Dissipation of Fungicide Residues during Winemaking and Their

Article pubs.acs.org/JAFC

Dissipation of Fungicide Residues during Winemaking and Their Effects on Fermentation and the Volatile Composition of Wines Raquel Noguerol-Pato,† Tania Fernández-Cruz,† Thais Sieiro-Sampedro,† Carmen González-Barreiro,† Beatriz Cancho-Grande,† Diego-Augusto Cilla-García,§ Marıá García-Pastor,§ ́ Marıa-Teresa Martínez-Soria,§ Jesús Sanz-Asensio,§ and Jesús Simal-Gándara*,† †

Nutrition and Bromatology Group, Analytical and Food Chemistry Department. Faculty of Food Science and Technology, University of Vigo, E-32004 Ourense, Spain § Analytical Chemistry Group, Chemistry Department, Faculty of Sciences, University of La Rioja, E-26006 Logroño, Spain ABSTRACT: The effects of four fungicides commonly used for the control of fungal diseases in vines and grapes in the course of winemaking were tested. The concentration of fungicide residues was monitored throughout the process to establish their kinetics of dissipation. In all cases the percentages of dissipation were >68%, which shows the detoxificant effect of the winemaking process. On the other hand, the effect of the fungicide residues on the aroma composition of Tempranillo red wines was tested. To evaluate possible modifications on the aroma profile of wines, seven odorant series (ripe fruits, fresh fruits, lactic, floral, spicy, vinous, and herbaceous) were built from the odor activity values (OAVs) obtained for each volatile compound. Ripe fruits and fresh fruits were the major aromatic attributes in all Tempranillo red wines. These two odorant series registered the highest variations in their total OAVs with respect to the control wine, especially with the application of boscalid + kresoximmethyl into vines, leading to a decrease in the ripe fruit and fresh fruit nuances of the resulting wines. Moreover, when the effect of these fungicides on the aroma of Tempranillo red wines was compared throughout two years (2012 and 2013), wines elaborated from grapes treated in the field with boscalid + kresoxim-methyl in 2013 displayed the highest variation in aroma profile with respect to control wine. KEYWORDS: boscalid, kresoxim-methyl, metrafenone, mepanipyrim, fenhexamid, volatile compounds, odorant series, Tempranillo red wines

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compounds, and/or terpene alcohols.24 These volatile compounds are produced through metabolic pathways during ripening and harvest of grapes (varietal and prefermentative aroma), during their fermentation (fermentative aroma), and/ or also during the storage of wines (postfermentative aroma).25 The content of aromatic compounds in wines varies depending on the variety of grape, as well as some factors related to climate and cultural factors,26−28 such as treatment with fungicides,29 which can alter the activity of micro-organisms during alcoholic fermentation. Vitis vinifera var. Tempranillo is the most characteristic variety of the Qualified Designation of Origin “Rioja” and the foundation of the identity of its red wines. This variety produces wines balanced in alcohol content, color, and acidity, which can be submitted to long aging. The objectives of the current study were, on the one hand, (i) to know the evolution of fungicides used against powdery mildew (boscalid, kresoximmethyl, and metrafenone) and gray mold (mepanipyrim and fenhexamid) throughout the winemaking process and (ii) to evaluate the influence of alcoholic fermentation in the dissipation of the residues of those compounds. On the other hand, this study also evaluated the effect of the application of

INTRODUCTION One of the main difficulties during the cultivation of grapes for winemaking is the fight against fungal deseases. Currently, the most efective method to counteract their negative effects on grape quality and yield is still the application of fungicides. The most common pesticides in viticulture are fungicides used to control gray mold (Botrytis cinerea), powdery mildew (Erisiphe necator, formerly Uncinula necator), and downy mildew (Plasmopara viticola), the common fungal diseases in grapevines. However, even when the application of fungicides is carried out according to Good Agricultural Practices (GAPs), fungicide residues may remain on grapes and be transferred to must and wine during winemaking.1−3 This fact can affect yeasts’ activity, resulting in stopped or sluggish fermentation, which is related to the negative effects of residues on the growth and metabolism of both yeasts and lactic acid bacteria.4−9 Consequently, the biosynthesis of volatile compounds and their concentrations in the final wine may also be affected.10−16 Luckily, various enological steps carried out in winemaking, such as clarification and filtration,17−22 can contribute to their reduction, as well as the activity of the microorganisms.17,23 On the other hand, yeasts can influence the levels of fungicide residues in the wine. Wine aroma is highly complex; in fact, it comprises more than a thousand volatile compounds belonging to a wide variety of chemical families such as acids, alcohols, aldehydes, esters, ethers, hydrocarbons, lactones, nitrogen compounds, sulfur © 2016 American Chemical Society

Received: Revised: Accepted: Published: 1344

October 29, 2015 January 11, 2016 January 25, 2016 January 25, 2016 DOI: 10.1021/acs.jafc.5b05187 J. Agric. Food Chem. 2016, 64, 1344−1354

