Water quality - Evaluation of the aerobic biodegradability of organic compounds at low concentrations - Part 2: Continuous flow river model with attached biomass

This part of ISO 14592 specifies a method for evaluating the biodegradability of organic test compounds by aerobic microorganisms in natural waters by means of a continuous flow river model with attached biomass. This part of ISO 14592 is applicable to organic test compounds present in lower concentrations than those of natural carbon substrates also present in the system. Under these conditions, the test compounds serve as a secondary substrate and the kinetics for biodegradation would be expected to be first order (“non-growth” kinetics). This part of ISO 14592 is applicable to organic test compounds, which under the conditions of the test and at the chosen test concentration, are:
- water soluble;
- quantitatively detectable with appropriate analytical methods or available in radiolabelled form;
- non-volatile from aqueous solution (e.g. Henry's law constant < 1 Pa⋅m3/mole);
- not significantly adsorbed;
- not photolyzed;
- not inhibitory to the microorganisms of the test system.
The test is not recommended for use as proof of ultimate biodegradability (mineralization) which is better assessed using other standardized tests (see ISO/TR 15462).

Qualité de l'eau - Évaluation de la biodégradabilité aérobie des composés organiques présents en faibles concentrations - Partie 2: Modèle de cours d'eau à courant continu avec biomasse associée

L'ISO 14592:2002 spécifie une méthode pour l'évaluation de la biodégradabilité de composés d'essai organiques par des micro-organismes aérobies dans les eaux naturelles au moyen d'un modèle de cours d'eau à courant continu avec biomasse associée.
L'ISO 14592:2002 s'applique aux composés d'essai organiques présents en concentrations plus faibles que celles des substrats carbonés naturels également présents dans le système. Dans ces conditions, les composés d'essai ont la fonction de substrat secondaire, et il est attendu que la cinétique de biodégradation soit du premier ordre (cinétique «sans croissance»).
L'ISO 14592:2002 s'applique aux composés d'essai organiques qui, dans les conditions d'essai et à la concentration choisie pour l'essai, sont solubles dans l'eau, détectables quantitativement par des méthodes d'analyse appropriées ou disponibles sous forme radio-marquée, non volatils en solution aqueuse, non adsorbés de manière significative, non photolysés et non inhibiteurs pour les micro-organismes du système d'essai.
L'utilisation de cette méthode d'essai n'est pas recommandée comme preuve de la biodégradation ultime (minéralisation), qui peut s'évaluer de manière plus fiable en utilisant d'autres essais normalisés.

Kakovost vode - Vrednotenje aerobne biorazgradljivosti organskih spojin pri nizkih koncentracijah - 2. del: Pretočni rečni model s pritrjeno biomaso

Ta del ISO 14592 določa metodo za vrednotenje biorazgradljivosti organskih preskusnih spojin z aerobnimi mikroorganizmi v naravnih vodah s pretočnim rečnim modelom s pritrjeno biomaso. Ta del ISO 14592 velja za organske preskusne spojine, prisotne pri nižjih koncentracijah kot so koncentracije naravnih substratov ogljika, ki so tudi prisotni v sistemu. Pod temi pogoji preskusne spojine služijo kot substrat drugotnega pomena, za kinetiko biorazgradljivosti pa se pričakuje, da je le-ta prva na vrsti (kinetika »ne rasti«). Ta del ISO 14592 velja za organske preskusne spojine, ki pod pogoji preskusa in pri izbrani preskusni koncentraciji:
- so topne v vodi;
- so kvantitativno zaznavne s primernimi analitičnimi metodami ali na voljo v obliki, ki je radioaktivno označena;
- ne hlapijo iz vodne raztopine (npr. konstanta Henryjevega zakona < 1 Pa⋅m3/mol);
- niso znatno absorbirane;  
- se ne uporabljajo pri fotolizi;
- niso zaviralne za mikroorganizme preskusnega sistema.
Ta preskusna metoda se ne priporoča za uporabo pri dokazovanju končne biorazgradljivosti (mineralizacije), ki se jo lažje določi z drugimi standardiziranimi preskusi (glej ISO/TR 15462).

General Information

Status
Published
Public Enquiry End Date
19-Jul-2009
Publication Date
16-Jun-2010
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
12-May-2010
Due Date
17-Jul-2010
Completion Date
17-Jun-2010

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INTERNATIONAL ISO
STANDARD 14592-2
First edition
2002-11-15
Corrected version
2003-08-01
Water quality — Evaluation of the aerobic
biodegradability of organic compounds at
low concentrations —
Part 2:
Continuous flow river model with attached
biomass
Qualité de l'eau — Évaluation de la biodégradabilité aérobie des composés
organiques présents en faibles concentrations —
Partie 2: Modèle de cours d'eau à courant continu avec biomasse associée
Reference number
ISO 14592-2:2002(E)
ISO 2002
---------------------- Page: 1 ----------------------
ISO 14592-2:2002(E)
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ii © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)
Contents Page

Foreword .................................................................................................................................................................... iv

Introduction................................................................................................................................................................. v

1 Scope.............................................................................................................................................................. 1

2 Normative reference...................................................................................................................................... 1

3 Terms, definitions and symbols .................................................................................................................. 2

4 Principle......................................................................................................................................................... 4

5 Reagents and media..................................................................................................................................... 4

6 Apparatus....................................................................................................................................................... 5

7 Test environment.......................................................................................................................................... 6

8 Procedure....................................................................................................................................................... 7

9 Calculation..................................................................................................................................................... 9

10 Validity of the test ....................................................................................................................................... 12

11 Test report.................................................................................................................................................... 12

Annex A (informative) Example of expression of results..................................................................................... 13

Bibliography.............................................................................................................................................................. 16

© ISO 2002 – All rights reserved iii
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ISO 14592-2:2002(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.

The main task of technical committees is to prepare International Standards. 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.

Attention is drawn to the possibility that some of the elements of this part of ISO 14592 may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 14592-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological

methods.

ISO 14592 consists of the following parts, under the general title Water quality — Evaluation of the aerobic

biodegradability of organic compounds at low concentrations:

 Part 1: Shake-flask batch test with surface water or surface water/sediment suspensions

 Part 2: Continuous flow river model with attached biomass
This corrected version of ISO 14592-2:2002 incorporates corrections to
 the term numbers 3.1.10 and 3.1.11;
 the reference given in the last line of 5.2;
 the reference given in the second line of 6.1.3;
 the reference given in the second line of 6.1.5;
 the reference given in the first line of 8.1.1;
 the reference given in the second line of 8.1.2;
 the reference given in the second line of the second paragraph of 8.2;
 the reference given in the third line of the second paragraph of 8.3
 the reference given in the fifth line of the first paragraph of 8.4;
 the reference given in the fourth line of the second paragraph of 8.4;
 the reference given in the second line of the first paragraph of 9.1;
 the reference given in the third line of the note under 9.2.
iv © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)
Introduction

This International Standard consists of two parts. Part 1 describes a die-away batch test for either surface water

with or without added sediment in suspension simulating either a pelagic aquatic environment or a

water-to-sediment interface. Part 2 describes a continuous flow system simulating a river with biomass attached to

stationary surfaces.

The test has been specifically designed to provide information on the biodegradation behaviour and kinetics for test

compounds at low concentrations, i.e. sufficiently low to simulate the biodegradation kinetics expected to occur in

natural environmental systems.

This method is designed to determine the primary biodegradation in a continuously operating test system

simulating a river. Before conducting this test, it is necessary to have information on the biodegradability behaviour

of the test compound (e.g. at usual test concentrations in standard biodegradation tests) and, if possible, on abiotic

degradability or elimination (e.g. photolysis, adsorption or stripping) under conditions which are comparable to

those of the river model and relevant physico-chemical data (e.g. water-solubility, adsorption coefficient K ) so as

to properly plan the experiment and interpret the results.
© ISO 2002 – All rights reserved v
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INTERNATIONAL STANDARD ISO 14592-2:2002(E)
Water quality — Evaluation of the aerobic biodegradability of
organic compounds at low concentrations —
Part 2:
Continuous flow river model with attached biomass

WARNING AND SAFETY PRECAUTIONS — Activated sludge, sewage and effluent contain potentially

pathogenic organisms. Therefore appropriate precautions should be taken when handling them. Toxic and

dangerous test compounds and those whose properties are unknown should be handled with care.

Radiolabelled compounds, if used, should be handled respecting existing rules and legislation.

1 Scope

This part of ISO 14592 specifies a method for evaluating the biodegradability of organic test compounds by aerobic

microorganisms in natural waters by means of a continuous flow river model with attached biomass.

This part of ISO 14592 is applicable to organic test compounds present in lower concentrations than those of

natural carbon substrates also present in the system. Under these conditions, the test compounds serve as a

secondary substrate and the kinetics for biodegradation would be expected to be first order (“non-growth” kinetics).

