Monitoring of residues of fungicides used in malting barley protection

During the last years strobilurines have become a very important group of efficacious substances used against the diseases of barley and hop. These are substances with a wide fungicide effect. Their characters, including the efficacy spectrum, are very variable. They affect mainly preventively, but also curatively and they even have eradicative effects. All of them act in contact, some of them also in depth or systemically.

1 INTRODUCTION

Today strobilurines represent a very important group of fungicidal efficacious substances. Their action is specific and they have a very wide efficacy spectrum.

They got into commercial use in 1996 as products of the companies Syngenta (azoxystrobin) and BASF (kresoxim-methyl). Currently following strobilurine effective substances have been used commercially (1) (Tab. 1).

Tab. 1 List of commercialy used strobilurines

Natural strobilurines (Strobilurus and Oudemansiella) were isolated from wood-rotting bracket fungi.These wood decay fungi produce fungicidially acting substances in defense against lower fungi. The name strobilurine comes from Strobilurus tanacellus, one of the first fungi from which they were isolated.

Strobilurines represent an important group of efficient fungicides and together with oxazoline-diones (famoxadone) and imidazolinones (fenamidone) they are Qo inhibitors (Quinone outside Inhibitors). Their action is based on the inhibition of mitochondrial respiration, they block Qo instead of cytochrom b, which is a part of cytochromal complex bc1 localized in the inner mitochondrial membrane of fungi. The transfer of electrons between cytochrom b and cytochrom c1 is blocked, and thus production of ATP is inhibited and the energy cycle of fungi is disrupted. Originally, strobilurines are natural substances produced by higher fungi, e.g. Strobilurus tanacellus (strobilurine A) Oudemansiella mucida (oudemansin A). Chemically they belong to methoxyacrylates (e.g. azoxystrobin and picoxystrobin), methoxycarbamates (pyraclostrobin), oximino acetates (kresoxim – methyl, trifloxystrobin), ozimino-acetamides (dimoxystrobin, etc.), and dihydrodioxazinones (fluoxastrobin). Their characters, including the efficacy spectrum, are very variable.They act mainly preventively, but also curatively and some even have eradicative effects. All of them act in contact, some of them in depth or translaminarily or systemically (azoxystrobin and picoxystrobin). They move acropetally in a plant. Their efficacy is very wide, they are active against sac fungi (Ascomycetes), bracket fungi (Basideomycetes), imperfect fungi (Anamorphic fungi) and oomycetes (Oomycetes).They are highly imperilled by the origin of resistance (cross resistance within the QoI fungicides) (1).

Resistance was proved in other pathogenic fungi, in cereals, powdery mildew (Blumeria graminis f. sp. tritici, Blumeria graminis f. sp. hordei), septoria blotch (Mycosphaerella graminicola), net blotch (Pyrenophora teres), helminthosporioses in wheat (Pyrenophora triticirepentis), and in the other crops, e.g. in apple, scab (Venturia inaequalis), grapevine downy mildew (Plasmopara viticola), and grapevine powdery mildew (Erysiphe necator). When applying strobilurines, observance of all provisions is highly recommended to avoid the development of resistance.

Following fungicides are used against fungal diseases of barley in the CR:azoxystrobin (Amistar), picoxystrobin (Acanto, Acanto Prima), trifloxystrobin (Sfera 267.5 EC), fluoxastrobin (Fandango 200 EC), and kresoxim-methyl (Juwel, Juwel Top). Azoxystrobin (Ortiva) is registered against fungal diseases of hop in the CR. Considering the increase in the extent of strobilurine use in the protection of barley and hop and their stability in a plant, it is necessary to introduce monitoring of their residues in raw materials, intermediaries and the final product.

As these substances can be potentially dangerous for human health and the environment (Tab. 2), the aim of our study was to optimize the method for the determination of azoxystrobin (Fig. 1), kresoxim-methyl (Fig. 2), picoxystrobin (Fig. 3) and trifloxystrobin (Fig. 4) in barley, malt and beer and to carry out monitoring of their occurrence in barley, malt and beer samples.

