report - ihcihc-platform.net/reportptschemeihc0113.pdf · thessaloniki, greece. in total,...

Proficiency Testing Scheme for Honey Pollen Analysis IHC01-13

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INTERNATIONAL HONEY COMMISSION

MELISSOPALYNOLOGY WORKING GROUP

LABORATORY OF APICULTURE, ARISTOTLE UNIVERSITY OF THESSALONIKI, GREECE

REPORT


October 2013


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Coordinators of the PT scheme

DimouMaria TananakiChrisoula ThrasyvoulouAndreas Laboratory of Apiculture&Sericulture Faculty of Agriculture Aristotle University of Thessaloniki Agroktima Panepistimiou Thermi 57001 Greece Tel.:+30 2310 472983 Fax:+30 2310 471939 E-mail: [email protected]

Thessaloniki, 26 October 2013

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

mailto:[email protected]


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Summary

This report describes the design, results and statistical analysis and of the inter-

comparison 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 IHC01-13. 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.


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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.


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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 ............................................................................................... 20

Botanical origin ................................................................................................................... 22

Geographical origin ............................................................................................................ 23

References ................................................................................................................... 26

Annex I. Participant Laboratories ................................................................................ 27

Annex II. Cover letter and Result Form ....................................................................... 30

Annex ΙIΙ. Methods and References ............................................................................. 34

Annex IV. Comments ................................................................................................... 38


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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 100 g sample

of honey in a plastic container labeled as IHC01-13. 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 15-07-2013. 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

14th of October 2013.

Statistical Analysis

For each numerical value, the following parameters were calculated according to ISO

13528:2005(E): median value, mean value, standard deviation, robust standard


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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 15 were accredited for

honey pollen analysis. Almost half of the analysts perform pollen analysis to more

than 500 honey samples per year (Figure 1 and Table 4).

Figure 1. Number of honey samples analysed per year from the analysts.

17

3

15

4

>500

200-500

50-200

<50


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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 1.

Table 1. Descriptive summary statistics

Parameter

HONEY POLLEN ANALYSIS

QUALITATIVE QUANTITATIVE

Brassicaceae Tilia Rosaceae Brassicaceae* Tilia* Rosaceae* Number of pollen

grains per 10g of honey

number of participants (n) 39 39 38 39 39 38 22

median value (x) 57 8 6 59 9 6 21000

mean value (m) 54,1 8,9 6,8 56,1 9,4 6,9 20383

standard deviation (sd) 11,9 3,3 3,1 12,0 3,4 3,0 9798

robust standard deviation(s*) 6,6 2,7 2,0 8,2 3,0 2,58 10741

assigned value X 57 8 6 59 9 6 21000

standard uncertainty of the assigned value (ux)

10,8 1,8 1,4 11,2 1,9 1,4 5432

target-value of the standard deviation

for proficiency assessment σ^

6,6 2,7 2,0 8,2 3,0 2,6 10741

relative standard deviation of

reproducibility RSDR%

12% 37% 45% 15% 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 2004. 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<0.001) and Tilia (paired t-test p<0.001) but


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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 1). 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).


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Figure2. Kernel density plot of the qualitative pollen analysis: a. Brassicaceae; b.

Tilia; c. Rosaceae.

0

0,01

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0,06

0,07

0 10 20 30 40 50 60 70 80 90

Kernel Density Plot a. Brassicaceae (%)

0

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-5 0 5 10 15 20 25

Kernel Density Plot b. Tilia (%)

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0,18

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Kernel Density Plot c. Rosaceae (%)


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Figure2. (continued): d. Brassicaceae* (nectarless excluded); e. Tilia*(nectarless

excluded); f. Rosaceae*(nectarless excluded).

0

0,01

0,02

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Kernel Density Plot d. Brassicaceae* (%)

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Kernel Density Plot e. Tilia* (%)

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Kernel Density Plot f. Rosaceae* (%)


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Table 2a. Results and z-scoresof the qualitative pollen analysis of the honey sample

(nectarless pollen sources were included in the analysis).

