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ICS 75.100

SR EN 17181

Standard Român Iulie 2019

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Titlu Lubrifianți
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Determinarea biodegradabilității aerobe, în

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soluție apoasă, a lubrifianților complet

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formulați

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Metodă de încercare bazată pe producerea de

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CO2 ro
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Lubricants - Determination of aerobic biological degradation of fully formulated

lubricants in an aqueous solution - Test method based on CO2-production
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Bio-lubrifiants - Détermination de la biodégradabilité aérobie, en solution

aqueuse, de lubrifiants complètement formulés - Méthode basée sur le
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dégagement de CO2
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Aprobare Aprobat de Directorul General al ASRO la 31 iulie 2019

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Standardul european EN 17181:2019 are statutul unui standard român

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Data publicării versiunii române: -

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Corespondență Acest standard este identic cu standardul european EN 17181:2019

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ASOCIAŢIA DE STANDARDIZARE DIN ROMÂNIA

 Str. Mendeleev nr. 21-25, cod 010362, București ,  www.asro.ro

© ASRO Reproducerea sau utilizarea integrală sau parțială a prezentului standard în orice
publicații și prin orice procedeu (electronic, mecanic, fotocopiere, microfilmare etc.) este
interzisă dacă nu există acordul scris prealabil al ASRO

Ref.: SR EN 17181:2019 Ediția 1

 EUROPEAN STANDARD EN 17181
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NORME EUROPÉENNE
EUROPÄISCHE NORM February 2019

ICS 75.100

English Version

Lubricants - Determination of aerobic biological

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degradation of fully formulated lubricants in an aqueous

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solution - Test method based on CO2-production

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Bio-lubrifiants - Détermination de la biodégradabilité Schmierstoffe - Bestimmung der aeroben biologischen
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aérobie, en solution aqueuse, de lubrifiants Abbaubarkeit ausformulierter Schmiermittel in
complètement formulés - Méthode basée sur le wässrigem Medium - Testmethode basierend auf CO2-

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dégagement de CO2 Bestimmung

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This European Standard was approved by CEN on 14 December 2018.

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CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
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European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
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member.
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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 CEN-CENELEC Management
Centre has the same status as the official versions.
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CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
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Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
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EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17181:2019 E
worldwide for CEN national Members.
 EN 17181:2019 (E)
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Contents Page

European foreword....................................................................................................................................................... 3
Introduction .................................................................................................................................................................... 4
1 Scope .................................................................................................................................................................... 5
2 Normative references .................................................................................................................................... 5
3 Terms and definitions ................................................................................................................................... 5
4 Principle ............................................................................................................................................................. 8

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5 Test environment ............................................................................................................................................ 8

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6 Reagents ............................................................................................................................................................. 8

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7 Apparatus........................................................................................................................................................... 9
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8 Procedure........................................................................................................................................................ 10

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8.1 Preparation of mineral medium ............................................................................................................. 10

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8.2 Preparation of the test volumes ............................................................................................................. 10
8.2.1 Test sample .................................................................................................................................................... 10

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8.2.2 Reference compound .................................................................................................................................. 11
8.2.3
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Inhibition control ......................................................................................................................................... 11
Elimination control ..................................................................................................................................... 11
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8.3 Preparation of the inoculum .................................................................................................................... 11
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8.3.1 General ............................................................................................................................................................. 11

8.3.2 Inoculum from an activated sludge plant ............................................................................................ 11
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8.3.3 Pre-conditioning........................................................................................................................................... 11
8.3.4 Pre-exposure ................................................................................................................................................. 11
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8.4 Test .................................................................................................................................................................... 12

Table 1 — Overview on the desired final test matrix .................................................................................... 12
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9 Calculation and reporting of results ..................................................................................................... 13

9.1 Theoretical amount of CO2 released from test sample .................................................................. 13
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9.2 Percentage biodegradation ...................................................................................................................... 13

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9.3 Expression of results................................................................................................................................... 14

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10 Validity and precision................................................................................................................................. 14

10.1 Validity ............................................................................................................................................................. 14
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10.2 Precision .......................................................................................................................................................... 15

10.2.1 General ............................................................................................................................................................. 15
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10.2.2 Repeatability.................................................................................................................................................. 15
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10.2.3 Reproducibility ............................................................................................................................................. 15

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11 Test report ...................................................................................................................................................... 15

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Annex A (informative) Comparison table of most common mineralisation methods ..................... 17

Annex B (informative) Sample insertion .......................................................................................................... 22
Annex C (informative) Specification of reference compound High Oleic Reference Oil
(HORO) ............................................................................................................................................................. 24
Annex D (informative) Example of degradation curves .............................................................................. 25
Figure D.1 — Example of degradation curve .................................................................................................... 25
Bibliography ................................................................................................................................................................. 26

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 EN 17181:2019 (E)
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European foreword

This document (EN 17181:2019) has been prepared by Technical Committee CEN/TC 19 “Gaseous and
liquid fuels, lubricants and related products of petroleum, synthetic and biological origin”, the
secretariat of which is held by NEN.

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 August 2019, and conflicting national standards shall
be withdrawn at the latest by August 2019.

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Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

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This document has been prepared under a mandate given to CEN by the European Commission and the

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European Free Trade Association.

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According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

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following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
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France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
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Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
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 EN 17181:2019 (E)
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Introduction

Existing test methods for determination of aerobic biodegradation of organic substances in an aqueous
medium (e.g. numerous ISO and OECD test methods) are widely used. Moreover, all actually existing
ecolabels, international and national regulations and recommendations are working with them.
Testing of organic substances requires a broad scope of preparation tools to maintain comparable
results for all different materials appearing liquid, solid, water-soluble, non-water-soluble or
emulsifiable.

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In consequence of this, results may differ regarding the choice of test method, preparation procedure or
inoculum thus leading to low levels of reproducibility and repeatability.

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Fully formulated lubricants (oils or greases or emulsions) are complex mixtures of mostly organic

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compounds that are mainly water insoluble. Based on ISO 9439 (resp. OECD 301B) this test method has
been aligned by modifying sample preparation and test procedure for such complex mixtures in order
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to achieve improved significance of test results. The test method described in this document, however,
was not assessed for greases. The precision statement therefore only applies for lubricating oils.