This part of ISO 14592 is applicable to organic test compounds, which under the conditions of the test and at the

chosen test concentration, are:
 water soluble;

 quantitatively detectable with appropriate analytical methods or available in radiolabelled form;

 non-volatile from aqueous solution (e.g. Henry's law constant < 1 Pa⋅m /mole);
 not significantly adsorbed;
 not photolyzed;
 not inhibitory to the microorganisms of the test system.

The test is not recommended for use as proof of ultimate biodegradability (mineralization) which is better assessed

using other standardized tests (see ISO/TR 15462).
2 Normative reference

The following normative document contains provisions which, through reference in this text, constitute provisions of

this part of ISO 14592. For dated references, subsequent amendments to, or revisions of, any of these publications

do not apply. However, parties to agreements based on this part of ISO 14592 are encouraged to investigate the

possibility of applying the most recent editions of the normative documents indicated below. For undated

references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain

registers of currently valid International Standards.
ISO/TR 15462, Water quality — Selection of tests for biodegradability
© ISO 2002 – All rights reserved 1
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ISO 14592-2:2002(E)
3 Terms, definitions and symbols
3.1 Terms and definitions

For the purposes of this part of ISO 14592, the following terms and definitions apply.

3.1.1
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

NOTE Total mineralization may be different from ultimate aerobic biodegradation in that total mineralization includes

secondary mineralization of biosynthesis products. The kinetics may therefore deviate from first-order kinetics in particular

towards the end of the experiment. In this part of ISO 14592, primary aerobic biodegradation is determined when using

substance specific analysis and total mineralization when using radiolabelled compounds.

3.1.2
primary biodegradation

structural change (transformation) of a chemical compound by microorganisms resulting in the loss of a specific

property of that compound
3.1.3
biochemical oxygen demand
BOD

mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological oxidation of

a chemical compound or organic matter in water

NOTE It is expressed in this case as milligrams of oxygen uptake per milligram or gram of test compound.

3.1.4
dissolved organic carbon
DOC

part of the organic carbon in a sample of water which cannot be removed by specified phase separation

NOTE Phase separation may be obtained, for example, by centrifugation of the water sample at 40 000 m/s for 15 min or

by membrane-filtration using membranes with pores of 0,45 µm diameter.
3.1.5
lag phase
lag

〈continuous flow-through test system〉 time from the start of a test until significant biodegradation (about 10 % of the

maximum level) can be measured
NOTE Lag phase is expressed in days.
3.1.6
degree of biodegradation

〈continuous flow-through test system〉 mean biodegraded amount of a test compound, calculated from the

measured concentrations in the inlet and the outlet of the system

NOTE The degree of biodegradation is determined when no further degradation can be measured and is expressed as a

percentage.
3.1.7
steady state

〈continuous flow-through test system with constant input〉 state where the concentration of a test compound

remains constant at any place and time
2 © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)
3.1.8
primary substrate

major carbon and energy source which is essential for growth or maintenance of microorganisms

3.1.9
secondary substrate

substrate component present at such low concentrations, that by its degradation, only insignificant amounts of

carbon and energy are supplied to the competent microorganisms, as compared to the carbon and energy supplied

by their degradation of primary substrates
3.1.10
degradation rate constant

rate constant for first order or pseudo first order kinetics which indicates the rate at which degradation processes

NOTE 1 The degradation rate constant is expressed as the inverse of days (d ).

NOTE 2 For a batch experiment, k is estimated from the initial part of the degradation curve obtained after the end of the lag

phase. For a continuously operating test system, k can be estimated from a mass balance for the reactor using data collected

under steady-state conditions.
3.1.11
degradation half-life
1/2

characteristic of the rate of a first-order reaction and corresponds to the time interval necessary for the

concentration to decrease by a factor of two
NOTE 1 The degradation half-life is expressed in days (d).

NOTE 2 The degradation half-life and the degradation rate constant are related by the following equation:

T = ln2/k
1/2

NOTE 3 The degradation half-life T for first-order reactions should not be confused with the half-life time, T , which is

1/2 50

often used to describe the environmental behaviour of pesticides and which is simply the time to reach 50 % of total

biodegradation. The half-life time T may be derived from degradation curves without making assumptions about the kinetics.

3.2 Symbols
Symbol Description Units
b width of a single tray metres (m)
c residual molar concentration of the test compound micromole per litre (µmol/l)

c initial molar concentration of the test compound in the inlet of tray 1 micromole per litre (µmol/l)

c final molar concentration of the test compound in the outlet of tray n micromole per litre (µmol/l)

D degree of biodegradation percentage

1) In ISO 31-8-13:1992, c is defined as the symbol for “molar concentration”, expressed in moles per litre and in

ISO 31-8-11.2:1992, ρ is defined as the symbol for “mass concentration”, expressed in kilograms per litre. Note that in ISO 31,

"concentration" of the test compound in solution is expressed in two ways:
 “ρ” refers to the mass of the test compound per unit volume;

 “c” is specifically used to mean the number of moles of the test compound per unit volume.

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ISO 14592-2:2002(E)
d depth of the layer of water above the glass beads metres (m)
r rate of biodegradation micrograms per litre per day
[µg/(l⋅d)]
k biodegradation rate constant inverse days (d )
eff
n number of the final tray
S free flow cross-section of a single tray square metres (m )
T degradation half-life days (d)
1/2
q volume flow rate cubic metres per day (m /d)
v axial flow speed metres per day (m/d)
x distance between tray 1 and tray n metres (m)
ρ residual mass concentration of the test compound micrograms per litre (µg/l)
ρ biomass mass concentration micrograms per litre (µg/l)

ρ initial mass concentration of the test compound in the inlet of tray 1 micrograms per litre (µg/l)

ρ final mass concentration of the test compound in the outlet of tray n micrograms per litre (µg/l)

ρ substrate mass concentration micrograms per litre (µg/l)
4 Principle

The test system consists of one or more test units (cascades) each usually containing seven trays. Each

continuously operating cascade is run with a mean hydraulic retention time of the test water of 24 h. The test water

containing organic carbon is used as the major carbon and energy source (primary substrate) for the

microorganisms. The organic test compound or the reference compound is added to the influent of the cascades as

a secondary substrate preferably at the lowest possible concentration after sufficient biomass has been developed.

The test mass concentration is dependent on the expected first order kinetics and the analytical tools (substance-

specific analyses or radiolabelled test compounds) used and usually should not exceed 200 µg/l. DOC

measurements are unsuitable for the determination of biodegradation as the test concentrations necessary are too

high. The test water is sampled regularly and the concentration of the test or the reference compound is measured.

Under steady state conditions, the difference between the inlet and outlet concentrations of the cascade is used to

determine the degree of biodegradation and to plot degradation curves (see annex A). The degradation rate

constant and the degradation half-life of the test and the reference compounds in this test system are calculated

using the measured data derived under steady-state conditions. These data, the degradation curves and any other

available information are used to evaluate the biodegradability of the test compound.

5 Reagents and media
5.1 Reagents

Use only reagents of recognized analytical grade and radiolabelled compounds of high radiochemical purity.

5.1.1 Deionized water, containing less than 1 mg/l DOC

5.1.2 Sodium hydrogen carbonate (NaHCO ) or any other suitable buffer (optional), for buffering the test

water. (A mass concentration of 50 mg/l has been shown to be suitable).
4 © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)

5.1.3 Mercury(II) chloride (HgCl ) (optional), mass concentration of 10 g/l, of which 20 ml/l is added to the water

sample containing the test or reference compound and used for stopping all biological activity.

5.1.4 Sodium azide (NaN ) (optional), added to a mass concentration of 10 g/l to 20 g/l in the water sample

containing the test or reference compound and used for stopping all biological activity.

5.2 Test water

Collect a sample of tap water or surface water (e.g. from ponds or rivers) and determine the hardness (6.2.11),

alkalinity (6.2.6), DOC (6.2.3), ammonium nitrogen (6.2.9) and phosphorus (6.2.10). This test water should have a

DOC mass concentration between 3 mg/l and 5 mg/l and mass concentrations of ammonium nitrogen (NH -N) and

phosphorus (P) < 1 mg/l each. Suitable surface water may be used directly.

In the case of tap water or if the DOC of the surface water is low, it is necessary to add organic medium to reach

the required DOC concentration. Obtain organic medium from either an effluent of a municipal wastewater

[11]

treatment plant or a laboratory treatment plant (for composition see for example ISO 11733 ). Fill a storage

vessel with the effluent of the secondary clarifier of this plant. Add the correct amount of effluent from the storage

vessel to the cascades. Do not use effluent that may have been pre-adapted to the test compound (e.g. from an

industrial wastewater treatment plant). Measure the DOC of the organic medium at appropriate intervals or with

each new batch.

Usually a ratio of water to organic medium between 1:1 and 10:1 is suitable. Use DOC-free tap water for dilution if

the DOC of the test water is too high. If the pH (6.2.6) of the test water is outside of the range of pH 6 to pH 9, take

suitable means to maintain the pH constant during the test, preferably at a pH of (7±1). For example, water of low

alkalinity could require buffering by the addition of sodium hydrogen carbonate (see 5.1.2).

