SIST EN ISO 14593:2005
(Main)Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test) (ISO 14593:1999)
Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test) (ISO 14593:1999)
This document specifies a method, by analysis of inorganic carbon, for the evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic substances at a given concentration of microorganisms.
Wasserbeschaffenheit - Bestimmung der vollständigen biologischen Abbaubarkeit organischer Substanzen im wässrigen Medium - Verfahren mittels Bestimmung des anorganischen Kohlenstoffs in geschlossenen Flaschen (CO2-Headspace-Test) (ISO 14593:1999)
Qualité de l'eau - Evaluation en milieu aqueux de la biodégradabilité aérobie ultime des composés organiques - Méthode par analyse du carbone inorganique dans des récipients hermétiquement clos (Essai au CO2 dans l'espace de tete) (ISO 14593:1999)
La présente Norme internationale prescrit une méthode pour l'évaluation, par analyse du carbone inorganique en milieu aqueux, de la biodégradabilité aérobie ultime de composés organiques présents à une concentration donnée sous l'action de micro-organismes.
La méthode de la présente Norme internationale s'applique à des composés organiques:
a) solubles dans l'eau dans les conditions de l'essai;
b) peu soluble dans l'eau dans les conditions de l'essai, auquel cas il peut être nécessaire de prendre des mesures particulières afin d'assurer une bonne dispersion du composé (voir par exemple l'ISO 10634);
c) volatils;
d) n'ayant pas d'effet inhibiteur sur les micro-organismes soumis à l'essai, à la concentration choisie pour l'essai. Au cours de cet essai, le carbone inorganique produit biologiquement est dosé in situ dans les récipients d'essai de telle manière que le taux mesuré soit presque égal au taux de production microbienne.
NOTE 1 Les conditions décrites dans la présente Norme internationale ne correspondent pas toujours aux conditions optimales permettant d'obtenir un degré maximal de biodégradation. Voir l'ISO 15462 pour d'autres méthodes de biodégradation.
NOTE 2 Pour des substances très volatiles, les pertes dans la phase gazeuse peuvent être minimisées en réduisant le volume de l'espace de tête. Cependant, il convient que la quantité d'oxygène présente dans le système d'essai soit suffisante pour éviter que le processus de biodégradation soit limité par un manque d'oxygène.
NOTE 3 L'existence d'un effet inhibiteur peut être mise en évidence suivant la méthode prescrite en 8.3, ou par toute autre méthode de détermination de l'effet inhibiteur d'une substance sur des bactéries (voir par exemple l'ISO 8192).
Kakovost vode – Vrednotenje aerobne biorazgradljivosti organskih spojin v vodnem mediju – Metoda z analizo anorganskega ogljika v zaprtih posodah (CO2 headspace test) (ISO 14593:1999)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 14593:2005
01-december-2005
Kakovost vode – Vrednotenje aerobne biorazgradljivosti organskih spojin v
vodnem mediju – Metoda z analizo anorganskega ogljika v zaprtih posodah (CO2
headspace test) (ISO 14593:1999)
Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in
aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2
headspace test) (ISO 14593:1999)
Wasserbeschaffenheit - Bestimmung der vollständigen biologischen Abbaubarkeit
organischer Substanzen im wässrigen Medium - Verfahren mittels Bestimmung des
anorganischen Kohlenstoffs in geschlossenen Flaschen (CO2-Headspace-Test) (ISO
14593:1999)
Qualité de l'eau - Evaluation en milieu aqueux de la biodégradabilité aérobie ultime des
composés organiques - Méthode par analyse du carbone inorganique dans des
récipients hermétiquement clos (Essai au CO2 dans l'espace de tete) (ISO 14593:1999)
Ta slovenski standard je istoveten z: EN ISO 14593:2005
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 14593:2005 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 14593:2005
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SIST EN ISO 14593:2005
EUROPEAN STANDARD
EN ISO 14593
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2005
ICS 13.060.70
English version
Water quality - Evaluation of ultimate aerobic biodegradability of
organic compounds in aqueous medium - Method by analysis of
inorganic carbon in sealed vessels (CO2 headspace test) (ISO
14593:1999)
Qualité de l'eau - Evaluation en milieu aqueux de la Wasserbeschaffenheit - Bestimmung der vollständigen
biodégradabilité aérobie ultime des composés organiques - biologischen Abbaubarkeit organischer Substanzen im
Méthode par analyse du carbone inorganique dans des wässrigen Medium - Verfahren mittels Bestimmung des
récipients hermétiquement clos (Essai au CO2 dans anorganischen Kohlenstoffs in geschlossenen Flaschen
l'espace de tête) (ISO 14593:1999) (CO2-Headspace-Test) (ISO 14593:1999)
This European Standard was approved by CEN on 25 May 2005.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions 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 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 translation
under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14593:2005: E
worldwide for CEN national Members.