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Finally, from customer’s point of view a biodegradation test method for the final product of improved

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significance will result in better market transparency, enhanced reputation of “bio-lubricants” and

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confidence of the customer in this product group. ro
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 EN 17181:2019 (E)
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1 Scope
This document specifies a procedure for determining the degree of aerobic degradation of fully
formulated lubricants. The organic material in a fully formulated lubricant is exposed in a synthetic
aqueous medium under laboratory conditions to an inoculum from activated sludge.
Biodegradation resulting in mineralisation of the organic material can be determined by measuring
released CO2 in a total organic carbon (TOC) analyser. In contrast to existing test methods measuring
released CO2 this method uses a precise preparation procedure for non-water soluble organic material.

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The above mentioned method applies to fully formulated lubricants which

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a) are water-soluble, non-water soluble or emulsifiable, and

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b) are not toxic and not inhibitory to the test microorganisms at the test concentration.
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The presence of inhibitory effects is determined as specified in this document.

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This test method is focused on fresh water as test medium. Tests in sea water are currently not included
in this method, but may be introduced later.

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2 Normative references
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The following documents are referred to in the text in such a way that some or all of their content
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constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
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ISO 9439, Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in
aqueous medium — Carbon dioxide evolution test
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3 Terms and definitions

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For the purposes of this document, the following terms and definitions apply.
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ISO and IEC maintain terminological databases for use in standardization at the following addresses:
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• IEC Electropedia: available at http://www.electropedia.org/

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• ISO Online browsing platform: available at http://www.iso.org/obp

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3.1
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organic material
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total amount of all organic compounds a fully formulated lubricant consists of

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3.2
aerobic biodegradation
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consumption of organic materials by microorganisms in a biochemical process under use of oxygen,

resulting in cleavage of chemical bonds and CO2 production providing energy and/or new biomass

3.3
primary biodegradation
first structural change (transformation) of an organic chemical compound by microorganisms resulting
in the change of a specific property

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3.4
mineralisation
aerobic biodegradation of organic material by microorganisms to yield carbon dioxide, water and
mineral salts of any other elements present and new biomass

3.5
activated sludge
biomass produced in the aerobic treatment of wastewater by the growth of bacteria and other
microorganisms in the presence of dissolved oxygen

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3.6
concentration of suspended solids of an activated sludge

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amount of solids obtained by filtration or centrifugation of a known volume of activated sludge and

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drying at about 105 °C to constant mass
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3.7

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inoculum

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sample of activated sludge for the purpose of this test method used in degradation procedures
described in this method

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3.8
reference compound
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organic compound of known biodegradability with a degradation degree of more than 60 %
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3.9
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dissolved organic carbon

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

Note 1 to entry For example by centrifugation at 4 000 rpm for 15 min or by membrane filtration using
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membranes with pores of 0,2 μm to 0,45 μm diameter.

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3.10
total inorganic carbon
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TIC
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total amount of inorganic carbon in a water sample arising from carbon dioxide and carbonates
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3.11
dissolved inorganic carbon
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DIC
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part of the inorganic carbon in water which cannot be removed by specified phase separation
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Note 1 to entry For example by centrifugation at 4 000 rpm for 15 min or by membrane filtration using
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membranes with pores of 0,2 μm to 0,45 μm diameter.

3.12
theoretically released amount of carbon dioxide
ThCO2
theoretical maximum amount of carbon dioxide released from a chemical compound, calculated from
TOC content

Note 1 to entry Expressed in this case as milligrams of carbon dioxide evolved per milligram or gram of test
compound.

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 EN 17181:2019 (E)
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3.13
total organic carbon
TOC
amount of carbon bound in an organic compound

Note 1 to entry Refer to ISO 8245 [17] for further details.

3.14
biochemical oxygen demand
BOD

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mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological
oxidation of a chemical compound or organic matter in water

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Note 1 to entry Expressed in this case as milligrams oxygen uptake per milligram or gram of test compound.

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3.15

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chemical oxygen demand
COD

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mass concentration of oxygen equivalent to the amount of a specified oxidant consumed by a chemical

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compound or organic matter when a water sample is treated with that oxidant under defined

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conditions ro
Note 1 to entry Expressed in this case as milligrams oxygen consumed per milligram or gram of test compound.
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3.16
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theoretical oxygen demand

ThOD
theoretical maximum amount of oxygen required to oxidize a chemical compound completely,
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calculated from elemental analysis

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Note 1 to entry Expressed in this case as milligrams of oxygen uptake per milligram or gram of test compound.

3.17
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lag phase
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time from the start of a test until adaptation and selection of the degrading microorganisms are
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achieved and the biodegradation degree of a chemical compound or organic matter has increased to
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about 10 % of the theoretical maximum biodegradation

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Note 1 to entry This is expressed in days.

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3.18
maximum level of biodegradation
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maximum biodegradation degree of a chemical compound in a test, above which no further

biodegradation takes place during the test
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Note 1 to entry This is expressed in percentage.

3.19
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 1 to entry This is expressed in days.

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3.20
plateau phase
time from the end of the biodegradation phase when the maximum level of biodegradation is reached
until the end of the test

3.21
pre-conditioning
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

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3.22

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pre-exposure

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pre-incubation of an inoculum in the presence of the test chemical compound or organic matter under
test, with the aim of enhancing the ability of the inoculum to biodegrade the test material by adaptation
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and/or selection of the microorganisms

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4 Principle

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Biodegradation is followed over a specified period by measuring the production of carbon dioxide CO2.

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The evolution of carbon dioxide (CO2) is determined and compared with the theoretical carbon dioxide
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evolution (ThCO2) using the CO2 evolution test as in ISO 9439.
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The mineralisation process of a fully formulated lubricant (see also Annex A) is determined using
aerobic microorganisms in a static aqueous test system. The test system contains of a mineral medium,
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activated sludge obtained from a waste water treatment plant of mainly domestic waste as the
inoculum and the organic material (the sole source of carbon and energy).
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This mixture is stirred in test flasks and aerated using CO2-free air for a maximum time of 28 days. The
amount of carbon dioxide evolved from biological degradation will be collected in external flasks,
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determined using a suitable analytical process, and expressed as a percentage of ThCO2.

