INTERNATIONAL HONEY COMMISSION MELISSOPALYNOLOGY WORKING GROUP LABORATORY OF APICULTURE, ARISTOTLE UNIVERSITY OF THESSALONIKI, GREECE REPORT

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1 INTERNATIONAL HONEY COMMISSION MELISSOPALYNOLOGY WORKING GROUP LABORATORY OF APICULTURE, ARISTOTLE UNIVERSITY OF THESSALONIKI, GREECE REPORT Proficiency Testing Scheme for Honey Pollen Analysis IHC0-3 October 203 Page/42

2 Coordinators of the PT scheme DimouMaria TananakiChrisoula ThrasyvoulouAndreas Laboratory of Apiculture&Sericulture Faculty of Agriculture Aristotle University of Thessaloniki Agroktima Panepistimiou Thermi 5700 Greece Tel.: Fax: Thessaloniki, 26 October 203 The use of data included in the present report for reasons other than the evaluation of laboratory performance is not allowed without the previous consent of the Laboratory of Apiculture-Sericulture of the Aristotle University of Thessaloniki in writing Page2/42

3 Summary This report describes the design, results and statistical analysis and of the intercomparison test on pollen analysis of honey which was held among the partners of the International Honey Commission Melissopalynology Working Group and organized by the Laboratory of Apiculture & Sericulture of the Aristotle University of Thessaloniki, Greece. In total, 39analysts from 36 laboratories from Europe, West Asia and South America participated in the PT scheme IHC0-3. Three pollen types were calculated: Brassicaceae, Tilia, Rosaceae. The relative standard deviation of reproducibility of the test results was slightly higher to the values stated in manuscript Harmonized methods of melissopalynology by the IHC in 2004, in all cases. Page3/42

4 Acknowledgements The coordinators of this test would like to thank the participants for their cooperation, the Famille Michaud Apiculteurs who kindly provided and dispatched the honey samples and Dr. Charis Alexopoulos from the General Chemical State Laboratory of Greece for his valuable information on the statistical analysis of the results. Page4/42

5 Table of contents Summary... 3 Acknowledgements... 4 Table of contents... 5 Introduction... 6 Materials and Methods... 6 Statistical Analysis... 6 Results... 7 Qualitative pollen analysis... 8 Quantitative pollen analysis Botanical origin Geographical origin References Annex I. Participant Laboratories Annex II. Cover letter and Result Form Annex ΙIΙ. Methods and References Annex IV. Comments Page5/42

6 Introduction This proficiency testing scheme was organized in order to strengthen the technical competency of the laboratories carrying out pollen analysis in honey and examine the harmonization among the methodologies and results given by the participant analysts of the International Honey Commission Melissopalynology Working Group. Materials and Methods In total, 39 analysts from 36 laboratories from Europe, West Asia and South America participated in the PT scheme(annex I). Each laboratory received one 00 g sample of honey in a plastic container labeled as IHC0-3. he participants ere recommended to store the sample in cool temperature 0-20 C until the analysis and analyze the sample as a routine sample, using the method that is regularly applied. The samples weredispatched by the Famille Michaud Apiculteurs from France on Each participant received also electronically the Result Form, the code number and a coverletter with instructions for the results submission. In particular, the participants were asked to define the percentage of three different pollen forms (Brassicaceae, Tilia, Rosaceae), the botanical and the geographical origin of the honey. Further details about the results which were asked by the participants to be filled in are mentioned in the Result Form and the cover letter (Annex II). The analytical methods which were used by the participants are presented in Annex II. Further comments made by the participants are mentioned in Annex IV. The closing date for submitting results -after a short extension-was the 4 th of October 203. Statistical Analysis For each numerical value, the following parameters were calculated according to ISO 3528:2005(E): median value, mean value, standard deviation, robust standard Page6/42

7 deviation, assigned value, standard uncertainty of the assigned value, target-value of the standard deviation for the proficiency assessment, relative standard deviation of reproducibility and z-score. The assigned value for each parameter was calculated from the median value of the results which were submitted by the participants. As target-value of the standard deviation for the proficiency assessment, the robust statistical parameter was used. The relative standard deviation of reproducibility was compared to the values given in the literature. The target-values of the standard deviation for the proficiency assessment were used in conjunction to the assigned values to derive the z-scores for participants results. The z-scores were considered satisfactory if z 2. he outliers ere checked by Grubbs and Hampel s tests. The normality was checked by the Kolmogorov-Smirnov test (the results follow the normal distribution in all cases). Kernel density diagrams were constructed and qualitatively evaluated. Results-Discussion From the 36 laboratories that participated in the PT Scheme 5 were accredited for honey pollen analysis. Almost half of the analysts perform pollen analysis to more than 500 honey samples per year (Figure and Table 4) > <50 Figure. Number of honey samples analysed per year from the analysts. Page7/42

8 The descriptive summary statistics for the qualitative and quantitative pollen analyses of the honey sample derived from the proficiency testing scheme are presented in Table. Table. Descriptive summary statistics HONEY POLLEN ANALYSIS Parameter QUALITATIVE Brassicaceae Tilia Rosaceae Brassicaceae* Tilia* Rosaceae* QUANTITATIVE Number of pollen grains per 0g of honey number of participants (n) median value (x) mean value (m) 54, 8,9 6,8 56, 9,4 6, standard deviation (sd),9 3,3 3, 2,0 3,4 3, robust standard deviation(s*) 6,6 2,7 2,0 8,2 3,0 2, assigned value X standard uncertainty of the assigned value (u x) target-value of the standard deviation for proficiency assessment σ^ relative standard deviation of reproducibility RSD R% 0,8,8,4,2,9, ,6 2,7 2,0 8,2 3,0 2, % 37% 45% 5% 32% 37% 53% *Nectarless pollen sources were excluded from the calculation of the results Qualitative Pollen Analysis All participants presented results about the qualitative pollen analysis according to the instructions. Almost half of the participants used the methodology proposed by the IHC in The rest used either another method alone or in combination with the method mentioned above (Annex III). In general, the values given with and without counting the nectarless taxa had statistically significant differences regarding Brassicaceae (paired t-test p<0.00) and Tilia (paired t-test p<0.00) but Page8/42

