1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 8692:2012Water quality Fresh wateralgal growth inhibition testwith unicellular green algae(ISO 8692:2012)BS EN ISO 8692:2012 BRITISH STANDARDNational forewordThis Briti
2、sh Standard is the UK implementation of EN ISO 8692:2012. It supersedes BS EN ISO 8692:2004, which is withdrawn.The UK participation in its preparation was entrusted to T e c h n i c a l C o m m i t t e e E H / 3 / 5 , B i o l o g i c a l M e t h o d s .A list of organizations represented on this co
3、mmittee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012Published by BSI Standards Limited 2012ISBN 978 0 580 68532 3 ICS 13.0
4、60.70 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 March 2012.Amendments issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPE
5、NNE EUROPISCHE NORM EN ISO 8692 February 2012 ICS 13.060.70 Supersedes EN ISO 8692:2004English Version Water quality - Fresh water algal growth inhibition test with unicellular green algae (ISO 8692:2012) Qualit de leau - Essai dinhibition de la croissance des algues deau douce avec des algues verte
6、s unicellulaires (ISO 8692:2012) Wasserbeschaffenheit - Swasseralgen-Wachstumshemmtest mit einzelligen Grnalgen (ISO 8692:2012) This European Standard was approved by CEN on 14 February 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for
7、 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 exists in three
8、 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 standards bodi
9、es of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and Un
10、ited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 8692:2
11、012: EBS EN ISO 8692:2012 EN ISO 8692:2012 (E) 3 Foreword This document (EN ISO 8692:2012) has been prepared by Technical Committee ISO/TC 147 “Water quality” in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard shall b
12、e given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2012, and conflicting national standards shall be withdrawn at the latest by August 2012. Attention is drawn to the possibility that some of the elements of this document
13、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 8692:2004. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound
14、 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, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sw
15、eden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 8692:2012 has been approved by CEN as a EN ISO 8692:2012 without any modification. BS EN ISO 8692:2012ISO 8692:2012(E) ISO 2012 All rights reserved iiiContents PageForeword iv1 Scope 12 Normative references . 13 Ter
16、ms and definitions . 14 Principle . 25 Reagents and media . 26 Apparatus 47 Procedure 57.1 Preparation of growth medium 57.2 Preparation of preculture and inoculum 57.3 Choice of test sample concentrations . 57.4 Preparation of test sample and stock solutions . 57.5 Preparation of test and control b
17、atches . 67.6 Incubation 67.7 Measurements 78 Validity criteria . 79 Calculation 79.1 Plotting of growth curves 79.2 Calculation of percentage inhibition 89.3 Determination of ErCx(e.g. ErC10and ErC50) . 810 Expression of results . 811 Interpretation of results . 912 Precision 913 Test report . 9Ann
18、ex A (informative) Rapid screening of waste water algal growth inhibition 11Annex B (informative) Test procedure with algae from algal beads, with direct measurement of algal growth in spectrophotometric cells .14Annex C (informative) Procedure for immobilization of algae in alginate beads 19Bibliog
19、raphy .21BS EN ISO 8692:2012ISO 8692:2012(E)ForewordISO (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
20、 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, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Elect
21、rotechnical 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
22、by the 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 ri
23、ghts. ISO shall not be held responsible for identifying any or all such patent rights.ISO 8692 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.This third edition cancels and replaces the second edition (ISO 8692:2004), which has been technically r
24、evised.iv ISO 2012 All rights reservedBS EN ISO 8692:2012INTERNATIONAL STANDARD ISO 8692:2012(E)Water quality Fresh water algal growth inhibition test with unicellular green algaeWARNING Persons using this International Standard should be familiar with normal laboratory practice. This International
25、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 practices and to ensure compliance with any national regulatory conditions.IMPORTANT It is absolutely essential that tests
26、 conducted in accordance with this International Standard be carried out by suitably qualified staff.1 ScopeThis International Standard specifies a method for the determination of the growth inhibition of unicellular green algae by substances and mixtures contained in water or by waste water. This m
27、ethod is applicable for substances that are easily soluble in water.With modifications to this method, as specified in ISO 14442 and ISO 5667-16, the inhibitory effects of poorly soluble organic and inorganic materials, volatile compounds, heavy metals and waste water can be tested.A rapid algal gro
28、wth inhibition screening test for waste water is described in Annex A.An alternative test procedure with algae from algal beads, with direct measurement of algal growth in spectrophotometric cells, is described in Annex B.2 Normative referencesThe following documents, in whole or in part, are normat
29、ively referenced in this document and are indispensable for its application. 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 quality Sampling Part 16: Guidance on biotest
30、ing of samplesISO/TR 11044, Water quality Scientific and technical aspects of batch algae growth inhibition testsISO 14442, Water quality Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste waterISO/TS 20281, Water quality Guidance on stat
