1、BS EN 16086-2:2011Soil improvers and growingmedia Determination ofplant responsePart 2: Petri dish test using cressBS EN 16086-2:2011 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16086-2:2011.BSI, as a member of CEN, is obliged to publish EN 16086-2:2011 as a
2、 British Standard. However, attention is drawn to the fact that the UK committee voted against its approval as a European Standard.The UK committee voted against the publication of this standard because, although the method is interesting for screening materials, the plant species specified for the
3、test are not sensitive to some of the pesticides of greatest concern currently in composted materials, and because it considered that the reproducibility obtained in inter-laboratory evaluation was not an adequate basis on which to estab-lish a reference method. This could cause problems in the even
4、t of dispute or litigation.The standard deviations of reproducibility in the inter-laboratory validation trials of this method were poor and there is a low prob-ability of getting the same result from two laboratories analysing the same sample. In the worst cases the standard deviation of reproducib
5、ility exceeded the mean analysis (after rejection of outliers), for example mean germination rate for compost 1 was 22.38 %, SR34.66; root length compost 1 0.98, SR1.51 cm.The UK committee advises that this standard will not detect inhibition by residues of some of the herbicides of greatest concern
6、, and that in addition the results are imprecise.The UK participation in its preparation was entrusted to Technical Committee AW/20, Top soil and other growing media.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to
7、 include all the necessary provisions of a contract. Users are responsible for its correct application. BSI 2011ISBN 978 0 580 71318 7ICS 65.080Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Po
8、licy and Strategy Committee on 30 November 2011.Amendments/corrigenda issued since publicationDateT e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16086-2 November 2011 ICS 65.080 English Version Soil improvers and growing media - Determination of plant response - Part 2:
9、Petri dish test using cress Amendements du sol et supports de culture - Dtermination de la rponse des plantes - Partie 2: Essai en bote de Ptri avec du cresson Bodenverbesserungsmittel und Kultursubstrate - Bestimmung der Pflanzenvertrglichkeit - Teil 2: Petrischalentest mit Kresse This European Sta
10、ndard was approved by CEN on 17 September 2011. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concern
11、ing such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into
12、its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland
13、, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17,
14、B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16086-2:2011: E2 Contents Page Foreword 31 Scope 42 Normative references 43 Terms and definitions .44 Principle 55 Choice of methodology .55.1 Contact method 55.2
15、 Extract method .56 Material .56.1 Cress seeds (Lepidium sativum) 56.2 Water of class 356.3 Sphagnum peat 56.4 Fertilized and limed Sphagnum peat .66.5 Petri dishes .66.6 Perlite 66.7 Testing facility 66.8 Sieve with 10 mm mesh size .66.9 Filter paper .66.10 Ground limestone 67 Contact method 67.1 G
16、eneral preparation .67.2 Sample storage and preparation 67.3 Procedure .77.4 Evaluation parameters 88 Extract method 108.1 Preparation of the sample extract . 108.2 Procedure 108.3 Evaluation parameters . 119 Test report . 11Annex A (informative) Validation 12Annex B (normative) Fist test, nutrient
17、solution . 14B.1 Fist test 14B.2 Composition of the nutrient solution 14Bibliography . 16BS EN 16086-2:2011 EN 16086-2:2011 (E)3 Foreword This document has been prepared by Technical Committee CEN/TC “Soil improvers and growing media”, the secretariat of which is held by ASI. This European Standard
18、shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2012, and conflicting national standards shall be withdrawn at the latest by May 2012. Attention is drawn to the possibility that some of the elements of this document
19、 may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. SAFETY PRECAUTIONS Care should be taken when handling samples that may contain sharps or is of a dusty nature. According to the CEN/CENELEC Internal Regulations, the
20、national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherla
21、nds, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 16086-2:2011 EN 16086-2:2011 (E)4 1 Scope This European Standard describes a method for the routine determination of the effect of soil improvers and growing media or constituents the
22、reof on the germination and early root development of cress. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (
23、including any amendments) applies. EN 13037, Soil improvers and growing media Determination of pH EN 13038, Soil improvers and growing media Determination of electrical conductivity EN 13040, Soil improvers and growing media Sample preparation for chemical and physical tests, determination of dry ma
24、tter content, moisture content and laboratory compacted bulk density EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 plant response variation in c
25、ress seed germination and/or growth when sown and grown in a growing medium, soil improver or constituent thereof NOTE Factors causing negative plant growth cannot be identified nor sufficiently quantified by applying this method. 3.2 germination for this method, the seed is said to have germinated
26、as soon as the radicle has emerged from the seed 3.3 root length index percentage difference of the root length of germinated cress seeds on the material under investigation compared to the root length of the control 3.4 Munoo-Liisa Vitality index index calculated from the germination rate and the r
27、oot length BS EN 16086-2:2011 EN 16086-2:2011 (E)5 4 Principle Cress seeds are exposed to the test material for a few days under controlled conditions. The germination and growth of young roots are measured and compared with a control sample. The inhibition of germination and growth of young roots m
28、ay be caused by phytotoxic substances. If the electrical conductivity (EC) in the diluted material is greater than 80 mS m-1according to EN 13038, the sample is diluted with sphagnum peat. In this case, the test does not measure the adverse effect of high EC of materials on germination and root deve