9 not Rosaceae (paired t-test p=0.440) -probably because of the small number of nectarless pollen types in the sample. The relative standard deviation of reproducibility of the test results was slightly higher than the values stated in manuscript Harmonized methods of melissopalynology by the IHC in 2004, in all cases (Table ). The results and z-scores of the qualitative pollen analysis per participant are presented in Table 2(a & b) and Figure 3(a-f). In more details, regarding Brassicaceae pollen, one outlier according to Grubbs test and t o according to Hampler s test were detected. The Kernel density plot showed one small secondary peak on the right side of the axis corresponding to the two higher values; and three more on the left side of the axis attributing to a lower value and two outliers (Figure 2a). One outlier was detected according to Grubbs test at Tilia pollen analysis; and a single peak distribution was observed in the Kernel density plot Figure 2b. One outlier according to Grubbs test and t o according to Hampler s test ere detected at Rosaceae pollen analysis. Small secondary peaks which attributed to the higher values and outliers were observed in the Kernel density plot (Figure 2c). When the nectarless pollen types were excluded from the analysis, the analyses showed similar results. In particular, Brassicaceae* (nectarless excluded) pollen statistical analysis showed one outlier according to Grubbs test and t o according to Hampler s test. Small secondary peaks were observed on the right and left side of the axis in the Kernel density plot attributing to the higher/lower values and outliers (Figure 2d).One outlier was detected according to Grubbs and Hampler s test at Tilia*(nectarless excluded) pollen analysis; and two small secondary peaks were observed on the right side of the axis in the Kernel density plot, attributing to the higher values and outlier (Figure 2e). Two outliers were detected at Rosaceae*(nectarless excluded) pollen analysis according to Hampler s test and one according to Grubbs test. Two small secondary peaks were observed on the right side of the axis in the Kernel density plot, attributing to the higher values and outliers(figure 2f). Page9/42

10 0,07 Kernel Density Plot a. Brassicaceae (%) 0,06 0,05 0,04 0,03 0,02 0, Kernel Density Plot b. Tilia (%) 0,4 0,2 0, 0,08 0,06 0,04 0, ,8 0,6 0,4 0,2 0, 0,08 0,06 0,04 0,02 Kernel Density Plot c. Rosaceae (%) Figure2. Kernel density plot of the qualitative pollen analysis: a. Brassicaceae; b. Tilia; c. Rosaceae. Page0/42

11 Kernel Density Plot d. Brassicaceae* (%) 0,06 0,05 0,04 0,03 0,02 0, ,4 0,2 0, 0,08 0,06 0,04 0,02 Kernel Density Plot e. Tilia* (%) Kernel Density Plot f. Rosaceae* (%) 0,8 0,6 0,4 0,2 0, 0,08 0,06 0,04 0, Figure2. (continued): d. Brassicaceae* (nectarless excluded); e. Tilia*(nectarless excluded); f. Rosaceae*(nectarless excluded). Page/42

12 Table 2a. Results and z-scoresof the qualitative pollen analysis of the honey sample (nectarless pollen sources were included in the analysis). Laboratory Code Brassicaceae assigned: 57 QUALITATIVE POLLEN ANALYSIS Tilia assigned: 8 Rosaceae assigned: 6 % z-score % z-score % z-score MEL02 4-2,4* 3,9 7 0,5 MEL ,2 0 0,7 6 0,0 MEL ,2 7-0,4 3 -,5 MEL05 6 0,6 6-0,7 3 -,5 MEL ,6 9 0,4 9,5 MEL0 55-0,3 8 0,0 5-0,5 MEL2 58 0,2 5 -, 6 0,0 MEL3 57 0,0 9 0,4 6 0,0 MEL5 63 0,9 5 -, 3 -,5 MEL8 8,2-7,4** 3,9 N/A - MEL2 53-0,6 5 -, 9,5 MEL ,5 0 0,7 3 -,5 MEL ,5 6-0,7 4 -,0 MEL ,0 2,5 8,0 MEL ,8 8 0,0 2,5 MEL ,3 6-0,7 7 0,5 MEL ,6, 9,5 MEL39 65,2 0 0,7 5-0,5 MEL ,5 8 0,0 5-0,5 MEL4 62 0,8 5 -, 4 -,0 MEL ,8 8 3,7** 3 3,4 MEL ,3 9 0,4 4 -,0 MEL ,3* 5 -, 2,5* MEL55 65,2, 2 2,9* MEL ,* 5 2, ,9** MEL62 65,2 8 0,0 6 0,0 MEL7 58 0,2 7-0,4 5-0,5 MEL ,3, 5-0,5 MEL ,6* 6 2,9* 7 0,5 MEL ,5 8 0,0 5-0,5 MEL8 54-0,5, 6 0,0 MEL ,4 9 0,4 6 0,0 MEL84 64, 8 0,0 5,2 4,4** MEL ,7* 6-0,7 7 0,5 MEL ,0 7-0,4 6 0,0 MEL ,3* 7-0,4 5-0,5 MEL95 5-0,9 8 0,0 7 0,5 MEL ,8** 4 -,5 5-0,5 MEL ,4 4 2,2 8,0 Outlier detection by Grubbs test 2 Outlier detection by Hampel stest *z-score value 2<IzI <3 **z-score value IzI 3 N/A: no answer Page2/42