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If a substance-specific analytical method is available then information on the primary degradability may
be obtained additionally for water-soluble, non-volatile lubricants only.
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5 Test environment
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Incubation shall take place in the dark or diffused light, at a temperature within a range 20 °C to 25 °C
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which shall not vary by more than ±1 °C during the test.

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6 Reagents
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Reagents of analytical grade should be used only.

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6.1 Water, preferably of distilled or de-ionized quality, with a DOC content of less than 1 mg/l.

6.2 Stock solutions for mineral medium, as follows:

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Stock solution (a)

Potassium dihydrogen orthophosphate, KH2PO4 8,50 g

Dipotassium hydrogen orthophosphate, K2HPO4 21,75 g

Disodium hydrogen orthophosphate dihydrate, Na2HPO4⋅2H2O 33,40 g
Ammonium chloride, NH4Cl 0,50 g

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Dissolve in water and make up to 1 l. The pH of the solution should be 7,4.

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Stock solution (b)

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Calcium chloride, anhydrous, CaCl2 27,50 g
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or
Calcium chloride dihydrate, CaCl2⋅2H2O 36,40 g

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Dissolve in water and make up to 1 l.

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Stock solution (c)
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Magnesium sulphate heptahydrate, MgSO4⋅7H2O 22,50 g
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Dissolve in water and make up to 1 l.

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Stock solution (d)

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Iron (III) chloride hexahydrate, FeCl3⋅6H2O 0,25 g

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Dissolve in water and make up to 1 l.

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NOTE Stock solution a) can be stabilized by the addition of one drop of concentrated HCl per litre. If any
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precipitate is formed during storage the stock solution is replaced by a freshly prepared one.
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7 Apparatus
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Ensure that all glassware is thoroughly cleaned and free from organic or toxic matter.
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7.1 Test flasks, allowing aeration, containing hose coupling impermeable to carbon dioxide, and a
method for agitation of the test sample such as shaking of the flasks or stirring inside the flasks.
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7.2 Water-bath or constant temperature room (to comply with Clause 5);
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7.3 Equipment for production of CO2-free air in order to constantly aerate all test flasks with a
flow rate of approximately 50 ml/min to 100 ml/min air for a maximum of 3 l medium;

7.4 Equipment for measurement of evolved carbon dioxide, consisting of a suitable instrument or
analytical process of sufficient sensitivity, e.g. CO2- or DIC-analysers or titrimetric determination of CO2
after absorption in alkaline solution.

7.5 Centrifuge or device for filtration, capable of producing an acceleration of 4 000 rpm.

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The filtration apparatus shall be equipped with filter material which do not adsorb or release organic
carbon. The pore size shall exhibit a nominal aperture diameter of 0,2 μm to 0,45 μm.
7.6 pH meter, optional

7.7 Equipment for measurement of dissolved organic carbon (DOC) (optional), of sufficient
sensitivity.

Only for sufficiently water-soluble organic material, removal of dissolved organic carbon (DOC) may be
determined (optionally) by measuring the concentration of DOC at the beginning and the end of
incubation to obtain additional information on the biodegradability.

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8 Procedure

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8.1 Preparation of mineral medium
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Mix 10 ml of solution (a) with 800 ml water, then add 1 ml of solutions (b), (c) and (d) and make up to
1 l with water.

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8.2 Preparation of the test volumes

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8.2.1 Test sample ro
Add a suitable amount of the lubricant to the test flasks to obtain an organic carbon concentration of
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10 mg/l to 20 mg/l (preferably 20 mg/l, if no inhibition is expected).

Prepare a stock solution of the lubricant in an organic solvent suitable to dissolve the lubricant
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completely, e.g. acetone, toluene, preferably hexane with regard to the complete removal of the
solvent.
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The stock solution in the solvent is slowly applied to glass fibre filters (diameter < 50 mm) under a
continuous air stream. The glass fibre filters should be washed with solvent before use in order to
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remove any organic traces.

After complete evaporation of the solvent, (check weight before and after application of the stock
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solution) the filter is incubated for 3 h at 40 °C to remove any traces of the solvent. Blank and reference
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vessels are treated in the same way by only applying solvent to the glass fibre filters. The filters are
directly applied in the test solutions. The concentration in the stock solution should be as such that
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0,5 ml of stock solution is applied per litre of final medium.

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NOTE 1 A comparison of several preparation procedures is listed in Annex B. For further details please see
DIN 51828-1 [4] or EN ISO 10634.
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Depending on the properties of the test sample and the purpose of the test, other concentrations may be
used. In any case of deviation the added amount needs to be determined precisely and stated in the
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report.
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Lubricants containing volatile compounds should be tested using closed respirometer method, provided
that suitable conditions are used. Additionally, the content of volatile compounds may be determined
using methods like e.g. ASTM D 5800 [1] or Noack-test DIN 51581 [2].
NOTE 2 ISO 9439 allows organic carbon concentrations between 10 mg/l and 40 mg/l. In case of fully
formulated lubricants, 10 mg/l can result in too low organic material in the test sample, but 40 mg/l can impede
the formation of homogenous test solutions with regard to limited water solubility of most lubricants.

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8.2.2 Reference compound

In order to check the procedure, a reference compound of known biodegradability should be tested in
parallel. Sodium benzoate or aniline may be used when testing water-soluble test compounds; for
poorly soluble test substances High Oleic Reference Oil (HORO) should be used. See Annex C for
detailed information.
Add the reference compound in the mineral medium (6.3) using the same procedure as for the organic
material in the test sample (8.2.1).
8.2.3 Inhibition control

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If the organic material is suspected to have toxic potential, use a solution of organic material (8.2.1) and

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reference compound (8.2.2) each at preferably a concentration of organic carbon of 20 mg/l in mineral
medium (8.1).

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8.2.4 Elimination control
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If required (e.g. chemical compounds suspected of hydrolysis or photolysis or volatile compounds etc.

