1、BS EN ISO 10253:2016Water quality Marinealgal growth inhibition testwith Skeletonema sp. andPhaeodactylum tricornutum(ISO 10253:2016)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 10253:2016 BRITISH STANDARDNational forewordThis British Standard is the UK
2、implementation of EN ISO 10253:2016. It supersedes BS EN ISO 10253:2006 which iswithdrawn.The UK participation in its preparation was entrusted to Technical Committee EH/3/5, Biological Methods.A list of organizations represented on this committee can be obtained on request to its secretary.This pub
3、lication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 87012 5 ICS 13.060.70 Compliance with a British Standard cannot confer immu
4、nity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 December 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 10253 November 201
5、6 ICS 13.060.70 Supersedes EN ISO 10253:2006English Version Water quality - Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum (ISO 10253:2016) Qualit de leau - Essai dinhibition de la croissance des algues marines avec Skeletonema sp. et Phaeodactylum tricornutum
6、 (ISO 10253:2016) Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema sp. und Phaeodactylum tricornutum (ISO 10253:2016) This European Standard was approved by CEN on 22 October 2016. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the con
7、ditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exis
8、ts in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national sta
9、ndards 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, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, S
10、lovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reser
11、ved worldwide for CEN national Members. Ref. No. EN ISO 10253:2016 EBS EN ISO 10253:2016EN ISO 10253:2016 (E) 3 European foreword This document (EN ISO 10253:2016) has been prepared by Technical Committee ISO/TC 147 “Water quality” in collaboration with Technical Committee CEN/TC 230 “Water analysis
12、” the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2017, and conflicting national standards shall be withdrawn at the latest by May 2017. Attention is d
13、rawn to the possibility that some of 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. This document supersedes EN ISO 10253:2006. According to the CEN-CENELEC Internal Regulations, the n
14、ational standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
15、Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 10253:2016 has been approved by CEN as EN ISO 10253:2016 without any modification. BS EN ISO 10253:2016ISO 1
16、0253:2016(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Materials . 35.1 Test organisms 35.2 Reagents 46 Apparatus . 57 Procedure. 67.1 Preparation of growth medium 67.2 Preparation of pre-culture and inoculum 67.3 Choice of test concentrations . 67.4 Prep
17、aration of test substance stock solutions . 67.5 Preparation of test and control batches 77.6 Incubation . 77.7 Measurements . 78 Validity criteria 89 Interpretation of data 89.1 Plotting growth curves . 89.2 Calculation of percentage inhibition 89.3 Determination of EC(r)x.910 Expression of results
18、 911 Interpretation of results . 912 Test report . 9Annex A (informative) Preparation of dilution series of mixtures in sea water(effluents or elutriates) 11Annex B (informative) Test procedure starting from stored algal inocula, and with direct measurement of algal growth in spectrophotometric cell
19、s 12Annex C (informative) Performance data .18Bibliography .19 ISO 2016 All rights reserved iiiContents PageBS EN ISO 10253:2016ISO 10253:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of pr
20、eparing International Standards is normally carried out through ISO technical committees. Each member body interested 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, i
21、n liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IE
22、C Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility t
23、hat some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent
24、declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as informati
25、on about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.This third edition ca
26、ncels and replaces the second edition (ISO 10253:2006), which has been technically revised.iv ISO 2016 All rights reservedBS EN ISO 10253:2016INTERNATIONAL STANDARD ISO 10253:2016(E)Water quality Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutumWARNING Persons us
27、ing this document should be familiar with normal laboratory practice. This document 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 practices and to ensure compliance with any nat
28、ional regulatory conditions.IMPORTANT It is absolutely essential that tests conducted according to this document be carried out by suitably trained staff.1 ScopeThis document specifies a method for the determination of the inhibition of growth of the unicellular marine algae Skeletonema sp. and Phae
29、odactylum tricornutum by substances and mixtures contained in sea water or by environmental water samples (effluents, elutriates, etc.).The method can be used for testing substances that are readily soluble in water and are not significantly degraded or eliminated in any other way from the test medi
30、um.NOTE With modifications, as described in ISO 14442 and ISO 566716, the inhibitory effects of poorly soluble organic and inorganic materials, volatile compounds, metal compounds, effluents, marine water samples and elutriates of sediments can be tested.2 Normative referencesThe following documents
31、 are referred to in the text in such a way that some or all of their content constitutes requirements 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 5667-16, Water qu
32、ality Sampling Part 16: Guidance on biotesting of samplesISO 14442, Water quality Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.
