Pest Management Science Pest Manag Sci 64:283–289 (2008)

Degradation kinetics andsafety evaluation of tetraconazole anddifenoconazole residues in grapeKaushik Banerjee,∗ Dasharath P Oulkar, Sangram H Patil, Soma Dasgupta andPandurang G AdsuleNational Research Centre for Grapes, PO Manjri Farm, PB No. 3, Solapur Road, Pune 412 307, Maharashtra, India

Abstract

BACKGROUND: Tetraconazole and difenoconazole are triazole fungicides with proven bioefficacy againstgrapevine powdery mildew disease. In the present work, the authors explored the residue dynamics of thesetwo compounds in grapes and determined their preharvest intervals (PHIs) corresponding to multiple fieldapplications at recommended and double rates considering the most critical use pattern in Indian viticulture. Aconfirmatory residue analysis method was validated for trace-level determination of both the compounds.

RESULTS: Dissipation of both the fungicides followed non-linear two-compartment first + first-order ratekinetics. Tetraconazole and difenoconazole dissipated with PHIs of 12.5 and 25.5 days at recommended rates andof 28.5 and 38.5 days at double application rates respectively. On all the sampling days, the residues were belowthe maximum permissible intake, indicating consumer safety. The residues in the grape samples drawn from thefarms where these two fungicides were applied, maintaining the above PHIs, were below their respective MRLs.

CONCLUSION: The rate of degradation of tetraconazole was faster than that of difenoconazole. Thus, thegrowers will have the choice of using these new chemicals for the management of powdery mildews in succession,difenoconazole at early growth stages, followed by tetraconazole during the last month before harvest. Therecommendations of PHIs proved to be effective in minimizing residues in farm grape samples. Thus, this workis of high significance to the grape industry of India, and will support the registration of these new fungicides foreffective management of powdery mildews with minimum residue problems. 2008 Society of Chemical Industry

Keywords: tetraconazole; difenoconazole; residues; grapes; preharvest interval; dissipation; validation; LC-MS/MS

1 INTRODUCTIONPowdery mildew, caused by the pathogenic parasiteUncinula necator (Schw.) Burr., is one of the mostimportant fungal diseases of grapevines all over theworld, and causes significant economic damage interms of yield and quality deterioration of grapeberries. In India, the management of powderymildew is challenging owing to frequent developmentof resistance against fungicides. This compels thegrape growers to apply the recommended chemicalsfrequently and sometimes at excessive doses in orderto achieve the desired level of disease control.However, when attempting any new chemical controlmeasure, the biggest apprehension a grower faces ispotential residue problems in the final produce. Thisis particularly important for grape, as it is consumedas fresh fruit without any pretreatment, e.g. peeling,cooking, etc. If the same chemical is repeatedlyapplied on grapevines, the probability of residuedetection in the final product increases because ofthe cumulative effect of multiple sprays. On the other

hand, if different chemicals are judiciously appliedin succession or rotation for the management ofany specific disease, the residue problem minimizes,as each compound is monitored separately for itsresidue level. Thus, grape growers always look for awider choice of newer and safer fungicides for diseasemanagement.

In the international market, the maximum residuelimit (MRL) regulations are stringent in mostcountries. Thus, the recommendations regarding thedose-specific preharvest interval (PHI) are essential toensure dissipation of any applied chemical below theprescribed MRL at harvest to provide safety to theconsumers.

Tetraconazole [(RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl 1,1,2,2-tetrafluoroethylether] and difenoconazole [cis, trans-3-chloro-4-[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl 4-chlorophenyl ether] are relatively broad-spectrum triazole fungicides, with proven bioeffi-cacy against powdery mildew and rust diseases of

∗ Correspondence to: Kaushik Banerjee, National Research Centre for Grapes, PO Manjri Farm, PB No. 3, Solapur Road, Pune 412 307, Maharashtra, IndiaE-mail: kbgrape@yahoo.com(Received 25 July 2007; revised version received 25 September 2007; accepted 27 September 2007)Published online 16 January 2008; DOI: 10.1002/ps.1524

