SIST-TS CEN/TS 16817-1:2016

Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen monitoring - Part 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-2-sampler

This standard describes a procedure for the combined use of the deposition-type Sigma-2 sampler and the PMF to sample pollen. Collected samples are used to analyse pollen input with regard to species and amount, and percentage of transgenic pollen. The Sigma-2 sampler here provides a standardized sampling method for direct microscopic pollen analysis. Using the PMF yields sufficient amounts of pollen to carry out molecular-biological diagnostics. As a basis document for the NWI VDI 4330 Part 3 is available.

Außenluft - Monitoring der Wirkungen gentechnisch veränderter Organismen (GVO) - Pollenmonitoring - Teil 1: Technische Pollensammlung mit Pollenmassenfilter (PMF) und Sigma-2-Sammler

Die vorliegende Technische Spezifikation beschreibt ein Verfahren zur Verwendung der Passivsammler Sigma-2 und PMF zur Sammlung von luftgetragenen Pollen. Beide Sammler sind auf die Sammlung grober Aerosolpartikel ausgelegt. Gesammelte Proben werden zur Analyse des Polleneintrags hinsichtlich Pollenart, Pollenmenge und dem Eintrag von transgenen Pollen verwendet. Der Sigma-2-Sammler dient hierbei der standardisierten Probenahme zur direkten mikroskopischen Pollenanalyse und der quantitativen Bestimmung des in der Luft vor Ort vorhandenen Polleneintrags. Durch den PMF können ausreichende Pollenmengen für zusätzliche molekularbiologische Diagnostiken zum GVO-Nachweis erhalten werden.
Wesentliche Grundlagen zur Durchführung des GVO-Monitorings werden in Richtlinie VDI 4330 Blatt 1 [4] vermittelt, basierend auf einer integrierten Beurteilung von zeitlichen und räumlichen Variationen des GVO-Anbaus (GVO-Quellen), der Umweltexposition sowie biologischen/ökologischen Wirkungen. Im Idealfall sollte die Pollensammlung mit technischen Sammlern für das GVO-Monitoring in Kombination mit der biologischen Pollensammlung durch Bienen vorgenommen werden (CEN/TS 16817-2).
Der Einsatz von technischen Passivsammlern und die Verwendung von Honigbienenvölkern als aktive biologische Sammler ergänzen sich beim Monitoring der Exposition zu GVO-Pollen in vielfältiger Weise. Während die technischen Sammler repräsentative Ergebnisse zum Polleneintrag am Standort liefern, werden bei der biologischen Sammlung durch die Honigbienen die Pollen der in einem Gebiet vorhandenen blühenden Pflanzen entsprechend der Sammelaktivität gesammelt. Hierbei wird die GVO-Exposition gegenüber umherstreifenden Insekten dargestellt. Die Kombination beider Sammelverfahren repräsentiert die beiden hauptsächlichen Grundsätze der Exposition. Darüber hinaus kann ein breiteres Spektrum an Pollenspezies erfasst werden.
Der Messplan richtet sich nach der vorgesehenen Messaufgabe. Siehe 6.2 für Beispiele.

Air ambiant - Surveillance des effets d'organismes génétiquement modifiés (OGM) - Surveillance du pollen - Partie 1 : Échantillonnage technique du pollen à l'aide d'un filtre de masse à pollen (FMP) et d'un échantillonneur Sigma-2

