CEN/TS 16817-2:2015

Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen monitoring - Part 2: Biological pollen sampling using bee colonies

This Technical Specification describes a procedure through which pollen – in particular pollen of genetically modified organisms (GMO) – can be sampled by means of bee colonies.
Bee colonies, especially the foraging bees, actively roam an area and are therefore area related samplers. Pollen sampling depends on the collection activity of the bees and the availability of pollen sources within the spatial zone according to the bees' preferences (supply of melliferous plants). A colony of bees normally forages over an area of up to 5 km radius (median 1,6 km, mean 2,2 km), in rare cases some bees may also forage in greater distances up to 10 km and more [26].
Foragers fix the gathered pollen on the outside of their hind legs (pollen loads, also known as pollen pellets). Inside the hive they place these pollen loads into comb cells close to the brood nest (bee bread). Furthermore, foragers gather nectar and honeydew. Nectar contains pollen which fell from the anthers of the blossom into the nectar drop, or pollen which was dispersed by the wind and sticks in the nectar of other blossoms or adheres to the sticky honeydew of plants. Nectar and honeydew are converted to honey and stored by the bees in the beehive.
Honey, pollen load and bee-bread may be used as sample matrices for the subsequent analysis of pollen as it is possible to concentrate sufficient amounts of pollen for microscopic and molecular biological diagnostics.
Microscopic analysis is used to identify the various pollen types and to quantify the exposure to the target pollen types in question. GMO exposure is analyzed by molecular-biological methods: For analysis of pollen DNA quantitative PCR methods are used and described here in this Technical Specification. The analysis of GMO specific proteins and toxins in pollen is possible, too, using ELISA, but to this date the method has not been evaluated enough in pollen matrices for standardization in this Technical Specification.

Außenluft - Monitoring der Wirkungen von gentechnisch veränderten Organismen (GVO) - Pollenmonitoring - Teil 2: Biologische Pollensammlung mit Bienenvölkern

Diese Technische Spezifikation beschreibt ein Verfahren, mit dem Pollen — insbesondere Pollen von gentechnisch veränderten Organismen (GVO) — mittels Bienenvölkern gesammelt werden können.
Bienenvölker, insbesondere die sammelnden Flugbienen, durchstreifen aktiv ein Gebiet und sind daher flächenbezogene Sammler. Die Pollensammlung steht in Abhängigkeit von der Sammelaktivität der Bienen sowie der Verfügbarkeit von Pollenquellen nach den Vorlieben der Bienen innerhalb des räumlichen Gebiets (Trachtangebot). Ein Bienenvolk befliegt in der Regel ein Gebiet mit einem Radius von bis zu 5 km (Medianwert 1,6 km, Mittelwert 2,2 km), in seltenen Fällen können einige Bienen auch Gebiete in größeren Entfernungen von bis zu 10 km und mehr anfliegen [26].
Sammelbienen befestigen die gesammelten Pollen auf der Außenseite ihrer Hinterbeine (Pollenladung, auch als Pollenhöschen bezeichnet). Im Inneren des Bienenstocks werden diese Pollenladungen in Wabenzellen in der Nähe des Brutnestes verbracht (Bienenbrot). Außerdem tragen Sammelbienen Nektar und Honigtau ein. Der Nektar enthält Pollen, der aus den Antheren der Blüte in den Nektartropfen gefallen ist, oder Pollen, der durch den Wind verbreitet im Nektar anderer Blüten bzw. auf klebrigem Honigtau an Pflanzen haften bleibt. Im Bienenstock werden Nektar und Honigtau zu Honig konvertiert und gelagert.
Honig, Pollenladung und Bienenbrot können als Probenmatrices für die anschließende Pollenanalyse genutzt werden, da es möglich ist, ausreichende Pollenmengen für eine molekularbiologische Diagnostik zu konzentrieren.
Die mikroskopische Analyse dient der Identifizierung der verschiedenen Pollenarten sowie zur quantitativen Bestimmung der Exposition gegenüber den betrachteten Zielpollenarten. Die GVO-Exposition wird durch molekularbiologische Verfahren ermittelt: Zur Analyse der Pollen-DNA werden quantitative PCR-Verfahren eingesetzt, die in der vorliegenden Technischen Spezifikation beschrieben werden. Durch Verwendung von ELISA können auch in Pollen befindliche GVO-spezifische Proteine und Toxine analysiert werden, jedoch wurde dieses Verfahren bis zum heutigen Datum noch nicht ausreichend für den Einsatz in Pollenmatrices und somit für eine Standardisierbarkeit im Rahmen dieser Technischen Spezifikation validiert.

