Stereoselective Metabolism of the Sterol Biosynthesis Inhibitor

Article pubs.acs.org/JAFC

Stereoselective Metabolism of the Sterol Biosynthesis Inhibitor Fungicides Fenpropidin, Fenpropimorph, and Spiroxamine in Grapes, Sugar Beets, and Wheat Ignaz J. Buerge,*,† Jürgen Krauss,† Rocío López-Cabeza,§ Werner Siegfried,† Michael Stüssi,† Felix E. Wettstein,‡ and Thomas Poiger† †

Institute for Plant Production Sciences, Agroscope, CH-8820 Wädenswil, Switzerland Institute for Sustainability Sciences, Agroscope, CH-8046 Zürich, Switzerland § IRNAS, Institute of Natural Resources and Agrobiology of Seville, E-41012 Seville, Spain ‡

S Supporting Information *

ABSTRACT: Metabolism of chiral pesticides in crops is typically studied using achiral analytical methods and, consequently, the stereoisomer composition of residues is unknown. In this study, we developed an enantioselective GC-MS/MS method to quantify residues of the fungicides fenpropidin, fenpropimorph, and spiroxamine in plant matrices. In field trials, the fungicides were applied to grapevines, sugar beets, or wheat. Fenpropidin was metabolized with no or only weak enantioselectivity. For fenpropimorph, slightly enantioselective metabolism was observed in wheat but more pronounced in sugar beets. This enantioselectivity was due to different rates of metabolism and not due to interconversion of enantiomers. The four stereoisomers of spiroxamine were also metabolized at different rates, but selectivity was only found between diastereomers and not between enantiomers. trans-Spiroxamine was preferentially degraded in grapes and cis-spiroxamine in wheat. These findings may affect the consumer dietary risk assessment because toxicological end points were determined using racemic test substances. KEYWORDS: fenpropidin, fenpropimorph, spiroxamine, sterol biosynthesis inhibitor, stereoselective metabolism, pesticide residues, enantioselective GC-MS

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INTRODUCTION The stereoisomers of chiral pesticides may show selective metabolism in plants, resulting in residues with a stereoisomer composition different from that in the applied product. However, because achiral analytical methods are routinely used in plant metabolism and residue studies, data for individual stereoisomers is available only for a few chiral pesticides (references are cited in refs 1,2). The triazole fungicide epoxiconazole is one rare example, where the stereoisomer composition was analyzed in food and feed commodities. Residues in cereal grains and maize forage were found to be clearly enriched with the (+)-enantiomer.3 This finding was then considered in the consumer dietary risk assessment,3 as principally required by the new EU legislation.4 Epoxiconazole and other triazole fungicides inhibit the enzyme C14-demethylase in the biosynthesis pathway of ergosterol.5 A second class of likewise chiral sterol biosynthesis inhibitors interferes with the enzymes sterol Δ14-reductase and Δ8 → Δ7isomerase. These fungicides are applied against powdery mildew and other fungal diseases in cereals, grapes, bananas, sugar beets, and other crops.5 The compounds all have a tertiary amine group in common, but apart from that, they are structurally quite diverse: fenpropidin is a piperidine, fenpropimorph a morpholine, and spiroxamine a spiroketal-amine (Figure 1). Fenpropidin and fenpropimorph consist of one pair of enantiomers (note that the two methyl groups in the morpholine ring of fenpropimorph have cis-configuration), and spiroxamine consists of two diastereomeric pairs of enantiomers. © XXXX American Chemical Society

Only a few studies have been published on chiral aspects of fenpropidin, fenpropimorph, and spiroxamine. For fenpropimorph, a higher fungicidal activity was observed than for the diastereomer with trans-configuration in the morpholine ring.6−8 Moreover, the enantiomer with S-(−)-configuration at the chiral center in the 2-methyl-trimethylene chain was clearly more active against mildew and brown rust of wheat than the R(+)-enantiomer.6,7 For spiroxamine, however, it was shown that all four stereoisomers contribute to the biological activity against powdery mildew of wheat or barley.9 In greenhouse tests, the trans-diastereomer was only slightly more active than the cisdiastereomer, and between enantiomers, no clear differences in activity were observed. In soil, spiroxamine was not degraded stereoselectively.10 Further stereoisomer-specific data on fungicidal activity, metabolism and human and environmental exposure and effects were not found in the open literature. In particular, studies on the stereoselectivity of metabolism in plants and the stereoisomer composition of residues are missing. For fenpropidin, fenpropimorph, and spiroxamine, the European Food Safety Authority (EFSA) identified several data gaps concerning these issues, with possible implications on the worker and consumer risk assessment.11−13 Received: February 25, 2016 Revised: May 19, 2016 Accepted: June 1, 2016

