1、BRITISH STANDARDBS EN ISO 20079:2006Incorporating amendment no. 1 (renumbers BS ISO 20079:2005 as BS EN ISO 20079:2006) BS 6068-5.39: 2005Water quality Determination of the toxic effect of water constituents and waste water on duckweed (Lemna minor) Duckweed growth inhibition testThe European Standa
2、rd EN ISO 20079:2006 has the status of a British StandardICS 13.060.70g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 20079:2006This Briti
3、sh Standard was published under the authority of the Standards Policy and Strategy Committee on 23 January 2006 BSI 2007ISBN 0 580 49761 5National forewordThis British Standard was published by BSI. It is the UK implementation of EN ISO 20079:2006. It is identical with ISO 20079:2005. The UK partici
4、pation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological Methods.A list of organizations represented on EH/3/5 can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a cont
5、ract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments issued since publicationAmd. No. Date Comments17054 30 March 2007 Renumbers BS ISO 20079:2005 as BS EN ISO 20079:2006EUROPEAN STANDARDNORME EUROPENNEEUR
6、OPISCHE NORMEN ISO 20079September 2006ICS 13.060.70English VersionWater quality - Determination of the toxic effect of waterconstituents and waste water on duckweed (Lemna minor) -Duckweed growth inhibition test (ISO 20079:2005)Qualit de leau - Dtermination de leffet toxique desconstituants de leau
7、et des eaux rsiduaires vis-vis deslentilles deau (Lemna minor) - Essai dinhibition de lacroissance des lentilles deau (ISO 20079:2005)Wasserbeschaffenheit - Bestimmung der toxischenWirkung von Wasserinhaltsstoffen und Abwassergegenber Wasserlinsen (kursiv(Lemna minor) -Wasserlinsen-Wachstumshemmtest
8、 (ISO 20079:2005)This European Standard was approved by CEN on 17 August 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibli
9、ographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a
10、CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
11、 Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 200
12、6 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 20079:2006: EForeword The text of ISO 20079:2005 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) an
13、d has been taken over as EN ISO 20079:2006 by Technical Committee CEN/TC 230 “Water analysis“, 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 March 2007,
14、and conflicting national standards shall be withdrawn at the latest by March 2007. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Esto
15、nia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 20079:2005 has been approved by CEN as
16、EN ISO 20079:2006 without any modifications. EN ISO 20079:2006Reference numberISO 20079:2005(E)INTERNATIONAL STANDARD ISO20079First edition2005-11-01Water quality Determination of the toxic effect of water constituents and waste water on duckweed (Lemna minor) Duckweed growth inhibition test Qualit
17、de leau Dtermination de leffet toxique des constituants de leau et des eaux rsiduaires vis-vis des lentilles deau (Lemna minor) Essai dinhibition de la croissance des lentilles deau EN ISO 20079:2006ii iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Terms and def
18、initions. 1 4 Principle. 3 5 Interferences . 4 6 Apparatus 4 7 Reagents 4 8 Test organisms . 7 9 Stock cultures and pre-cultures 7 10 Procedure 8 11 Validity criteria 10 12 Expression of results . 10 13 Estimation of EC(r)xvalues for frond number and the second observation parameter . 12 14 Document
19、ation of results 12 15 Precision 12 16 Test report . 13 Annex A (informative) Preparation of the nutrient media 14 Annex B (informative) Measurement of the lowest ineffective dilution (LID) of a waste water A simplified evaluation for testing of waste water 19 Annex C (informative) Suppliers of Lemn
20、a species . 22 Bibliography . 23 EN ISO 20079:2006iv 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. E
21、ach 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, in liaison with ISO, also take part in the work. ISO collaborates closely with the Internati
22、onal Electrotechnical 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 Standa
23、rds adopted 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
24、 of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 20079 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. EN ISO 20079:2006vIntroduction The duckweed species Lemna minor is used as model organis
25、m for higher water plants. Duckweeds are monocotyledonous, free-floating angiosperms and belong to the Arales within the subclass of Aridae. Duckweeds are fast growing higher plants, spreading from the tropic to the arctic zone. As primary producers they are a food source for waterfowl, fish and sma
26、ll animals and serve as physical support for a variety of small invertebrates. Duckweed can be damaged by water constituents and effluents (see Annex B). The subsequent inhibition of growth is calculated from the observation parameters (frond number, frond area, chlorophyll, dry weight) by a number
27、of defined calculation methods. EC values are determined to allow for an assessment of toxic effects of water constituents (e.g. chemicals, plant protection products). The evaluation for at least two observation parameters is based on the average specific growth-rates. The test is designed for measu
28、rement of response of substances dissolved in water. This includes the definition of a fixed dilution step, or a concentration of the test sample at which a parameter of observation (endpoint) is inhibited relative to a control for a defined percentage. EN ISO 20079:2006blank1Water quality Determina
29、tion of the toxic effect of water constituents and waste water on duckweed (Lemna minor) Duckweed growth inhibition test WARNING Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any,
