Tracking, Behavior and Fate of 58 Pesticides Originated from Hops

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Tracking, Behavior and Fate of 58 Pesticides Originated from Hops during Beer Brewing. Martin Dusek, Vladimíra Jandovská, and Jana Olšovská J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03416 • Publication Date (Web): 03 Sep 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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Journal of Agricultural and Food Chemistry

Tracking, Behavior and Fate of 58 Pesticides Originated from Hops during Beer Brewing Martin Dušek1*, Vladimíra Jandovská1,2, Jana Olšovská1 1

Research Institute of Brewing and Malting, Lípová 15, CZ–120 44 Prague 2, Czech Republic

2

Faculty of Science, Charles University, Albertov 6, CZ–128 43 Prague 2, Czech Republic

*Corresponding author, e-mail [email protected], telephone +420 224 900 184

1 ACS Paragon Plus Environment


ABSTRACT The study presents tracking of 58 pesticide residues associated with hops to estimate their carryover into brewed beer. The pesticides were spiked onto organic hops at a concentration of 15 mg/kg, and the wort was boiled with the artificially contaminated hops and fermented on a laboratory scale. Samples were collected during the whole brewing process and pesticide residues were extracted using a method known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). An HPLC-HR-MS/MS method was developed and validated to identify and quantitate pesticide residues in treated hops, spent hops, hopped wort, green beer, and beer samples. Quantitation was achieved using standard addition with isotopically labeled standards. The carryover percentages into hopped wort and the percentages of decay reduction relative to the amount spiked on hops were calculated. The relationship between the partition coefficients n-octanol–water (log P values) and the residual ratios (RW and RB) of a pesticide were evaluated to predict their behavior during hopping of wort and fermentation. Pesticides with a high log P values (>3.75) tended to remain in spent hops. The pesticides that have a low log P value up to approximately 3 could represent the demarcation lines of appreciable transfer rate of pesticides from hops to beer. Consequently, the pesticides were divided into three categories depending upon their fate during the brewing process. The most potential risk category represents a group involving the thermostable pesticides, such as azoxystrobin, boscalid, dimethomorph, flonicamid, imidacloprid, mandipropamid, myclobutanil, and thiamethoxam, which were transferred at high rates from the pesticide enriched hops into beer during the laboratory brewing trial. These results can be used as a guideline in the application of pesticides on hop plants that would reduce the level of pesticide residues in beer and their exposure in humans.

Key words: Pesticide residues, fate, thermal stability, hops, wort, green beer, beer


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1

INTRODUCTION

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Beer is made from just four main ingredients: barley, hops, water and yeast. Barley is the

3

most commonly used grain for malting, but malts can be also made from wheat, rye, and some

4

other cereal. The hops, Humulus lupulus, is a flowering plant belonging to the family

5

Cannabaceae, whose non-fertilized flowers, that are also called seed cones or strobiles, have

6

on the inside of the flower a cluster of yellow lupulin glands. The strobiles store acids and oils

7

that give beer its bitterness and aroma and represent an irreplaceable key ingredient for beer

8

production. An agricultural production of barley and hops is frequently negatively affected

9

due to bacterial diseases, fungus and mildew, virus diseases, as well as pests and parasitic

10

attacks. For this reason, agrochemical usage in various combinations on barley and hop plants

11

cultivation makes it possible to reach good yields and reduce the losses during storage. Hop

12

plant belongs to crops with intensive chemical protection and pesticides such as azoxystrobin,

13

boscalid, cyhalothrin, dithianon, dimethomorph, flonicamid, hexythiazox, imidacloprid,

14

mandipropamid, myclobutanil, pyraclostrobin, triadimenol, trifloxystrobin represent pesticide

15

residues typically found in hops.1

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Agrochemicals applied on crops can persist in the plants for a long time and could be

17

carried over to processed food - beer from raw materials - malt and hops.2 Malt is germinated

18

barley and the behavior of pesticides during malting operations (steeping, germinating,

19

kilning and drying) was studied and published.3,4 The fate of pesticide residues or some

20

fungicidally active metabolites was described in several publications 5-7 including an extensive