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DOI: 10.1021/acs.jafc.6b00919 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

epoxiconazole, BASF, Ludwigshafen, Germany), Input Xpro (emulsifiable concentrate with 250 g/L spiroxamine, 100 g/L prothioconazole, and 50 g/L bixafen, Bayer, Monheim, Germany), Opus Top (suspoemulsion with 250 g/L fenpropimorph and 84 g/L epoxiconazole, Syngenta), Orius Top (emulsifiable concentrate with 150 g/L fenpropidin, 200 g/L prochloraz, and 100 g/L tebuconazole, Schneiter, Seon, Switzerland), Prosper (emulsifiable concentrate with 500 g/L spiroxamine, Bayer), and Spyrale (emulsifiable concentrate with 375 g/ L fenpropidin and 100 g/L difenoconazol, Syngenta) were purchased from local distributors in Switzerland. Field Trials and Greenhouse Experiments. Seven commercially available fungicide formulations were applied to grapevines, sugar beets, or wheat, cultivated under field conditions. Because fenpropimorph is not registered for use in grapes and spiroxamine not in sugar beets, no corresponding trials were possible for these crop−pesticide combinations. Details on crop cultivation, pesticide applications, sampling, and weather data are available in the Supporting Information. In addition, pure enantiomers of fenpropidin and fenpropimorph were applied to single sugar beet plants in the greenhouse to study the possible interconversion of enantiomers (see Supporting Information). Homogenization of Plant Material. Plant samples from the field trials were stored at −20 °C for no longer than 5 months before they were homogenized and extracted. For registration of the three fungicides in Europe, a number of storage stability studies were performed with different plant matrices.14−16 These studies demonstrated that the compounds are stable at ≈−20 °C for at least 1−2 years. Only for fenpropimorph in sugar beet leaves, a loss of ≈20% was registered after one year,15 which was considered acceptable. To facilitate homogenization, ≈500 g of frozen plant material was further cooled in liquid nitrogen. A knife mill (Grindomix GM 300, Retsch, Haan, Germany) was then used for grinding. It was first operated in antirotation at 2500 rpm during 10 s (with two intervals) to smash the frozen plants into smaller pieces, followed by normal rotation at 4000 rpm during 30 s (five intervals) to cut and grind the plants to a fine powder. The grinding sequence was run twice. Thereafter, the homogenized plants were filled into PETG wide mouth bottles, closed, and stored in the freezer at −20 °C until extraction within the next 24 h. Extraction. The extraction procedure was based on the QuEChERS multiresidue method,17 with some commonly used modifications (use of sodium acetate instead of sodium chloride18 and ethyl acetate instead of acetonitrile19). In detail, 10 g of homogenized plant material (or 5 g of wheat) were weighed into a 50 mL extraction tube containing 6 g of magnesium sulfate and 1.5 g of sodium acetate (DisQuE, Waters, Drinagh, Ireland). After addition of ≈100 μg of the appropriate internal standard, dissolved in 100 μL methanol, the samples were extracted with ethyl acetate (15 mL, or 10 mL for grapes) by vigorous shaking during 5 min. Note that longer extraction times of 15 or 60 min, or 5 min sonication, did not result in higher extraction efficiencies. The tubes were centrifuged at 1000g (centrifuge, 5804 R, Eppendorf, Hamburg, Germany) and a 1 mL aliquot of the centrifugate was transferred into a 2 mL cleanup tube containing 150 mg of magnesium sulfate and 50 mg of PSA sorbent (DisQuE, Waters; the primary secondary amine sorbent removes various polar, organic compounds such as acids, pigments, and sugars). After 1 min of shaking, the tubes were again centrifuged at 1000g (Mini Spin, Eppendorf) and the supernatant was transferred into an autosampler vial. Extracts were stored at 4 °C, and GC-MS/MS analysis was performed within the next days (except for achiral analysis of spiroxamine extracts, 2−4 months after extraction, see below). Sugar beet leaves from the greenhouse experiments with pure enantiomers could not be homogenized in the same way because only two leaves were harvested per sampling time. The fresh leaves were first cut into small pieces with a knife. The amount of 2 g was then weighed into a 50 mL extraction tube containing 1.2 g of magnesium sulfate and 0.3 g of sodium acetate. Thereafter, ≈100 μg of the internal standard amorolfine, dissolved in 100 μL methanol, were added, and the samples were extracted with 7 mL of ethyl acetate by vigorous shaking during 5 min. Extraction was followed by the same cleanup procedure that was described above for plant samples from the field trials. Enantioselective GC-MS/MS Analysis. The enantiomers of amorolfine, fenpropidin, fenpropimorph, and cis- and trans-spiroxamine

Figure 1. Chemical structures of the fungicides fenpropimorph, fenpropidin, and spiroxamine, and the antifungal drug amorolfine. The asterisks indicate the asymmetrically substituted C atoms of the chiral compounds.

The present study was aimed at filling some of these gaps. We developed an enantioselective analytical method based on gas chromatography−tandem mass spectrometry (GC-MS/MS) to quantify residues of the three fungicides in plant matrices. In field trials, we applied the fungicides to grapevines, sugar beets, or wheat, all cultivated according to good agricultural practice. Plant samples were taken immediately after application until harvest, and residue decline curves were measured in grape and sugar beet leaves and in wheat forage. At harvest, grapes were analyzed as well. In all samples, the stereoisomer composition was determined. In addition, we prepared milligram quantities of the pure enantiomers of fenpropidin and fenpropimorph using chiral HPLC. In a greenhouse, we then treated single sugar beet plants with the pure enantiomers to study the possible interconversion of enantiomers. Our results show that the stereoselectivity of metabolism in plants differs between the three fungicides but also between crops.