29、lopment. NOTE 1 In the case of composted materials, these phytotoxic substances can be for instance ammonia, ethylene oxide or short chain fatty acids. NOTE 2 The test can also be used as an indication of the instability and “immaturity” of the material. If required (for example to fulfil certain qu
30、ality certification requirements or legislation), materials can be tested without checking the EC. For testing of specific effects, the use of additional plant species such as Chinese cabbage or lettuce may be considered. 5 Choice of methodology 5.1 Contact method For most of growing media or soil i
31、mprovers, the petri dish test can be carried out with the seeds in physical contact with the material to be tested. 5.2 Extract method Coarse samples such as bark, expanded clay, lava, mineral wool, perlite, polyurethane and pumice, used at 100 % as a growing medium, are not suitable for this proced
32、ure. For these materials, the seeds should be in contact with a filter paper thoroughly wetted with an extract of the material to be tested. 6 Material 6.1 Cress seeds (Lepidium sativum) Germination capacity 95 %. 6.2 Water of class 3 According to EN ISO 3696. 6.3 Sphagnum peat Sphagnum peat with a
33、degree of humification H3 H5 according to von Post scale, a pH between 3,0 and 4,5 (measured according to EN 13037), an EC of between 1 and 5 mS m-1(measured according to EN 13038) and a particle size 80 mS m-1, the sample shall be diluted with a sufficient amount of sphagnum peat (see 6.3) until th
34、e EC does not exceed 80 mS m-1. The pH according to EN 13037 is ideally within the range between 5,5 and 6,5. If it is below, the pH shall be adjusted by adding limestone (see 6.10). After adding limestone, the sample shall be equilibrated for 24 h. NOTE Usually, 2 g to 3 g of limestone per litre sh
35、ould be sufficient. If required (for example to fulfil certain quality certification requirements or legislation), materials can be tested without checking the EC. 7.3 Procedure Fill the petri dish completely and level the surface (for example with a spatula) without heavy compression. Where the see
36、d is being placed, remove any particles 5 mm. Sow 10 cress seeds per dish evenly spaced on the test material 10 mm to 20 mm from the top and press the seed gently into the surface of the test material. It is important that there is good contact with the test material. To ensure this, a drop of water
37、 shall be added to each seed using a pipette. Perform the procedure in at least three replicates. NOTE 1 A higher number of replicates can be used. The number of replicates should be taken into account for the calculation of the results. As a control sample, perform the same procedure with limed and
38、 fertilized sphagnum peat (see 6.4), in three replicates. NOTE 2 As a “positive” reference, the procedure can be performed using fertilized Sphagnum peat (see 6.4) wetted with a solution of acetic acid resulting in a final concentration of approximately 350 mg acetic acid per litre of sphagnum peat.
39、 This should give a reduction of the germination rate and/or the root length of about 50 %. Close the dishes with their covers and incubate with the Petri dish placed 70to 80 to the horizontal with the end where the seeds are placed uppermost and with the substrate on the lower surface in the dark a
40、t (25 5) C (see Figure 1). Incubate as described for 72 h. Determine the percentage germination (germination rate) root development (root score) by measuring the length in mm. If the average germination rate (see 7.4) in the reference material is below 85 %, the test is invalid. NOTE 3 The covers ca
41、n be fixed by using a rubber band or wrapping them in aluminium foil. If the Petri dish is completely covered by the foil, it can be incubated without additional darkening. NOTE 4 A digital photograph can be taken and an image analysis can be prepared using an image analysis programme. This will giv
42、e root length and root diameter and the results can be reported as percentage of control. BS EN 16086-2:2011 EN 16086-2:2011 (E)8 Key A side view B front view 1 cress seed 2 substrate/perlite Figure 1 Petri dish placement for incubation Roots can be stored in bottles containing 50 % V/V ethanol at (
43、5 3) C and measured later. 7.4 Evaluation parameters Calculate the coefficient of variance for the germination rate and the root length for the test sample and the control according to Equations (1), (2), (3), (4) and (5). 33) (dish2) (dish1) (dishGRGRGR=AGR+(1) where AGR is the average germination
44、rate; GR is the germination rate. 1002)(2AGRAGRGR=CVG (2) where CVG is the coefficient of variance for the germination rate. BS EN 16086-2:2011 EN 16086-2:2011 (E)9 NGSRL=RLP(3) where RLP is the root length, per plant; RL is the root length; NGS is the number of germinated seed. 33) (dish2) (dish1)
45、(dishRLPRLPRLP=ARLP+(4) where ARLP is the average root length, per plant. 1002)(2ARLPARLPRLP=CVR (5) where CVR is the coefficient of variance for the root length. The root length index (RI) is expressed as a percentage difference of the root length of germinated cress seeds on the tested material pe
46、r dish compared to the average root length of all the control samples and is calculated according to Equation (6) 1003(%)321+=cscscsRLRLRLRLRLRLRI (6) where RI is the root length index; RLs1is the average root length of first replicate; RLs2is the average root length of second replicate; RLs3is the
47、average root length of third replicate; RLcis the average root length of the control samples. The Munoo Liisa vitality index (MLV) compares the product of germination of seeds in the tested material (%) and the average root length in the test and control samples and is calculated according to Equati
48、on (7). ()()()1003(%)332211+=ccssssssRLGRRLGRRLGRRLGRMLV (7) where MLV is the Munoo Liisa vitality index of the sample (% compared with control); GRs1is the germination rate in first replicate, in %; GRs2is the germination rate in second replicate, in %; BS EN 16086-2:2011 EN 16086-2:2011 (E)10 GRs3
49、is the germination rate in third replicate, in %; GRcis the average germination rate of the control samples, in %; RLs1is the average root length of first replicate; RLs2is the average root length of second replicate; RLs3is the average root length of third replicate; RLcis the average root length of the control samples. 8 Extract method 8.1 Preparation of the sample extract Fill a watertight container of approximately 1 litre volume to the r