21

study that investigated the fates of more than 300 pesticides during beer brewing.8 These

22

studies were predominantly based on spiking malt with a mixture of pesticides and

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observation of their carryover to beer during brewing process via the steps such as mashing,

24

lautering, boiling, and fermentation. The authors of these studies concluded that the

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agrochemicals applied on barley crops remain in malt only in case that their residues have log

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P values between 2 and 4.3,4 On the contrary, during malting the pesticide residues with

27

higher log P than 2 tended to be absorbed into spent grain and they were not dominantly

28

carried over into sweet wort.8 Thus, the variation of the content of pesticide residues during

29

the whole brewing process is predominantly influenced by the input of agrochemicals from

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hops. The fate of pesticide residues coming from the agrochemicals applied in a hop yard

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during beer brewing was thoroughly studied only for a few mostly GC-amenable pesticides,

32

such as chlorfenapyr, quinoxyfen, tebuconazole, fenarimol, or dimethomorph.9,10

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In our initial set of experiments, we focused on laboratory scale beer brewing from hops

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artificially spiked with the mixture of 58 pesticides, including fungicides, insecticides, and 3 ACS Paragon Plus Environment


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their metabolites. The list of compounds included 16 pesticides registered for the use on hops

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in the Czech Republic by the Central Institute for Supervising and Testing in Agriculture.

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These 16 pesticides (abamectin, azoxystrobin, boscalid, cymoxanil, fenpyroximate,

38

flonicamid,

39

quinoxyfen, spirotetramat, tebuconazole, thiamethoxam, triflumizole) represent the most

40

commonly used agrochemicals applied on the hop yards over the world. The laboratory

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brewing trial of bottom fermented Pilsner-type of beer was conducted to maximally simulate

42

in the industrial scale production. Our attention then focused on determination and

43

quantitation of pesticide residues using HPLC-HR-MS/MS in the samples collected within the

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whole brewing process.

hexythiazox,

imidacloprid,

mandipropamid,

metalaxyl,

pyraclostrobin,

45 46

MATERIALS AND METHODS

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Chemicals and Material

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Acetonitrile, methanol, formic acid and ammonium formate (all LC-MS grade), sodium

49

citrate tribasic dihydrate and sodium hydrogencitrate sesquihydrate were purchased from

50

Sigma-Aldrich (Steinheim, Germany). Sodium chloride (anal. grade) was obtained from

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Lach-Ner (Neratovice, Czech Republic). Magnesium sulfate (anal. grade, >98%) was

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obtained from Penta (Prague, Czech Republic). Pure water was obtained from a Milli-Q

53

purification system (MilliporeSigma, Burlington, MA, USA).

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Pesticide standards abamectin, acephate, acetamiprid, ametoctradin, azoxystrobin,

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bifenthrin, boscalid, bupirimate, carbendazim, chlorantraniliprole, chlorpyrifos, clothianidin,

56

cyazofamid,

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fenpyroximate, flonicamid, fludioxonil, fluopicolide, fluopyram, hexythiazox, imazalil,

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imidacloprid, indoxacarb, malaoxon, malathion, mandipropamid, mepanipyrim, metalaxyl,

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methoxyfenozide, metrafenone, myclobutanil, oxadiazon, penconazol, pendimethalin,

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pirimicarb, propamocarb, propargite, propiconazol, pyraclostrobin, pyridaben, quinoxyfen,

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spirodiclofen,

spirotetramat,

spiroxamine,

tebuconazole,

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thiabendazole,

thiacloprid,

thiamethoxam,

triadimefon,

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triflumizole and internal standards azoxystrobin-d4, probenecid, thiamethoxam-d3 and

64

triphenyl phosphate (TPP) were purchased from Sigma Aldrich (St. Louis, MO, USA).

cymoxanil,

diflubenzuron,

dimethomorph,

etoxazole,

tebufenozide, triadimenol,

fenpropimorph,

tebufenpyrad, trifloxystrobin,

65

Standard and internal standard stocks solutions (1.0 mg/mL for all except 0.2 mg/mL for

66

ametoctradin, carbendazim and chlorantraniliprole) were prepared in acetonitrile or, in case of