6 Apparatus

6.1 Test system, consisting of at least one test cascade and the required storage vessels and dosing facilities.

Additional cascades are required if several test compounds or concentrations or the reference compound are

tested in parallel.

6.1.1 Cascade, each normally consisting of seven trays (6.1.1.1) installed in the form of an aquatic staircase

model.

On the short side of each tray (6.1.1.1), in the middle, downstream, is a hole fitted with a small tube (6.1.1.2) for

leading the test water containing the test or reference compound from one tray to the next in the cascade. The

bottom of each tray is covered with about 1 kg of glass beads (6.1.1.3) as artificial sediment serving as a support

for the growth of biofilm in the test system. The hole is fixed in such a way that the depth of the water is about 1 cm

above the glass beads and the volume of the water is 2 l ± 0,2 l.

This system of cascades is one type of river model for determining biodegradation kinetics, which has been shown

to be suitable during test development. It is also possible to use other test systems (e.g. different size and shape of

the trays, other sediments or different surface-volume relations) and other test conditions (e.g. flowrate of water,

hydraulic load, illumination, inoculation). In this case, all the relevant parameters of a different test system have to

be documented and taken into consideration for the test performance and the calculation of the test results.

6.1.1.1 Trays, shallow and rectangular, of about 3 l capacity, each placed at a vertical distance of about 5 cm

higher than the next one, e.g. plastic photographic washing tanks with side lengths of about 45 cm × 31 cm and a

water depth of about 2 cm.

On the short side of each tray, in the middle, downstream, is a hole for transferring the test water.

6.1.1.2 Tubes, fitted to each tray for leading the test water from one tray to the next.

6.1.1.3 Glass beads, 5 mm in diameter.
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ISO 14592-2:2002(E)

6.1.2 Storage vessels, for storing suitable water samples and organic medium (see clause 7) for one or several

days and fitted with outlets for tubes, which can be connected to the tray of the cascade.

6.1.3 Pumps, dispensers or diluters, for dosing test water, organic medium (see clause 7) and stock solution of

the test or the reference compound (see 8.1.2) in such a way that the required amounts and concentrations in the

system are obtained.

6.1.4 Means of mixing, in a separate mixing vessel, in the tubes or in the first trays.

6.1.5 Fluorescent tubes, in a row installed about 50 cm above the cascades and required if the system is to be

artificially lighted (see clause 7).
6.2 Analytical equipment, consisting of the following:

6.2.1 Equipment suitable for specific analyses, for determining primary biodegradation, depending on the

characteristics of the test and the reference compound.
6.2.2 Equipment for counting radioactivity (e.g. liquid scintillation counter).
[7]
6.2.3 Laboratory carbon analyser, for determining DOC (see e.g. ISO 8245 ).
6.2.4 Filtration apparatus or centrifuge.
6.2.5 Temperature measurement apparatus.
6.2.6 pH meter.
[3]

6.2.7 Biochemical oxygen demand (BOD) measurement equipment, using e.g. ISO 5815 .

[2]
6.2.8 Oxygen measurement apparatus, using e.g. ISO 5814 .
[10]
6.2.9 Ammonia concentration measurement equipment, using e.g. ISO 11732 .
[5]
6.2.10 Phosphorous measurement equipment, using e.g. ISO 6878 .
6.2.11 Water hardness, measurement equipment.
7 Test environment

The test should normally take place at a given room temperature in an enclosure free from vapours toxic to

microorganisms and without direct sunlight on the surface of the cascades. If the test is to occur at a certain

temperature, the test system has to be set up in a temperature-controlled room.

Algae are present in natural aquatic environments. Therefore, they should also be present in this test system. To

allow sufficient algal growth in the test system but to prevent excess growth, it is recommended that the test be

performed under controlled illumination. Depending on the locality, either diffuse daylight or illumination using a

white light for not more than 8 h per day may be appropriate. The intensity of the light reaching the surface of the

trays should be measured and adjusted to be about 2 300 lx and the wavelength of the light should be within the

range of 400 nm to 700 nm. Lamps with almost no ultraviolet light should be used to prevent photolysis. Usual

daylight fluorescent tubes fastened in a distance of about 1 m to 1,5 m above the trays have shown to be suitable.

6 © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)
8 Procedure
8.1 Preparation of test and reference compound stock solutions
8.1.1 Test compounds

Prepare a stock solution in deionized water (5.1.1) to a suitable concentration. A suitable concentration is one that

when used with the dosing apparatus, provides the desired test compound concentration throughout the test. In the

case of poorly water-soluble test compounds, a stock solution is usually prepared at the level of solubility. In this

case, make sure that the test concentration obtained in the test system can be adequately detected by the chosen

analytical means. If this is not the case, the compound cannot be tested. Store the stock solution in storage vessels

(6.1.2) of sufficient size throughout the test in a refrigerator to prevent biodegradation.

Determine the concentration of the test compound in the stock solution by specific analysis and compare it with the

expected theoretical value so as to ascertain whether the analytical recovery is acceptable (normally > 80 %).

Measure the concentration for each new batch of stock solution. Determine the pH of the stock solution. Extreme

pH values outside a range of, for example pH 4 to pH 10, indicate that the test compound at higher concentrations

may influence the pH of the test water. In this case, neutralize the stock solution [pH (7 ± 0,5)] but ensure that the

test compound does not precipitate out of solution. If this is unavoidable, even at reduced concentrations, the

compound cannot be tested.

The test compound should not be toxic to the organisms in the test system, especially to bacteria that are of most

importance for biodegradation processes. Inhibitory effects of the test compound on bacteria can be determined

[6]

using test methods such as the respiration inhibition test with activated sludge (see ISO 8192 ) or the cell

[9]

multiplication inhibition test with a pure culture (Pseudomonas) (see ISO 10712 ) or with activated sludge

[13]

microorganisms (see ISO 15522 ). Low test concentrations, such as the levels used in this test, should not

produce effects toxic to bacteria.
8.1.2 Reference compound (optional)

To check the quality of the biomass and the biodegradable potential in the system and as validity criterion (see

clause 10) a pre-test or a parallel test with a reference compound should be performed. Preferably use aniline,

which has shown to be suitable for the test system developed for this part of ISO 14592. Follow the biodegradation

by substance-specific analysis or use radiolabelled substance in the same concentration range as the test

compound.

Prepare a suitable stock solution, handle and dose the reference compound as described for the test compound.

8.2 Addition of test or reference compounds to the test water

Assemble the test system and fill it with test water (5.2). Connect the storage vessels to the first trays of the

respective cascade. It is recommended that the volumes in the storage vessels be measured at regular intervals to

determine the exact amount of test compound or reference compound delivered to the system.

Add the stock solution of the test or reference compound to the first tray of the system so as to obtain the desired

test concentration (see 8.1.1). The concentration of the test or reference compounds depends on their chemical

and physical properties (e.g. water-solubility, volatility), the analytical tools used and the necessity to test in a

concentration range where first order kinetics are expected. To ensure obtaining first order biodegradation kinetics,

reduce the test concentration t
...

SLOVENSKI STANDARD
SIST ISO 14592-2:2010
01-september-2010
.DNRYRVWYRGH9UHGQRWHQMHDHUREQHELRUD]JUDGOMLYRVWLRUJDQVNLKVSRMLQSUL
QL]NLKNRQFHQWUDFLMDKGHO3UHWRþQLUHþQLPRGHOVSULWUMHQRELRPDVR

Water quality - Evaluation of the aerobic biodegradability of organic compounds at low

concentrations - Part 2: Continuous flow river model with attached biomass

Qualité de l'eau - Évaluation de la biodégradabilité aérobie des composés organiques

présents en faibles concentrations - Partie 2: Modèle de cours d'eau à courant continu

avec biomasse associée
Ta slovenski standard je istoveten z: ISO 14592-2:2002
ICS:
13.060.10 Voda iz naravnih virov Water of natural resources
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 14592-2:2010 en,fr

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST ISO 14592-2:2010
---------------------- Page: 2 ----------------------
SIST ISO 14592-2:2010
INTERNATIONAL ISO
STANDARD 14592-2
First edition
2002-11-15
Corrected version
2003-08-01
Water quality — Evaluation of the aerobic
biodegradability of organic compounds at
low concentrations —
Part 2:
Continuous flow river model with attached
biomass
Qualité de l'eau — Évaluation de la biodégradabilité aérobie des composés
organiques présents en faibles concentrations —
Partie 2: Modèle de cours d'eau à courant continu avec biomasse associée
Reference number
ISO 14592-2:2002(E)
ISO 2002
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SIST ISO 14592-2:2010
ISO 14592-2:2002(E)
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© ISO 2002

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 either ISO at the address below or ISO's member body

in the country of the requester.
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Printed in Switzerland
ii © ISO 2002 – All rights reserved
---------------------- Page: 4 ----------------------
SIST ISO 14592-2:2010
ISO 14592-2:2002(E)
Contents Page

Foreword .................................................................................................................................................................... iv

Introduction................................................................................................................................................................. v

1 Scope.............................................................................................................................................................. 1

2 Normative reference...................................................................................................................................... 1

3 Terms, definitions and symbols .................................................................................................................. 2

4 Principle......................................................................................................................................................... 4

5 Reagents and media..................................................................................................................................... 4

6 Apparatus....................................................................................................................................................... 5

7 Test environment.......................................................................................................................................... 6

8 Procedure....................................................................................................................................................... 7

9 Calculation..................................................................................................................................................... 9

10 Validity of the test ....................................................................................................................................... 12

11 Test report.................................................................................................................................................... 12

Annex A (informative) Example of expression of results..................................................................................... 13

Bibliography.............................................................................................................................................................. 16

© ISO 2002 – All rights reserved iii
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SIST ISO 14592-2:2010
ISO 14592-2:2002(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.