Laboratory Code

QUALITATIVE POLLEN ANALYSIS

Brassicaceae assigned: 57

Tilia assigned: 8

Rosaceae assigned: 6

% z-score % z-score % z-score

MEL02 41 -2,4* 13 1,9 7 0,5 MEL03 56 -0,2 10 0,7 6 0,0 MEL04 58 0,2 7 -0,4 3 -1,5 MEL05 61 0,6 6 -0,7 3 -1,5 MEL09 53 -0,6 9 0,4 9 1,5 MEL10 55 -0,3 8 0,0 5 -0,5 MEL12 58 0,2 5 -1,1 6 0,0 MEL13 57 0,0 9 0,4 6 0,0 MEL15 63 0,9 5 -1,1 3 -1,5 MEL18 81,2 -7,4** 13 1,9 N/A - MEL21 53 -0,6 5 -1,1 9 1,5 MEL23 54 -0,5 10 0,7 3 -1,5 MEL25 60 0,5 6 -0,7 4 -1,0 MEL29 57 0,0 12 1,5 8 1,0 MEL30 52 -0,8 8 0,0 11 2,5 MEL34 59 0,3 6 -0,7 7 0,5 MEL37 53 -0,6 11 1,1 9 1,5 MEL39 65 1,2 10 0,7 5 -0,5 MEL40 60 0,5 8 0,0 5 -0,5 MEL41 62 0,8 5 -1,1 4 -1,0 MEL44 45 -1,8 181 3,7** 13 3,4 MEL45 59 0,3 9 0,4 4 -1,0 MEL50 72 2,3* 5 -1,1 11 2,5* MEL55 65 1,2 11 1,1 12 2,9* MEL59 43 -2,1* 15 2,6 142 3,9** MEL62 65 1,2 8 0,0 6 0,0 MEL71 58 0,2 7 -0,4 5 -0,5 MEL74 55 -0,3 11 1,1 5 -0,5 MEL75 40 -2,6* 16 2,9* 7 0,5 MEL78 60 0,5 8 0,0 5 -0,5 MEL81 54 -0,5 11 1,1 6 0,0 MEL82 48 -1,4 9 0,4 6 0,0 MEL84 64 1,1 8 0,0 151,2 4,4** MEL89 39 -2,7* 6 -0,7 7 0,5 MEL90 57 0,0 7 -0,4 6 0,0 MEL93 72 2,3* 7 -0,4 5 -0,5 MEL95 51 -0,9 8 0,0 7 0,5 MEL97 252 -4,8** 4 -1,5 5 -0,5

MEL98 54 -0,4 14 2,2 8 1,0 1Outlier detection by Grubbs’ test 2Outlier detection by Hampel ‘stest *z-score value 2<IzI <3 **z-score value IzI≥3 N/A: no answer


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Table 2b. Results and z-scoresof the qualitative pollen analysis of the honey sample

(*nectarless pollen sources were excludedfrom the analysis).

