Food Chemistry 117 (2009) 634640

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The fate of fungicide and insecticide residues in Australian wine grape by-products following eld application
Gavin Rose a,*, Simon Lane b, Robert Jordan b
a b

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

a r t i c l e

i n f o

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 oil solubility. Twenty-four samples of grape marc, representing a range of red and white wine grape varieties, were collected and analysed for selected fungicides and insecticides. Fifteen of the 24 samples were matched with insecticide and fungicide application diaries. Residue concentrations of the fungicides, procymidone, iprodione, cyprodinil, fenhexamid, udioxinil, pyrimethanil and trioxystrobin, and the insecticides, indoxacarb and tebufenozide, were higher in grape seed oil and grape seed meal than in the fruit and the marc. The relative concentrations were approximately proportional to the octanol to water partition coefcients, log Kow. A range of other fungicides and insecticides were detected but were not signicantly concentrated in the oil and seed meal relative to fruit and marc. The presence of pesticide residues in grape seed oil and grape seed meal will impact on the possibility of producing these wine by-products. Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.

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

Keywords: Grape seed Grape marc Pesticide residues Systemic pesticides Gas chromatography Liquid chromatographytandem mass spectrometry

1. Introduction Large quantities of grape marc (solids remaining after juice extraction) are produced by regional wineries in Victoria, Australia (Jordan, 2002). Currently, the marc is either fed to livestock or consigned to land ll as the costs for transport and reprocessing into distilled alcohol, tartaric acid or polyphenolic supplements make these options uneconomic. Alternatively, grape seed oil (for human consumption) and seed meal (for animal feed), could be produced from the marc. However, previous work (Cabras & Angioni, 2000; Navarro, Barba, Oliva, Navarro, & Pardo, 1999; Teixeira, Aguiar, Afonso, Alves, & Bastos, 2004; Tsiropolous, Aplada-Sarlis, & Miliadis, 1999) suggests that the grape seed oil and the seed meal may contain unacceptable levels of systemic pesticide residues that were applied to the vines prior to harvest. Residue levels of ve out of six pesticides (penconazole, chlorpyrifos, vinclozolin, fenarimol and metalaxyl) applied to grape vines prior to harvest were higher in grape marc than in the crushed grapes during elaboration of red wines. Also, the residue levels in must, 4 days after vinication, were signicantly lower than in crushed grapes for the six pesticides (including mancozeb) (Navarro et al., 1999). Residue levels of 10 out of 12 fungicides applied to vines prior to harvest were lower in must and wine than in the grapes. Six out of
* Corresponding author. Tel.: +61 3 9742 8750; fax: +61 3 97428700. E-mail address: gavin.rose@dpi.vic.gov.au (G. Rose).

eight of these fungicides had reduced levels in claried must, suggesting that the residues tend to be retained in the solid portion of the must (seeds, skins and stalks). Similarly, the levels of six out of nine organophosphate insecticides were lower in the wine than in the grapes and the levels of ve of these organophosphate insecticides were lower in claried must (after solids removal) than in the must. However, dimethoate and methidathion, the compounds with lowest log Kow (more soluble in water and ethanol), were at higher concentrations in the must and wine than in the marc (Cabras & Angioni, 2000). Another study by Tsiropolous et al. (1999) showed signicantly lower levels of the insecticide teflubenzuron in wine than in the grapes. Claried must residue levels were also signicantly lower than in must suggesting that teflubenzuron residues were associated with the solid portion of seed, skins and stalks rather than the juice of the grape. It was suggested by Teixeira et al. (2004) that systemic pesticides, such as oxydixyl, were preferentially found in the grape pulp, whilst contact pesticides, such as folpet, were present only in the skin. The relationship between the levels in different parts of the grape was also thought to be dependent on the time between application and sampling. This study investigates the potential concentration of applied fungicides and insecticides in grape seed produced from grape marc collected from selected wineries in Victoria, Australia. Additionally, the relationship between the octanol to water coefcient (log Kow) and the concentration of each fungicide/ insecticide in grape seed was explored.