The main task of technical committees is to prepare International Standards. 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.

Attention is drawn to the possibility that some of the elements of this part of ISO 14592 may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 14592-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological

methods.

ISO 14592 consists of the following parts, under the general title Water quality — Evaluation of the aerobic

biodegradability of organic compounds at low concentrations:

 Part 1: Shake-flask batch test with surface water or surface water/sediment suspensions

 Part 2: Continuous flow river model with attached biomass
This corrected version of ISO 14592-2:2002 incorporates corrections to
 the term numbers 3.1.10 and 3.1.11;
 the reference given in the last line of 5.2;
 the reference given in the second line of 6.1.3;
 the reference given in the second line of 6.1.5;
 the reference given in the first line of 8.1.1;
 the reference given in the second line of 8.1.2;
 the reference given in the second line of the second paragraph of 8.2;
 the reference given in the third line of the second paragraph of 8.3
 the reference given in the fifth line of the first paragraph of 8.4;
 the reference given in the fourth line of the second paragraph of 8.4;
 the reference given in the second line of the first paragraph of 9.1;
 the reference given in the third line of the note under 9.2.
iv © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)
Introduction

This International Standard consists of two parts. Part 1 describes a die-away batch test for either surface water

with or without added sediment in suspension simulating either a pelagic aquatic environment or a

water-to-sediment interface. Part 2 describes a continuous flow system simulating a river with biomass attached to

stationary surfaces.

The test has been specifically designed to provide information on the biodegradation behaviour and kinetics for test

compounds at low concentrations, i.e. sufficiently low to simulate the biodegradation kinetics expected to occur in

natural environmental systems.

This method is designed to determine the primary biodegradation in a continuously operating test system

simulating a river. Before conducting this test, it is necessary to have information on the biodegradability behaviour

of the test compound (e.g. at usual test concentrations in standard biodegradation tests) and, if possible, on abiotic

degradability or elimination (e.g. photolysis, adsorption or stripping) under conditions which are comparable to

those of the river model and relevant physico-chemical data (e.g. water-solubility, adsorption coefficient K ) so as

to properly plan the experiment and interpret the results.
© ISO 2002 – All rights reserved v
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SIST ISO 14592-2:2010
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SIST ISO 14592-2:2010
INTERNATIONAL STANDARD ISO 14592-2:2002(E)
Water quality — Evaluation of the aerobic biodegradability of
organic compounds at low concentrations —
Part 2:
Continuous flow river model with attached biomass

WARNING AND SAFETY PRECAUTIONS — Activated sludge, sewage and effluent contain potentially

pathogenic organisms. Therefore appropriate precautions should be taken when handling them. Toxic and

dangerous test compounds and those whose properties are unknown should be handled with care.

Radiolabelled compounds, if used, should be handled respecting existing rules and legislation.

1 Scope

This part of ISO 14592 specifies a method for evaluating the biodegradability of organic test compounds by aerobic

microorganisms in natural waters by means of a continuous flow river model with attached biomass.

This part of ISO 14592 is applicable to organic test compounds present in lower concentrations than those of

natural carbon substrates also present in the system. Under these conditions, the test compounds serve as a

secondary substrate and the kinetics for biodegradation would be expected to be first order (“non-growth” kinetics).

This part of ISO 14592 is applicable to organic test compounds, which under the conditions of the test and at the

chosen test concentration, are:
 water soluble;

 quantitatively detectable with appropriate analytical methods or available in radiolabelled form;

 non-volatile from aqueous solution (e.g. Henry's law constant < 1 Pa⋅m /mole);
 not significantly adsorbed;
 not photolyzed;
 not inhibitory to the microorganisms of the test system.

The test is not recommended for use as proof of ultimate biodegradability (mineralization) which is better assessed

using other standardized tests (see ISO/TR 15462).
2 Normative reference

The following normative document contains provisions which, through reference in this text, constitute provisions of

this part of ISO 14592. For dated references, subsequent amendments to, or revisions of, any of these publications

do not apply. However, parties to agreements based on this part of ISO 14592 are encouraged to investigate the

possibility of applying the most recent editions of the normative documents indicated below. For undated

references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain

registers of currently valid International Standards.
ISO/TR 15462, Water quality — Selection of tests for biodegradability
© ISO 2002 – All rights reserved 1
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ISO 14592-2:2002(E)
3 Terms, definitions and symbols
3.1 Terms and definitions

For the purposes of this part of ISO 14592, the following terms and definitions apply.

3.1.1
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

NOTE Total mineralization may be different from ultimate aerobic biodegradation in that total mineralization includes

secondary mineralization of biosynthesis products. The kinetics may therefore deviate from first-order kinetics in particular

towards the end of the experiment. In this part of ISO 14592, primary aerobic biodegradation is determined when using

substance specific analysis and total mineralization when using radiolabelled compounds.

3.1.2
primary biodegradation

structural change (transformation) of a chemical compound by microorganisms resulting in the loss of a specific

property of that compound
3.1.3
biochemical oxygen demand
BOD

mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological oxidation of

a chemical compound or organic matter in water

NOTE It is expressed in this case as milligrams of oxygen uptake per milligram or gram of test compound.

3.1.4
dissolved organic carbon
DOC

part of the organic carbon in a sample of water which cannot be removed by specified phase separation

NOTE Phase separation may be obtained, for example, by centrifugation of the water sample at 40 000 m/s for 15 min or

by membrane-filtration using membranes with pores of 0,45 µm diameter.
3.1.5
lag phase
lag

〈continuous flow-through test system〉 time from the start of a test until significant biodegradation (about 10 % of the

maximum level) can be measured
NOTE Lag phase is expressed in days.
3.1.6
degree of biodegradation

〈continuous flow-through test system〉 mean biodegraded amount of a test compound, calculated from the

measured concentrations in the inlet and the outlet of the system

NOTE The degree of biodegradation is determined when no further degradation can be measured and is expressed as a

percentage.
3.1.7
steady state

〈continuous flow-through test system with constant input〉 state where the concentration of a test compound

remains constant at any place and time
2 © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)
3.1.8
primary substrate

major carbon and energy source which is essential for growth or maintenance of microorganisms

3.1.9
secondary substrate

substrate component present at such low concentrations, that by its degradation, only insignificant amounts of

carbon and energy are supplied to the competent microorganisms, as compared to the carbon and energy supplied

by their degradation of primary substrates
3.1.10
degradation rate constant

rate constant for first order or pseudo first order kinetics which indicates the rate at which degradation processes

NOTE 1 The degradation rate constant is expressed as the inverse of days (d ).

NOTE 2 For a batch experiment, k is estimated from the initial part of the degradation curve obtained after the end of the lag

phase. For a continuously operating test system, k can be estimated from a mass balance for the reactor using data collected

under steady-state conditions.
3.1.11
degradation half-life
1/2

characteristic of the rate of a first-order reaction and corresponds to the time interval necessary for the

concentration to decrease by a factor of two
NOTE 1 The degradation half-life is expressed in days (d).

NOTE 2 The degradation half-life and the degradation rate constant are related by the following equation:

T = ln2/k
1/2

NOTE 3 The degradation half-life T for first-order reactions should not be confused with the half-life time, T , which is

1/2 50

often used to describe the environmental behaviour of pesticides and which is simply the time to reach 50 % of total

biodegradation. The half-life time T may be derived from degradation curves without making assumptions about the kinetics.

3.2 Symbols
Symbol Description Units
b width of a single tray metres (m)
c residual molar concentration of the test compound micromole per litre (µmol/l)

c initial molar concentration of the test compound in the inlet of tray 1 micromole per litre (µmol/l)

c final molar concentration of the test compound in the outlet of tray n micromole per litre (µmol/l)

D degree of biodegradation percentage

1) In ISO 31-8-13:1992, c is defined as the symbol for “molar concentration”, expressed in moles per litre and in

ISO 31-8-11.2:1992, ρ is defined as the symbol for “mass concentration”, expressed in kilograms per litre. Note that in ISO 31,

"concentration" of the test compound in solution is expressed in two ways:
 “ρ” refers to the mass of the test compound per unit volume;

 “c” is specifically used to mean the number of moles of the test compound per unit volume.