Laboratory Code

QUALITATIVE POLLEN ANALYSIS

Brassicaceae* assigned: 59

Tilia* assigned: 9

Rosaceae* assigned: 6

% z-score % z-score % z-score

MEL02 42 -2,1* 13 1,3 7 0,4 MEL03 58 -0,1 10 0,3 6 0,0 MEL04 61 0,2 7 -0,7 3 -1,2 MEL05 63 0,5 6 -1,0 3 -1,2 MEL09 54 -0,6 10 0,3 10 1,6 MEL10 58 -0,1 8 -0,3 5 -0,4 MEL12 60 0,1 6 -1,0 6 0,0 MEL13 64 0,6 10 0,3 7 0,4 MEL15 66 0,9 6 -1,0 3 -1,2 MEL18 81,2 -6,2** 13 1,3 N/A - MEL21 54 -0,6 5 -1,3 9 1,2 MEL23 55 -0,5 11 0,7 3 -1,2 MEL25 62 0,4 6 -1,0 4 -0,8 MEL29 58 -0,1 12 1,0 8 0,8 MEL30 55 -0,5 9 0,0 11 1,9 MEL34 64 0,6 7 -0,7 8 0,8 MEL37 54 -0,6 11 0,7 9 1,2 MEL39 65 0,7 10 0,3 5 -0,4 MEL40 63 0,5 9 0,0 6 0,0 MEL41 63 0,5 5 -1,3 4 -0,8 MEL44 46 -1,6 201,2 3,7** 151,2 3,5** MEL45 60 0,1 9 0,0 4 -0,8 MEL50 72 1,6 5 -1,3 11 1,9 MEL55 67 1,0 12 1,0 13 2,7 MEL59 44 -1,8 16 2,4* 142 3,1** MEL62 62 0,4 7 -0,7 6 0,0 MEL71 59 0,0 7 -0,7 5 -0,4 MEL74 56 -0,4 11 0,7 5 -0,4 MEL75 43 -1,9 17 2,7* 7 0,4 MEL78 62 0,4 8 -0,3 5 -0,4 MEL81 56 -0,4 11 0,7 7 0,4 MEL82 47 -1,5 9 0,0 6 0,0 MEL84 71 1,5 9 0,0 11 1,9 MEL89 44 -1,8 7 -0,7 7 0,4 MEL90 59 0,0 7 -0,7 6 0,0 MEL93 73 1,7 8 -0,3 5 -0,4 MEL95 53 -0,7 9 0,0 7 0,4 MEL97 282 -3,8** 5 -1,3 5 -0,4

MEL98 59 0 14 1,7 8 0,8 1Outlier detection by Grubbs’ test 2Outlier detection by Hampel ‘stest *z-score value 2<IzI <3 **z-score value IzI≥3 N/A: no answer


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Figure 3a.Z-score histogram of the qualitative pollen analysis of Brassicaceae pollen.

-8

-7

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z-sc

ore

Laboratory code

Brassicaceae


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Figure 3b.Z-score histogram of the qualitative pollen analysis ofTilia pollen.

-2

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97

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Laboratory code

Tilia


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Figure 3c.Z-score histogram of the qualitative pollen analysis of Rosaceae pollen.

-2

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Laboratory code

Rosaceae


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Figure 3d. Z-score histogram of the qualitative pollen analysis of Brassicaceae pollen (*nectarless excluded).

-7

-6

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Laboratory code

Brassicaceae*


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Figure 3e. Z-score histogram of the qualitative pollen analysis of Tilia pollen (*nectarless excluded).

-2

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Laboratory code

Tilia*


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Figure 3f. Z-score histogram of the qualitative pollen analysis of Rosaceae pollen (*nectarless excluded).

-2

-1

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Laboratory code

Rosaceae*


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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 (1540±862); while

centrifuging lasted at least 10 minutes in all cases (15 ± 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 1).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=21000).

QUANTITATIVE POLLEN ANALYSIS

Laboratory Code

Number of pollen

grains/10g of honey

z-score Laboratory

Code

Number of pollen

grains/10g of honey

z-score

MEL02 5500 -1,4 MEL39 21000 0,0 MEL05 28420 0,7 MEL40 8640 -1,2 MEL09 15500 -0,5 MEL44 23400 0,2 MEL10 30000 0,8 MEL45 17700 -0,3 MEL12 25000 0,4 MEL50 28957 0,7 MEL15 34000 1,2 MEL62 21000 0,0 MEL21 14400 -0,6 MEL74 17180 -0,4 MEL25 18000 -0,3 MEL75 35764 1,4 MEL30 28084 0,7 MEL90 35000 1,3 MEL34 22000 0,1 MEL95 5128 -1,5 MEL37 9800 -1,0 MEL97 3957 -1,6

1Outlier detection by Grubbs’ test

2Outlier detection by Hampel’s test

*z-score value 2<IzI <3 **z-score value IzI≥3


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Figure 4.Kernel density plot of the quantitative pollen analysis.

Figure 5.Z-score histogram of the quantitative pollen analysis.

0

0,000005

0,00001

0,000015

0,00002

0,000025

0,00003

0,000035

0,00004

-20000 -10000 0 10000 20000 30000 40000 50000 60000

Kernel Density Plot (number of pollengrains per 10g of honey)

-2

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MEL

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Laboratory code


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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 15% as Tilia at the first case; and at the second case,

the percentages were 14% for Brassicaceae and 14% for Tilia. The results of the

botanical origin per participant are presented in Table 4.

