1021/acs.jafc.7b02622 J. Agric. Food Chem. 2017, 65, 7653−7660

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Journal of Agricultural and Food Chemistry fate and efficacy in the field but also to provide valuable input for improving risk and impact assessment models.12,13 Although extensive data exist on photolysis of agrochemicals in water and soil media, a very limited number of studies have assessed their photoreactivity at the surface of plant leaf models such as cuticular waxes.14−18 Using model systems such as isolated cuticles or thin paraffinic wax films, it was reported that the photolysis rates of some pesticides at the surface of plant waxes can be significantly faster or slower compared with corresponding photolysis half-lives in water and soil.15,18,19 It has also been shown that some formulation agents, particularly surfactants and photosensitizing herbicides, can promote photodegradation of active ingredients when used in a mixture.16 These studies have highlighted the need for systematic and realistic assessment of the photodegradation of agrochemicals on the surface of plants. In the present work, we investigated the effect of key factors that can influence the photolysis rate of BTH, such as the formulation, the nature of the surface, and the deposit characteristics (e.g., crystalline vs amorphous, shape, and dispersal). Three surfaces were selected for this study: glass for its ease of use and hydrophilicity, paraffin wax as a surrogate for the epicuticular wax of plants, and apple leaf as one of the target plants treated with BTH and also one of the crops most treated with agrochemicals. In addition, efforts were made to fully characterize the surface and potential gaseous photoproducts by developing a small-scale chamber coupled with mass spectrometry analyses. Our main objective was to better understand the roles that the surface and formulation play in the kinetics and pathways of photodegradation. The results were validated by comparing experiments conducted on model systems using wax films with experiments conducted on detached apple leaves. Moreover, the role of reactive oxygen species (ROS) produced by abiotic stress and their contribution were assessed. Finally, the environmental relevance and implications of this heterogeneous photochemistry under realistic conditions are discussed.

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to dry overnight at room temperature in the dark before use in irradiation tests. Finally, apple leaves of equivalent surfaces (6−9 cm2) were cut just before the experiment and then immediately treated with BION solution following the same procedure. Using this procedure, the leaves remained fresh after drying and only a fraction (30−50%) of the leaf surface was covered. After drying, the surface concentration of BTH on each sample was 11 μg cm−2, corresponding to an application rate of 1100 g ha−1. This rate is higher than the recommended application rates in the field (50−200 g ha−1), but it was chosen to allow better monitoring of BTH decay and enhanced detection of BTH photoproducts. A set of experiments was also carried out at an application rate of 140 g ha−1 for microscopic measurements and also to compare the nature of the photoproducts with that of the photoproducts obtained in the experiments carried out at a high application rate (1100 g ha−1). Irradiation. After drying, treated samples were irradiated inside a Suntest CPS photosimulator (Atlas Material Testing Solutions, Élancourt, France) equipped with a xenon lamp filtered below 290 nm (irradiance within the range of 290−800 nm). The intensity of the lamp was set at 500 W m−2 to simulate the sunlight average intensity in the summer in France. To avoid overheating and potential thermal reactions, 10 °C cooled water flowed through the bottom of the sample holder to maintain the surface temperature as low as possible. The surface temperature of the samples was measured with an infrared thermometer gun and remained in the range of 25−35 °C during irradiation. Control samples were run in the Suntest using dishes wrapped with an aluminum foil for protection from light. Monitoring of BTH and Formation of Surface Photoproducts. Three dishes were taken after each selected irradiation time. A total of 2 mL of acetonitrile was then used to rinse each dish and recover the remaining BTH and its photoproducts. This same method was used with BTH and BION deposits, allowing satisfactory recovery yields of BTH prior to irradiation (93−99%) based on performing three repetitions. However, 2 mL of methanol was also used in some experiments for photoproduct identification as it was more efficient to extract specific photoproducts such as photoproduct A. Extracts were analyzed by liquid chromatography coupled with diode array detection (HPLC−DAD; Waters, Saint-Quentin-enYvelines, France) to monitor BTH decay and by ultrafast HPLC coupled with electrospray ionization and Orbitrap high-resolution mass spectrometry (UHPLC−ESI-Orbitrap-HRMS; Thermo Scientific, Waltham, MA) for photoproduct identification. HPLC−DAD separation was conducted using a reversed-phase column, MachereyNagel C18 (150 mm length, 4.6 mm i.d., 5 μm particle size), and a 60:40 acetonitrile/water mobile phase at a flow rate of 1 mL min−1. The injection volume was 10 μL, the flow cell volume was 12 μL, and the detection wavelength was 330 nm. Mass spectrometry was performed on a UHPLC Ultimate 3000 RSLC (Thermo Scientific) equipped with an electrospray ionization source and a high-resolution Orbitrap Q-Exactive. Analyses were performed in both negative and positive modes at capillary voltages of 3.2 kV (ESI+) and 3.2 kV (ESI−) with detection in the 80−1200 m/z range. UHPLC chromatographic separation was conducted using a Phenomenex (Le Peck, France) Kinetex Cl8 LC column (100 mm × 2.1 mm, 1.7 μm particle size). The binary solvent system used was composed of acetonitrile (ACN) and water acidified with 0.5‰ (v/v) formic acid. The gradient program was 5% ACN for the 7 first min, followed by a linear gradient to 99% in 7.5 min, which was kept constant until 20 min. The flow rate was set at 0.45 mL/min, and the injection volume was 5 μL. Identification of photoproducts was based on structural elucidation of mass spectra and the use of accurate mass determination obtained with the high-resolution Orbitrap. Quantification of dimethyl disulfide (DMDS) was based on calibration with dimethyl sulfide. UV−visible spectra were recorded using a Cary 3 (Varian, Palo Alto, CA) spectrophotometer. Solid-state spectra of BTH deposited on quartz plates were recorded using a DRA-CA-30I integrating sphere accessory (Varian) and a BaSO4 reflectance standard (Spectralon). Volatile Emissions. To explore the formation of volatile byproducts during irradiation, the BTH samples described above were inserted into a stainless steel batch reactor (volume 400 mL)