Article

Journal of Agricultural and Food Chemistry

and tetradifon from Riedel-de-Haën with a purity of 99.5% were used as homogenization and extraction, respectively, internal standards (ISTD) for QuEChERS sample preparation. Triphenylphosphate (TPP) from Riedel-de-Haën with a purity of 99.5% was used as quantification injection internal standard (I-ISTD) to compensate for any sample and/or injection volume changes and to correct the variability in gas chromatographic injection and mass spectrometric detection response. Fungicide stock and intermediary standard solutions were prepared in methanol. Standards for determining volatile compounds were purchased from Sigma-Aldrich (Steinheim, Germany) and used to prepare stock standard solutions in ethanol.31 All standard solutions were stored at −20 °C in the dark. Extraction, Separation, Identification, and Quantification Procedures. Fungicides. A modified QuEChERS extraction procedure was developed for must and wine.32 For each sample, 25 mL was accurately weighed into a 50 mL polypropylene centrifuge tube. A volume of homogenization internal standard solution (ISTD diazinon) was added at concentration of 0.5 mg L−1. These were shaken vigorously by vortex for 1 min. Two aliquots of 10 mL of the homogenized sample were weighed (in duplicate) in a 50 mL polypropylene centrifuge tube. Ten milliliters of dichloromethane (1% HOAc) was added as extraction solvent, and the resulting solution was shaken vigorously by vortex (1 min) to ensure that the solvent interacted well with the matrix. Next, 1 g of sodium chloride and 4 g of magnesium sulfate anhydrous were added, and the final mixture was also shaken by vortex (10 min) to prevent salt agglomeration, and the extraction internal standard (ISTD tetradifon) was added at a concentration of 0.5 mg L−1. The extracts were then centrifuged for 6 min at 5000 rpm. A volume of 5 mL of an aliquot was sampled from the upper layer into another centrifuge tube, cleaned by dispersive solid-phase extraction with 250 mg of PSA and 750 mg of magnesium sulfate anhydrous, and the mixture was shaken for 1 min. Then, centrifugation was carried out at 5000 rpm for 6 min. Following centrifugation, the IISTD (TPP) solution was added at a concentration of 0.5 mg L−1 and homogenized by agitation. The final extract was filtered through a 0.22 μm pore membrane filter, transferred into a 2.0 mL glass vial, and analyzed by GC-MS. GC separation was performed using an Agilent Technologies GC 6890N coupled to a 5975C MS mass selective detector. The analytical column used was a HP-5MS with a 30 m length, a 0.25 mm internal diameter, and a 0.25 mm film thickness. The initial oven temperature was set at 100 °C, increased to 150 °C at 40 °C min−1, subsequently increased to 250 °C at 5 °C min−1, held for 5 min, and finally increased at rate of 40 °C min−1 to 300 °C for 10 min. The volume of sample was 2 μL, injected in splitless mode with a splitless time of 0.9 min. The injector temperature was set at 250 °C. Helium (99.999% purity) was used as carrier gas at constant flow rate of 1.5 mL min−1. The mass spectrometer was operated in electron impact (70 eV of ion energy), with 6 min of solvent delay; the interface temperature was kept at 310 °C, and the ion source temperature was kept at 230 °C. The dwell time for ion monitoring was 100 ms per ion. A mass range of m/z 50−550 was scanned to confirm the retention times of analytes. For the determination of fungicide residues, selected ion monitoring (SIM) mode was used. For each target compound the most abundant ions of higher m/z were selected (m/z 223 and 223 for mepanipyrim; m/z 116, 131, and 207 for kresoxim-methyl; m/z 97 and 177 for fenhexamid; m/z 325 and 326 for TPP (I-ISTD); m/z 111, 159, and 356 for tetradifon (extraction ISTD); m/z 377 and 393 for metrafenone; m/z 112, 140, and 342 for boscalid; m/z 137, 152, and 179 for diazinon (homogenization ISTD)), which were the most characteristic ones. Confirmation criteria were that the retention times of the compounds in the sample be within ±0.5% of the respective retention times in matrix-matched calibration standards and the intensity ratios of the target and qualifiers ions in the sample be within ±10%. To ascertain the dissipation rate of residues in each winemaking process, the experimental data have been fitted to the following mathematical model:33

those fungicides on the volatile composition and aroma profile of Tempranillo red wines (iii) elaborated from grapes treated under GAPs with boscalid + kresoxim-methyl and metrafenone and (iv) elaborated from crushed grapes directly supplied with mepanipyrim and fenhexamid. Additionally, the aroma profiles of Tempranillo wines obtained in 2012 under the same experimental conditions were compared to those from 2013 to add further value to the conclusions reached.

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MATERIALS AND METHODS

Fungicide Treatments, Winemaking Process, and Wine Samples. Trials were conducted in an experimental vineyard located in Aldeanueva de Ebro, La Rioja (northern Spain), belonging to the Qualified Designation of Origin “Rioja” (DOC Rioja), during two consecutive years (2012 and 2013). The vineyard produced the red grape V. vinifera L. cv. Tempranillo. It was 3000 m2 area, approximately, and contained 20 rows with 40−50 vines each. The gaps between rows and grapevines were 2.6 and 1.2 m, respectively. All vineyard managements were carried out under the same conditions for all vines belonging to the vineyard to guarantee sample homogeneity. The vineyard was split into three experimental plots (A−C). Plot A was left untreated; plots B and C were treated under GAPs with Collis (boscalid 20% and kresoxim-methyl 10%) and Vivando (metrafenone 50%), respectively. Grapes from each plot were harvested separately in September 2012 and October 2013, but only those from the two midle rows were used for the vinifications to avoid contaminations between treatments. Grapes from plot A, in turn, were divided into batches A, D, and E. Batch A was left untreated to use as a control, and batches D and E were spiked with the commercial products Frupica (mepanipyrim 50%) and Teldor (fenhexamid 50%), respectively; both were added at the corresponding amounts to the maximum residue levels (MRLs) of each active ingredient, 3 and 5 mg/kg, respectively. As a result, five vinification experiments were performed in duplicate with the grapes from each batch (A, containing grapes from plot A; B, containig grapes from plot B previously treated with boscalid + kresoxim-methyl; C, containing grapes from plot C, previously treated with metrafenone; D, containing grapes from plot A spiked with mepanypirim; and E, containing grapes from plot A spiked with fenhexamid). The winemaking process was carried out in the experimental cellar of the University of La Rioja, and it was identical in all vinification experiments following the winemaking procedure carried out by Noguerol-Pato et al.15 The general parameters (pH, titratable and volatile acidity, reducing sugars, and alcoholic content) of the final wines were measured according to the Organisation of Vine and Wine (OIV) International Methods of Analysis.30 Chemicals and Small Apparatuses. The solvents used included dichloromethane, methanol, and water (HPLC quality), which were purchased from Sigma-Aldrich (Steinheim, Germany), and ethanol absolute (HPLC grade), which was acquired from Scharlau (Barcelona, Spain). HPLC grade acetic acid (HOAc) was supplied by Scharlab (Barcelona, Spain). Sodium chloride, magnesium sulfate, syringes with 10 mL barrels, and glass wool were purchased from Scharlau (Barcelona, Spain). Bondesil-PSA (primary−secondary amine) was supplied by Sigma-Aldrich (Madrid, Spain). Teflon polytetrafluoroethylene (PTFE) filters, 0.22 μm, were purchased from Sigma-Aldrich (Madrid, Spain). Anhydrous sodium sulfate for residue analysis were obtained from Panreac (Barcelona, Spain). The sorbent material used for solid-phase extraction (SPE) of volatile compounds was Strata-X, 33 μm polymeric reversed phase (500 mg, 6 mL size) from Phenomenex (Torrance, CA, USA). Small apparatuses such as an Ultrasons-H ultrasound bath (JP Selecta, Barcelona, Spain), a Reax Top vortex (Heidolph, Schwabach, Germany), a Visiprep SPE vacuum manifold (Supelco, Bellefonte, PA, USA), and a Turbo Vap LV evaporator (Caliper Life Sciences, Hopkinton, MA, USA) were also used. Pesticide analytical standards of boscalid, kresoxim-methyl, mapanipyrim, fenhexamid, and metrafenone with purity >99.0% were purchased from Riedel-de-Haën (Seelze, Germany). Diazinon 1345