Figure 6.Botanical origin of the honey sample according to the melissopalynological

analysis.

23 10

6

multifloral/blossom

Brassicaceae/ Brassica

Tilia


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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, 31analysts 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.

21

4

4

multifloral/blossom

Brassicaceae

Tilia


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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 * botanical origin** physicochemical

sensory geographical origin

MEL02 >500 Brassicaceae Flower honey

(brassicaceae& tilia) x x Roumanie?

MEL03 50-200 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

MEL10 50-200 multifloral N/A

N/A


x East Europe

MEL13 200-500 Brassicaceae Honey Brassicaceae Honey x x Southern Europe


x East Europe

MEL18 <50 heterofloral N/A

France



unifloral honey x x


Europe

MEL23 >500 Polyfloral Polyfloral x x N/A

MEL25 50-200 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

MEL34 50-200 Brassica N/A

N/A

MEL37 >500 Polyflora Polyflora

x Eastern Europe

MEL39 200-500 Tilia sp. Brassica sp. x probably Central

Europe

MEL40 50-200 Tilia &Brassica type (B.napus)&Malus

Pyrus-Type

Tilia (Lime tree honey)

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


Page25/42

Table 4.(continued).

Laboratory Code

number of

samples analysed per year

botanical origin *

botanical origin** physicochemical

sensory geographical origin

MEL41 >500 Blossom Honey Blossom x x South-East-Europe

MEL44 50-200 Tilia Tilia

x Eastern Europe



unifloral honey x x


Europe

MEL50 >500 Tilia Tillia and Frangula

alnus x x Temperate zone

MEL55 50-200 wildflower honey N/A

N/A

MEL59 50-200 Brassica spp - Brassicaceae

Brassica spp - Brassicaceae

x

Southern Europe

MEL62 50-200 multifloral multifloral

x Eastern Europe

MEL71 50-200 multifloral multifloral x x Southeastern Europe,

Italy, Poland

MEL74 50-200 Brassica napus+Tilia mixture Brassica napus and others

x x possible France

MEL75 50-200 Brassicaceae N/A

Europe

MEL78 >500 multifloral multifloral x x

Central Europe, France, also Eastern

Europe cannot be excluded

MEL81 <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

MEL89 50-200 floral floral x x N/A

MEL90 50-200 tilia tilia

x Europe (middle

western)

MEL93 >500 Brassica Polyfloral

x Eastern Europe

MEL95 200-500 Brassica napus N/A

Southern Europe

MEL97 200-500 Multifloral honey

from Brassicaceae, Prunus …

multifloral

x France

MEL98 >500 Summer flowers Summer flowers

x Mediterranean


Page26/42

References

Eurachem -2011, Selection, Use and Interpretation of Proficiency Testing Schemes by Laboratories.

ISO 13528:2005, Statistical methods for the use of proficiency testing by inter-laboratory comparisons.

ISO 15725:1994, Accuracy of Measurement Methods and Results.

ISO17043:2010, 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. 1): 18-25.


Page27/42

ANNEX I

Participant Laboratories


Page28/42

Country Institution

AUSTRIA Austrian Agency for Health and Food Safety (AGES), Institute for Seed and Propagation Material, Phytosanitary Service and Apiculture, Department for Apiculture and Bee Protection

BELGIUM CARI asbl

BRAZIL Instituto Oswaldo Cruz Fiocruz

CROATIA Faculty of Agriculture, J.J. Strossmayer University in Osijek

CROATIA University of Zagreb Faculty of Agriculture Department of Fisheries, Beekeeping Game management and Special Zoology

FRANCE Famille Michaud Apiculteurs

FRANCE Naturalim France Miel

GERMANY Breitsamer + Ulrich GmbH & Co KG

GERMANY Eurofins Analytik GmbH

GERMANY Intertek Food Services GmbH

GERMANY Länderinstitut für Bienenkunde Hohen Neuendorf e.V.