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are present in a lubricant) use a solution containing test sample (8.2.1) but no inoculum (8.3). The

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solution needs to be sterilized by using a suitable toxic substance to avoid microbial activity.
8.3 Preparation of the inoculum
8.3.1 General du
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Prepare the inoculum to obtain a microbial population that offers sufficient biodegradation activity.
Validate the activity of the inoculum using a suitable reference compound (8.2.2).
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8.3.2 Inoculum from an activated sludge plant

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Take a sample of activated sludge collected from the aeration tank of a waste-water treatment plant
dealing predominantly domestic sewage. If necessary, concentrate the sludge by settling, so that the
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volume of sludge added to the test assay is minimal but nevertheless fulfils the criteria of 8.2.1.
Mix well and determine the concentration of suspended solids of the activated sludge (e.g. using
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ISO 11923).
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If it is suspected that the sludge contains inhibiting matter, centrifuge, wash with mineral medium (8.1),
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centrifuge again and suspend in the medium. Keep the sample under aerobic conditions and use
preferably on the day of collection. Use a suitable volume to obtain 30 mg/l of suspended solids with
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respect to dry weight in the final mixture.

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NOTE Based on experience, suitable volume means:

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— sufficient to give a microbial population which offers enough biodegradation activity;

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— degrades the reference compound by the stipulated percentage (10.1);

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— 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.

8.3.3 Pre-conditioning

It may be helpful to precondition the inoculum to reduce the influence of the blank, e.g. by aerating it up
to one week before use.
8.3.4 Pre-exposure

Inoculum shall not be pre-exposed to the test compound.

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8.4 Test

Set up the equipment for CO2-free air (7.3) and assemble a sufficient number of test flasks in order to
have
— at least 2 test flasks (FT) for the test sample (8.2.1);

— at least 2 test flasks (Fc) for positive control (8.2.2) containing the reference compound;

— at least 2 test flasks (FB) for blank control containing mineral medium (8.1) and inoculum (8.3.2)

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only;

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and optionally, if needed,

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— 1 test flask (FI) for inhibition control (8.2.3);
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— 1 test flask (FS) for abiotic elimination control (see 8.2.4).

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Table 1 gives an overview on the desired final test matrix.

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Add appropriate amounts of mineral medium (8.1) and inoculum (8.3) to the test flasks to obtain the
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desired final test volume as shown in Table 1. Other test volumes than 3 l are acceptable, in that case all
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relevant parameters as well as calculation need to be adjusted. Connect the test flasks with the
equipment for the production of CO2-free air.
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Table 1 — Overview on the desired final test matrix

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Mineral Organic Reference

Inoculum
medium material compound
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Test flask
8.1 8.2.1 8.2.2 8.3
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Test sample
FT1 + + - +
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FT2 + + - +
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Reference control
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Fc1 + - + +
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Fc2 + - + +
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Blank control
F B1 + - - +
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F B2 + - - +
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Inhibition control (optional)

FI + + + +

Abiotic elimination control (optional)

FS + + - -

Place all test flasks in a constant temperature room, allow them to reach the desired temperature and
aerate them for 24 h using CO2-free air in order to flush out CO2 already present in the vessels. Seal the
flasks and agitate constantly by either shaking or stirring. In case foaming is observed, keep on agitating

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without flushing with CO2-free air. After the pre-aeration process, connect the air exit of each flask with
the equipment to trap CO2 for further determination.
Add the test compound (8.2.1) and the reference compound (8.2.2) at the desired concentrations to the
respective flasks in accordance with Table 1 and start the test by bubbling CO2-free air through each
flask at a rate of between 50 ml/min and 100 ml/min.
Measure CO2 production using an appropriate analytical procedure. If a nearly constant level of CO2
production is attained (plateau phase) and no further biodegradation is expected, the test is considered
to be completed. Usually the maximum test period shall not exceed 28 days. Optionally the

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measurement period can be extended to 35 days or 42 days, if degradation has obviously started but
not reached a plateau phase.

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Determine the pH of all test flasks on the last day of test, acidify all the bottles with between 1 and 10 ml

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of concentrated hydrochloric acid to decompose the carbonates and bicarbonates. Continue aeration for
another 24 h, followed by the determination of CO2 for each flask.
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9 Calculation and reporting of results

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9.1 Theoretical amount of CO2 released from test sample

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The theoretical amount of released CO2 (ThCO2) in mg shall be calculated according to Formula (1):
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 44 
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ThCO 2 =  ρ ⋅ c ⋅ V L ⋅   (1)
 12 
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where
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ρc is the concentration of organic carbon of the test sample in sample FT in mg / l, either measured
or calculated from the stock solution of the test sample (8.2.1);
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VL is the volume of the test sample in flask FT in litres.

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ThCO2 from reference sample and inhibition solution shall be calculated in the same manner.
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9.2 Percentage biodegradation

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The percentage biodegradation Dm (%) shall be calculated for each test flask FT for every test period
td

following Formula (2):

n

∑ mTt − ∑ mBt
me

Dm = (2)
ThCO 2
cu

where
Do

∑ mTt is the mass of carbon dioxide in mg released between start and testing time t;

∑ mBt is the average mass of carbon dioxide in mg in the blank control released between start and
testing time t;
The percentage degradation of reference compound and, if included, abiotic elimination control and/or
inhibition control, shall be calculated in the same manner.