DOI: 10.1021/acs.jafc.5b05187 J. Agric. Food Chem. 2016, 64, 1344−1354

Article

Journal of Agricultural and Food Chemistry Table 1. Classification of Volatile Compounds into Odorant Series According to their Odor Descriptors volatile compound

odor threshold (μg/L)

monoterpenes (±)-linalol α-terpineol (±)-β-citronellol geraniol trans,trans-farnesol C13 norisoprenoids β-damascenone β-ionone C6 alcohols 1-hexanol trans-3-hexen-1-ol cis-3-hexen-1-ol aliphatic alcohols 2-methyl-1-propanol 1-butanol isoamyl alcohols 4-methyl-1-pentanol 3-methyl-1-pentanol 1-octanol aromatic alcohols benzyl alcohol 2-phenylethanol esters ethyl isovalerate ethyl (±)-2methylbutyrate isopentyl acetate ethyl caproate hexyl acetate ethyl lactate ethyl caprylate ethyl 3hydroxybutyrate

b

15 250c 100d 30b 1000e

odorant seriesa

odor descriptors orange flowers, citrus lilac rose, citrus geranium, rose, citric floral, balsamic

2, 4 4 2, 4 2, 4 4

dry plum violets

1 4

grass green grass

7 7 7

40000d 150000d 30000b 50000f 50000f 10000f

alcohol alcohol alcohol almond, toasted vinous, herbaceous rose, jasmine, citrus

5 5 5 2, 6 5, 7 2, 4

200000d 10000b

walnut, fruity rose

2 4

0.05b 0.09c 8000b 1000e 400b

c

3 18c 30c 14d 1500c 154636c 5d 20000d

berry, blackberry strawberry, green apple banana green apple, banana apple, pear, banana strawberry, raspberry, buttery pineapple, strawberry grape-like

R t = R 0 e−Kt

volatile compound diethyl malonate ethyl decanoate ethyl laurate 2-phenylethyl acetate diethyl succinate diethyl malate acids acetic acid isobutyric acid butanoic acid isovaleric acid caproic acid caprylic acid capric acid volatile phenols guaiacol eugenol syringol vanillin ethyl vanillate acetovanillone lactones γ-nonalactone

odorant seriesa

100000g 200c 500e 250b

fruity, sweet sweet, fruity fruity, floral rose

1 1 2, 4 4

200000d 760000d

wine-like over-ripe, peach

5 1

200000b 2300d 173d 33c 420c 500c 1000d

pungent, vinegar rancid, butter, cheese rancid, butter, sweat acid, rancid sweat sweat, cheese rancid fat

3 3 3 3 3 3 3

sweet, smoky clove, liquorice smoky vanilla honey, vanillin vanilla, clove

6 6 6 6 6 6

coconut

1

10c 6c 570h 60d 990d 1000d 30c

1, ripe fruits; 2, fresh fruits; 3, lactic; 4, floral; 5, vinous; 6, spicy; 7, herbacoeus. bGuth.36 The matrix was 10% water/ethanol solution. c Ferreira et al.37 The matrix was a 11% water/ethanol solution containing 7 g/L glycerol and 5 g/L tartaric acid, with the pH adjusted to 3.4 with 1 M NaOH. dEtiévant.25 The matrix was a 12% water/ ethanol solution. eMoyano et al.38 The matrix was a 14% ethanol solution adjusted to pH 3.5 with tartaric acid.. fMoreno et al.39 The matrix was a 14% water/ethanol solution. gDetermined by authors, the matrix was a 10% water/ethanol solution at pH 3.4. hLópez et al.40 The matrix was a 10% water/ethanol solution at pH 3.2.