67

a solubility problem, in methanol or acetone and stored at –20°C. A standard mixture

68

solution, with all 58 pesticides, was prepared in acetonitrile at 1 mg/L of each pesticide 4 ACS Paragon Plus Environment

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Preparation Hops Spiked with Pesticides

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The sample of organically grown, pesticide-free, dried hops cones (Saaz variety) was minced,

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placed in a tightly closed jar and stored in a freezer. A 10 g sample of ground hop cones was

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spread into a thin layer on a Petri dish (20 cm I.D.) and subsequently spiked by a pesticide

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mixture containing 200 µg of each pesticide that corresponds with the spiking concentration

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20 mg/kg. A 100 mL glass bottle with spray head was used for spiking. After application of

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the pesticides, 3 mL of acetonitrile was added to the bottle to rinse the rest of pesticides. The

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spiked hops was dried at room temperature overnight and dry hops sample was gently minced

78

to ensure full homogenization.

79 80

Lab-scale Beer Brewing

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An infusion mash was employed using a mashing device controlled via PC that allowed

82

setting up individual temperature, time and temperature gradient. In all, 5 batches of 75 g of

83

ground malt were mixed with 400 mL of 44 °C hot brewing water and then heated (1°C/min)

84

to 50°C for a 20-min. protein rest. Next, the mash was heated (1°C/min) to 62°C and rested

85

for 30 minutes. In the next step the mash was heated (1°C/min) to 71°C for a 30-min.

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saccharification rest. Finally, the mash was heated (1°C/min) to 78°C for 20 min in a mash-

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off rest. The mash was stirred during the whole process. The hot wort was individually

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lautered by using a filter paper (approximately 10 min). Prior to the end of filtration 100 ml of

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65°C hot water was added into each filtration funnel for sparging the spent grain. The worts

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from all batches were combined to a final volume of approximately 2 L of cold wort, with an

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original extract of 12.25°P. The original gravity of the sweet wort was adjusted to 10°P

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(%Plato) by mixing 1 620 mL with 380 mL of water, a 2 L portion of wort was transferred to

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a 4 L boiling flask and heated to a boil. A 5 g portion (2.5 g per liter of sweet wort) of

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pesticide enriched hops was added after the boiling started and the boiling was continued for

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another 90 minutes under atmospheric conditions under a water-cooled reflux condenser. The

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hopped wort was cooled down for 30 min at laboratory temperature with occasional circular

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stirring and the spent hops were removed by using a filtration paper rinsed with hot water

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before filtering. Filtrated hopped wort was rapidly cooled down to the pitching temperature of

99

14°C. Bottom fermenting yeast (10 g, Saccharomyces pastorianus, RIMB 95) was added and

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the fermentation was conducted pressureless at a constant temperature of 12°C for 7 days.

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The green beer, free of foam and sedimented yeast, was carefully transferred into a 2 L plastic

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bottle, well tightened and fermented at 3°C for further six weeks. 5 ACS Paragon Plus Environment


103 104

Extraction and Sample Preparation

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The spent hops removed by filtration were spread into a thin layer on a Perish dish,

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covered by filtration paper and left to dry at room temperature for two days. Dry material was

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gently minced to crush lumps, weighed and 2 g was used for the determination of percent dry

108

matter using the moisture analyzer HR83 (Mettler Toledo, Columbus, OH, USA). Sample

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preparation procedure for pesticide residue analysis in hops, spent hops, beer and wort was

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carried out by the citrate QuEChERS method.11 Hops or spent hops sample (1 g, dry weight

111

equivalent) or a portion of 10 mL of liquid samples was placed into a 50 mL centrifuge tube

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and 10 mL of water was added. The sample was vortexed for 1 minute and left to soak for 30

113

minutes. A 10 ml portion of acetonitrile and 50 µL of internal standard spiking solution

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(1 mg/L) were added to each tube. After vortexing the sample for 1 minute, the mixture of 4 g

115

of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g trisodium citrate dehydrate, and

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0.5 g disodium hydrogen citrate sesquihydrate were added, the tubes were capped, shaken