13
 EN 17181:2019 (E)
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9.3 Expression of results

Compile a table of measured CO2 release (ΣmTt and ΣmBt) and the percentages of biodegradation Dm
for each test flask and for each measuring interval. Plot a biodegradation curve, in percent, as a function
of time, and indicate lag phase and degradation phase. If comparable results are obtained for the
duplicate test flasks FT (8.4), plot a mean curve, otherwise plot curves for each flask. Similarly plot a
biodegradation curve of the reference compound Fc and, if included, of the abiotic elimination check FS
and the inhibition control FI.
An example of degradation curves is given in Annex D.

sa
Determine the mean value of percent biodegradation in the plateau phase or use the highest value, e.g.

rzi
when the curve decreases in the plateau phase, and indicate this maximum level of biodegradation as
“degree of biodegradation of the test sample” in the test report.

nte
Information on the toxicity of the test sample may be useful in the interpretation of test results showing
uz intern

ai
a low biodegradation. If in flask FI the degradation percentage is < 25 % and insufficient degradation of
the test sample is observed in flasks FT, it can be assumed that the test sample is inhibitory. In this case

re
the test shall be repeated using a lower test concentration. If in the flask FS (abiotic elimination check if

ce
included) a significant amount (> 10 %) of CO2 release is observed, abiotic degradation processes may
have taken place.
du
ro
10 Validity and precision
ep

10.1 Validity
-R

Test is considered as valid if

RO

a) the percentage degradation of the positive control Fc is greater than 60 % and not higher than
110 % after 28 d in case of HORO as reference compound
AS

or
ru

the positive control Fc is greater than 60 % after the 14th day in case sodium benzoate or aniline is
uc

used as reference compound, and;

uz intern

el

b) the CO2 production in the blank control FB at the end of the test, which is usually at around
td

40 mg/l, does not exceed 70 mg/l after 28 d.

n
me

If either a) or b) is not met, the test should be repeated using another freshly-prepared inoculum.
For water-soluble formulations the test is considered valid if:
cu

c) the amount of DIC does not exceed 5 % of the organic carbon content of the test compound at the
Do

beginning of the test.

If c) is not met, verify that the air for aerating the flasks is definitely free from CO2.
If inhibition control FI (if included) shows degradation lower than 25 % at the end of the test, the test
sample is assumed to be inhibiting. In this case, it is advisable to repeat the test with a lower
concentration of test sample.
If pH value is out of the range of 6 to 8,5 at the end of the test and the percentage degradation of the test
sample is less than 60 %, it is advisable to repeat the test with a lower concentration of the test sample,
and either using a non-nitrifying activated sludge or increasing buffer capacity of the mineral medium.

14
 EN 17181:2019 (E)
uz intern
10.2 Precision
10.2.1 General

The precision data have been obtained by statistical examination in accordance with the EN ISO 4259
series of interlaboratory test results from a European study organized in 2017.
10.2.2 Repeatability

The difference between two independent results obtained in the normal and correct operation of the
same method, for test material considered to be the same, within a short interval of time, under the

sa
same test conditions, that is expected to be exceeded with a probability of 5 % due to random variation,
shall not be greater than:

rzi
r = 16, 73
 % (3)

nte
10.2.3 Reproducibility
uz intern

ai
The difference between two independent results obtained in the normal and correct operation of the

re
same method, for test material considered to be the same, under different test conditions, that is

ce
expected to be exceeded with a probability of 5 % due to random variation, shall not be greater than:

du
R = 35, 90
 % ro (4)

11 Test report
ep

The test report shall contain at least the following information:

-R

a) a reference to this European Standard, i.e. EN 17181;

RO

b) all necessary information for the identification of the test sample;

AS

c) all measured and calculated data obtained, as well as the degradation curve;

d) degradation after 28 days (see 9.2);

ru
uc

e) time until the plateau phase is reached (see 9.3);

uz intern

el

f) the concentration, ThCO2 and/or DOC of the test sample (see 9.1) and the reference compound
td

used;
n

g) the name of the reference compound used and the degradation obtained with this compound;
me

h) the source, the characteristics, the concentration of the volume of the inoculum used and
cu

information on any pre-treatment;

Do

i) the main characteristics of the CO2 analysis used;

j) the incubation temperature of the test;

k) if determined, the percentage of DOC removal or primary biodegradation;

l) if determined, the percentage of degradation obtained in flask FS (abiotic elimination);

15
 EN 17181:2019 (E)
uz intern
m) if determined, the percentage of degradation in flask FI (inhibition check) and a statement on the
toxicity of the test compound;

n) the reasons in the event of the rejection of the test (see 10.1);

o) any alteration of the standard procedure or any circumstance that may have affected the results.

sa
rzi
nte
uz intern

ai
re
ce
du
ro
ep
-R
RO
AS
ru
uc
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el
n td
me
cu
Do

16
uz intern EN 17181:2019 (E)

Annex A

sa
(informative)

rzi
nte
Comparison table of most common mineralisation methods

eai
er
This annex provides an overview of widely used test methods for evaluation of ultimate aerobic biodegradation of organic compounds in an
aqueous medium, including a summary of advantages and disadvantages.

c
du
OECD 301B OECD 301C OECD 301F OECD 310 OECD 306

ro
Latest version 07.07.1992 17.07.1992 17.07.1992 23.03.2006 17.07.1992

ep
Interlaboratory 1988 1988 1988 1997 1987
Study (ILS)

-R
uz intern

Repeatability 5 % to 10 %
Reproducibility 12 % to 16 %

RO
Organic compound as single Organic compound as single Organic compound as single Organic compound as Parallel to OECD 301D,E but
source for biodegradation source for biodegradation source for biodegradation single source for using sea water instead of

AS
biodegradation stock solution/ inoculum
Test principles (DOC inoculum < < DOC test (DOC inoculum < < DOC test (DOC inoculum < < DOC test Headspace as oxygen Degradation = f(DOC) or
substance) substance) substance) reservoir f(dissolved O2)
Degradation = f(CO2) ru
Degradation = f(O2 loss) Degradation = f(O2 loss) Degradation = f(CO2)
luc
Suitable for: Suitable for: Suitable for: Suitable for: Suitable for:
- Water-soluble - Water-soluble - Water-soluble - Water-soluble - Water-soluble
de

- poorly water-soluble - poorly water-soluble - poorly water-soluble - poorly water-soluble Moderately suitable:
Test compound
- adsorbing - adsorbing - adsorbing - adsorbing - volatile compounds
nt

Not suitable: Moderately suitable: Moderately suitable: Moderately suitable: - poorly water-soluble
me

- volatile compounds - volatile compounds - volatile compounds - volatile compounds - adsorbing

10 mg to 20 mg DOC/l or 100 mg/l 100 mg/l or 50 mg to 100 mg 20 mg/l (limits: 2 mg to 2 mg/l to 10 mg/l
cu