0308-8146/$ - see front matter Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.061

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2. Materials and methods 2.1. Reagents Pesticide hexanes (nanograde 95% n-hexane), nanograde dichloromethane (DCM), and HPLC grade methanol, were all purchased from Mallinkrodt, Kentucky, USA. Acetone was redistilled from AR grade solvent (BDH/Merck, Kilsyth, Australia). HPLC grade acetonitrile 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 mufe furnace at 550 C for 2 h prior to use. Anhydrous AR grade sodium sulphate and anhydrous AR grade ammonium acetate were purchased from Ajax Fine Chemicals, Sydney Australia. Water was Type 1 (18 Megohm resistivity). 2.2. Certied standards Cyprodinil (98.5% purity), fenhexamid (99.5%), udioxinil (98.0%), metalaxyl (99.5%), pyraclostrobin (99.7%), trioxystrobin (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. Ehrenstorfer-Schafers, Augsburg, Germany. Indoxacarb (99.3%), pyrimethanil (99.6%), triadimenol (97.7%) and tebufenozide (99.9%) were purchased from the National Measurement Institute, Pymble, Australia. Penconazole (99%) and myclobutanil (99.9%) were purchased from 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 samples Grape 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 were matched with application diaries. A target list of fungicides and insecticides was developed from examination of the available diaries. Fungicides represented the highest proportion of chemicals applied to the vines prior to harvest. There was no record of organophosphate or synthetic pyrethroid insecticide usage. Central Victorian marc samples were collected fresh and transferred to Stoney Creek Oils (SCO) located at Talbot in central Victoria. The seed was wet-separated immediately at SCO and the seed dried on forced air drying screens, sieved and stored for pressing. Yarra Valley marc samples were collected and immediately frozen prior to transfer to SCO. The frozen marc samples were thawed and the seed wetseparated, dried, sieved and stored for pressing. Where possible, matching fruit samples were collected from harvest fruit bins at the wineries. However, due to harvesting patterns, there is no guarantee that the fruit samples were representative of chemical usage throughout harvest blocks. A standard linseed oil press was used to cold-press the seed. Residue free seed oil was used to clean the oil press between samples. Seed meal samples were also collected from each seed batch processed and analysed for organic residue content. 2.3.2. Fruit and marc extraction Fifty grammes of marc or homogenised grape berry were extracted with 200 ml of acetone using a probe homogeniser. The sample was vacuum-ltered, the ltrate mixed with 650 ml of saturated aqueous sodium sulphate and extracted with 1 100 ml and 2 50 ml volumes of DCM. The extracts were combined, passed through an anhydrous sodium sulphate drying column and inverted into 10 ml of hexane.

2.3.3. Seed oil extraction Two grammes of oil were diluted with 25 ml of hexane and extracted with 1 50 ml and a further 2 25 ml of hexane saturated acetonitrile. The acetonitrile layers were combined, dried, and inverted into 2 ml of dichloromethane (DCM). 2.3.4. Seed meal extraction Approximately 25 g of meal were mixed with 200 ml of 35% water/65% acetone and shaken for 30 min. The sample was vacuum-ltered, the ltrate mixed with 650 ml of saturated aqueous sodium sulphate and extracted with 1 100 ml and 2 50 ml volumes of DCM. The DCM volumes were combined, dried through an anhydrous sodium sulphate drying column and inverted into 10 ml of hexane. 2.4. Apparatus 2.4.1. Gas chromatographynitrogen phosphorus detection (GCNPD) The fungicides, chlorothalonil, penconazole, procymidone, iprodione, triadimenol and myclobutanil were determined using a Varian 3400 CX capillary gas chromatograph tted with nitrogenphosphorus detector (Varian, Mulgrave, Australia). An aliquot (2 ll) of hexane (derived for each sample as described above) was simultaneously injected onto parallel columns (15 m, 0.32 ID dimethyl-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 of 1:20. The GC oven was temperature-programmed (120 C 0 2 min, 120300 C at 20 C/min, held 300 C for 1 min) for optimum separation efciency. The injector and detector temperatures were set at 280 and 320 C, respectively. Helium was used as carrier gas. Varian Star software (V6.0) was used to manage the chromatographic data. The organic residues were quantied by comparison with external standards. 2.4.2. Gas chromatographyelectron capture detection (GCECD) The fungicide captan was determined using a Varian 3800 capillary gas chromatograph tted with an electron capture detector (Varian, Mulgrave, Australia). The hexane extract obtained from sample processing, as described above, was further puried by adding 0.5 ml of the extract to 3.0 g of activated orisil, followed by elution with 25 ml of 1% acetonitrile/49% hexane/50% DCM. This was inverted to 2 ml of hexane. An aliquot (2 ll) of hexane was injected simultaneously onto parallel capillary columns (15 m, 0.32 ID 14% cyanopropyl-phenyl on dimethyl-polysiloxane stationary phase, J&W DB-1701) and a 15 m, 0.32 ID, 25% cyanopropyl, 25% diphenyl, 50% dimethyl-polysiloxane stationary phase (Restek, RTX-225) via a split/splitless injector with a split ratio of 1:20. The GC oven temperature was set at 200 C. The injector and detector temperatures were set at 280 and 350 C, respectively. Helium was used as carrier gas. Varian Star software (V6.0) was used to manage the chromatographic data. The organic residues were quantied by comparison with external standards. 2.4.3. Liquid chromatographytandem mass spectrometry (LCMS/ MS) The fungicides, cyprodinil, fenhexamid, metalaxyl, udioxinil, pyraclostrobin, quinoxyfen, trioxystrobin and pyrimethanil, and the insecticides, spinosad, spiroxamine, indoxacarb and tebufenozide, were determined using a Waters 2975 separation module interfaced with a Waters Micromass Quattro Micro tandem mass spectrometer operating in the positive ion electrospray mode (Waters Micromass, Manchester, UK). An aliquot (0.2 ml) of the hexane extract, obtained as described above, was inverted into methanol (4 ml) containing 0.06 lg/ml of tributylphosphate (TBP) as internal standard. The compounds were separated with