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MATERIALS AND METHODS

Chemicals. Amorolfine hydrochloride (purity, ≥98%, cis-4-[(RS)-3(4-tert-pentylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine), fenpropidin (96.6%, 1-[(RS)-3-(4-tert-butylphenyl)-2-methylpropyl]piperidine), fenpropimorph (98.3%, cis-4-[(RS)-3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine), and spiroxamine (98.5%, 8-tert-butyl-1,4-dioxaspiro[4.5]decan-2-ylmethyl(ethyl)(propyl)amine) were from Sigma-Aldrich, Steinheim, Germany. Amorolfine was used as the internal standard for fenpropimorph, fenpropidin for trans- and cis-spiroxamine, and trans-spiroxamine for fenpropidin. Small quantities of pure enantiomers of fenpropidin and fenpropimorph and pure diastereomers of spiroxamine were isolated by HPLC (for details, see Supporting Information). The commercial fungicides Astor (an emulsifiable concentrate containing 750 g/L fenpropidin, from Syngenta, Basel, Switzerland), Capalo (suspoemulsion with 200 g/L fenpropimorph, 75 g/L metrafenone, and 62.5 g/L B


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Journal of Agricultural and Food Chemistry were separated on a chiral GC column coated with tert-butyldimethylsilyl-β-cyclodextrin (BSCD, 20%) in 15% phenyl-, 85% methylpolysiloxane (BGB 172, 15 m, 0.25 mm i.d., 0.12 μm film, BGB, Boeckten, Switzerland). GC conditions were as follows: instrument, Agilent 6890N (Santa Clara, CA) with a COMBI PAL autosampler (CTC Analytics, Zwingen, Switzerland); 1 μL split/splitless injection (220 °C, initial 60 s splitless); temperature program, 70 °C, 2 min isothermal, 10 °C/min to 160 °C, 1 °C/min to 185 °C, isothermal hold at 220 °C; constant pressure, 100 kPa helium. The GC was coupled to a Quattro Micro triple quadrupole mass spectrometer (Micromass, Manchester, UK), operated in electron impact ionization (70 eV, 180 °C) and multiple-reaction-monitoring mode. Amorolfine and fenpropimorph were quantified using the ion transition m/z 128 → 70 (317 → 128 and 303 → 128 for confirmatory purposes, respectively), fenpropidin at 273 → 98 (145 → 117), and spiroxamine at 100 → 72 (100 → 58). Collision energies were 5 eV, except for the transitions 128 → 70 and 100 → 58 (9 eV). Quantification was based on peak area ratios relative to the internal standards and in reference to suitable standard solutions of the racemic compounds in ethyl acetate. Determination of the Diastereomer Composition of Spiroxamine with Achiral GC-MS/MS. Cis- and trans-spiroxamine were quantified using an achiral BGB 5 column (5% phenyl-, 95% methylpolysiloxane, 30 m, 0.32 mm i.d., 0.25 μm film). GC conditions were as follows: 1 μL split/splitless injection (260 °C, initial 60 s splitless); temperature program, 70 °C, 2 min isothermal, 25 °C/min to 150 °C, 10 °C/min to 250 °C, isothermal hold at 250 °C; constant flow, 1.6 mL/min helium. MS conditions were identical to those with the enantioselective GC column. For quantification, matrix matched standard solutions were prepared. Untreated, homogenized sugar beet leaves were extracted as described above (including cleanup) but without prior addition of the internal standard. This extract was then used to prepare the standards (with fenpropidin as internal standard). This achiral GC-MS/MS method allowed for a more accurate determination of the diastereomer composition because the two diastereomers eluted within less than 1 min, whereas on the chiral column, the individual stereoisomers were separated by almost 9 min. Spiroxamine was also analyzed on a GC instrument equipped with a flame ionization detector (FID, Agilent 6890N, chromatographic parameters as described above). This method was only used to determine the diastereomer composition in the fungicide formulations Input Xpro and Prosper, and in the reference standard from SigmaAldrich, assuming equal response of both diastereomers with this system.

Figure 2. Enantioselective GC separation of fenpropimorph (trace A, m/z 128 → 70; extract from sugar beet leaves 11 d after application, with elution of the internal standard amorolfine), fenpropidin (B, m/z 273 → 98; extract from spring wheat 14 d after application), and trans- and cisspiroxamine (C, m/z 100 → 72; extract from grape leaves 50 d after application) using the chiral BSCD column with MS/MS detection (arbitrary scales). Trace D shows the elution of trans- and cisspiroxamine (same sample) on the achiral BGB-5 column. Note that in the three plant extracts, the stereoisomer composition was different from that of the applied products.

Recoveries, Precision, Carryover, and Limits of Quantification. Recovery experiments were performed at three fortification levels with untreated grapes, sugar beet leaves, and grass (Tables 1−3). For that, racemic reference compounds were spiked to homogenized plant material prior to extraction. Recoveries of the two enantiomers of fenpropimorph, relative to the internal standard amorolfine, were 94−103% (Table 1), confirming that amorolfine is an excellent internal standard for fenpropimorph (as expected from their structural similarity, Figure 1). Consequently, also the enantiomer fractions, determined in the plant extracts, were very close to the expected value of 0.50 for the racemic compound (0.49−0.51, Table 1; the enantiomer fraction is defined here as EF = [+]/([+] + [−]), where [+] and [−] are the concentrations of the (+)- and (−)-enantiomer, respectively). Recoveries of fenpropidin, relative to the internal standard trans-spiroxamine, were good for sugar beet leaves and grass (88−105%) and moderate for grapes (111−149%, Table 2). Because grapes were analyzed only at harvest, this was considered acceptable. The primary aim of the study was to determine the stereoisomer composition of residues rather than the absolute concentrations, and for fenpropidin, the enantiomer fractions recovered from all three matrices were very close to the expected value (0.49−0.51, Table 2). Recoveries of cis-spiroxamine were insufficient when using standards that were prepared in ethyl acetate (data not shown). Therefore, matrix matched standards were prepared (see Materials and Methods). Trans- and cis-spiroxamine were quantified on the achiral BGB-5 column with recoveries, relative to the internal standard fenpropidin, ranging from 91 to 130% for the three matrices (Table 3). The fraction of the transdiastereomer recovered from the plants varied from 0.48 to 0.50. The enantiomer fractions of trans- and cis-spiroxamine were then determined using the chiral BSCD column, yielding EF values of 0.50−0.52 and 0.48−0.51, respectively (Table 3). The precision of the analytical procedure was investigated by four replicate extractions of representative, homogenized plant