La présente Spécification technique décrit un mode opératoire relatif à l’utilisation des échantillonneurs passifs Sigma 2 et FMP pour échantillonner le pollen en suspension dans l’air. Tous deux sont conçus pour échantillonner les grosses particules d’aérosols. Les échantillons prélevés sont utilisés pour analyser l'apport de pollen en termes de type et de quantité de pollen, ainsi que l’apport de pollen transgénique. L’échantillonneur passif Sigma-2 offre ici une méthode d’échantillonnage standardisée pour l’analyse microscopique directe du pollen et la quantification de la dispersion aérienne du pollen sur le site. Le FMP permet d’obtenir des quantités suffisantes de pollen pour effectuer en plus des diagnostics par biologie moléculaire pour la détection des OGM.
Des informations générales essentielles sur la surveillance des OGM sont données dans la ligne directrice VDI 4330 Part 1 [4], qui repose sur une évaluation intégrée de la variation spatio-temporelle de la culture d’OGM (sources d’OGM), de l’exposition dans l’environnement et des effets biologiques/écologiques. Dans l’idéal, il convient d’effectuer l’échantillonnage de pollen à l’aide d’échantillonneurs techniques pour la surveillance des OGM en association avec la récolte biologique de pollen par les abeilles (FprCEN/TS 16817­2).
L’application d’échantillonneurs techniques passifs et l’utilisation de colonies d’abeilles mellifiques comme échantillonneurs biologiques actifs se complètent à plusieurs niveaux lors de la surveillance de l’exposition au pollen génétiquement modifié. Les échantillonneurs techniques fournissent des résultats représentatifs concernant l’apport de pollen sur le site d’échantillonnage, alors qu’avec les échantillonneurs biologiques tels que les colonies d’abeilles mellifiques, le pollen des plantes à fleurs de la zone est récolté en fonction de l’activité de récolte des abeilles. Cette méthode représente ainsi l’exposition des OGM aux insectes itinérants. En combinant les deux méthodes d’échantillonnage, ces deux principes majeurs d’exposition sont représentés. En outre, une vaste gamme d’espèces de pollen est couverte.
Le plan d’échantillonnage dépend de l’objectif de mesurage prévu. Des exemples sont donnés en 6.2.

Zunanji zrak - Monitoring učinkov gensko spremenjenih organizmov (GSO) - Monitoring peloda - 1. del: Tehnično vzorčenje peloda z uporabo masnega filtra peloda in vzorčevalnika Sigma-2

Ta standard opisuje postopek za združeno uporabo vzorčevalnika Sigma-2 za določanje vrste usedanja in masnega filtra peloda (PMF) za vzorčenje peloda. Na podlagi zbranih vzorcev se izvede analiza vnosa peloda glede na vrsto in količino ter odstotek transgenega peloda. Vzorčevalnik Sigma-2 v tem primeru omogoča standardizirano metodo vzorčenja za namen neposredne mikroskopske analize peloda. Z uporabo filtra PMF se zagotovijo zadostne količine peloda za izvedbo molekularno-biološke diagnostike. Za podlago je na voljo dokument NWI VDI 4330, 3. del.

General Information

• Status: Published
• Publication Date: 21-Dec-2015
• ICS: 13.040.20 - Ambient atmospheres
• Technical Committee: KAZ - Air quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 10-Dec-2015
• Due Date: 14-Feb-2016
• Completion Date: 22-Dec-2015
• Ref Project: CEN/TS 16817-1:2015 - Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen monitoring - Part 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-2-sampler

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TS CEN/TS 16817-1:2016
01-februar-2016
=XQDQML]UDN0RQLWRULQJXþLQNRYJHQVNRVSUHPHQMHQLKRUJDQL]PRY*62
0RQLWRULQJSHORGDGHO7HKQLþQRY]RUþHQMHSHORGD]XSRUDERPDVQHJDILOWUD
SHORGDLQY]RUþHYDOQLND6LJPD
Ambient air - Monitoring the effects of genetically modified organisms (GMO) - Pollen
monitoring - Part 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-
2-sampler
Außenluft - Monitoring der Wirkungen gentechnisch veränderter Organismen (GVO) -
Pollenmonitoring - Teil 1: Technische Pollensammlung mit Pollenmassenfilter (PMF) und
Sigma-2-Sammler
Air ambiant - Surveillance des effets d'organismes génétiquement modifiés (OGM) -
Surveillance du pollen - Partie 1 : Échantillonnage technique du pollen à l'aide d'un filtre
de masse à pollen (FMP) et d'un échantillonneur Sigma-2
Ta slovenski standard je istoveten z: CEN/TS 16817-1:2015
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
SIST-TS CEN/TS 16817-1:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 16817-1:2016

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SIST-TS CEN/TS 16817-1:2016


CEN/TS 16817-1
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 1:
Technical pollen sampling using pollen mass filter (PMF)
and Sigma-2-sampler
Air ambiant - Surveillance des effets d'organismes Außenluft - Monitoring der Wirkungen von
génétiquement modifiés (OGM) - Surveillance du gentechnisch veränderten Organismen (GVO) -
pollen - Partie 1 : Échantillonnage technique du pollen Pollenmonitoring - Teil 1: Technische Pollensammlung
à l'aide d'un filtre de masse à pollen (PMF) et d'un mit Pollenmassenfilter (PMF) und Sigma-2-Sammler
échantillonneur Sigma-2
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 16817-1:2015 E
worldwide for CEN national Members.