MATERIALS AND METHODS

Chemicals. BTH (Pestanal; 99.9%), paraffin wax (mp 70−80 °C), terephthalic acid (TA; 98%), hydroxyterephthalic acid (TAOH; 97%), and dimethyl sulfide (98%) were purchased from Sigma-Aldrich (St. Louis, MO). BION 50WG water-dispersible granular formulation containing 50% active ingredient in weight and an ionic surfactant (sodium dibutylnaphthalenesulfonate) was supplied by Syngenta (Stein, Switzerland). Methanol (99%, HPLC grade) and acetonitrile (99%, HPLC grade) were provided by Riedel de Haën (Saint-Quentin Fallavier, France). Water was purified using a Millipore Milli-Q system (Millipore αQ, Darmstadt, Germany; resistivity 18 MΩ cm, DOC < 0.1 mg L−1). All other chemicals were of the highest grade available. Deposit of BTH and BION. BTH and BION solutions were deposited using a micropipet (Eppendorf, Montesson, France) as fine droplets of 5 μL on the surface of glass Petri dishes, paraffin wax films, and freshly cut apple leaves. The pure wax films were made by adding directly 0.8 g of paraffin wax to polypropylene Petri dishes and by heating it at 90 °C to achieve film formation according to the protocol previously described.18 The BTH solution was prepared by dissolving pure BTH powder in acetonitrile at 0.14 g L−1. The BION suspension was diluted in acetonitrile to obtain a BTH final concentration of 0.29 g L−1. Deposition of the BTH solution was as follows: 144 droplets of 5 μL of a BTH solution were deposited on each glass dish and wax film of 9 cm2 total surface. When BION was used instead of pure BTH, 340 μL of BION suspension was deposited as droplets of 5 μL to obtain identical BTH total amounts per dish (100 μg). Three dishes were used as replicates for each experiment. The samples were then allowed 7654