2 2 1 1, 2 1, 2 2, 3 1, 2 2, 5

González-Á lvarez et al.35 with slight modifications.30 Wine samples (50 mL) containing 20 μL of 4-nonanol (50 mg L−1 in ethanol) as surrogate standard were loaded in a Strata-X cartridge previously conditioned with methanol (17 mL) and water (20 mL at pH 3.7). A cleaning step with water (20 mL at pH 3.7) was performed after the sample loading. Subsequently, the sorbent was dried by passing N2 for 45 min, and then volatiles were eluted with dichloromethane (10 mL). The eluate was dried over anhydrous sodium sulfate, concentrated to a volume 0.9044 for alcoholic fermentation. Data obtained indicate that there was statistically significant correlation between the quantity of residues remaining and the time with

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RESULTS AND DISCUSSION Vinification. The evolution of the vinifications was controlled by the measurement of the density (% sugar). In both years, 2012 and 2013, the application of fungicide treatments did not modify the evolution of the fermentation process, which lasted between 15 and 17 days for all assays. In particular, wines elaborated from grapes treated with boscalid + kresoxim-methyl in 2013 showed a delay in the evolution of its fermentation, although it was not significant, and the process concluded at the same time as the rest of the vinifications. Also, there were no significant differences between treatments and years with respect to the enological parameters determined in the wines elaborated (Table 2A). pH values were between 3.5 and 3.8 and alcoholic strengths between 13.7 and 15.0 (% 1347


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Table 3. Votalile Compounds in the Final Tempranillo Red Wines Elaborated in 2013 (Mean Values and SD, n = 3 Replicates) concentration ± SD (μg/L) b

volatile compound

wine Aa (control)

wine B (boscalid + kresoximmethyl)

wine Cc (metrafenone)

wine Dd (mepanipyrim)

wine Ee (fenhexamid)

Varietal Compounds monoterpenes (±)-linalool α-terpineol (±)-β-citronellol geraniol trans,trans-farnesol sum C13-norisoprenoids β-damascenone β-ionone sum

3.2 ± 0.37 0.58 ± 0.10 3.8 ± 0.53

C6 alcohols 1-hexanol trans-3-hexen-1-ol cis-3-hexen-1-ol sum

1611 42 440 2094

± ± ± ±

244 5.5 99 247

622 542 143071 45 183 18 144483

± ± ± ± ± ± ±

153 263 26557 11 48 3.4 20452

aliphatic alcohols 2-methyl-1-propanol 1-butanol isoamyl alcohols 4-methyl-1-pentanol 3-methyl-1-pentanol 1-octanol sum aromatic alcohols benzyl alcohol 2-phenylethanol sum esters ethyl isovalerate ethyl (±)-2-methylbutyrate isopentyl acetate ethyl caproate hexyl acetate ethyl lactate ethyl caprylate ethyl 3-hydroxybutyrate diethyl malonate ethyl decanoate ethyl laurate 2-phenylethyl acetate diethyl succinate diethyl malate sumf fatty acids acetic acid isobutyric acid butanoic acid isovaleric acid caproic acid caprylic acid capric acid sum volatile phenols guaiacol eugenol

2.7 0.53 7.7 7.8 8.0 27

± ± ± ± ± ±

0.28 0.20 1.4 1.4 1.6 2.9

115 ± 26 40983 ± 4339 41099 ± 43642

2.2 0.17 4.0 5.2 5.6 17

± ± ± ± ± ±

0.26 0.033 0.82 1.0 1.4 1.2

2.2 0.79 7.7 7.1 8.4 26

± ± ± ± ± ±

0.22 0.16 2.0 0.66 1.0 6.0

2.3 0.39 5.2 10 5.8 24

± ± ± ± ± ±

0.32 0.035 0.73 2.8 0.66 6.9

3.0 ± 0.47 3.8 ± 0.62 0.66 ± 0.16 0.77 ± 0.080 3.7 ± 0.56 4.6 ± 0.55 Prefermentative Compounds

4.2 ± 0.38 0.90 ± 0.17 5.1 ± 0.50

1340 ± 259 1591 ± 253 35 ± 6.9 45 ± 12 390 ± 75 540 ± 93 1765 ± 337 2176 ± 349 Fermentative Compounds

1854 51 452 2357

± ± ± ±

473 5.0 43 605

409 279 105752 55 199 13 106710

± ± ± ± ± ± ±

85 29 29531 17 60 3.7 29618

670 349 101911 36 134 13 103114

± ± ± ± ± ± ±

188 88 22881 4.9 18 2.3 23516

392 258 137522 48 224 15 138460

118 ± 27 41161 ± 4650 41279 ± 4663

± ± ± ± ± ± ±

54 25 14432 5.0 22 2.2 14567

146 ± 24 50821 ± 7583 50967 ± 7597

157 ± 30 39572 ± 4918 39729 ± 4948

2.3 0.52 6.9 8.9 7.2 25

± ± ± ± ± ±

0.44 0.082 1.9 0.96 0.082 4.4

3.8 ± 0.28 0.80 ± 0.12 4.6 ± 0.53

1513 40 369 1921

± ± ± ±

314 3.6 65 380

456 335 159664 57 200 16 160731

± ± ± ± ± ± ±

80 83 45661 2.9 25 2.6 45640

121 ± 11 41072 ± 11392 41193 ± 11387

4.4 4.1 995 760 4.6 10359 515 579 11 24 5.5 30 1877 511 15680

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.9 0.86 373 221 1.3 1299 218 179 1.7 10 1.0 8.4 674 224 2186 b

6.6 5.7 562 256 4.9 2263 231 170 7.7 11 6.5 38 830 716 5107

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.2 0.94 113 64 1.9 600 76 54 0.75 1.4 2.2 3.9 298 63 2648 a

8.4 7.6 818 441 6.0 5787 350 328 9.0 16 3.9 37 1158 634 9605

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.7 3.3 127 184 1.5 2471 137 93 2.2 3.0 0.34 2.9 462 41 3708 b

3.6 3.2 694 581 2.1 3485 314 460 8.3 17 7.8 30 1172 648 7426

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.7 0.93 54 121 0.98 623 80 54 2.0 5.6 3.8 5.4 434 145 1252 a

3.7 3.4 987 531 2.5 3140 315 358 11 21 8.8 36 1143 454 7014

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.61 0.75 189 309 1.5 1119 141 81 0.84 0.20 4.6 0.84 183 21 2698 a

