Food Chemistry 117 (2009) 634–640

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Food Chemistry

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The fate of fungicide and insecticide residues in Australian wine grapeby-products following field application

Gavin Rose a,*, Simon Lane b, Robert Jordan b

a Future Farming Systems Research Division, DPI-Werribee Centre, 621 Sneydes Road, Werribee, 3030 Victoria, Australiab Stoney Creek Oil Products, 145 Davies Road, Talbot, 3371 Victoria, Australia

a r t i c l e i n f o

Article history:Received 8 April 2008Received in revised form 15 April 2009Accepted 17 April 2009

Keywords:Grape seedGrape marcPesticide residuesSystemic pesticidesGas chromatographyLiquid chromatography–tandem massspectrometry

0308-8146/$ - see front matter Crown Copyright � 2doi:10.1016/j.foodchem.2009.04.061

* Corresponding author. Tel.: +61 3 9742 8750; faxE-mail address: gavin.rose@dpi.vic.gov.au (G. Rose

a b s t r a c t

Pesticides applied to grape vines before harvest may concentrate in the grape seed due to their high oilsolubility. Twenty-four samples of grape marc, representing a range of red and white wine grape varie-ties, were collected and analysed for selected fungicides and insecticides. Fifteen of the 24 samples werematched with insecticide and fungicide application diaries. Residue concentrations of the fungicides,procymidone, iprodione, cyprodinil, fenhexamid, fludioxinil, pyrimethanil and trifloxystrobin, and theinsecticides, indoxacarb and tebufenozide, were higher in grape seed oil and grape seed meal than inthe fruit and the marc. The relative concentrations were approximately proportional to the octanol towater partition coefficients, log Kow. A range of other fungicides and insecticides were detected but werenot significantly concentrated in the oil and seed meal relative to fruit and marc. The presence of pesti-cide residues in grape seed oil and grape seed meal will impact on the possibility of producing these wineby-products.

Crown Copyright � 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Large quantities of grape marc (solids remaining after juiceextraction) are produced by regional wineries in Victoria, Australia(Jordan, 2002). Currently, the marc is either fed to livestock or con-signed to land fill as the costs for transport and reprocessing intodistilled alcohol, tartaric acid or polyphenolic supplements makethese options uneconomic. Alternatively, grape seed oil (for humanconsumption) and seed meal (for animal feed), could be producedfrom the marc. However, previous work (Cabras & Angioni, 2000;Navarro, Barba, Oliva, Navarro, & Pardo, 1999; Teixeira, Aguiar,Afonso, Alves, & Bastos, 2004; Tsiropolous, Aplada-Sarlis, & Milia-dis, 1999) suggests that the grape seed oil and the seed mealmay contain unacceptable levels of systemic pesticide residuesthat were applied to the vines prior to harvest. Residue levels offive out of six pesticides (penconazole, chlorpyrifos, vinclozolin,fenarimol and metalaxyl) applied to grape vines prior to harvestwere higher in grape marc than in the crushed grapes during elab-oration of red wines. Also, the residue levels in must, 4 days aftervinification, were significantly lower than in crushed grapes forthe six pesticides (including mancozeb) (Navarro et al., 1999). Res-idue levels of 10 out of 12 fungicides applied to vines prior to har-vest were lower in must and wine than in the grapes. Six out of

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eight of these fungicides had reduced levels in clarified must, sug-gesting that the residues tend to be retained in the solid portion ofthe must (seeds, skins and stalks). Similarly, the levels of six out ofnine organophosphate insecticides were lower in the wine than inthe grapes and the levels of five of these organophosphate insecti-cides were lower in clarified must (after solids removal) than in themust. However, dimethoate and methidathion, the compoundswith lowest log Kow (more soluble in water and ethanol), were athigher concentrations in the must and wine than in the marc(Cabras & Angioni, 2000). Another study by Tsiropolous et al.(1999) showed significantly lower levels of the insecticide tef-lubenzuron in wine than in the grapes. Clarified must residue levelswere also significantly lower than in must suggesting that tef-lubenzuron residues were associated with the solid portion of seed,skins and stalks rather than the juice of the grape. It was suggested byTeixeira et al. (2004) that systemic pesticides, such as oxydixyl, werepreferentially found in the grape pulp, whilst contact pesticides,such as folpet, were present only in the skin. The relationship be-tween the levels in different parts of the grape was also thought tobe dependent on the time between application and sampling.

This study investigates the potential concentration of appliedfungicides and insecticides in grape seed produced from grapemarc collected from selected wineries in Victoria, Australia. Addi-tionally, the relationship between the octanol to water coefficient(log Kow) and the concentration of each fungicide/ insecticide ingrape seed was explored.

