1、May 2014 Translation by DIN-Sprachendienst.English price group 12No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13
2、.080.30!%2 the seeds bearing these secondary roots are then used for the purpose of the test.This germination step of the Vicia faba seeds, necessary in both ways of exposure, can be started four days and eight days respectively for solid-phase and liquid-phase exposure before beginning the test.1)
3、LUFA soils are an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.9DIN ISO 29200:2014-05 6.3 Conducting of the test6.3.1 Soils and soil materialsT
4、he dilutions of the test mixture are chosen within a geometric series with a factor not exceeding two and shall cover a large range of concentrations (e.g. from 0,01 % to 100 % ). These mixtures are prepared by diluting the soil with a reference soil.Each test shall include a negative control withou
5、t any test sample and a positive control (see 5.3).The direct exposure of the plant organisms to the different concentrations of the soil is performed by placing the germinated seeds (at least three per dilution) in a plastic pot containing 200 g of the tested soil and/or mixtures (see Figure 1) thr
6、oughout the exposure time between three and five days, according to obtain at least ten roots of 1 cm length.Figure 1 Method of direct exposure of the Vicia faba seeds6.3.2 Water extracts of soilThe concentrations of the sample under test are chosen within a geometric series with a factor not exceed
7、ing two and shall cover a large range of concentrations (e.g. from 0,01 % to 100 % for matrices). This range of concentrations is prepared by diluting the sample with the previously oxygenated Hoaglands medium (see Annex A).Each test shall include a negative control without any test sample and a pos
8、itive control (see 5.3).At the time of the test, the different solutions to be tested are extemporaneously brought up to a temperature of 24 C 1 C and well homogenised before exposure of plant organisms. This is carried out by placing the germinated seeds (at least three per concentration) in a glas
9、s container having a sufficient diameter in order to prevent, as far as possible, contact between the root tips and the container wall throughout the exposure time. The roots are immersed in a minimum volume of 200 ml of test solution (see Figure 2).10DIN ISO 29200:2014-05 Figure 2 Method of aqueous
10、 exposure of the Vicia faba seedsThe exposure time shall be at least 30 h, which corresponds to the approximate duration of the cell cycle. However the optimal exposure time recommended to detect genotoxic effects is 48 h to be sure that the cell cycle is ended and for a better practicability.6.4 Te
11、st environmentThe tests are performed in a climatic chamber (with an intensity of at least 5 000 lx and 16/8 photoperiod) at a temperature of 24 C 1 C. The liquid-phase test can also be carried out in darkness if necessary (e.g. maleic hydrazide).6.5 Cell preparationAt the end of the exposure period
12、, the roots are simply removed from the water extract, or carefully extracted from the soil. Then they are cleaned with deionised water and the last two centimeters of the secondary roots (ten or so roots per seed, chosen at random) are sampled and placed at 4 C for a minimum duration of one night i
13、n Carnoys solution. These root tips can then be stored on a long term basis in 70 % ethanol for a deferred observation or else can be hydrolysed in the case of an immediate observation.The root tips are then placed in distilled water for 10 min, hydrolysed in the hydrolysis solution at 60 C for 6 mi
14、n and retransferred into distilled water for a few minutes.For root cell observation, place a root tip on a slide after wiping it cautiously. Remove the first millimeter corresponding to the root cap and the meristematic region and, with the help of a scalpel, retain only the second millimeter which
15、 is made up of the subsequent generation of cells obtained after mitosis. Micronuclei scoring shall be done in this particular cells region (see Figure 3). All these steps may be done on a black background to see the different cell regions.The staining of the DNA is carried out by crushing the root
16、tips after addition of a drop of orcein; the coverslip can then be placed in position and squeezed to obtain a single layer of cells.11DIN ISO 29200:2014-05 It is recommended to carry out at least two cell spreadings (obtained with two different roots) per seed.1 mm 1 mmRoot capMeristem Cells formic
17、ronucleiscoringFigure 3 Longitudinal section of Vicia faba root showing the root cap, the meristem and cells region to be selected for micronuclei scoringIt is preferable to perform the scoring under blindfold conditions prior to their examination so that the person conducting the test is not influe