2008 Society of Chemical Industry. Pest Manag Sci 1526–498X/2008/$30.00

K Banerjee et al.

various agricultural crops.1–5 Although the field testbioefficacy results of both the fungicides are promisingin various agroclimatic zones of the country (unpub-lished in-house experimental results), none of thesefungicides is currently recommended for grape culti-vation in India6 because of the lack of informationregarding their residue dynamics. The manufacturingindustries are conducting multilocation residue trialsfor registration purposes, but to date no publishedinformation is available to recommend safe usage ofthese chemicals to grape growers. Thus, it was feltnecessary to study the dissipation pattern of these twofungicides in grapes at different doses to determinetheir PHIs, so that the residue data can be utilizedfor registration of these products for usage in Indianviticulture and the growers can have a wider choiceof fungicides for management of powdery mildewsalong with recommendations to minimize residueproblems.

This research paper presents the rate of degradationof tetraconazole and difenoconazole in grapes atrecommended and double doses, with frequencyof application based on the most critical usepattern pertaining to Indian viticulture. Differentkinetic models were applied to assess the dissipationpattern of the residues for determination of themost realistic PHIs to assure the safe use ofthese fungicides. The residues found in grapeberries on different sampling dates were furtherassessed in relation to their maximum permissibleintake (MPI), calculated on the basis of thecorresponding acceptable daily intake (ADI) toevaluate the consumer risk out of the residues.Field surveys were also conducted to evaluate thePHI results in farmers’ vineyards in minimizing theresidues.

2 MATERIALS AND METHODS2.1 Field experimentField experiments were conducted on Vitis viniferaL. (cv. Thompson Seedless) at the vineyard ofthe National Research Centre for Grape (NRCG),located on the western peninsular of India in thecity of Pune. Tetraconazole 40 g L−1 EW (IsagroAsia Agrochemicals Pvt. Ltd) was applied at 30 and60 g AI ha−1 in separate plots, and difenoconazole250 g L−1 SC (Score 25 SC; Syngenta India Ltd) wasapplied at 125 and 250 g AI ha−1. Both these pesticideswere sprayed in 1000 L water ha−1 in separateplots at 15 days interval on 90, 75, 60 and 45 daysbefore harvest (DBH) during January to March 2006.Each treatment, including the untreated control, wasreplicated 3 times in randomized blocks.

The average maximum and minimum temperaturesduring the experiment were 34.8 and 7.7 ◦C, withaverage relative humidity ranging between 47 and75%. There was no rainfall during the study. Thecrop was grown under drip irrigation following arecommended package of practices.

2.2 Sampling for residue dissipation studiesThe berry samples (5 kg) were collected at randomfrom each replicate of the treated and control plotsseparately at regular time interval on 0 (1 h afterspraying), 1, 3, 5, 7, 10, 15, 25, 30 and 45 days afterthe final foliar spray. Bunches hidden inside the canopyor those showing infestation of insect pests, diseasesor any physiological disorder were not included in thesamples. The grape berries were separated from thepedicels and directly analysed without any washing orany kind of pretreatment.

2.3 Sampling from growers’ vineyards formonitoring of the residues in fresh grapesField samples were collected at random from majorgrape-growing regions of India in the subsequent grapeseason. A total of 50 samples were collected fromvineyards that had received application of pesticides asper the dose and PHI recommendations. Out of these50 samples, 30 samples were collected from vineyardsof the Maharashtra state alone, which accounts formore than 60% of total production of the country. Thiswas followed by the collection of ten samples each fromthe other two important grape-growing states of India,Andhra Pradesh and Karnataka. At first, 2% grapesby weight pertaining to the total harvest of a particularplot were drawn at random. A 12 kg bulk sample wasprepared out of this by randomly selecting the grapebunches. From the bulk sample, a laboratory sample(3 kg) was prepared following a standard reductionprinciple.

2.4 Sample preparationThe entire laboratory sample (berries only) wascrushed thoroughly in a blender. Approximately 200 gof the crushed sample was further homogenized, and,from this, a 10 g sample was drawn and extractedwith ethyl acetate (1 × 20 mL) plus anhydroussodium sulfate (10 g) by homogenization followed bycentrifugation at 2000 rpm for 5 min.