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RESULTS AND DISCUSSION Stereoselective Chromatography and Optical Rotation. Typical GC chromatograms obtained with the chiral BSCD column are shown in Figure 2 (traces A−C). Baseline separation of the enantiomers was achieved for all analytes, except for transspiroxamine, with a still satisfactory enantiomer resolution of ≈1.3 (defined as R = 2 (t2 − t1)/(w1 + w2), where ti is the retention time of enantiomer i and wi is the width at the base of peak i). Elution of the two diastereomers of spiroxamine on the achiral BGB-5 column is shown in Figure 2 (trace D). transSpiroxamine has a higher vapor pressure (0.006 Pa)13 than cisspiroxamine (0.004 Pa), and it was thus concluded that the first peak is that of the trans-diastereomer. Note that the elution temperature of the diastereomers differed by ≈5 °C. The optical rotation of the individual stereoisomers was determined after separation on a chiral HPLC column. On the particular column used, the (+)-enantiomers of fenpropidin, fenpropimorph, and trans-spiroxamine eluted first, for cisspiroxamine the (−)-enantiomer. HPLC isolates were then analyzed by GC-MS/MS. Under GC conditions, the (−)-enantiomers of all compounds eluted first (Figure 2). C


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Journal of Agricultural and Food Chemistry Table 1. Enantioselective Residue Analysis of Fenpropimorph in Plant Matrices: Recoveries, Precision, and Limits of Quantification Recovery: Relative to Internal Standard Amorolfine concentration [mg/kg fresh weight]a

(+)-enantiomer [%]

grapes

5 94 0.5 95 0.05 101 sugar beet leaves 5 98 0.5 97 0.05 97 grass 10 103 1 100 0.1 94 Precision: Relative Standard Deviation from Four Replicate Extractions of a Homogenized Sample concentration [mg/kg fresh weight]b

(+)-enantiomer [%]

sugar beet leaves, day 14 0.11, 0.27 wheat (forage), day 14 0.11, 0.17 Limit of Quantification: Signal-to-Noise Ratio of Primary Mass Transition 6:1

fraction of (+)-enantiomer [−]

94 94 98 97 101 97 101 100 97

0.50 0.50 0.51 0.50 0.49 0.50 0.50 0.50 0.49

(−)-enantiomer [%]

fraction of (+)-enantiomer [%]

14 10

0.6 1.6

(+)-enantiomer [mg/kg fresh weight]

(−)-enantiomer [mg/kg fresh weight]

0.003 0.003 0.001

0.003 0.003 0.001

grapes sugar beet leaves grass/wheat (forage) a

14 14


Fortification level of an individual enantiomer. b(+)-Enantiomer, (−)-enantiomer.

Table 2. Enantioselective Residue Analysis of Fenpropidin in Plant Matrices: Recoveries, Precision, Limits of Quantification Recovery: Relative to Internal Standard Spiroxamine (trans-Diastereomer) concentration [mg/kg fresh weight]a

(+)-enantiomer [%]

grapes

5 144 0.5 111 0.05 143 sugar beet leaves 5 105 0.5 96 0.05 88 grass 10 101 1 94 0.1 92 Precision: Relative Standard Deviation from Four Replicate Extractions of a Homogenized Sample concentration [mg/kg fresh weight]b

(+)-enantiomer [%]

grapes, day 76 0.15, 0.15 grape leaves, day 14 2.4, 2.6 sugar beet leaves, day 14 0.88, 0.95 wheat (forage), day 14 0.08, 0.12 Limit of Quantification: Signal-to-Noise Ratio of Primary Mass Transition 6:1

fraction of (+)-enantiomer [−]

149 116 137 105 101 90 103 97 88

0.49 0.49 0.51 0.50 0.49 0.50 0.50 0.49 0.51


fraction of (+)-enantiomer [%]

1.9 12 9.2 8.8

0.6 1.0 0.6 0.6

(+)-enantiomer [mg/kg fresh weight]

(−)-enantiomer [mg/kg fresh weight]

0.02 0.02 0.01

0.02 0.02 0.01

grapes sugar beet leaves grass/wheat (forage) a

3.0 11 10 9.9


Fortification level of an individual enantiomer. b(+)-enantiomer, (−)-enantiomer.