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a 150 mm 2.0 mm Luna 5 lm C18(2) analytical HPLC column tted with a C18 guard column. The HPLC column was maintained at 25 C. The mobile phase consisted of (A) 20% methanol in 5 mM ammonium acetate and (B) 90% methanol in 5 mM ammonium acetate with the following gradient: 100% A100% B (015 min), 100% B (1528 min), 100% B100% A (2830 min) with a ow rate of 0.2 ml/min. Masslynx Software (V4.0) was used for data processing. Residues were quantied using external standards and each standard set was assayed a minimum of three times during each sample batch run. Each standard also contained TBP (0.06 lg/ml) as the internal standard. Sample and recovery concentrations were calculated from a linear regression of the standards. Samples and standards were corrected for internal standard (IS) response. The tandem mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The precursor ions, product ions, conrmatory product ions and retention times for each compound are listed in Table 1. 2.5. Quality control Spiked recoveries for each residue were included in every sample extraction batch and the recoveries determined as described for samples. Samples were spiked in the range of 0.051.2 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 oil and 51% in seed meal. The reported data were not corrected for batch recoveries. The lower recovery of LCMS/MS analytes in grapes is related to the matrix suppression of mass spectrum ion response routinely observed in extracts from fruit high in moisture and soluble sugars, such as grapes and pome fruit. This effect was also observed in LCMS/MS in the extracts of seed oil and seed meal. Matrix enhancement of the LC.MS/MS response was observed for pyrimethanil, and the strobilurin fungicides, pyraclostrobin and trioxystrobin. 3. Results and discussion 3.1. General Table 2 shows the residue levels present in fruit, marc, seed oil and 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 Australia and New Zealand (FSANZ) Code (Food Standards Australia New

Zealand, 2006). FSANZ MRLs for agricultural produce are applied to derived foods, such as seed oils, where there is no separate MRL listed. 3.2. Cyprodinil and udioxinil The anilinopyrimidine systemic fungicide cyprodinil and the phenylpyrrole non-systemic fungicide udioxinil are applied in a mixture formulated to control grey mould, Botrytis cinerea (Australian Pesticide and Veterinary Medicine Authority (APVMA), 2008). The formulated mixture has a withholding period of four weeks and 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 have been above the MRL at the withholding period. Label instructions limit applications prior to grape veraison at the latest, so it is unlikely that any crop would be harvested at the minimum withholding period. The relative concentrations of the fungicides in the samples reect the composition of the formulation (37.5% cyprodinil and 25% udioxinil). 3.3. Procymidone The systemic dicarboximide fungicide procymidone is registered for control of grey mould in wine grapes and has a withholding period of 7 days (APVMA, 2008). Seven out of nine seed oil samples contained residues above the Australian MRL of 2 mg/kg (Food Standards Australia New Zealand (FSANZ), 2006). Four out of nine marc samples also contained residues above the MRL. The residues for grape sample CH4 was more than twice the MRL for grapes, indicating that the withholding period may be too short, especially where multiple applications have been made prior to harvest. There were no application diary records for samples CH8, CH14 and Ref#14. The data suggest that it is likely that sample CH15 had an unrecorded application close to the withholding period. Sample CH15 also reported a grape sample above the MRL, supporting the previous observation that the withholding period was too short for the listed MRL. Results show that procymidone is a high-risk fungicide for seed oil, marc, seed meal and grapes in these samples. In 2005, following numerous international reports of its toxicity, additional application and usage controls for procymidone, to control grey mould in wine grapes, were implemented by the Australian Pesticide and Veterinary Medicine Authority. The revised controls have resulted in the phase-out of procymidone use on wine grapes in Australia.