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Contents Page
European foreword . 5
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Basic principle of the procedure . 9
5 Sampling . 9
5.1 Instruments and materials . 9
5.1.1 General . 9
5.1.2 Sigma-2 passive sampler . 10
5.1.3 Pollen mass filter PMF . 10
5.2 Technical implementation . 12
6 Sampling procedure . 13
6.1 General . 13
6.2 Sampling design . 13
6.2.1 General . 13
6.2.2 Exposure assessment of pollen input in the vicinity of fields with genetically
modified crop (gm-fields) related to a specific GMO and region . 14
6.2.3 Exposure assessment of pollen input for validating and/or calibrating dispersal
models . 15
6.2.4 General monitoring of pollen exposure at larger scales . 15
6.2.5 Assessment of standardized and acceptor specific pollen deposition . 15
6.3 Site conditions . 15
6.4 Installing the equipment . 16
6.5 Exposure time . 17
6.6 Sampling at site . 17
6.6.1 Sigma-2 passive sampler . 17
6.6.2 PMF . 17
6.7 Sample preparation . 18
6.7.1 Preparation of slides for microscopy . 18
6.7.2 Preparation of PMF samples . 19
7 Microscopic pollen analysis . 20
7.1 General . 20
7.2 Sigma-2 passive sampler . 20
7.2.1 Microscopic imaging methods . 20
7.2.2 Qualitative analysis of the pollen diversity . 21
7.2.3 Quantitative analysis of the pollen . 21
7.3 PMF . 21
7.3.1 Microscopic analysis . 21
7.3.2 Qualitative analysis of the pollen (diversity) . 21
7.3.3 Quantitative microscopic analysis of pollen . 21
8 Molecular-biological analyses of GMO . 23
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9 Determination of the target parameters for GMO monitoring and representation of
the results . 24
9.1 General . 24
9.2 Sigma-2 passive sampler . 24
9.2.1 Determination of pollen deposition per sampling period . 24
9.2.2 Determination of the daily mean pollen deposition rate per sampling period . 25
9.2.3 Determination of yearly pollen deposition . 25
9.2.4 References to pollen dispersal models . 25
9.3 PMF . 25
9.3.1 Pollen count per sample N . 25
i,PMF
9.3.2 Relative frequency of pollen species i . 26
9.3.3 Determination of pollen flux per sampling period . 26
9.3.4 Determination of the daily mean pollen flux rate per sampling period . 26
9.3.5 Determination of the yearly pollen flux . 27
9.3.6 Assessment of results from molecular-biological analyses . 27
9.3.7 References to pollen dispersal models . 28
10 Performance characteristics of the methods. 28
10.1 General . 28
10.2 Validation . 28
10.3 Distribution of measured values . 29
10.4 Methodical approach and determination of basic parameters. 29
10.5 Sigma-2 passive sampler . 31
10.5.1 Sensitivity, detection limit and reproducibility . 31
10.5.2 Detection confidence level and required numbers of cases . 33
10.6 PMF . 35
10.6.1 Sensitivity, detection limit and reproducibility . 35
10.6.2 Detection confidence level and required numbers of cases . 38
10.7 Parallel measurements. 40
10.8 Comparative measurements using a standard volumetric pollen trap (Hirst type) . 42
10.9 Pollen diversity . 43
11 Quality assurance and quality control . 44
11.1 General monitoring strategy and terms of reference of pollen monitoring with
technical samplers . 44
11.2 Site protocol . 44
11.3 Accompanying documentation for samples . 45
11.4 Parallel measurements. 45
11.5 Comparative measurements using active samplers as calibration bases . 45
11.6 Quality assurance and reference materials . 45
11.7 Qualification . 46
Annex A (normative) Maize-specific requirements . 47
A.1 Scope . 47
A.2 Basic principles . 47
A.3 Sampling . 48
A.4 Sample preparation . 49
A.5 Quantitative microscopic pollen analysis . 50
A.6 Molecular-biological analysis of maize DNA using PCR . 51
A.6.1 General . 51
A.6.2 DNA extraction . 51
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A.6.3 Real-time PCR analysis . 51
A.7 Determination of the target parameters for GMO monitoring and assessment of the
results . 52
Bibliography . 53