© ISO 2002 – All rights reserved 3
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SIST ISO 14592-2:2010
ISO 14592-2:2002(E)
d depth of the layer of water above the glass beads metres (m)
r rate of biodegradation micrograms per litre per day
[µg/(l⋅d)]
k biodegradation rate constant inverse days (d )
eff
n number of the final tray
S free flow cross-section of a single tray square metres (m )
T degradation half-life days (d)
1/2
q volume flow rate cubic metres per day (m /d)
v axial flow speed metres per day (m/d)
x distance between tray 1 and tray n metres (m)
ρ residual mass concentration of the test compound micrograms per litre (µg/l)
ρ biomass mass concentration micrograms per litre (µg/l)

ρ initial mass concentration of the test compound in the inlet of tray 1 micrograms per litre (µg/l)

ρ final mass concentration of the test compound in the outlet of tray n micrograms per litre (µg/l)

ρ substrate mass concentration micrograms per litre (µg/l)
4 Principle

The test system consists of one or more test units (cascades) each usually containing seven trays. Each

continuously operating cascade is run with a mean hydraulic retention time of the test water of 24 h. The test water

containing organic carbon is used as the major carbon and energy source (primary substrate) for the

microorganisms. The organic test compound or the reference compound is added to the influent of the cascades as

a secondary substrate preferably at the lowest possible concentration after sufficient biomass has been developed.

The test mass concentration is dependent on the expected first order kinetics and the analytical tools (substance-

specific analyses or radiolabelled test compounds) used and usually should not exceed 200 µg/l. DOC

measurements are unsuitable for the determination of biodegradation as the test concentrations necessary are too

high. The test water is sampled regularly and the concentration of the test or the reference compound is measured.

Under steady state conditions, the difference between the inlet and outlet concentrations of the cascade is used to

determine the degree of biodegradation and to plot degradation curves (see annex A). The degradation rate

constant and the degradation half-life of the test and the reference compounds in this test system are calculated

using the measured data derived under steady-state conditions. These data, the degradation curves and any other

available information are used to evaluate the biodegradability of the test compound.

5 Reagents and media
5.1 Reagents

Use only reagents of recognized analytical grade and radiolabelled compounds of high radiochemical purity.

5.1.1 Deionized water, containing less than 1 mg/l DOC

5.1.2 Sodium hydrogen carbonate (NaHCO ) or any other suitable buffer (optional), for buffering the test

water. (A mass concentration of 50 mg/l has been shown to be suitable).
4 © ISO 2002 – All rights reserved
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ISO 14592-2:2002(E)

5.1.3 Mercury(II) chloride (HgCl ) (optional), mass concentration of 10 g/l, of which 20 ml/l is added to the water

sample containing the test or reference compound and used for stopping all biological activity.

5.1.4 Sodium azide (NaN ) (optional), added to a mass concentration of 10 g/l to 20 g/l in the water sample

containing the test or reference compound and used for stopping all biological activity.

5.2 Test water

Collect a sample of tap water or surface water (e.g. from ponds or rivers) and determine the hardness (6.2.11),

alkalinity (6.2.6), DOC (6.2.3), ammonium nitrogen (6.2.9) and phosphorus (6.2.10). This test water should have a

DOC mass concentration between 3 mg/l and 5 mg/l and mass concentrations of ammonium nitrogen (NH -N) and

phosphorus (P) < 1 mg/l each. Suitable surface water may be used directly.

In the case of tap water or if the DOC of the surface water is low, it is necessary to add organic medium to reach

the required DOC concentration. Obtain organic medium from either an effluent of a municipal wastewater

[11]

treatment plant or a laboratory treatment plant (for composition see for example ISO 11733 ). Fill a storage

vessel with the effluent of the secondary clarifier of this plant. Add the correct amount of effluent from the storage

vessel to the cascades. Do not use effluent that may have been pre-adapted to the test compound (e.g. from an

industrial wastewater treatment plant). Measure the DOC of the organic medium at appropriate intervals or with

each new batch.

Usually a ratio of water to organic medium between 1:1 and 10:1 is suitable. Use DOC-free tap water for dilution if

the DOC of the test water is too high. If the pH (6.2.6) of the test water is outside of the range of pH 6 to pH 9, take

suitable means to maintain the pH constant during the test, preferably at a pH of (7±1). For example, water of low

alkalinity could require buffering by the addition of sodium hydrogen carbonate (see 5.1.2).

6 Apparatus

6.1 Test system, consisting of at least one test cascade and the required storage vessels and dosing facilities.

Additional cascades are required if several test compounds or concentrations or the reference compound are

tested in parallel.

6.1.1 Cascade, each normally consisting of seven trays (6.1.1.1) installed in the form of an aquatic staircase

model.

On the short side of each tray (6.1.1.1), in the middle, downstream, is a hole fitted with a small tube (6.1.1.2) for

leading the test water containing the test or reference compound from one tray to the next in the cascade. The

bottom of each tray is covered with about 1 kg of glass beads (6.1.1.3) as artificial sediment serving as a support

for the growth of biofilm in the test system. The hole is fixed in such a way that the depth of the water is about 1 cm

above the glass beads and the volume of the water is 2 l ± 0,2 l.

This system of cascades is one type of river model for determining biodegradation kinetics, which has been shown

to be suitable during test development. It is also possible to use other test systems (e.g. different size and shape of

the trays, other sediments or different surface-volume relations) and other test conditions (e.g. flowrate of water,

hydraulic load, illumination, inoculation). In this case, all the relevant parameters of a different test system have to

be documented and taken into consideration for the test performance and the calculation of the test results.

6.1.1.1 Trays, shallow and rectangular, of about 3 l capacity, each placed at a vertical distance of about 5 cm

higher than the next one, e.g. plastic photographic washing tanks with side lengths of about 45 cm × 31 cm and a

water depth of about 2 cm.

On the short side of each tray, in the middle, downstream, is a hole for transferring the test water.

6.1.1.2 Tubes, fitted to each tray for leading the test water from one tray to the next.

6.1.1.3 Glass beads, 5 mm in diameter.
© ISO 2002 – All rights reserved 5
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SIST ISO 14592-2:2010
ISO 14592-2:2002(E)

6.1.2 Storage vessels, for storing suitable water samples and organic medium (see clause 7) for one or several

days and fitted with outlets for tubes, which can be connected to the tray of the cascade.

6.1.3 Pumps, dispensers or diluters, for dosing test water, organic medium (see clause 7) and stock solution of

the test or the reference compound (see 8.1.2) in such a way that the required amounts and concentrations in the

system are obtained.

6.1.4 Means of mixing, in a separate mixing vessel, in the tubes or in the first trays.

6.1.5 Fluorescent tubes, in a row installed about 50 cm above the cascades and required if the system is to be

artificially lighted (see clause 7).
6.2 Analytical equipment, consisting of the following:

6.2.1 Equipment suitable for specific analyses, for determining primary biodegradation, depending on the

characteristics of the test and the reference compound.
6.2.2 Equipment for counting radioactivity (e.g. liquid scintillation counter).
[7]
6.2.3 Laboratory carbon analyser, for determining DOC (see e.g. ISO 8245 ).
6.2.4 Filtration apparatus or centrifuge.
6.2.5 Temperature measurement apparatus.
6.2.6 pH meter.
[3]

6.2.7 Biochemical oxygen demand (BOD) measurement equipment, using e.g. ISO 5815 .

[2]
6.2.8 Oxygen measurement apparatus, using e.g. ISO 5814 .
[10]
6.2.9 Ammonia concentration measurement equipment, using e.g. ISO 11732 .
[5]
6.2.10 Phosphorous measurement equipment, using e.g. ISO 6878 .
6.2.11 Water hardness, measurement equipment.
7 Test environment

The test should normally take place at a given room temperature in an enclosure free from vapours toxic to

microorganisms and without direct sunlight on the surface of the cascades. If the test is to occur at a certain

temperature, the test system has to be set up in a temperature-controlled room.

Algae are present in natural aquatic environments. Therefore, they should also be present in this test system. To

allow sufficient algal growth in the test system but to prevent excess growth, it is recommended that the test be

performed under controlled illumination. Depending on the locality, either diffuse daylight or illumination using a

white light for not more than 8 h per day may be appropriate. The intensity of the light reaching the surface of the

trays should be measured and adjusted to be about 2 300 lx and the wavelength of the light should be within the

range of 400 nm to 700 nm. Lamps with almost no ultraviolet light should be used to prevent photolysis. Usual

daylight fluorescent tubes fastened in a distance of about 1 m to 1,5 m above the trays have shown to be suitable.

6 © ISO 2002 – All rights reserved
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SIST ISO 14592-2:2010
ISO 14592-2:2002(E)
8 Procedure
8.1 Preparation of test and reference compound stock solutions
8.1.1 Test compounds

Prepare a stock solution in deionized water (5.1.1) to a suitable concentration. A suitable concentration is one that

when used with the dosing apparatus, provides the desired test compound concentration throughout the test. In the

case of poorly water-soluble test compounds, a stock solution is usually prepared at the level of solubility. In this

case, make sure that the test concentration obtained in the test system can be adequately detected by the chosen

analytical means. If this is not the case, the compound cannot be tested. Store the stock solution in storage vessels

(6.1.2) of sufficient size throughout the test in a refrigerator to prevent biodegradation.