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G. Rose et al. / Food Chemistry 117 (2009) 634–640 635

2. Materials and methods

2.1. Reagents

Pesticide hexanes (nanograde 95% n-hexane), nanogradedichloromethane (DCM), and HPLC grade methanol, were all pur-chased from Mallinkrodt, Kentucky, USA. Acetone was redistilledfrom AR grade solvent (BDH/Merck, Kilsyth, Australia). HPLC gradeacetonitrile was purchased from OmniSolv, EM Science NJ, USA.Labchem grade Florisil was purchased from Ajax Fine Chemicals,Sydney Australia, and activated by placing in a muffle furnace at550 �C for 2 h prior to use. Anhydrous AR grade sodium sulphateand anhydrous AR grade ammonium acetate were purchased fromAjax Fine Chemicals, Sydney Australia. Water was Type 1 (18 Meg-ohm resistivity).

2.2. Certified standards

Cyprodinil (98.5% purity), fenhexamid (99.5%), fludioxinil(98.0%), metalaxyl (99.5%), pyraclostrobin (99.7%), trifloxystrobin(99.0%), quinoxyfen (99.0%), spinosad A + D (95%), spiroxamine(97.5%), captan (99.5%), chlorothalonil (98.5%), procymidone(98.0%) and iprodione (99.0%) were purchased from Dr. Ehrenstor-fer-Schafers, Augsburg, Germany. Indoxacarb (99.3%), pyrimethanil(99.6%), triadimenol (97.7%) and tebufenozide (99.9%) were pur-chased from the National Measurement Institute, Pymble, Austra-lia. Penconazole (99%) and myclobutanil (99.9%) were purchasedfrom Riedel de Haan, Hanover, Germany and tributyl phosphate(TBP) was purchased from Chem Service, PA, USA.

2.3. Sampling and sample preparation

2.3.1. Fruit and by-product samplesGrape marc and, where possible, matching fruit samples were

collected from regional Victorian wineries during the autumn,2004 vintage. Of a total of 24 samples of marc, 15 samples werematched with application diaries. A target list of fungicides andinsecticides was developed from examination of the available dia-ries. Fungicides represented the highest proportion of chemicalsapplied to the vines prior to harvest. There was no record of orga-nophosphate or synthetic pyrethroid insecticide usage. Central Vic-torian marc samples were collected fresh and transferred to StoneyCreek Oils (SCO) located at Talbot in central Victoria. The seed waswet-separated immediately at SCO and the seed dried on forced airdrying screens, sieved and stored for pressing. Yarra Valley marcsamples were collected and immediately frozen prior to transferto SCO. The frozen marc samples were thawed and the seed wet-separated, dried, sieved and stored for pressing. Where possible,matching fruit samples were collected from harvest fruit bins atthe wineries. However, due to harvesting patterns, there is noguarantee that the fruit samples were representative of chemicalusage throughout harvest blocks. A standard linseed oil presswas used to cold-press the seed. Residue free seed oil was usedto clean the oil press between samples. Seed meal samples werealso collected from each seed batch processed and analysed for or-ganic residue content.

2.3.2. Fruit and marc extractionFifty grammes of marc or homogenised grape berry were ex-

tracted with 200 ml of acetone using a probe homogeniser. Thesample was vacuum-filtered, the filtrate mixed with 650 ml of sat-urated aqueous sodium sulphate and extracted with 1 � 100 mland 2 � 50 ml volumes of DCM. The extracts were combined,passed through an anhydrous sodium sulphate drying columnand inverted into 10 ml of hexane.

2.3.3. Seed oil extractionTwo grammes of oil were diluted with 25 ml of hexane and ex-

tracted with 1 � 50 ml and a further 2 � 25 ml of hexane saturatedacetonitrile. The acetonitrile layers were combined, dried, and in-verted into 2 ml of dichloromethane (DCM).

2.3.4. Seed meal extractionApproximately 25 g of meal were mixed with 200 ml of 35%

water/65% acetone and shaken for 30 min. The sample was vac-uum-filtered, the filtrate mixed with 650 ml of saturated aqueoussodium sulphate and extracted with 1 � 100 ml and 2 � 50 ml vol-umes of DCM. The DCM volumes were combined, dried through ananhydrous sodium sulphate drying column and inverted into 10 mlof hexane.

