1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58measurement of the induction of micronuclei Part 1: Evaluation of genotoxicity using amphibian larv
2、aeICS 13.060.70Water quality Evaluation of genotoxicity by BRITISH STANDARDBS ISO 21427-1:2006BS 6068-5.44:2006BS ISO 21427-1:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2006 BSI 2006ISBN 0 580 48927 2Cross-referencesThe Bri
3、tish Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online.This pu
4、blication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations.Summary of pagesThis document comprises a front cover, an inside front cov
5、er, the ISO title page, pages ii to v, a blank page, pages 1 to 15 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsA list of organizations represented on this subcommittee can be
6、 obtained on request to its secretary. present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.National forewordThis
7、 British Standard reproduces verbatim ISO 21427-1:2006 and implements it as the UK national standard. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods, which has the responsibility to: aid enquirers to unders
8、tand the text;Reference numberISO 21427-1:2006(E)INTERNATIONAL STANDARD ISO21427-1First edition2006-08-01Water quality Evaluation of genotoxicity by measurement of the induction of micronuclei Part 1: Evaluation of genotoxicity using amphibian larvae Qualit de leau valuation de la gnotoxicit par le
9、mesurage de linduction de micronoyaux Partie 1: valuation de la gnotoxicit laide de larves damphibiens BS ISO 21427-1:2006ii iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 2 4 Principle. 2 5 Test environment 3 6 Reagents 3 7 Apparatus 4 8
10、Treatment and preparation of samples 4 8.1 Waters, effluents, leachates and eluates to be tested 4 8.2 Substances to be tested 5 9 Procedure 5 9.1 Selection of concentrations. 5 9.2 Conducting the test 6 9.3 Reading of the histological preparations. 6 10 Expression of results . 6 10.1 Presentation o
11、f results. 6 10.2 Processing of results . 7 10.3 Interpretation of results 7 11 Validity of the test. 7 12 Test report . 7 Annex A (normative) Data specific to Pleurodeles waltl 9 Annex B (normative) Breeding . 10 Annex C (informative) Example of a statistical method for processing results 11 Biblio
12、graphy . 14 BS ISO 21427-1:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body int
13、erested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnic
14、al Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the
15、technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights.
16、ISO shall not be held responsible for identifying any or all such patent rights. ISO 21427-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. ISO 21427 consists of the following parts, under the general title Water quality Evaluation of genotoxici
17、ty by measurement of the induction of micronuclei: Part 1: Evaluation of genotoxicity using amphibian larvae Part 2: Mixed population method using the cell line V79 BS ISO 21427-1:2006vIntroduction Environmental protection calls for taking into account the genotoxic hazards likely to concern the dif
18、ferent populations making up ecosystems. Since such hazards may emerge within waters, it is essential to assess them by means of laboratory tests that allow the evaluation, within aqueous environments, of the genotoxicity of a water, effluent, substance or preparation with respect to organisms livin
19、g in aquatic environments. This part of ISO 21427 describes a test method that is likely to provide information in this field. It allows the highlighting, within aqueous environments, of the genotoxic effects on larvae of the two amphibian species, Xenopus laevis and Pleurodeles waltl. The choice of
20、 the Xenopus is recommended because of several advantages offered by this species: high hatching rates, available all year round after hormone treatment of the breeders, rapid larval development, easy feeding of larvae, widespread distribution within research or breeding centres. However, the descri
21、bed method is, basically, also applicable to Pleurodeles larvae. The provisions specific to Pleurodeles are described in Annex A. BS ISO 21427-1:2006blank1Water quality Evaluation of genotoxicity by measurement of the induction of micronuclei Part 1: Evaluation of genotoxicity using amphibian larvae
22、 WARNING Persons using this part of ISO 21427 should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health pract
23、ices and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted according to this part of ISO 21427 be carried out by suitably trained staff. 1 Scope This part of ISO 21427 specifies a method for assessing genotoxicity using Amphibia l
24、arvae (Xenopus laevis and Pleurodeles waltl). The provisions specific to Pleurodeles are described in Annex A. The method is applicable to: aqueous effluents; aqueous leachates; eluates of soils; eluates of industrial waste; eluates of sludges from sewage treatment; surface and ground water; water-s
25、oluble substances. 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 (including any amendments) applies. ISO 566
26、7-16, Water quality Sampling Part 16: Guidance on biotesting of samples ISO 7346-1, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 1: Static method BS ISO 21427-1:20062 ISO 7346-2, Water qua
27、lity Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 2: Semi-static method ISO 7346-3, Water quality Determination of the acute lethal toxicity of substances to a freshwater fish Brachydanio rerio Hamilton
28、-Buchanan (Teleostei, Cyprinidae) Part 3: Flow-through method EN 12457-2, Characterization of waste Leaching Compliance test for leaching of granular waste materials and sludges Part 2: One stage batch test at a liquid to solid ratio of 10 l/kg with particle size below 4 mm (without or with size red
29、uction) NF T 90-305, Testing water Determination of the acute toxicity of a substance to Salmo gairdneri Static and flow through methods 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 micronuclei small particles consisting of acentric fragme