Table 1 LCMS/MS precursor ions, product ions, conrmatory product ions and retention times for the fungicides and pesticides measured in this study. Fungicide/insecticide Cyprodinil Fenhexamid Fludioxinil Pyraclostrobin Quinoxyfen Trioxystrobin Pyrimethanil Spinosad A Spinosad D Spiroxamine Indoxacarb Tebufenozide Metalaxyl Tributylphosphate (TBP)a na = not available. a TBP is the internal standard. Precursor ion (m/z) 226.10 301.97 248.98 388.06 307.90 409.18 200.11 732.36 746.67 298.23 528.15 353.40 280.35 267.44 Production quantitative (m/z) 92.64 96.79 157.86 162.96 196.88 185.94 106.92 141.91 141.89 143.92 202.91 297.03 220.10 98.78 Production conrmatory (m/z) 117.86 142.70 185.00 132.80 161.85 144.81 81.90 na na 99.97 149.82 na na na Retention time (min) 18.99 17.70 17.13 19.04 21.32 19.62 16.94 24.87 26.58 21.09 19.42 18.35 15.47 19.83

G. Rose et al. / Food Chemistry 117 (2009) 634640 Table 2 MRL, label withholding periods and fungicide residues in fruit, marc, seed oil and seed meal. Fungicide (MRL) Label WHP (days) Cyprodinil (2 mg/kg) Label WHP = 28 days Application: days prior to harvest nr 72 81 88 nr nr 81 88 nr 9, 30, 84 28, 105 nr 34 40 43 nr 77 nr 115 132 110 95 101 nr 119 140 120 nr nr nr 51, 84, 140 72, 105, 161 46, 96 77 52 90 77 nr nr nr nr nr nr nr nr 102 108 120 nr 145 nr nr nr 152 110 nr 68 74 64 nr nr 72 115 122 Sample/varietya CH4 (C) CH8 (C) CH9 (C) CH10 (P) CH14 (M) Ref#14 (S) CH9 (C) CH10 (P) Ref#14 (S) CH4 (C) CH5 (S) CH8 (C) CH9 (C) CH10 (P) CH11 (P) CH14 (M) CH15 (S) Ref#14 (S) CH4 (C) CH5 (S) CH8 (C) CH9 (C) CH10 (P) Ref#14 (S) CH4 (C) CH5 (S) CH11 (P) CH14 (M) CH15 (S) Ref#14 (S) CH4 (C) CH5 (S) CH8 (C) CH9 (C) CH10 (P) CH14 (M) CH15 (S) Ref#1 (R) Ref#6 (S) Ref#7 (S) Ref#8 (CS) Ref#9 (M) Ref#10 (CS) Ref #17 (S) Ref #18 (S) CH9 (C) CH10 (P) CH11 (P) Ref#7 (S) Ref#8 (CS) Ref#9 (M) Ref#10 (CS) Ref#14 (S) Ref#16 (S) CH8 (C) CH15 (S) CH9 (C) CH10 (P) CH11 (P) Ref#14 (S) CH4 (C) CH8 (C) Ref#10 (CS) Ref#16 (S) Fruit (mg/kg) <0.01 0.01 0.08 0.20 <0.01 n/s 0.10 Trace n/s 4.5 0.46 <0.05 0.39 0.90 0.41 0.10 2.2 n/s <0.01 0.01 <0.01 0.03 0.03 n/s 0.12 0.04 0.03 0.02 0.08 n/s 0.29 0.16 0.25 0.14 0.07 0.06 0.66 1.1 n/s <0.05 <0.05 <0.05 n/s n/s n/s 0.008 0.005 0.006 0.011 0.012 0.016 n/s n/s n/s <0.01 0.02 <0.01 <0.01 0.01 n/s <0.01 <0.01 <0.01 n/s Marc (mg/kg) <0.01 0.46 0.20 0.35 <0.01 0.10 0.12 0.21 0.11 8.5 2.1 0.11 1.0 1.8 2.3 0.36 10 0.78 0.06 0.05 0.03 0.13 0.07 0.06 0.47 0.06 0.11 0.05 0.15 0.01 0.16 <0.01 1.6 0.11 <0.01 0.03 <0.01 1.9 <0.05 <0.05 <0.05 n/s <0.05 n/s n/s 0.011 0.006 0.014 0.008 0.025 n/s 0.010 0.016 n/s 0.06 0.07 <0.01 <0.01 0.01 Trace <0.01 0.07 Trace n/s Seed oil (mg/kg) 0.36 0.90 0.63 1.6 0.09 0.18 0.23 0.56 0.27 17 11 1.7 7.0 11 13 3.9 30 1.8 0.49 0.10 0.06 0.25 0.18 0.09 0.29 0.13 0.30 0.11 0.34 0.02 1.2 0.10 0.45 1.0 0.04 0.12 0.11 16 2.2 0.56 0.60 0.52 2.0 16 0.56 0.050 0.020 0.030 0.010 0.150 0.130 0.068 0.042 0.010 0.10 0.20 Trace 0.01 0.02 <0.01 0.04 0.10 0.030 Trace