144 997 1054 1147 1916 1801 382 7744

± ± ± ± ± ± ± ±

25 254 321 287 337 335 63 1199

194 1316 586 1049 926 898 236 5205

± ± ± ± ± ± ± ±

32 177 79 191 174 142 36 6243

127 1427 790 1530 1291 1164 323 6652

± ± ± ± ± ± ± ±

23 423 175 233 130 162 83 687

44 833 787 869 2136 1625 218 6513

± ± ± ± ± ± ± ±

19 138 113 275 323 208 102 634

86 920 739 767 1762 1475 192 5941

± ± ± ± ± ± ± ±

19 114 30 112 263 437 107 548

10 ± 2.2 4.9 ± 0.52

7.8 ± 1.7 4.0 ± 0.70

12 ± 2.9 5.8 ± 1.2 1348

10 ± 1.3 4.6 ± 0.90

11 ± 1.0 4.9 ± 0.34


Article

Journal of Agricultural and Food Chemistry Table 3. continued concentration ± SD (μg/L) b

volatile compound syringol vanillin ethyl vanillate acetovanillone sum lactones γ-nonalactone sum

wine Aa (control) 4.7 42 150 44 256

± ± ± ± ±

1.1 8.7 33 9.8 65

6.3 ± 2.0 6.3 ± 2.0

wine B (boscalid + kresoximmethyl)

wine Cc (metrafenone)

wine Dd (mepanipyrim)

Fermentative Compounds 6.4 ± 1.4 5.3 ± 0.65 29 ± 2.2 17 ± 0.90 111 ± 7.5 143 ± 11 43 ± 4.8 44 ± 3.0 202 ± 15 227 ± 16

2.8 17 113 54 202

6.6 ± 0.88 6.6 ± 0.88

8.0 ± 1.7 8.0 ± 1.7

7.4 ± 1.0 7.4 ± 1.0

± ± ± ± ±

0.39 3.9 24 11 44

wine Ee (fenhexamid) 4.5 33 126 53 232

± ± ± ± ±

0.94 3.5 15 6.5 25

6.3 ± 0.61 6.3 ± 0.61

a Control wine. bWine elaborated from grapes treated under GAPs with boscalid + kresoxim-methyl. cWine elaborated from grapes treated under GAPs with metrafenone. dWine elaborated with grapes supplied with mepanipyrim after harvesting. eWine elaborated with grapes supplied with fenhexamid after harvesting. fDifferent letters within the same row indicate means significantly different at p < 0.05 (Student’s t test).

t values > theoretical t value at the 95% confidence level (t (n = 4) = 2.78) for alcoholic fermentation. The K values for alcoholic fermentation were very similar between products and years, ranging between 0.147 and 0.122 days−1 for boscalid and between 0.129 and 0.179 days−1 for metrafenone. Kresoximmethyl showed very different K values, 0.068 and 0.105 days−1 in 2012 and 2013, respectively. The half-life values, calculated as pseudo-first-order kinetics, were very similar for boscalid and metrafenone between years: 4.7 and 5.7 days and 5.4 and 3.9 days in 2012 and 2013, respectively, whereas they were very different for kresoxim-methyl, 10.2 and 6.6 days, respectively. As can be seen, in 2013 boscalid and kresoxim-methyl showed similar kinetics with t1/2 of 5.7 and 6.6 days, respectively. In 2013, K values for kresoxim-methyl were higher (approximately 2 times) than 2012; therefore, t1/2 was higher, 10.2 days, and different from that for boscalid, 4.7 days. Cellar Treatments at MRLs. To establish the worst initial conditions in the grape for vinification, fenhexamid and mepanipyrim were directly added to the crushed grapes in the corresponding concentrations to their MRLs in years 2012 and 2013. Once again, there were no significant differences in the concentration of fungicide residues in must after the addition of fungicides (mepanipyrim, 2.39 ± 0.15 mg L−1 in 2012 and 2.43 ± 0.04 mg L−1 in 2013; and fenhexamid, 3.91 ± 0.04 mg L−1 in 2012 and 3.01 ± 0.24 mg L−1 in 2013). As alcoholic fermentation progresses, the dissipation of fungicide residues began, reaching at the end of the process reductions of 83 and 92% in 2012 and 2013, respectively (Table 2B). The linear correlation between Ln Rt and the time was good in all assays, with r2 > 0.885 for alcoholic fermentation. Data obtained indicate that there was statistically significant correlation between the quantity of residues remaining and the time with t values > theoretical t value at the 95% confidence level (t (n = 4) = 2.78) for alcoholic fermentation. The K values for alcoholic fermentation were very similar between products and years, ranging between 0.126 and 0.139 days−1 for mepanipyrim and between 0.107 and 0.154 days−1 for fenhexamid. The half-life values, calculated as pseudo-firstorder kinetics, were very similar for mepanipyrim and fenhexamid between years: 5.5 and 6.5 days and 5.0 and 4.5 days in 2012 and 2013, respectively. Volatile Compounds and Aroma Profile of Tempranillo Red Wines by Using Odorant Series. Table 3 lists the average concentrations of the volatile compounds detected in the final Tempranillo red wines obtained from the different