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Journal of Agricultural and Food Chemistry equipped with a quartz window (diameter 6.4 cm). The reactor was then placed inside the solar simulator for duration ranging between 0.5 and 6 h. After each irradiation, the reactor was connected via a heated transfer line (60 °C) to a thermal desorption unit (SRA Instruments, Marcy-l’Étoile, France) equipped with a Tenax sorbent trap and coupled to an Agilent 6890 gas chromatograph and an Agilent 5973 mass spectrometry detector. The reactor’s gas phase was sampled for 3 min at a sampling rate of 100 mL min−1 into the Tenax trap that was held at 5 °C during sampling. After sampling, the trap was instantly heated to 200 °C for 30 s. Separation of desorbed volatiles was carried out using a DB-5 ms column (25 m × 0.25 mm × 0.25 μm) operated initially at 50 °C over 1 min, followed by a 20 °C min−1 ramp to reach 230 °C. Mass spectra were scanned between m/z 35 and m/z 300 with the source temperature set at 230 °C. Additional analyses of volatile products were conducted using a headspace apparatus coupled with a GC−MS system (HS-20, Shimadzu, Kyoto, Japan). This was performed by depositing 0.5 mL of BTH (10−3 M in acetonitrile) inside custom-made Quartz 20 mL vials. After drying, the HS vials were either heated at 60 °C using the HS-20 system or irradiated in the Suntest to verify if the volatile photoproducts are due to thermal or photochemical reactions. GC analyses were performed using the same column and temperature program described above. OH Radical Formation. A 100 μL volume of TA solution prepared in water buffered at pH 8.9 at 7 × 10−3 M was deposited as fine droplets of 5 μL using a manual micropipet adjustable from 2 to 20 μL (Eppendorf, Montesson, France) on the surface of an apple leaf of 4 cm2. As a control experiment, 100 μL of TA (7 × 10−3 M) was deposited on a glass Petri dish and on apple leaves. After drying, the samples were irradiated in the Suntest for 3 h. TA and its fluorescent photoproduct TAOH were recovered from the surface using 2 mL of acetonitrile. The recovery efficiency ranged between 94% and 98% based on TA. The detection of OH radicals at the surface of apple leaves was based on their reaction with TA and on the generation of TAOH. The photogenerated TAOH was measured by HPLC equipped with fluorescence detection (λexc = 330 nm and λem = 430 nm) after calibration with TAOH standard solutions. Due to the high TA concentration used, we assumed that TA traps all the OH radicals formed, and we postulated that the initial rate of •OH formation was equal to the rate of TAOH formation divided by the chemical yield of TAOH (η), which is around 0.2, as previously reported.20 Less than 0.1% of TA was oxidized into TAOH in the dark after several hours of storage, suggesting that no significant oxidation occurs without light or via potential metabolism of TA. Moreover, TAOH was shown to be stable when deposited on the leaf surface since no significant degradation occurred during storage or under light exposure for 2 h. Microscopic Analysis. Several 5 μL droplets of BTH solution and BION suspension were deposited on glass dishes, paraffin wax films, and apple leaves. After being dried overnight at room temperature, the deposits were then imaged with a Shimadzu micro hardness tester (HMV-2E) using a 40× objective lens. For each sample, an average of three deposit spots was measured. Measurement of the Contact Angle. Static contact angle measurements were performed using an Attension Theta Lite optical tensiometer (Biolin Scientific, Västra Frölunda, Sweden) associated with an imaging source camera. The contact angle was measured using the sessile drop method: a 5 μL droplet of ultrapure water was gently deposited on each surface (glass, wax, and apple leaf) using an adjustable micropipet (2−20 μL, Eppendorf, Montesson, France). Images captured to measure the angle formed between the liquid and support were treated using imageJ, an open-source Java-based software developed for the digital processing of scientific images. The contact angle is the angle, conventionally measured through the liquid, where a liquid−vapor interface meets a solid surface. It quantifies the wettability of a solid surface by a liquid via the Young equation.

Figure 1. Time course of acibenzolar-S-methyl photolysis on glass and wax surfaces in a solar simulator at an irradiance of 500 W m−2. The BTH application rate was 1100 g ha−1. BION is the commercial formulation of BTH. Error bars represent the uncertainty obtained for triplicate samples.

