Preface Fundamental understanding of the uptake, translocation, and distribution of agrochemicals is of great interest among scientists in industry and academia, because biological activities of pesticides against their target species can be significantlyinfluencedbythebiokineticsofthepesticides. Biologicalactivityof pesticides is initially identified during the courses of in vitro bioassays, but the activemoleculesoftenlosetheirbiologicalactivityingreenhousetests. Thelack oftranslationofactivitybetweeninvitroassaysandgreenhousetestsisgenerally associated with many factors, including poor retention on plant surface, lack of foliar or root uptake, and limited systemicity within plants. Therefore, a clear understanding of the factors that govern the effectiveness of pesticides is key to overcome certain barriers for the expression of biological activity, and this can leadtoastrategytoimprovebiologicalperformance. This ACS symposium book is based on a symposium that was held at the 246thAmericanChemicalSocietyNationalMeeting&ExpositioninIndianapolis, Indiana from September 8-12, 2013. Although uptake, translocation, and distribution of agrochemicals in plants have been extensively studied over the years, there are still many unanswered questions that need to be addressed. This book aims to update current knowledge with new studies that contain new findingsontheuptake,translocation,anddistributionofagrochemicalsinplants as well as provide review-style chapters that summarize existing information on specificsubjects. It is hoped that this book will serve as a valuable resource for researchers who study uptake, translocation, and distribution of pesticides in plants. As researchers involved in discovery and development of agrochemicals want to understand a broad range of biological factors, it is also hoped that this book promotes researchers in other scientific disciplines to generate new ideas and technologies in the process of new product development. Knowledge of the biokineticswillhelpusfurtherunderstandtheuseofagrochemicalsonourplanet. We thank the presenters and authors for their invaluable contributions to the symposium and this book. We gratefully acknowledge ACS division of agrochemicalsandDowAgroSciencesforfinancialassistanceforthesymposium. We would like to express our sincere thanks to many other colleagues who reviewed chapters for their timely and critical assessment. We are also thankful forexcellentsupportsofstaffmembersinACSBooksDivision. ix In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; KyungMyung CropProtectionBiology,DiscoveryResearch,DowAgroSciences 9330ZionsvilleRoad,Indianapolis,IN46268,USA 317-337-7104(telephone) 317-337-3205(fax) [email protected](e-mail) NorbertM.Satchivi CropProtectionBiology,DiscoveryResearch,DowAgroSciences 9330ZionsvilleRoad,Indianapolis,IN46268,USA 317-337-4128(telephone) 317-337-3265(fax) [email protected](e-mail) ColeenK.Kingston DuPontCropProtection ElktonRoad,Newark,DE19714,USA 302-451-3335(telephone) 302-451-3570(fax) [email protected](e-mail) x In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; Editors’ Biographies Kyung Myung KyungMyungreceivedhisB.S.andM.S.degreesinHorticulturefromKyung HeeUniversity,SouthKoreaandPennsylvaniaStateUniversity,respectively,and his Ph.D. degree in Plant Physiology from University of Kentucky. Since then hehasworkedforNorthernIllinoisUniversityandUSDA-ARSandthenjoined DiscoveryResearchDepartmentatDowAgroSciences. Heiscurrentlyengaged in research in biokinetics of pesticides in biological matrices. He has published over20researcharticlesinthefieldofagriculturalbiologyandchemistry. Norbert M. Satchivi Norbert M. Satchivi obtained his Bachelor degree in Plant Physiology from the Université d’Abidjan (Cote-d’Ivoire) and his Doctorate from the Université de Perpignan in France (Agrochemistry). He was a Postdoctoral Scientist at the UniversityofIllinoisatUrbana-Champaignwhereheworkedonthedevelopment ofcomputersimulationmodelsforthemovementofxenobioticsinplant. Healso worked at the University of Guelph (Ontario, Canada) as a Research Scientist focusing on the mechanisms of herbicide antagonism. He is currently a