Air ambiant - Surveillance des effets d'organismes génétiquement modifiés (OGM) - Surveillance du pollen - Partie 2 : Échantillonnage biologique du pollen à l'aide de colonies d'abeilles

La présente Spécification technique décrit un mode opératoire avec lequel le pollen, en particulier le pollen d’organismes génétiquement modifiés (OGM), peut être échantillonné à l’aide de colonies d’abeilles.
Les colonies d’abeilles, notamment les abeilles butineuses, se déplacent activement sur une zone et sont donc des échantillonneurs de zone. L’échantillonnage du pollen dépend de l’activité de récolte des abeilles et de la disponibilité des sources de pollen dans la zone spatiale selon les préférences des abeilles (abondance des plantes mellifères). Une colonie d’abeilles butine normalement sur une zone de 5 km de rayon (médiane de 1,6 km, moyenne de 2,2 km), dans de rares cas certaines abeilles peuvent également butiner à des distances supérieures à 10 km et plus [26].
Les butineuses fixent le pollen récolté sur la partie extérieure de leurs pattes arrières (charges de pollen, également appelées pelotes de pollen). Une fois dans la ruche, elles placent ces charges de pollen dans les alvéoles qui entourent le nid à couvain (pain d’abeille). Par ailleurs, les butineuses récoltent le nectar et le miellat. Le nectar contient du pollen tombé des anthères de la fleur dans la goutte de nectar, ou du pollen qui a été dispersé par le vent et se colle au nectar d’autres fleurs ou adhère au miellat collant de plantes. Le nectar et le miellat sont transformés en miel et stockés par les abeilles dans la ruche.
Le miel, la charge de pollen et le pain d’abeille peuvent être utilisés comme matrices d’échantillons pour l’analyse ultérieure du pollen car il est possible de concentrer des quantités suffisantes de pollen pour les diagnostics microscopiques et par biologie moléculaire.
L’analyse microscopique est utilisée pour identifier les différents types de pollen et pour quantifier l’exposition aux types de pollen cibles étudiés. L’analyse de l’exposition aux OGM utilise des méthodes par biologie moléculaire. Pour l’analyse de l’ADN du pollen, des méthodes de PCR quantitative sont utilisées et décrites dans la présente Spécification technique. Il est également possible d’analyser les protéines et les toxines spécifiques des OGM dans le pollen à l’aide de la méthode ELISA. Toutefois, la méthode n’a pas encore été suffisamment évaluée dans les matrices de pollen pour être normalisée dans la présente Spécification technique.

Zunanji zrak - Monitoring učinkov gensko spremenjenih organizmov (GSO) - Monitoring peloda - 2. del: Biološko vzorčenje peloda z uporabo čebelje družine

Načrtovani standard opisuje postopek, v katerem je mogoče pelod – zlasti pelod gensko spremenjenih organizmov – vzorčiti s pomočjo kolonij čebel. Aktivne pašne čebele nabirajo pelod in nektar. Med in cvetni prah se lahko uporabita kot vzorca v naknadnih analizah peloda, saj je mogoče zbrati zadostne količine peloda za molekularno-biološko diagnostiko. Vzorčenje peloda v medu in cvetnem prahu je treba obravnavati skupaj s tehničnim vzorčenjem monitoringa gensko spremenjenih organizmov (NWI št. 264088).

General Information

• Status: Published
• Publication Date: 20-Oct-2015
• ICS: 07.080 - Biology. Botany. Zoology
• ICS: 13.020.99 - Other standards related to environmental protection
• Technical Committee: CEN/TC 264 - Air quality
• Drafting Committee: CEN/TC 264/WG 29 - Ambient air - Monitoring of genetically modified organisms (GMO)
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 08-Feb-2018
• Completion Date: 14-Apr-2025
• Ref Project: SIST-TS CEN/TS 16817-2:2016 - Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen monitoring - Part 2: Biological pollen sampling using bee colonies

Overview

CEN/TS 16817-2:2015 - Ambient air: Monitoring the effects of genetically modified organisms (GMO) - Pollen monitoring, Part 2: Biological pollen sampling using bee colonies describes a standardized procedure for sampling pollen (including pollen from GMOs) using honey bee colonies as biological samplers. The Technical Specification explains how foraging bees collect and concentrate pollen in hive matrices - honey, pollen loads, and bee‑bread - and how these matrices can be used for palynological (microscopic) and molecular‑biological (DNA/qPCR) analyses to detect and quantify GMO pollen exposure across a landscape.