surfaces. As seen, the photolysis rate constant (k) can vary by up to a factor of 20 depending on the combination of solution/ surface used. This finding highlights the importance of experimental conditions in the evaluation of the surface photochemistry of agrochemicals. The half-life of BTH on glass is 1.70 ± 0.05 h compared to 5.0 ± 0.7 h on wax. On the other hand, the half-life of BION is 16 ± 4 h on glass vs 36 ± 14 h on wax. When the same surface is used, it appears that the photolysis half-life can be up to 10 times faster when using a pure BTH solution than when uisng BION. In contrast, when the same solution is used, the half-life is 2−3 times shorter on glass than on wax. Analysis of the UV spectra of pure BTH and BION dissolved in water did not reveal the presence of any UV absorber able to reduce light absorption of BTH above 290 nm (see Figure S1, Supporting Information). Furthermore, no band shift or broad broadening of the BTH spectrum was observed when BTH was deposited on glass or paraffinic wax, suggesting that BTH does not bind or interact strongly with the substrate. On the other hand, contact angle measurements revealed that a water droplet deposited on glass forms an angle of 70−80° whereas on paraffin wax the angle is in the range of 90−100°. This translates into a smaller coverage (spreading) area after water evaporation on the wax surface, leading to a higher concentration per surface and thus a larger light screening effect. Similarly, when a droplet of acetonitrile is deposited on glass, the droplet spreads better than water and forms a small contact angle in the range of 35−40°. Since the BTH solution was prepared in acetonitrile whereas BION contained water, the higher dispersal of the BTH deposit due to the smaller contact angle could be responsible for reducing the light screening effect and resulting in a more effective and faster photolysis. Hence, the composition of the solution in the case of wet deposition is a key factor that impacts the rate of photolysis. Given the moderate vapor pressure of BTH (4.6 × 10−4 Pa), it is also possible that a small volatilization could have occurred during long irradiation periods as suggested by the reported volatilization data for other agrochemicals with similar vapor

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RESULTS AND DISCUSSION Kinetics of BTH Photolysis on Model Supports. Figure 1 presents the first-order kinetic curves and corresponding rate constants (k) for BTH and BION photolysis on glass and wax 7655

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Journal of Agricultural and Food Chemistry pressures.21 Nevertheless, no significant volatilization was found during overnight drying as measured by the very high recoveries, and no BTH was detected in the gas-phase samples taken during irradiation. This can be due to the fact that BTH was irradiated in solid form and as is well-known volatilization from the solid phase (sublimation) is of lesser importance. Effect of the Surface and Adjuvants on the BTH Photodegradability. The characteristics of BTH deposits on the surface may have an impact on the photodegradability. To better understand this effect, micrographs of BTH and BION dry deposits on glass, parrafin wax, and an apple leaf were recorded, as illustrated in Figure 2. The BTH deposition

known that the physical type of formulation influences the deposit characteristics and distribution patterns on leaf surfaces.24−26 Pesticides formulated as wettable powders or granules as in the case of BION yield deposits with a greater median diameter than liquid formulations due to the low solubility and presence of particle agglomerates.27 Interestingly, the nature of the BION deposit on wax is not crystalline as in the case of BTH on wax. This can also be due to the presence of surfactant and other adjuvants such as the silica particles that are used in the formulation as flow aid and anticaking agents. The melting point also influences the physical form of an active ingredient (ai) within a dried deposit. Baker et al. have reported in an extensive study with 26 ai’s that chemicals with a melting point of greater than 200 °C formed crystalline deposits whether formulated with or without surfactant.28 Chemicals with a lower melting point between 135 and 200 °C similar to BTH (mp 135 °C) formed crystalline deposits in the absence of surfactant but amorphous deposits in the presence of surfactant. The formation of amorphous and highly dense spreading of BTH can likely explain the large increase in the half-life (36 h) of BION due to the light screening effect. Finally, the BION deposit on an apple leaf appears more uniform and well spread than that on a wax surface, which could lead to a more efficient light absorption and photodegradability, as shown in the next section. This improvement in BTH spreading can be attributed to combined effects of the leaf shape, surface roughness, and presence of stomata and trichomes which can influence the BTH mobility but cannot be mimicked in the model paraffinic wax. BTH Photolysis on Apple Leaves. To verify if BTH photolysis occurs similarly on apple leaves and paraffinic wax, dried deposits of the BION aqueous suspension on wax and leaves cut from an apple seedling (10−15 leaves) just before deposition of BION were irradiated in the stainless steel chamber using the solar simulator. To reduce leaf dehydration, a small volume of water (0.5 mL) was added underneath the unexposed side of the leaf. An identical volume of water was also deposited on a clean corner of the wax surface to reproduce the same condition. The initial relative humidity inside the chamber was around 55−60% at 21 °C as measured with a hygrometer (Testo 174H, France). Extraction of BTH residues using 2 mL of acetonitrile from both samples showed very high recovery (94% ± 5%). No significant transformation of BTH occurred on the apple leaf during the night in the absence of light, indicating that no uptake and metabolism occurred in the dark. Monitoring of BTH decay during irradiation revealed that it is more rapidly degraded on the apple leaf than on wax, with a half-life of 2.8 ± 0.4 h. About 75% of BTH was transformed within 4 h on the apple leaf, whereas only 10% of BTH disappeared on wax for the same duration. Our initial hypothesis was that uptake and metabolism in the apple leaf promoted by the irradiation could be responsible for a fraction of the observed fast BTH disappearance. LC−ESI-MS analysis of the residue revealed the presence of benzo-1,2,3-thiadiazole-5-carboxylic acid (product G), a known major metabolite of BTH, suggesting that a small fraction of BTH does indeed undergo metabolism during irradiation. Nevertheless, other factors may be involved, such as the nature and form of the deposit as discussed above but also the chemistry of the apple leaf cuticle and the possibility of formation of reactive oxygen species (ROS) induced by abiotic stress.14,29 Moreover, the microscopic view of the deposit on the apple leaf indicates clearly a good spreading of BTH, which