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Seed meal (mg/kg) 0.01 0.48 0.41 0.84 <0.01 0.29 Trace 0.19 <0.1 14 3.0 0.10 0.64 2.0 2.8 0.48 9.0 0.52 0.08 0.15 0.04 0.14 0.10 0.13 1.1 0.18 0.25 0.14 0.35 0.05 0.14 0.01 0.05 0.02 <0.01 0.02 0.02 2.6 <0.10 <0.10 <0.10 <0.10 <0.10 4.2 <0.10 0.002 0.008 0.005 0.01 0.027 0.020 0.010 0.005 0.003 0.020 0.09 <0.01 0.01 0.04 0.03 <0.01 0.02 Trace <0.01

Fludioxinil (2 mg/kg) Label WHP = 28 days Procymidone (2 mg/kg) Label WHP = 7 days

Fenhexamid (10 mg/kg) Label WHP = 21 days

Pyrimethanil (5 mg/kg) Label WHP = 7 days

Captan (10 mg/kg) Label WHP = 30 days

Iprodione (20 mg/kg) Label WHP = 7 days

Trioxystrobin (0.5 mg/kg) Label WHP = 35 days

Pyraclostrobin (2 mg/kg) Label WHP = 21 days Metalaxyl (MRL = 1 mg/kg) Label WHP = 7 days

Quinoxyfen (2 mg/kg) Label WHP = 14 days

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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3.4. Fenhexamid Fenhexamid is a contact (non-systemic) hydroxyanilide fungicide, used for controlling grey mould. Fenhexamid is registered for a 21-day withholding period on grapes (APVMA, 2008). There is a label restriction with a maximum of two applications per season. Application diary records show one application 34 months prior to harvest. Seed oil samples showed the highest level of residues but these were less than 10% of the Australian MRL. Given the existing usage patterns, fenhexamid is a low risk fungicide. 3.5. Pyrimethanil Pyrimethanil is a contact anilinopyrimidine fungicide used for control of grey mould. The withholding period for pyrimethanil application on grapes is 7 days (APVMA, 2008). All samples were reported at less than 25% of the Australian MRL. Three of the six reported samples had application diary records which indicated application four months prior to harvest. It is likely that the three samples without application diary records were also treated 34 months prior to harvest. Following this usage pattern, pyrimethanil is a low risk fungicide for residues in seed oil and marc. 3.6. Captan Captan is a contact multi-site phthalimide fungicide. The registered product has a withholding period of 7 days on grapes and is used for control of grey mould, black spot (Elsinoe ampelina), downy mildew (Plasmopara viticola) and phomopsis leaf blight (Phomopsis viticola) (APVMA, 2008). Up to ve applications are allowed per season. Three of the seven samples showed captan use more than once in the season. All applications were at a minimum of 45 days withholding period and reported residues were all less than 20% of the Australian MRL. As expected, the sample with the highest residue level (1.6 mg/kg in seed oil) was the sample with the shortest actual withholding period. Captan is a low risk fungicide in the samples tested with the reported usage patterns. 3.7. Iprodione Iprodione is a contact dicarboximide fungicide used for controlling grey mould (B. cinerea). The withholding period is 7 days with a recommendation to limit application to a maximum of two sprays per season (APVMA, 2008). None of the available application diaries showed applications of iprodione; however, iprodione residues were detected in samples for which application diaries were not available. Two of the reported seed oil samples contained 16 mg/kg of iprodione, or 75% of the Australian MRL. It is likely that iprodione was used with an application pattern similar to procymidone. Results show that iprodione is a high-risk fungicide for seed oil, marc, seed meal and grapes in these samples. 3.8. Trioxystrobin