13 Table 2b. Results and z-scoresof the qualitative pollen analysis of the honey sample (*nectarless pollen sources were excludedfrom the analysis). Laboratory Code Brassicaceae* assigned: 59 QUALITATIVE POLLEN ANALYSIS Tilia* assigned: 9 Rosaceae* assigned: 6 % z-score % z-score % z-score MEL ,* 3,3 7 0,4 MEL , 0 0,3 6 0,0 MEL04 6 0,2 7-0,7 3 -,2 MEL ,5 6 -,0 3 -,2 MEL ,6 0 0,3 0,6 MEL0 58-0, 8-0,3 5-0,4 MEL2 60 0, 6 -,0 6 0,0 MEL3 64 0,6 0 0,3 7 0,4 MEL5 66 0,9 6 -,0 3 -,2 MEL8 8,2-6,2** 3,3 N/A - MEL2 54-0,6 5 -,3 9,2 MEL ,5 0,7 3 -,2 MEL ,4 6 -,0 4-0,8 MEL , 2,0 8 0,8 MEL ,5 9 0,0,9 MEL ,6 7-0,7 8 0,8 MEL ,6 0,7 9,2 MEL ,7 0 0,3 5-0,4 MEL ,5 9 0,0 6 0,0 MEL4 63 0,5 5 -,3 4-0,8 MEL ,6 20,2 3,7** 5,2 3,5** MEL , 9 0,0 4-0,8 MEL50 72,6 5 -,3,9 MEL55 67,0 2,0 3 2,7 MEL ,8 6 2,4* 4 2 3,** MEL ,4 7-0,7 6 0,0 MEL7 59 0,0 7-0,7 5-0,4 MEL ,4 0,7 5-0,4 MEL ,9 7 2,7* 7 0,4 MEL ,4 8-0,3 5-0,4 MEL8 56-0,4 0,7 7 0,4 MEL ,5 9 0,0 6 0,0 MEL84 7,5 9 0,0,9 MEL ,8 7-0,7 7 0,4 MEL ,0 7-0,7 6 0,0 MEL93 73,7 8-0,3 5-0,4 MEL ,7 9 0,0 7 0,4 MEL ,8** 5 -,3 5-0,4 MEL ,7 8 0,8 Outlier detection by Grubbs test 2 Outlier detection by Hampel stest *z-score value 2<IzI <3 **z-score value IzI 3 N/A: no answer Page3/42

14 MEL8 MEL97 MEL89 MEL75 MEL02 MEL59 MEL44 MEL82 MEL95 MEL30 MEL37 MEL09 MEL2 MEL8 MEL23 MEL98 MEL0 MEL74 MEL03 MEL29 MEL90 MEL3 MEL2 MEL04 MEL7 MEL45 MEL34 MEL78 MEL25 MEL40 MEL05 MEL4 MEL5 MEL84 MEL62 MEL55 MEL39 MEL50 MEL93 z-score 3 Brassicaceae Laboratory code Figure 3a.Z-score histogram of the qualitative pollen analysis of Brassicaceae pollen. Page4/42

15 MEL97 MEL2 MEL50 MEL4 MEL5 MEL2 MEL05 MEL25 MEL34 MEL89 MEL90 MEL04 MEL93 MEL7 MEL0 MEL78 MEL84 MEL62 MEL30 MEL95 MEL40 MEL09 MEL45 MEL82 MEL3 MEL23 MEL39 MEL03 MEL8 MEL74 MEL37 MEL55 MEL29 MEL02 MEL8 MEL98 MEL59 MEL75 MEL44 z-score 4 Tilia Laboratory code Figure 3b.Z-score histogram of the qualitative pollen analysis oftilia pollen. Page5/42

16 MEL04 MEL05 MEL5 MEL23 MEL4 MEL25 MEL45 MEL0 MEL78 MEL74 MEL93 MEL7 MEL39 MEL40 MEL97 MEL2 MEL90 MEL8 MEL62 MEL82 MEL3 MEL03 MEL75 MEL02 MEL34 MEL95 MEL89 MEL29 MEL98 MEL37 MEL09 MEL2 MEL50 MEL30 MEL55 MEL44 MEL59 MEL84 z-score 4 Rosaceae Laboratory code Figure 3c.Z-score histogram of the qualitative pollen analysis of Rosaceae pollen. Page6/42

17 MEL8 MEL97 MEL02 MEL75 MEL59 MEL89 MEL44 MEL82 MEL95 MEL37 MEL09 MEL2 MEL23 MEL30 MEL8 MEL74 MEL0 MEL29 MEL03 MEL90 MEL98 MEL7 MEL2 MEL45 MEL04 MEL78 MEL25 MEL62 MEL4 MEL05 MEL40 MEL34 MEL3 MEL39 MEL5 MEL55 MEL84 MEL50 MEL93 z-score 2 Brassicaceae* Laboratory code Figure 3d. Z-score histogram of the qualitative pollen analysis of Brassicaceae pollen (*nectarless excluded). Page7/42

18 MEL50 MEL4 MEL2 MEL97 MEL2 MEL05 MEL5 MEL25 MEL90 MEL04 MEL62 MEL34 MEL7 MEL89 MEL78 MEL93 MEL0 MEL84 MEL45 MEL30 MEL95 MEL82 MEL40 MEL09 MEL3 MEL39 MEL03 MEL8 MEL74 MEL37 MEL23 MEL29 MEL55 MEL02 MEL8 MEL98 MEL59 MEL75 MEL44 z-score 4 Tilia* Laboratory code Figure 3e. Z-score histogram of the qualitative pollen analysis of Tilia pollen (*nectarless excluded). Page8/42

19 MEL04 MEL05 MEL5 MEL23 MEL4 MEL25 MEL45 MEL78 MEL74 MEL93 MEL7 MEL39 MEL97 MEL0 MEL2 MEL90 MEL62 MEL82 MEL03 MEL40 MEL8 MEL75 MEL02 MEL95 MEL3 MEL89 MEL29 MEL98 MEL34 MEL37 MEL2 MEL09 MEL50 MEL84 MEL30 MEL55 MEL59 MEL44 z-score 4 Rosaceae* Laboratory code Figure 3f. Z-score histogram of the qualitative pollen analysis of Rosaceae pollen (*nectarless excluded). Page9/42

