Method development for determination of contaminants in honey and trueness verification in the proficiency testing scheme

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1 ORIGINAL SCIENTIFIC PAPER Method development for determination of contaminants in honey and trueness verification in the proficiency testing scheme Veronika Kmecl, Helena Baša Česnik Kmetijski inštitut Slovenije, Hacquetova ulica 17, Ljubljana, Slovenija Abstract Two methods for determination of contaminants in honey samples were developed in Central Laboratories of Agricultural Institute of Slovenia. The trueness of methods was verified by participation in the French proficiency testing scheme Bipea (Bureau InterProfessionnel d'etude Analytique, France). The first method comprises determination of acaricides from apicultural sources and the second comprises determination of pesticides from the sources in the environment. Key words: honey, pesticides, chromatography, verification, quality assurance Introduction Pollution of bee products with contaminants arises from apicultural and environmental sources. Many accaricides are used in beekeeping to control the number of mite Varroa destructor, which causes a bee disease varroasis. Most of the time, accaricide treatments lead to residues in hives. Several surveys have already monitored residues in honey, such as bromopropylate, coumaphos, amitraz, tau-fluvalinate, flumethrin (Chauzat et. al., 2007). However, less references concerning honey contamination related to crop treatments are available in the literature. Pesticides are aplied world wide to a broad variety of crops for field and post harvest protection. Honey bees are largely affected by insecticides as well as pesticides. As a consequence, residues of certain pesticides could appear in apiarian products (Rissato et. al, 2004). In the European Union, Maximal Residue Limits (MRLs) are defined for residues of pharmacologically active substances and pesticides in honey. In Agricultural Institute of Slovenia we developed and verified analytical methods to control the excessive contamination of honey. Materials and methods Method development The simultaneous determination of acaricides: bromopropylate, coumaphos, fluvalinate and flumethrin from apicultural sources is achieved by liquid-liquid extraction (LLE) of honey sample with the solution of n-hexane, 2-propanol and 0.28% ammonia. The combined extract is decanted to separating funnel and the filtrate is separated. The upper n- hexane phase is filtered into evaporation flask and evaporated to dryness under vacuum at 40 C. The residue is dissolved in acetone for GC analysis (Martel et al., 2002). Quantification of bromopropylate, coumaphos, fluvalinate and flumethrin in honey samples was conducted by gas chromatography with electron capture detection (GC-ECD), according to Bogdanov et al. (1998). Multiresidual method for simultaneous determination of ten pesticides: acrinathrin, bifenthrin, boscalide, bromopropylate, chlorpyrifos-ethyl, cyhalothrin-lambda, cypermethrin, deltamethrin, malathion and tetradifon, comprises liquid-liquid extraction with the mixture of solvents (acetone, petroleum ether and dichloromethane with ratio 1:2:2), separation in separatory funnel and evaporation of organic phase to dryness. Extract is dissolved in mixture of cyclohexane and ethyl acetate with ratio 1:1 and cleaned on gel permeation chromatograph th Croatian & 8 th International Symposium on Agriculture Dubrovnik Croatia

2 Ribarstvo, lovstvo i pčelarstvo The eluate is evaporated to dryness and dissolved in a mixture of cyclohexane and ethyl acetate with ratio 1:1 (GC grade). Qualitative and quantitative determination is performed using gas chromatograph coupled with mass spectrometer (GC-MS) under conditions described in Table 2 (Baša et al., 2003, Baša et. al., 2006). Method verification The trueness of analytical results was verified by participation in proficiency testing scheme organized by Bipea (Bureau InterProfessionnel d'etude Analytique). Results and discussion Method development The GC-ECD chromatogram of acaricide residues: bromopropylate, coumaphos, fluvalinate, flumethrin as a matrix match standard is given in Graph 1. Separation of four active substances was achieved under the chromatographic conditions showed in Table 1. Graph 1. GC-ECD chromatogram of four active substances in honey matrix match standard Graph 2. GC-MS chromatogram of ten active substances in honey matrix match standard Graph 2 presents the GC-MS chromatogram of ten active substances in honey matrix match standard. The separation of acrinathrin, bifenthrin, boscalide, bromopropylate, cypermethrin, chlorpyrifos ethyl, cyhalothrin lambda, deltamethrin, malathion and tetradifon was achived under the chromatographic conditions showed in Table 2. Method verification Trueness of the results was verified in the proficiency testing scheme. The statistical treatment in Bipea is carried out in accordance with ISO standard Statistical methods for use in proficiency testing by inter-laboratory comparisons. The Minimum and the Maximum values (Mean ± 2 Standard Deviation) define the tolerance interval inside which the result can be accepted as correct. The mean, tolerance, minimal and maximal value and results obtained in our laboratory are presented in Table hrvatski i 8. međunarodni simpozij agronoma Dubrovnik Hrvatska 697