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SIST EN ISO 14593:2005
EN ISO 14593:2005 (E)
Foreword
The text of ISO 14593:1999 has been prepared by Technical Committee ISO/TC 147 "Water
quality” of the International Organization for Standardization (ISO) and has been taken over as
EN ISO 14593:2005 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 December 2005, and conflicting national
standards shall be withdrawn at the latest by December 2005.
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, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 14593:1999 has been approved by CEN as EN ISO 14593:2005 without any
modifications.
2
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SIST EN ISO 14593:2005
INTERNATIONAL ISO
STANDARD 14593
First edition
1999-03-15
Water quality — Evaluation of ultimate
aerobic biodegradability of organic
compounds in aqueous medium — Method
by analysis of inorganic carbon in sealed
vessels (CO headspace test)
2
Qualité de l’eau — Évaluation en milieu aqueux de la biodégradabilité
aérobie ultime des composés organiques — Méthode par analyse du
carbone inorganique dans des récipients hermétiquement clos (Essai
au CO dans l'espace de tête)
2
A
Reference number
ISO 14593:1999(E)
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SIST EN ISO 14593:2005
ISO 14593:1999(E)
Contents
1 Scope .1
2 Normative reference .1
3 Terms and definitions .2
4 Principle.3
5 Test environment.4
6 Reagents.4
7 Apparatus .5
8 Procedure .5
9 Calculation and expression of results.9
10 Expression of results .10
11 Validity of results.10
12 Precision of the method.11
13 Test report .11
Annex A (informative) Example of a biodegradation curve .12
Annex B (informative) Statistical treatment of results.13
Annex C (informative) Interlaboratory ring test. 14
Bibliography.15
© ISO 1999
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
ii
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SIST EN ISO 14593:2005
© ISO
ISO 14593:1999(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. 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, 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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards 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.
International Standard ISO 14593 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee
SC 5, Biological methods.
Annexes A, B and C of this International Standard are for information only.
iii
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SIST EN ISO 14593:2005
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SIST EN ISO 14593:2005
INTERNATIONAL STANDARD © ISO ISO 14593:1999(E)
Water quality — Evaluation of ultimate aerobic biodegradability
of organic compounds in aqueous medium — Method by analysis
of inorganic carbon in sealed vessels (CO headspace test)
2
WARNING — Activated sludge and sewage may contain potentially pathogenic organisms and should be
handled with appropriate precautions. Hazardous test compounds and those whose properties are
unknown should be handled with care.
1 Scope
This International Standard specifies a method, by analysis of inorganic carbon, for the evaluation in an aqueous
medium of the ultimate aerobic biodegradability of organic substances at a given concentration of microorganisms.
This International Standard is applicable to organic compounds which are:
a) water-soluble under the test conditions;
b) poorly water-soluble under the test conditions, in which case special measures may be necessary to achieve a
good dispersion of the compound (see ISO 10634);
c) volatile;
d) not inhibitory to the test microorganisms at the concentration chosen for the test.
In this test, biogenically produced inorganic carbon is measured in situ in the test vessels in such a manner that the
rate measured nearly equals the rate of microbial production.
NOTE 1 The conditions described in this International Standard do not always correspond to the optimal conditions for
allowing the maximum degree of biodegradation to occur. For alternative biodegradation methods see ISO 15462.