20 Quantitative Pollen Analysis In total, 22analysts performed quantitative pollen analysis. The methods used for the analysis varied significantly among the participants (Annex III). The relative centrifugal force also varied and ranged between357 and 3220 g (540±862); while centrifuging lasted at least 0 minutes in all cases (5 ± 7).The relative standard deviation of reproducibility of the test results was much higher than the values stated in manuscript Harmonized methods of melissopalynology by the IHC in 2004 (Table ).This could be explained due to the great variety of RCF, time and methods used by the participants. No outlier was detected either by Hampler s or Grubbs test and a single peak distribution was observed in the Kernel density plot (Figure 4). The results and z-scores of the quantitative pollen analysis per participant are presented in Table 3 and Figure 5. Table 3. Results and z-scores of the quantitative pollen analysis of the honey sample derived from the participants (assigned value=2000). Laboratory Code QUANTITATIVE POLLEN ANALYSIS Number of pollen grains/0g of honey z-score Laboratory Code Number of pollen grains/0g of honey z-score MEL ,4 MEL ,0 MEL ,7 MEL ,2 MEL ,5 MEL ,2 MEL ,8 MEL ,3 MEL ,4 MEL ,7 MEL ,2 MEL ,0 MEL ,6 MEL ,4 MEL ,3 MEL ,4 MEL ,7 MEL ,3 MEL , MEL ,5 MEL ,0 MEL ,6 Outlier detection by Grubbs test 2 Outlier detection by Hampel s test *z-score value 2<IzI <3 **z-score value IzI 3 Page20/42

21 MEL97 MEL95 MEL02 MEL40 MEL37 MEL2 MEL09 MEL74 MEL45 MEL25 MEL39 MEL62 MEL34 MEL44 MEL2 MEL30 MEL05 MEL50 MEL0 MEL5 MEL90 MEL75 z-score Kernel Density Plot (number of pollengrains per 0g of honey) 0, , , , , , ,0000 0, Figure 4.Kernel density plot of the quantitative pollen analysis Laboratory code Figure 5.Z-score histogram of the quantitative pollen analysis. Page2/42

22 Botanical Origin All the participants stated the botanical origin of the honey based single on the melissopalynological analysis and 29stated additionally the botanical origin of the sample based both on melissopalynological analysis and physicochemical and/or sensory analysis. In most cases the analysts used the Main European honeys: descriptive sheets published in Apidologie in 2004 to conclude to the botanical origin of the honey sample however, it must be noticed that in several cases, although the participants found similar percentages, they did not conclude to the same result. Additional references were also used usually in combination (Annex III). In total, 59% of the participants characterized the sample as multifloral honey when only the melissopalynological analysis was taken into account; and 72% when both the melissopalynological and the physicochemical and/or sensory analysis were considered (Figure 6 and 7). Correspondingly, 26% of the participants characterized the sample as Brassicaceae and 5% as Tilia at the first case; and at the second case, the percentages were 4% for Brassicaceae and 4% for Tilia. The results of the botanical origin per participant are presented in Table multifloral/blossom Brassicaceae/ Brassica Tilia Figure 6.Botanical origin of the honey sample according to the melissopalynological analysis. Page22/42

23 4 4 2 multifloral/blossom Brassicaceae Tilia Figure 7.Botanical origin of the honey sample according to the melissopalynological and physicochemical and/or sensory analysis. Geographical Origin The honey was harvested in Eastern Europe. In total, 3analysts provided data about the geographical origin of the honey sample. The references used for the results are mentioned in Annex III. Several analysts stated correctly the geographical origin of the sample. The results of the geographical origin per participant are presented in Table 4. Page23/42

24 Table 4. Results of the determination of the botanical and geographical origin of the honey sample derived from the participants. Laboratory Code number of samples analysed per year botanical origin * MEL02 >500 Brassicaceae botanical origin** Flower honey (brassicaceae& tilia) physicoc hemical sensory geographical origin x x Roumanie? MEL Brassica spp. N/A N/A MEL04 <50 Brassica spp. Brassica spp. x Central Europe? MEL05 >500 multifloral multifloral x East Europe MEL09 >500 Blossom, no unifloral honey Blossom, no unifloral honey x x Western/Central Europe or Eastern Europe MEL multifloral N/A N/A MEL2 >500 multifloral multifloral x East Europe MEL Brassicaceae Honey Brassicaceae Honey x x Southern Europe MEL5 >500 multifloral multifloral x East Europe MEL8 <50 heterofloral N/A France MEL2 >500 Blossom, no unifloral honey Blossom, no unifloral honey x x Western/Central Europe or Eastern Europe MEL23 >500 Polyfloral Polyfloral x x N/A MEL multifloral multifloral x x France (maybe also Germany is possible) MEL29 >500 multifloral multifloral x x Central Europe MEL30 >500 Lime/Tilia Lime x x France MEL Brassica N/A N/A MEL37 >500 Polyflora Polyflora x Eastern Europe MEL Tilia sp. Brassica sp. x MEL Tilia &Brassica type (B.napus)&Malus Pyrus-Type Tilia (Lime tree honey) probably Central Europe x x Mediterranean-France *only melissopalynological analysis was considered for the botanical origin **melissopalynological and physicochemical or/and sensory analysis were considered for the botanical origin N/A: no answer Page24/42