For final analysis by liquid chromatography–tandemmass spectroscopy (LC-MS/MS), 4 mL of the aliquotwas evaporated to near dryness in a low-volume con-centrator (TurboVap LV; Caliper Life Sciences, USA)at 35 ◦C in the presence of 200 µL of 10% diethyleneglycol. The residue was redissolved in 1 mL methanol+ 1 mL 0.1% acetic acid and analysed by LC-MS/MS.

2.5 LC-MS/MSThe LC-MS/MS analysis was done with a PerkinElmer HPLC linked to an API 2000 (ABS Sciex) massspectrometer equipped with an electrospray ionization(ESI) probe. The HPLC separation was carried outusing a Perkin Elmer C18 column (150 mm × 2.0 mmID, 5 µm). The mobile phase was composed of(A) methanol with 5 mM ammonium formate and(B) water with 5 mM ammonium formate; gradient0–1 min 10% A phase, 1–2 min 10–95% A phase,2–6 min 95% A phase, 6–6.5 min 95–10% A phaseand 6.5–12 min 10% A phase. Tetraconazole and

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difenoconazole were eluted at retention times of5.86 and 6.47 min successively. The column oventemperature was maintained at 24 ◦C, and the flowratewas maintained at 1.0 mL min−1. An aliquot of20 µL was injected using a PE-200 Perkin Elmerautosampler.

The estimation was performed in positive modeby multiple reaction monitoring (MRM) with masstransition [M + H]+ 372.0/159.0 and 372.0/70.0for tetraconazole and [M + H]+ 406.0/251.0 and406.0/337.0 for difenoconazole with a scan time of70 ms (Fig. 1). The declustering potential (DP) fortetraconazole was 28 V with collision energies (CEs)of 38 and 47 V, whereas for difenoconazole the DP was10 V with a CE of 33 and 25 V respectively for the firstand second mass transitions. The first mass transitionwas used for quantification, while the second masstransition was used for confirmation of the residues.The ratio of the peak area of these two daughter ionswas 0.45 and 0.2 for tetraconazole and difenoconazolerespectively. The corresponding ratio in the positivesamples was determined and confirmed in accordancewith the EC guidelines.7

2.6 Analysis by gas chromatography (GC)As both tetraconazole and difenoconazole are GC-amenable compounds, the authors also explored theirfinal analysis by GC (Shimadzu 2010) with electroncapture detection (ECD) and mass spectrometry (GC-MS) for comparison and further confirmation of theresults. For GC analysis, 1 mL of the sample extractwas cleaned with 25 mg of the clean-up agent PSA(primary secondary amine, Bondelut; Varian Inc.)by centrifugation at 10 000 rpm for 5 min. The clearsupernatant was decanted, filtered through a 0.2 µmPTFE membrane filter and then injected to the GC.The effect of solvent exchange in toluene on GCresponse was also evaluated, where the ethyl acetateextract was evaporated to complete dryness under agentle stream of nitrogen and then reconstituted in1 mL toluene by sonication.

GC analysis was done on a DB 5MS capillarycolumn (30 m × 0.25 mm, 0.25 µm), and the carriergas (nitrogen) flowrate was maintained at 1 mL min−1.The oven temperature was programmed with an initialtemperature of 120 ◦C and held for 1 min, increased to200 ◦C at 40 ◦C min−1 and held for 1 min, increasedto 210 ◦C at 5 ◦C min−1 and held for 1 min, increased

to 220 ◦C at 5 ◦C min−1 and, finally, increased to270 ◦C at 15 ◦C min−1 and held for 18 min (runtime = 30.33 min). Tetraconazole was eluted at anRT of 9.47 min. Difenoconazole was eluted as twopeaks pertaining to its two stereoisomers at 22.82 and23.11 min. GC-MS confirmation of the residues wasaccomplished by electron impact ionization at full scanmode.