GERMANY LAVES Institut für Bienenkunde Celle

GERMANY Quality Services International GmbH

GREECE ATTIKI Bee Culturing Co. Alexandros Pittas S.A

GREECE Laboratory of Apiculture and Sericulture,Faculty of Agriculture, Aristotle University of Thessaloniki

GREECE Laboratory of Residue Analysis in Food Of Animal Origin

GREECE Mediterranean Agronomic Institute of Chania

GREECE Region of South Aegean, General Directorate of Rural Economy & Veterinary, Directorate of Rural Economy

ITALY A.S.S.A.M. Centro Agrochimico Regionale

ITALY CRA-API Honey bee and Silkworm Research Unit of the Italian Agricultural research Council

ITALY Dipartimento di Scienze Agrarie, Forestali e Alimentari - Università di orino

ITALY Ispettorato Centrale della tutela della qualità e repressione frodi dei prodotti agroalimentari Laboratorio di Perugia

ITALY Laboratorio biologico, Agenzia provinciale per l'ambiente di Bolzano

ITALY Piana Ricerca e Consulenza srl

ITALY Unità Operativa di Apicoltura - Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana


Page29/42

Country Institution

NETHERLANDS Jacob Kerkvliet

POLAND Agricultural and Food Quality Inspection Main Inspectorate Warsaw

POLAND Bee Product Quality Testing Laboratory, Research Institute of Horticulture, Apiculture Division

POLAND Mazurskie Miody

POLAND Office of Competition and Consumer Protection, Laboratory in Olsztyn

SERBIA Faculty of Agriculture, University of Belgrade, Center for Food Analysis

SLOVENIA Slovenian Beekeepers Association

SPAIN Casa de la Miel. Cabildo de Tenerife

SWITZERLAND Biologisches Institut für Pollenanalyse K. Bieri GmbH

TURKEY Altıparmak Gıda Sanayi ve ic. A.Ş.


Page30/42

ANNEX II

Cover Letter and Result Form


Page31/42



RING TEST FOR HONEY QUALITATIVE AND QUANTITATIVE

MELISSOPALYNOLOGICALANALYSIS

Date 24/07/2013

Dear participant,

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 1-15) and fill in the

attached Reporting Form.

The Reporting Form must be sent back until 30 September 2013 to this e-mail

address: [email protected].

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. 1): 18-25.http://dx.doi.org/10.1051/apido:2004050

Persano Oddo L., Piro R. (2004) Main European unifloral honeys: descriptive sheets, Apidologie 35

(Suppl. 1):38-81.http://dx.doi.org/10.1051/apido:2004049

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, 45:1, 71-72. http://dx.doi.org/10.1080/00173130500520479

mailto:[email protected]

http://dx.doi.org/10.1051/apido:2004050

http://dx.doi.org/10.1051/apido:2004049


Page32/42

1 4

LABORATORY CODE NUMBER ACCREDITATION2

DATE OF RECEIPT NUMBER OF SAMPLES ANALYSED PER YEAR

3 <50 50-200 200-500 >500 5

STATE OF SAMPLE

SAMPLE CODE

METHODOLOGY 6

1 QUALITATIVE POLLEN ANALYSIS Reference: Harmonized methods of melissopalynology(Apidologie, 2004)

Other (please specify)…………………………………………………………………

all pollen

types

nectarless

exluded

Value(%) Value(%)

1 Brassicaceae Brassicaceae

2 Tiliaceae Tilia

3 Rosaceae Rosaceae

4

0 0

METHODOLOGY 6

2 QUANTITATIVE POLLEN ANALYSIS Reference: Harmonized methods of melissopalynology(Apidologie, 2004)

9 Other (please specify)…………………………………………………………………

Number of pollen grains/10g of honey: RCF10

minutes11

6

3 BOTANICAL ORIGIN (only pollen) Reference: European unifloral honeys (Apidologie, 2004)

12 Other (please specify)…………………………………………………………………

Honey from:

4 BOTANICAL ORIGIN (combination sensory, pollen, physicochemical) 6

9, 13 Reference: European unifloral honeys (Apidologie, 2004)

Honey from: Other (please specify)………………………………………………………

14

physicochemical

15

sensory

6

5 GEOGRAPHICAL ORIGIN Reference: (please specify)…………………………………………………………………

9

Honey from:

6 COMMENTS

Other


Pollen FormFamily

IHC 01-13


INTER-LABORATORY RING TEST FOR HONEY QUALITATIVE AND QUANTITATIVE

MELISSOPALYNOLOGICAL ANALYSIS

……………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………………………….....................................................................