25 Table 4.(continued). Laboratory Code number of samples analysed per year botanical origin * botanical origin** physicoc hemical sensory geographical origin MEL4 >500 Blossom Honey Blossom x x South-East-Europe MEL Tilia Tilia x Eastern Europe MEL45 >500 Blossom, no unifloral honey MEL50 >500 Tilia Blossom, no unifloral honey Tillia and Frangula alnus x x Western/Central Europe or Eastern Europe x x Temperate zone MEL wildflower honey N/A N/A MEL Brassica spp - Brassicaceae Brassica spp - Brassicaceae x Southern Europe MEL multifloral multifloral x Eastern Europe MEL multifloral multifloral x x Southeastern Europe, Italy, Poland MEL Brassica napus+tilia mixture Brassica napus and others x x possible France MEL Brassicaceae N/A Europe MEL78 >500 multifloral multifloral x x Central Europe, France, also Eastern Europe cannot be excluded MEL8 <50 Tilia N/A N/A MEL82 >500 polyfloral honey with parts of rape and lime N/A Central-East Europe MEL84 <50 Brassica N/A N/A MEL floral floral x x N/A MEL tilia tilia x Europe (middle western) MEL93 >500 Brassica Polyfloral x Eastern Europe MEL Brassica napus N/A Southern Europe MEL Multifloral honey from Brassicaceae, Prunus multifloral x France MEL98 >500 Summer flowers Summer flowers x Mediterranean Page25/42

26 References Eurachem -20, Selection, Use and Interpretation of Proficiency Testing Schemes by Laboratories. ISO 3528:2005, Statistical methods for the use of proficiency testing by interlaboratory comparisons. ISO 5725:994, Accuracy of Measurement Methods and Results. ISO7043:200, Conformity assessment-general requirements for proficiency testing. Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004) Harmonized methods of melissopalynology, Apidologie (Suppl. ): Page26/42

27 ANNEX I Participant Laboratories Page27/42

28 Country AUSTRIA BELGIUM BRAZIL CROATIA CROATIA FRANCE FRANCE GERMANY GERMANY GERMANY GERMANY GERMANY GERMANY GREECE GREECE GREECE GREECE GREECE ITALY ITALY ITALY ITALY ITALY ITALY ITALY Institution Austrian Agency for Health and Food Safety (AGES), Institute for Seed and Propagation Material, Phytosanitary Service and Apiculture, Department for Apiculture and Bee Protection CARI asbl Instituto Oswaldo Cruz Fiocruz Faculty of Agriculture, J.J. Strossmayer University in Osijek University of Zagreb Faculty of Agriculture Department of Fisheries, Beekeeping Game management and Special Zoology Famille Michaud Apiculteurs Naturalim France Miel Breitsamer + Ulrich GmbH & Co KG Eurofins Analytik GmbH Intertek Food Services GmbH Länderinstitut für Bienenkunde Hohen Neuendorf e.v. LAVES Institut für Bienenkunde Celle Quality Services International GmbH ATTIKI Bee Culturing Co. Alexandros Pittas S.A Laboratory of Apiculture and Sericulture,Faculty of Agriculture, Aristotle University of Thessaloniki Laboratory of Residue Analysis in Food Of Animal Origin Mediterranean Agronomic Institute of Chania Region of South Aegean, General Directorate of Rural Economy & Veterinary, Directorate of Rural Economy A.S.S.A.M. Centro Agrochimico Regionale CRA-API Honey bee and Silkworm Research Unit of the Italian Agricultural research Council Dipartimento di Scienze Agrarie, Forestali e Alimentari - Università di orino Ispettorato Centrale della tutela della qualità e repressione frodi dei prodotti agroalimentari Laboratorio di Perugia Laboratorio biologico, Agenzia provinciale per l'ambiente di Bolzano Piana Ricerca e Consulenza srl Unità Operativa di Apicoltura - Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana Page28/42

29 Country NETHERLANDS POLAND POLAND POLAND POLAND SERBIA SLOVENIA SPAIN SWITZERLAND TURKEY Institution Jacob Kerkvliet Agricultural and Food Quality Inspection Main Inspectorate Warsaw Bee Product Quality Testing Laboratory, Research Institute of Horticulture, Apiculture Division Mazurskie Miody Office of Competition and Consumer Protection, Laboratory in Olsztyn Faculty of Agriculture, University of Belgrade, Center for Food Analysis Slovenian Beekeepers Association Casa de la Miel. Cabildo de Tenerife Biologisches Institut für Pollenanalyse K. Bieri GmbH Altıparmak Gıda Sanayi ve ic. A.Ş. Page29/42

30 ANNEX II Cover Letter and Result Form Page30/42

31 INTERNATIONAL HONEY COMMISSION MELISSOPALYNOLOGY WORKING GROUP RING TEST FOR HONEY QUALITATIVE AND QUANTITATIVE MELISSOPALYNOLOGICALANALYSIS Dear participant, Date 24/07/203 thank you very much for participating in the present inter-laboratory ring test (proficiency scheme test) on the melissopalynological analysis of honey organized by the International Honey Commission under the coordination of the Laboratory of Apiculture of the Aristotle University of Thessaloniki. Within - - Analyze the sample as if was a routine sample, using the method that you regularly apply. Read the instructions given in the end of the form (notes -5) and fill in the attached Reporting Form. The Reporting Form must be sent back until 30 September 203 to this address: mdimou@agro.auth.gr. Please name the file using your laboratory code at the end e.g. IHCPOLLENTEST_REPORTING_FORM_MEL25. When the test is completed, a Final Result Report using the laboratories codes will be sent to all the participants along with the statistical evaluation of the results (z-scores, e.t.c). Thank you for your cooperation best regards, Maria Dimou Recommended bibliography Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004) Harmonized methods of melissopalynology, Apidologie (Suppl. ): Persano Oddo L., Piro R. (2004) Main European unifloral honeys: descriptive sheets, Apidologie 35 (Suppl. ): Pendleton M. (2006) Descriptions of melissopalynological methods involving centrifugation should include data for calculating Relative Centrifugal Force (RCF) or should express data in units of RCF or gravities (g),grana, 45:, Page3/42

