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Article
Heterogeneous Photochemistry of Agrochemicals at Leaf Surface: a Case Study of Plant Activator Acibenzolar-S-methyl Mohamad Sleiman, Pascal de Sainte Claire, and Claire Richard J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02622 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 14, 2017
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Journal of Agricultural and Food Chemistry
Heterogeneous Photochemistry of Agrochemicals at Leaf Surface: a Case Study of Plant Activator Acibenzolar-S-methyl
M. Sleiman1,2*, P. De Sainte Claire1,2, C Richard1,2
1
Equipe Photochimie CNRS, UMR 6296, ICCF, 63178 Aubière, France.
2
Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de Clermont-
Ferrand, F-63000 Clermont–Ferrand, France.
Corresponding author: M Sleiman Email:
[email protected] Tel: +33 (0)4 73 40 76 35
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Abstract.
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The photoreactivity of plant activator benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl
3
ester (BTH) commonly named acibenzolar-S-methyl was studied on surfaces of glass,
4
paraffinic wax films and apple leaves. Experiments were carried out in a solar simulator using
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pure and formulated BTH (BION®). Surface photoproducts were identified using liquid
6
chromatography coupled with electrospray ionization and high resolution Orbitrap mass
7
spectrometer, while volatile photoproducts were characterized using an online thermal
8
desorption system coupled to gas chromatography-mass spectrometry (GC-MS). Pure BTH
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degraded quickly on wax surfaces with a half-life of 5.0 ± 0.5 h, whereas photolysis of
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formulated BTH was seven times slower (t1/2 = 36 ± 14 h). On the other hand, formulated
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BTH was found to photolyze quickly on detached apple leaves with a half-life of 2.8 h ± 0.4
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h. This drastic difference in photoreactivity was attributed to the nature and spreading of BTH
13
deposit, as influenced by surfactant and surface characteristics. Abiotic stress of irradiated
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apple leaf was also shown to produce OH radicals which might contribute to the enhanced
15
photodegradability. Eight surface photoproducts were identified whereas GC-MS analyses
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revealed the formation of gaseous dimethyl disulfide and methanthiol. The yield of dimethyl
17
disulfide ranged between 1.5% and 12%, and a significant fraction of dimethyl disulfide
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produced was found to be absorbed by the leaf. This is the first study to report on formation
19
of volatile chemicals and OH radicals during agrochemical photolysis on plant surfaces. The
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developed experimental approach can provide valuable insights on the heterogeneous
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photoreactivity of sprayed agrochemicals and could help improve dissipation models.
Keywords: benzothiadiazole, pesticides, apple, half-life, volatile, dimethyldisulfide.
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Introduction
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With a projected increase in the world population from 7.4 billion in 2016 to 9.7 billion in
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2050, the pressure to achieve high crop yields will be ever growing.1 Thus, the use of
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pesticides will likely continue to rise, despite concerns over environmental pollution and
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associated health risks. In recent years, technological advances in biochemical and molecular
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biological tools have improved our understanding of plant-pathogen interactions.2-4 This led
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to the discovery of promising alternatives, in particular a new generation of plant protection
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products, termed activators or synthetic elicitors, as illustrated in Table 1.5 Exogenous
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application of elicitors could trigger systemic acquired resistance (SAR)-like responses that
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mimic pathogen-induced resistance in plants. One of the most frequently used elicitors is
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benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) commonly named as
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acibenzolar S-methyl, which was introduced by Syngenta.6-7 BTH is ananalog of salicylic acid
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that is naturally produced by plants.5 The product is commercialized in the United States as
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Actigard® 50WG, in Europe as BION®, and in other parts of the world as Blockade® and
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Boost® to control downey mildew on vegetables and to manage a range of fungal, bacterial,
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and viral diseases of crops such as tomato, cucumber, broccoli, tobacco, melon, apple and
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pear trees.8-11 BTH and other synthetic elicitors are typically applied by foliar spray. Upon
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spraying on crops, the active ingredients remain at the surface of leaves as solid deposit after
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water evaporation. If they absorb solar radiations, photolysis can take place and accelerate the
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dissipation of active ingredients, reducing the efficiency of the treatment and increasing
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production costs due to the need for repeated applications. Thus, it is essential to study the
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photoreactivty of such chemicals under environmentally relevant conditions, as an important
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step to evaluate their fate and efficacy in the field but also to provide valuable input for
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improving risk and impact assessment models. 12-13
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Although extensive data exist on photolysis of agrochemicals in water and soil media, a very
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limited number of studies have assessed their photoreactivity at the surface of plant leaf
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models such as cuticular waxes.14-18 Using model systems such as isolated cuticles or thin
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paraffinic wax films, it was reported that photolysis rates of some pesticides at the surface of
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plant waxes can be significantly faster or slower compared with corresponding photolysis
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half-lives in water and soil.15,
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particularly surfactants and photosensitizing herbicides can promote photodegradation of
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active ingredients when used in mixture.16 These studies have highlighted the need for
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systematic and realistic assessment of the photodegradation of agrochemicals on the surface
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of plants.
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In the present work, we investigated the effect of key factors that can influence the photolysis
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rate of BTH such as the formulation, the nature of the surface and the deposit characteristics
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(e.g. crystalline vs. amorphous, shape and dispersal). Three surfaces were selected for this
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study: glass for its ease of use and hydrophilicity, paraffin wax as surrogate for epicuticular
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wax of plants and apple leaf as one of the target plants treated with BTH and also one of the
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most treated crops with agrochemicals. In addition, efforts were made to fully characterize
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surface and potential gaseous photoproducts by developing a small scale chamber coupled
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with mass spectrometry analyses. Our main objective was to better understand the roles that
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surface and formulation play in the kinetic and pathways of photodegradation. The results
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were validated by comparing model systems using wax films with experiments conducted on
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detached apple leaves. Moreover, the role of reactive oxygen species (ROS) produced by
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abiotic stress and their contribution was assessed. Finally, the environmental relevance and
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implications of this heterogeneous photochemistry under realistic conditions are discussed.
18-19
It has also been shown that some formulation agents
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MATERIALS AND METHODS
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Chemicals. BTH (Pestanal, 99.9%), paraffin wax (mp 70-80°C), terephthalic acid (TA, 98%),
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hydroxyterephthalic acid (TAOH, 97%), dimethylsulfide (98%) were purchased from Sigma-
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Aldrich (St. Louis, Missouri, USA). BION® 50WG water-dispersible granular formulation
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containing 50% of active ingredient in weight and an ionic surfactant (Sodium
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dibutylnaphthalenesulphonate) was supplied by Syngenta (Stein, Switzerland). Methanol
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(99%, HPLC grade), acetonitrile (99%, HPLC grade) were provided by Riedel de Haën
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(Saint-Quentin Fallavier, France). Water was purified using a Millipore Milli-Q system
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(Millipore αQ, Darmstadt, Germany, resistivity 18 MΩ cm, DOC < 0.1 mg L-1). All other
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chemicals were of the highest grade available.