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European foreword
This document (CEN/TS 16817-1:2015) 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 [the present
document];
— Part 2: Biological pollen sampling using bee colonies.
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.
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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 [5; 6]. 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). A
procedure is described for GMO monitoring that enables quantification and documentation of GMO
input and spread through pollen in a nationwide monitoring network which represents natural
landscapes. Technical and biological pollen sampling (the present Technical Specification and
CEN/TS 16817-2) and molecular biological analysis methods (polymerase chain reaction (PCR) for
DNA; Enzyme-linked immunosorbent assay (ELISA) for proteins) are used for the detection of GMO
input.
It is reasonable to use both technical and biological sampling of pollen, thus they supplement each other
in manifold ways. The technical sampling (i.e. the present document) is conducted with stationary
point-samplers. They give a record of pollen input at the sample site that correlates with the prevailing
wind direction and relative position to the surrounding pollen sources. Bee colonies actively roam an
area and are therefore area related samplers. Further, pollen sampling depends here on the collection
activity of the bees and the availability of pollen sources within the roaming area according to the bees'
preferences and supply of melliferous plants [32].
Presently known pollen traps are only partially suited for GMO monitoring, since they can neither be
standardized nor is the instrumentation designed for exposure times that are suitable for this purpose.
Another limitation of commonly used pollen samplers is the requirement for a power supply, e.g. as for
the Hirst type trap. The use of these instruments is therefore restricted to a limited exposure area.
For these reasons, a new type of passive pollen sampler, the pollen mass filter (PMF), was developed.
The PMF is used either in combination with the Sigma-2 passive sampler or solely.
The present Technical Specification is largely based on German VDI/Guideline 4330 Part 3 [31].
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1 Scope
This Technical Specification describes a procedure for the use of the passive samplers Sigma-2 and PMF
to sample airborne pollen. Both are designed to sample coarse aerosol particles. Collected samples are
used to analyse pollen input with regard to pollen type and amount, and input of transgenic pollen. The
Sigma-2 passive sampler here provides a standardized sampling method for direct microscopic pollen
analysis and quantifying the input of airborne pollen at the site. The PMF yields sufficient amounts of
pollen to additionally carry out molecular-biological diagnostics for detection of GMO.
Essential background information on performing GMO monitoring is given in VDI/Guideline 4330
Part 1 [4], which is based on an integrated assessment of temporal and spatial variation of GMO
cultivation (sources of GMO), the exposure in the environment and biological/ecological effects. Ideally,
the pollen sampling using technical samplers for GMO monitoring should be undertaken in combination
with the biological collection of pollen by bees (CEN/TS 16817-2).
The application of technical passive samplers and the use of honey bee colonies as active biological
collectors complement each other in a manifold way when monitoring the exposure to GMO pollen.
Technical samplers provide results regarding the pollen input at the sampling site in a representative
way, whereas with biological sampling by honey bee colonies, pollen from flowering plants in the area
is collected according to the bees' collection activity. Thus, this method represents GMO exposure to
roaming insects. By combining the two sampling methods these two main principles of exposure are
represented. Furthermore, a broad range of pollen species is covered.
The sample design depends on the intended sampling objective. Some examples are given in 6.2.
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.
1)
VDI 2119:2013-06 , Ambient air measurements — Sampling of atmospheric particles > 2,5 µm on an
acceptor surface using the Sigma-2 passive sampler — Characterisation by optical microscopy and
calculation of number settling rate and mass concentration
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
acceptor surface
natural or manmade collection surface for airborne particles
3.2
concentration
number concentration
3
number of particles per unit air volume; here number of pollen per m air
3.3
deposition
pollen deposition
deposition of atmospheric particles; here pollen on an acceptor surface