2.7 Method validation2.7.1 Standards and calibrationThe certified reference standards of tetraconazole anddifenoconazole were purchased from Dr EhrenstorferGmbH, Germany, and were of >99% purity. All thesolvents were of HPLC grade or equivalent quality.The calibration standards (five calibration points)ranging from 0.01 to 1.0 mg kg−1 were prepared inmethanol for LC analysis and in ethyl acetate andtoluene for GC analyses.

The calibration curves for both the compoundswere obtained by plotting the peak area againstthe concentration of the corresponding calibrationstandards. The limit of detection (LOD) of the testcompounds was determined by considering a signal-to-noise ratio of 3 with reference to the backgroundnoise obtained for the blank sample, whereas thelimits of quantification (LOQ) were determined byconsidering a signal-to-noise ratio of 10.

2.7.2 Evaluation of matrix effectThe matrix effect was assessed by employing matrix-matched standards prepared in a similar fashion tothe above with untreated green seedless grape berriesof the same variety. The matrix extracts were firstanalysed to confirm the absence of the test pesticidesin them before spiking. All five calibration standardswere prepared in the blank extracts and analysed.Further, detector response to different levels of matrixin final solution was evaluated (see Fig. 2).

2.7.3 PrecisionThe precision in the conditions of repeatability (forsix analyses in a single day) and the intermediateprecision (for six analyses in six different days) weredetermined separately for a standard concentrationof 0.05 mg kg−1 of both the analytes. The Horwitzratio (HorRat) pertaining to intralaboratory precision,which indicates the acceptability of a method with

Figure 1. LC-MS/MS MRM transitions for tetraconazole and difenoconazole.

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0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Matrix level in 1 mL mobile phase (mL)

Fin

al c

on

c.(n

g/m

L)

Difenconazole

Tetraconazole

Figure 2. Effect of different level of matrix on the response oftetraconazole and difenoconazole at the 50 ng mL−1 level.

respect to precision,8 was calculated for six differentdays with six measurements on each day for both thepesticides at a fortification level of 50 µg kg−1 in thefollowing way:

HorRat = RSD/PRSD (1)

where RSD is the relative standard deviation andPRSD is the predicted relative standard deviation =2C−0.15, where C is the concentration expressed asmass fraction (50 µg kg−1 = 50 × 10−9).

2.7.4 Accuracy – recovery experimentsThe recovery experiments were carried out on freshuntreated grape berries by fortifying the samples (10 g)in triplicate with tetraconazole and difenoconazoleseparately at three concentration levels. For bothtetraconazole and difenoconazole the fortificationlevels were 0.025, 0.1 and 1.0 mg kg−1.

2.8 Data analysisA number of recent publications have shown thatsimple first-order kinetics cannot adequately explainthe degradation behaviour of pesticides in naturalsystems like soils, where the degradation patternmay follow a non-linear path.9 A non-linear two-compartment first- + first-order model can adequatelyfit to the degradation pattern of many pesticides in soil,and could also predict the DT50 in a more realisticmanner than a linear first-order model. In view of this,the authors attempted to analyse the timewise residuedata of both the pesticides in grapes by linear as wellas non-linear regression analysis with the followingmathematical expressions:

First-order model

[A]t = [A]1 exp(−k1t) (2)

First + first-order model

[A]t = [A]1 exp(−k1t) + [A]2 exp(−k2t) (3)

where [A]t is the concentration (mg kg−1 grape) ofA at time t (days), [A]1 and [A]2 are the initial

concentrations of A at time 0 degraded throughfirst-order processes 1 and 2 and k1 and k2 are thedegradation rate constants for 1 and 2. The units of kdepend on the model used.

The half-life (DT50), which is the time at which theconcentration of initial deposits reaches the 50% level,was determined by the following equation:

First-order model

DT50 = ln(2) × k−11 (4)

DT50 is an important parameter that signifies thespeed of degradation.