Senior Research Scientist with Dow AgroSciences with a focus in herbicide discovery. Publications included the areas of herbicide characterization, xenobiotic uptake andtransport,andmodelingxenobioticmovement. Coleen K. Kingston Coleen K. Kingston obtained her Bachelor degree from the University of Wisconsin (Biochemistry, Math) and her Doctorate from Purdue University in Indiana (Analytical Chemistry). Subsequent posts have included research and development scientific positions within the pharmaceutical and agrochemical industries. SheiscurrentlyaseniorscientistwithDuPontCropProtectionwitha specialty in crop residue human safety assessment. Publications over the years haveincludedtheareasofpharmaceuticalanalysismethoddevelopmentinhuman matrices and the prediction of residues in food and feedstuffs yielded following foliarandsystemicdeliveryapplicationofcropprotectionchemicals. ©2014AmericanChemicalSociety In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; Chapter 1 Spray Retention of Crop Protection Agrochemicals on the Plant Surface ChenglinYao,*KyungMyung,NickWang,andAlexandriaJohnson DowAgroSciences,9330ZionsvilleRoad,Indianapolis,Indiana46268 *E-mail: [email protected]. Retention efficiency of crop protection products, one of the importantattributesfordeliveringbiologicalefficacy,ismainly determined by the physicochemical properties of the spray formulations and the surface characteristics of target plants. In this chapter, effects of plant leaf surface characteristics, developmental stages and canopy density on leaf wettability and spray retention are reviewed. Factors modifying the physicochemical properties of spray liquids, including active ingredients, formulation types, and a variety of adjuvants are also discussed. Using a quantitative structure-activity relationship (QSAR) analysis, we have developed a model based on four calculated physicochemical properties of a training set of 17 fungicides to predict compound retention rates on wheat seedling plants. The model was validated by a strongcorrelationbetweenexperimentallydeterminedretention ratesandpredictedvaluesofasmalltestset,whichincludedsix additional fungicide compounds. Retention efficiency of three epoxiconazoleformulationswasalsoevaluated,andsignificant differences in retention were observed for the products Ignite (8 % EC, w/w), Opus (12.1% SC, w/w), and a generic lab SC (10%w/w). ©2014AmericanChemicalSociety In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; Introduction Highefficiencysprayretentionontheplantsurfaceisrequiredformaximizing activitydeliveryofcropprotectionagrochemicalsbecauseitincreasestheamount ofactiveingredientpotentiallyavailableforreachingthebiologicalsiteofaction. Retention is the overall capture of spray droplets by a plant and determines the amountofactiveingredientonaplant. Itisdependentonthecomplexinterfacial interaction of spray droplets and the plant surface. The factors considered to be important for spray liquid adhesion and retention include: 1) physicochemical properties of the spray solutions; 2) diameter spectra and impaction velocity of spray droplets; and 3) characteristics of plant surface, shape and orientation of the target leaves and density of plant canopy. While the impact of adjuvants on theinteractionofsprayliquidsandleafsurfacehasbeenextensivelystudiedand reviewed(1–4),specificallytheeffectofmajoradditivesinthesprayformulations such as solvents, surfactants, oil and polymer adjuvants, attention to the active ingredients has not been the focus for the majority of the studies. For plants, wettabilityoftheleafsurfaceistypicallygovernedbysurfaceroughnesscaused bydifferentmicrostructures(trichomes,cuticularfoldsandwaxcrystals),together with the hydrophobic properties of the epicuticular wax. Hydrophobicity of epicuticular wax and the microstructures can efficiently reduce the deposition andretentionofspraydropletsbyincreasingcontactanglesandreducingcontact areawithplantleafsurface(4,5). Greenhousegrownseedlingsandyoungplants havebeenthemajortargetsinearlierstudies. Surfacepropertiesandplantcanopy structures change dramatically as plants grow, and ultimately affect the quantity