2.4. Apparatus

2.4.1. Gas chromatography–nitrogen phosphorus detection (GC–NPD)The fungicides, chlorothalonil, penconazole, procymidone,

iprodione, triadimenol and myclobutanil were determined usinga Varian 3400 CX capillary gas chromatograph fitted with nitro-gen–phosphorus detector (Varian, Mulgrave, Australia). An aliquot(2 ll) of hexane (derived for each sample as described above) wassimultaneously injected onto parallel columns (15 m, 0.32 ID di-methyl-polysiloxane stationary phase, J&W� DB-1) and a 15 m,0.32 ID 50% diphenyl-dimethyl-polysiloxane stationary phase(J&W� DB-17) via a split/splitless injector with a split ratio of1:20. The GC oven was temperature-programmed (120 �C 0–2 min, 120–300 �C at 20 �C/min, held 300 �C for 1 min) for opti-mum separation efficiency. The injector and detector temperatureswere set at 280 and 320 �C, respectively. Helium was used as car-rier gas. Varian Star software (V6.0) was used to manage the chro-matographic data. The organic residues were quantified bycomparison with external standards.

2.4.2. Gas chromatography–electron capture detection (GC–ECD)The fungicide captan was determined using a Varian 3800 cap-

illary gas chromatograph fitted with an electron capture detector(Varian, Mulgrave, Australia). The hexane extract obtained fromsample processing, as described above, was further purified byadding 0.5 ml of the extract to 3.0 g of activated florisil, followedby elution with 25 ml of 1% acetonitrile/49% hexane/50% DCM. Thiswas inverted to 2 ml of hexane. An aliquot (2 ll) of hexane was in-jected simultaneously onto parallel capillary columns (15 m, 0.32ID 14% cyanopropyl-phenyl on dimethyl-polysiloxane stationaryphase, J&W� DB-1701) and a 15 m, 0.32 ID, 25% cyanopropyl,25% diphenyl, 50% dimethyl-polysiloxane stationary phase (Res-tek�, RTX-225) via a split/splitless injector with a split ratio of1:20. The GC oven temperature was set at 200 �C. The injectorand detector temperatures were set at 280 and 350 �C, respec-tively. Helium was used as carrier gas. Varian Star software(V6.0) was used to manage the chromatographic data. The organicresidues were quantified by comparison with external standards.

2.4.3. Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

The fungicides, cyprodinil, fenhexamid, metalaxyl, fludioxinil,pyraclostrobin, quinoxyfen, trifloxystrobin and pyrimethanil, andthe insecticides, spinosad, spiroxamine, indoxacarb and tebufenoz-ide, were determined using a Waters 2975 separation moduleinterfaced with a Waters Micromass Quattro Micro tandem massspectrometer operating in the positive ion electrospray mode(Waters Micromass, Manchester, UK). An aliquot (0.2 ml) of thehexane extract, obtained as described above, was inverted intomethanol (4 ml) containing 0.06 lg/ml of tributylphosphate(TBP) as internal standard. The compounds were separated with

636 G. Rose et al. / Food Chemistry 117 (2009) 634–640

a 150 mm � 2.0 mm Luna� 5 lm C18(2) analytical HPLC columnfitted with a C18 guard column. The HPLC column was maintainedat 25 �C. The mobile phase consisted of (A) 20% methanol in 5 mMammonium acetate and (B) 90% methanol in 5 mM ammoniumacetate with the following gradient: 100% A–100% B (0–15 min),100% B (15–28 min), 100% B–100% A (28–30 min) with a flow rateof 0.2 ml/min. Masslynx Software (V4.0) was used for data process-ing. Residues were quantified using external standards and eachstandard set was assayed a minimum of three times during eachsample batch run. Each standard also contained TBP (0.06 lg/ml)as the internal standard. Sample and recovery concentrations werecalculated from a linear regression of the standards. Samples andstandards were corrected for internal standard (IS) response. Thetandem mass spectrometer was operated in the multiple reactionmonitoring (MRM) mode. The precursor ions, product ions, confir-matory product ions and retention times for each compound arelisted in Table 1.

2.5. Quality control

Spiked recoveries for each residue were included in every sam-ple extraction batch and the recoveries determined as described forsamples. Samples were spiked in the range of 0.05–1.2 mg/kg.Mean recoveries for GC residues were; 79% in fruit, 92% in marc,89% in seed oil and 85% in seed meal. Mean recoveries for LC–MS/MS residues were: 65% in fruit, 91% in marc, 69% in seed oiland 51% in seed meal. The reported data were not corrected forbatch recoveries. The lower recovery of LC–MS/MS analytes ingrapes is related to the matrix suppression of mass spectrum ionresponse routinely observed in extracts from fruit high in moistureand soluble sugars, such as grapes and pome fruit. This effect wasalso observed in LC–MS/MS in the extracts of seed oil and seedmeal. Matrix enhancement of the LC.MS/MS response wasobserved for pyrimethanil, and the strobilurin fungicides, pyrac-lostrobin and trifloxystrobin.

