EN 29408:1993
(Main)Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method of determining the oxygen demand in a closed respirometer (ISO 9408:1991)
Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method of determining the oxygen demand in a closed respirometer (ISO 9408:1991)
Wasserbeschaffenheit - Bestimmung der vollständigen aerobiologischen Abbaubarkeit eines organischen Stoffes in einem wäßrigen Medium über die Bestimmung des Sauerstoffbedarfs in einem geschlossenen Respirometer (ISO 9408:1991)
Qualité de l'eau - Evaluation, en milieu aqueux, de la biodégradabilité aérobie "ultime" des composés organiques - Méthode par détermination de la demande en oxygène dans un respiromètre fermé (ISO 9408:1991)
Kakovost vode - Vrednotenje popolne aerobne biološke razgradljivosti organskih snovi v vodnem okolju - Metoda določanja potrebe po kisiku v zaprtem raspirometru (ISO 9408:1991)
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.WHPWasserbeschaffenheit - Bestimmung der vollständigen aerobiologischen Abbaubarkeit eines organischen Stoffes in einem wäßrigen Medium über die Bestimmung des Sauerstoffbedarfs in einem geschlossenen Respirometer (ISO 9408:1991)Qualité de l'eau - Evaluation, en milieu aqueux, de la biodégradabilité aérobie "ultime" des composés organiques - Méthode par détermination de la demande en oxygene dans un respirometre fermé (ISO 9408:1991)Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method of determining the oxygen demand in a closed respirometer (ISO 9408:1991)13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of waterICS:Ta slovenski standard je istoveten z:EN 29408:1993SIST EN 29408:1998en01-januar-1998SIST EN 29408:1998SLOVENSKI
STANDARDSIST ISO 9408:1997/C1:1997SIST ISO 9408:19971DGRPHãþD
SIST EN 29408:1998
SIST EN 29408:1998
SIST EN 29408:1998
INTERNATIONAL STANDARD ISO 9408 First edition 1991-02-15 Water quality - Evaluation in an aqueous medium of the “ultimate” aerobic biodegradability of organic compounds - Method by determining the Oxygen demand in a closed respirometer Qualife de l’eau - Evaluation, en milieu agueux, de la biod&gradabilit& aerobie “ultime” des composes organiques - Methode par determination de la demande en oxygene dans un respirom6tre ferm6 Reference number ISO 9408:1991(E) SIST EN 29408:1998
ISO 9408:1991(E) 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. Esch 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, govern- mental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an Inter- national Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard ISO 9408 was prepared by Technical Committee ISO/TC 147, Water quality. Annexes A, 6, C and D of this International Standard are for information only. 0 ISO 1991 All rights reserved. No part of this publication may be reproduced or utilized in any form or by any means, electronie or mechanical, including photocopying and microfilm, without Permission in writing from the publisher. International Organization for Standardization Case Postale 56 l CH-1211 Geneve 20 l Switzerland Printed in Switzerland ii SIST EN 29408:1998
INTERNATIONAL STANDARD ISO 9408:1991 (E) Water quality - Evaluation in an aqueous medium of the “ultimate” aerobic biodegradability of organic compounds - Method by determining the Oxygen demand in a closed respirometer 1 Scope This International Standard specifies a method, by determination of the Oxygen demand in a closed respirometer, for the evaluation in an aqueous me- dium of the “ultimate” biodegradability of organic compounds at a given concentration by aerobic micro-organisms. The conditions described in this International Stan- dard do not always correspond to the optimal con- ditions for allowing the maximum degree of biodegradation to occur. The method applies to organic compounds which a) are soluble in the test conditions; b) are insoluble in the test conditions, in which case special measures may be necessary to achieve good dispersion of the compound; c) do not resch and react with the CO, absorbant; d) are volatile 1 prov pirometer is used; ided that a suitable res- e) are not inhibitory to the test micro-organisms at the concentration Chosen for the test. The pres- ence of inhibitory effects tan be determined as specified in 8.3, or by using any other method for determining the inhibitory effect of a compound on bacteria (See, for example, ISO 8192). 