Key topics and requirements

• Biological sampling principle: Honey bees act as area‑related samplers; typical foraging range is up to 5 km radius (median ~1.6 km, mean ~2.2 km), with occasional foraging at greater distances.
• Sample matrices: Guidance on collecting and processing honey, pollen loads (pollen pellets on hind legs), and bee‑bread for downstream analysis.
• Sampling procedure elements:
  • Selection and management of bee colonies and hives
  • Site placement and documentation
  • Preparation, exposure time, sampling dates
  • Extraction, transport and storage of samples
• Analytical methods:
  • Palynology (microscopic identification and quantification of pollen types)
  • Molecular‑biological analysis, primarily quantitative PCR (qPCR) for detection of GMO‑specific DNA
  • Note: ELISA for proteins/toxins is mentioned but not standardized for pollen in this document
• Performance and QA: Sections on method performance characteristics, foraging variability, quality assurance, sampling protocols, and required accompanying documentation.
• Crop‑specific annexes: Normative annexes provide maize‑ and rapeseed‑specific sampling and PCR guidance; an informative annex covers good beekeeping practice.

Applications and users

This Technical Specification is intended for:

• Environmental and regulatory authorities implementing GMO post‑market environmental monitoring
• Air quality and biosurveillance programs monitoring pollen dispersal and GMO exposure
• Laboratories performing palynology and qPCR analyses for GMO detection
• Researchers studying pollen-mediated gene flow or ecological impacts of GMOs
• Beekeepers and citizen science networks participating in biological sampling networks

Practical applications include landscape‑scale monitoring of GMO pollen, complementing stationary technical samplers, assessing exposure risks to surrounding ecosystems, and supporting compliance with regulatory post‑marketing monitoring.

Related standards

• CEN/TS 16817‑1 - Technical pollen sampling using pollen mass filter (PMF) and Sigma‑2 sampler (complements the biological approach in Part 2).

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2016
=XQDQML]UDN0RQLWRULQJXþLQNRYJHQVNRVSUHPHQMHQLKRUJDQL]PRY*62
0RQLWRULQJSHORGDGHO%LRORãNRY]RUþHQMHSHORGD]XSRUDERþHEHOMHGUXåLQH
Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen
monitoring - Part 2: Biological pollen sampling using bee colonies
Außenluft - Monitoring der Wirkungen gentechnisch veränderter Organismen (GVO) -
Pollenmonitoring - Teil 2: Biologische Pollensammlung mit Bienenvölkern
Air ambiant - Surveillance des effets d'organismes génétiquement modifiés (OGM) -
Surveillance du pollen - Partie 2 : Échantillonnage biologique du pollen à l'aide de
colonies d'abeilles
Ta slovenski standard je istoveten z: CEN/TS 16817-2:2015
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 16817-2
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
October 2015
TECHNISCHE SPEZIFIKATION
ICS 07.080; 13.020.99
English Version
Ambient air - Monitoring the effects of genetically
modified organisms (GMO) - Pollen monitoring - Part 2:
Biological pollen sampling using bee colonies
Air ambiant - Surveillance des effets d'organismes Außenluft - Monitoring der Wirkungen von
génétiquement modifiés (OGM) - Surveillance du gentechnisch veränderten Organismen (GVO) -
pollen - Partie 2 : Échantillonnage biologique du pollen Pollenmonitoring - Teil 2: Biologische Pollensammlung
à l'aide de colonies d'abeilles mit Bienenvölkern
This Technical Specification (CEN/TS) was approved by CEN on 16 May 2015 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16817-2:2015 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Basic principle of the procedure . 8
5 Sample matrices . 8
5.1 Honey . 8
5.2 Pollen load . 9
5.3 Bee-bread . 9
6 Sampling procedure . 9
6.1 General . 9
6.2 Bee colony and hive . 9
6.3 Sample site . 9
6.4 Preparation and assembly . 10
6.5 Exposure time . 10
6.6 Sampling dates . 10
6.7 Extraction, transport and storage . 10
7 Palynology . 11
7.1 General . 11
7.2 From sample preparation to embedded slide preparation . 11
7.2.1 General . 11
7.2.2 Honey . 11
7.2.3 Pollen load . 11
7.2.4 Bee bread . 11
7.3 Microscopic analysis . 12
7.4 Pollen diversity . 12
8 Molecular-biological analysis . 13
8.1 General . 13
8.2 Sample preparation . 13
8.2.1 Honey . 13
8.2.2 Pollen loads . 13
8.2.3 Bee bread . 14
9 Determination of the target parameters for GMO monitoring and representation of
the results . 14
9.1 Microscopic pollen analysis . 14
9.1.1 General . 14
9.1.2 Concentration in counts per gram of sample mass . 14
9.1.3 Total number of pollen collected per exposure time and season. 15
9.1.4 Relative frequency . 15
9.2 Molecular-biological analysis . 16
10 Performance characteristics of the methods . 16
10.1 General . 16
10.2 Bee colony . 16
10.3 Foraging distance . 17
10.4 Honey samples, bee-bread samples and pollen load samples . 17
10.5 Microscopic pollen analysis . 17
10.6 Molecular-biological analysis. 17
11 Quality assurance and quality control . 18
11.1 General measurement strategy and task of pollen monitoring with biological
samplers . 18
11.2 Site protocol . 18
11.3 Accompanying documentation for samples . 19
11.4 Parallel measurements. 19
11.5 Quality assurance and reference materials . 19
Annex A (normative) Maize-specific requirements . 20
A.1 Scope . 20
A.2 Basic principles . 20
A.3 Sampling . 21
A.4 Sample preparation . 22
A.5 Molecular-biological analysis of maize DNA using PCR . 23
A.5.1 General . 23
A.5.2 DNA extraction . 23
A.5.3 Real-time PCR analysis . 24
A.6 Determination of the target parameters for GMO monitoring and assessment of the
results . 24
Annex B (normative) Rapeseed specific requirements . 25
B.1 Scope . 25
B.2 Basic principles . 25