Sum

7A 7B

8


Page33/42

1

2

3

4

5

6

7A

7B

8

9

10

11

12

13

14

15 Please mark if you performed physicochemical analysis e.g. electrical conductivity, sugars e.t.c.

Please write the date when you received the honey sample

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

Please mark if your laboratory is accredited for honey pollen analysis

Please mark the approximate number of honey samples that you perform pollen analysis per year

Please mark or write the method you use for pollen anaysis / botanical / geographical determination

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.

Please mark if you performed sensory analysis

Please fill in the botanical origin of the honey considering ONLY the results from pollen analysis

Please fill in the botanical origin of the honey considering also sensory and/or physicochemical analysis

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

no decimals.

This field is optional

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)

This field is automatically filled to sum 100%

Please write the code number of your laboratory


Page34/42

ANNEX III

Methods and References


Page35/42

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 Number of participants

Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004) Harmonized methods of melissopalynology, Apidologie 35

24

DIN Deutsches Institut für Normung 2002 Untersuchung von Honig – Bestimmung der relative Pollenhäufigkeit, DIN 10760: 2002-05, Berlin ASU § 64 LFGB L40.00-11)

8

Louveaux J., Maurizio A., Vorwohl G. (1978) Methods of Melissopalynology, Bee World 59

5

UNI-11299/2008 OR internal method based on UNI-11299/2008 (Miele - Analisi microscopica o melissopalinologica) (2008)

4

Erdtman (1969) Handbook of Palynology, Munksgaard, Copenhagen

1

Croatian Regulation of the Minister of Agriculture and Forestry NN20/2000

1

Polska Norma PN-88/A-77626 – Miód pszczeli 998 1

Rozp. MRiRW z dnia 4 stycznia 2009r. Zał. do rozp. pkt. VI (Dz.U. z 2009r. Nr 17; poz.94

1

Other Internal method 1

Quantitative Melissopalynological Analysis

The methodology applied by the participants for the quantitative pollen analysis of

the honey sample.

Methods Number of participants


Page36/42

Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004) Harmonized methods of melissopalynology, Apidologie 35

6

Internal method based on Lycopodium spores tablets (Stockmarr J.(1971) Tablets with Spores used in Absolute Pollen Analysis, Pollen et Spores 13)

4

DIN Deutsches Institut für Normung 2002 Untersuchung von Honig – Bestimmung der relative Pollenhäufigkeit, DIN 10760: 2002-05, Berlin ASU § 64 LFGB L40.00-11)

3

Louveaux J., Maurizio A., Vorwohl G. (1978) Methods of Melissopalynology, Bee World 59

3

UNI-11299/2008 OR internal method based on UNI-11299/2008 (Miele - Analisi microscopica o melissopalinologica) (2008)

3

Erdtman (1969) Handbook of Palynology. Munksgaard, Copenhagen

1

Other Internal method 1

Botanical Origin

The references used by the by the participants for the determination of the botanical

origin of the honey sample.

References Number of participants


25

Beckh G., Camps G.(2009) Neue Spezifikationen fur Trachthonige, Deutsche Lebensmittel-Rundschau 105

5

Polish Standard „Honey” Polska Norma PN-88/A-77626 – Miód pszczeli (1998)

3

Polish standard - Regulation of the Minister of Agriculture and Rural Development of 03.10.2003r (Journal of Laws 03.181.1773) Dz.U. nr 8 , poz. 773 z późn. zm.