Concentration
TOC/l ThOD/l 40 mg Carbon/l) (solubility > 2 mg/l)
Do

17
uz intern EN 17181:2019 (E)

sa
OECD 301B OECD 301C OECD 301F OECD 310 OECD 306

rzi
Poorly soluble: Solvent and emulsifier Poorly soluble: Poorly soluble: Water soluble:
- direct addition forbidden - direct addition - direct addition - direct addition

nte
- use emulsifier - direct addition - use emulsifier - use ultra-sonic - stock solution
- use solvent - use ultra-sonic - use solvent - use emulsifier Poorly soluble:
Preparation of

ai
test compound - use solid carrier - finely solid carrier - use solid carrier - use solvent - ultrasonic
(but not for adsorbing

e
Adsorbing: Adsorbing: - use solid carrier - others ? (not stated)
material)

er
- do not use solid carrier - do not use solid carrier Adsorbing:

c
do not use solid carrier

du
- activated sludge - mixture of sewage effluent - activated sludge - activated sludge Pure sea water
from 10 sites

ro
- sewage effluent - sewage effluent - sewage effluent No inoculum
Inoculum - surface water - exact preparation - surface water - surface water (necessity to add N and P for

ep
source - soil - exact preconditioning - soil - soil diet)
- limited

-R
or mixture of above or mixture of above or mixture of above
uz intern

- store life
Colony

RO
102 to 103 CFU/ml (final
Formung Units 107 to 108 107 to 108 107 to 108 ???
(CFU) mixture)

AS
Test conditions
Temperature 22 °C ± 2 °C 25 °C ± 1 °C 22 °C ± 2 °C 20 °C ± 1 °C 15 °C to 20 °C
pH 7,4 ± 0,2 7 ru 7,4 ± 0,2 7,4 ± 0,2 ?
luc
Pre-adaption No No No No No
Pre-
Allowed Unnecessary Allowed Allowed Yes
de

conditioning
Controlling
nt

Blank control Yes Yes Yes Yes Yes

me

Reference Yes Yes Yes Yes Yes

control
cu

Abiotic control Preferably Yes Preferably If needed Optional

Do

18
uz intern EN 17181:2019 (E)

sa
OECD 301B OECD 301C OECD 301F OECD 310 OECD 306

rzi
CO2 determination step-wise O2 loss continuously by O2 loss continuously by CO2 determination step- Dissolved O2 determination
as IC from trapped CO2 in manometric respirometer manometric respirometer wise as IC from treatment step-wise chemically or

nte
BaOH with NaOH or with acid electrochemically
% degradation % degradation % degradation % degradation % degradation

ai
Data and ICtest − ICinoculum
Reporting ICtest − ICinoculum BOD   
test − BOD inoculum BOD   
test − BOD inoculum = BOD   
test − BOD inoculum
= = = =
( ) ( )

e
ThCO 2  
or TOC ThOD * ThOD    or COD * TOC ThOD   (
or COD * )

c er
*COD less accurate due to *COD less accurate due to

du
*Calculation via DOC possible incomplete oxidation
incomplete oxidation

ro
- IC test substance < 5 % TC - c(O2) in blank not to exceed - c(O2) in blank not to exceed - % degradation - O2 depletion in blank not
at beginning reference > 60 % after

ep
60 mg/l in 28 d 60 mg/l in 28 d to exceed 30 %
- CO2 from blank 14 d TIC
- Difference in % - residual O2 > 0,5 mg/l - Reference degradation
blank < 3 mg/l at the

-R
control < 40 mg/l at end degradation < 20 % compliant with ring test
uz intern

- Difference in % end of test

of test between 2 flasks
Validation degradation < 20 %
- Difference in % - O2 consumption of between 2 flasks

RO
degradation < 20 % aniline > 40 % after 7 d - % degradation
between 2 flasks and > 65 % after 14 d reference > 60 % after

- % degradation
reference > 60 % after
14 d
CO2 production easily related
to carbon content of test
substance (TOC)
Easy-to-handle procedure
AS
14 d
ru
Inoculum mixture probably Continuous measurement of Uses head-space – Using seawater:
luc
leading to less variation in O2 – less error-prone than improves collection of "real" approach
results detection of CO2 CO2; CO2 detection in
Closed-bottle-method easy
Continuous measurement of liquid and gaseous phase to handle, even for volatiles;
de

General Easy-to-handle procedure

O2 – less error-prone than Continuous shaking – inhibitory effects low
advantages
detection of CO2 supports testing of
nt

insoluble material
Methods of preparation
me

restricted – probably leading

to less variation in results
cu
Do

19
uz intern EN 17181:2019 (E)
OECD 301B
Detection of CO2 influenced
by adsorption, volatilisation,
precipitation or hydrolysis
Also microbial or non-
biological reaction
Measurement is cumbersome,
General despite problem of sealing
disadvantages
uz intern
ISO 9439 (1999)
- More test substance
possible
(10 mg/l to 40 mg/l)
- Inoculum better defined
- Adoption of different
kinetics (no 10-d window)
ISO 14852 (1999)
Corresponding - Similar to OECD 301B
ISO methods /ISO 9439 but for testing
of plastics
- Other reference materials:
aniline, microcrystalline
cellulose powder, ashless
cellulose filters or poly- β -
hydroxy-butyrate
- More test compound
possible (100 mg TOC/l)
Do
20
sa
OECD 301C OECD 301F OECD 310 OECD 306
rzi
Preparation of inoculum Using chemical oxygen Too many preparation No "unique" sea water
cumbersome: demand (COD) leads to wrong methods for water- definition
nte
- complex mixture difficult to degradation due to less insoluble material with No inoculum used – results
prepare and store carbon found in this analytics respect to variation of not comparable to other
compared to TOC results OECD methods
ai
- resulting inoculum mix
more or less inactive High complexity of test Method difficult for non-
e
equipment; water-soluble test samples,
Test medium completely
er
different to other methods If not tested in-line, a lot especially formulations
of bottles necessary – Shake-flask-method needs
c
Single use in Japan
limited number of test C-analyser, inhibitory effects
du
rigs; problem of sealing of possible due to high content
bottles of test substance
ro
Low demand for this test Closed-bottle-method shows
ep
lack of agitation, probably
nitrification
-R
None ISO 9408 (1999) ISO 14593 (1999) ISO 16221 (2001)
- Optimized test substance
RO
concentration 100 mg/l
ThOD
AS
- Inoculum better defined
- Adoption of different
kinetics (no 10-d window)
ru ISO 14851 (1999)
luc
- Similar to
OECD 301F/ISO 9408 but
for testing of plastics
de
- Other reference materials:
aniline, microcrystalline
nt
cellulose powder, ashless
cellulose filters or poly-  β –
me
hydroxylbutyrate
- More test substance
cu
possible (170 mg ThOD/l)
uz intern EN 17181:2019 (E)