Liquid nitrogen, used for flash freezing of the plants, was replaced for each set of fungicide/crop but not within a particular time series. Therefore, there is potential carryover through dissolution of the test substance in liquid nitrogen or transfer of plant fragments from the previous sample. To minimize impact of carryover, the samples were flash frozen in reverse order of time (and thus increasing concentrations). Additionally, to estimate worst-case carryover, untreated plant material was flash frozen and homogenized after the day 0 sample (with the highest residue level). Carryover, determined with these samples in

samples from the field experiments. In Tables 1−3, relative standard deviations (RSD) are listed for the individual stereoisomers of the three compounds, extracted from grapes, grape leaves, sugar beet leaves, or wheat forage. The precision was acceptable with RSD values of 2−14%, depending on compound and plant. Determination of the enantiomer fractions of fenpropimorph and fenpropidin was highly reproducible, with RSD values of 0.6−1.6% (Tables 1, 2). Diastereomer fractions and enantiomer fractions for spiroxamine showed adequate precision as well (RSD, 0.5−4.9%, Table 3). D


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Table 3. Stereoselective Residue Analysis of Spiroxamine in Plant Matrices: Recoveries, Precision, and Limits of Quantification Recovery: Relative to Internal Standard Fenpropidin concentration [mg/kg fresh weight]a

trans-diastereomer [%]

cis-diastereomer [%]

fraction of trans-diastereomer [−]

fraction of (+)-enantiomer, trans [−]

fraction of (+)-enantiomer, cis [−]

5 0.5 0.05 5

91 108 99 101

94 112 106 102

0.49 0.49 0.48 0.50

0.50 0.51 0.52 0.50

0.50 0.51 0.50 0.50

0.5 114 116 0.50 0.05 97 101 0.49 grass 10 106 107 0.50 1 125 130 0.49 0.1 121 126 0.49 Precision: Relative Standard Deviation from 4 Replicate Extractions of a Homogenized Sample

0.50 0.50 0.50 0.50 0.50

0.50 0.49 0.50 0.51 0.48

grapes

sugar beet leaves

concentration [mg/kg fresh weight]b

trans-diastereomer [%]

cis-diastereomer [%]

fraction of trans-diastereomer [%]

grapes, day 76 0.005, 0.029 9.4 12 3.5 grape leaves, 1.1, 2.0 5.5 7.4 1.7 day 14 wheat (forage), 0.25, 0.28 9.2 8.9 0.9 day 14 Limit of Quantification: Signal-to-Noise Ratio of Primary Mass Transition 6:1 (for Single Enantiomers) grapes sugar beet leaves grass/wheat (forage) a

fraction of (+)-enantiomer, trans [%]

fraction of (+)-enantiomer, cis [%]

4.9 2.6

2.0 0.5

1.8

1.2

trans-diastereomer [mg/kg fresh weight]

cis-diastereomer[mg/kg fresh weight]

0.01 0.01 0.01

0.01 0.01 0.01

Fortification level of an individual diastereomer. btrans-Diastereomer, cis-diastereomer.

relation to the day 0 samples, was low for grape leaves and wheat forage (1.3−3.5%) and somewhat higher for sugar beet leaves (3.9−4.6% for fenpropimorph enantiomers, 10% for fenpropidin enantiomers, Tables 1−3). Limits of quantification were defined by a signal-to-noise ratio of ≈6:1 of the primary mass transitions found in plant extracts. Quantification of the individual stereoisomers was thus possible down to 0.001−0.003 mg/kg fresh weight for fenpropimorph and 0.01−0.02 mg/kg for fenpropidin and spiroxamine (Tables 1−3). Residues in Grape Leaves and Grapes: Unchanged Enantiomer Composition of Fenpropidin and Spiroxamine, Changed Diastereomer Composition of Spiroxamine. Grapevines were treated in July with the fungicides fenpropidin or spiroxamine. In the leaves, residues of fenpropidin decreased continuously from 14 mg/kg fresh weight immediately after application to 2.2 mg/kg at harvest, 76 days later (Figure 3A). Fenpropidin is rapidly absorbed by leaves,14 and as no precipitation was recorded during the first 5 days following application (Supporting Information, Table S2), washing off from leaves probably was negligible. Translocation occurs acropetally in the xylem.5 Young leaves, which developed after fungicide application, were thus expected to have low residues and were therefore not sampled. Consequently, the residue decline in the treated leaves was primarily due to actual metabolism and much less due to plant growth. Mature grapes contained much lower residues than leaves (0.29 mg fenpropidin/kg fresh weight). In Switzerland, fenpropidin is approved for use until mid-August with a maximum of four applications. In our study, the fungicide was applied only once in mid-July and the residues in grapes were thus well below the current maximum residue level of 2 mg/kg

Figure 3. Residues and stereoisomer composition of fenpropidin (A) and spiroxamine (B) in grape leaves (and mature grapes) 0−76 d after a single spray application at BBCH growth stage 77. Error bars indicate the standard deviation of four replicate extractions (day 14 for leaves, day 76 for grapes).

