1、BSI Standards PublicationAmbient air Monitoring the effects of genetically modified organisms (GMO) Pollen monitoringPart 2: Biological pollen sampling using bee coloniesPD CEN/TS 16817-2:2015National forewordThis Published Document is the UK implementation of CEN/TS 16817-2:2015.The UK participatio
2、n 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 necessary provisions ofa contr
3、act. Users are responsible for its correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 87462 8ICS 07.080; 13.020.99Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under
4、 the authority of theStandards Policy and Strategy Committee on 31 October 2015.Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 16817-2:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16817-2 October 2015 ICS 07.080; 13.0
5、20.99 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 dorganismes gntiquement modifis (OGM) - Surveillance du pollen - Partie 2 : chantillonn
6、age biologique du pollen laide de colonies dabeilles Auenluft - Monitoring der Wirkungen von gentechnisch vernderten Organismen (GVO) - Pollenmonitoring - Teil 2: Biologische Pollensammlung mit Bienenvlkern This Technical Specification (CEN/TS) was approved by CEN on 16 May 2015 for provisional appl
7、ication. 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 exist
8、ence 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
9、 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, Netherla
10、nds, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights
11、of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16817-2:2015 EPD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 2 Contents Page European foreword . 4 Introduction 5 1 Scope 6 2 Normative references 6 3 Terms and definitions . 6 4 Basic principle
12、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 Paly
13、nology 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.
14、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 colle
15、cted 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 PD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 3 10.2 Bee colony . 16 10.3 Foraging distance . 17 10.4 Honey samples, bee-bread
16、 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 documenta
17、tion 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
18、.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 pre
19、paration . 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 be
20、ekeeping practice . 30 Bibliography . 31 PD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 4 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 o
21、f 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 followin
22、g 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 a
23、nnounce 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,
24、 Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 5 Introduction The European Parliament and the European Council require an environmental risk assessment and a post-marketing monitoring for any GMO released to the
25、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
26、 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 syst
27、em 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
28、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 sampl
29、ing (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 ind
30、icator 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
31、using both sampling methods with the procedures complementing each other across the vegetation period 21. PD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 6 1 Scope This Technical Specification describes a procedure through which pollen in particular pollen of genetically modified organisms (GMO) can b
32、e 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 preferenc
33、es (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,
34、 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 b
35、y 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
36、 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 anal
37、ysis 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
38、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
39、 (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 col
40、ony colony of the honeybee species Apis mellifera 3.3 beehive hive container in which honeybees are kept by beekeepers PD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 7 3.4 event unique DNA recombination event that took place in one plant cell, which was then used to generate entire transgenic plants
41、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, modi
42、fied 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
43、 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
44、 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 PD CEN/TS 16817-2:2015CEN/TS 16817-2:2015 (E) 8 3.15 pollen type pol
45、len 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 po
46、llen. 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 ra
47、w 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 b
48、ees 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 req
49、uire 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