33、ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp3.1cell densitynumber of cells per unit volume of mediumNote 1 to entry: The
34、 cell density is expressed as x cells/ml. ISO 2016 All rights reserved 1BS EN ISO 10253:2016ISO 10253:2016(E)3.2specific growth rateproportional rate of increase in cell density per unit of time: =11xxtddday(/ )wherex is the cell density, expressed in cells per millilitre;t is the time, expressed in
35、 days.Note 1 to entry: Specific growth rate is expressed in inverse days (day1).3.3growth mediummixture of sea water and nutrients which is used for pre-cultures and controls3.4test mediummixture of sea water, nutrients growth medium (3.3) and test material in which algal cells are incubated3.5test
36、batchmixture of sea water, nutrients and test material test medium (3.4) inoculated with algae3.6controlmixture of sea water, nutrients growth medium (3.3) without test material, inoculated with algae3.7effective concentrationEC(r)xconcentration of test substance which results in an x % reduction in
37、 specific growth rate relative to the controls4 PrincipleMono-specific algal strains are cultured for several generations in a defined medium containing a range of concentrations of the test substance, prepared by mixing appropriate quantities of nutrient concentrate, sea water, stock solutions of t
38、he test substance, and an inoculum of exponentially growing algal cells. The test solutions are incubated for a period of (72 2) h, during which the cell density in each is measured at intervals of at least every (24 2) h. Inhibition is measured as a reduction in specific growth rate, relative to co
39、ntrol cultures grown under identical conditions.2 ISO 2016 All rights reservedBS EN ISO 10253:2016ISO 10253:2016(E)5 Materials5.1 Test organismsUse either of the following marine algae:a) Skeletonema sp.1)(CCAP 1077/1C, NIVA BAC 1); orb) Phaeodactylum tricornutum Bohlin (CCAP 1052/1A, SAG 1090-1a, N
40、IVA BAC 2).These algae are important and widely distributed phytoplankton species (phylum Bacillariophyta) in estuarine and coastal areas.The recommended algae are available in unialgal, non-axenic cultures from the following sources.NIVANorwegian Institute for Water ResearchGaustadalen 21N 0349 Osl
41、oNorwayCCAPDunstaffnage Marine LaboratoryP O Box 3 ObanArgyll PA37 1QAUnited KingdomSAGCollection of Algal CulturesUniversity of GttingenAlbrecht-von-Haller Institute for Plant ScienceUntere Karsple 237073 GttingenGermanyStock cultures may be maintained in the medium described in 7.1. Regular subcul
42、turing is necessary. Weekly intervals may be necessary for Skeletonema sp., every two or three weeks may be sufficient for Phaeodactylum tricornutum. The stock cultures may also be maintained for extended periods on richer algal media such as those recommended by the culture collection. It is recomm
43、ended to keep the 1) The previous editions of this document suggested the use of two strains of Skeletonema costatum. Following ataxonomic review of the Skeletonema genus, several strains originally identified as S. costatum may in fact be other species. In light of this and to enable continuity in
44、the use of previously accepted strains, the present revision of this document has changed the reference from Skeletonema costatum to Skeletonema sp. to avoid non-compliance for labs that may be using different strains. ISO 2016 All rights reserved 3BS EN ISO 10253:2016ISO 10253:2016(E)stock culture
45、in the medium described in 7.1 and in an exponential growth phase immediately before preparing the pre-culture for testing as described in 7.2.NOTE Concentrated cultures of the diatom Phaeodactylum tricornutum can also be stored for several months without losing their viability. Stock cultures for t
46、he toxicity tests can easily be prepared from the stored concentrated cultures2).5.2 Reagents5.2.1 WaterAll water used in the preparation of the synthetic sea water, growth medium and test substance solutions shall be deionized or of equivalent purity. Take special care to avoid contamination of the
47、 water by inorganic or organic substances during preparation and storage. Equipment made of copper shall not be used.5.2.2 Sea waterFor culturing and testing Phaeodactylum tricornutum, the growth medium (7.1) is made up by adding nutrients to either natural salinity = (30 5) g/kg or synthetic sea wa
48、ter (approximate salinity = 33 g/kg). For Skeletonema sp., the use of natural sea water may be necessary for the long-term maintenance of cultures and may also be necessary for the test medium, because a synthetic sea water medium may not always support sufficient growth to meet the test quality cri
49、teria. If natural sea water is used, care shall be taken to ensure that it is not polluted.Prepare synthetic sea water with the composition given in Table 1 (approximate salinity = 33 g/kg). All the chemicals used shall be of analytical grade.Table 1 Synthetic sea waterSaltConcentration of salt in synthetic sea waterg/lNaCl 22MgCl26H2O 9,7Na2SO4(anhydrous) 3,7CaCl2(anhydrous) 1,0KCl 0,65NaHCO30,20H3BO30,023Filter the sea water (synthetic as well as natural one) through a 0,45 m membrane filter in order to remove par
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