B.3 Sampling . 26
B.4 Sample preparation . 27
B.5 Molecular-biological analysis of rapeseed DNA using PCR for GMO detection . 28
B.5.1 General . 28
B.5.2 DNA extraction . 28
B.5.3 Real-time PCR analysis . 29
B.6 Determination of the target parameters for GMO monitoring and assessment of the
results . 29
Annex C (informative) Good beekeeping practice . 30
Bibliography . 31

European foreword
This document (CEN/TS 16817-2:2015) has been prepared by Technical Committee CEN/TC 264 “Air
quality”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
CEN/TS 16817, Ambient air — Monitoring the effects of genetically modified organisms (GMO) — Pollen
monitoring, is composed of the following parts:
— Part 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-2-sampler;
— Part 2: Biological pollen sampling using bee colonies [the present document].
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
The European Parliament and the European Council require an environmental risk assessment and a
post-marketing monitoring for any GMO released to the environment [6; 7]. This had to be implied in
national law in any member state of the EC by date.
Pollen dispersal plays a significant role in the dissemination of genetically modified organisms (GMO).
Hence, a monitoring procedure that involves recording and documentation of input and distribution of
GMO via pollen in a monitoring network mirroring the natural environment is required. For this,
technical (CEN/TS 16817-1) and biological sampling of pollen as well as PCR-screening (polymerase
chain reaction) procedures are employed to provide evidence of GMO-exposure. The biological
sampling system using honey bee colonies is described in the present Technical Specification.
VDI/Guideline 4330 Part 1 [3] presents the necessary fundamentals for the understanding of this
Technical Specification. The sampling of pollen in the sample matrices honey, pollen load and bee-bread
[5] needs to be viewed in conjunction with the technical sampling for the GMO-monitoring [4].
The use of the biological, actively foraging honeybee and the technical passive samplers complement
each other in a manifold and positive way for pollen monitoring of GMO. Therefore it is reasonable to
use both. The technical sampling (CEN/TS 16817-1) is based on stationary point-samplers [1]. They
give a record of pollen exposure in the air at the sample site that correlates with the prevailing wind
direction and relative position to the surrounding pollen sources. The biological sampling using honey
bee colonies serves as indicator for GMO exposure in an area and for exposure to roaming insects. Bees
display a spatially averaging sampling activity, which represents a cross section of the established,
blossoming plants in the area according to the bees collection activities. A wide spectrum of pollen
species is recorded using both sampling methods with the procedures complementing each other
across the vegetation period [21].
1 Scope
This Technical Specification describes a procedure through which pollen – in particular pollen of
genetically modified organisms (GMO) – can be sampled by means of bee colonies.
Bee colonies, especially the foraging bees, actively roam an area and are therefore area related
samplers. Pollen sampling depends on the collection activity of the bees and the availability of pollen
sources within the spatial zone according to the bees' preferences (supply of melliferous plants). A
colony of bees normally forages over an area of up to 5 km radius (median 1,6 km, mean 2,2 km), in rare
cases some bees may also forage in greater distances up to 10 km and more [26].
Foragers fix the gathered pollen on the outside of their hind legs (pollen loads, also known as pollen
pellets). Inside the hive they place these pollen loads into comb cells close to the brood nest (bee
bread). Furthermore, foragers gather nectar and honeydew. Nectar contains pollen which fell from the
anthers of the blossom into the nectar drop, or pollen which was dispersed by the wind and sticks in the
nectar of other blossoms or adheres to the sticky honeydew of plants. Nectar and honeydew are
converted to honey and stored by the bees in the beehive.
Honey, pollen load and bee-bread may be used as sample matrices for the subsequent analysis of pollen
as it is possible to concentrate sufficient amounts of pollen for microscopic and molecular biological
diagnostics.
Microscopic analysis is used to identify the various pollen types and to quantify the exposure to the
target pollen types in question. GMO exposure is analysed by molecular-biological methods: For
analysis of pollen DNA quantitative PCR methods are used and described here in this Technical
Specification. The analysis of GMO specific proteins and toxins in pollen is possible, too, using ELISA, but
to this date the method has not been evaluated enough in pollen matrices for standardization in this
Technical Specification.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
DIN 10760, Analysis of honey — Determination of the relative frequency of pollen
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
bee bread
pollen load stored in comb cells close to the brood nest
3.2
bee colony
colony of the honeybee species Apis mellifera
3.3
beehive
hive
container in which honeybees are kept by beekeepers
3.4
event