2 Normative references The following Standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publi- cation, the editions indicated were valid. All stan- dards are subject to revision, and Parties to agreements based on this International Standard are encouraged to investigate the possibility of ap- plying the most recent editions of the Standards in- dicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 60603989, Water quality - Determination of the Chemical Oxygen demand. ISO 6107-2:1989, Water quality - Vocabulary - Part 2. ISO 7827:1984, Water quality - Evaluation in an aqueous medium of the ” ul tima te” aerobic biodegradability of organic compounds - Method by analysis of dissolved organic carbon (DOC). ISO 8192:1986, Water quality - Test for inhibition of Oxygen consumption by activated sludge. 3 Definitions For the purposes of this International Standard, the following definitions apply. 3.1 ultimate biodegradation: The level of degra- dation achieved when the test compound is totally utilized by micro-organisms resulting in the pro- duction of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass). 3.2 biochemical Oxygen demand (BOD): The mass concentration of dissolved Oxygen consumed under specified conditions by the biological Oxidation of organic and/or inorganic matter in water (see ISO 6107-2;. 3.3 suspended solids (of an activated sludge): Sol- ids removed by filtration or centrifuging of a known volume of sludge under specified conditions, and, 1 SIST EN 29408:1998
ISO 9408:1991(E) for the purpose of this International Standard, drying at about 100 “C. 3.4 pre-exposure (or pre-adaptation): The pre- incubation of an inoculum in the presence of the test compound, with the aim of enhancing the ability of the inoculum to degrade the test compound. 3.5 pre-conditioning (or pre-acclimatization): The pre-incubation of an inoculum under the conditions of the test in the absence of the test compound, to improve the Performance of the test. 4 Principle Determination of the biodegradation of organic compounds by aerobic micro-organisms, using a test medium. The organic compound is the sole Source of carbon and energy in the medium. The concentration of the test compound is normally 100 mg/l, but its theor- etical Oxygen demand (ThOD) shall be at least 100 mg/l. The inoculated medium is stirred in a closed flask and the consumption of Oxygen is determined either by measuring the amount of Oxygen required to maintain a constant gas volume in the respirometer flask, or by measuring the Change in volume or pressure (or a combination of the two) in the appar- atus. Evolved carbon dioxide is absorbed in a suitable substan ce in the test vessel. The degradation is followed over a period of 28 days, or longer if necessary, by determining the consumption of Oxygen either automatically or manually. The amount of Oxygen consumed by the organic compound (after correction by comparison with the blank test) is expressed as a percentage of the theoretical Oxygen demand (ThOD) calculated from the formula of the compound or the Chemical Oxygen demand (COD). Additionally, the degree of biodegradation may also be calculated from sup- plemental Chemical analyses, for example, dis- solved organic carbon (DOC) for sufficiently water-soluble compounds or specific analysis (con- cerning only primary biodegradation), made at the beginning and the end of incubation. Evaluation of the biodegradability of the test compound is made on the basis of these data. 5 Test environment lncubation shall take place in the dark or in diffused light, in an enclosure which is maintained at a con- stant temperature (within at least + 1 OC) between 20 OC and 25 “C and which is free from toxic va- pours. 6 Reagents Use only reagents of recognized analytical grade. 6.1 Distilled or de-ionized water. Containing less than 10 % of the initial DOC content introduced by the organic compound to be tested. 6.2 Test medium 6.2.1 Composition 6.2.1.1 Solution (a). Anhydrous potassium dihydrogenphosphate (KH,PO,) 875 g Anhydrous dipotassium hydrogenphosphate (K,HPO,) 21,75 g Disodium hydrogenphosphate dihydrate (Na,HP0,.2H,O) 3x4 g Ammonium chloride (NH,CI) 095 g Water (6.1) (quantity necessary to make uP to) 1000 ml The pH of this solution should be about 7,4. 6.2.4.2 Solution (b). Dissolve 22,5 g of magnesium sulphate hepta- hydrate (MgSO,.7H,O) in 1000 ml of the water (6.1). 6.2.1.3 Solution (c). Dissolve 27,5 g of anhydrous Calcium chloride (CaCI,) or 36,4 g of Calcium chloride dihydrate (CaCl,.2H,O) in 1000 ml of the water (6.1). 6.2.4.4 Solution (d). Dissolve 0,25 g of iron(lll) chloride hexahydrate (FeCI,.GH,O) in 1000 ml of the water (6.1). Prepare this Solution just before use. NOTE 1 It is not necessary to prepare this solution just before use if a drop of concentrated hydrochloric acid (I-ICI) or 0,4 g/l of ethylenediamine-tetraacetic acid (EDTA) is added. 6.2.2 Preparation of the test medium. For 1 litre of test medium, just before use add to 800 ml of the water (6.1) - 10 ml of Solution (a); - and then 1 ml of each Solution (b), (c) and (d), (to avoid the formation of turbidity in the final me- dium). Make up to 1000 ml with the water (6.1). 2 SIST EN 29408:1998