NOTE 2 With highly volatile substances, losses to the gaseous phase can be minimized by reducing the volume of the
headspace. However, there should be sufficient oxygen in the test system to prevent biodegradation being oxygen-limited.
NOTE 3 The presence of an inhibitory effect can be determined as specified in 8.3, or by using any other method for
determining the inhibitory effect of a substance on bacteria (see, for example, ISO 8192).
2 Normative reference
The following normative document contains provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent edition of the normative document indicated below. For
undated reference, the latest edition of the normative document referred to applies. Members of IEC and ISO
maintain registers of currently valid International Standards.
ISO 10634:1995, Water quality — Guidance for the preparation and treatment of poorly water-soluble organic
compounds for the subsequent evaluation of their biodegradability in aqueous medium.
1
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SIST EN ISO 14593:2005
© ISO
ISO 14593:1999(E)
3 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
3.1
ultimate aerobic biodegradation
breakdown of a chemical compound or organic matter by microorganisms in the presence of oxygen to carbon
dioxide, water and mineral salts of any other elements present (mineralization) and the production of new biomass
3.2
primary biodegradation
structural change (transformation) of a chemical compound by microorganisms resulting in the loss of a specific
property
3.3
activated sludge
biomass produced in the aerobic treatment of wastewater by the growth of bacteria and other microorganisms in the
presence of dissolved oxygen
3.4
concentration of suspended solids
amount of solids obtained by filtration or centrifugation of a known volume of activated sludge
and drying at about 105 �C to constant mass
3.5
total organic carbon
TOC
all that carbon present in organic matter which is dissolved and suspended in the water sample
3.6
dissolved organic carbon
DOC
that part of the organic carbon in the water sample which cannot be removed by specified phase separation
-2
NOTE For example, by centrifugation at 40 000 m⋅s for 15 min or by membrane-filtration using membranes with pores of
diameter 0,2 mm to 0,45 mm.
3.7
total inorganic carbon
TIC
all that inorganic carbon in the water deriving from carbon dioxide and carbonate
3.8
dissolved inorganic carbon
DIC
that part of the inorganic carbon in water which cannot be removed by specified phase separation
-2
NOTE For example, by centrifugation at 40 000 m⋅s for 15 min or by membrane-filtration using membranes with pores of
diameter 0,2 mm to 0,45 mm.
3.9
theoretical amount of inorganic carbon
ThIC
theoretical maximum amount of inorganic carbon formed after oxidizing a chemical compound completely
NOTE It is calculated from the molecular formula and expressed in this case as milligrams carbon per milligram (or gram)
test compound.
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SIST EN ISO 14593:2005
© ISO
ISO 14593:1999(E)
3.10
lag phase
time from the start of a test until adaptation and/or selection of the degrading microorganisms are achieved and the
biodegradation degree of a chemical compound or organic matter has increased to about 10 % of the maximum
level of biodegradation
NOTE It is normally recorded in days.
3.11
maximum level of biodegradation
maximum biodegradation degree of a chemical compound or organic matter in a test, above which no further
biodegradation takes place during the test
NOTE It is normally recorded in percent.
3.12
biodegradation phase
time from the end of the lag phase of a test until about 90 % of the maximum level of biodegradation has been
reached
NOTE It is normally recorded in days.
3.13
plateau phase
time from the end of the biodegradation phase until the end of the test
NOTE It is normally recorded in days.
3.14
pre-exposure
pre-incubation of an inoculum in the presence of the test chemical compound or organic matter, with the aim of
enhancing the ability of this inoculum to biodegrade the test material by adaptation and/or selection of the
microorganisms
3.15
preconditioning
pre-incubation of an inoculum under the conditions of the subsequent test in the absence of the test chemical
compound or organic matter, with the aim of improving the performance of the test by acclimatization of the
microorganisms to the test conditions
4 Principle
The test compound, as the sole source of carbon and energy, is added to a mineral salts medium inoculated with a
mixed population of microorganisms and incubated in sealed vessels with a headspace of air. The concentration of
the compound used normally yields an initial organic carbon concentration in the medium of 2 mg/l to 40 mg/l,
usually 20 mg/l. Biodegradation (mineralization to carbon dioxide) is determined by measuring the net increase in
total inorganic carbon (TIC) levels over time compared with unamended blanks. The test generally runs for 28 d.