winemaking assays in 2013. Although the concentration of volatile compounds may have undergone variations as a consequence of the application of fungicides both in field and in the cellar, from a practical point of view, it is important to know whether these possible modifications in the concentration can be sensorily detectable or not. The calculation of OAVs allowed the quick and easy identification of the major volatile compounds in odorant terms, because those compounds displaying OAVs >0.2 are deemed to contribute to the overall wine aroma.41 However, the evaluation of OAV variations one by one introduces certain complexity in the interpretation and presentation of the results as a large number of volatile compounds with different odor descriptors were identified. To solve these setbacks, volatile compounds with similar odor descriptors were grouped into seven odorant series characterized by a generic descriptor: ripe fruits, fresh fruits, lactic, floral, vinous, spicy, and herbaceous. The approach based on odorant series and OAVs allows an indirect evaluation of the organoleptic profile of wines from quantitative data of volatile compounds provided by the chromatographic analysis. Therefore, changes in the total OAVs can offer a comprehensive overview of sensory variations in wines as a result of the fungicide treatment. Although the use of OAVs to estimate the importance of each volatile compounds in the wine is a valuable tool, it must be pointed out that it should be considered an approximation, because additive, synergic, and antagonistic effects are not considered in the calculation. Figure 1a shows the contribution of each odorant series to the aroma profile of the final wines. In addition, percentages of variation in the OAVs of wines by effect of each active substance with respect to the control are depicted in Figure 1b. Only the odorant series fresh fruits showed statistically significant differences in the OAVs among the Tempranillo wines analyzed (Figure 1b). Therefore, it could be assumed that the application of fungicides either in the field or in the cellar barely affected the aroma profile of the final wines. However, trends can be estimated by considering two levels of variation: 20% of variation with respect to the control wine for the major aromatic series (ripe fruits, fresh fruits, and lactic) and 40% of variation with respect to the control wine for the minor ones (floral, spicy, vinous, and herbaceous). Ripe Fruits. Ripe fruit odorant series displayed the highest OAV; hence, it was quantitatively the most important in all wines. Wine B elaborated from grapes treated with boscalid + kresoxim-methyl in the field showed a great decrease by >40% 1349


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Figure 1. (a) Aromatic profile of Tempranillo red wines according to their odorant series (∗ indicates OAVs significantly different at p < 0.05, Student’s t test); (b) percentages of variation with respect to the control wine on the OAV of Tempranillo red wines.

mepanipyrim to Tempranillo ecological musts did lessen the fruity nuances, which were related to a decrease in the concentration of the ester group. In addition, Garcı ́a et al.42 observed a decrease in the levels of ethyl caproate, ethyl caprylate, and ethyl decanoate when cyprodinil, fludioxonil, and pyrimethanil were added to V. vinifera white wines (var. Airén). On the contrary, significant increments in relation to the control wine were observed for ethyl esters (ethyl butyrate, ethyl caprylate, and ethyl caproate) in Monastrell red wines treated with quinoxyfen, kresoxim-methyl, and trifloxystrobin applied under GAP, leading to an increase in the fruity nuances of the resulting wines.11 Fresh Fruits. Fresh fruit notes were decreased in all of the studied wines (>20%) with respect to the control wine,

in the ripe fruit nuances with respect to the control wine (wine A). Among the volatile compounds that belong to this odorant series, β-damascenone (dry plum), isopentyl acetate (banana), ethyl caproate (banana), and ethyl caprylate (pineapple) were the only ones that showed OAVs >1. All of them, except βdamascenone, showed remarkably lower concentrations with respect to wine A and, therefore, OAVs of these compounds are lower in wine B. These are volatile compounds of fermentative origin, which are synthesized enzymatically by yeasts during alcoholic fermentation, so the presence of fungicide residues of boscalid and kresoxim-methyl during winemaking may have affected yeast metabolism, leading to a decrease of the ripe fruit notes in the resulting wine. Recently, Noguerol-Pato et al.9 found the addition of ametoctradin, dimethomorph, and 1350


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Figure 2. Percentages of variation with respect to the control wine on the OAV of Tempranillo red wines of 2012 and 2013 (∗ indicates OAVs significantly different at p < 0.05, Student’s t test): (a) treatment with boscalid + kresoxim-methyl in the field; (b) treatment with metrafenone in the field; (c) addition of mepanipyrim to grapes in the cellar; (d) addition of fenhexamid to grapes in the cellar.

than five carbon atoms (caproic, caprylic, and capric acid) are between 4 and 10 mg/L, they contribute positively to the aroma of the wine, leading to wines with a rounded, smooth taste.43 The presence of fungicide residues in the studied wines seemed not to cause variations in the lactic notes >20% with respect to the control wine; only wine E (fenhexamid) slightly exceeded 25% of variation in the OAV for the lactic series with respect to wine A. Similarly, Oliva et al.11 found that the application of kresoxim-methyl under both GAPs and CAPs also decreased the fatty character of Monastrell red wines. Although the OAVs of this odorant series remained nearly the same, substantial decreases in the concentrations of caproic and caprylic acids were observed in wine B with respect to wine A. This is consistent with previous results from González-Á lvarez et al.,13 who observed a decrease in the concentration of both volatile fatty acids in Godello white wines with the application of other fungicides. Floral. Floral nuances did not apparently experience great changes in Tempranillo red wines whether fungicides were applied in the field or directly added to crushed grapes. Floral odorant series is related to the grape variety from which wines

especially in wine B (boscalid + kresoxim-methyl), which showed a decrease in the OAV for this odorant series of >40% that was statistically significant. Nuances such as citrus, blackberry, strawberry, green apple, or grape-like are included in this odorant series and bestow complexity and freshness to wines. Therefore, the diminution of these attributes in the aroma profile of wines obtained from treated grapes, both field and cellar, detracted from the sensory quality of the wines. Similar to what happened with the ripe fruits series, the main contributors to fresh fruits series were two fermentative volatile compounds, ethyl caproate (green apple) and ethyl caprylate (strawberry); therefore, changes in the concentrations of these volatile compounds were responsible for the decrease of the fresh fruit notes of the studied wines. Because they are synthesized by yeast during alcoholic fermentation, it seems the presence of residues of fungicides may affect yeast metabolism as previously reported by other authors.8,9,11 Lactic. All fatty acids along with ethyl lactate were grouped under the lactic aromatic label. Most fatty acids are related with unpleasant odor descriptors such as rancid, sweat, vinegar, or cheese. However, when the concentrations of acids with more 1351