3. Results and discussion

3.1. General

Table 2 shows the residue levels present in fruit, marc, seed oiland seed meal for fungicides that are routinely used in Victoria,Australia. At the time of the study, the maximum residue limit(MRL) for grapes was taken from the Food Standards Australiaand New Zealand (FSANZ) Code (Food Standards Australia New

Table 1LC–MS/MS precursor ions, product ions, confirmatory product ions and retention times fo

Fungicide/insecticide Precursor ion (m/z) Production quantitat

Cyprodinil 226.10 92.64Fenhexamid 301.97 96.79Fludioxinil 248.98 157.86Pyraclostrobin 388.06 162.96Quinoxyfen 307.90 196.88Trifloxystrobin 409.18 185.94Pyrimethanil 200.11 106.92Spinosad A 732.36 141.91Spinosad D 746.67 141.89Spiroxamine 298.23 143.92Indoxacarb 528.15 202.91Tebufenozide 353.40 297.03Metalaxyl 280.35 220.10Tributylphosphate (TBP)a 267.44 98.78

na = not available.a TBP is the internal standard.

Zealand, 2006). FSANZ MRLs for agricultural produce are appliedto derived foods, such as seed oils, where there is no separateMRL listed.

3.2. Cyprodinil and fludioxinil

The anilinopyrimidine systemic fungicide cyprodinil and thephenylpyrrole non-systemic fungicide fludioxinil are applied in amixture formulated to control grey mould, Botrytis cinerea (Austra-lian Pesticide and Veterinary Medicine Authority (APVMA), 2008).The formulated mixture has a withholding period of four weeksand a restriction of a maximum of two applications per season.No samples contained either fungicide above the Australian MRL;however, the residues in the seed oil from sample CH10 may havebeen above the MRL at the withholding period. Label instructionslimit applications prior to grape veraison at the latest, so it is unli-kely that any crop would be harvested at the minimum withhold-ing period. The relative concentrations of the fungicides in thesamples reflect the composition of the formulation (37.5% cyprod-inil and 25% fludioxinil).

3.3. Procymidone

The systemic dicarboximide fungicide procymidone is regis-tered for control of grey mould in wine grapes and has a withhold-ing period of 7 days (APVMA, 2008). Seven out of nine seed oilsamples contained residues above the Australian MRL of 2 mg/kg(Food Standards Australia New Zealand (FSANZ), 2006). Four outof nine marc samples also contained residues above the MRL. Theresidues for grape sample CH4 was more than twice the MRL forgrapes, indicating that the withholding period may be too short,especially where multiple applications have been made prior toharvest. There were no application diary records for samplesCH8, CH14 and Ref#14. The data suggest that it is likely that sam-ple CH15 had an unrecorded application close to the withholdingperiod. Sample CH15 also reported a grape sample above theMRL, supporting the previous observation that the withholdingperiod was too short for the listed MRL. Results show that procymi-done is a high-risk fungicide for seed oil, marc, seed meal andgrapes in these samples. In 2005, following numerous internationalreports of its toxicity, additional application and usage controls forprocymidone, to control grey mould in wine grapes, were imple-mented by the Australian Pesticide and Veterinary MedicineAuthority. The revised controls have resulted in the phase-out ofprocymidone use on wine grapes in Australia.

r the fungicides and pesticides measured in this study.

ive (m/z) Production confirmatory (m/z) Retention time (min)

117.86 18.99142.70 17.70185.00 17.13132.80 19.04161.85 21.32144.81 19.62

81.90 16.94na 24.87na 26.58

99.97 21.09149.82 19.42na 18.35na 15.47na 19.83

Table 2MRL, label withholding periods and fungicide residues in fruit, marc, seed oil and seed meal.