marc, seed oil or meal would be at 50% of the Australian MRL, so trioxystrobin is a low risk fungicide for the production of grape seed oil and seed meal for commercial purposes. The reported levels are lower than for most fungicides, due to the exceptional sensitivity of the LCMS/MS instrument for the determination of trioxystrobin. 3.9. Pyraclostrobin (log Kow = 4.0) Pyraclostrobin is used for controlling powdery mildew and downy mildew. The label withholding period is 21 days. The label stipulates a maximum of three applications at 1014 day intervals, commencing at owering (APVMA, 2008), therefore it is unlikely to be applied within three months of harvest. Two samples of seed oil contained pyraclostrobin residues at 10% of the Australian MRL. Pyraclostrobin is a low risk fungicide providing label usage is followed. Pyraclostrobin is considered to be a lower risk fungicide than trioxystrobin, as the MRL for this similar fungicide is four times lower (0.5 mg/kg). 3.10. Metalaxyl (log Kow = 1.75) Metalaxyl is a phenylamideacylalanine systemic fungicide with protective and curative properties with a label withholding period of 7 days (APVMA, 2008). It is registered for use in controlling downy mildew and is absorbed through leaves, stems and roots. Label instructions limit application to a maximum of four per season. Application diary records for three samples showed one application at two months minimum prior to harvest. Residues were less than 10% of the Australian MRL for grapes and by-products. Metalaxyl is low risk given current usage patterns. 3.11. Quinoxyfen (log Kow = 4.7) Quinoxyfen is registered for protective (not curative) action against powdery mildew and has a withholding period of 14 days (APVMA, 2008). Label advice limits applications to three per season. Reported levels are less than 10% of the Australian MRL. Quinoxyfen is low risk given the current usage pattern. 3.12. Chlorothalonil (log Kow = 2.9) Chlorothalonil is a multi site-chloronitrile non-systemic foliar fungicide 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 to at least seven samples and records show that the minimal actual withholding period was 100 days. It was detected in three of the grape samples at low levels but not detected in seed oil, marc or seed meal samples. It is extremely unlikely that the Australian MRL of 10 mg/kg would be contravened in grapes or grape byproducts under these application conditions. 3.13. Penconazole (log Kow = 3.7)

Trioxystrobin is a strobilurin type fungicide, used for controlling powdery mildew (Uncinula necator) and suppression of downy mildew. This fungicide is mesostemic in action and it also has translaminar activity and can be redistributed by supercial vapour movement (Tomlin, 2003). The label withholding period is 35 days. Label instructions limit usage prior to grape bunch closure (APVMA, 2008), thus it is unlikely that application would be made three months prior to harvest. This would include the samples for which no application diary records were available. Five samples had application diary records indicating application had actual withholding periods of more than one hundred days prior to harvest. With these application patterns, it is unlikely that grape,

Penconazole is a DMI:triazole systemic fungicide registered for control of powdery mildew. The label withholding period is 14 days. Application is permitted up to three times per season (APVMA, 2008). The Australian MRL is 0.1 mg/kg. It was applied to a minimum of four samples but not detected in any of the samples analysed. Records showed that the minimum actual withholding period was 84 days with no repeat applications. Due to the low MRL, penconazole would represent a risk of contravening MRL residue levels in grape and grape by-products; however, for this reason, growers probably use the chemical with a maximum possible withholding period.