Tab. 2 Maximum limits of residues in barley (Notice no. 381/2007 Coll. of 19 December 2007)

Fig. 1 Azoxystrobin; Fig. 2 Kresoxim-methyl; Fig. 3 Picoxystrobin; Fig. 4 Trifloxystrobin

2 MATERIAL AND METHODS

2.1 Standards and chemicals

Standards: azoxystrobin (99.5 %, Dr. Ehrenstorfeer GmbH, Germany), picoxystrobin (99.9 %, Riedel-de Haën, Germany), kresoxymmethyl (96.6 %, Riedel-de Haën, Germany), trifloxistrobin (99.2 %, Riedel-de Haën, Germany).

Columns: ENVITMCarbII/PSA 500/500 mg (Supelco, USA), LiChrolut EN 200 mg (Merck, Germany), Discovery DSC-18 (Supelco, USA).

Chemicals: NaOH (Merck, Germany), methanol, acetone, acetonitrile, toluene, ethyl acetate (Sigma Aldrich, USA), distilled water.

2.2 Samples of barley, malt and beer

Total of 50 samples of malting barley acquired from various growing areas of the CR were analysed. In addition, 50 samples of malt produced from the acquired barley samples were analysed. The analysed beer samples were randomly selected and bought in retail shops (25 samples).

2.2.1 Preparation of samples of barley and malt

60 ml of methanol/acetone mixture (8:2) was added to 20 g of ground matrix. Extraction of the mixture was performed in an ultrasound bath for 30 minutes. The extracted mixture was centrifuged at 6500 RPM for 15 min at 10–15 °C. The obtained solutions were transferred to boiling flasks (250 ml) and evaporated to dryness on a vacuum evaporator. The obtained dry residue was dissolved with 5 ml of acetonitrile in the ultrasound bath for 1 minute. The mixture was purified through the SPE column (2). Three types of SPE columns were tested for purification – ENVITMCarbII/PSA, LiChrolut EN and Discovery DSC-18.

The ENVITMCarbII/PSA column was conditioned with the 5 ml acetonitrile: toluene mixture (3:1). 5 ml of barley (malt) extract in acetonitrile was transferred to the conditioned column. Analytes were eluted with 3 ml of an acetonitrile:toluene mixture (3:1). The obtained eluate was concentrated on a vacuum rotary evaporator, the evaporation residue was transferred to ethyl acetate (0.5 ml).

The LiChrolut EN and Discovery DSC-18 columns were conditioned with 5 ml of methanol and 5 ml of distilled water. 5 ml of barley (malt) extract was transferred to the conditioned columns and the columns were washed with distilled water (1 ml). After drying with nitrogen (15 min) the analytes were eluted with 2 x 5 ml mixture of ethyl-acetate:water (1:1). The obtained eluates were concentrated on the vacuum rotary evaporator, the evaporation residues were transferred to 0.5 ml of ethyl acetate.

2.2.2 Preparation of beer samples

pH of the beer sample (100 ml) was adjusted to pH 6 by adding NaOH solution and the sample was then purified through the SPE column. For purification two types of SPE columns – ENVITMCarbII/PSA and LiChrolut EN were tested.

The ENVITMCarbII/PSA column was conditioned with 5ml of acetonitrile: toluene mixture (3:1). 100 ml of beer was transferred to the conditionated column. The analytes were eluted with 3 ml of acetonitrile: toluene mixture (3:1). The obtained eluate was concentrated on the on the vacuum rotary evaporator, the evaporation residue was transferred to 0.5 ml of ethyl acetate.

The LiChrolut EN column was conditioned with 5 ml of methanol and 5 ml of distilled water. 100 ml of beer was transferred to the conditionated column and the column was washed with 1 ml of distilled water. After drying with nitrogen (15 min) the analytes were eluted with 2x5 ml mixture of ethylacetate:water (1:1). The obtained eluate was concentrated on the vacuum rotary evaporator, the evaporation residue was transferred to 0.5 ml of ethyl acetate.