The PHI, i.e. the time period (in days) required fordissipation of the initial residue deposits to below themaximum residue limit (MRL) for first-order kinetics,was determined by the equation

PHI = [log(intercept) − log(MRL)]/slope of

first-order equation (5)

Since the first + first-order model cannot bedescribed in a differential form, DT50 and PHI couldonly be calculated by an iterative procedure. Theequation parameters were calculated by use of acommercially available program OriginPro,10 whichallows the defining of new equations and statisticalparameters. For tetraconazole an MRL of 0.1 mg kg−1

was used, which is recommended for wine by theAustralian Wine Research Institute MRL Database,11

while for difenoconazole an MRL of 0.05 mg kg−1 wasconsidered, which is the value applicable for berriesand small fruits for the European Union12 and in theNetherlands.13

2.9 Safety evaluationComparing the dietary exposure of each sample withMPI provided a safety evaluation of the pesticideresidues. The prescribed ADI values of tetraconazoleand difenoconazole are 0.004 and 0.01 mg kg−1

body weight day−1.14,15 Multiplying the ADI by thebody weight of an average child (16 kg), the MPIswere found to be 0.064 and 0.16 mg person−1 day−1

for tetraconazole and difenoconazole respectively.The values of dietary exposure were calculated bymultiplying the residue levels by the average per capitadaily consumption of 9 g of fruit.16

2.10 Transfer of technology and impactassessmentThe PHI data generated by the above field experimentswere communicated to the grape growers through fieldvisits, seminars and extension activities. The growerswere asked to apply the above pesticides at the dosesand frequencies as used in the present experiment andwere specifically advised strictly to maintain the PHIin order to minimize residues. Representative sampleswere collected after harvest from the pack-houses tomonitor the residues of the two test chemicals in the

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final produce. The main objective of this endeavourwas to validate the PHI data under a real farmsituation.

3 RESULTS AND DISCUSSION3.1 Method validationThe linearity of the calibration curve was establishedin the range 0.01–1.0 mg kg−1 for both the chemicalswith a correlation coefficient (R2) of the calibrationcurve of >0.99. For matrix calibration, the R2 wasalso >0.99 for both the compounds. The LOD andLOQ of the two compounds were 0.01 and 0.025 mgkg−1 respectively. The coefficients of variation (CV)regarding the repeatability and intermediate precisionfor tetraconazole at 0.05 mg kg−1 were 2.1 and7.2%, and for difenoconazole the corresponding CVswere 3.5 and 7.1% respectively. The average HorRatvalues for tetraconazole and difenoconazole were 0.27and 0.21 respectively, which indicates satisfactoryreproducibility of the analytical method.8 The matrixeffect was prominent for both the pesticides. Therewas an overall suppression of the detector response by5–22% at different levels of matrix in the final solution.The matrix effects at 50 ng mL−1 are presented inFig. 2. It was observed that the matrix effect wassimilar for both pesticides and the effect increasedwith increasing proportion of matrix, resulting in lessrecovery. In view of this, the matrix calibration wasused for quantification of the residues of both thefungicides.

GC could also be used to analyse the residues ofboth the compounds. In the case of tetraconazole, theresponse of the ECD significantly increased by a factorof almost 2.8 in signal-to-noise ratio as a consequenceof solvent exchange of the final residues into toluene.

The average recoveries (%) of tetraconazole anddifenoconazole at the 0.025 (LOQ), 0.10 and 1.0 mgkg−1 levels of fortification were 80% ± 5.1, 91% ± 3.8and 83% ± 2.1 and 81.5% ± 5.6, 93.4% ± 4.4 and95.5% ± 3.1 respectively. Thus, the EU DG SANCOcriterion in this regard, which requires mean recoverieswithin the range 70–110%, is met.17 The extractableresidues of both the fungicides were quantifiedin comparison with the control. The residue datawere fitted against the sampling day and analysedstatistically18 to determine PHI pertaining to all thetreatments.

