1、BSI Standards PublicationAmbient air Monitoring the effects of genetically modified organisms (GMO) Pollen monitoringPart 1: Technical pollen sampling using pollen mass filter (PMF) and Sigma-2-samplerPD CEN/TS 16817-1:2015National forewordThis Published Document is the UK implementation of CEN/TS 1
2、6817-1:2015.The UK participation in its preparation was entrusted by TechnicalCommittee EH/2, Air quality, to Subcommittee EH/2/3, Ambient atmospheres.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the
3、 necessary provisions ofa contract. Users are responsible for its correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 87461 1ICS 07.080; 13.020.99Compliance with a British Standard cannot confer immunity fromlegal obligations.This Publish
4、ed Document was published under the authority of theStandards Policy and Strategy Committee on 31 October 2015.Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 16817-1:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16817-
5、1 October 2015 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 dorganismes gntiquement m
6、odifis (OGM) - Surveillance du pollen - Partie 1 : chantillonnage technique du pollen laide dun filtre de masse pollen (PMF) et dun chantillonneur Sigma-2 Auenluft - Monitoring der Wirkungen von gentechnisch vernderten Organismen (GVO) - Pollenmonitoring - Teil 1: Technische Pollensammlung mit Polle
7、nmassenfilter (PMF) und Sigma-2-Sammler 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, part
8、icularly 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 conflict
9、ing 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 Yugo
10、slav 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 COMIT EUROPEN
11、 DE NORMALISATION EUROPISCHES KOMITEE FR 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 worldwide for CEN national Members. Ref. No. CEN/TS 16817-1:2015 EPD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E)
12、2 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 i
13、mplementation 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 val
14、idating 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
15、 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 analy
16、sis 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 PD CEN/TS 16817-1:2015CEN/TS
17、 16817-1:2015 (E) 3 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 samp
18、ling 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 Ni,PMF25 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 o
19、f 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 Val
20、idation 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 Se
21、nsitivity, 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 cont
22、rol 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 Qualit
23、y 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
24、PCR 51 A.6.1 General . 51 A.6.2 DNA extraction . 51 PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 4 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 PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 5 Eur
25、opean 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 shal
26、l 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-sample
27、r 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 Rep
28、ublic, 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. PD
29、CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 6 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 da
30、te. 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 lan
31、dscapes. 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 reason
32、able 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 wi
33、nd 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 t
34、he 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 po
35、llen 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 combi
36、nation with the Sigma-2 passive sampler or solely. The present Technical Specification is largely based on German VDI/Guideline 4330 Part 3 31. PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 7 1 Scope This Technical Specification describes a procedure for the use of the passive samplers Sigma-2 and P
37、MF 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 microscopi
38、c 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
39、 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 t
40、he 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 regardin
41、g 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
42、 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 nor
43、matively 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. VDI 2119:2013-061), Ambient air measurements Sampling of atmosph
44、eric particles 2,5 m 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 surfa
45、ce natural or manmade collection surface for airborne particles 3.2 concentration number concentration number of particles per unit air volume; here number of pollen per m3air 3.3 deposition pollen deposition deposition of atmospheric particles; here pollen on an acceptor surface 1) For application
46、of the Sigma-2. PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 8 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 tran
47、sgenic 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: Directiv
48、e 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 pol
49、len 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 PD CEN/TS 16817-1:2015CEN/TS 16817-1:2015 (E) 9 4 Basic principle of the procedure For the technical pollen sampling, two passive sam