sa
OECD 301B OECD 301C OECD 301F OECD 310 OECD 306

rzi
D5864–11 (2011) none D6731–01 (2011) none D6691 (2009)
- Suggestion of low erucic - Suggestion of low erucic - For testing of plastics

nte
acid rapeseed oil as acid rapeseed oil as - Evaluation of
reference to water- reference to water- biodegradation in
insoluble material insoluble material

ai
respirometer and
- Reference material should - Reference material should determination of CO2

e
be added in same manner be added in same manner
- Other reference materials:

er
as test substance as test substance
cellulose, chitin or Kraft
Corresponding - Sonication allowed as - ThOD of test substance not

c
paper
ASTM methods sample preparation less than 50 mg O2/l but

du
- Pre-adaption allowed not higher than

ro
D6139–11 (2011) 200 mg O2/l;

ep
- Modification of medium - Optimum: 50 mg/l to
- Special flask type 100 mg/l

-R
- Sonication allowed as
uz intern

- Pre-adaption allowed
sample preparation
- Only direct addition
- Pre-adaption allowed

RO
allowed

AS
ru
luc
de
nt
me
cu
Do

21
uz intern EN 17181:2019 (E)

Annex B

sa
(informative)

rzi
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Sample insertion

e ai
er
This annex provides an overview of possible preparation procedures for non-water-soluble lubricants, including a summary of advantages and
disadvantages.

c
du
Adsorption on an inert
Direct insertion using an Production of Production of
Direct insertion carrier (fibre mat, silica

ro
Direct insertion inert carrier (glass fibre, dispersion using dispersion using
using a solvent gel, silica sand, amorphous
aluminium plate etc.) ultra-sonic sound emulsifiers

ep
spherical nanoparticles)

Advantage No adulteration of Proper dosage Dosage easier to handle Better dispersion oft e test Proper dosage and Proper dosage achievable

-R
uz intern

results due to achievable compared to direct sample in the test flask bio-availability Test sample will
biodegradation of insertion Sample spread over a large possible as long as a homogeneously be
impurities stable dispersion is

RO
Weighted sample record surface dispersed in the test
more precise formed volume
case 1: creation of a

AS
pre-solution using
ultra-sonic sound
case 2: preparation
ru of the whole sample
luc
short before
addition of inoculum
Disadvantage Dosage of test Low volatility of a Reduced bio-availability of Low bio-availability due to Stability of Choice of feasible
de

sample difficult to biodegradable ingredients due to low strong adsorption or dispersion probably emulsifiers quite difficult.
handle. solvent might water solubility absorption not satisfying. Question of comparability
nt

Reduced bio- influence Glass plates might stick on Insoluble ingredients might Test substance of results regarding the
degradation test
me

availability of the flask wall remain unabsorbed. might modify under use of different
ingredients due to results using influence of shearing emulsifiers.
Potential loss of Silica gel of high porosity
low water solubility respirometric forces.
lubricants, which show should not be used for
cu

methods.
extraordinary wetting on greases and possibly for oil
glass surfaces samples.
Do

Determination of CSB
difficult for greases.

22
uz intern EN 17181:2019 (E)

sa
Adsorption on an inert
Direct insertion using an Production of Production of
Direct insertion carrier (fibre mat, silica
Direct insertion inert carrier (glass fibre, dispersion using dispersion using

rzi
using a solvent gel, silica sand, amorphous
aluminium plate etc.) ultra-sonic sound emulsifiers
spherical nanoparticles)

nte
Standard Measurement of Assure to work with Inert carrier shall be free Strong adsorption on porous Applicable only in Content of emulsifier
proceedings weight needs to be real solution, stable of residual carbon. carrier shall be avoided. combination with should not exceed 10

ai
done with sufficient suspension or stable Perform blind test. Result Inert carrier shall be free of emulsifiers or mass-% of test sample
precision. dispersion. of blind test shall not solvents. quantity.

e
residual carbon. Perform
Applicable only for No use of aliquots. exceed the limit of the blind test. Result of blind test Duration and energy Resulting emulsion

er
liquid samples by Total amount of correspondent test shall not exceed the limit of input need to be should be dilutable with

c
use of differential solvent and sample procedure. the correspondent test defined. water in any ratio.

du
measurement of is added. procedure. Stability of Emulsion should be
weight. In case of clear, Test material need to be dispersion need to stable during the whole

ro
transparent, homogeneously dispersed on be ensured test procedure. (control

ep
possibility to use inert carrier. Concentration using turbidimetry)
aliquots (ensure of aliquot of test sample
transparency by use should be done using CSB,

-R
uz intern

of formazin method) TOC or calcination.