3.2 Persistence and dissipationThe dissipation behaviours of tetraconazole anddifenoconazole pertaining to recommended anddouble-dose treatments are presented in Figs 3aand 3b respectively. In all cases, the dissipationrate was faster at the beginning and slowed downwith the passage of time. This indicated a non-linearpattern of degradation and implies that simple first-order kinetics might not be adequate to explain thedissipation behaviour of these pesticides in grapes.The PHIs estimated by the first-order kinetics were

0

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0 10 20 30 40

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idu

es (

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Recommended dose

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5.0

6.0

0 10 20 30 40 50

Sampling days

Res

idu

es (

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Recommended dose

Double dose

(b)

Figure 3. (a) Dissipation of tetraconazole residues (mean of threereplicates ± SD) in grapes; (b) dissipation of difenoconazole residues(mean of three replicates ± SD) in grapes.

found to be inadequate for both the pesticides inreducing the residue load to the MRL, indicatingthe inappropriateness of this model to explain thedissipation behaviour of tetraconazole as well asdifenoconazole residues. Hence, the kinetics of theresidue data of both the pesticides was simultaneouslyevaluated by fitting the data into a non-linear first +first-order order model to estimate the parametersDT50 and PHI (Table 1). In all cases, the fit ofdata to the first-order kinetics was also relativelypoor compared with the first + first-order model.The typical nature of dissipation suggests the fitof data to a two-compartment first + first-orderkinetics model, where one part of the added pesticide,which is immediately available in solution phase,degrades rapidly, leaving the other part possiblyremaining in dynamic equilibrium as an adsorbedfraction on cellular components. On comparing thePHI values obtained by the first-order model, itis concluded that PHI as per the first + first-order model is more realistic and is almost exactlythat expected after this time period; the residuelevel also goes down to below the MRL. In thecase of tetraconazole, the rate of dissipation wasrelatively slower at double dose, so the PHI atdouble dose was considerably longer than expected ascompared with the recommended dose. This indicatesthat PHI may not increase proportionately withincrease in application rate, and further establishes theimportance of adhering to the dose recommendationsin the management of pesticide residues. In the caseof difenoconazole, the initial rate of dissipation was

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Table 1. Degradation parameters for the residue dissipation of tetraconazole and difenoconazole in grapea

First-order First + first-order

Pesticide Parameter Unit T1bc T2bc T1b T2b

Tetraconazole [A]1 µg kg−1 0.36 0.64 0.29 0.15k1 day−1 0.09 0.07 0.20 0.02[A]2 µg kg−1 N/A N/A 0.22 0.73k2 day−1 N/A N/A 0.05 0.20R2 – 0.97 0.91 0.99 0.99DT50 day 7.8 9.5 5.5 4.5PHI day 6.3 11.0 12.5 28.5

Difenoconazole [A]1 µg kg−1 2.81 4.97 1.44 0.45k1 day−1 0.16 0.14 0.16 0.79[A]2 µg kg−1 N/A N/A 1.44 4.29k2 day−1 N/A N/A 0.16 0.12R2 – 0.98 0.99 0.99 0.99DT50 day 4.4 5.1 4.5 5.5PHI day 11.2 14.7 25.5 38.5

a Parameters are defined in Section 2.8.b T1 = recommended dose, T2 = double dose.c N/A = not applicable.

also relatively slower at double dose, but the rateof degradation increased in the later phase and thusthe residue levels after 30 days were similar at boththe doses, which are further reflected in terms ofsimilar DT50 values. The relative appraisal of the PHIfor tetraconazole and difenoconazole suggests thatfarmers should choose these chemicals for powderymildew management on different timescales in astaggered fashion, with difenoconazole applicationfollowed by tetraconazole application. Such anapproach can also effectively minimize the chancesof resistance development. At the recommended dose,the application of difenoconazole should be avoidedfor a full month before harvest to ensure the dissipationof residues below MRL, whereas application oftetraconazole can be continued up to 15 days beforeharvest.

3.3 Safety evaluationThe dietary exposure calculated for the residuescorresponding to each sampling date was comparedwith the MPI. The dietary exposure was less than theMPI for both the fungicides at both the treatmentlevels.

A critical perusal of the samples collected throughsurvey from the growers’ vineyards reveals a goodimpact of the transfer of technology in minimizingthe residues under real farm situations. Out ofthe 50 samples monitored, none of the samplesfailed for MRL compliance for tetraconazole anddifenoconazole. In all the samples, in fact, the residuelevels were below the limit of quantification. Thus, thePHI recommendations out of these experiments weresuccessfully transferred to the growers and proved tohave significant impact in minimizing their residues intable grapes at harvest.

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