Fungicide (MRL) Application: days priorto harvest

Sample/varietya Fruit (mg/kg) Marc (mg/kg) Seed oil (mg/kg) Seed meal (mg/kg)Label WHP (days)

Cyprodinil (2 mg/kg) nr CH4 (C) <0.01 <0.01 0.36 0.01Label WHP = 28 days 72 CH8 (C) 0.01 0.46 0.90 0.48

81 CH9 (C) 0.08 0.20 0.63 0.4188 CH10 (P) 0.20 0.35 1.6 0.84nr CH14 (M) <0.01 <0.01 0.09 <0.01nr Ref#14 (S) n/s 0.10 0.18 0.29

Fludioxinil (2 mg/kg) 81 CH9 (C) 0.10 0.12 0.23 TraceLabel WHP = 28 days 88 CH10 (P) Trace 0.21 0.56 0.19

nr Ref#14 (S) n/s 0.11 0.27 <0.1

Procymidone (2 mg/kg) 9, 30, 84 CH4 (C) 4.5 8.5 17 14Label WHP = 7 days 28, 105 CH5 (S) 0.46 2.1 11 3.0

nr CH8 (C) <0.05 0.11 1.7 0.1034 CH9 (C) 0.39 1.0 7.0 0.6440 CH10 (P) 0.90 1.8 11 2.043 CH11 (P) 0.41 2.3 13 2.8nr CH14 (M) 0.10 0.36 3.9 0.4877 CH15 (S) 2.2 10 30 9.0nr Ref#14 (S) n/s 0.78 1.8 0.52

Fenhexamid (10 mg/kg) 115 CH4 (C) <0.01 0.06 0.49 0.08Label WHP = 21 days 132 CH5 (S) 0.01 0.05 0.10 0.15

110 CH8 (C) <0.01 0.03 0.06 0.0495 CH9 (C) 0.03 0.13 0.25 0.14101 CH10 (P) 0.03 0.07 0.18 0.10nr Ref#14 (S) n/s 0.06 0.09 0.13

Pyrimethanil (5 mg/kg) 119 CH4 (C) 0.12 0.47 0.29 1.1Label WHP = 7 days 140 CH5 (S) 0.04 0.06 0.13 0.18

120 CH11 (P) 0.03 0.11 0.30 0.25nr CH14 (M) 0.02 0.05 0.11 0.14nr CH15 (S) 0.08 0.15 0.34 0.35nr Ref#14 (S) n/s 0.01 0.02 0.05

Captan (10 mg/kg) 51, 84, 140 CH4 (C) 0.29 0.16 1.2 0.14Label WHP = 30 days 72, 105, 161 CH5 (S) 0.16 <0.01 0.10 0.01

46, 96 CH8 (C) 0.25 1.6 0.45 0.0577 CH9 (C) 0.14 0.11 1.0 0.0252 CH10 (P) 0.07 <0.01 0.04 <0.0190 CH14 (M) 0.06 0.03 0.12 0.0277 CH15 (S) 0.66 <0.01 0.11 0.02

Iprodione (20 mg/kg) nr Ref#1 (R) 1.1 1.9 16 2.6Label WHP = 7 days nr Ref#6 (S) n/s <0.05 2.2 <0.10

nr Ref#7 (S) <0.05 <0.05 0.56 <0.10nr Ref#8 (CS) <0.05 <0.05 0.60 <0.10nr Ref#9 (M) <0.05 n/s 0.52 <0.10nr Ref#10 (CS) n/s <0.05 2.0 <0.10nr Ref #17 (S) n/s n/s 16 4.2nr Ref #18 (S) n/s n/s 0.56 <0.10

Trifloxystrobin (0.5 mg/kg) 102 CH9 (C) 0.008 0.011 0.050 0.002Label WHP = 35 days 108 CH10 (P) 0.005 0.006 0.020 0.008

120 CH11 (P) 0.006 0.014 0.030 0.005nr Ref#7 (S) 0.011 0.008 0.010 0.01145 Ref#8 (CS) 0.012 0.025 0.150 0.027nr Ref#9 (M) 0.016 n/s 0.130 0.020nr Ref#10 (CS) n/s 0.010 0.068 0.010nr Ref#14 (S) n/s 0.016 0.042 0.005152 Ref#16 (S) n/s n/s 0.010 0.003

Pyraclostrobin (2 mg/kg) 110 CH8 (C) <0.01 0.06 0.10 0.020Label WHP = 21 days nr CH15 (S) 0.02 0.07 0.20 0.09

Metalaxyl (MRL = 1 mg/kg) 68 CH9 (C) <0.01 <0.01 Trace <0.01Label WHP = 7 days 74 CH10 (P) <0.01 <0.01 0.01 0.01

64 CH11 (P) 0.01 0.01 0.02 0.04nr Ref#14 (S) n/s Trace <0.01 0.03

Quinoxyfen (2 mg/kg) nr CH4 (C) <0.01 <0.01 0.04 <0.01Label WHP = 14 days 72 CH8 (C) <0.01 0.07 0.10 0.02

115 Ref#10 (CS) <0.01 Trace 0.030 Trace122 Ref#16 (S) n/s n/s Trace <0.01

n/s = no sample.nr = no record(diary).

a C = chardonnay, P = pinot, M = merlot, S = shiraz, R = Riesling, CS = cabernet sauvignon.