The extent of biodegradation is expressed as a percentage of the theoretical amount of inorganic carbon (ThIC)
based on the amount of test compound added initially.
For sufficiently water-soluble substances, dissolved organic carbon (DOC) removal during the test may also be
determined (see for example ISO 7827).
If a suitable analytical method is available, the primary biodegradation of the test compound during the test may
also be determined.
3
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SIST EN ISO 14593:2005
© ISO
ISO 14593:1999(E)
5 Test environment
o o
Incubation shall take place in the dark or in diffused light, at a temperature within the range 20 C to 25 C which
o
shall not vary by more than – 2 C during the test.
6 Reagents
Use only reagents of recognized analytical grade.
6.1 Distilled or deionized water, containing less than 1 mg/l total organic carbon.
6.2 Test medium.
6.2.1 Composition
a) Solution a)
Dissolve:
Anhydrous potassium dihydrogenphosphate (KH PO ) 8,50 g
2 4
Anhydrous dipotassium hydrogenphosphate (K HPO ) 21,75 g
2 4
Disodium hydrogenphosphate dihydrate (Na HPO ⋅ 2H O) 33,40 g
2 4 2
Ammonium chloride (NH Cl) 0,50 g
4
in water (6.1), quantity necessary to make up to 1 000 ml
In order to check this buffer solution, it is recommended to measure the pH, which should be at about 7,4. If this is
not the case, prepare a new solution.
b) Solution b)
Dissolve 36,40 g of calcium chloride dihydrate (CaCl ⋅2H O) in water (6.1), quantity necessary to make up to
2 2
1 000 ml.
c) Solution c)
Dissolve 22,50 g of magnesium sulfate heptahydrate (MgSO ⋅ 7H O) in water (6.1), quantity necessary to make up
4 2
to 1 000 ml.
d) Solution d)
Dissolve 0,25 g iron(III) chloride hexahydrate (FeCl ⋅6H O) in water (6.1), quantity necessary to make up to
3 2
1 000 ml. To avoid precipitation, prepare this solution freshly before use or add a drop of concentrated hydrochloric
acid (HCl).
6.2.2 Preparation of the test medium
For 1 l of test medium, mix 10 ml of solution a) with 800 ml water (6.1) and then add 1 ml each of solutions b), c)
and d) and make up to 1 000 ml with water (6.1).
6.3 Concentrated orthophosphoric acid (H PO ) (> 85 % mass per volume).
3 4
4
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SIST EN ISO 14593:2005
© ISO
ISO 14593:1999(E)
6.4 Sodium hydroxide solution.
Dissolve 280 g of sodium hydroxide (NaOH) in 1 l of water (6.1) to obtain a solution of concentration c = 7 mol/l.
Use either a freshly prepared solution or determine the concentration of dissolved inorganic carbon (DIC) of this
solution and consider this value when calculating the test result (see clause 9).
7 Apparatus
Ensure that all glassware is thoroughly cleaned and, in particular, free from both organic and toxic matter. Use usual
laboratory equipment, and the following apparatus.
7.1 Carbon analyzer, instrument for measuring inorganic and (optionally) organic carbon, such as the appropriate
parts of a total organic carbon (TOC) analyser or a gas chromatograph.
7.2 Gas-tight glass vessels of known volume, for example, serum bottles of 160 ml capacity sealed with butyl
rubber septa and aluminium crimp seals or any other gas-tight system.
7.3 Orbital shaker.
7.4 Syringes of high precision for aqueous and gaseous samples.
7.5 Glass bottles to take, for example, 5 l medium.
7.6 Centrifuge.
7.7 pH-meter.
7.8 Device for filtration, with membrane filters of suitable porosity (nominal aperture diameter between 0,20 μm
and 0,45 μm) which adsorb organic substances or release organic carbon to a minimum degree.
7.9 CO -free air production system.
2
Prepared by passing air through soda lime granules or a solution of sodium hydroxide (see, for example, ISO 9439).