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3.14. Triadimenol (log Kow = 3.2) Triadimenol is a systemic DMI:triazole fungicide with protective, curative and eradicant action used for control of powdery mildew. Its label withholding period is 7 days (APVMA, 2008) and the Australian MRL is 0.5 mg/kg. Application is listed on the label for the period until the fruit is set, so it is unlikely that residues would be reported at harvest. Application diaries indicated that triadimenol was applied to two samples with withholding periods listed as 125 and 146 days, however, triadimenol was not detected in any of the samples analysed. 3.15. Myclobutanil (log Kow = 2.9)

seed oil for this sample would be at the MRL at the minimum withholding period. 3.18. Indoxacarb This was listed in four sample application diaries and was reported in these samples in addition to three other samples. The actual withholding periods are listed as 81140 days, in excess of the minimum label withholding period of 56 days. Two samples of seed oil without application diary records show residues at 40% of the MRL. It is possible that MRL levels of indoxacarb would occur at the minimum withholding period in the seed oil. 3.19. Spinosad

Myclobutanil is a systemic DMI:triazole fungicide with protective and curative action, used to control powdery mildew. The label withholding period is 14 days (APVMA, 2008) and the Australian MRL 1 mg/kg. Applications are restricted to three per season; however, it is unlikely that more than one application was made. Application diaries indicate that myclobutanil was applied to one sample; however, it was not detected in any of the samples analysed. 3.16. Spiroxamine Spiroxamine is a morpholine:spiroketalamine systemic fungicide with protective, curative and eradicant action, used to control powdery mildew. It is composed of two isomers, A and B, with a log Kow = 2.8 and log Kow = 2.9, respectively. The label withholding period is 28 days (APVMA, 2008) and application was listed in three 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, tebufenozide, and the oxydiazine insecticide, indoxacarb. These compounds are used to control light brown apple moth. Withholding periods are 21 days and 8 weeks, respectively. 3.17. Tebufenozide This was listed in two application diaries (100 and 115 days prior to harvest) and residues were detected in six samples. For the samples with application diaries, the residues were detected in the seed oil up to 10% of the MRL. However, for sample CH14, where the application was not recorded, the residue level in the seed oil is 30% of MRL. It is possible that tebufenozide residues in

Spinosad is a biopesticide used for controlling light brown apple moth, comprising a mixture of two isomers, A and D. The log Kow is 4.0 for the A isomer and 4.5 for the D isomer at pH 7 (Tomlin, 2003). The log Kow for both isomers decreases with decreasing pH. The Australian MRL is 0.5 mg/kg. Spinosad was identied in two 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 of the listed pesticides with acceptable sample sets, are signicantly higher in seed oil than in fruit. The residues are also higher in marc than in fruit and by calculation, higher in seed oil than marc. A similar pattern of residue levels for the fungicides, udioxinil, quinoxyfen and pyraclostrobin, was also evident for a smaller number of samples. The two systemic fungicides (procymidone and cyprodinil) have the highest concentration factor in seed oil relative to fruit and marc. This suggests that there is a partitioning of the highly oilsoluble (log Kow > 2.5) fungicides into the oily components of the

Table 4 Mean concentration ratios of pesticides in oil/fruit and marc/fruit. Pesticide Cyprodinil Procymidone Tebufenozide Indoxacarb Fenhexamid Trioxystrobin Pyrimethanil Captan log Kow 3.9 3.1 4.2 4.6 3.5 4.5 2.8 2.8 Number of samples 6 9 4 7 6 6/5 5 7 Oil/fruit 27 20 10 8.5 8 6.1 5 2.4 Marc/fruit 12 3.6 5.0 2.5 3.7 1.6 2.7 2.1

Table 3 MRL, label withholding periods and pesticide residues in fruit, marc, seed oil and seed meal. Pesticide (MRL) Label WHP (days) Tebufenozide (2 mg/kg) Label WHP = 21 days Application: days prior to harvest 115 nr 110 nr nr nr 81 88 107 nr nr nr 140 Sample/varietya CH4 (C) CH5 (S) CH8 (C) CH9 (C) CH11 (P) CH14 (M) CH9 (C) CH10 (P) CH11 (P) Ref#7 (S) Ref#8 (CS) Ref#9 (M) Ref#10 (CS) Fruit (mg/kg) 0.02 0.03 0.01 <0.01 <0.01 0.05 0.03 0.05 0.02 0.05 0.07 0.07 n/s Marc (mg/kg) 0.13 0.05 0.05 <0.01 <0.01 0.42 0.06 0.13 0.07 0.09 0.17 n/s 0.05 Seed oil (mg/kg) 0.18 0.14 0.21 0.03 0.02 0.63 0.15 0.27 0.13 0.41 0.94 0.82 0.27 Seed meal (mg/kg) 0.13 0.10 0.03 <0.01 <0.01 0.36 0.09 0.19 0.17 0.01 0.03 0.02 0.01