Figure 2. Microscopic view of the surface deposit of pure BTH (140 g ha−1) on a glass surface (A) and on a wax film (B) and of formulated BTH (BION) on a wax film (C) and on an apple leaf (D).

pattern and form were very different depending on the substrate but also the presence of formulants. On glass, BTH residues were uniformly distributed with random shapes of small-sized particles (1−2 μm) which are mostly amorphous. In contrast, BTH on paraffin wax formed crystalline deposits of needle shape (10−30 μm). The growth of the crystalline form of BTH on wax could be due to its surface roughness and hydrophobicity causing a delay in droplet evaporation and favoring the crystallization.22,23 These factors resulted also in a highly localized surface coverage of BTH, leading to a stronger light screening effect which can explain the slower photolysis of BTH on paraffin wax. However, one cannot rule out a possible higher stability of crystallized BTH that can be due to intermolecular interactions or an autodeactivation in the crystal lattice. When looking at the deposit of water-dispersible granules of BION containing BTH, the difference is even more striking with the formation of large and highly dense coverage. This behavior is not surprising given the presence of a surfactant (sodium dibutylnaphthalenesulfonate) in the formulation, identified by HRMS. In fact, surfactants are generally designed to improve the dispersing, spreading, and sticking properties of active ingredients. Besides, it is relatively well7656