unique DNA recombination event that took place in one plant cell, which was then used to
generate entire transgenic plants
3.5
flying bee
foraging bee
forager
worker bee of a colony which is active outside the hive
3.6
genetically modified organism
GMO
organism in which the genetic material has been altered in a way that does not occur naturally by
mating and/or natural recombination
[SOURCE: Directive 2001/18/EC [6], modified — The content of the definition was changed.]
3.7
honey
product generated by bees from the raw materials nectar and honey dew
3.8
honeydew
sugar containing secretion of aphids and cicadas sucking on plants
3.9
melliferous plant
plant from which nectar, honey dew and/or pollen is offered as sources of food for bees
3.10
monitoring
environmental monitoring
characterizing the state and quality of the environment and its changes by measurements/observations
in regard to defined objectives
3.11
nectar
sugar containing secretion of the nectar glands in or from blossoms
3.12
pollen
male gametophyte of the flowering plant
3.13
pollen and honey flow
food supply within the environment (foraging area) of a bee colony
3.14
pollen load
pollen pellets
pollen brought into the bee colony by the pollen foraging bees at their hind legs
3.15
pollen type
pollen species
class of pollen being distinguished by microscopic means on species, family or other order level
3.16
sampling
pollen sampling
collection of particles, here pollen by technical or biological means
4 Basic principle of the procedure
The bee colony serves as biological active sampler of pollen. The bee colonies are positioned within the
area under investigation i.e. relocated bee colonies are used or bee colonies which are already present,
i.e. permanent apiaries by local beekeepers.
Flying bees forage for food sources (supply of melliferous plants) and if successful, bring in the raw
materials nectar, honey dew and pollen. By gathering nectar, honeydew and pollen, bees collect a
fraction of the pollen present at the time in the area. These pollen are stored in wax combs as honey and
bee-bread and are available for future analyses. Further on, the collected pollen load of the bees may be
used directly as sample matrix gained by pollen traps at the hive entrance. Advantages and
disadvantages of the different matrices are given in Clause 5.
Depending on the supply from melliferous plants, if there is a shortage bees also gather pollen from
anemophilous plants. Bees also require water which they collect from numerous sources (dew, open
bodies of water, etc.). Pollen is produced in the anthers of the flowers. Anthers burst apart after
reaching maturity, making pollen available. Pollen, released from the anthers of the same flower, also
stick to the nectar of this flower. Anemophilous pollen is distributed by wind and can stick to honeydew
or nectar. So anemophilous pollen can be collected indirectly by the bees as well as by flying through
the air.
The area used by the bees depends on various factors (weather, availability of melliferous plants,
utilization of landscape and landscape structure, etc.). The main foraging distances are [26]: modal
distance from hive to forage site 0-0,7 km, median distance 1,6 km, mean distance 2,2 km, maximum
10 km.
Exposure time may be flexibly specified from a minimum of five days up to several weeks. For exposure
times of more than a week, sampling in intervals is also possible.
The pollen samples are analysed using light microscopy (palynology) and by molecular biological
analysis (e.g. PCR).
5 Sample matrices
5.1 Honey
Honey is produced by bees from the gathered raw materials nectar or honeydew and is stored in special
combs (honeycombs). Both raw materials contain pollen among other things. Centrifuges extract honey
from the honeycombs.
Honey yield is more reliable than bee-bread or pollen load and is thus clearly preferable. The available
amount of pollen load and beebread depends a lot more on the supply of plants and the consumption by
the bees. The matrix honey is significantly better suited for light microscopic and molecular biological
analyses [10; 25; 32]. But the amount of anemophilous pollen grains in honey is small.
For comparative studies, extracted honeys are preferable to the other matrices (pollen load and bee
bread) as a strong homogenization occurs from the type of extraction. Extracted honey possesses a
better spatial and time representation. Basically, two sampling dates per bee site may be assumed for
each region (spring and summer honey). Summer honey is not collected by all bee-keepers regularly.
5.2 Pollen load
Pollen load is the pollen brought in separately by the foraging bees. Pollen load can be taken off the hind
legs of homecoming bees using special pollen traps. Pollen traps are devices which can be attached to
the front of the hive (front pollen trap) or inside between bottom and first hive body (inside pollen
trap). These traps have a hole pattern. Passing through these holes incoming pollen foragers will lose
their pollen load. The removed pollen loads will drop in a collection vessel. Using pollen traps negative
effects on foraging behaviour can occur. These effects are less when using inside pollen traps than front
pollen traps.
Pollen load has advantages over honey under specific circumstances: differentiations of flowering and