Indoxacarb (2 mg/kg) Label WHP = 56 days

n/s = no sample. nr = no record (diary). a C = chardonnay, P = pinot, M = merlot, S = shiraz, CS = cabernet sauvignon.

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grape vine, and that systemic activity facilitates transfer to the seed oil. This supports the hypothesis that oil-soluble pesticides, removed from must by clarication, are present in the seed oil. Alternatively, polar (water-soluble) pesticides are more likely to be retained in the wine and metabolised by fermentation. For cyprodinil (Table 2), the increase in residue levels from fruit to seed oil ranged from 8 (CH9, CH10) to 64 (CH8). Similarly, for procymidone (Table 2), the increase in residue levels from fruit to seed oil ranged from 4 (CH4) to 40 (CH8, CH14). 4. Conclusion This study conrms that oil-soluble (log Kow > 2.5) fungicides and insecticides applied to wine grapevines are concentrated in the grape seed oil. The concentrations of the pesticide residues in the seed oil relative to marc and fruit were highest for the systemic fungicides, procymidone and cyprodinil. Thus oil-solubility and systemic action are conrmed as key risk factors for residue contamination in grape marc and seed oil. Thus the pattern of minimal applied pesticide residues in wine reported in the literature is logical, as these mainly oil-soluble pesticides are concentrated in the seeds, a major component of marc. Acknowledgements The authors would like to thank Greg Jarratt, Jamie Hewett, Andrew Fleming and Richard Sheneld, of Fosters Ltd., Fred Davies of Stoney Creek Oil Products P/L for supplying samples, Dr. Jun Du, Pei

Zhang and Colin Cook of DPI-Werribee for technical support and Dr. Craige Trenerry for assistance in preparing the manuscript. This project was funded by the Department of Primary Industries (Victoria) through the Naturally Victorian Initiative. References
Australian Pesticide and Veterinary Medicine Authority (APVMA). (2008). Web address: <http://services.apvma.gov.au/PubcrisWebClient/welcome. do; jsessionid=vskyFtjLZKvxGrpbpnfpZXLRLqj9Z390Z9Gk5JWF2nQBccpBXFFw! 546591743>. (The pesticide label is accessed by typing the pesticide Active Constituent 1 box in the PUBCRIS database search page). Cabras, P., & Angioni, A. (2000). Pesticide residues in grapes, wine and their processing products. Journal of Agriculture and Food Chemistry, 48, 967973. Food Standards Australia New Zealand. (2006). Web address: <http://www. foodstandards.gov.au/thecode/foodstandardscode/standard142maximumre4244. cfm>. Jordan, R. (2002). Grape marc utilisation-cold pressed grapeseed oil and meal. Technical report for the Cooperative Research Centre for International Food Manufacture and Packaging Science, Melbourne, Australia. Navarro, S., Barba, A., Oliva, J., Navarro, G., & Pardo, F. (1999). Evolution of residual levels of six pesticides during elaboration of red wines. Effect of wine-making procedures in their disappearance. Journal of Agriculture and Food Chemistry, 47, 264270. Teixeira, M. J., Aguiar, A., Afonso, A. M. M., Alves, A., & Bastos, M. S. M. (2004). Comparison of pesticides levels in grape skin and in the whole grape by a new liquid chromatographic multiresidue methodology. Analytica Chimica Acta, 513, 333340. Tomlin, C. D. S. (2003). The pesticide manual (13th ed.). Alton, UK: British Crop Protection Council. Tsiropolous, N. G., Aplada-Sarlis, P. G., & Miliadis, G. E. (1999). Evaluation of teubenzuron residue levels in grapes exposed to eld treatments and in the must and wine produced from them. Journal of Agriculture and Food Chemistry, 47, 45834586.