2.3 Instrumentation and chromatographic determination

The analyses of samples were performed on the gas chromatograph (Trace GC Ultra, Thermo Finnigan) connected to the mass detector (Trace DSQ, Thermo Finnigan).To separate the analysed substances the DB5-MS capillary column (30m x 0.25mm i.d., 0.25 μm) with following thermal program was used: initial temperature 70 °C for 1 min, increase in temperature 10 °C.min⁻¹ to 280 °C, maintained for 5 min. Programmed flow of carrying gas He was from 1.5 ml.min⁻¹ to 3 ml.min⁻¹. Temperature of PTV injector 280 °C, splitless regime for 0.8 min. Temperature in the transfer line between GC and MSD was 200 °C. The mass spectrometer was adjusted in SCAN (50–450 m/z) and SIM (Selected Ion Monitoring) mode (EI+ – positive electron ionisation) and selected values (m/z) for relevant analytes were as follows:

• azoxystrobin – 344, 388 (m/z)
• trifloxyistrobin – 116, 131, 222 (m/z)
• picoxystrobin – 145, 335 (m/z)
• kresoxim-methyl – 116, 131, 222 (m/z)

Identification of the strobilurines analysed was performed on the basis of retention times and specific ions m/z (Fig. 5), quantification was carried out using calibration curves (Fig.6).

Fig. 5 Mass spectra of strobilurines

Fig. 6 Calibration curves of strobilurines

3 RESULTS AND DISCUSSION

Three types of extraction SPE columns – ENVITMCarbII/PSA, LiChrolut EN and Discovery DSC-18 for samples of barley and malt and two types of columns – ENVITMCarbII/PSA and LiChrolut EN for samples of beer were tested.

The ENVITMCarbII/PSA column provided very good results for barley and malt samples.To optimize the column, the analytes were eluted using various elution volumes (10 ml, 6 ml, 4 ml and 3 ml) of the acetonitrile:toluene mixture (3:1). The 3 ml volume was found as the most suitable; it provided the least elution of interferents and the highest recovery. But this column was not suitable for the analyses of beer samples due to washing off the analytes and elution of the interfering substances.

The LiChrolut EN and Discovery DSC-18 columns were not suitable for the analyses of barley and malt. When using these columns, the interfering substances were insufficiently separated and at chromatographic analyses they had the same retention times as the determined analytes.

The LiChrolut EN column proved to be the most suitable for the analyses of beer samples. This column is suitable for concentration and purification of bigger volumes and in the beer samples, the interfering substances were sufficiently separated.

The method for determination of strobilurines in barley, malt and beer was optimized (3). Validation parameters for the individual matrices and SPE columns are given in tables (Tab. 3, 4).

Tab. 3 and malt (SPE – EnviTMCarbII/PSA)

Tab. 4 Beer (SPE – LiChrolut EN))

Results of the determination of individual substances in barley, malt and beer are given in tab. 5.

Tab. 5 Content of strobilurine residues in the analysed samples

4 CONCLUSION

Strobilurine pesticides represent a group of pesticides with a quite wide spectrum of efficiency against fungal diseases. A relatively new group of substances is regarded here which has not been perfectly investigated and possible long-term consequences of their residues have not been sufficiently described. These substances may therefore represent health risk for the human organism.

The aim of this study was to determine possible traces of strobilurine pesticides in barley, malt and beer. Possible excessive occurrence of resiudes could endanger a consumer, for this reason it is necessary to monitor them. Acceptable limits of residues are given by the Notice no. 381/2007 Coll. of 19th December 2007 on determination of maximum limit of strobilurine residues in foods and raw materials.

Selected strobilurines, the most frequently applied in protection of malting barley in the CR, were analysed – azoxystrobin, kresoximmethyl, picoxystrobin, trifloxystrobin.

So far only few methods for the determination of strobilurines in cereals or beer have been described (4, 5, 6, 7, 8). Majority of the published studies have been focused on the assessment of these substances in fruit and wine grapes. Therefore, it was necessary to optimize the method for the determination in cereals and beer. Cereals, i.e. determined barley, contain a considerable amount of dyes, first of all chlorophylls, which considerably hamper the analysis with the GC-MSD method as they can overlap the determined analytes and they further load the ion source. Similar situation is also with beer samples where it is necessary to remove interfering ballast substances.

The method for the determination of selected strobilurines in barley, malt and beer was optimized and validated. For purification of extracts from barley and malt and beer samples the SPE method was optimized by selection of suitable columns for the individual matrixes. The most appropriate column for barley and malt was ENVITMCarbII/PSA and the LiChrolut EN column for the beer samples.

Strobilurine contents in all the analysed samples were below the detection limit and thus also below the maximum limit of residues (MLR) given by the Notice no. 381/2007 Coll. of 19th December 2007 on determination of maximum residue limits in foods and raw material.