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Deposit of BTH and BION®. BTH and BION® solutions were deposited using a micropipette
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(Eppendorf®, Montesson, France) as fine droplets of 5 µL on the surface of glass petri dishes,
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paraffin wax films and freshly cut apple leaves. The pure wax films were made by adding
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directly 0.8 g of paraffin wax in polypropylene petri dishes and by heating it at 90° C to
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achieve film formation according to the protocol previously described.18 BTH solution was
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prepared by dissolving pure BTH powder in acetonitrile at 0.14 g L-1. BION® suspension was
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diluted in acetonitrile to obtain a BTH final concentration of 0.29 g L-1. Deposit of BTH
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solution was as follows: 144 droplets of 5 µl of a BTH solution were deposited on each glass
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dish and wax film of 9 cm2 total surface. When BION® was used instead of pure BTH, 340 µl
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of BION® suspension were deposited as droplets of 5 µL to obtain identical BTH total amount
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per dish (100 µg). Three dishes were used as replicates for each experiment. Samples were
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then allowed to dry overnight at room temperature in dark before use in irradiation tests.
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Finally, apple leaves of equivalent surfaces (6-9 cm2) were cut just before the experiment,
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then immediately treated with BION® solution following the same procedure. Using this
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procedure, the leaves remained fresh after drying and only a fraction (30-50%) of the leaf
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surface was covered. After drying, the surface concentration of BTH on each sample was 11
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µg cm-2 corresponding to an application rate of 1100 g ha-1. This rate is higher than the
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recommended application rates in the field (50 - 200 g ha-1) but it was chosen to allow better
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monitoring of BTH decay and enhanced detection of BTH photoproducts. A set of
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experiments was also carried out at an application rate of 140 g ha-1 for microscopic
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measurements and also to compare the nature of photoproducts with the experiments carried
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at high application rate (1100 g ha-1).
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Irradiation. After drying, treated samples were irradiated inside a Suntest CPS
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photosimulator (Atlas Material Testing Solutions, Élancourt, France) equipped with a xenon
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lamp filtered below 290 nm (irradiance within the range of 290−800 nm). The intensity of the
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lamp was set at 500 W m−2 to simulate the sunlight average intensity in summer in France. To
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avoid overheating and potential thermal reactions, a 10° C cooled water flowed through the
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bottom of the sample holder to maintain the surface temperature as low as possible. Surface
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temperature of the samples was measured with an infrared thermometer gun and remained in
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the range of 25° – 35° C during irradiation. Control samples were run in the Suntest using
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dishes wrapped with an aluminium foil for protection from light.
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Monitoring of BTH and formation of surface photoproducts. Three dishes were taken
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after each selected irradiation time. A total of 2 mL of acetonitrile was then used to rinse each
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dish and recover remaining BTH and its photoproducts. This same method was used with
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BTH and BION® deposits, allowing satisfactory recovery yields of BTH prior to irradiation
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(93-99%) based on performing three repetitions. However, 2 mL of methanol was also used in
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some experiments for photoproducts identification as it was more efficient to extract specific
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photoproducts such as photoproduct A. Extracts were analyzed by liquid chromatography
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coupled with diode array detector (HPLC-DAD, Waters, Saint-Quentin-en-Yvelines, France)
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to monitor BTH decay and by ultrafast HPLC coupled with electrospray ionization and
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Orbitrap high resolution mass spectrometer (UHPLC-ESI-Orbitrap-HRMS, Thermo
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Scientific, Waltham, Massachusetts, USA) for photoproducts identification. HPLC-DAD
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separation was conducted using a reversed phase column, Macherey-Nagel C18, (150 mm
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length, 4.6 mm i.d., 5 µm particle size) and 60:40 acetonitrile: water mobile phase, at a flow
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rate of 1 ml min-1. Injection volume was 10 µL, flow cell volume was 12 µL and detection
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wavelength was 330 nm. Mass spectrometry was performed on a UHPLC Ultimate 3000
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RSLC (Thermo Scientific, Waltham, Massachusetts, USA) equipped with electrospray
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ionisation source and a high resolution Orbitrap Q-Exactive. Analyses were performed in both
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negative and positive modes at a capillary voltage of 3.2 kV (ESI+) and 3.2 kV (ESI-) with a
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detection of 80-1200 m/z range. UHPLC chromatographic separation was conducted using a
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Phenomenex (Le Peck, France) Kinetex™ Cl8 LC column (100 mm × 2.1 mm, 1.7 µm
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particle size). The binary solvent system used was composed of acetonitrile (ACN) and water
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acidified with 0.5‰ v/v formic acid. The gradient program was 5% ACN for the 7 first min,
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followed by a linear gradient to 99% in 7.5min and kept constant until 20 min. The flow rate
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was set at 0.45 mL/min and injection volume was 5 µL. Identification of photproducts was
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based on structural elucidation of mass spectra and the use of accurate mass determination
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obtained with the Orbitrap high resolution. Quantification of dimethyldisulfide (DMDS) was
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based on calibration with dimethyl sulfide.
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UV-visible spectra were recorded using a Cary 3 (Varian, Palo Alto, California, USA)
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spectrophotometer. Solid state spectra of BTH deposited on quartz plates were recorded using
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a DRA-CA-30I integrating sphere accessory (Varian, Palo Alto, California, USA) and BaSO4
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reflectance standard (Spectralon).
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Volatile emissions. To explore the formation of volatile by-products during irradiation, BTH
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samples described above were inserted into a stainless steel batch reactor (volume: 400 mL),
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equipped with a quartz window (diameter 6.4 cm). The reactor was then placed inside the
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solar simulator for duration ranging between 0.5 and 6 hours. After each irradiation, the
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reactor was connected via a heated transfer line (60° C) to a thermal desorption unit (SRA
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instruments, Marcy-l'Étoile, France) equipped with a Tenax sorbent trap and coupled to a
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Agilent 6890 gas chromatograph and Agilent 5973 mass-spectrometry detector . The reactor's
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gas phase was sampled for 3 min at a sampling rate of 100 mL min-1 into the Tenax trap that
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was held at 5°C during sampling. After sampling, the trap was instantly heated to 200°C for
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30 seconds. Separation of desorbed volatiles was carried out using a DB-5 ms column (25
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m×0.25 mm×0.25 µm) operated initially at 50°C over 1 min, followed by a 20° C min-1 ramp
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to reach 230° C. Mass spectra were scanned between m/z 35 and 300 with a source
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temperature set at 230° C. Additional analyses of volatile products were conducted using a
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head space apparatus coupled with a GC-MS (HS-20, Shimadzu, Kyoto, Japan). This was
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performed by depositing 0.5 mL of BTH (10-3 M in acetonitrile) inside custom-made Quartz
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20-mL vials. After drying, the HS vials were either heated at 60° C using the HS-20 system or
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irradiated in the Suntest to verify if the volatile photoproducts are due to thermal or
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photochemical reactions. GC analyses were performed using the same column and
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temperature program described above.