2

DIN Deutsches Institut für Normung 2002 Untersuchung von Honig – Bestimmung der relative Pollenhäufigkeit, DIN 10760: 2002-05, Berlin

2

Ricciardelli del Arbore (1998) Mediterranean melissopalynology, Perugia

2

Croatian Regulation of the Minister of Agriculture and Forestry NN20/2000

1

Maurizio A., Louveaux J. 965. Pollens de plantes mellifères d’Europe. Union des Groupements apicoles français, Paris 1


Page37/42

Persano Oddo L., Piazza M.G., Sabatini A.G., Accorti M. (1995) Characterization of unifloral honeys, Apidologie 26, 453-465

1

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

1

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: 438-452

1

Ricciardelli del Arbore, v.d.Ohe,Bucher (2000)Il Meli regionali Italiani

1

Ricciardelli del Arbore (1997) Textbook of melissopalynology, Apimondia

1

Internal method based on ISO 6658/2005 and 8586/2012 1

Geographical Origin

The references used by the participants for the determination of the geographical

origin of the honey sample.

References Number of participants

Ricciardelli d’Albore G. 997) Textbook of melissopalynology, Apimondia

3

Ricciardelli del Arbore (1998) Mediterranean melissopalynology, Perugia

2

Battaglini M., Ricciardelli D'albore G. (1972) 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 1972. Apimondia, Bucarest, Romania: 96-111

1

Beckh G., Camps G.(2009) Neue Spezifikationen fur Trachthonige, Deutsche Lebensmittel-Rundschau 105

1

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

1


1

Ricciardelli del Arbore, v.d.Ohe,Bucher (2000) Il Meli regionali Italiani

1

http://www.researchgate.net/researcher/34595901_Kaspar_Ruoff/

http://www.researchgate.net/researcher/11930012_Werner_Luginbuehl/

http://www.researchgate.net/researcher/78316842_Verena_Kilchenmann/

http://www.researchgate.net/researcher/2030306653_Jacques_Olivier_Bosset/

http://www.researchgate.net/researcher/12941441_Katharina_von_der_Ohe/

http://www.researchgate.net/researcher/12941441_Katharina_von_der_Ohe/

http://www.researchgate.net/researcher/2006053778_Werner_von_der_Ohe/

http://www.researchgate.net/researcher/9502950_Renato_Amado/


Page38/42

Polish standard - Regulation of the Minister of Agriculture and Rural Development of 03.10.2003r (Dz.U. nr 181, poz. 1773 z późn. zm.

1

Sawyer R. (1988) - Honey identyfication. Cardiff Academic Press 1


Page39/42

ANNEX IV

Comments


Page40/42

Further information about the melissopalynological analysis stated by the

participants at the6th paragraph of the Report Formis summarized in random order

bellow:

1. 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, Vicia-t/ Pinaceae: Pinus/ Rhamnaceae/ Rosaceae: Pyrus-Prunus-t/ Salicaceae:Salix/ Simoaroubaceae: Ailanthus/ Umbelliferae-Apiaceae: Umbelliferae-Apiaceae, Coriandrum, Foeniculum

10. Other pollen forms: Asteraceae form H, Trifolium pratense gr., Apiaceae, Vicia, Robinia, Asteraceae form S, Cornus sanguinea, Rhamnaceae, Campanulaceae. Pollen not nectariferous: Cupressaceae, Chenopodiaceae

11. Other (Rhamnaceae, Apiaceae, Salix, Trifolium, Helianthus) 12. 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

13. nectraless: Oleaceae,Graminae, Cyperaceae, Chenopodiaceae andhoneydew elements

14. 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

15. 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,


Page41/42

Betulaceae, Filipendula ulmeria, Oleaceae 16. The geographical origin of honey was determined based on presence of

Castanea sativa and Cistus pollen 17. Considering the organoleptical characteristics (taste, colour, consistency)

there are no monofloral characteristics. So the part of rape is overrepresented 18. 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)

19. 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.

21. 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


Page42/42

(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 (1989) Nomenclatura Melissopalinologica, Apicoltura, 5: 63-72)

24. Other pollens: Amorpha, Papaver, Helianthus, Trifolium alexandrinum, Papaver, Vicia, Cynoglossum, Lotus, Robinia,Vitis, Rumex, Plantago, Sambucus, Gramineae

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