foraging time, foraging of nectarless melliferous and anemophilous plants. However, to implement
these advantages, many more sampling dates and efforts are necessary. Molecular-biological analysis of
the matrix pollen load needs more preparatory steps than the matrix honey. Advantage: greater
amounts of pollen.
5.3 Bee-bread
Bee-bread is the pollen brought in separately by the bees which is stored in special areas of the combs.
Bee-bread may be extracted by cutting out corresponding areas of the combs.
Bee bread has advantages over honey under specific circumstances: differentiations of flowering and
foraging time, foraging of nectarless melliferous and anemophilous plants. To implement these
advantages, many more sampling dates and efforts are necessary though. PCR analysis of the matrix bee
bread is much more complicated than of the matrix honey. In addition, bee bread is sometimes not
available due to consumption by nurse bees.
6 Sampling procedure
6.1 General
For the sampling procedure including site conditions, placing the colonies and sampling the pollen
matrices the “Good Beekeeping Practice” shall be regarded (see Annex C). Some general aspects and
specific requirements in the scope of this TS for GMO-monitoring are stated here.
6.2 Bee colony and hive
The bee colony includes the hive (box in the broader sense as housing for the bees), frames with wax
combs, a queen, 10 000 to 40 000 worker bees as well as several hundreds of drones in certain months.
It is managed by the beekeeper according to good beekeeping practice (see Annex C).
Modern hives are multiple-storey hives (one or up to five storeys or bodies) made of wood or
Polystyrene (PS) foam with wooden frames and mostly wax foundation. These types of hives are
predominant. Within the frames honeybees build their combs. Due to the type of hive number and size
of the frames are different. According to good beekeeping practice size of multiple-storey hives can be
adapted to the size of the colony or the space needed by the colony.
6.3 Sample site
At least one bee colony is positioned at a fixed site. Exact positioning takes place according to good
beekeeping practice (protection against flooding, storm, branch lashing, etc.) [22; 33]. Regular
attendance to the colony of bees needs to be guaranteed (approximately every 9 d to 14 d).
6.4 Preparation and assembly
No further activity is required regarding the assembly of previously installed colonies of bees
(permanent sites).
Newly migrated bee colonies shall be placed at the specified site. Migration shall take place outside the
flying times of the bees from late in the evening till early in the morning. The previous site shall be at
least five kilometres away from the new location in order to exclude a return flight of the bees back to
the old location.
Stationary as well as migrating bee colonies should be harvested beforehand when there is a surplus of
honey (more than required for bees' needs).
6.5 Exposure time
Exposure time shall be defined depending on the task of the monitoring. For example, should the pollen
distribution of a plant species such as oil seed rape be recorded, it is reasonable to define the exposure
time covering the flowering period, e.g. at least from beginning of the flowering (5 % to 10 % open
blossoms, BBCH code 61 [18]) until withering of the last blossoms.
For newly placed bee colonies, exposure starts after putting up the bee colonies. After approximately
two days, the foraging bees have explored the area and are familiar with the local environment. After
five days at the earliest, the first samples of honey could be taken of combs.
Depending on honey flow larger amounts of honey may be extracted after approximately two weeks or
later by removal of entire combs.
Where there is insufficient supply of food sources with long exposure times, honey or bee bread placed
in storage might be consumed by the bees.
The colonies shall be regularly attended during longer exposure times (more than nine days) according
to good beekeeping practice [22; 33 and Annex C].
6.6 Sampling dates
Sampling dates are to a large extent defined by the exposure time (see 6.5). For longer exposure times
samples may be taken at intervals.
For the matrix honey, based on the amount available, either pieces of comb or entire combs may be
removed.
For pollen loads: Front pollen traps (see 5.2) have a significant effect on foraging behaviour. Pollen load
can only be taken from one colony for a short time (e.g. one day). If longer exposure time is necessary,
more colonies should be placed at the site so one colony after the other can be used for pollen
collection. This can be avoided by using inside pollen traps that has less effects. They are therefore
better suited to cover a flowering period by daily sampling without intervals.
6.7 Extraction, transport and storage
Complete honeycombs are stored inaccessibly for bees after removal and, following completion of the
necessary beekeeping tasks, are immediately taken to the bee-keeper's apiary for extraction according
to good beekeeping practice.
Alternatively honey can be obtained by scraping out of honey combs (preferably capped, ripe areas). A
representative sample (at least 500 g) of the stirred honey of all bee colonies at one site is sealed in a jar
and kept cool (<8°C). For further analysis, the honey is frozen on arrival at the laboratory (<-18°C).