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Journal of Agricultural and Food Chemistry can enhance the photodegradability, as discussed above. While these leaf physical attributes can influence the photodeagradability, it remains difficult to confirm if they can be solely responsible for the 7-fold increase in the photolysis rate. Other studies have also reported the enhancement of the photolysis of fungicides when plant wax was used as a support. As an example, on a bean leaf, more S-oxidation of carboxin proceeded as compared with that on a glass surface.30,31 Accelerated photolysis was also reported for 2,4-D (2,4dichlorophenoxyacetic acid) on Zea mays leaves.32 Moreover, photoinduced oxidation was reported for guazatine on dwarf apple trees.33 It was proposed that the induced photodegradation proceeds via reaction with the hydroxyl radical or sensitizers in the wax components. To explore if such a mechanism can be involved in our conditions, an attempt was made to measure OH radicals on the surface of an apple leaf. Using TA (7 × 10−3 M) as a molecular probe deposited as drops like BTH, we found that irradiation of an apple leaf for 3 h in the solar simulator results in the formation of (3 ± 0.4) × 10−6 M fluorescent TAOH in the final extract (3 mL). This value corresponds to a •OH produced surface concentration of (2.2 ± 0.3) × 10−9 mol cm−2. It is important to note that this value was obtained after subtraction of the signal of TAOH formed when TA was deposited on the leaf surface for 3 h in dark conditions. On the other hand, TAOH was also formed when TA was irradiated on the glass surface; however, only (0.3 ± 0.2) × 10−6 M was measured. Thus, OH radical production on the leaf surface was about 10 times higher. Moreover, our method for measuring OH radicals might underestimate the real concentration of •OH produced since only a fraction of OH radicals produced by the leaf are effectively scavenged by TA, given their short lifetime and the absence of solvent in our conditions. If we assume a TAOH yield from •OH reaction with TA of 20% as reported by Charbouillot et al. in aqueous solution, the actual OH radical cumulative production can reach (1.50 ± 0.04) × 10−5 M in solution corresponding to a surface concentration of (1.13 ± 0.03) × 10−8 mol cm−2.20 This gives (4.50 ± 0.12) × 10−8 mol of OH radicals produced on the surface of the leaf assuming a leaf surface of 4 cm2. During the same period of irradiation, (1.0 ± 0.1) × 10−7 mol of BTH in BION was phototransformed. Hence, the levels of OH radicals produced by the leaf are relatively comparable to that of converted BTH in BION. Moreover, the detection of hydroxylated photoproducts such as H and E (see Table 2) on the surface of the leaf supports a potential implication of OH radicals in the phototransformation of BTH. The mechanism by which OH radicals are produced is currently unclear and requires further investigation. One possible source could be oxidative burst as well as direct photolysis of the leaf chemical constituents, in particular phenolic compounds such as rutin and phloridzin.34,35 This is the first time that OH radicals have been detected using the terephthalic method on the surface of plant leaves. Despite the relatively high uncertainty of the method (0.3 × 10−9 mol cm−2), it can provide useful insights into the contribution of OH radicals in the degradation of deposited agrochemicals. Characterization of Surface Photoproducts. Table 2 summarizes the identified surface photoproducts obtained by high-resolution mass spectrometry (ESI-Orbitrap-HRMS) analyses at around 55−70% conversion of irradiated BTH and BION on glass, wax, and apple leaf surfaces. The same photoproducts identified on apple leaves were detected at BION application rates of 140 and 1100 g ha−1.

Table 2. Proposed Structures and Relative Abundance of BTH Surface Photoproducts, Identified by High-Resolution Orbitrap Electrospray Mass Spectrometrya

a

Gaseous dimethyl disulfide (DMDS) and methyl mercaptan were identified by gas chromatoghraphy coupled to mass spectrometry. The abundance on different supports is illustrated by “minor” and “major”; n.d. = not detected.

This indicates that the concentration of BTH did not influence the nature of the photoproducts. Given the lack of reference standards, quantification was not possible. Instead, the peak area of the pseudomolecular ion (m/z) for each product was compared between the three samples. Overall, eight major photoproducts were identified. Seven of the photoproducts have lost the N2 fragment, which indicates that N2 elimination is the primary photolytic step. This is consistent with previous studies reporting that the absorbing moiety of the BTH (1,2,3benzothiadiazole) functionality can undergo photodissociation by cleavage of the heteroatomic ring and N2 loss that yields a diradical from which a variety of photoproducts can be formed, in particular dimeric species.36,37 Photoproduct A is produced by cyclization of the diradical generated after N2 elimination and photocleavage of the C−S bond followed by oxidation of the acyl radical. The photochemical scission of thioesters is a well-known reaction as well as the oxidation of the acyl radical into a carboxylic acid.38,39 Interestingly, product A was only observed when methanol was used for extraction, suggesting that either acetonitrile is not effective for recovering A or it might be unstable in acetonitrile. Photoproduct B seems to undergo similar oxidation of the acyl group with the difference that the S atom is oxidized into sulfonic acid. The formation of this compound appears to be more pronounced on the wax surface than on the glass surface. Similarly, products C, D, and E are formed through oxidation of the S atom into sulfinic (C) 7657

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Journal of Agricultural and Food Chemistry

analysis. The same volatiles observed with the thermal desorption system were also found, and DMDS was in both cases the major volatile released. Figure 3 shows the yields of DMDS after 2 h of irradiation of BTH and BION on glass and wax surfaces as well as on apple leaves.