3 Table 1. The trueness of a certain active substance of pesticides verified by Bipea Acrinathrin (GC-MS) Sample Unit Mean Tolerance Minimum Maximum Our result Trueness 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Bifenthrin (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Boscalide (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Bromopropylate (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Bromopropylate (GC-ECD) 1 mg/kg mg/kg mg/kg mg/kg Cyhalothrin-lambda (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Cypermethrin (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg Chlorpyrifos-ethyl (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Coumaphos (GC-ECD) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Deltamethrin (GC-MS) 1 mg/kg mg/kg mg/kg th Croatian & 8 th International Symposium on Agriculture Dubrovnik Croatia

4 Ribarstvo, lovstvo i pčelarstvo Fluvalinate (GC-ECD) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Malathion (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Tertadifon (GC-MS) 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Notice: Bipea did not treat the results of flumethrin statisticaly due to the insufficient number of cooperated laboratories. As we can see from Table 1, the results of twelve active substances achieved by GC-MS or GC-ECD chromatography are within the tolerance interval (Mean 2 Standard deviation). In this respect we can confirm the trueness of developing multiresidual methods. Prescribed Maximum residues levels (MRLs) for certain active substances are presented in Table 2. Values of Bipea testing scheme are within the range of MRLs (below and above MRLs) for all compounds, except for bromopropylate and cyhalothrin-lambda, for which the MRL values are slightly higher. Table 2. Maximum Residues Levels (MRL) for active substances determined in honey Active substance MRL (mg/kg) Reference Acrinathrin 0.05 Reg. (EC) No. 839/2008 Bifenthrin 0.01 Reg. (EU) No. 441/2012 Boscalide 0.5 Reg. (EU) No. 441/2012 Bromopropylate 0.01 Reg. (EU) No. 310/2011 Cyhalothrin-lambda 0.02 Reg. (EC) No. 459/2010 Cypermethrin 0.05 Reg. (EU) No. 520/2011 Chlorpyrifos-ethyl MRL set at LOQ Reg. (EC) No. 839/2008 Coumaphos 0.1 Reg. (EU) No. 37/2010 Deltamethrin 0.03 Reg. (EU) No. 441/2012 Flumethrin no MRL required Reg. (EU) No. 37/2010 Fluvalinate no MRL required Reg. (EU) No. 37/2010 Malathion 0.02 Reg. (EU) No. 270/2012 Tertadifon 0.05 Reg. (EU) No. 310/2011 Conclusions At Agricultural Institute of Slovenia we developed and verified two methods to control the contaminants in honey. GC-ECD method was applied for determination of four acaricides used in beekeeping: bromopropylate, coumaphos, fluvalinate and flumethrin. Multiresidual GC-MS method was used to define the content of environmental pesticides: acrinathrin, bifenthrin, boscalide, bromopropylate, chlorpyrifos-ethyl, cyhalothrin-lambda, 48. hrvatski i 8. međunarodni simpozij agronoma Dubrovnik Hrvatska 699

5 cypermethrin, deltamethrin, malathion, and tetradifon. Both methods were verified with successful participation in proficiency testing scheme Bipea. Based on the results we can confirm the competence of our laboratory in this area. References Baša Č.H., Gregorčič A. (2003). Multirezidualna analizna metoda za določevanje ostankov pesticidov v sadju in zelenjavi. Research Reports Biotechnical Faculty University of Ljubljana. Agriculture. Zootechny. 82: Baša Č. H., Gregorčič A., Velikonja B. Š., Kmecl V. (2006). Monitoring of pesticide residues in apples, lettuce and potato of the Slovene origin in Food Additives and Contaminants. 23: Bogdanov S., Kilchenmann V., Imdorf A. (1998). Acaricide residues in some bee products. Journal of Apicultural Research. 37 (2): Chauzat M.P., Faucon J.P. (2007). Pesticides residues in beeswax samples collected from honey bee colonies (Apis mellifera L.) in France. Pest Management Science. 63: Martel A.C., Zeggane S. (2002). Determination of acaricides in honey by high-performance liquid chromatography with photodiode array detection. Journal of Chromatography A. 954: Rissato S.R., Galhiane M.S., Knoll F.R.N., Apon B.M. (2004). Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection. Journal of Chromatography A. 1048: th Croatian & 8 th International Symposium on Agriculture Dubrovnik Croatia