32 INTERNATIONAL HONEY COMMISSION MELISSOPALYNOLOGY WORKING GROUP INTER-LABORATORY RING TEST FOR HONEY QUALITATIVE AND QUANTITATIVE MELISSOPALYNOLOGICAL ANALYSIS LABORATORY CODE NUMBER DATE OF RECEIPT STATE OF SAMPLE 4 ACCREDITATION 2 NUMBER OF SAMPLES ANALYSED PER YEAR 3 < >500 5 SAMPLE CODE IHC 0-3 METHODOLOGY 6 QUALITATIVE POLLEN ANALYSIS Reference:Harmonized methods of melissopalynology(apidologie, 2004) Other (please specify) Family Pollen Form Brassicaceae Brassicaceae 2 Tiliaceae Tilia 3 Rosaceae Rosaceae 4 Other Sum all pollen types Value(%) 7A nectarless exluded Value(%) 7B METHODOLOGY 6 2 QUANTITATIVE POLLEN ANALYSIS Reference:Harmonized methods of melissopalynology(apidologie, 2004) 9 Other (please specify) 0 Number of pollen grains/0g of honey: RCF minutes 3 BOTANICAL ORIGIN (only pollen) Reference:European unifloral honeys (Apidologie, 2004) 6 Honey from: 2 Other (please specify) 4 BOTANICAL ORIGIN (combination sensory, pollen, physicochemical) 6 9, 3 Reference:European unifloral honeys (Apidologie, 2004) Honey from: Other (please specify) physicochemical sensory GEOGRAPHICAL ORIGIN Reference: (please specify) Honey from: 9 6 COMMENTS... Page32/42

33 Please write the code number of your laboratory 2 Please write the date when you received the honey sample 3 Please describe the state of the delivered sample e.g. good, damaged, liquid, crystallised e.t.c. If the sample is inappropriate for analysis please contact us 4 Please mark if your laboratory is accredited for honey pollen analysis 5 Please mark the approximate number of honey samples that you perform pollen analysis per year 6 Please mark or write the method you use for pollen anaysis / botanical / geographical determination 7A Fill in the the presence (%) of the specific pollen forms in the honey sample.to calculate their percentage INCLUDE also all nectarless taxa.use NUMERICAL data with no decimals. Fill in the the presence (%) of the specific pollen forms in the honey sample.to calculate their percentage EXLUDE all nectarless taxa.use NUMERICAL data with 7B no decimals. 8 This field is automatically filled to sum 00% 9 This field is optional 0 Please write the relative centrifugal force (for more information see Pendleton, Grana, 2006) Please write the minutes of centrifuging (for more information see Pendleton, Grana, 2006) 2 Please fill in the botanical origin of the honey considering ONLY the results from pollen analysis 3 Please fill in the botanical origin of the honey considering also sensory and/or physicochemical analysis 4 Please mark if you performed sensory analysis 5 Please mark if you performed physicochemical analysis e.g. electrical conductivity, sugars e.t.c. Page33/42

34 ANNEX III Methods and References Page34/42

35 The methods applied by the participants per analysis are mentioned below-wherever the data was available. In some cases, the analyst companied more than one method/reference to perform the analysis or to conclude to the results. Qualitative Melissopalynological Analysis The methodology applied by the participants for the qualitative pollen analysis of the honey sample. Methods Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004) Harmonized methods of melissopalynology, Apidologie 35 DIN Deutsches Institut für Normung 2002 Untersuchung von Honig Bestimmung der relative Pollenhäufigkeit, DIN 0760: , Berlin ASU 64 LFGB L40.00-) Louveaux J., Maurizio A., Vorwohl G. (978) Methods of Melissopalynology, Bee World 59 UNI-299/2008 OR internal method based on UNI-299/2008 (Miele - Analisi microscopica o melissopalinologica) (2008) Erdtman (969) Handbook of Palynology, Munksgaard, Copenhagen Croatian Regulation of the Minister of Agriculture and Forestry NN20/2000 Number of participants Polska Norma PN-88/A Miód pszczeli 998 Rozp. MRiRW z dnia 4 stycznia 2009r. Zał. do rozp. pkt. VI (Dz.U. z 2009r. Nr 7; poz.94 Other Internal method Quantitative Melissopalynological Analysis The methodology applied by the participants for the quantitative pollen analysis of the honey sample. Methods Number of participants Page35/42

36 Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004) Harmonized methods of melissopalynology, Apidologie 6 35 Internal method based on Lycopodium spores tablets (Stockmarr J.(97) Tablets with Spores used in Absolute Pollen 4 Analysis, Pollen et Spores 3) DIN Deutsches Institut für Normung 2002 Untersuchung von Honig Bestimmung der relative Pollenhäufigkeit, DIN : , Berlin ASU 64 LFGB L40.00-) Louveaux J., Maurizio A., Vorwohl G. (978) Methods of Melissopalynology, Bee World 59 3 UNI-299/2008 OR internal method based on UNI-299/2008 (Miele - Analisi microscopica o melissopalinologica) (2008) 3 Erdtman (969) Handbook of Palynology. Munksgaard, Copenhagen Other Internal method Botanical Origin The references used by the by the participants for the determination of the botanical origin of the honey sample. References Persano Oddo L., Piro R. (2004) Main European unifloral honeys: descriptive sheets, Apidologie 35 Beckh G., Camps G.(2009) Neue Spezifikationen fur Trachthonige, Deutsche Lebensmittel-Rundschau 05 Polish Standard Honey Polska Norma PN-88/A Miód pszczeli (998) Polish standard - Regulation of the Minister of Agriculture and Rural Development of r (Journal of Laws ) Dz.U. nr 8, poz. 773 z późn. zm. DIN Deutsches Institut für Normung 2002 Untersuchung von Honig Bestimmung der relative Pollenhäufigkeit, DIN 0760: , Berlin Ricciardelli del Arbore (998) Mediterranean melissopalynology, Perugia Croatian Regulation of the Minister of Agriculture and Forestry NN20/2000 Maurizio A., Louveaux J Pollens de plantes mellifères d Europe. Union des Groupements apicoles français, Paris Number of participants Page36/42