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Table 4. Contribution of Each Odorant Series (Percent) to the Overall Aroma for Tempranillo Wines Elaborated in 2012 and 2013 wine A

wine B

wine C

wine D

wine E

odorant series

2012

2013

2012

2013

2012

2013

2012

2013

2012

2013

ripe fruits fresh fruits lactic floral vinous spicy herbaceous

48 35 12 2.6 1.6 0.27 0.25

51 34 10 2.7 1.0 0.52 0.26

51 35 10 2.3 1.4 0.24 0.32

53 25 15 4.2 1.3 0.78 0.44

50 33 12 2.9 1.5 0.50 0.33

52 27 15 3.7 1.2 0.67 0.38

51 34 11 2.1 1.5 0.24 0.24

56 28 11 3.9 0.9 0.59 0.37

51 35 10 2.4 1.3 0.31 0.26

57 28 10 3.7 1.0 0.71 0.31

for the herbaceous odorant series. The phenomenon of cellular rupture involved in the prefermentative steps (viz., harvesting, transport, crushing, and pressing of grapes), besides the oxygen incorporation to the medium, allows the enzyme systems (acylhydrolase, lipoxygenase, peroxide cleavage enzyme, and alcohol dehydrogenase) to come in contact with the substrates (linolenic and linoleic acids) present in the berry, starting the mechanism of formation of C6 alcohols.49 Therefore, the application of fungicides seemed not to alter the level of precursors in the grapes or the enzymatic activity of the enzymes implied in the formation of C6 alcohols. No substantial changes in the concentration of 1-hexanol (the major prefermentative alcohol) were found with the application of kresoxim-methyl and fenhexamid to Monastrell red grapes.11 Changes on the Aroma Profile of Tempranillo Red Wines between 2012 and 2013. In 2012, V. vinifera Tempranillo var. grapes harvested from the same experimental vineyard and in a state of maturity similar to those used for the elaboration of the wines studied in this paper were submitted to the same vineyard managements and winemaking assays.15,16 Figure 2 depicts the percentages of variation of the OAVs for each odorant series by active substance for both Tempranillo wines obtained in 2012 as in 2013. In general terms, throughout the two vintages the aroma profile of wines elaborated from grapes treated under GAPs with boscalid + kresoxim-methyl and metrafenone registered more erratic behaviors, whereas wines from grapes spiked with mepanipyrim and fenhexamid showed more similar and shorter variations in the odorant series with respect to the control wines. The application of boscalid + kresoxim-methyl under GAPs in 2012 barely modified the aroma profile of the obtained wine; however, in 2013, fruity attributes (ripe and, especially, fresh fruits) were greatly lessened (>40%) in the presence of these active substances (Figure 2a). This fact could support the hypothesis that the course of fermentation depends on fungicide alongside that of the substrate to ferment. Therefore, this behavior should be checked in the future to establish recommendations for the wine sector with respect to the use of these active substances. None of the odorant series were modified by >40% with the application of metrafenone, mepanipyrim, and fenhexamid in Tempranillo wines from 2012 and 2013 (Figure 2b−d), although it should be noted that the aromatic profiles of wines from 2013 showed a slight decline in the major odorant series (>20% of variation with respect to the control). Nevertheless, because only in 2013 did the odorant series fresh fruits in wines with residues of boscalid + kresoximmethyl show significant variations with respect to the control wine, it should be considered that the presence of residues of these active substances during alcoholic fermentation does not

are obtained because it mainly includes varietal compounds such as monoterpenes and C13-norisoprenoids. Besides, 2phenylethanol, which belongs to the aromatic alcohols group, also contributed to floral notes as it showed concentrations above its odor threshold. Although this volatile compound is mainly produced during fermentation, it is already present in grapes.44 Violets (β-ionone) and roses (2-phenylethanol) were the dominant nuances in Tempranillo red wines as the volatile compounds related to these odor descriptors registered the highest OAVs in the floral odorant series. In Monastrell and Mencı ́a red wines, floral notes were increased with most fungicides assessed.11,45 On the other hand, GonzálezRodrı ́guez et al.46 observed that the presence of fungicide residues lowered the levels of monoterpenes in Godello white wines. Aubert et al.29,47 reported that the total amount of free monoterpenols decreased significantly in grapes and in the resulting wines, as well as the levels of C13-norisoprenoids in the final wines. Vinous. The vinous character of the studied wines remained almost unchanged regardless of the fungal treatment as no variations >40% were detected in any case. Fermentative compounds, mainly aliphatic alcohols and some esters, were the volatile compounds included in this odorant series, although only isoamyl alcohols showed OAVs >1. Therefore, the behavior of this odorant series was determined by the concentration of isoamyl alcohols in wines, which remained virtually constant. Isoamyl alcohols (3-methyl-1-butanol and 2methyl-1-butanol) are formed from leucine and isoleucine, respectively, by decarboxylation and later reduction of the αketo acids obtained by their transamination (Ehrlich’s pathway).48 Therefore, it could be considered that application of fungicides, both in the field and in the cellar, does not affect either the assimilation of these amino acids by yeast or the activity of the enzymes implied in this catabolic mechanism. A similar outcome was previously found in wines from Godello white grapes treated under GAPs46 or CAPs13 with other newgeneration fungicides. Spicy. Although in quantitative terms this odorant series barely contributed to the aroma profile of wines, spicy nuances endow personality to wines and can be used as distinguishing elements. Spicy odorant series is mainly formed by volatile phenols, guaiacol (sweet, smoky), eugenol (clove, liquorice), and vanillin (vanilla) being the main contributors to this odorant series. None of the studied wines displayed variations in their spicy notes >40% with respect to the control wine. Therefore, the spicy character of Tempranillo wines was not compromised by the application of fungicides. Herbaceous. C6 alcohols were the volatile compounds that provided herbaceous notes in greater extent. No variations >40% with respect to the control wine were found in any wine 1352