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vigorously for 1 min by hand and finally centrifuged at 4500 rpm for 7 min. A 6 mL aliquot

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of the upper acetonitrile layer was transferred into a 15 ml centrifuge tube containing 0.9 g of

119

magnesium sulfate (150 mg per 1 mL). The tubes were tightly capped, vortexed for 30 s and

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centrifuged at 4500 rpm for 5 minutes. A 2 mL aliquot of the acetonitrile extract of samples

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hops and spent hops obtained in the previous step was diluted in a 20 mL volumetric flask and

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filled up to the mark using acetonitrile. Samples of sweet wort, hopped wort, green beer and

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beer were analyzed without dilution.

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Quantitation was done using a standard addition method. Two replicates were measured

125

for each sample. The diluted acetonitrile extract of hops (200 µL each) was pipetted into four

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2 mL glass vials for each sample, three of which were fortified with the pesticide standard

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solution (1 mg/L) at the concentration levels of 0.033, 0.067 and 0.100 mg/kg to make the

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standard addition calibration curve corresponding to 50, 100 and 150% of expected

129

concentration of pesticide residues in the sample extract. The diluted extract of spent hops

130

(300 µL) was fortified at concentrations of 0.25, 0.50 and 0.75 mg/kg for calibration.

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Undiluted extract of liquid samples (400 µL) was fortified at concentrations of 0.017, 0.033

132

and 0.050 mg/L for calibration. The appropriate volume of acetonitrile was added to each vial

133

to make the final volume of 600 µL.

134 135

Thermal Stability of Pesticides during Boiling of Sweet Wort


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The solutions of pesticides in sweet wort were prepared by taking 100 µL (500 µL) aliquots of

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a stock solution (1 mg/mL) of each pesticide and diluting it in a 1 L volumetric flask with

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sweet wort (10°P). This resulted in a 100 mg/L mixed solution. The pesticide spiked sweet

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wort was brought to boil in a 2 L 2-neck round bottom flask under a water-cooled reflux

140

condenser and boiled for 2 hours. The samples were collected prior to boiling, after 35

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minutes when the boiling began and subsequently after every 15 minutes of boiling. Samples

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(approx. 15 mL) were pipetted into a 50 mL centrifuge tube immediately cooled on dry ice

143

and placed in the freezer. Before sample preparation the samples were melted down,

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centrifuged at 4500 rpm for 7 minutes, and 10 mL of supernatant was transferred into 50 ml

145

centrifuge tube, spiked with 50 µL of internal standard solution and processed using citrate

146

QuEChERS method such as described above.

147

Quantitation of pesticide residues was achieved using matrix-matched standard calibration

148

curves with isotopically labeled standards at a concentration of 0.050 mg/L and the calibration

149

curves consisted of four points (0.010, 0.020, 0.050, and 0.100 mg/L, equivalent in a sample).

150 151

LC-MS/MS Analysis

152

HPLC-HR-MS/MS was carried out using a Dionex UltiMate 3000 UHPLC system (Thermo

153

Scientific, Germering, Germany) consisting of a binary pump (HPG-3400RS), an autosampler

154

(WPS-3000TRS), a degasser (SRD-3400) and a column oven (TCC-3000RS). Detection was

155

carried out by a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo

156

Scientific, Waltham, MA, USA). Analytes were separated on a reversed-phase C18 Atlantis

157

T3 column (2.1×100 mm, 3 µm) from Waters (Milford, MA, USA) with a corresponding

158

guard C18 column (SecurityGuard ULTRA) from Phenomenex (Aschaffenburg, Germany).

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The LC-MS system was equipped with a heated electrospray ionization source (HESI-II) and

160

TraceFinder software version 4.1. Chromatographic separation was accomplished using

161

gradient elution with 2 mM ammonium formate containing 0.1% formic acid in water as

162

solvent A and methanol as solvent B; LC gradient: 0 min: 85% of solvent A + 15% of solvent

163

B, 0.5 min: 85% A + 15% B, 9 min: 5% A + 95% B, 15 min: 95% A + 5% B with a flow rate

164

of 340 µL per minute was used. The column oven was heated to 35°C and injection volume

165

was 2 µL.