18、nced when counting the micronuclei (see Figure 4). The micronuclei observed in cells in division shall not be taken into account when determining micronucleus frequency. The results are expressed in number of micronuclei per 1 000 cells in interphase.Figure 4 Micronuclei in the cells of the root tip
19、s of Vicia faba (x 400)During the micronuclei scoring, it is highly recommended to verify the proper progress of the cell division (an essential condition for the micronuclei formation) by determining the proportion of cells in mitotic division (commonly called mitotic index). A genotoxic effect can
20、 be masked by a cytotoxic one (toxic with respect to the cell functioning) which would induce an underestimation of the genotoxic potential of the sample under test. The results are expressed in number of cells in division per 1 000 cells observed. All the stages of the mitosis are taken into accoun
21、t, from the prophase (when the chromosomes begin to condense) up to the telophase (when the chromatin of the two nuclei formed at each pole of the cell finishes decondensing).The microscopic examination of the slides is carried out under a magnification of 400. A minimum number of two slides is prep
22、ared for each of the three replicates for each concentration. Considering that 1 000 cells per slide are observed, the averages and standard deviations are therefore calculated on a minimum of 6 000 cells for each concentration.12DIN ISO 29200:2014-05 7 Assessment of the results7.1 Presentation of t
23、he dataThe results of the negative and positive controls and of the concentrations under test are expressed in terms of average number of micronuclei per 1 000 cells observed.7.2 Statistical analysisThe use of a non-parametric method (e.g. the Kruskal-Wallis test followed by Dunns multiple compariso
24、n test) is recommended in order to highlight the significant differences between the control and test concentrations.7.3 Interpretation of the results7.3.1 Positive testThe test is considered as positive if a statistically significant result with respect to the negative control is detected for at le
25、ast one of the test concentration.7.3.2 Negative testThe test is considered as negative if, with respect to the negative control, no statistically significant positive response is observed for the tested concentrations. It is noteworthy that the absence of micronuclei in root cells may be due to a d
26、ysfunction of mitosis: in that case, DNA breakages are not excluded from the nucleus and do not form any micronucleus. Consequently, it is important to check if the mitotic index (number of cells in division) of each spreading is greater than 20 for 1000 cells, otherwise results of micronucleus freq
27、uency shall not be taken into account.8 Validity criteriaThe test is considered as valid if a positive response is obtained with the reference substance.For each concentration, the micronucleus frequency is reliable if the mean mitotic index is greater than 20 cells in division for 1 000 observed ce
28、lls.9 Test reportThe test report shall include the following information:a) a reference to this International Standard;b) a full description of the experimental methodology and procedures;c) the origin of the broad bean seeds;d) the test environment (temperature, photoperiod, lighting, etc.);e) the
29、characteristics of test soil (if appropriate);f) the characteristics of test material: compost, sludge, waste (if appropriate);g) the method of preparation of water extract of soils (if appropriate);h) the characteristics of control soil;i) the exposure time;13DIN ISO 29200:2014-05 j) the observatio
30、ns of acute toxicity made during the course of the test (visible damage of the organisms: blackening of the roots, necrosis of certain parts of the plants, etc.);k) the results as indicated in Clause 7, specifying the statistical methods used.14DIN ISO 29200:2014-05 Annex A (informative) Composition
31、 of Hoaglands mediumSee Table A.1.Table A.1 Composition of Hoaglands mediumProducts Stock solutionQuantity of stock solution per 1 l of mediumFinal concentrationKNO350,5 g/l 10 ml 5 mmol/lCa(NO3)24H2O 118,0 g/l 10 ml 5 mmol/lMgSO4K,7H2O 123,2 g/l 10 ml 5 mmol/lKH2PO413,6 g/l 10 ml 1 mmol/lIron tartr
32、ate 500 mg/l 10 ml 9 mol/lThe stock solutions of Hoaglands medium can be stored for three months at 4 C. It is recommended to prepare the Hoaglands medium at the time of use and to aerate the medium by bubbling through.15DIN ISO 29200:2014-05 Annex B (informative) Testing chemicals added to soilsThe
33、 stock solution of the chemical substance to be experimented is prepared by dissolving a known quantity of substance in a defined volume of deionised water. It shall be prepared at the time of use.NOTE If it is shown analytically that the substance is stable in the dark and at 4 C, it may be prepare