or sulfonic (D) acid, but these products conserve the COSCH3 group, whereas compounds E and H likely arise from ring hydroxylation of intermediate D. In contrast with product B, higher concentrations of compounds D and E were found on the glass and apple leaf surfaces than on the wax surface. These findings indicate that the surface can have an impact on the nature and distribution of the photoproducts. For example, hydroxylation of product D into product E on the glass surface can be rationalized by the presence of OH groups on glass as silanols or adsorbed molecular water and the presence of water or OH radicals produced on the leaf surface, whereas this is not possible on the wax surface, which is highly hydrophobic. Besides, the detection of photoproduct F (CH3SO3H) supports the hypothesis of C−S photocleavage as it can only be formed via oxidation of the •SCH3 radical released. Finally, it is noteworthy that many of the photoproducts recently reported in solution, in particular coupling products, are not detected here, and similarly, some of the photoproducts found on glass and wax (A−C) were not detected on the leaf surface.40 This provides an additional reason for carrying out heterogeneous photolysis experiments on plant surfaces, for a better evaluation of the agrochemical fate and risk assessment of teh byproducts. Analysis of Volatile Photoproducts. In addition to characterizing the surface composition during photolysis of BTH, we attempted to measure volatile photoproducts. Hence, we carried out irradiations of BTH and BION samples deposited on glass, wax, and apple leaf surfaces inside an airtight stainless steel small chamber. Periodic measurements of the gas-phase composition every 30 min to 1 h were performed. Two products were successfully detected in all experiments: DMDS [CH3SSCH3] predominantly and traces of methanethiol [CH3SH]. These two compounds can arise from recombination of two SCH3 radicals and H-abstraction, respectively. The detection of these compounds confirms the cleavage of the CO−SCH3 bond and the release of the SCH3 radical. This is in accordance with the observation of products B and F on the surface after irradiation and in line with our recently published work on the photolysis of BTH in solution.40 Density functional theory calculations performed at the B3LYP/6-311++G(d,p) level indicated that the electronic dissociation energy of the S−N bond is 27.9 kcal/ mol, therefore much lower than that of the C−SMe bond (69.0 kcal/mol). The release of MeSSMe is however still possible because the N2 elimination from the diradical involves a significant activation barrier (27.9 kcal/mol). This makes possible the back-reaction through the process of the ring closure reaction (the respective activation barrier is less than 1 kcal/mol in that case) with regeneration of BTH and thus increasing the probability of the C−SMe bond photocleavage occurring.40 On the contrary, the C−S homolytic dissociation leads to separated fragments (radicals). In that case, recombination is prevented. Thus, in the absence of O2, C− SMe bond dissociation is favored. This oxygen effect was confirmed experimentally where the concentration of DMDS increased by a factor of 2.8 when solid BTH was irradiated in an oxygen-purged quartz headspace vial compared with the saturated air condition. It is worth noting that the identified volatiles were only formed during the photochemical reaction and not as thermal products during thermal desorption. This was confirmed by headspace analysis of a sealed glass vial containing deposited dry residues of BTH irradiated for 2 h in the solar simulator. The vial was incubated at 60 °C for 2 min followed by GC−MS

Figure 3. Yields of gaseous dimethyl disulfide (DMDS) produced after 2 h of irradiation of pure BTH and BION on glass, a paraffin wax film, and an apple leaf, applied at the rate of 1100 g ha−1 in a solar simulator at 500 W m−2. The far right data bar (light gray) was obtained by irradiating BTH on the wax surface in the presence of two surrounding apple leaves inside the reactor.

The first observation is that the DMDS yield was about 6 times smaller on the glass surface (2%) than on wax (12%). On the other hand, the yields were identical for pure BTH and the BION formulation deposited on wax. It is unclear why the DMDS yield is smaller on glass, but one possible explanation is that the higher density and stacking of BTH molecules on the wax surface enhances the probability of recombination of two released SCH3 radicals to yield DMDS. Considering that 12% of the converted BTH yields DMDS, this is a relatively important degradation pathway. Thus, more systematic analysis of volatile products during agrochemicals photodegradation should be included in future studies for better understanding of their environmental fate on plants, soils, and other surfaces. Analysis of volatile products during photolysis of BION deposited on an apple leaf revealed that only 1.5% of BTH transformed yields DMDS, compared to 12% on a wax surface. While the difference in deposit characteristics might have played a role, we investigated if a fraction of DMDS produced was absorbed by the leaf itself. Thus, we repeated the experiment with BION deposited on wax, and we added two apple leaves surrounding the internal walls of the reactor. The measurement of DMDS showed that the yield was reduced from 12% in the absence of leaves to 3% in the presence of leaves, suggesting that plant leaves are indeed capturing DMDS. Plants are known to absorb and metabolize gaseous organic compounds such as formaldehyde and carbonyl sulfide (COS), which can enter plant leaves through the stomata.41,42 Data on DMDS uptake by plant leaves or its impact are however lacking despite its use as a soil fumigant.43,44 In addition, mass spectrometry analyses of the BION formulation before and after irradiation on the leaf revealed the formation of adducts resulting from the addition of CH3S• radical on the structures 7658