Determine the concentration of the test compound in the stock solution by specific analysis and compare it with the

expected theoretical value so as to ascertain whether the analytical recovery is acceptable (normally > 80 %).

Measure the concentration for each new batch of stock solution. Determine the pH of the stock solution. Extreme

pH values outside a range of, for example pH 4 to pH 10, indicate that the test compound at higher concentrations

may influence the pH of the test water. In this case, neutralize the stock solution [pH (7 ± 0,5)] but ensure that the

test compound does not precipitate out of solution. If this is unavoidable, even at reduced concentrations, the

compound cannot be tested.

The test compound should not be toxic to the organisms in the test system, especially to bacteria that are of most

importance for biodegradation processes. Inhibitory effects of the test compound on bacteria can be determined

[6]

using test methods such as the respiration inhibition test with activated sludge (see ISO 8192 ) or the cell

[9]

multiplication inhibition test with a pure culture (Pseudomonas) (see ISO 10712 ) or with activated sludge

[13]

microorganisms (see ISO 15522 ). Low test concentrations, such as the levels used in this test, should not

produce effects toxic to bacteria.
8.1.2 Reference compound (optional)
To check the quality of th
...

NORME ISO
INTERNATIONALE 14592-2
Première édition
2002-11-15
Qualité de l'eau — Évaluation de la
biodégradabilité aérobie des composés
organiques présents en faibles
concentrations —
Partie 2:
Modèle de cours d'eau à courant continu
avec biomasse associée
Water quality — Evaluation of the aerobic biodegradability of organic
compounds at low concentrations —
Part 2: Continuous flow river model with attached biomass
Numéro de référence
ISO 14592-2:2002(F)
ISO 2002
---------------------- Page: 1 ----------------------
ISO 14592-2:2002(F)
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Version française parue en 2003
Publié en Suisse
ii © ISO 2002 — Tous droits réservés
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ISO 14592-2:2002(F)
Sommaire Page

Avant-propos..................................................................................................................................................... iv

Introduction ........................................................................................................................................................ v

1 Domaine d'application.......................................................................................................................... 1

2 Référence normative............................................................................................................................. 2

3 Termes, définitions et symboles ......................................................................................................... 2

4 Principe .................................................................................................................................................. 5

5 Réactifs et milieux................................................................................................................................. 5

6 Appareillage........................................................................................................................................... 6

7 Environnement d'essai......................................................................................................................... 7

8 Mode opératoire .................................................................................................................................... 7

9 Calculs.................................................................................................................................................. 10

10 Validité de l'essai ................................................................................................................................ 13

11 Rapport d'essai ................................................................................................................................... 13

Annexe A (informative) Exemple d'expression des résultats ...................................................................... 14

Bibliographie .................................................................................................................................................... 17

© ISO 2002 — Tous droits réservés iii
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ISO 14592-2:2002(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de

normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée

aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du

comité technique créé à cet effet. Les organisations internationales, gouvernementales et non

gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec

la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.

Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,

Partie 3.

Les projets de Normes internationales adoptés par les comités techniques sont soumis aux comités membres

pour vote. Leur publication comme Normes internationales requiert l'approbation de 75 % au moins des

comités membres votants.

L'attention est appelée sur le fait que certains des éléments de la présente partie de l'ISO 14592 peuvent faire

l'objet de droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour

responsable de ne pas avoir identifié de tels droits de propriété et averti de leur existence.

La Norme internationale ISO 14592-2 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau,

sous-comité SC 5, Méthodes biologiques.

L'ISO 14592 comprend les parties suivantes, présentées sous le titre général Qualité de l'eau — Évaluation

de la biodégradabilité aérobie des composés organiques présents en faibles concentrations:

— Partie 1: Essai en lots de flacons agités avec des eaux de surface ou des suspensions eaux de

surface/sédiments
— Partie 2: Modèle de cours d'eau à courant continu avec biomasse associée
L'annexe A est donnée uniquement à titre d'information.
iv © ISO 2002 — Tous droits réservés
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ISO 14592-2:2002(F)
Introduction

La présente Norme internationale comprend deux parties. La Partie 1 décrit un essai de disparition par lots

avec des eaux de surface avec ou sans suspension de sédiments, simulant soit un environnement aquatique

pélagique, soit une interface eau/sédiment. La Partie 2 décrit un système à courant continu simulant un cours

d'eau avec une biomasse associée à des surfaces stationnaires.

L'essai a été spécifiquement mis au point pour fournir des informations sur le comportement de

biodégradation et les cinétiques de dégradation de composés d'essai présents en faibles concentrations,

c'est-à-dire suffisamment basses pour assurer qu'elles simulent des cinétiques de biodégradation que l'on

rencontrerait dans des systèmes environnementaux naturels.

La méthode de la présente norme vise à déterminer la biodégradation primaire dans un système d'essai en

mode continu simulant un cours d'eau. Des informations sur le comportement de biodégradabilité du composé

d'essai (par exemple à des concentrations d'essai usuelles pour les essais de biodégradation normalisés) et,

si possible, des informations sur la dégradabilité abiotique ou l'élimination (photolyse, adsorption ou

élimination par exemple) dans des conditions comparables à celles du modèle de cours d'eau, ainsi que les

données physico-chimiques correspondantes (par exemple solubilité dans l'eau, coefficient d'adsorption K )

doivent être disponibles avant de procéder à l'essai afin de planifier l'expérience et d'interpréter les résultats

convenablement.
© ISO 2002 — Tous droits réservés v
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NORME INTERNATIONALE ISO 14592-2:2002(F)
Qualité de l'eau — Évaluation de la biodégradabilité aérobie des
composés organiques présents en faibles concentrations —
Partie 2:
Modèle de cours d'eau à courant continu avec biomasse
associée

AVERTISSEMENT ET PRÉCAUTIONS DE SÉCURITÉ — Les boues activées, les eaux usées et les

effluents contiennent des organismes potentiellement pathogènes. C'est pourquoi il convient de

prendre les mesures de précaution appropriées lors de leur manipulation. Il est recommandé de faire

preuve de prudence lors de la manipulation de composés d'essai toxiques et dangereux et de ceux

dont les propriétés sont inconnues. Il convient de respecter les règles et réglementations en vigueur

en cas de manipulation de composés radio-marqués.
1 Domaine d'application

La présente partie de l'ISO 14592 spécifie une méthode pour l'évaluation de la biodégradabilité de composés

d'essai organiques par des micro-organismes aérobies dans les eaux naturelles au moyen d'un modèle de

cours d'eau à courant continu avec biomasse associée.

La présente partie de l'ISO 14592 s'applique aux composés d'essai organiques présents en concentrations

plus faibles que celles des substrats carbonés naturels également présents dans le système. Dans ces

conditions, les composés d'essai ont la fonction de substrat secondaire, et il est attendu que la cinétique de

biodégradation soit du premier ordre (cinétique «sans croissance»).

La présente partie de l'ISO 14592 s'applique aux composés d'essai organiques qui, dans les conditions

d'essai et à la concentration choisie pour l'essai, sont
 solubles dans l'eau;

 détectables quantitativement par des méthodes d'analyse appropriées ou disponibles sous forme radio-

marquée;

 non volatils en solution aqueuse (par exemple, constante de Henry < 1 Pa⋅m /mole);

 non adsorbés de manière significative;
 non photolysés;
 non inhibiteurs pour les micro-organismes du système d'essai.

L'utilisation de cette méthode d'essai n'est pas recommandée comme preuve de la biodégradation ultime

(minéralisation), qui peut s'évaluer de manière plus fiable en utilisant d'autres essais normalisés (voir

l'ISO/TR 15462).
© ISO 2002 — Tous droits réservés 1
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ISO 14592-2:2002(F)
2 Référence normative

Le document normatif suivant contient des dispositions qui, par suite de la référence qui y est faite,

constituent des dispositions valables pour la présente partie de l'ISO 14592. Pour les références datées, les

amendements ultérieurs ou les révisions de ces publications ne s'appliquent pas. Toutefois, les parties

prenantes aux accords fondés sur la présente partie de l'ISO 14592 sont invitées à rechercher la possibilité

d'appliquer l'édition la plus récente du document normatif indiqué ci-après. Pour les références non datées, la

dernière édition du document normatif en référence s'applique. Les membres de l'ISO et de la CEI possèdent

le registre des Normes internationales en vigueur.
ISO/TR 15462 Qualité de l'eau — Sélection d'essais de biodégradabilité
3 Termes, définitions et symboles
3.1 Termes et définitions

Pour les besoins de la présente partie de l'ISO 14592, les termes et définitions suivants s'appliquent.