34、d in advance and stored under these conditions.In aqueous exposure, the test solutions are prepared just prior to use by dissolving the stock solution in the dilution medium (Hoaglands medium, see Annex A) in order to obtain the necessary concentrations. In direct exposure, the different solutions a
35、re used to spike the reference soil.In the case of barely water-soluble substances or substances that are insoluble in water, an intermediate water-miscible solvent (see (5.4.5) can be used. In this case, the concentration of the solvent in each container shall be constant and shall not exceed 100 l
36、/l. The dilution medium is shaken at the time of introduction of the intermediate solution, which generally leads to the forming of a microsuspension. If a precipitate appears, the test cannot be carried out. If the use of an intermediate solvent cannot be avoided, a negative control containing the
37、same concentration of solvent shall be included in the test.16DIN ISO 29200:2014-05 17DIN ISO 29200:2014-05 Annex C (informative) Results of the interlaboratory test conducted within the framework of NF T 90-327C.1 Results with CdCl2See Tables C.1 and C.2.Table C.1 Proportion of micronuclei (number
38、of micronuclei/1 000 cells) according to the concentration of CdCl2Laboratory Negative control 7,5.10-8mol/l 10-7mol/l 2,5.10-7mol/lTest 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2France A 3,83 1,33 4,67 1,75 17,83 4,66a11,50 3,27a30,00 5,29a16,17 3,97a39,33 10,01a25,67 5,05aFrance B 0,33 0,5
39、2 0,17 0,41 7,83 3,25a5,33 2,58a15,17 4,02a11,33 1,75a8,83 6,62a5,83 3,25aFrance C 0,33 0,52 0,17 0,41 12,50 2,43a14,67 2,66a16,83 3,43a15,83 2,93a20,17 3,76a27,17 3,19aaStatistically significant difference from negative control (Kruskal Wallis test, p0,05).Table C.2 Mitotic index (number of cells i
40、n division/1 000 cells) according to the concentration of CdCl2Laboratory Negative control 7,5.10-8mol/l 10-7mol/l 2,5.10-7mol/lTest 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2France A 51,17 7,83 61,33 14,78 54,50 8,73 53,83 11,36 79,50 11,04 55,83 15,22 70,33 19,47 59,00 14,18France B 29,83
41、7,94 24,00 2,68 27,83 8,13 22,83 6,68 30,00 12,30 24,67 5,68 15,33 5,20 10,33 4,50France C 36,83 5,56 43,33 4,37 38,50 3,27 42,17 4,53 34,50 3,73 42,33 4,18 38,33 4,97 39,67 6,47C.2 Results with maleic hydrazideSee Tables C.3 and C.4.Table C.3 Proportion of micronuclei (number of micronuclei/1 000 c
42、ells) according to the concentration of maleic hydrazideLaboratory Negative control 5.10-6mol/l 10-5mol/l 2.10-5mol/lTest 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2France A 5,33 1,21 1,33 1,97 10,00 1,41a12,67 5,20a39,17 15,52a20,33 3,72a10,33 4,93a12,50 3,56aFrance B 0,33 0,52 17,33 8,91a19
43、,33 5,28a9,17 6,31aFrance C 0,50 0,84 0,67 0,82 13,67 2,66a7,50 1,22a57,83 20,60a62,50 13,58a9,33 3,61a13,33 3,56aFrance D 0,50 0,84 0,50 0,84 38,67 20,25a32,25 8,02a73,67 27,53a67,00 27,53a48,50 24,3a44,33 9,22aFrance E 0,33 0,52 0,50 0,84 13,50 2,74a10,83 2,93a44,17 6,18a40,67 8,14a11,17 2,23a12,8
44、3 3,13aaStatistically significant difference from negative control (Kruskal Wallis test, p0,05).Table C.4 Mitotic index (number of cells in division/1 000 cells) according to the concentration of maleic hydrazideLaboratory Negative control 5.10-6mol/l 10-5mol/l 2.10-5mol/lTest 1 Test 2 Test 1 Test 2
45、 Test 1 Test 2 Test 1 Test 2France A 40,00 5,97 29,00 10,94 43,83 9,30 23,17 8,95 40,67 8,55 20,67 7,26 19,67 7,63 25,00 12,03France B 79,50 6,47 50,50 5,47 45,50 10,93 22,00 6,07France C 23,50 2,51 22,00 2,61 22,50 1,87 20,50 2,26 18,00 3,35 16,67 2,94 7,33 5,99 3,17 2,86France D 40,83 20,93 52,33
46、7,23 42,00 12,46 24,75 11,18 33,50 8,31 25,50 14,43 17,83 13,61 17,83 9,58France E 47,67 9,00 46,33 8,41 48,33 11,43 44,83 8,13 44,83 9,06 47,00 9,84 12,50 3,39 14,33 4,4118DIN ISO 29200:2014-05 19DIN ISO 29200:2014-05 Annex D (informative) Results of the interlaboratory test conducted on the refere
47、nce substance and an industrial contaminated soilThe aim of this ring test, organized by LIEBE-CNRS 7146 (University of Metz, France) was to check if the Vicia-micronucleus test could be used in different countries, so each participant used its own reference soil and bought its own seeds. The most i
48、mportant variability sources were chosen to assess the robustness of the test rather than the reproducibility. Maleic hydrazide, recommended as a reference substance, was tested by the two ways of exposure: respectively 10 mol/kg (1,12 mg/kg) and 10 mol/l (1,12 mg/l) for solid-phase and liquid-phase
49、 exposures. Each participant had to prepare the spiked soil mixing its own reference soil and the maleic hydrazide.D.1 Reference substance resultsSee Tables D.1 to D.4.Table D.1 Micronucleus frequency (number of micronuclei/1 000 cells) for the maleic hydrazide (1,12 mg/kg) in solid phasenegative control positive