166

In the positive electrospray ionization (ESI) mode, the ion spray voltage was set at 2.8 kV,

167

the sheath gas flow was at 32 arbitrary units, the auxiliary gas flow rate was kept at 7 arbitrary

168

units, the capillary temperature was set at 295°C and the auxiliary gas heater temperature was



169

set at 295°C. In the negative ESI mode, the ion spray voltage was set at –2.5 kV. Nitrogen

170

was used as both sheath and auxiliary gas.

171

The mass spectrometer was generally operated in parallel reaction monitoring (PRM). The

172

precursor ions from scheduled inclusion list were, within the retention time window ±0.3 min,

173

filtered in the quadrupole at isolation window (target m/z ± 0.7 amu), fragmented in HCD

174

collision cell, product ions were collected in the C-trap at 17,500 resolution (FWHM, full

175

width at half maximum, at m/z 200), AGC target value of 2e5, and maximum ion injection

176

time of 40 ms and finally two specific pairs of precursor-product ion transitions were

177

monitored for each compound of interest. A mass tolerance of 5 ppm was employed. The

178

normalized collision energy (NCE) was optimized for each compound. Table 1 shows

179

monitored precursor and two daughter ions, retention times and normalized collision energies

180

(NCE). The instrument was externally calibrated prior to each measurement using the mixture

181

of mass calibrants.

182 183

RESULTS AND DISCUSSION

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Pesticide Residue Analysis in Hops, Spent Hops, Wort, Hopped Wort and Beer

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The QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation method

186

applied for the determination of compound of interest in this study, was originally developed

187

for the multiresidue analysis of pesticides in produce.12 The method involves an extraction of

188

a sample with acetonitrile, followed by liquid–liquid partitioning using an appropriate mixture

189

of salts, and then, usually a final cleanup step using dispersive solid-phase extraction (dSPE).

190

This sample preparation approach was previously modified and validated for the hops

191

matrix13 and successfully applied for the determination of compounds of interest in the

192

pesticide enriched hops sample and also in spent hops. Sample dilution approach14 as an easy

193

and effective method to overcome massive matrix effect typical for this type of matrix was

194

advantageously applied in view of the fact that organic hops for brewing trial was enriched at

195

a relatively high level (15 mg/kg). Percentage recovery values for all analytes were calculated

196

for 6 replicates at a fortification level of 0.050 mg/kg and most of mean recoveries ranged

197

between 70 and 120% (average 85.5%), with relative standard deviations (RSDs) in the range

198

of 1.4 to 10.4% (average 3.4%). The recoveries outside the range of 70–120% were calculated

199

for imazalil (127.6%), malathion (61.2%), pyridaben (68.6%), and thiabendazole (63.5%).

200

The liquid samples of hopped wort, green beer and beer were prepared using QuEChERS

201

method without the dilution step or additional clean-up step. Pesticide recoveries in beer

202

matrix spiked at the level of 0.050 mg/kg ranging from 80 to 113% (average 95.5%) for all 8 ACS Paragon Plus Environment

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compounds of interest, and RSD values were lower than 10% for all analytes. The limit of

204

detection (LOD) and the limit of quantitation (LOQ) were estimated for spiked samples on the

205

basis of a signal-to-noise ratio of 3:1 and 10:1, respectively. The limit of quantitation was

206

mostly 0.2 mg/kg for ten times diluted acetonitrile extract of hops or spent hops, and 0.5 µg/L

207

for samples of hopped wort, green beer and beer. The limits of quantitation for pesticide

208

residues, such as acephate, abamectin, bifenthrin, boscalid, cyazofamid, dimethomorph,

209

flonicamid, hexythiazox, myclobutanil, oxadiazon, pendimethalin, propiconazol, quinoxyfen,

210

spirodiclofen, and triadimenol were 0.5 mg/kg or 1 µg/L, respectively. Considering the

211

complexity of these matrices and the need of using calibration in the matrix to compensate

212

matrix effects, a standard addition method based on three level standard additions in the

213

samples was employed to quantitate pesticide residues in all samples collected during the

214

laboratory brewing trial.