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OH Radical Formation. 100 µL of TA solution prepared in water buffered at pH 8.9 at 7 x
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10-3 M were deposited as fine droplets of 5 µL using a manual micropipette adjustable from 2
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to 20 µL (Eppendorf®, Montesson, France) on the surface of an apple leaf of 4 cm2. As a
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control experiment, 100 µL of TA (7 10-3 M) was deposited on a glass petri dish and on apple
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leaves. After drying, samples were irradiated in the Suntest for 3h. TA and its fluorescent
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photoproduct TAOH were recovered from the surface using 2 mL of acetonitrile. Recovery
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efficiency ranged between 94 and 98% based on TA. The detection of OH radicals at the
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surface of apple leaves was based on their reaction with TA and on the generation of TAOH.
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The photogenerated TAOH was measured by HPLC equipped with fluorescence detection
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(λexc= 330 nm and λem= 430 nm) after calibration with TAOH standard solutions. Due to the
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high TA concentration used, we assumed that TA traps all the OH radicals formed and we
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postulated that the initial rate of •OH formation was equal to the rate of TAOH formation
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divided by the chemical yield of TAOH (η), which is around 0.2, as previously reported.20
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Less than 0.1% of TA was oxidized into TAOH in dark after several hours of storage
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suggesting that no significant oxidation occurs without light or via potential metabolism of
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TA. Moreover, TAOH was shown to be stable when deposited on leaf surface since no
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significant degradation occurred during storage or under light exposure for 2 hours.
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Microscopic analysis. Several 5 µL droplets of BTH solution, and BION® suspension were
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deposited on glass dishes, paraffin wax films and apple leaves. After drying overnight at room
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temperature, the deposits were then imaged with a Shimadzu micro hardness tester HMV-2E,
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using a 40x objective lens. For each sample, an average of 3 deposit spots was measured.
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Measurement of contact angle. Static contact angle measurements were performed using an
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Attension Theta Lite optical tensiometer (Biolin Scientific, Västra Frölunda, Sweden)
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associated with an imaging source camera. The contact angle was measured using the sessile
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drop method: a 5 µL droplet of ultrapure water was gently deposit on the surface of each
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surface (glass, wax and apple leaf) using an adjustable micropipette (2-20 µL, Eppendorf®,
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Montesson, France). Images captured to measure the angle formed between liquid and support
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were treated using imageJ, an open source java-based software developed for the digital
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processing of scientific image. A contact angle is the angle, conventionally measured through
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the liquid, where a liquid–vapor interface meets a solid surface. It quantifies the wettability of
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a solid surface by a liquid via the Young equation.
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RESULTS AND DISCUSSION
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Kinetics of BTH Photolysis on model supports. Figure 1 presents the first-order kinetic
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curves and corresponding rate constants (k) for BTH and BION® photolysis on glass and wax
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surfaces. As seen, the photolysis rate constant (k) can vary by up to a factor of 20 depending
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on the combination of solution/surface used. This finding highlights the importance of
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experimental conditions in the evaluation of surface photochemistry of agrochemicals. The
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half-life of BTH on glass is 1.70 ± 0.05 compared to 5.0 ± 0.7 on wax. On the other hand,
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half-life of BION® is 16 ± 4 on glass vs. 36 ± 14 on wax. When the same surface is used, it
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appears that the photolysis half-life can be up to 10 times faster when using pure BTH
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solution than when uisng BION®. In contrast, when the same solution is used, half-life is 2-3
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times shorter on glass than on wax. Analysis of UV spectra of pure BTH and BION®
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dissolved in water didn't reveal the presence of any UV absorber able to reduce light
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absorption of BTH above 290 nm (see Figure S1, supporting info). Furthermore, no band shift
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or broad broadening of BTH spectrum was observed when BTH is deposited on glass or
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paraffinic wax, suggesting that BTH does not bind or interact strongly with the substrate. On
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the other hand, contact angle measurements revealed that a water droplet deposited on glass
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forms an angle of 70°-80° whereas on paraffin wax the angle is in the range of 90°-100°. This
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translates into smaller coverage (spreading) area after water evaporation on wax surface
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leading to a higher concentration per surface and thus a larger light screening effect.
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Similarly, when a droplet of acetonitrile is deposited on glass, the droplet spreads better than
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water and forms a small contact angle in the range 35°-40°. Since BTH solution was prepared
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in acetonitrile whereas BION® contained water, the higher dispersal of BTH deposit due to
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the smaller contact angle, could be responsible for reducing the light screening effect and
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resulting in a more effective and faster photolysis. Hence, the composition of the solution in
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the case of wet deposition is a key factor that impact the rate of photolysis.
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Given the moderate vapor pressure of BTH (4.6 10-4 Pa), it is also possible that a small
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volatilization could have occurred during long irradiation periods as suggested by the reported
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volatilization data for other agrochemicals with similar vapor pressure.21 Nevertheless, no
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significant volatilization was found during overnight drying as measured by the very high
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recoveries and no BTH was detected in the gas phase samples taken during irradiation. This
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can be due to the fact that BTH was irradiated in solid form and as it is well known
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volatilization from the solid phase (sublimation) is of lesser importance.
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Effect of Surface and Adjuvants on BTH Photodegradability. The characteristics of BTH
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deposits on the surface may have an impact on the photodegradability. To better understand
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this effect, micrographs of BTH and BION® dry deposits on glass, parrafin wax and on an
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apple leaf were recorded, as illustrated in Figure 2. BTH deposition pattern and form were
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very different depending on the substrate but also the presence of formulants. On glass, BTH
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residues were uniformly distributed with random shapes of small size particles (1-2 µm)
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which are mostly amorphous. In contrast BTH on paraffin wax formed crystalline deposits of
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needle shape (10-30 µm). The growth of crystalline form of BTH on wax could be due to its
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surface roughness and hydrophobicity causing a delay in droplet evaporation and favoring the
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crystallization.22-23 These factors resulted also in a highly localized surface coverage of BTH
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leading to a stronger light screening effect which can explain the slower photolysis of BTH on
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paraffin wax. However, one cannot rule out a possible higher stability of crystallized BTH
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that can be due to intermolecular interactions or an auto-deactivation in the crystal lattice.