At the extraction site, the pieces of comb for bee-bread are placed in sufficiently large containers or
plastic bags carefully sealed and then labelled. In a cold chain (<8°C), samples are delivered to the
laboratory and frozen for further processing (<-18°C).
If possible, each day the pollen load should be taken out the traps and transferred in sufficiently large
containers or plastic bags carefully sealed and then labelled. In a cold chain (<8°C), samples are
delivered to the laboratory and frozen for further processing (<-18°C).
7 Palynology
7.1 General
In principle, extracted honey is the sample matrix to be preferred (see Clause 5) for primary pollen
types that are abundantly collected and present in honey. For some other pollen types relevant for
GMO-monitoring, like for example maize, pollen loads are preferably used. In case of non-primary
pollen types that are underrepresented in the samples an up-concentration may be advisable by using
separation techniques [for example see Annex A for maize pollen].
7.2 From sample preparation to embedded slide preparation
7.2.1 General
The sample S is the amount of matrix collected by a bee colony during the collection period. The total
sample defines the amount collected over the whole flowering season.
7.2.2 Honey
From the collected honey sample S a subsample U of 5 g of honey dissolved in 10 ml distilled water is
centrifuged [DIN 10760; [20]].
Excess liquid is thoroughly rinsed off on absorbent paper. 0,5 ml distilled water is added to the
sediment (in the centrifuge tube), while it is thoroughly stirred. An aliquot A with 0,1 ml of the
suspension is spread evenly over the marked area of 22 mm × 22 mm (mark off area beforehand) of the
slide. The slide is dried for approximately one hour and then immersed in glycerol-gelatine (size of the
cover slip 22 mm × 22 mm; see DIN 10760).
For molecular-biological analysis the sample preparation is described in Clause 8.
7.2.3 Pollen load
The pollen load should be cleaned from other material like leaves, etc. which have fallen in the pollen
traps.
For each daily pollen load the weight is determined.
For representative analysis the sample shall be homogenized. The pollen load is diluted 1:1 (w/w) with
distilled water and stirred for approximately one hour until total homogenization (magnetic stirrer).
For quantitative microscopic pollen analysis an aliquot A with 15 µl of this solution are transferred onto
a microscopic slide, 30 µl of distilled water added, homogenized again and spread over an area of
22 mm × 22 mm. After drying on a warming plate at 40°C embedding takes place with the mounting
medium Kaiser’s Glycerine jelly and a 22 mm × 22 mm cover slip.
For molecular-biological analysis the sample preparation is described in Clause 8.
7.2.4 Bee bread
The bee-bread is extracted from the pieces of honeycomb (10 cm × 10 cm) with a spatula. Subsequently
bee-bread is diluted 1:1 (w/w) with distilled water and stirred for approximately two hours until total
homogenization (magnetic stirrer).
An aliquot A with 15 µl ml of this solution is transferred onto a microscopic slide, 30 µl of distilled water
added, homogenized again and spread over an area of 22 mm × 22 mm. After drying on a warming plate
at 40°C embedding takes place with the mounting medium Kaiser’s Glycerine jelly and a
22 mm × 22 mm cover slip.
For molecular-biological analysis the sample preparation is described in Clause 8.
7.3 Microscopic analysis
Light microscopic analysis for honey takes place according to DIN 10760 in the laboratory [9; 19; 23;
24; 29; 30; 31]. The procedure for the other matrices is analogous. Analysis takes place at 400-times
magnification. In a first general check, quality of the microscopic mount and in particular the
homogeneous distribution of the pollen is tested. The pollen should be identified by the established
literature for determination [19; 29; 31; 32].
Microscopic analysis is done mainly at 400-times and in some cases at 1 000-times magnification:
Pollen grains of every 5th row (cross slide) are identified and counted for each pollen type present to
not less than 1 000 pollen grains in the total sum.
In case that for any target pollen type less than 5 counts have been detected, then successively another
6 rows shall be counted (only the targeted pollen type) until either 5 counts have been reached or the
whole sample has been analysed (dynamic counting method).
The total pollen counts over all pollen types are stated as well as the pollen counts for the target pollen
type(s) i in question.
First the sum of pollen counts of each pollen type i is extrapolated to the sample aliquot on the mount
by taking the ratio of the number of counted rows (counted area) to the total number of rows (total area
of the cover slide on the mount) into account. The number of rows may vary depending on the
microscope (depends on the size of field of vision. The count to at least 1 000 pollen enables a statement
on a detection limit of 0,1 % (detecting 1 pollen).
z
total rows K
N=sum pollen counts ×=nn⋅ pollen (1)
[ ]
i ∑ ik,
counted rows k
k=1
where
N
is the number of pollen counts of type i on mount;
i
n
are the pollen counts of type i in row (k = 1, 2, 3, …., K);
i,k
i is the pollen type;
k is the index of row on microscopic mount (k = 1, 2, 3…, K);