ISO 9408:1991 (E) 6.3 Carbon dioxide absorber, Potassium hydroxide Solution (about 10 mol/l), sodium carbonate pellets or another suitable absorbant. 7 Apparatus Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or toxic matter. Usual laboratory equipment, and 7.1 Closed respirometer. The principle of a closed respirometer is given in annex D. When testing volatile compounds, the ap- paratus used shall be appropriate or adapted to this particular purpose. Care shall be taken that there is no loss of compound due to the apparatus. 7.2 Water-bath or constant temperature room (to comply with clause 5). 7.3 Equipment for measurement of dissolved or- ganic carbon, instrument of sufficient sensitivity for the measurement of dissolved organic carbon (DOC). 7.4 Device for determining Chemical Oxygen de- mand (COD). 7.5 Device for filtration, with membrane filters of suitable porosity (nominal aperture diameter be- tween 0,2 pm and 0,45 um) which adsorb organic compounds or release organic carbon to a minimum degree (see note 3 to 8.3). 7.6 Centrifuge. 7.7 pH=meter. 8 Procedure 8.1 Preparation of the test solutions Prepare the following solutions: a) Solution of the test compound in the test medium (6.2) to obtain a concentration of 100 mg/l, but at least 100 mg/1 ThOD; b) solution of a known organic compound (“refer- ence” compound), for example, sodium acetate, sodium benzoate, aniline, in the test medium (6.2) to obtain a concentration of 100 mg/l; c) Solution containing, in the test medium (6.2), the Same concentrations of the test compound and the reference compound as used in a) and b). NOTE 2 Compounds of low water solubility may be added directly, in solid or liquid form, to the medium in the appropriate flask; an ISO International Standard will be prepared for guidance. 8.2 Preparation of the inoculum Take a Sample of activated sludge from the aeration tank of a biological wastewater treatment plant, or a laboratory unit, dealing with predominantly dom- estic sewage. If the sludge is actively respiring on external Substrate, bring it to the “endogenous” Phase (i.e. having no external Substrate remaining) as follows: - either aerate for a few hours before use, or - centrifuge, wash with medium (6.2), recentrifuge and resuspend in the medium (this treatment is recommended if it is suspected that the sludge contains inhibiting matter). When the sludge is judged to be in the “endogenous” Phase, or free from inhibiting matter, mix Welf, maintain in an aerobic state by stirring or aeration at the required temperature, and use on the day of collection or one day Iater. Just before use, determine the concentration of suspended solids. If required, concentrate the sludge by settling, so that the volume of sludge added to obtain 30 mg/1 of dry matter in the reaction mixture is less than or equal to 1 % of the mixture, that is, the sludge should contain at least 3 g/l of dry solids. NOTES 3 A concentration of 30 mg/l of suspended solids in the final medium has been found suitable for concentrations of test compound in the range of 50 mg/1 to 150 mg/l. The Oxygen consumption of the blank Solution shall not be greater than 60 mg/l in 28 days and should normally be in the range of 20 mg/l to 30 mg/l. In Order to reduce the in- fluence of the blank, the sludge may be pre-conditioned (see 3.5) by aeration for up to one week before it is used. 4 Secondary effluent and used as inoculum, but these centrated by filtration or biomass. surface water may also be inocula may have to be con- centrifugation to get more 5 Pre-exposed inocula may be used for certain purposes. When such inocula are used this should be clearly stated in the test results (e.g. percentage biodegradation = x %, using pre-exposed inocula) and the method of pre-exposure detailed in the test report. Pre-exposed inocula tan be obtained from laboratory bio- degradation tests run under a variety of conditions (e.g. Zahn-WeIlens and SCAS tests) or from samples collected from locations where relevant environmental conditions exist (e.g. treatment plants dealing with identical com- pounds, contaminated areas, etc.). SIST EN 29408:1998
ISO 9408:1991 (E) 8.3 Test Set up the closed respirometer (see 7.1 and the ex- ample described in annex 0). Assemble a sufficient number of reaction vessels in Order to have at least a) two test flasks (Symbol FT), containing the ap- propriate volume of test solution [8.1, item a)]; b) two flasks for the blank test (Symbol Fg), con- taining the appropriate volume of the test me- dium (6.2); c) if needed, one flask (Symbol FC), for checki
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