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638 G. Rose et al. / Food Chemistry 117 (2009) 634–640

3.4. Fenhexamid

Fenhexamid is a contact (non-systemic) hydroxyanilide fungi-cide, used for controlling grey mould. Fenhexamid is registeredfor a 21-day withholding period on grapes (APVMA, 2008). Thereis a label restriction with a maximum of two applications per sea-son. Application diary records show one application 3–4 monthsprior to harvest. Seed oil samples showed the highest level of res-idues but these were less than 10% of the Australian MRL. Giventhe existing usage patterns, fenhexamid is a low risk fungicide.

3.5. Pyrimethanil

Pyrimethanil is a contact anilinopyrimidine fungicide used forcontrol of grey mould. The withholding period for pyrimethanilapplication on grapes is 7 days (APVMA, 2008). All samples werereported at less than 25% of the Australian MRL. Three of the six re-ported samples had application diary records which indicatedapplication four months prior to harvest. It is likely that the threesamples without application diary records were also treated3–4 months prior to harvest. Following this usage pattern, pyri-methanil is a low risk fungicide for residues in seed oil and marc.

3.6. Captan

Captan is a contact multi-site phthalimide fungicide. The regis-tered product has a withholding period of 7 days on grapes and isused for control of grey mould, black spot (Elsinoe ampelina),downy mildew (Plasmopara viticola) and phomopsis leaf blight(Phomopsis viticola) (APVMA, 2008). Up to five applications are al-lowed per season. Three of the seven samples showed captan usemore than once in the season. All applications were at a minimumof 45 days withholding period and reported residues were all lessthan 20% of the Australian MRL. As expected, the sample withthe highest residue level (1.6 mg/kg in seed oil) was the samplewith the shortest actual withholding period. Captan is a low riskfungicide in the samples tested with the reported usage patterns.

3.7. Iprodione

Iprodione is a contact dicarboximide fungicide used for control-ling grey mould (B. cinerea). The withholding period is 7 days witha recommendation to limit application to a maximum of twosprays per season (APVMA, 2008). None of the available applicationdiaries showed applications of iprodione; however, iprodione res-idues were detected in samples for which application diaries werenot available. Two of the reported seed oil samples contained16 mg/kg of iprodione, or 75% of the Australian MRL. It is likely thatiprodione was used with an application pattern similar to procymi-done. Results show that iprodione is a high-risk fungicide for seedoil, marc, seed meal and grapes in these samples.

3.8. Trifloxystrobin

Trifloxystrobin is a strobilurin type fungicide, used for control-ling powdery mildew (Uncinula necator) and suppression of downymildew. This fungicide is mesostemic in action and it also hastranslaminar activity and can be redistributed by superficial va-pour movement (Tomlin, 2003). The label withholding period is35 days. Label instructions limit usage prior to grape bunch closure(APVMA, 2008), thus it is unlikely that application would be madethree months prior to harvest. This would include the samples forwhich no application diary records were available. Five sampleshad application diary records indicating application had actualwithholding periods of more than one hundred days prior to har-vest. With these application patterns, it is unlikely that grape,

marc, seed oil or meal would be at 50% of the Australian MRL, sotrifloxystrobin is a low risk fungicide for the production of grapeseed oil and seed meal for commercial purposes. The reported lev-els are lower than for most fungicides, due to the exceptional sen-sitivity of the LC–MS/MS instrument for the determination oftrifloxystrobin.

3.9. Pyraclostrobin (log Kow = 4.0)

Pyraclostrobin is used for controlling powdery mildew anddowny mildew. The label withholding period is 21 days. The labelstipulates a maximum of three applications at 10–14 day intervals,commencing at flowering (APVMA, 2008), therefore it is unlikely tobe applied within three months of harvest. Two samples of seed oilcontained pyraclostrobin residues at 10% of the Australian MRL.Pyraclostrobin is a low risk fungicide providing label usage is fol-lowed. Pyraclostrobin is considered to be a lower risk fungicidethan trifloxystrobin, as the MRL for this similar fungicide is fourtimes lower (0.5 mg/kg).

3.10. Metalaxyl (log Kow = 1.75)

Metalaxyl is a phenylamide–acylalanine systemic fungicidewith protective and curative properties with a label withholdingperiod of 7 days (APVMA, 2008). It is registered for use in control-ling downy mildew and is absorbed through leaves, stems androots. Label instructions limit application to a maximum of fourper season. Application diary records for three samples showedone application at two months minimum prior to harvest. Residueswere less than 10% of the Australian MRL for grapes and by-prod-ucts. Metalaxyl is low risk given current usage patterns.