1) For application of the Sigma-2.
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3.4
dispersal
pollen dispersal
spread of pollen from the flower/field into the surrounding environment by wind drift
3.5
event
unique DNA recombination event that took place in one plant cell, which was then used to
generate entire transgenic plants
3.6
flux
horizontal flux
number of particles (here pollen) that are drifted horizontally per wind
3.7
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 [5], modified — The content of the definition was changed.]
3.8
monitoring
environmental monitoring
characterizing the state and quality of the environment and its changes by measurements/observations
in regard to defined objectives
3.9
pollen
male gametophyte of the flowering plant
3.10
pollen type
species
class of pollen being distinguished by microscopic means on species, family or other order level
3.11
sampler
device for sampling here of pollen
3.12
sampling
pollen sampling
collection of particles, here pollen by technical or biological means
3.13
sedimentation
directed particle movement by gravity (here pollen in the air), which consists in a vertical flux towards
the ground
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4 Basic principle of the procedure
For the technical pollen sampling, two passive samplers are used, the PMF and Sigma-2 passive
sampler, either in combination or the PMF solely:
The Sigma-2 passive sampler is designed for determining the pollen deposition rate (dry deposition by
sedimentation). Wind-dispersed pollen grains enter the interior through the laterally shifted slits of the
sampler. The pollen are deposited on an adhesive tray as acceptor (tape, foil, slide) at the bottom of the
sampler. Thus, the deposition takes place in the turbulence-depleted interior of the sampler which
provides protection from wind and rain. Pollen adhering to the adhesive tray are directly analysed with
regard to pollen type and counts by means of light microscopy. For the purpose of GMO monitoring, an
exposure time in the range of four weeks is recommended to be able to cover the main flowering period
of the target plant species with as few sampling periods as possible (the rationale for this is given in
6.5). The microscopic single-particle analysis yields an average pollen deposition rate for the respective
pollen species and time period. Summing up the deposition rates of all sampling periods in the season
yields the total pollen deposition per season/year as target parameter.
The pollen mass filter (PMF) exhibits a 10 times to 100 times higher sampling efficiency, so that pollen
samples can be analysed both microscopically to quantify pollen input and further on, with regard to
possible GMO input by using molecular-biological based methods (e.g. PCR for DNA, ELISA for
proteins/toxins). The PMF consists of a layered hollow filter that is constructed in such a way as to let
the air pass through nearly unopposed. However, coarse aerosol particles bigger than 10 µm, such as
pollen, are retained. A laterally mounted collection flask is used for collecting rainwater. For the PMF,
an exposure time of four weeks is recommended (see 6.5) so that only a few samples are needed to
cover the relevant flowering period. In order to cover a complete blooming period of one or more target
plant species a respective number of exposure (sampling) periods lasting four weeks each can be
carried out.
The Sigma-2 passive sampler collects aerosol particles bigger than 1 µm covering the size range of most
pollen and fungal spores. Its sampling efficiency reaches its limitation towards bigger and heavier
aerosol particles over 60 µm diameter, like e.g. maize pollen. In such cases, the evaluation of pollen
deposition shall be based on the PMF solely.
Field experiments have shown that the method is well suited for environmental monitoring of GMO [14;
17; 20].
5 Sampling
5.1 Instruments and materials
5.1.1 General
The combined sampling equipment consisting of a Sigma-2 passive sampler and PMF is described in
Figure 1. For some tasks the PMF sampler is used solely, e.g. for maize pollen, and/or when it is
necessary to increase the amount of sampled pollen at a site within a certain period (see 10.5.2, e.g. for
keeping detection limits for PCR-analysis of pollen DNA). For such tasks, stacked versions of the PMF
sampler with more than one PMF-unit per sampler are additionally available as shown in Figure 2. The
2 3
) )
complete sampling equipment is available.

2) TIEM technic GbR, Hohenzollernstr. 20, 44135 Dortmund. Samplers are manufactured by the supplier mentioned above. It
is an example of a suitable product. This information is given for the convenience of users of this European Technical
Specification and does not constitute an endorsement by CEN of this product. Equivalent products may be used if
...