37 Persano Oddo L., Piazza M.G., Sabatini A.G., Accorti M. (995) Characterization of unifloral honeys, Apidologie 26, Persano Oddo L., Sabatini A.G., Accorti M., Colombo R., Marcazzan G.L., Piana M.L., Piazza M.G:, Pulcini P. (2000) I mieli uniflorali italiani. Nuove schede di caratterizzazione. Ministero delle Politiche Agricole e Forestali, Istituto Sperimentale per la Zoologia Agraria, Sezione di Apicoltura, Roma Ruoff K., Luginbühl W., Kilchenmann V., Olivier Bosset J., von der Ohe K., von der Ohe W., Amadò R. (2007) Authentication of the botanical origin of honey using profiles of classical measurands and discriminant analysis. Apidologie 38: Ricciardelli del Arbore, v.d.ohe,bucher (2000)Il Meli regionali Italiani Ricciardelli del Arbore (997) Textbook of melissopalynology, Apimondia Internal method based on ISO 6658/2005 and 8586/202 Geographical Origin The references used by the participants for the determination of the geographical origin of the honey sample. References Ricciardelli d Albore G. 997) Textbook of melissopalynology, Apimondia Ricciardelli del Arbore (998) Mediterranean melissopalynology, Perugia Battaglini M., Ricciardelli D'albore G. (972) Differenziazione dei mieli italiani e stranieri in base allo spettro pollinico. Simposio internazionale di Apicoltura: problemi di Flora mellifera e Impollinazione. Torino, Italy, 2-6 ottobre 972. Apimondia, Bucarest, Romania: 96- Beckh G., Camps G.(2009) Neue Spezifikationen fur Trachthonige, Deutsche Lebensmittel-Rundschau 05 Louveaux J. 970 Annexes microphotographiques aux méthodes officielles d analyse. ome III. Atlas photographique d analyse pollinique des miels. Ministère de l Agriculture, Service de la Répression des fraudes et du Control de la Qualité, Paris Persano Oddo L., Piro R. (2004) Main European unifloral honeys: descriptive sheets, Apidologie 35 Ricciardelli del Arbore, v.d.ohe,bucher (2000) Il Meli regionali Italiani Number of participants 3 2 Page37/42

38 Polish standard - Regulation of the Minister of Agriculture and Rural Development of r (Dz.U. nr 8, poz. 773 z późn. zm. Sawyer R. (988) - Honey identyfication. Cardiff Academic Press Page38/42

39 ANNEX IV Comments Page39/42

40 Further information about the melissopalynological analysis stated by the participants at the6 th paragraph of the Report Formis summarized in random order bellow:. great variety of pollen types/forms (over 70) in the honey sediment 2. detection of pollen grains from Fraxinus ornus, Vitis and probable Cotinus 3. missing of Mediterranean elements 4. many calcium oxalate rosettes (crystalls) - typical for Tilia 5. nectarless of Oleaceae,Graminae, Cyperaceae, Chenopodiaceae 6. presence of starch grains 7. presence of Helianthus annus, Trifolium, Robinia, Rhamnaceae, Salix and isolatedrumex, Caesalpiniaceae, Amorpha,Lotus,Melilotus,Castanea, Cornus, Fraxinus, Carduus, Caryophyllaceae, Poaceae, Quercus, Apiaceae, Vicia 8. the analysed pollen spectrum lacks diversity: Cruciferae, Rosaceae and Tilia are the mainly identified pollen grains and the other identified pollen grains are not distinctive enough for a certain origin (there are no marker pollen indicating a definite origin) 9. other pollen types: Aceraceae: Acer /Boraginaceae /Boraginaceae: Echium/ Caesalpinaceae: Gleditsia/ Compositae-Asteraceae: Compositae-Asteraceae, Centaurea cyanus, Helianthus-t, Serratula, Taraxacum/ Convolvulaceae/ Cornaceae/ Fagaceae: Quercus/ Graminae: Zea/ Hippocsatanaceae: Aesculus/ Labitae/ Leguminosae: Amorpha, Lotus, Robinia, Trifolium-t, Viciat/ Pinaceae: Pinus/ Rhamnaceae/ Rosaceae: Pyrus-Prunus-t/ Salicaceae:Salix/ Simoaroubaceae: Ailanthus/ Umbelliferae-Apiaceae: Umbelliferae-Apiaceae, Coriandrum, Foeniculum 0. Other pollen forms: Asteraceae form H, Trifolium pratense gr., Apiaceae, Vicia, Robinia, Asteraceae form S, Cornus sanguinea, Rhamnaceae, Campanulaceae. Pollen not nectariferous: Cupressaceae, Chenopodiaceae. Other (Rhamnaceae, Apiaceae, Salix, Trifolium, Helianthus) 2. great variety of pollen types/forms (over 70) in the honey sediment; detection of pollen grains from Fraxinus ornus, Vitis and probable Cotinus might indicate, presumably, an origin to the more Southern part of central Europe; missing Mediterranean elements 3. nectraless: Oleaceae,Graminae, Cyperaceae, Chenopodiaceae andhoneydew elements 4. other pollens: Rhamnus alaternus, Trifolium, Robinia pseudoacacia, Salix, Gleditschia, Amorpha, Helianthus, Acer, Vicia, Lotus, Artemisia, Lappa, Centaurea Cyanus, Cerinthe, Castanea sativa, Quercus, Chenopodium, Cornus, Cupressaceae, Linium, Convolvulus, Liliaceae, Aesculus, Labiaceae, Mercurilis, Millepertus, Verbascum, Rhamnus, Oleacea, Apiaceae, Papaveraceae, Plantago, Reseda lutea, Salix, Schinus, Tamaris, Vitis, Viburnum 5. Other pollen types: Rhamnaceae, Helianthus annuus, Umbelliferae, Trifolium repens, robina pseudoacacia, Salix, Polygonaceae, Melilotus spp., Vicia, Campanula, Compositae, Castanea, Lotus corniculatus, Labiatae, Betulaceae, Plantago, Trifolium pratense, Caryophyllaceae, Quercus, Zea mays, Page40/42