ofspraydepositedandredistributedovertheentireplantcanopy(6,7). In this paper, we present the results of our investigations on the effect of the physicochemical properties of fungicides on retention by seedling wheat plants,andthedevelopmentandvalidationofaQSARmodeltopredictfungicide retention. The impact of three epoxiconazole formulations on retention and the relationship to fungicidal activity were also investigated. The relationship betweenretentionofspraydropletsandthewettingcharacteristicsoftargetplants and their canopy structure, as well as the physicochemical properties of spray liquids,arereviewedbasedonthepublishedliterature. Effect of Plant Surface Characteristics and Canopy Structures on Spray Retention ThePlantCuticleandLeafWettability Allaerialsurfacesofterrestrialplantsarecoveredbyacuticle,whichserves astheinterfaceofplantstotheirabovegroundenvironment. Theprimaryfunction ofthecuticleistopreventwaterlossfromplantssothatphysiologicalprocesses can proceed under water-limiting conditions. In addition, the cuticle acts as an effectivebarriertotheentryofxenobioticsandmicroorganismsintotheplants. Theplantcuticleconsistsoftwomajorcomponents: cutinandwaxes. Cutin isapolymercomplexconsistingofmanylong-chainfattyacidsthatareattachedto oneanotherbyesterlinkages,whichcreatearigidthree-dimensionalnetwork(8). 2 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; Cutinishydrophobic,butcontainssomehydrophilicmoietiesattachedtothechain, suchashydroxylorepoxidegroups. Waxesarehydrophobiccomplexmixturesof long-chainacyllipids. Themostcommoncomponentsofwaxesarestraight-chain alkanes and alcohols of 25 to 35 carbon atoms. Long-chain aldehydes, ketones, esters,andfreefattyacidsarealsofoundinwaxes. Thewaxesofthecuticleare synthesizedbyepidermalcells,whichareembeddedinthecutinpolymermatrix (intracuticular)andalsodepositedonthesurfaceofcuticularlayer(epicuticular). The epicuticular waxes often crystallize in an intricate pattern of rods, tubes, or plates (9–11). Table1. LeafSurfaceCharacteristicsofSelectedPlantSpecies Common name Latinname Wettability Surfacemorphology Micropapillae Super superimposedbywaxy Rice Oryzasativa hydrophobic nanobumps Crystalline plates, Wheat Triticumaestivum Difficult trichomes Crystaline plates, Barley Hordeumvulgare Difficult trichomes Corn Zeamays Difficult Crystallineplates Crystallinetubes,plates Oilseedrape Brassicanapus Difficult anddendrites Dense arrangement of Pea Pisiumsativum Difficult crystallineplates Dense arrangement of Whiteclover Trifoliumrepens Difficult crystallineplates Thickfilmoverlaidbyfine Strawberry Fragariaananassa Difficult waxribbons Soybean Glycinemax Difficult Crystalline,trichomes Thinfilm;occasionalwax Sugarbeet Betavulgaris Easy mounds Extremely thin film, Drybean Phaseolusvulgaris Easy trichomes Curlydock Rumexcrispus Easy Smooth Apple Malusdomestica Easy Smooth Cucumber Cucumissativus Easy Thinfilm,trichomes Leaf surface characteristics are critical factors affecting leaf wettability and retention of spray droplets (4, 12–14). Leaf surface wetting is dependent on microroughness of epicuticular wax crystals (5, 9, 15–17). Table 1 lists the leaf 3 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; surfacecharacteristicsofselectedplantspeciesoftenusedinsprayretentionand deposition studies of crop protection products. Generally, smooth leaf surfaces withoutcrystallineepicuticularwaxesorhydrophobictrichomesareeasy-to-wet, withawatercontactanglelessthan110degrees(15). Leafsurfacescoveredwith crystalline epicuticular waxes are difficult-to-wet and give a contact angle with water droplets greater than 110 degrees (15). An easy to use and quick way to determineleafsurfacewettabilityistopipettea2-μLwaterdropletontotheplant surface; plants with easy-to-wet surfaces are very receptive to the placement of thewaterdroplet,butplantswithmicroscopicallyroughsurfacesmakeplacement ofthewaterdropletextremelydifficult(Yao,unpublisheddata). Figure1. Retentionofherbicidemetsulfuronbydifficulty-to-wetpeaand easy-to-wetcurlydockplants,withorwithouttheadditionofanadjuvant. Data fromNichollsetal. (18). Intracksprayapplications,Nichollsetal. (18)(Figure1)observedthateasy- to-wetcurlydockretainedtheherbicidemetsulfuronmethylveryefficiently,and