3.1.1
biodégradation aérobie ultime

décomposition d'un composé chimique ou d'une matière organique par des micro-organismes en présence

d'oxygène, en dioxyde de carbone, eau et sels minéraux des autres éléments éventuellement présents

(minéralisation), et production d'une nouvelle biomasse

NOTE La minéralisation totale peut être différente de la biodégradation aérobie ultime car la minéralisation totale

inclut la minéralisation secondaire des produits de biosynthèse. En conséquence, la cinétique peut dévier de la cinétique

du premier ordre, en particulier vers la fin de l'essai. Dans la présente partie de l'ISO 14592, la biodégradation aérobie

primaire est déterminée lorsqu'une analyse spécifique de la substance est utilisée, et la minéralisation totale est

déterminée lorsque des composés radio-marqués sont utilisés.
3.1.2
biodégradation primaire

modification structurelle (transformation) d'un composé chimique par des micro-organismes résultant en la

perte d'une propriété spécifique de ce composé
3.1.3
demande biochimique en oxygène
DBO

concentration massique d'oxygène dissous consommé dans des conditions déterminées par l'oxydation

biologique aérobie d'un composé chimique ou d'une matière organique dans l'eau

NOTE Elle est, dans ce cas, exprimée en milligrammes d'oxygène absorbé par milligramme ou gramme de composé

d'essai.
3.1.4
carbone organique dissous
COD

partie du carbone organique présent dans un échantillon d'eau qui ne peut être éliminée par une séparation

spécifiée des phases

NOTE La séparation des phases peut être obtenue, par exemple, par centrifugation de l'échantillon d'eau à

40 000 m/s pendant 15 min ou par filtration sur membrane dont le diamètre des pores est de 0,45 µm.

2 © ISO 2002 — Tous droits réservés
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ISO 14592-2:2002(F)
3.1.5
phase de latence
latence

〈système d'essai à circulation continue〉 période entre le début d'un essai et le moment où une dégradation

significative (environ 10 % du niveau maximal de dégradation) peut être mesurée
NOTE La phase de latence est exprimée en jours (j).
3.1.6
degré de biodégradation

〈système d'essai à circulation continue〉 quantité biodégradée moyenne d'un composé d'essai, calculée à

partir des concentrations mesurées à l'entrée et la sortie du système

NOTE Le degré de biodégradation est déterminé lorsque aucune dégradation ne peut plus être mesurée et est

exprimé en pourcentage.
3.1.7
état permanent

〈système d'essai à circulation continue avec alimentation constante〉 état où la concentration d'un composé

d'essai reste constante en tout lieu et à tout moment
3.1.8
substrat primaire

source majeure de carbone et d'énergie essentielle à la croissance ou au maintien des micro-organismes

3.1.9
substrat secondaire

élément de substrat présent à des concentrations si faibles que, par sa dégradation, seules des quantités

insignifiantes de carbone et d'énergie sont fournies aux micro-organismes, par comparaison avec le carbone

et l'énergie fournis par la dégradation des substrats primaires
3.1.10
constante de vitesse de dégradation

constante de vitesse pour cinétique de premier ordre ou de pseudo-premier ordre, qui indique la vitesse des

processus de dégradation

NOTE 1 La constante de vitesse de dégradation est exprimée en inverse de jours (j ).

NOTE 2 Dans le cadre d'une expérience par lots, k est estimée à partir de la partie initiale de la courbe de dégradation

obtenue après la fin de la phase de latence. Dans le cadre de systèmes d'essai fonctionnant en continu, k peut être

estimée à partir d'un bilan massique sur le réacteur en utilisant les données recueillies en conditions d'état permanent.

3.1.11
demi-vie de dégradation
1/2

caractéristique de la vitesse d'une réaction du premier ordre: intervalle de temps nécessaire à une réduction

de la concentration par un facteur de deux
NOTE 1 La demi-vie de dégradation est exprimée en jours (j).

NOTE 2 La demi-vie de dégradation et la constante de vitesse de dégradation sont liées par l'équation suivante:

T = ln2/k
1/2

NOTE 3 Il convient de ne pas confondre la demi-vie de dégradation T pour les réactions du premier ordre avec la

1/2

durée de demi-vie, T , qui est souvent utilisée pour décrire le comportement environnemental des pesticides, et qui

correspond simplement au temps nécessaire pour atteindre 50 % de la biodégradation totale. La durée de demi-vie T

peut être dérivée de courbes de dégradation sans faire d'hypothèses sur les cinétiques.

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ISO 14592-2:2002(F)
3.2 Symboles
Symbole Description Unité
b largeur d'un plateau mètres (m)

c concentration molaire résiduelle du composé d'essai micromoles par litre (µmol/l)

c concentration molaire initiale du composé d'essai à l'entrée micromoles par litre (µmol/l)

du plateau 1

c concentration molaire finale du composé d'essai à la sortie micromoles par litre (µmol/l)

du plateau n
D degré de biodégradation pourcentage
d profondeur de la couche d'eau au-dessus des billes de verre mètres (m)
r vitesse de biodégradation microgrammes par litre
par jour [µg/(l⋅j)]
k constante de vitesse de biodégradation inverse de jours (j )
eff
n numéro du dernier plateau
S section d'écoulement libre d'un plateau mètres carrés (m )
T demi-vie de dégradation jours (j)
1/2
q débit mètres cubes par jour (m /j)
v vitesse d'écoulement axial mètres par jour (m/j)
x distance entre le plateau 1 et le plateau n mètres (m)

ρ concentration massique résiduelle du composé d'essai microgrammes par litre (µg/l)

ρ concentration massique de la biomasse microgrammes par litre (µg/l)

ρ concentration massique initiale du composé d'essai à l'entrée microgrammes par litre (µg/l)

du plateau 1

ρ concentration massique finale du composé d'essai à la sortie microgrammes par litre (µg/l)

du plateau n
ρ concentration massique du substrat microgrammes par litre (µg/l)

1) Dans l'ISO 31-8-13:1992, c est défini comme le symbole pour la «concentration molaire», exprimée en moles par litre,

et dans l'ISO 31-8-11.2:1992, ρ est défini comme le symbole pour la «concentration massique», exprimée en kilogrammes

par litre. On remarque que dans l'ISO 31, la «concentration» du composé d'essai dans la solution est exprimée de deux

manières:
 «ρ» est la masse du composé d'essai par unité de volume;

 «c» est utilisé spécifiquement pour le nombre de moles du composé d'essai par unité de volume.

4 © ISO 2002 — Tous droits réservés
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ISO 14592-2:2002(F)
4 Principe

Le système d'essai consiste en une ou plusieurs unités d'essai (cascades) contenant chacune sept plateaux.

Une cascade à action continue fonctionne sur la base d'un temps de rétention hydraulique moyen de l'eau

d'essai de 24 h. L'eau d'essai contenant du carbone organique est employée comme source principale de

carbone et d'énergie (substrat primaire) pour les micro-organismes. Le composé d'essai organique ou la

substance de référence est ajouté à l'influent des cascades comme substrat secondaire, de préférence à la

plus faible concentration possible, après le développement d'une biomasse suffisante. La concentration

massique d'essai est dépendante des cinétiques du premier ordre escomptées et des outils d'analyse

(analyses spécifiques de la substance ou composés d'essai radio-marqués) employés. Il convient qu'elle ne

dépasse pas 200 µg/l. Les dosages du COD ne sont pas appropriés pour la détermination de la

biodégradation car les concentrations d'essai nécessaires sont trop élevées. L'eau d'essai est échantillonnée

à intervalles réguliers, et la concentration du composé d'essai ou de la substance de référence est mesurée.

Dans des conditions d'état permanent, la différence entre les concentrations en entrée et en sortie de la

cascade est utilisée pour déterminer le degré de biodégradation et pour tracer des courbes de dégradation

(voir l'annexe A). Dans ce système d'essai, la constante de vitesse de dégradation et la demi-vie de

dégradation du composé d'essai et de la substance de référence sont calculées en utilisant les mesures

dérivées en conditions d'état permanent. Ces données, les courbes de dégradation et toute autre information

disponible sont utilisées pour évaluer la biodégradabilité du composé d'essai.
5 Réactifs et milieux
5.1 Réactifs

Utiliser exclusivement des réactifs de qualité analytique reconnue et des composés radio-marqués de grande

pureté radiochimique.
5.1.1 Eau déionisée, contenant moins de 1 mg/l de COD.

5.1.2 Bicarbonate de sodium (NaHCO ) ou tout autre tampon approprié (facultatif), pour tamponner l'eau

d'essai. (Une concentration massique de 50 mg/l s'est révélée appropriée.)

5.1.3 Chlorure mercurique (HgCl ) (facultatif), à une concentration massique de 10 g/l, dont 20 ml/l est

ajouté à l'échantillon d'eau contenant le composé d'essai ou la substance de référence afin de stopper toute

activité biologique.