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When looking at the deposit of water dispersible granules of BION® containing BTH, the
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difference is even more striking with the formation of large and highly dense coverage.
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This
behavior is
not surprising given
the
presence
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dibutylnaphthalenesulphonate) in the formulation, identified by HR-MS. In fact, surfactants
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surfactant (Sodium
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are generally designed to improve the dispersing, spreading, and sticking properties of active
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ingredients. Besides, it is relatively well known that the physical type of formulation
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influences deposit characteristics and distribution patterns on leaf surfaces.24-26 Pesticides
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formulated as wettable powders or granules as in the case of BION® yield deposits with
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greater median diameter than liquid formulations due to the low solubility and presence of
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particle agglomerates.27 Interestingly, the nature of BION® deposit on wax is not crystalline
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as in the case of BTH on wax. This can also be due to the presence of surfactant and other
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adjuvants such as the silica particles that are used in the formulation as flow aid and
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anticaking agent. The melting point also influences the physical form of an active ingredient
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(a.i.) within a dried deposit. Baker et al. have reported in an extensive study with 26 a.i.’s that
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chemicals with a melting point of greater than 200°C formed crystalline deposits whether
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formulated with or without surfactant.28 Chemicals with lower melting point between 135°
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and 200°C similar to BTH (m.p. 135 °C) formed crystalline deposits in the absence of
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surfactant but amorphous deposits in the presence of surfactant. The formation of amorphous
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and highly dense spreading of BTH can likely explain the large increase in half-life (36 h) of
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BION® due to light screening effect. Finally, BION® deposit on an apple leaf appears more
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uniform and well spread than that on wax surface which could lead to a more efficient light
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absorption and photodegradability, as shown in the next section. This improvement in BTH
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spreading can be attributed to combined effects of leaf shape, surface roughness and presence
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of stomata and trichomes which can influence BTH mobility but cannot be mimicked in the
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model paraffinic wax.
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BTH photolysis on apple leaves. To verify if BTH photolysis occurs similarly on apple
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leaves and paraffinic wax, dried deposits of BION® aqueous suspension on wax and leaves
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cut from an apple seedling (10 to 15 leaves) just before depositing BION® were irradiated in
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the stainless steel chamber using the solar simulator. To reduce leaf dehydration, a small
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volume of water (0.5 mL) was added underneath the unexposed side of the leaf. Identical
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volume of water was also deposited on a clean corner of the wax surface to reproduce the
270
same condition. Initial Relative humidity inside the chamber was around 55-60% at 21° C as
271
measured with a hygrometer (Testo 174H, France). Extraction of BTH residues using 2 mL
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acetonitrile from both samples showed very high recovery (94 ± 5%). No significant
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transformation of BTH occurred on apple leaf during the night in the absence of light,
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indicating that no uptake and metabolism occurred in dark. Monitoring of BTH decay during
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irradiation revealed that it is more rapidly degraded on apple leaf than on wax, with a half-life
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of 2.8 ± 0.4 hours. About 75% of BTH was transformed within 4 hours on apple leaf whereas
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only 10% of BTH disappeared on wax for the same duration. Our initial hypothesis was that
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uptake and metabolism in the apple leaf promoted by the irradiation could be responsible for a
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fraction of the observed fast BTH disappearance. LC-ESI-MS analysis of the residue revealed
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the presence of Benzo-1,2,3-thiadiazole-5-carboxylic acid (product G), a known major
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metabolite of BTH, suggesting that a small fraction of BTH does indeed undergo metabolism
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during irradiation. Nevertheless, other factors may be involved such as the nature and form of
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deposit as discussed above but also the chemistry of apple leaf cuticle and the possibility of
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formation of reactive oxygen species (ROS) induced by abiotic stress.14,
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microscopic view of the the deposit on apple leaf indicates clearly a good spreading of BTH
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which can enhance photodegradability, as discussed above. While these leaf physical
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attributes can influence photodeagradability, it remains difficult to confirm if they can be
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solely responsible for the 7-fold increase in photolysis rate. Other studies have also reported
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enhancement of photolysis of fungicides when plant wax was used as support. As an example,
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on bean leaf, more S-oxidation of carboxin proceeded as compared with that on a glass
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surface.30-31 Accelerated photolysis was also reported for 2,4-D on Zea mays leaves.32
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Moreover, photoinduced oxidation was reported for guazatine on dwarf apple trees.33 It was
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proposed that the induced photodegradation proceeds via reaction with the hydroxyl radical or
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sensitizers in wax components. To explore if such mechanism can be involved in our
295
conditions, an attempt was made to measure OH radicals on the surface of an apple leaf.
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Using TA (7 10-3 M) as a molecular probe deposited as drops like BTH, we found that
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irradiation of an apple leaf for 3 hours in the solar simulator results in the formation of 3 ± 0.4
298
10-6 M of fluorescent TA-OH in the final extract (3mL). This value corresponds to an •OH
299
produced surface concentration of 2.2 ± 0.3 10-9 moles cm-2. It is important to note that this
300
value was obtained after subtracting the signal of TA-OH formed when TA is deposited on
301
leaf surface for 3 hours in dark conditions. On the other hand, TAOH was also formed when
302
TA was irradiated on glass surface, however only 0.3 ± 0.2 10-6 M was measured. Thus, OH
303
radical production on leaf surface was about 10 times higher. Moreover, our method for
304
measuring OH radicals might underestimate the real concentration of •OH produced since
305
only a fraction of OH radicals produced by the leaf are effectively scavenged by TA, given
306
their short life-time and the absence of solvent in our conditions. If we assume a TA-OH yield
307
from •OH reaction with TA of 20% as reported by Charbouillot et al. in aqueous solution, the
308
actual OH radical cumulative production can reach 1.50 ± 0.04 10-5 M in solution
309
corresponding to a surface concentration of 1.13 ± 0.03 10-8 moles cm-2.20 This gives 4.50 ±
310
0.12 10-8 moles of OH radicals produced on the surface of the leaf assuming a leaf surface of
311
4 cm2. During the same period of irradiation, 1.0 ± 0.1 10-7 moles of BTH in BION® was
312
phototransformed. Hence, the levels of OH radicals produced by the leaf are relatively
313
comparable to that of converted BTH in BION®. Moreover, the detection of hydroxylated
314
photoproducts such as H, and E (see Table 2) on the surface of leaf supports a potential
315
implication of OH radicals in the phototransformation of BTH. The mechanism by which OH
316
radicals are produced is currently unclear and requires further investigation. One possible
317
source could be oxidative burst as well as direct photolysis of leave chemical constituents in
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particular phenolic compounds such as rutin and phloridzin.34-35 This is the first time that OH
319
radicals are detected using the terephthalic method on the surface of plant leaves. Despite the
320
relatively high uncertainty of the method (0.3 10-9 moles cm-2), it can provide useful insights
321
on the contribution of OH radicals in the degradation of deposited agrochemicals.