E


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Journal of Agricultural and Food Chemistry for wine grapes.20 The enantiomer composition of fenpropidin did not change during the study, neither in leaves nor in grapes, with EF values ranging from 0.48 to 0.50 (Figure 3A). Residues of spiroxamine decreased somewhat faster than those of fenpropidin, from 19 mg/kg fresh weight in leaves after application to 0.59 mg/kg at harvest and 0.03 mg/kg in mature grapes (Figure 3B; maximum residue level in Switzerland for wine grapes, 1 mg/kg,20 approved are up to four applications until mid-August). Spiroxamine also rapidly penetrates into plants,16 followed by acropetal translocation to the leaf tips,5 and as for fenpropidin, it was concluded that metabolism was primarily responsible for the residue decline. As for fenpropidin, no significant change of the enantiomer fractions of trans- and cisspiroxamine was observed, with EF values of 0.45−0.51 in leaves and grapes (Figure 3B). Figure 2 (trace C) shows a chromatogram of an extract from day 50, measured with the chiral BSCD column, which illustrates that the enantiomer composition of trans- and cis-spiroxamine was still racemic. However, the diastereomer composition of spiroxamine clearly changed with time. In the leaves, the fraction of transspiroxamine decreased from 0.43 to 0.29 at harvest (Figure 3B). Note that the diastereomer composition of spiroxamine in the applied product was not exactly 1:1 (with GC-FID, we determined a diastereomer fraction of 0.48, which is within the specification of the active substance of 0.44−0.5116). In Figure 2 (trace D), we also show the chromatogram of the same extract from day 50, analyzed with the achiral BGB-5 column. The fraction of trans-spiroxamine was 0.30 in this sample. In mature grapes, the fraction of trans-spiroxamine was even lower and amounted to 0.14. In acidic, aqueous solution, spiroxamine is slowly hydrolyzed with a somewhat faster degradation of trans-spiroxamine. At pH 4 and 20 °C, half-lives of 120 and 790 d were reported for transand cis-spiroxamine, respectively, whereas at pH 7−9, no hydrolysis was observed.16 We prepared small amounts of the pure diastereomers to investigate whether the preferential hydrolysis of trans-spiroxamine in acidic water may also be the result of a conversion to cis-spiroxamine. However, this is not the case. Hydrolysis experiments at pH 4 and 50 °C showed that the diastereomers do not interconvert but confirmed the faster degradation of trans-spiroxamine, with half-lives of 2.8 and 7.5 d for trans- and cis-spiroxamine, respectively (first-order kinetics, determined in experiments with the pure diastereomers as well as with the diastereomer mixture, data not shown). When extrapolated to 20 °C, these half-lives probably are similar to above-reported data. This preferential hydrolysis may thus, in part, explain the low fractions of trans-spiroxamine measured in grapes and grape leaves. Aqueous suspensions of homogenized plant material (ratio, 2.5:1) were quite acidic, with pH values of 3.7 and 4.0, respectively (76 d after application). Of course, enzymemediated metabolism is also expected to contribute to the observed stereoselectivity. Residues in Sugar Beet Leaves: Changed Enantiomer Composition of Fenpropimorph, Unchanged Enantiomer Composition of Fenpropidin. Two field trials were performed with sugar beets that were treated in July with fenpropimorph or fenpropidin. The residues of fenpropimorph in leaves decreased from 1.6 mg/kg fresh weight 1 day after application to 0.11 mg/kg at harvest, on day 77 (Figure 4A). This decrease was not only due to metabolism but also due to plant growth and development of new leaves (note that, in contrast to grapevines, all leaves were sampled). Washing off from leaves was

Figure 4. Residues and stereoisomer composition of fenpropimorph (A) and fenpropidin (B) in sugar beet leaves 0−77 d after a single spray application at BBCH growth stage 45. Error bars indicate the standard deviation of four replicate extractions (day 14).

probably of minor importance, even though between day 3 to 5 after application, 52 mm of precipitation were recorded (Supporting Information, Table S2). Metabolism of fenpropimorph was enantioselective in sugar beets. The enantiomer composition changed rapidly within the first days (Figure 4A). Already 20 h after application, the enantiomer fraction slightly deviated from racemic, with an EF value of 0.45, and then further decreased to 0.25 on day 11. The corresponding chromatogram of this sample is depicted in Figure 2 (trace A). In the following days until harvest, however, the enantiomer composition remained more or less constant (EF ≈0.3). In contrast to fenpropimorph, fenpropidin exhibited only a slight decrease of the enantiomer fraction, from 0.50 after application to 0.44 on day 62 (Figure 4B). Within two months, the residues declined from 4.8 to 0.48 mg/kg fresh weight. No Interconversion of Fenpropimorph and Fenpropidin Enantiomers in Sugar Beet Leaves. The relatively fast change in enantiomer composition of residues of fenpropimorph in sugar beet leaves may, in principle, also be the result of an interconversion of enantiomers rather than preferential metabolism. For example, in soils, the S-enantiomer of the herbicide haloxyfop is converted within less than 1 day and almost completely into R-haloxyfop.21 To investigate a possible interconversion of enantiomers in our study, we performed further trials with the pure enantiomers of fenpropimorph (and also with fenpropidin, for which no pronounced enantioselectivity was observed). For that, we prepared milligram quantities of the pure enantiomers, using chiral HPLC as described in the F