30、 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 conducted according to this International Standard be carried out by s
31、uitably trained staff. 1 Scope This International Standard specifies a method for the determination of the growth-inhibiting response of duckweed (Lemna minor) to substances and mixtures contained in water, treated municipal wastewater and industrial effluents. 2 Normative references The following r
32、eferenced 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 5667-16, Water quality Sampling Part 16: Guidance on biotest
33、ing of samples ISO 10260, Water quality Measurement of biochemical parameters Spectrometric determination of the chlorophyll-a concentration 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 axenic cultures monocultures of organisms from a sing
34、le species, free from fungi, algae and other macrophyte species 3.2 calculation parameters parameters for the estimation of toxicity derived from any parameters of observation by different methods of calculation EXAMPLE Growth-rates derived from frond number, frond area, chlorophyll and dry weight a
35、re calculation parameters in this International Standard. EN ISO 20079:20062 3.3 chlorosis loss of pigment (yellowing of frond tissue) 3.4 colony aggregate of mother and daughter fronds, attached to each other, sometimes referred to as a plant 3.5 control batch control medium, including organisms us
36、ed for testing 3.6 control medium combination of dilution water and/or nutrient medium used in the test 3.7 dilution water water added to the test sample to prepare a series of defined dilutions 3.8 doubling time quotient of natural logarithm of 2 (ln 2) divided by average specific growth-rate 3.9 e
37、ffective concentration concentration of the test sample (ECx) at which an effect of x % is measured, if compared to the control NOTE To unambiguously denote an EC value deriving from growth-rate, it is proposed to use the symbol “EC(r)”, followed by the observation parameter used, e.g. EC(r) (frond
38、number). 3.10 frond individual leaf-like structure on a duckweed colony; the smallest unit (i.e. individual), capable of reproducing 3.11 frond area total area of all fronds visible from vertically above 3.12 frond number all fronds protruding from a mother frond which are directly visible from abov
39、e without magnification 3.13 growth increase in biomass over time as the result of proliferation of new tissues NOTE In this test it refers to any parameter of observation. 3.14 growth-rate calculation parameter defined as quotient of the difference of the natural logarithms of a parameter of observ
40、ation and the respective time period NOTE If the time period comprises the total duration of the test, the term is referred to as average specific growth-rate. If the period between two measurements within the test period is used, the term is named segmented growth-rate (see 12.1.2). EN ISO 20079:20
41、0633.15 inoculum number of fronds (colonies) added to the test batch at the beginning of the test 3.16 necrosis localised dead frond tissue (i.e. brown or white) 3.17 nutrient medium solution of nutrients and micronutrients in water which are essential for the growth of duckweed 3.18 observation par
42、ameters observed or measured biomass parameters like frond number, frond area, chlorophyll, dry weight, which are measured or counted once or repeatedly by observation or measurement NOTE These parameters are relevant for the assessment of growth and vitality of the test organisms (e.g. frond number
43、, frond area or chlorophyll content, dry weight). 3.19 pre-culture culture of duckweed used for acclimatisation of test plants to the test conditions and for the growing of the plants to be used in the inoculum 3.20 root that part of the Lemna plant that assumes a root-like structure 3.21 stock cult
44、ure culture of a single species of duckweed to conserve the original defined Lemna species in the laboratory and to provide inoculum for the pre-culture NOTE It is necessary to use defined and verified strains, because of possible insecurities in species taxonomy. An address list of suppliers is giv
45、en in Annex C. 3.22 test batch test medium including organisms used for testing 3.23 test medium combination of test sample, dilution water and/or nutrient medium used in the test 3.24 test sample discrete portion of a sample (taken from i.e. receiving water, waste water, dissolved chemical substanc
46、es or mixtures, products and compounds) pretreated according to the needs of this test (e.g. dissolution, filtering, neutralisation) 4 Principle Plants of the species Lemna minor are allowed to grow as monocultures in different concentrations of the test sample over a period of seven days. The objec
47、tive of the test is to quantify substance-related effects on vegetative growth over this period based on assessments of frond number, and also on assessments on biomass (total frond area, dry weight or chlorophyll). To quantify substance-related effects, the growth-rate in the test solutions is comp
48、ared with that of the controls and the concentration resulting in a specified x % inhibition of growth-rate is determined and expressed as the EC(r)x. EN ISO 20079:20064 5 Interferences Non-soluble, poorly soluble, volatile, bio- or photodegradable substances or substances reacting with the dilution
49、 water or the nutrient medium or changing their state during the test, may falsify or reduce the reproducibility of the results (see ISO 5667-16).Special consideration is necessary in the case of substances accumulated at the water surface as this may increase the effects on duckweed. 6 Apparatus The test design determines the requirements for the apparatus. 6.1 Cylindrical vessels, (glass beakers, crystallising dishes, Petri dishes). Minimum volume of 150 ml (for 2/3 of total volume, i.e. 100 ml of test solution). 6.2 Uniform glas