322
Characterization of surface photoproducts. Table 2 summarizes the identified surface
323
photoproducts obtained by high resolution mass spectrometry (HR-ESI-Orbitrap-MS)
324
analyses at around 55-70% conversion of irradiated BTH and BION® on glass, wax and apple
325
leaf surfaces.
326 327
The same photoproducts identified on apple leaves were detected at BION® application rate
328
of 140 g ha-1 and at 1100 g ha-1. This indicates that concentration of BTH did not influence
329
the nature of photoproducts. Given the lack of reference standards, quantification was not
330
possible. Instead, the peak area of pseudo-molecular ion (m/z) for each product was compared
331
between the three samples. Overall, eight major photoproducts were identified. Seven of the
332
photorpducts have lost the N2 fragment which indicates that N2 elimination is the primary
333
photolytic step. This is consistent with previous studies reporting that the absorbing moiety of
334
BTH (1,2,3-benzothiadiazole) functionality can undergo photodissociation by cleavage of the
335
heteroatomic ring and N2 loss that yields a diradical from which a variety of photoproducts
336
can be formed, in particular dimeric species.36-37 Photoproduct A is produced by cyclization
337
of the diradical generated after N2 elimination and photocleavage of the C-S bond followed by
338
oxidation of the acyl radical. The photochemical scission of thioesters is a well-known
339
reaction as well as the oxidation of the acyl radical into a carboxylic acid. 38-39 Interestingly,
340
product A was only observed when methanol was used for extraction, suggesting that either
341
acetonitrile is not effective for recovering A or that it might be unstable in acetonitrile.
342
Photoproduct B seems to undergo similar oxidation of the acyl group with the difference that
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343
the S-atom is oxidized into sulfonic acid. The formation of this compound appears to be more
344
pronounced on wax than on glass surface. Similarly, products C, D and E, are formed through
345
oxidation of S-atom into sulfinic (C) or sulfonic acid (D) but these products conserve the
346
COSCH3 group, whereas compounds E and H likely arise from ring hydroxylation of
347
intermediate D. In contrast with product B, higher concentrations of compounds D and E were
348
found on glass and apple leaf than on wax surface. These findings indicate that the surface can
349
have an impact on the nature and distribution of the photoproducts. For example,
350
hydroxylation of product D into E on glass surface can be rationalized by the presence of OH
351
groups on glass as silanols or adsorbed molecular water and presence of water or OH radicals
352
produced on leaf surface, whereas this is not possible on wax surface which is highly
353
hydrophobic. Besides, the detection of photoproduct F (CH3SO3H) support the hypothesis of
354
C-S photocleavage as it can only be formed via oxidation of the •SCH3 radical released.
355
Finally, it is noteworthy that many of the photoproducts recently reported in solution in
356
particular coupling products are not detected here, and similarly some of the photoproducts
357
found on glass and wax (A-C) were not detected on leaf surface.40 This provides an additional
358
reason for carrying out heterogeneous photolysis experiments on plant surfaces, for a better
359
evaluation of agrochemical fate and risk assessment of by-products.
360
Analysis of volatile photoproducts. In addition to characterizing surface composition during
361
photolysis of BTH, we attempted to measure volatile photoproducts. Hence, we carried out
362
irradiations of BTH and BION® samples deposited on glass,wax and apple leaf surfaces inside
363
an air-tight stainless steel small chamber. Periodic measurements of gas phase composition
364
every 30 min to 1h was performed. Two products were successfully detected in all
365
experiments: DMDS [CH3S-SCH3] predominantly and traces of methanthiol [CH3S-H].
366
These two compounds can arise from recombination of two SCH3 radicals and H-abstraction
367
respectively. The detection of these compounds confirms the cleavage of the CO-SCH3 bond
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and the release of the SCH3 radical. This is in accordance with the observation of products B
369
and F on the surface after irradiation and in line with our recently published work on the
370
photolysis of BTH in solution.40 Density Functional Theory calculations performed at the
371
B3LYP/6–311+ +G(d,p) level indicated that the electronic dissociation energy of the S-N
372
bond is 27.9 kcal/mol, therefore much lower than that of the C-SMe bond (69.0 kcal/mol).
373
The release of Me-S-S-Me is however still possible because the N2 elimination from the
374
diradical involves a significant activation barrier (27.9 kcal/mol). This makes possible the
375
back reaction through the process of ring closure reaction (the respective activation barrier is
376
less than 1 kcal/mol in that case) with regeneration of BTH and thus increasing the probability
377
of the C-SMe bond photocleavage to occur.40 On the contrary, the C-S homolytic dissociation
378
leads to separated fragments (radicals). In that case, recombination is prevented. Thus, in the
379
absence of O2, C-SMe bond dissociation is favored. This oxygen effect was confirmed
380
experimentally where the concentration of DMDS increased by a factor of 2.8 when solid
381
BTH was irradiated in an oxygen purged quartz headspace vial compared with saturated air
382
condition.
383
It is worthy to note that the identified volatiles were only formed during the photochemical
384
reaction and not as thermal products during thermal desorption. This was confirmed by
385
headspace analysis of a sealed glass vial containing deposited dry residues of BTH irradiated
386
for 2h in the solar simulator. The vial was incubated at 60°C for 2 min followed by GC-MS
387
analysis. The same volatiles observed with the thermal desorption system were also found,
388
and DMDS was in both cases the major volatile released. Figure 3 shows the yields of
389
DMDS after 2 hours of irradiation of BTH and BION® on glass and wax surfaces as well as
390
on apple leaves.