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(8) Noguerol-Pato, R.; Torrado-Agrasar, A.; González-Barreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Influence of new generation fungicides on Saccharomyces cerevisiae growth, grape must fermentation and aroma biosynthesis. Food Chem. 2014, 146, 234−241. (9) Sarris, D.; Kotseridis, Y.; Linga, M.; Galiotou-Panayotou, M.; Papanikolaou, S. Enhanced ethanol production, volatile compound biosynthesis and fungicide removal during growth of a newly isolated Saccharomyces cerevisiae strain on enriched pasteurized grape musts. Eng. Life Sci. 2009, 9, 29−37. (10) Oliva, J.; Navarro, S.; Barba, A.; Navarro, G.; Salinas, M. R. Effect of pesticide residues on the aromatic composition of red wines. J. Agric. Food Chem. 1999, 47, 2830−2836. (11) Oliva, J.; Zalacaín, A.; Payá, P.; Salinas, M. R.; Barba, A. Effect of the use of recent commercial fungicides, under good and critical agricultural practices, on the aroma composition of Monastrell red wines. Anal. Chim. Acta 2008, 617, 107−118. (12) González-Rodríguez, R. M.; Noguerol-Pato, R.; GonzálezBarreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Application of new fungicides under good agricultural practices and their effects on the volatile profile of white wines. Food Res. Int. 2011, 44, 397−403. (13) González-Á lvarez, M.; González-Barreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Impact of phytosanitary treatments with fungicides (cyzofamid, famoxadone, mandipropamid and valifenalate) on aroma compounds of Godello white wines. Food Chem. 2012, 131, 826−836. (14) Noguerol-Pato, R.; González-Barreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Influence of tebuconazole residues on the aroma composition of Mencı ́a red wines. Food Chem. 2011, 124, 1525−1532. (15) Noguerol-Pato, R.; Sieiro-Sampedro, T.; González-Barreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Effect on the aroma profile of Graciano and Tempranillo red wines of the application of two antifungal treatments onto vines. Molecules 2014, 19, 12173−12193. (16) Noguerol-Pato, R.; Sieiro-Sampedro, T.; González-Barreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Evaluation of the effect of fenhexamid and mepanipyrim in the volatile composition of Tempranillo and Graciano wines. Food Res. Int. 2015, 71, 108−117. (17) Cabras, P.; Angioni, A.; Garau, V. L. Fate of quinoxyfen residues in grapes, wine, and their processing products. J. Agric. Food Chem. 2000, 48, 6128−6131. (18) Fernández, M. J.; Oliva, J.; Barba, A.; Cámara, M. A. Effects of clarification and filtration processes on the removal of fungicide residues in red wines (var. Monastrell). J. Agric. Food Chem. 2005, 53, 6156−6161. (19) De Melo Abreu, S.; Caboni, P.; Pirisi, F. M.; Cabras, P.; Alves, A.; Garau, V. L. Residues of the fungicide famoxadone in grapes and its fate during wine production. Food Addit. Contam. 2006, 23, 289−294. (20) Oliva, J.; Barba, A.; Payá, P.; Cámara, M. A. Disappearance of fenhexamid residues during wine-making process. Commun. Agric. Appl. Biol. Sci. 2006, 71, 65−74. (21) Oliva, J.; Payá, P.; Cámara, M. A.; Barba, A. Removal of famoxadone, fluquinconazole and trifloxystrobin residues in red wines: effects of clarification and filtration processes. J. Environ. Sci. Health, Part B 2007, 42, 775−781. (22) Garau, V. L.; De Melo Abreu, S.; Caboni, P.; Angioni, A.; Alves, A.; Cabras, P. Residue-free wines: fate of some quinone outside inhibitor (QoI) fungicides in the winemaking process. J. Agric. Food Chem. 2009, 57, 2329−2333. (23) Ruediger, G. A.; Pardon, K. H.; Godden, P.; Pollnitz, A. P. Removal of pesticides from red and white wine by the use of fining and filter agents. Aust. J. Grape Wine Res. 2004, 10, 8−16. (24) Cabredo-Pinillos, S.; Cedron-Fernandez, T.; Saenz-Barrio, C. Differentiation of “Claret”, Rose, Red and blend wines based on the content of volatile compounds by HS-SPME and gas chromatography. Eur. Food Res. Technol. 2008, 226, 1317−1323. (25) Etiévant, P. X. Volatile compounds in foods and beverages. In Wine; Maarse, H., Ed.; Dekker: New York, 1991; pp 483−546. (26) Bureau, S. M.; Razungles, A. L.; Baumes, R. L. The aroma of Muscat of Frontignan grapes: effect of the light environment of vine or

induce remarkable changes in the aroma profile of the resulting wines. In fact, both 2012 and 2013 Tempranillo wines showed very similar aromatic profiles: ripe fruits was always the highest contributor to the overall aroma of Tempranillo wines (48− 57%), followed by fresh fruits (25−35%), and lactic (10−15%) as major odorant series; on the other hand, floral, vinous, spicy, and herbaceous nuances represented all together percentages >7% of the total (Table 4). In summary, there were no significant differences in the concentrations of fungicide residues at the beginning of the fermentation between years 2012 and 2013. Moreover, the concentration of fungicide residues was reduced throughout the fermentation process, reaching dissipation percentages >68% in all cases. On the other hand, the application of fungicides both in the field during the grape growth and directly on the crushed grapes seemingly did not induce important changes in the aroma profile of the resulting Tempranillo-based wines. However, some trends in the aroma profile of the wines can be pointed out, such as the effects on ripe fruit and fresh fruit odorant series, which were the most affected by the application of fungicides, especially boscalid + kresoxim-methyl, mainly as a consequence of the decrease in the synthesis of esters. When compared with the results obtained in 2012, the aroma profiles of wines from the treatment with boscalid + kresoxim-methyl under GAPs displayed the biggest differences in their behaviors with respect to the control wine.

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AUTHOR INFORMATION

Corresponding Author

*(J.S.-G.) Phone: + 34-988-387000. Fax: +34-988-387001. Email: [email protected]. Funding

This work was granted by EU FEDER funds and by the Spanish Ministry of Education and Science (AGL2011-30378-C03 and The Economic Development Agency for La Rioja (ADER 2012-I-IDD-00103). Notes

The authors declare no competing financial interest.

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REFERENCES

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