3.11. Quinoxyfen (log Kow = 4.7)

Quinoxyfen is registered for protective (not curative) actionagainst powdery mildew and has a withholding period of 14 days(APVMA, 2008). Label advice limits applications to three per sea-son. Reported levels are less than 10% of the Australian MRL. Quin-oxyfen is low risk given the current usage pattern.

3.12. Chlorothalonil (log Kow = 2.9)

Chlorothalonil is a multi site-chloronitrile non-systemic foliarfungicide registered for protective action against downy mildew,black spot and grey mould. The label withholding period is 14 days(APVMA, 2008). Application diaries indicate that it was applied toat least seven samples and records show that the minimal actualwithholding period was 100 days. It was detected in three of thegrape samples at low levels but not detected in seed oil, marc orseed meal samples. It is extremely unlikely that the AustralianMRL of 10 mg/kg would be contravened in grapes or grape by-products under these application conditions.

3.13. Penconazole (log Kow = 3.7)

Penconazole is a DMI:triazole systemic fungicide registered forcontrol of powdery mildew. The label withholding period is14 days. Application is permitted up to three times per season(APVMA, 2008). The Australian MRL is 0.1 mg/kg. It was appliedto a minimum of four samples but not detected in any of the sam-ples analysed. Records showed that the minimum actual withhold-ing period was 84 days with no repeat applications. Due to the lowMRL, penconazole would represent a risk of contravening MRL res-idue levels in grape and grape by-products; however, for this rea-son, growers probably use the chemical with a maximum possiblewithholding period.

Table 4Mean concentration ratios of pesticides in oil/fruit and marc/fruit.

Pesticide log Kow Number of samples Oil/fruit Marc/fruit

Cyprodinil 3.9 6 27 12Procymidone 3.1 9 20 3.6Tebufenozide 4.2 4 10 5.0Indoxacarb 4.6 7 8.5 2.5Fenhexamid 3.5 6 8 3.7Trifloxystrobin 4.5 6/5 6.1 1.6Pyrimethanil 2.8 5 5 2.7Captan 2.8 7 2.4 2.1

G. Rose et al. / Food Chemistry 117 (2009) 634–640 639

3.14. Triadimenol (log Kow = 3.2)

Triadimenol is a systemic DMI:triazole fungicide with protec-tive, curative and eradicant action used for control of powdery mil-dew. Its label withholding period is 7 days (APVMA, 2008) and theAustralian MRL is 0.5 mg/kg. Application is listed on the label forthe period until the fruit is set, so it is unlikely that residues wouldbe reported at harvest. Application diaries indicated that triadime-nol was applied to two samples with withholding periods listed as125 and 146 days, however, triadimenol was not detected in any ofthe samples analysed.

3.15. Myclobutanil (log Kow = 2.9)

Myclobutanil is a systemic DMI:triazole fungicide with protec-tive and curative action, used to control powdery mildew. The labelwithholding period is 14 days (APVMA, 2008) and the AustralianMRL 1 mg/kg. Applications are restricted to three per season; how-ever, it is unlikely that more than one application was made. Appli-cation diaries indicate that myclobutanil was applied to onesample; however, it was not detected in any of the samplesanalysed.

3.16. Spiroxamine

Spiroxamine is a morpholine:spiroketalamine systemic fungi-cide with protective, curative and eradicant action, used to controlpowdery mildew. It is composed of two isomers, A and B, with alog Kow = 2.8 and log Kow = 2.9, respectively. The label withholdingperiod is 28 days (APVMA, 2008) and application was listed inthree application diaries at actual withholding periods of 119,161 and 168 days. Spiroxamine was not detected in any samples.

Table 3 shows data for the diacylhydrazine insecticide, tebufe-nozide, and the oxydiazine insecticide, indoxacarb. These com-pounds are used to control light brown apple moth. Withholdingperiods are 21 days and 8 weeks, respectively.