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Journal of Agricultural and Food Chemistry Supporting Information. These amounts were sufficient to treat one sugar beet plant each under greenhouse conditions. Residues of fenpropimorph rapidly decreased by more than 92% and 97%, 31 and 49 d after application, respectively (data not shown), which is consistent with the field trial. However, no formation of the opposite enantiomer was observed, suggesting that the enantioselectivity observed in the field trial was due to different rates of metabolism and not the result of interconversion of enantiomers. Experiments with fenpropidin confirmed the findings with fenpropimorph: residues declined by more than 94% within 51 d after application, without interconversion of enantiomers. Residues in Wheat: Fast Decline, Diastereomer Composition of Spiroxamine Opposite to that in Grapes. In the three field trials with spring wheat, we observed by far the fastest decline of the residues of all three fungicides. On day 57, residues were up to 3 orders of magnitude lower than directly after application (Figure 5, note the logarithmic scale). Several factors may explain these findings. The fungicides were applied at the earliest growth stage of wheat for the intended use against powdery mildew. The plants were about 30 cm high and then grew to 60 and 90 cm, 14 and 57 d after application, respectively. Even more pronounced was the increase in plant biomass, by a factor of ≈10 on day 57. Plant growth was thus responsible for 1 order of magnitude of residue decline. Furthermore, 2 days after application, heavy rainfall of 35 mm may have washed off a certain amount of the fungicides from the leaves. Nevertheless, we concluded that metabolism still considerably contributed to the observed residue decline in wheat. The early timing of fungicide application was not due to agronomic necessity but allowed to follow metabolism and, in particular, the stereoisomer composition of the residues over a longer time period. In Figure 2 (trace B), we show a chromatogram of an extract of fenpropidin-treated wheat at day 14, illustrating that metabolism was slightly enantioselective. However, in the following days, the enantiomer composition did not change anymore (enantiomer fractions ≈0.4; Figure 5B). For fenpropimorph, the same, weak enantioselectivity was observed (Figure 5A), whereas trans- and cis-spiroxamine were metabolized without any enantioselectivity (Figure 5C). Metabolism of spiroxamine was, however, selective with regard to its diastereomers. The fraction of trans-spiroxamine continuously increased from 0.43 immediately after application to 0.67 on day 57 (0.46 was measured in the applied product). This trend was just in the opposite direction to that observed in grape leaves (and grapes). As discussed above, preferential hydrolysis of trans-spiroxamine at low pH may, in part, explain the findings in grapes. In wheat forage, the pH was clearly higher (pH 6.2 in an aqueous suspension of homogenized wheat forage) and abiotic hydrolysis probably was insignificant. The preferential metabolism of cis-spiroxamine in wheat may therefore have other reasons such as diastereomer-selective enzymatic degradation. Stereoselectivity of Metabolism Differs between Compounds and Crops: Regulatory Considerations. Levels and decline of residues in the various crops were comparable to data reported in studies submitted for registration in Europe.14−16 Our study now provides further information regarding stereoselectivity of metabolism. Fenpropidin was applied to three crops and was metabolized with no or only weak enantioselectivity. For fenpropimorph, a minimal enantioselectivity was observed in wheat but more pronounced in sugar beets. As shown with separate experiments using pure

Figure 5. Residues and stereoisomer composition of fenpropimorph (A), fenpropidin (B), and spiroxamine (C) in the aerial parts of spring wheat 0−57 d after a single spray application at BBCH growth stage 31. Error bars indicate the standard deviation of four replicate extractions (day 14). Note the logarithmic scale for concentrations (left axis) and the linear scale for stereoisomer fractions (right axis).

enantiomers, this enantioselectivity was due to different rates of metabolism and not due to interconversion of enantiomers. The four stereoisomers of spiroxamine were also metabolized at different rates, but selectivity was only found between diastereomers and not between enantiomers. Trans-Spiroxamine was preferentially degraded in grapes, cis-spiroxamine in wheat. As discussed above, the pH of the plant material may, in part, help to rationalize these differing trends, however, this would need to be confirmed with other crops. Overall, our results show that the stereoselectivity of metabolism and, consequently, stereoisomer composition of residues differs between the three fungicides but also between crops. Despite the high crop interception in the field studies, a certain fraction of the fungicides may have reached the soil, where they may be degraded stereoselectively. For spiroxamine, the available data indicates nonstereoselective degradation in soil.10 For fenpropidin and fenpropimorph, no corresponding studies were G


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(Agroscope) for agronomic issues, and S. Huntscha (Agroscope) for support in the laboratory.

found. However, possible stereoselective degradation in soil is expected to have minimal influence on residues in plants. Because the compounds show strong sorption to soil,14−16 transfer to the root zone and thus possibility for plant uptake will be limited. Nevertheless, we cannot fully exclude that stereoselective degradation in soil, followed by plant uptake, may have affected the stereoisomer composition in crops to some extent. In Switzerland (and the European Union), the current maximum residue level (MRL) for spiroxamine in wine and table grapes is 1 mg/kg.20 EFSA proposed an acute reference dose (ARfD) of 0.1 mg/kg body weight that was derived from a neurotoxicity study with rats.13 For a simple consumer dietary risk assessment, we used the pesticide residue intake model PRIMo,22 which, based on this MRL, calculates an acceptable acute risk for children as well as for adults, corresponding to an intake of 66% and 32% of the ARfD, respectively. The model assumes consumption of a large portion of 212 g of table grapes by a child of 16 kg or 400 g by an adult of 63 kg and considers a variability factor of 5 in the IESTI equation. In our study, residues in mature grapes were composed of 14% trans-spiroxamine and 86% cis-spiroxamine, whereas toxicological studies were probably performed with a diastereomer composition of ≈1:1. If cis-spiroxamine was primarily responsible for the observed neurotoxicity, the consumption of table grapes, containing residues of 1 mg/kg spiroxamine, may then be unacceptable for children. An additional safety factor of ≈2 may thus be justified in this case, without further knowledge on the toxicity of the individual diastereomers. In the consumer dietary risk assessment of the fungicide epoxiconazole, EFSA also considered such a conversion factor that was deduced from the median enantiomer composition of residues in cereal grains.3 For registration, applicants should thus routinely determine residues of chiral pesticides with stereoselective analytical methods, at least for the risk assessment, not necessarily for monitoring purposes.