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of the surfactant used (sodium dibutylnaphthalenesulfonate), as shown in the Supporting Information, Table S1. On the basis of an application rate of 150 g ha−1 of BTH on apples and on an estimated chemical yield of DMDS of 1.5%, a maximum emission level of 2.2 g ha−1 of DMDS could be generated via BTH photolysis. Recent studies have identified DMDS as an elicitor of induced systemic resistance in Nicotiana benthamiana against Botrytis cinerea.45 Others have also found that DMDS promotes growth of plants by enhancing sulfur nutrition and shows antimicrobial and nematicidal activity.46−48 It can also help plants to fight against nonspecialist herbivore feeding and functions as both an oviposition repellent and an attractant to different natural enemies of some plants.49 Given the role of DMDS in plant signaling and growth, further studies on the effect of gaseous DMDS on plants are needed to evaluate its potential impact. These findings along with our observation imply that DMDS and potentially volatile degradation byproducts of other agrochemicals should be characterized and their impacts on plant physiology and air quality merit further attention. In summary, our study shows that a better understanding of the nature and form of agrochemical deposits is key to evaluating their photochemical reactivity on plant surfaces. The discrepancy between the half-life of BTH on paraffin wax and apple leaves suggests that paraffin wax is not an adequate model to mimic the photolysis on the apple leaf surface. A simple comparison between the irradiance of the Suntest photosimulator (46 W m−2 between 300 and 400 nm) and sunlight irradiance in central Europe in the summertime (22 W m−2) over the 24 h of a day allows us to estimate that the half-life of BTH on apple leaves would not exceed 6 h. This very short time scale demonstrates that photolysis on crops is a major dissipation process for BTH, and it raises the question of the efficiency of BTH treatment and whether photoproducts formed on the surface and in the gas phase can contribute to the observed elicitor activity. Due to experimental constraints, our validation experiments were carried out using freshly cut dead leaves and not attached live leaves. We acknowledge that the results might differ when using live leaves because other factors such as metabolism, transport, antioxidant activity, and microbe load could contribute to the observed transformation of BTH. Therefore, a systematic investigation of formulated pesticide photolysis on dead and live leaves should be performed to test the validity of the results using wax surrogates or extrapolation based on soil studies. This will also improve the assessment of the photolysis contribution to the overall pesticide dissipation from plants and will be very valuable for risk and impact assessment models. Moreover, our study demonstrates that the formation of volatile degradation products can possibly occur during agrochemicals photolysis; however, this process is overlooked. Our experimental methodology presented here can help to assess the role of heterogeneous photochemistry in the production of volatile pollutants that may be toxic. For instance, farmers could be exposed to released volatile photoproducts during and after field application of pesticides, especially in a confined atmosphere such as that in agricultural greenhouses.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02622. Absorption spectra of BTH in water and BION and their difference (Figure S1), UV spectrum of a BTH deposit on a quartz plate (Figure S2), and structures of formulants detected in BION before and after irradiation on wax and on detached leaves, identified by highresolution electrospray mass spectrometry (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +33 (0)4 73 40 76 35. ORCID

M. Sleiman: 0000-0002-2273-1053 Funding

This work was partially supported by the European Regional Development Fund and a new investigator award from the region of Auvergne-Rhône-Alpes. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Malgorzata Stawinoga, who prepared the paraffinic wax films, Dr. Loic̈ Della Puppa for the contact angle measurements, and Dr. Pascal Goupil for providing the apple seedlings and for her valuable comments and suggestions.

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REFERENCES

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