3.17. Tebufenozide

This was listed in two application diaries (100 and 115 daysprior to harvest) and residues were detected in six samples. Forthe samples with application diaries, the residues were detectedin the seed oil up to 10% of the MRL. However, for sample CH14,where the application was not recorded, the residue level in theseed oil is 30% of MRL. It is possible that tebufenozide residues in

Table 3MRL, label withholding periods and pesticide residues in fruit, marc, seed oil and seed me

Pesticide (MRL) Application: days prior to harvest Sample/varietya

Label WHP (days)

Tebufenozide (2 mg/kg) 115 CH4 (C)Label WHP = 21 days nr CH5 (S)

110 CH8 (C)nr CH9 (C)nr CH11 (P)nr CH14 (M)

Indoxacarb (2 mg/kg) 81 CH9 (C)Label WHP = 56 days 88 CH10 (P)

107 CH11 (P)nr Ref#7 (S)nr Ref#8 (CS)nr Ref#9 (M)140 Ref#10 (CS)

n/s = no sample.nr = no record (diary).

a C = chardonnay, P = pinot, M = merlot, S = shiraz, CS = cabernet sauvignon.

seed oil for this sample would be at the MRL at the minimum with-holding period.

3.18. Indoxacarb

This was listed in four sample application diaries and was re-ported in these samples in addition to three other samples. The ac-tual withholding periods are listed as 81–140 days, in excess of theminimum label withholding period of 56 days. Two samples ofseed oil without application diary records show residues at 40%of the MRL. It is possible that MRL levels of indoxacarb would occurat the minimum withholding period in the seed oil.

3.19. Spinosad

Spinosad is a biopesticide used for controlling light brown applemoth, comprising a mixture of two isomers, A and D. The log Kow is4.0 for the A isomer and 4.5 for the D isomer at pH 7 (Tomlin,2003). The log Kow for both isomers decreases with decreasingpH. The Australian MRL is 0.5 mg/kg. Spinosad was identified intwo application diaries but was not detected in any of the samples.Applications were at 84 and 105 days prior to harvest.

The data in Table 4 show that residue concentrations, for eight ofthe listed pesticides with acceptable sample sets, are significantlyhigher in seed oil than in fruit. The residues are also higher in marcthan in fruit and by calculation, higher in seed oil than marc. A sim-ilar pattern of residue levels for the fungicides, fludioxinil, quinoxy-fen and pyraclostrobin, was also evident for a smaller number ofsamples. The two systemic fungicides (procymidone and cyprodi-nil) have the highest concentration factor in seed oil relative to fruitand marc. This suggests that there is a partitioning of the highly oil-soluble (log Kow > 2.5) fungicides into the oily components of the

al.

Fruit (mg/kg) Marc (mg/kg) Seed oil (mg/kg) Seed meal (mg/kg)

0.02 0.13 0.18 0.130.03 0.05 0.14 0.100.01 0.05 0.21 0.03

<0.01 <0.01 0.03 <0.01<0.01 <0.01 0.02 <0.01

0.05 0.42 0.63 0.36

0.03 0.06 0.15 0.090.05 0.13 0.27 0.190.02 0.07 0.13 0.170.05 0.09 0.41 0.010.07 0.17 0.94 0.030.07 n/s 0.82 0.02

n/s 0.05 0.27 0.01

640 G. Rose et al. / Food Chemistry 117 (2009) 634–640

grape vine, and that systemic activity facilitates transfer to the seedoil. This supports the hypothesis that oil-soluble pesticides, re-moved from must by clarification, are present in the seed oil. Alter-natively, polar (water-soluble) pesticides are more likely to beretained in the wine and metabolised by fermentation.

For cyprodinil (Table 2), the increase in residue levels from fruitto seed oil ranged from 8 (CH9, CH10) to 64 (CH8). Similarly, forprocymidone (Table 2), the increase in residue levels from fruitto seed oil ranged from 4 (CH4) to 40 (CH8, CH14).

4. Conclusion

This study confirms that oil-soluble (log Kow > 2.5) fungicidesand insecticides applied to wine grapevines are concentrated inthe grape seed oil. The concentrations of the pesticide residues inthe seed oil relative to marc and fruit were highest for the systemicfungicides, procymidone and cyprodinil. Thus oil-solubility andsystemic action are confirmed as key risk factors for residue con-tamination in grape marc and seed oil. Thus the pattern of minimalapplied pesticide residues in wine reported in the literature is log-ical, as these mainly oil-soluble pesticides are concentrated in theseeds, a major component of marc.

Acknowledgements

The authors would like to thank Greg Jarratt, Jamie Hewett, An-drew Fleming and Richard Shenfield, of Fosters Ltd., Fred Davies ofStoney Creek Oil Products P/L for supplying samples, Dr. Jun Du, Pei

Zhang and Colin Cook of DPI-Werribee for technical support andDr. Craige Trenerry for assistance in preparing the manuscript. Thisproject was funded by the Department of Primary Industries (Vic-toria) through the Naturally Victorian Initiative.

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