31、istical interpretation of ecotoxicity data3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1cell densitynnumber of cells per volume of mediumNOTE Cell density is expressed in cells per millilitre. ISO 2012 All rights reserved 1BS EN ISO 8692:2012I
32、SO 8692:2012(E)3.2effective concentrationconcentration of the test sample (ECx) at which an effect of x % is measured, if compared to the controlNOTE To unambiguously denote an EC value deriving from growth rate, it is proposed to use the symbol “ErC”.3.3lowest ineffective dilutionLIDdilution level
33、at which no inhibition, or only effects not exceeding the test-specific variability, are observedNOTE Adapted from ISO 15088:200713, 3.5.3.4specific growth ratemproportional rate of increase in cell density per time: =1nntddwheren is the cell density, expressed in cells per millilitre;t is the time,
34、 expressed in days.NOTE Specific growth rate is expressed in reciprocal days (day1).ISO/TR 11044:2008, 3.24 PrincipleMonospecies algal strains are cultured for several generations in a defined medium containing a range of concentrations of the test sample, prepared by mixing appropriate quantities o
35、f growth medium, test sample, and an inoculum of exponentially growing algal cells. The test batches are incubated for a period of (72 2) h during which the cell density in each test solution is measured at least every 24 h.Inhibition is measured as a reduction in specific growth rate relative to co
36、ntrol cultures grown under identical conditions.5 Reagents and media5.1 Test organism, using either of the following planktonic fresh water algae species:a) Desmodesmus subspicatus (R. Chodat) E. Hegewald et A. Schmidt1)(86.81 SAG2);b) Pseudokirchneriella subcapitata (Korshikov) Hindak3)(ATCC 22662T
37、M,2)CCAP 278/42)or 61.81 SAG2).NOTE 1 The two species do not show identical responses to toxic agents.NOTE 2 Both algae species are planktonic green algae belonging to the order of Sphaeropleales (Chlorophyta, Chlorophyceae) and are usually unicellular in culture.1) This species is formerly known as
38、 Scenedesmus subspicatus Chodat.2) Trade names of strains are examples of suitable strains available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of these products.3) This species is formerly known as Selenastrum
39、capricornutum Prinz. The new name is currently cited by culture collections.2 ISO 2012 All rights reservedBS EN ISO 8692:2012ISO 8692:2012(E)The strains recommended are available in unialgal, non-axenic cultures from the following collections: SAG Sammlung von Algenkulturen Gttingen Gttingen Algal C
40、ulture Collection, Germanywww.epsag.uni-goettingen.de (viewed 2012-01-30); ATCC American Type Culture Collection, USAwww.atcc.org (viewed 2012-01-30); CCAP Culture Collection of Algae and Protozoa, UKwww.ccap.ac.uk (viewed 2012-01-30); ALCP Algothque du Laboratoire de Cryptogamie, Francewww.mnhn.fr
41、(viewed 2012-01-30).Stock cultures can be maintained in the medium specified in 5.3. and 7.1. However, frequent subculturing is necessary (once a week) to prevent failure of growth. The stock culture can be maintained for extended periods on richer algal media such as those recommended by the cultur
42、e collection.Alternatively, algae can be stored for several months on agar plates or in alginate beads4)without losing their viability1. The algae can be easily recovered from the agar or liberated from the algal beads (see Annex C) when needed to perform the toxicity tests.The appearance of the cel
43、ls and the identity of the test organisms should be confirmed by microscopy.5.2 Water, deionized or of equivalent purity (conductivity 10 S/cm), for use in the preparation of the growth medium and test substance solutions.Take special care to avoid contamination of the water by inorganic or organic
44、substances during preparation and storage. Do not use equipment made of copper.5.3 NutrientsPrepare four nutrient stock solutions in water, according to the compositions given in Table 1.These solutions are eventually diluted (see 7.1 and 7.4) to achieve the final nutrient concentrations in the test
45、 solutions. However, the macronutrients may instead be added directly to the water.All chemicals used shall be of reagent-grade quality.Sterilize the stock solutions by membrane filtration (mean pore diameter 0,2 m) or by autoclaving (120 2) C, 15 min. Store the solutions in the dark at 4 C.Do not a
46、utoclave stock solution 4 in order to avoid evaporative loss of NaHCO3, but sterilize it by membrane filtration.4) The algal beads supplied by MicroBioTests Inc., Mariakerke-Gent, Belgium are an example of a suitable product available commercially. This information is given for the convenience of us
47、ers of this document and does not constitute an endorsement by ISO of this product. Equivalent products may be used if the validity criteria specified in this document are fulfilled. ISO 2012 All rights reserved 3BS EN ISO 8692:2012ISO 8692:2012(E)Table 1 Mass concentrations of nutrients in the test
48、 solutionStock solution NutrientMass concentration in stock solutionFinal mass concentration in test solution1: MacronutrientsNH4ClMgCl26H2OCaCl22H2OMgSO47H2OKH2PO41,5 g/l1,2 g/l1,8 g/l1,5 g/l0,16 g/l15 mg/l (N: 3,9 mg/l)12 mg/l (Mg: 2,9 mg/l)18 mg/l (Ca: 4,9 mg/l15 mg/l (S: 1,95 mg/l)1,6 mg/l (P: 0
49、,36 mg/l)2: Fe-EDTAFeCl36H2ONa2EDTA2H2O64 mg/l100 mg/l64 g/l (Fe: 13 g/l)100 g/l3: Trace elementsH3BO3aMnCl24H2OZnCl2CoCl26H2OCuCl22H2ONa2MoO42H2O185 mg/l415 mg/l3 mg/l1,5 mg/l0,01 mg/l7 mg/l185 g/l (B: 32 g/l)415 g/l (Mn: 115 g/l)3 g/l (Zn: 1,4 g/l)1,5 g/l (Co: 0,37 g/l)0,01 g/l (Cu: 3,7 ng/l)7 g/l (Mo: 2,8 g/l)4: NaHCO3NaHCO350 g/l 50 mg/l (C: 7,14 mg/l)aH3BO3can be dissolved by the addition of 0,1 mol/l NaOH.6 ApparatusAll equipment that comes in contact with the test medium