Alternatively, a mixture of 80 % N /20 % O may be used. The absence of CO in the air can be confirmed by
2 2 2
passage through barium hydroxide solution (a white precipitate indicates the presence of CO ).
2
8 Procedure
8.1 Preparation of the test solutions
8.1.1 Test compound
Prepare a stock solution of a sufficiently water-soluble test compound in water (6.1) or the test medium (6.2.2) at a
concentration preferably 100-fold greater than the final concentration to be used in the test. Add the stock solution
to the test medium (6.2.2) to give a final TOC concentration of between 2 mg/l and 40 mg/l (see 8.3), preferably
20 mg/l TOC.
Add substances of low water-solubility directly, in solid or liquid form, to the inoculated medium in the appropriate
test vessels. Liquid test compounds, including those which are volatile, may be injected directly into sealed vessels
using a high-precision microsyringe. Determine the added amount exactly.
Poorly soluble substances can often be dispensed as a dispersion produced using a non-biodegradable emulsifying
agent and/or ultrasonication. Refer to ISO 10634 for guidance on the preparation and treatment of poorly water-
soluble substances.
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SIST EN ISO 14593:2005
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ISO 14593:1999(E)
8.1.2 Reference substance
Prepare a stock solution of the reference substance (an organic substance of known biodegradability such as
aniline or sodium benzoate) in water (6.1) in the same way as in 8.1.1, and then dilute in the test medium (6.2.2) to
give a final TOC concentration of 20 mg/l.
8.1.3 Solution to check inhibition
If required (when e.g. no information on the toxicity of test compound is available), prepare a solution containing, in the
test medium (6.2.2), both the test compound (8.1.1) and the reference compound (8.1.2) preferably at concentrations
of organic carbon of 20 mg/l for each.
8.2 Preparation of the inoculum
8.2.1 General
Prepare the inoculum using the sources described in 8.2.2, 8.2.3 and 8.2.4, or using a mixture of these sources, to
obtain a microbial population that offers sufficient biodegradative activity. Check this activity by means of the
reference substance (8.1.2). Based on experience (clause 12 and annex C), the usual inoculum is activated sludge
at a dry solids concentration of 4 mg/l. The carbon dioxide production of the blanks should be as low as possible
(see clause 11). To reduce the influence of the blanks, it may be helpful to precondition the inoculum by aeration for
up to one week before it is used. Use a suitable volume for inoculation (see note 2 below).
Normally the inoculum should not be pre-exposed to the test compound, to allow a general prediction of the
degradation behaviour in the environment. In certain circumstances, depending on the purpose of the test, pre-
exposed inocula may be used, provided that this is clearly stated in the test results (e.g. biodegradation = x %,
using pre-exposed inocula) and the method of the pre-exposure is detailed in the test report.
NOTE 1 Pre-exposed inocula can be obtained from laboratory biodegradation tests conducted under a variety of conditions
(e.g. ISO 9888 Zahn-Wellens test; ISO 9887 SCAS test) or from samples collected from locations where relevant
environmental conditions exist (for example treatment plants dealing with similar substances or contaminated areas).
NOTE 2 Based on experience, suitable volume means:
sufficient to give a population which offers enough biodegradation activity;
degrades the reference compound by the stipulated percentage (see clause 11);
2 5
gives between 10
to 10 colony forming units per millilitre in the final mixture;
gives normally a concentration of 4 mg/l suspended solids of activated sludge in the final mixture (higher concentrations
up to 30 mg/l are generally possible but may influence significantly the CO production of the blanks and are therefore not
2
recommended);
the quantity of dissolved organic carbon provided by the inoculum should be less than 10 % of the initial concentration of
organic carbon introduced by the test compound;
generally 1 ml to 10 ml of inoculum are sufficient for 1 000 ml of test solution.