K is the total number of rows on microscopic mount;
z is the number of counted rows.
7.4 Pollen diversity
The pollen diversity for each sample site and matrix shall be noted in form of a table. The results may be
presented qualitatively (detected) or quantitatively. Quantitative results are required for at least the
target pollen types and total pollen content.
8 Molecular-biological analysis
8.1 General
Molecular-biological analysis is used for identifying GMO-exposure.
The analysis of the DNA may be done using PCR (Polymerase-Chain-Reaction). Here the state of the art
is given by quantitative PCR methods, even though the results of environmental samples – especially in
case of pollen samples – may often be restricted to be semiquantitative. For optimal results in PCR
analysis the sample preparation shall be performed specifically for any pollen type and matrix. Further
screening methods for more than one GMO may be used, too, which are, in general, less sensitive. For
the molecular-biological analysis of pollen DNA using PCR a sufficient high amount of target pollen is
necessary to meet the detection limits. For performing the analysis with a detection limit of 0,1 % GM-
content a number of at least in the order of 10 000 pollen of the target pollen type is required.
Therefore, the counting should precede the PCR analysis.
The rationale behind this is that for PCR analysis the sample gets divided into aliquots and that any
single aliquot needs at least 3 DNA copies of the target GMO to be detected. 0,1 % detection limit of the
target GMO means 1 GM-pollen out of 1,000 pollen of that pollen type. Each pollen contains 2-3 DNA
copies. The extraction efficacy of pollen DNA depends on the pollen type and extraction method used
and ranges between 10 to 50 % for are commonly good performance. Per single PCR-analysis only a
portion, between 5 to 10 % of the extracted DNA (aliquot) is used, this allowing repeated analysis and /
or the analysis of more than one event/GMO. Considering the Poisson distribution for the probability of
at least 1 extracted target DNA copy in a single aliquot, this results in a minimum pollen content in the
magnitude of 10 000 pollen of the target pollen type in the sub-sample taken for the PCR. For screening
methods like the analysis of the promoter 35S the calculation is the same taking here the minimum
percentage into account to which a GM pollen type shall be present in the sample to be detected.
NOTE GMO are usually single copy events.
After DNA-extraction, various methods of quantitative PCR are possible. By the state of the art this
includes single event analysis to multiple event detection. An example for Bt-maize is given in Annex A.
Some GMO express specific proteins like for example the Bt-toxin, that can be analysed using ELISA
(Enzyme-linked immunosorbent assay). This method will not be described further in this Technical
Specification.
In the following the general sample preparation steps are given which are necessary independently of
GMO and pollen type and prior to the specific preparatory steps.
8.2 Sample preparation
8.2.1 Honey
The amount of honey used for the analysis depends on the pollen counts detected in 7.3 for the
respective pollen types questioned in regard to keep detection limits.
EXAMPLE A honey sample shall be tested specifically for GM-oil seed rape. The quantitative
microscopic pollen analyses resulted in 1 300 Brassica pollen per g honey. For the GM-specific analysis
the required minimum sample amount to achieve 10 000 target pollen would be > 50 g and > 200 g for
optimal performance.
In case of non-primary pollen types with frequencies H lower 10 %, for optimal performance of the PCR
the target pollen types shall be concentrated by using separation techniques. An example is given in
Annex A for maize pollen shown with the matrix pollen load.
8.2.2 Pollen loads
The homogenized daily pollen load samples from 7.2.3 are splitted in two halve parts.
One part (A) is retained for any potential day specific analysis.
The second part of all daily pollen samples are combined to one pooled sample B for overall analysis.
The pooled sample shall be homogenized using a magnetic stirrer or mixer.
Halve of the pooled sample B1 (25 % of the original sample) containing all pollen types will be stored as
retain sample and for any potential future screening purposes.
The other halve B2 will be further prepared for GMO-/pollen type specific analysis.
In case of non-primary pollen types with frequencies H lower 10 %, for optimal performance of the PCR
the target pollen types shall be concentrated by using separation techniques. An example is given in
Annex A for maize pollen shown with the matrix pollen load.
8.2.3 Bee bread
The homogenized bee bred sample from 7.2.4 is splitted in two halve parts.
One part B1 (50 % of the original sample) containing all pollen types will be stored as retain sample.
The other halve B2 will be further prepared for GMO-/pollen type specific analysis.
In case of non-primary pollen types with frequencies H lower 10 %, for optimal performance of the PCR
the target pollen types shall be concentrated by using separation techniques. An example is given in
Annex A for maize pollen shown with the matrix pollen load.
9 Determination of the target parameters for
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