41 Betulaceae, Filipendula ulmeria, Oleaceae 6. The geographical origin of honey was determined based on presence of Castanea sativa and Cistus pollen 7. Considering the organoleptical characteristics (taste, colour, consistency) there are no monofloral characteristics. So the part of rape is overrepresented 8. Apiaceae: Coriandrum, Foeniculum, Heracleum/ Asparagaceae: Asparagus/ Boraginaceae: Cynoglossum, different Boraginaceae/ Campanulaceae: Jasione/ Hypericaceae: Hypericum/ Asteraceae: Achillea, Cichorium, Centaurea cyanus/ Convolvulaceae: Convolvulus arvensis/ Cornaceae: Cornus sanguisorba/ Fabaceae: Amorpha fructicosa, Gleditsia, Lotus corniculatus, Robinia pseudoacacia, Trifolium repens, Vicia/ Fagaceae: Quercus (nectarless)/ Lamiaceae: Thymus/ Oleaceae: Ligustrum/ Papaveraceae: different (nectarless)/ Plantaginaceae: Plantago (nectarless)/ Poaceae:wild grasses (nectarless)/ Polygonaceae: Rumex (nectarless)/ Ranunculaceae: different Ranunculaceae/ Resedaceae: Reseda/ Rhamnaceae: different/ Rosaceae: Potentilla,Fragaria, Prunus (Amygdaleae), Pyrus (Pyreae)/ Rubiaceae: Galium/ Salicaceae: Populus/ Ulmaceae: Ulmus/ Vitaceae: Vitis (nectarless) 9. Apiaceae/ Asteraceae: Helianthus annuus, Achillea spp., Cirsium spp./ Campanulaceae: Campanula spp./ Fabaceae: Amorpha fruticosa, Trifolium pratense gr., Lotus spp., Robinia pseudoacacia, Vicia spp., Trifolium repens, Quercus spp. (nectarless)/ Hydrophyllaceae: Phacelia tanacetifolia/ Lamiaceae/ Plantaginaceae: Plantago spp. (nectarless)/ Poaceae (nectarless)/ Polygonaceae: Rumex spp. (nectarless)/ Rhamnaceae: Rhamnus spp./ Salicaceae: Salix spp. 20. According to sensory properties the test sample is not dominated by rapeseed nectar, despite the high percentage of the rapeseed pollen grains determinated by melissoplynologycal analysis. 2. Apiaceae, Chenopodiaceae, Fabaceae-Faboideae Vicia type, Poaceae, Asteraceae, Myrtaceae, Caesalpiniaceae 22. Acer Form/ Amaranthaceae-Chenopodiaceae/ Boraginaceceae: Echium, Symphytum/ Caryophyllaceae/ Asteraceae: Artemisia, Centaurea jacea, Compositae A,Compositae H, Compositae S, Compositae T/ Cornaceae: Cornus sanguinea/ Dipsacaceae/ Fagaceae: Castanea, Quercus robur/ Graminaceae-Poaceae: Graminaceae-Poaceae, Zea/ Jugladaceae: Juglans/ Lauraceae/ Fabaceae: Amorpha, Lotus, Melilotus, Robinia, Trifolium pretense, Trifolium repens, Vicia faba/ Oleaceae: Fraxinus ornus/ Plantaginaceae: Plantago/ Ranunculaceae: Clematis/ Rhamnaceae: Frangula/Salicaceae: Salix/ Umbelliferae/Apiaceae 23. Hippocrepis spp. (Coronilla-Hippocrepis form)/ Myrtus communis (Myrtus- Syzygium form)/ Amorpha fruticosa (Amorpha form)/ Salix spp. (Salix)/ Helianthus annuus (H form)/ Daucus carota (Forma Astrantia (A) >25u Apiaceae)/ Robinia pseudoaccacia (Robinia form)/ Vicia spp. (Vicia)/ Bryonia dioica ( Bryonia)/ Trifolium repens (Trifolium repens group)/ Trifolium pratense (Trifolium pratense group)/ Lathyrus spp. (Lathyrus)/ Castanea sativa (Castanea)/ Melilotus spp. (Melilotus)/ Lotus corniculatus (Lotus corniculatus group)/ Medicago spp. (Medicago)/ Heracleum sphondylium Page4/42

42 (Forma Heracleum (H) Apiaceae)/ Scrophularia nodosa (Scrophularia form)/ Ligustrum spp. (Ligustrum form)/ Ailanthus altissima(ailanthus)/ Eucalyptus spp. (Eucalyptus form)/ Caryophyllaceae (Saponaria form)/ Euonymus europaeus (Euonymus)/ Taraxacum officinale (Forma T Asteraceae)& nectarless:hypericum spp. (Hypericum)/ Vitis vinifera (Vitis)/ Betula spp. (Betula)/ Plantago lanceolata (Plantago)/ Frangula alnus (Frangula)/ Fraxinus ornus (Fraxinus ornus)/ Xanthium spp. (Xanthium)/Papaver rhoeas (Papaver)/ Sambucus nigra (Sambucus nigra/ Rosaceae (Filipendula spp., Aruncus dioicus (Walter) Fernald) (Pollen type according to Livia Persano Oddo & Giancarslo Ricciardelli D Albore (989) Nomenclatura Melissopalinologica, Apicoltura, 5: 63-72) 24. Other pollens: Amorpha, Papaver, Helianthus, Trifolium alexandrinum, Papaver, Vicia, Cynoglossum, Lotus, Robinia,Vitis, Rumex, Plantago, Sambucus, Gramineae Page42/42