215 216

Transfer of Pesticide Residues from Hops to Wort During Hopping

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The pesticide residue levels in pesticide enriched hops were determined (see Table 3) prior to

218

the brewing trial and the dose of hops (2.5 g/L) was modified to set up the concentration of

219

pesticide residue to 35-40 mg per liter of hopped wort. Laboratory‐scale brewing trials of a

220

10°P pale “Pilsner” type of beer were conducted to study the fate of pesticide residues during

221

wort hopping. To verify that the wort and beer which were produced on the lab scale fitted the

222

specifications for this type of beer15 some characteristic parameters were checked and are

223

depicted in Table 2. The amount of pesticide residues in spent hops and hopped wort was

224

quantified as described in Material and Methods; the variation of pesticide residues in both

225

materials allowed us to classify these pesticides based on their behavior. Table 3 shows that

226

all pesticides involved in this study were separated into three groups (A, B and C), the amount

227

of pesticide residues determined in spent hops (mg/kg), hopped wort (mg/kg) and the

228

corresponding transfer rates (%) represent the amount of pesticide that was found in each

229

matrix related to the initial concentration in the pesticide spiked hops before hopping wort

230

(see Table 2, columns 4 and 6). The sorting into these three groups was done based on the

231

behavior during boiling wort and includes the following sorts: (A) the pesticide carryover

232

percentages into hopped wort against the amount spiked on hops were at least 50%; (B)

233

pesticides remained in spent hops or were extracted from less than 50%; (C) pesticides which

234

were not detected at all or were detected only at trace level. The results clearly showed that

235

boiling for 90 minutes has a significant influence on the amount of a few pesticides that were

236

sorted into group A and B. Total amount of pesticides such as triflumizole (71%), bupirimate 9 ACS Paragon Plus Environment


237

(57%), chlorantraniliprole (56%) and mepanipyrim (51%) was reduced more than about 50%

238

due to unspecified thermal decomposition, pyrolysis, hydrolysis or/and adsorption onto

239

insoluble components, which probably represent the dominant and common reason for

240

pesticide losses during wort hopping.8 The percentages of these losses were calculated and are

241

listed in Table 3 (column 7) for each pesticide.

242

The ability of pesticides to be carried over into hopped wort was expressed as residual

243

ratio (RW) and calculated on the basin of pesticide amount in hopped wort compared to the

244

sum of amounts of the pesticide in spent hops and hopped wort. This approach is different

245

from the calculation used by Inoue at al.8, who calculated the residual ratio solely on the basis

246

of a comparison with the amount of pesticide initially spiked. This approach is not able to

247

distinguish whether the pesticide was not transferred due to its poor extraction ability or its

248

low thermal stability or it was adsorbed onto insoluble components. Thus, this inaccurate

249

expression of the residual ratio of some pesticides could affect their right correlation with the

250

log P values. On the contrary, the calculation used in this study allowed us to express the

251

residual ratio (RW) more precisely without influences due to pesticide losses as described

252

above. The behavior of pesticides during wort hopping can thus be newly described based on

253

two parameters: (1) the residual ratio (RW) that expresses the efficiency of extraction from raw

254

material (see Table 3, column 10) and (2) the decay percentages during boiling of wort (see

255

Table 3, column 7). The carryover of pesticides could be related to their partition coefficients

256

between n-octanol and water (log P values) as published by Navarro et al.4 and Miyake et al.3

257

for the pesticide fate during barley malting or by Miyake and Tajima7 and Inoue et al.8 for

258

pesticides carried over into sweet wort from malt. The graphs in Figure 1 show a correlation

259

between residual ratios (RW) and log P values for all pesticides sorted in groups A and B (see

260

Table 3, column 13). The relationship between these values was assessed by LOWESS

261

(Locally Weighted Scatterplot Smoothing) regression analysis and is represented by a smooth

262

curve through the data points. The results show that water soluble pesticides (log P < 3) were

263

extracted at >70% and pesticides that have low log P value of

Tracking, Behavior and Fate of 58 Pesticides Originated from Hops

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