5.1.4 Azoture de sodium (NaN ), (facultatif), ajouté à une concentration massique comprise entre 10 g/l et

20 g/l à l'échantillon d'eau contenant le composé d'essai ou la substance de référence afin de stopper toute

activité biologique.
5.2 Eau d'essai

Prélever un échantillon d'eau de distribution ou d'eau de surface (provenant d'étangs ou de rivières, par

exemple) et en déterminer la dureté (6.2.11), l'alcalinité (6.2.6), le COD (6.2.3), l'azote ammoniacal (6.2.9) et

le phosphore (6.2.10). Il convient que cette eau d'essai ait une concentration massique en COD comprise

entre 3 mg/l et 5 mg/l, et des concentrations massiques en azote ammoniacal (NH -N) et en phosphore

(P) < 1 mg/l chacune. De l'eau de surface appropriée peut être utilisée directement.

En cas d'utilisation d'eau de distribution, ou si la concentration en COD de l'eau de surface est trop faible, il

est nécessaire d'ajouter un milieu organique pour atteindre la concentration requise en COD. Se procurer le

milieu organique à partir d'un effluent d'une station d'épuration municipale ou d'une station de traitement en

[11]

laboratoire (pour la composition, voir par exemple l'ISO 11733 ). Remplir un récipient de stockage avec

l'effluent provenant du clarificateur secondaire de cette station. Ajouter la quantité correcte d'effluent du

récipient de stockage dans les cascades. Ne pas utiliser un effluent qui aurait pu être préadapté au composé

d'essai (provenant d'une station d'épuration industrielle, par exemple). Mesurer la teneur en COD du milieu

organique à intervalles appropriés ou à chaque nouveau lot.
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ISO 14592-2:2002(F)

Habituellement, une proportion eau/milieu organique comprise entre 1:1 et 10:1 est appropriée. Utiliser de

l'eau de distribution exempte de COD pour la dilution si le COD de l'eau d'essai est trop élevé. Si le pH (6.2.6)

de l'eau d'essai est en dehors de la plage pH 6 à pH 9, prendre des moyens appropriés pour maintenir le pH

constant durant l'essai, de préférence à un pH de (7 ± 1). Par exemple, une eau à faible alcalinité peut

nécessiter un tamponnage par l'ajout de bicarbonate de sodium (voir 5.1.2).
6 Appareillage

6.1 Système d'essai, comprenant au moins une cascade d'essai, ainsi que les récipients de stockage et le

matériel de dosage requis.

Il est nécessaire de prévoir des cascades supplémentaires dans le cas où plusieurs composés d'essai ou

concentrations de la substance de référence sont analysés en parallèle.

6.1.1 Cascade, comprenant normalement sept plateaux (6.1.1.1) installés en escalier.

Au milieu du petit côté aval de chaque plateau (6.1.1.1) se trouve un orifice traversé par un tube de petite

taille (6.1.1.2) qui permet d'acheminer d'un plateau à l'autre l'eau d'essai contenant le composé d'essai ou la

substance de référence. Le fond de chaque plateau est recouvert d'environ 1 kg de billes de verre (6.1.1.3)

jouant le rôle de sédiment artificiel et de support de croissance du biofilm utilisé dans le système d'essai.

L'orifice est positionné de manière à ce que le niveau de l'eau soit à environ 1 cm au-dessus des billes de

verre et à ce que le volume d'eau soit de 2 l ± 0,2 l.

Ce système de cascade est un type de modèle de cours d'eau pour la détermination de la cinétique de

biodégradation qui s'est révélé approprié au cours de la mise au point de l'essai. Il est également possible

d'utiliser d'autres systèmes d'essai (par exemple, des formes et des tailles de plateaux différentes, d'autres

sédiments ou des rapports surface/volume différents) ou des conditions d'essai différentes (par exemple,

débit d'eau, charge hydraulique, éclairage, ensemencement). En cas d'utilisation d'un système d'essai

différent, il faut indiquer et prendre en considération tous les paramètres applicables pour la performance et le

calcul des résultats de l'essai.

6.1.1.1 Plateaux, rectangulaires et peu profonds, d'une capacité d'environ 3 l, placés chacun à une

distance verticale d'environ 5 cm au-dessus du suivant, par exemple des bacs de rinçage photographique

d'environ 45 cm × 31 cm de côtés avec une profondeur d'eau d'environ 2 cm.

Au milieu du petit côté aval de chaque plateau se trouve un orifice pour le transfert de l'eau d'essai.

6.1.1.2 Tubes, disposés sur chaque plateau pour guider l'eau d'essai d'un plateau au suivant.

6.1.1.3 Billes de verre, de 5 mm de diamètre.

6.1.2 Récipients de stockage, pour le stockage d'échantillons d'eau appropriés et du milieu organique

(voir l'article 7) pendant un ou plusieurs jours et dotés d'orifices de sortie pour tubes, qui peuvent être reliés

au plateau de la cascade.

6.1.3 Pompes, distributeurs ou dilueurs, pour doser l'eau d'essai, le milieu organique (voir l'article 7) et

une solution mère du composé d'essai ou de la substance de référence (voir 8.1.2) de façon à obtenir les

quantités et les concentrations requises dans le système.

6.1.4 Mélangeur, dans un réservoir de mélange séparé, dans les tubes ou dans les premiers plateaux.

6.1.5 Tubes fluorescents, positionnés en batterie à environ 50 cm au-dessus des cascades, nécessaires

si le système doit être éclairé artificiellement (voir l'article 7).
6.2 Matériel d'analyse, consistant en ce qui suit:

6.2.1 Matériel approprié pour les analyses spécifiques, visant à déterminer la biodégradation primaire,

selon les caractéristiques du composé d'essai et de la substance de référence.
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ISO 14592-2:2002(F)

6.2.2 Matériel pour tracer la radioactivité (par exemple, compteur à scintillation en phase liquide).

6.2.3 Analyseur de carbone de laboratoire, pour déterminer la concentration en COD (voir par exemple

[7]
l'ISO 8245 ).
6.2.4 Appareillage de filtration ou centrifugeuse.
6.2.5 Appareillage de mesure de température.
6.2.6 pH-mètre.

6.2.7 Matériel pour déterminer la demande biochimique en oxygène (DBO), en utilisant par exemple

[3]
l'ISO 5815 .
[2]
6.2.8 Appareillage de mesure de l'oxygène, en utilisant par exemple l'ISO 5814 .
[10]

6.2.9 Matériel de mesure de la concentration en ammonium, en utilisant par exemple l'ISO 11732 .

[5]
6.2.10 Matériel de mesure du phosphore, en utilisant par exemple l'ISO 6878 .
6.2.11 Matériel de mesure de la dureté de l'eau.
7 Environnement d'essai

Il convient normalement que l'essai soit réalisé à une température ambiante donnée, dans une enceinte

exempte de vapeurs toxiques pour les micro-organismes et sans éclairage direct du soleil sur les surfaces

des cascades. Si l'essai doit être conduit à une température spécifique, le système d'essai doit être établi

dans une salle à température régulée.

Dans les environnements aquatiques naturels, des algues sont présentes. Par conséquent, il convient qu'elles

soient aussi présentes dans ce système d'essai. Pour permettre une croissance suffisante des algues dans le

système d'essai, sans qu'elle soit excessive, il est recommandé de conduire l'essai sous éclairage contrôlé.

Selon la situation locale, une lumière de jour diffuse ou un éclairage à la lumière blanche pendant au plus 8 h

par jour peuvent être appropriés. Il convient de mesurer et d'ajuster à 2 300 lx l'intensité de la lumière à la

surface de l'eau des plateaux. Il est recommandé que la longueur d'onde de la lumière soit comprise entre

400 nm et 700 nm. Il convient d'utiliser des lampes quasiment sans lumière ultraviolette pour éviter la

photolyse. Des tubes fluorescents d'éclairage usuels fixés à une distance d'environ 1 m à 1,5 m au-dessus

des plateaux se sont révélés appropriés.
8 Mode opératoire

8.1 Préparation de solutions mères pour les composés d'essai et la substance de référence

8.1.1 Composés d'essai

Préparer une solution mère dans de l'eau déionisée (5.1.1) à une concentration appropriée. Une

concentration appropriée est une concentration qui permet d'obtenir, avec l'appareillage de dosage utilisé, la

concentration souhaitée du composé d'essai pendant toute la durée de l'essai. En cas de composé d'essai

faiblement soluble dans l'eau, la solution mère est généralement préparée au niveau de solubilité. Dans ce

cas, il faut s'assurer que la concentration obtenue dans le système d'essai soit effectivement détectable par la

méthode d'analyse choisie. Dans le cas contraire, il est impossible d'analyser le composé. Conserver la

solution mère dans un récipient de stockage (6.1.2) de taille suffisante pendant l'essai, afin d'éviter tout risque

de biodégradation.

Déterminer la concentration du composé d'essai dans la solution mère en appliquant une analyse spécifique,

puis la comparer à la valeur théorique escomptée afin de déterminer si la récupération analytique est

© ISO 2002 — Tous droits réservés 7
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ISO 14592-2:2002(F)
acceptable (normalem
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