inclusion of three adjuvants in the spray solutions showed little effect on spray formulationretention. Incontrast,thesamethreeadjuvants,BS1000inparticular, improved the herbicide deposition significantly on the difficult-to-wet field pea plants. Manyinvestigationshaveobservedthesamegeneraltrend: onplantswith smooth, easy-to-wet leaf surfaces, retention is high and not greatly affected by spray application and solution properties (19–21). For monocotyledonous plant leaves with crystalline vertical wax plates such as barley and maize, 95% of the outmostleafsurfaceconsistedofairbecausetheexposededgesofwaxplatesonly representedapproximately5%oftheleafsurface(5). Thepresenceofairatthe interfacebetweenthedropletandthehydrophobiccrystallineepicuticularwaxon theleafsurfacecauseslargecontactanglesandpoorspraydropletadhesion(5,13, 15). Thecontactareaofdropletsondifficult-to-wetplants,suchasoilseedrape, 4 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; maize, and strawberry, was smaller than the contact area on plants with smooth surfaces,suchassugarbeetanddrybean(Figure2)(12). Bakeretal. (12)have observedthattheimpactiondiameterofdropletsonoilseedrapeisapproximately one sixth of those on dry bean (Figure 2). To achieve good retention on micro- roughsurfaces,alowdynamicsurfacetensionatthemomentofimpacthastobe guaranteedsonotonlytheexposededgesbutalsothelargestpossiblesurfacearea ofthewaxiswetted(5). Figure2. Impactiondiametersfordroplets(in-flightdiameter175μm)of0.3% aqueoussolutionofUvitex2Bontoleavesoffiveplants. DatafromBakeret al. (12). DevelopmentStageandCanopyDensity Greenhouse grown plants are more difficult to wet due to the intact epicuticular wax when compared to plants grown outdoor, which are subjected totheeffectsofphysicalwaxabrasioncausedbyrainwatersandleavesrubbing againsteachother,andotherenvironmentalconditions(16). Taylor(4)hasshown that outdoor grown spring barley plants retained more surfactant solutions than those grown in the greenhouse. Scanning electron micrographs of the sprayed leavesshowedthatallofthedepositsobservedwereinclosecontactwithatleast oneofthewax-abradedareas. Another factor influencing leaf wettability is plant developmental stage. Leaves at different growth stages may have different wettability. Moran Puente and Baur (7) found that soybean leaves at growth stage (GS) 16 were 30 times more wettable with water than leaves at GS 11. A dense layer of epicuticular wax crystals was observed uniformly on the adaxial and abaxial leaf surfaces fromGS11. Howevertheuniformlayerofdensewaxcrystalwasonlyobserved in the abaxial leaf surface but not on the adaxial surface in the GS 15 to 17, which were found to be only partially covered by epicuticular wax crystals. 5 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; In another study, Moran and Baur (22) showed that soybean leaves developed at early growth stages have low wettability and also the lowest tebuconazole penetration,indicatingthatgooduptakeofcropprotectionproductsrequiresgood contact of the spray droplets to the leaf surface. This was true even when using spray solutions with added wetting agents (22). It has also been demonstrated that maize leaves prior to GS 15 were densely covered with crystalline waxes, withwaterretentionatpracticallyzero. Incontrast,atGS18,maizeleaveswere covered with an amorphous wax film due to wax biosynthesis modifications at latergrowthstages,resultinginawaterretentionvalueof85%(23). Ellis et al. (6) have shown that wheat plant size and canopy density have a significant impact on spray retention of three spray liquids- tallow amine, EC blank, and water. When the plant density was at 1540 stems/m2, there was a clearrankingoftheretentionabilityofthethreesprayliquids,withtallowamine retention higher than EC, and EC retention higher than water (Table 2). At the highest canopy density of 2640 stems/m2, with a leaf area index higher than seven,theeffectsofliquidpropertiesonwholeplantretentionbecameverysmall and retention rates were almost identical for the three liquids (Table 2). This indicatedthatahighpercentageofthesprayisretainedontheplants, withlittle liquid deposition bouncing off the plants to the ground. Because plants at high densities and more advanced growth stages have significant more biomass, it was not surprising to observe that larger plants or those grown in high densities received less spray liquid when retention rates were based on a unit rather than thetotalplantdryweight(Table2). Table2. RetentionRates(μL/g)ofThreeSprayLiquidsonOutdoor-Grown WheatPlantsatDifferentGrowthStagesandDensities. TheNumberinthe ParenthesisIstheStandardErroroftheMean. DatafromEllisetal. (6). 