391
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The first observation is that DMDS yield was about six times smaller on glass surface (2%)
393
than on wax (12%). On the other hand, the yield was identical for pure BTH and BION®
394
formulation deposited on wax. It is unclear why DMDS yield is smaller on glass but one
395
possible explanation is that the higher density and stacking of BTH molecules on the wax
396
surface enhances the probability of recombination of two released SCH3 radicals to yield
397
DMDS. Considering that 12% of converted BTH yield DMDS, this is a relatively important
398
degradation pathway. Thus, more systematic analysis of volatile products during
399
agrochemicals photodegradation should be included in future studies for better understanding
400
of their environmental fate on plants, soils and other surfaces.
401
Analysis of volatile products during photolysis of BION® deposited on an apple leaf revealed
402
that only 1.5% of BTH transformed yield DMDS, compared to 12% on wax surface. While
403
the difference in deposit characteristics might have played a role, we investigated if a fraction
404
of DMDS produced was absorbed by the leaf itself. Thus, we repeated the experiment with
405
BION® deposited on wax and we added 2 apple leaves surrounding the internal walls of the
406
reactor. The measurement of DMDS showed that the yield was reduced from 12% in absence
407
of leaves to 3% in presence of leaves suggesting that plant leaves are indeed capturing
408
DMDS. Plants are known to absorb and metabolize gaseous organic compounds such as
409
formaldehyde and carbonyl sulfide (COS) which can enter plant leaves through stomata.41-42
410
Data on DMDS uptake by plant leaves or its impact is however lacking despite its use as soil
411
fumigant.43-44 In addition, mass spectrometry analyses of BION® formulation before and after
412
irradiation on the leaf revealed the formation of adducts resulting from the addition of CH3S•
413
radical on structures of the surfactant used (Sodium dibutylnaphthalenesulphonate), as shown
414
in SI, Table S1. Based on an application rate of 150 g ha-1 of BTH on apples and on an
415
estimated chemical yield of DMDS of 1.5%, a maximum emission level of 2.2 g ha-1 of
416
DMDS could be generated via BTH photolysis.
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Recent studies have identified DMDS as an elicitor of induced systemic resistance in N.
418
Benthamiana against Botrytiscinerea.45 Others have also found that DMDS promotes growth
419
of plants by enhancing sulfur nutrition and shows antimicrobial and nematicidal activity.46-48
420
It can also help plants to fight against non-specialist herbivores feeding and functions as both
421
an oviposition repellent and as an attractant to different natural enemies of some plants.49
422
Giving the role of DMDS in plant signaling and growth, further studies on the effect of
423
gaseous DMDS on plants are needed to evaluate its potential impact. These findings along
424
with our observation imply that DMDS and potentially volatile degradation by-products of
425
other agrochemicals should be characterized and their impacts on plant physiology and air
426
quality merit further attention.
427
In summary, our study shows that a better understanding of the nature and form of
428
agrochemicals deposit is key to evaluate their photochemical reactivity on plant surfaces. The
429
discrepancy between half-life of BTH on paraffin wax and apple leaves suggests that paraffin
430
wax is not an adequate model to mimic the photolysis on apple leaf surface. A simple
431
comparison between irradiance of the Suntest photosimulator (46 W m-2 between 300 and
432
400 nm) and sunlight irradiance in central Europe in summertime (22 W m-2) over 24h of a
433
day, allows us to estimate that half-lifetime of BTH on apple leaves would not exceed 6h.
434
This very short time scale demonstrates that photolysis on crops is a major dissipation process
435
for BTH and it raises the question of the efficiency of BTH treatment and whether
436
photoproducts formed on surface and in gas phase can contribute to the observed elicitor
437
activity. Due to experimental constraints, our validation experiments were carried out using
438
freshly cut dead leaves and not attached live leaves. We acknowledge that the results might
439
differ when using live leaves because other factors such as metabolism, transport, antioxidant
440
activity, microbe load could contribute to the observed transformation of BTH. Therefore, a
441
systematic investigation of formulated pesticides photolysis on dead and live leaves should be
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442
performed to test the validity of results using wax surrogates or extrapolation based on soil
443
studies. This will also improve the assessment of photolysis contribution to overall pesticide
444
dissipation from plants and will be very valuable for risk and impact assessment models.
445
Moreover, our study demonstrates that formation of volatile degradation products can
446
possibly occur during agrochemicals photolysis, however this process is overlooked. Our
447
experimental methodology presented here can help to assess the role of heterogeneous
448
photochemistry in the production of volatile pollutants that may be toxic. For instance,
449
farmers could be exposed to released volatile photoproducts during and after field application
450
of pesticides, especially in confined atmosphere like in agricultural greenhouses.
451
AKNOWLEDGMENTS. This work was partially supported by the European Regional
452
Development Fund and a new investigator award from the regional of Auvergne-Rhône-
453
Alpes. The authors gratefully acknowledge Malgorzata Stawinoga, who prepared the
454
paraffinic wax films, Dr. Loïc Della Puppa for the contact angle measurements and Dr. Pascal
455
Goupil for providing the apple seedlings and for her valuable comments and suggestions.
456 457 458 459
ASSOCIATED CONTENT Supporting information Absorption spectrum of BTH in water, BION® and their difference (Figure S1), UV spectrum
460
of BTH deposit on a quartz plate (Figure S2), and structures of formulants detected in BION®
461
before and after irradiation on wax and on detached leave, identified by high resolution
462
electrospray mass spectrometry (Table S1).