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(1) Katagi, T. Isomerization of chiral pesticides in the environment. J. Pestic. Sci. 2012, 37, 1−14. (2) Pérez-Fernández, V.; García, M. Á .; Marina, M. L. Chiral separation of agricultural fungicides. J. Chromatogr. A 2011, 1218, 6561−6582. (3) European Food Safety Authority (EFSA). Peer review of the pesticide risk assessment for the active substance epoxiconazole in light of confirmatory data submitted. EFSA J. 2015, 13, 4123. (4) Commission Regulation (EU) No 283/2013 of 1 March 2013 setting out the data requirements for active substances, in accordance with regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market. Off. J. Eur. Union 2013, 03.04.2013, L 93/1−84. (5) Mac Bean, C., Ed., A World CompendiumThe Pesticide Manual. 16th ed.; The British Crop Protection Council, Alton, UK, 2012. (6) Himmele, W.; Pommer, E.-H. 3-Phenylpropylamines, a new class of systemic fungicides. Angew. Chem., Int. Ed. Engl. 1980, 19, 184−189. (7) Pommer, E.-H. Chemical structure-fungicidal activity relationships in substituted morpholines. Pestic. Sci. 1984, 15, 285−295. (8) Baloch, R. I.; Mercer, E. I. Inhibition of sterol Δ8 → Δ7-isomerase and Δ14-reductase by fenpropimorph, tridemorph and fenpropidin in cell-free enzyme systems from Saccharomyces cerevisiae. Phytochemistry 1987, 26, 663−668. (9) Krämer, W.; Berg, D.; Dutzmann, S.; Etzel, W. A.; Gau, W.; Stelzer, U.; Weissmüller, J. Chemistry, stereochemistry and biological properties of KWG 4168. Pestic. Sci. 1999, 55, 610−614. (10) Sukul, P.; Zühlke, S.; Lamshöft, M.; Rosales-Conrado, N.; Spiteller, M. Dissipation and metabolism of 14C-spiroxamine in soil under laboratory condition. Environ. Pollut. 2010, 158, 1542−1550. (11) Conclusion on the Peer Review of Fenpropimorph; European Food Safety Authority (EFSA), 2008; pp 1−89, http://www.efsa.europa.eu/ en/efsajournal/pub/144r (accessed on May 13, 2016). (12) European Food Safety Authority (EFSA). Review of the existing maximum residue levels (MRLs) for fenpropidin according to Article 12 of Regulation (EC) No 396/2005. EFSA J. 2011, 9, 2333. (13) European Food Safety Authority (EFSA). Conclusion on the peer review of the pesticide risk assessment of the active substance spiroxamine. EFSA J. 2010, 8, 1719. (14) Draft Assessment Report (DAR): Initial Risk Assessment for the Existing Active Substance Fenpropidin; Rapporteur Member State Sweden, 2005; http://dar.efsa.europa.eu/dar-web/provision (accessed on May 13, 2016). (15) Draft Assessment Report (DAR): Initial Risk Assessment for the Existing Active Substance Fenpropimorph; Rapporteur Member State Germany, 2005; http://dar.efsa.europa.eu/dar-web/provision (accessed on May 13, 2016). (16) Assessment Report: Initial risk assessment for the existing active substance spiroxamine; Rapporteur Member State Germany, 2009; http://dar.efsa.europa.eu/dar-web/provision (accessed on May 13, 2016). (17) Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F. J. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J. AOAC Int. 2003, 86, 412−431. (18) Lehotay, S. J.; Mastovska, K.; Lightfield, A. R. Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables. J. AOAC Int. 2005, 88, 615−629. (19) Analysis of Pesticide Residues in Fruit and Vegetables with Ethyl Acetate Extraction Using Gas and Liquid Chromatography with Tandem Mass Spectrometric Detection; EU Reference Laboratories for Residues of Pesticides; www.eurl-pesticides.eu (accessed January 19, 2015). (20) Verordnung des EDI über Fremd- und Inhaltsstoffe in Lebensmitteln; The Federal Department of Home Affairs (FDHA), 2015; https:// www.admin.ch/opc/de/classified-compilation/19950193/index.html (accessed on May 13, 2016).

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00919. Separation of stereoisomers by chiral HPLC, hydrolysis of spiroxamine diastereoisomers in acidic water, field trials with grapes, wheat, and sugar beets, application of pure enantiomers of fenpropidin and fenpropimorph to sugar beets in the greenhouse, enantioselective HPLC separation of fenpropidin and fenpropimorph; pesticide application data; weather data (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +41 58 460 6383. Fax: +41 58 460 6341. E-mail: ignaz. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the field teams of Agroscope in Wädenswil (D. Malo, R. Schmon, C. Total, T. Wins) and Zürich (P. Walther, F. Käser) for performing and supervising the trials, A. Schürmann (Official Food Control Authority of the Canton of Zurich), E. Arrigoni and D. Baumgartner (Agroscope) for helpful discussions concerning homogenization and extraction of plants, M. Keller H


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Journal of Agricultural and Food Chemistry (21) Poiger, T.; Mü ller, M. D.; Buser, H.-R.; Buerge, I. J. Environmental behavior of the chiral herbicide haloxyfop. 1. Rapid and preferential interconversion of the enantiomers in soil. J. Agric. Food Chem. 2015, 63, 2583−2590. (22) Pesticide Residue Intake Model “PRIMo”, revision 2; European Food Safety Authority (EFSA), 2013; http://www.efsa.europa.eu/en/ mrls/mrlteam.htm (accessed on April 6, 2016).

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Stereoselective Metabolism of the Sterol Biosynthesis Inhibitor

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