1540stems/m2 1540stems/m2 2640stems/m2 Liquid (GS22to26) (GS30) (GS22to26) Tallowamine 53(±4.32) 34(±2.35) 37(±3.10) EC 48(±3.13) 27(±1.93) 37(±1.52) Water 38(±2.01) 20(±0.94) 35(±1.30) Forfieldcrops, thetopcanopywillmostlikelyinterceptmoresprayliquids than the lower canopy, either due to more receptive leaf surface characteristics or closer proximity to the spray nozzles, resulting in less canopy penetration andlessavailablesprayformulationforlowerleaves. Thisisnotabigissuefor crop protection products which have good systemicity, particularly those with phloemtranslocation. Forproductswithlimitedornosystemicity,however,less canopy penetration may lead to unsatisfactory commercial performance. More efforts should be directed to better understand the interception of spray droplets onleavesatdifferentpositionsintheplantcanopy. Thisinformationwouldbeof practical use in catering the formulation of crop protection products to specific fieldsituationsandspecificactiveingredients. 6 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; Effect of Physicochemical Properties of Spray Solutions on Spray Retention The physicochemical property of a spray formulation is the most important factordeterminingtheoutcomeoftheinteractionbetweenplantsurfaceandspray solutions. Formulation types and the components such as active ingredients, adjuvants, and additives, define the overall spray solution properties. While the effect of adjuvants and formulation types on spray droplet deposition and adhesionhasbeenextensivelyinvestigated,littleefforthasbeendirectedtoward understandingtheimpactofactiveingredients. ActiveIngredients ExperimentalStudyoftheRetentionRates We have investigated the retention rate of fungicides on greenhouse grown wheat seedlings and the relationship of retention rates with physicochemical propertiesofthecompounds. Fungicidesastechnicalmaterialswereformulated as 10% EC formulations (N-methyl pyrrolidone:Aromatic 200:Agrimul Lipo-D, 47:47:6 by volume). Spray formulations were applied onto 9-day old wheat seedlings(variety‘Yuma’)usingatracksprayerfittedwithaTeeJet8003Enozzle operatedat32psi. Thefungicideratewas100gai/Hawithanapplicationvolume of 200 L/Ha. Each fungicide treatment had four pots of five wheat plants, each potrepresentinganindividualreplicate. Onehourafterspraying,plantswerecut rightabovethesoillevelandweighed. Thecompoundwaswashedfromtheplant surface by immersing the plants in chloroform for 1 min. Plants were removed from the chloroform, allowed to dry, and frozen for future use. The chloroform was evaporated from the vials with nitrogen gas, then 1 mL of acetonitrile was addedtoeachvial. ThesampleswerequantifiedwithLC-DAD/MStodetermine fungicideretentionrateinng/mgfreshweightofplants. Theinitialretentionstudyincluded17fungicidecompounds: epoxiconazole, azoxystrobin, tebuconazole, myclobutanil, isopyrazam, penthiopyrad, cyproconazole, picoxystrobin, fluxapyroxad, pyraclostrobin, fluoxastrobin, fenbuconazole, bixafen, and four Dow AgroSciences experimental fungicides (Compounds I to IV). The 17 fungicide compounds exhibited different levels of retentionrateonwheatseedlings(Figure3). Afterthecompoundswereremovedfromtheplantsurfacebythechloroform wash, the plants treated with epoxiconazole, azoxystrobin, and Compounds I, II and IV were extracted with acetonitrile to determine the amount of material partitioned into the plant tissue. The amount of fungicide detected inside the plants was minimal, and would not significantly affect the surface retention rate (Table 3). 7 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.;