463
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464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
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References 1. United Nations 2017 Revision of World Population Prospects. https://esa.un.org/unpd/wpp/ (accessed August 03, 2017). 2. Barakate, A.; Stephens, J., An Overview of CRISPR-Based Tools and Their Improvements: New Opportunities in Understanding Plant-Pathogen Interactions for Better Crop Protection. Front. Plant Sci. 2016, 7. 3. Rojas, C. M.; Senthil-Kumar, M.; Tzin, V.; Mysore, K. S., Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense. Front. Plant Sci. 2014, 5. 4. Welling, L. L., Induced resistance: from the basic to the applied. Trends Plant Sci. 2001, 6, 445-7. 5. Bektas, Y.; Eulgem, T., Synthetic plant defense elicitors. Front. Plant Sci. 2015, 5. 6. Cole, D. L., The efficacy of acibenzolar-S-methyl, an inducer of systemic acquired resistance, against bacterial and fungal diseases of tobacco. Crop Prot. 1999, 18, 267-273. 7. Friedrich, L.; Lawton, K.; Ruess, W.; Masner, P.; Specker, N.; Rella, M. G.; Meier, B.; Dincher, S.; Staub, T.; Uknes, S.; Metraux, J. P.; Kessmann, H.; Ryals, J., A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant J. 1996, 10, 61-70. 8. Jiang, S.; Park, P.; Ishii, H., Ultrastructural study on acibenzolar-S-methyl-induced scab resistance in epidermal pectin layers of Japanese pear leaves. Phytopathology 2008, 98 (5), 585-91. 9. Pajot, E.; Silue, D., Evidence that DL-3-aminobutyric acid and acibenzolar-S-methyl induce resistance against bacterial head rot disease of broccoli. Pest Manag. Sci. 2005, 61, 1110-4. 10. Scarponi, L.; Buonaurio, R.; Martinetti, L., Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato. Pest Manag. Sci. 2001, 57, 262-8. 11. Zavareh, A. H.; Tehrani, A. S.; Mohammadi, M., Effects of Acibenzolar-S-methyl on the specific activities of peroxidase, chitinase and phenylalanine ammonia-lyase and phenolic content of host leaves in cucumber-powdery mildew interaction. Commun. Agric. Appl. Biol. Sci. 2004, 69, 555-63. 12. Fantke, P.; Gillespie, B. W.; Juraske, R.; Jolliet, O., Estimating half-lives for pesticide dissipation from plants. Environ. Sci. Technol. 2014, 48, 8588-602. 13. Jacobsen, R. E.; Fantke, P.; Trapp, S., Analysing half-lives for pesticide dissipation in plants. SAR QSAR Environ. Res. 2015, 26, 325-42. 14. Angioni, A.; Cabizza, M.; Cabras, M.; Melis, M.; Tuberoso, C.; Cabras, P., Effect of the epicuticular waxes of fruits and vegetables on the photodegradation of rotenone. J. Agric. Food Chem. 2004, 52, 3451-5. 15. Monadjemi, S.; El Roz, M.; Richard, C.; Ter Halle, A., Photoreduction of Chlorothalonil Fungicide on Plant Leaf Models. Environ. Sci. Technol. 2011, 45, 9582-9589. 16. Monadjemi, S.; ter Halle, A.; Richard, C., Accelerated dissipation of the herbicide cycloxydim on wax films in the presence of the fungicide chlorothalonil and under the action of solar light. J. Agric. Food Chem. 2014, 62, 4846-51. 17. Sinderhauf, K.; Schwack, W., Photodegradation chemistry of the insecticide phosmet in lipid models and in the presence of wool wax, employing a 15N-labeled compound. J. Agric. Food Chem. 2004, 52, 8046-52. 18. Ter Halle, A.; Drncova, D.; Richard, C., Phototransformation of the herbicide sulcotrione on maize cuticular wax. Environ. Sci. Technol. 2006, 40, 2989-95.
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19. Lavieille, D.; Ter Halle, A.; Bussiere, P. O.; Richard, C., Effect of a Spreading Adjuvant on Mesotrione Photolysis on Wax Films. J. Agric. Food Chem. 2009, 57, 96249628. 20. Charbouillot, T.; Brigante, M.; Mailhot, G.; Maddigapu, P. R.; Minero, C.; Vione, D., Performance and selectivity of the terephthalic acid probe for (OH)-O-center dot as a function of temperature, pH and composition of atmospherically relevant aqueous media. J. Photochem. Photobiol., A 2011, 222, 70-76. 21. European Commission, health & consumer protection directorate-general, pesticides in air: considerations for exposure asssessment. http://esdac.jrc.ec.europa.eu/public_path/projects_data/focus/air/docs/FOCUS_AIR_GROUP _REPORT-FINAL.pdf (accessed on August 07, 2017). 22. Bukovac, M. J.; Whitmoyer, R. E.; Reichard, D. R., Spray Droplet - Leaf Surface Interaction - Droplet Drying Characteristics and Nature of Growth-Regulator Deposits as Revealed by Dispersive-X-Ray Analysis. Hortscience 1984, 19, 577-577. 23. Krämer, T., Deposit characteristics, penetration and biological efficacy of selected agrochemicals as affected by surfactants and plant micromorphology. Cuvillier Verlag: Göttingen, Germany, 2009. 24. Lownds, N. K.; Bukovac, M. J., Foliar Penetration of Growth-Regulators from Droplets - Physical Parameters and Their Modification by Surfactants. Hortscience 1982, 17, 496-496. 25. Stevens, P. J. G.; Baker, E. A., Factors Affecting the Foliar Absorption and Redistribution of Pesticides .1. Properties of Leaf Surfaces and Their Interactions with Spray Droplets. Pestic. Sci. 1987, 19, 265-281. 26. Stevens, P. J. G.; Baker, E. A.; Anderson, N. H., Factors Affecting the Foliar Absorption and Redistribution of Pesticides .2. Physicochemical Properties of the Active Ingredient and the Role of Surfactant. Pestic. Sci. 1988, 24, 31-53. 27. Kudsk, P.; Mathiassen, S. K.; Kirknel, E., Influence of Formulations and Adjuvants on the Rainfastness of Maneb and Mancozeb on Pea and Potato. Pestic. Sci. 1991, 33, 57-71. 28. Baker, E. A.; Hayes, A. L.; Butler, R. C., Physicochemical Properties of Agrochemicals - Their Effects on Foliar Penetration. Pestic. Sci. 1992, 34, 167-182. 29. Katagi, T., Photodegradation of pesticides on plant and soil surfaces. Rev. Environ. Contam. Toxicol. 2004, 182, 1-189. 30. Buchenauer, H., Inactivation of Triforine by Uv and Sunlight on Glass and on Leaves of Bean-Plants. Pestic. Sci. 1975, 6, 553-559. 31. Buchenauer, H., Differences in Light Stability of Some Carboxylic-Acid Anilide Fungicides in Relation to Their Applicability for Seed and Foliar Treatment. Pestic. Sci. 1975, 6, 525-535. 32. Venkatesh, R.; Harrison, S. K., Photolytic degradation of 2,4-D on Zea mays leaves. Weed Sci. 1999, 47, 262-269. 33. Sato, K.; Kato, Y.; Maki, S.; Matano, O.; Goto, S., Penetration, Translocation and Metabolism of Fungicide Guazatine in Dwarf Apple-Trees. J. Pestic. Sci. 1985, 10, 81-90. 34. Bhattacharjee, S., Reactive oxygen species and oxidative burst: Roles in stress, senescence and signal transduction in plants. Curr. Sci. 2005, 89, 1113-1121. 35. Shao, X.; Bai, N. S.; He, K.; Ho, C. T.; Yang, C. S.; Sang, S. M., Apple Polyphenols, Phloretin and Phloridzin: New Trapping Agents of Reactive Dicarbonyl Species. Chem. Res. Toxicol. 2008, 21, 2042-2050. 36. European Food Safety Authority., Conclusion on the peer review of the pesticide risk assessment of the active substance acibenzolar-S-methyl. EFSA J. 2014, 12(8), 3691-3764. 37. Krantz, A.; Laureni, J., Matrix Photolysis of 1,2,3-Thiadiazole - on Possible Involvement of Thiirene. J. Am. Chem. Soc. 1974, 96, 6768-6770.