8.2.2 Inoculum from an activated sludge plant
Collect activated sludge from the aeration tank of a full-scale or a laboratory wastewater treatment plant which
treats predominantly domestic sewage. If necessary, remove coarse particles by filtration through a sieve (for
2
example, 1 mm mesh size) and keep the sludge aerobic thereafter. Since it is necessary for the inoculum blank to
have as low an evolution of CO as possible, the sludge may need further treatment. For example, settle or
2
-2
centrifuge (e.g. at 10 800 m⋅s for 10 min) the sludge, discard the supernatant and resuspend the settled or
centrifuged solids in test medium (6.2.2) to give a suspended solids concentration of about 3 g/l (see, for example,
ISO 11923). Alternatively or additionally, aerate the sludge overnight before use. To reduce the blank value still
further, the sludge can be preconditioned to the test conditions prior to use by diluting in test medium (6.2.2) to give
the final concentration and aerating with moist air for up to one week at the test temperature. Use 4 mg/l dry solids
as the concentration of the inoculum in a test (see note 2 in 8.2.1).
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SIST EN ISO 14593:2005
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ISO 14593:1999(E)
8.2.3 Inoculum from wastewater
Take a sample of the influent or the effluent from a full-scale or laboratory wastewater treatment plant dealing with
predominantly domestic sewage. Keep this sample under aerobic conditions and use on the day of collection (or
precondition if necessary). Coarse-filter the effluent to remove gross particulate matter and measure its pH. Before
use, sparge the filtrate with CO -free air (7.9) for about 1 h while maintaining the pH at 6,5 using orthophosphoric
2
acid (6.3), restore the pH to its original value and finally let the sample settle for 1 h and take a suitable volume of
the supernatant for inoculation.
NOTE This sparging procedure reduces the TIC content of the inoculum. For example, when the maximum of 100 ml
filtered effluent per litre test volume is used as the inoculum, the amount of TIC produced in blank vessels in 28 d should be in
the range 0,4 mg/l to 1,3 mg/l (see annex D in [9]). The TIC values in the blanks may be different for the various inocula used.
8.2.4 Inoculum from a surface water
Take a sample of an appropriate surface water. Keep under aerobic conditions and use on the day of collection. If
necessary, concentrate the surface water by filtration or centrifugation. Use a suitable volume as inoculum.
8.3 Test procedure
8.3.1 Provide a number of test vessels (7.2) sufficient for requirements in order to have:
a) test vessels (denoted F ) for the test compound;
T
b) blank vessels (denoted F ) containing test medium and inoculum;
B
c) vessels for checking the procedure (denoted F ) containing the reference compound;
C
d) if needed, vessels for checking a possible inhibitory effect of the test compound (denoted F );
I
e) if needed, vessels for checking a possible abiotic elimination (denoted F ) containing the test compound but no
S
inoculum, sterilized by autoclaving or by the addition of a suitable inorganic toxic compound to prevent
microbial activity. Use, for example, 5 ml/l of a solution containing 10 g/l of mercury(II) chloride (HgCl ). Add the
2
same amount of the toxic substance two weeks after the test was begun.
To separate large glass bottles (7.5), make the additions as indicated in Table 1.
The number of vessels needed will depend on the frequency of analysis and the confidence limits required for the
final extent of biodegradation (see annex B). Usually at least five vessels from sets F , F and F are analysed at
T B C
the end of the test.
8.3.2 Thoroughly mix the contents of each of the large bottles (7.5) in turn and dispense suitable aliquots (for
example, 100 ml) into labelled test vessels (7.2). Add water-soluble test compounds from stock solutions and poorly
water-soluble test compounds directly to the test vessels (see 8.1.1) and add water to give the same volume in
each vessel. Ensure that the liquid to headspace ratio and the test compound concentration are such that sufficient
oxygen is available in the headspace to allow for complete biodegradation (for example, avoid using a high
substrate concentration and a small headspace). A headspace to liquid ratio of 1:2 is usual.
Seal the vessels when all additions have been made, for example, with butyl rubber septa and aluminium caps,
place on an orbital shaker in the dark or diffuse light at the test temperature (see clause 5) and start the shaker at a
speed sufficient to ensure good mixing of the vessel contents (for example, 150 r/min to 200 r/min).
Calibrate the carbon analyser (7.1) as required (8.4) on the days when analysis is to be made. Sacrifice vessels for
analysis on the day of sampling but at least weekly or more frequently if a complete degradation curve is re
...
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