30、nts of chromosomes and/or entire chromosomes which lag behind at anaphase stage of cell division and form, after telophase, single or multiple micronuclei in the cytoplasm 3.2 sample under test substance, effluent, surface water, ground water, aqueous effluent, leachate, percolate or eluate submitte
31、d to testing 3.3 test medium mixture of test water (6.2), the sample under test and food 3.4 test solution mixture of test water (6.2) and the sample under test 4 Principle The test organisms are exposed during 12 d 3to a range of concentrations of the sample under test. A control test without a sam
32、ple (negative control) and a control test with a genotoxic reference substance (positive control) are carried out in parallel under the same conditions. The positive control allows the quality of the biological reagent to be checked and the test to be validated. During cell division, chromatid fragm
33、ents without centromers do not move to the nuclei of the daughter cells and stay within the cytoplasma. Some of the chromosomal aberrations induced by the test material are due to chromatid fragments without centromer and are therefore not incorporated in the nuclei of the daughter cells. In additio
34、n, spindle disorders may lead to chromosomes which are not incorporated into the nucleus. They are not nuclei and they are formed in the cell cytoplasm not in the plasma. The rate (per thousand ) of erythrocytes with micronuclei is determined for each concentration of the sample under test and for t
35、he control solutions. The rate of erythrocytes with micronuclei for each concentration is compared to that of the negative control in order to determine the concentrations that induce a positive genotoxic effect. NOTE The rate of erythrocytes with micronuclei is defined as the level of erythrocytes
36、including one or more micronuclei scored in a sample of 1 000 erythrocytes observed in one blood smear. BS ISO 21427-1:200635 Test environment All of the tests and handling operations as well as the breeding of the adults and larvae shall be performed in a room in which the atmosphere is free from t
37、oxic dusts and vapours. The test is conducted under lighting (artificial light or natural light without direct sunshine) according to a 12 h light/12 h dark cycle, in a thermostatically-controlled chamber (7.3) so as to maintain the bottles at a temperature of 22 C 1 C. 6 Reagents 6.1 Biological rea
38、gent, Xenopus laevis1)designated hereafter by the common name Xenopus. The tests shall start on larvae at stage 50 of the Nieuwkoop and Faber 1chronological table of development. At this stage, the animals measure 20 mm to 27 mm in length and present a constriction at the base of the hind leg. The l
39、arvae shall be kept in vessels (7.2) for a period of at least 8 d before the start of the test under temperature and lighting conditions identical to those of the test. The larvae shall be free of diseases and malformations. For a given test, the batches of treated larvae and control larvae shall st
40、em from the same hatching process. Each batch is made up of at least 15 test vessels (7.2). 6.2 Test water. The water used for the test shall meet the criteria described in the first paragraph of Annex B. If water other than the breeding water is used, the organisms shall be maintained in this water
41、 during at least 8 d before starting the test. 6.3 Food. The food employed is that usually intended for aquarium fish2). It shall be ground to a powder just prior to use. Freeze-dried watercress powder may also be used. 6.4 Reference substance, Cyclophosphamide monohydrate (C7H15Cl2N2O2P,H2O), of re
42、cognized analytical quality, at a concentration of 20 mg/l. 6.5 Intermediate solvent, dimethyl sulfoxide (DMSO) or any other appropriate water-miscible solvent. The genotoxicity and general toxicity of the solvent being used shall be established beforehand. 6.6 Anaesthetic, tricaine methane sulfonat
43、e. 6.7 Heparin, 200 g/l solution of powdered heparin in a 7 g/l aqueous solution of sodium chloride. 6.8 Methanol, CH3OH, of recognized analytical quality. 1) Xenopus can be obtained from the Service dlevage de Xnopes du C.N.R.S., UPRES A 6026 C.N.R.S., Universit de Rennes I, Avenue du Gnral Leclerc
44、, 35042 Rennes Cedex, France. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product. 2) Tetraphyll is an example of a suitable product available commercially. This information is given for the convenience of users of thi
45、s document and does not constitute an endorsement by ISO of this product. BS ISO 21427-1:20064 6.9 Stains166.9.1 Massons haemalum. Composition Haematin 2 mg/ml Potassium alum 10 % 6.9.2 Groats haematoxylin. Composition Ammonium ferric sulfate (Iron alum) 1,0 g Haematoxylin 0,5 g Sulfuric acid concen
46、trated 0,8 ml Ethanol 50 mlWater ml7 Apparatus Ordinary laboratory apparatus in glass or chemically inert material and in particular the following. 7.1 Aquariums, 25 l or 50 l capacity. 7.2 Glass containers, e.g. bottles, 5 l capacity, capable of being hermetically closed. Conical flasks with ground
47、-glass stoppers or rubber bungs covered with a tetrafluorocarbon film are suitable. 7.3 Thermostatically-controlled chamber, of sufficient capacity to hold all the bottles corresponding to the different batches of one complete test test sample, reference substance and negative control and able to ma
48、intain the bottles (7.2) at a temperature of 22 C 1 C. 7.4 Binocular magnifying glass. 7.5 Microscope, fitted with an immersion lens (total magnification 1 500). 7.6 Heparinized micropipettes. NOTE To be heparinized, the micropipettes are stretched and opened, then the sharp size of the micropipette
49、 is filled with a 200 g/ml solution of heparin in a 7 g/l NaCl solution. 8 Treatment and preparation of samples 8.1 Waters, effluents, leachates and eluates to be tested It is recommended to carry out the sampling, transportation and storage of the samples in accordance with the general procedures as specified in ISO 5667-16. The interval between the collection of the sample and its receipt by the laboratory shall not exceed 24 h. BS ISO