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38. Chatgilialoglu, C.; Crich, D.; Komatsu, M.; Ryu, I., Chemistry of acyl radicals. Chem. Rev. 1999, 99, 1991-2069. 39. Grunwell, J. R.; Marron, N. A.; Hanhan, S. I., Photochemistry of Aromatic Thiol Esters. J. Org. Chem. 1973, 38, 1559-1562. 40. Sleiman, M.; Stawinoga, M.; Wang, S.; de Sainte-Claire, P.; Goupil, P.; Richard, C., Photochemical transformation of the plant activator Acibenzolar-S-methyl in solution. J. Photochem. Photobiol., A 2017, 333, 79-86. 41. ProtoschillKrebs, G.; Wilhelm, C.; Kesselmeier, J., Consumption of carbonyl sulphide (COS) by higher plant carbonic anhydrase (CA). Atmos. Environ. 1996, 30, 3151-3156. 42. Stimler, K.; Nelson, D.; Yakir, D., High precision measurements of atmospheric concentrations and plant exchange rates of carbonyl sulfide using mid-IR quantum cascade laser. Glob. Change Biol. 2010, 16, 2496-2503. 43. Le Bechec, M.; Costarramone, N.; Fouillet, T.; Charles, P.; Pigot, T.; Begue, D.; Lacombe, S., Photocatalytic films for soil fumigation: Control of dimethyl disulfide concentration after fumigation. Appl. Catal., B 2015, 178, 192-200. 44. McAvoy, T.; Freeman, J. H.; Reiter, M., Soil Persistence of Dimethyl Disulfide Fumigant due to Application Rate, Chemical Formulation, and Plastic Mulch Type. Hortscience 2010, 45, 502-503. 45. Huang, C. J.; Tsay, J. F.; Chang, S. Y.; Yang, H. P.; Wu, W. S.; Chen, C. Y., Dimethyl disulfide is an induced systemic resistance elicitor produced by Bacillus cereus C1L. Pest Manag. Sci. 2012, 68, 1306-1310. 46. Dandurishvili, N.; Toklikishvili, N.; Ovadis, M.; Eliashvili, P.; Giorgobiani, N.; Keshelava, R.; Tediashvili, M.; Vainstein, A.; Khmel, I.; Szegedi, E.; Chernin, L., Broadrange antagonistic rhizobacteria Pseudomonas fluorescens and Serratia plymuthica suppress Agrobacterium crown gall tumours on tomato plants. J. Appl. Microbiol. 2011, 110, 341-352. 47. Kai, M.; Haustein, M.; Molina, F.; Petri, A.; Scholz, B.; Piechulla, B., Bacterial volatiles and their action potential. Appl. Microbiol. Biotechnol. 2009, 81 (6), 1001-1012. 48. Meldau, D. G.; Meldau, S.; Hoang, L. H.; Underberg, S.; Wunsche, H.; Baldwin, I. T., Dimethyl Disulfide Produced by the Naturally Associated Bacterium Bacillus sp B55 Promotes Nicotiana attenuata Growth by Enhancing Sulfur Nutrition. Plant Cell 2013, 25, 2731-2747. 49. Ferry, A.; Le Tron, S.; Dugravot, S.; Cortesero, A. M., Field evaluation of the combined deterrent and attractive effects of dimethyl disulfide on Delia radicum and its natural enemies. Biol. Control 2009, 49, 219-226.
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Figure Captions 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. BTH application rate was 1100 g ha-1. BION® is the commercial formulation of BTH. Error bars represent uncertainty obtained for triplicate samples. Figure 2. Microscopic view of surface deposit of pure BTH (140 g ha-1) on glass surface (A), on wax film (B), and of formulated BTH (BION®) on wax film (C) and on an apple leaf (D Figure 3. Yields of gaseous dimethyl disulfide (DMDS) produced after 2h of irradiation of pure BTH and BION® on glass, paraffin wax film and 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 grey) was obtained by irradiating BTH on wax surface in the presence of two surrounding apple leaves inside the reactor.
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Figure 1
1 0.9
k = 0.40 ± 0.01 R² = 0.989
0.8
h-1 k = 0.14 ± 0.02 h-1 R² = 0.988
BTH/glass BION®/glass BTH/wax BION®/wax
Ln [BTH]0/[BTH]
0.7 k = 0.044 ± 0.010 h-1 R² = 0.989
0.6 0.5
k = 0.019 ± 0.008 h-1 R² = 0.976
0.4 0.3 0.2 0.1 0 0
2
4
6
8 10 12 14 16 18 20 22 24 Irradiation time (hours)
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Figure 2
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Figure 3
16
DMDS yield (%)
14 12 10 8 6 4 2 0
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Table 1: Synthetic plant defense elicitors, commercialized (*) or emerging Tiadinil Benzo(1,2,3)thi N N H adiazole-7N N N carbothioacid SS S methyl ester O BTH *
O
SCH3
2,6-dichloroisonicotinic acid
Cl
N
Cl
Isotianil*
N
Cl
Cl
Cl
H N
CO2H 3,5dichoroanthra nilic acid
Cl
N S O
Cl
Sulfamethoxazole
NH2 CO2H
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S
O
N H NH2
28
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Table 2. Proposed structures and relative abundance of BTH surface photoproducts, identified by high resolution Orbitrap electrospray mass spectrometry. Gaseous dimethyldisulfide (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.
Structure
m/z in negative mode
glass
Paraffin wax
Apple leaf
S
A
150.9852
major
major
n.d.
minor
minor
n.d.
minor
minor
n.d.
230.9795
major
minor
major
246.9742
minor
minor
major
94.9721
major
major
n.d.
178.9911
n.d.
n.d.
minor
262.9692
n.d.
n.d.
minor
CO2H
200.9857
B
SO3H CO2H
214.9812
C
SO2H COSCH3
D
SO 3H COSCH3
HO
E
SO3H COSCH3
F
CH3SO3H N N
G
S CO2H OH
H
HO SO 3H COSCH3
I
CH3S-SCH3
94 (100), 79
minor
major
minor
J
CH3SH
48
minor
minor
minor
ACS Paragon Plus Environment
29
Journal of Agricultural and Food Chemistry
Page 30 of 30
For Table of Contents Only
TOC graphic
ACS Paragon Plus Environment
30