In case of Assure the test sample not to

RO
respirometric be overheated during
methods: grinding process.
Ensure complete

AS
evaporation of
solvent.
Direct insertion Direct insertion ru
Direct insertion using an
inert carrier (glass fibre,
Adsorption on an inert
carrier (fibre mat, silica gel,
Production of
dispersion using
luc
using a solvent
aluminium plate etc.) silica sand, amorphous ultra-sonic sound
spherical nano-particles)
de
nt
me
cu
Do

23
 EN 17181:2019 (E)
uz intern
Annex C
(informative)

Specification of reference compound High Oleic Reference Oil (HORO)

This annex provides a quality definition of a high oleic reference oil (HORO). HORO in the sense of this
standard means a blend of 99,7 % High Oleic Sunflower Oil and 0,3 % (1,4-Di-tert.-butyl-hydroxy-

sa
toluene (BHT). HORO fulfils the following minimum specifications:

rzi
Analytical Test Characteristic Specification limits Units
Method

nte
lower upper
uz intern

EN ISO 3675 /
kg/m3

ai
Density at 15 °C 900,0 930,0
EN ISO 12185

re
EN ISO 3104 Kinematic viscosity at 40 °C 36,0 44,0 mm2/s

ce
EN 14111 or EN 16300 Iodine number — 90,0 g Iodine / 100 g
EN 14104 Acid number —
du 0,2 mg KOH / g
ro
Fatty acid distribution:
ep

cis-9-Octadecanoic acid C18:1 cis 90 — % (m/m)

EN ISO 12966-4
-R

Other unsaturated fatty acids — 4 % (m/m)

Saturated fatty acids — 6 % (m/m)
RO

Oxidation stability (Rancimat) at

EN 14112 — 30 h
110 °C, airflow 10 l/h
AS

ISO 3960 Peroxide value — 10 meq O2/kg

ru

EN ISO 12937 Water content — 750 mg/kg

uc

EN ISO 20884 or
Sulfur content — 10,0 mg/kg
EN ISO 20846
uz intern

el

DIN 51627-6 Phosphorous content — 3,0 mg/kg

td

DIN 51627-6 Calcium content — 1,0 mg/kg

n

DIN 51627-6 Magnesium content — 1,0 mg/kg

me

Certificate of Analysis for material stored in unopened containers and stored in cool dry conditions
cu

under exclusion of light should be valid in the moment of using the material as reference for in the
biodegradation method.
Do

The HORO is available from company FUCHS Schmierstoffe GmbH, Mannheim, Germany, or can be
obtained from other suppliers providing it meets the specification described above. Alternatively, if
desired, HORO meeting the above minimum specification can be prepared in-house.
The quality of the HORO is checked prior to use:
a) if the indicated shelf life is exceeded, or

b) if the certificate was issued 12 months before use, or

c) if the in-house produced HORO was prepared more than 12 months before use.

24
 EN 17181:2019 (E)
uz intern
Annex D
(informative)

Example of degradation curves

Figure D.1 shows an example of a degradation curve.

sa
rzi
nte
uz intern

ai
re
ce
du
ro
ep
-R
RO
AS
ru
uc
uz intern

el
n td
me

Key
cu

1 maximum level of biodegradation

Do

2 lag phase
3 biodegradation phase
4 plateau phase
X duration (days)
Y degradation rate (%)
high biodegradation rate
low biodegradation rate

Figure D.1 — Example of degradation curve

25
 EN 17181:2019 (E)
uz intern
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[1] ASTM D 5800, Standard test method for evaporation loss of lubricating oils by the Noack method

[2] DIN 51581, Testing of lubricants — determination of evaporation loss of lubricating oils (Noack
test)

[3] DIN 51627-6, Automotive Fuels — Test methods — Part 6: Direct determination of trace elements

sa
in vegetable oils by inductively coupled plasma optical emission spectroscopy (ICP OES)

rzi
[4] DIN 51828-1, Testing of lubricants and related products — Determination of the rapid

nte
biodegradability — Part 1: General working principles
uz intern

EN 14104, Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of acid value

ai
[5]

re
[6] EN 14111, Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of iodine
value

ce
[7]
stability (accelerated oxidation test) du
EN 14112, Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of oxidation
ro
ep

[8] EN 16300, Automotive fuels - Determination of iodine value in fatty acid methyl esters (FAME) -
Calculation method from gas chromatographic data
-R

[9] EN ISO 3104, Petroleum products - Transparent and opaque liquids - Determination of kinematic
RO

viscosity and calculation of dynamic viscosity (ISO 3104)

[10] EN ISO 3675, Crude petroleum and liquid petroleum products - Laboratory determination of
AS

density - Hydrometer method (ISO 3675)

ru

[11] EN ISO 12966-4, Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl
esters — Part 4: Determination by capillary gas chromatography (ISO 12966-4)
uc
uz intern

el

[12] EN ISO 12185, Crude petroleum and petroleum products - Determination of density - Oscillating U-
tube method (ISO 12185)
td

[13] EN ISO 12937, Petroleum products - Determination of water - Coulometric Karl Fischer titration
n
me

method (ISO 12937)

[14] EN ISO 20846, Petroleum products - Determination of sulfur content of automotive fuels -
cu

Ultraviolet fluorescence method (ISO 20846)

Do

[15] EN ISO 20884, Petroleum products - Determination of sulfur content of automotive fuels -
Wavelength-dispersive X-ray fluorescence spectrometry (ISO 20884)

[16] ISO 3960, Animal and vegetable fats and oils — Determination of peroxide value — Iodometric
(visual) endpoint determination

[17] ISO 8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and
dissolved organic carbon (DOC)

26
 EN 17181:2019 (E)
uz intern
[18] EN ISO 10634, Water quality - Guidance for the preparation and treatment of poorly water-soluble
organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium
(ISO 10634)

[19] EN ISO 4259-1, Petroleum and related products - Precision of measurement methods and results -
Part 1: Determination of precision data in relation to methods of test (ISO 4259-1)

[20] ISO 11923, Water quality — Determination of suspended solids by filtration through glass-fibre
filters

sa
[21] ISO 14851, Determination of the ultimate aerobic biodegradability of plastic materials in an
aqueous medium - Method by measuring the oxygen demand in a closed respirometer

rzi
[22] ISO 14852, Determination of the ultimate aerobic biodegradability of plastic materials in an

nte
aqueous medium — Method by analysis of evolved carbon dioxide
uz intern

ai
[23] ISO 9408, Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds

re
in aqueous medium by determination of oxygen demand in a closed respirometer

ce
[24] ISO 9439, Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds

du
in aqueous medium — Carbon dioxide evolution testro
[25] ISO 14593, Water quality — Evaluation of ultimate aerobic biodegradability of organic
compounds in aqueous medium — Method by analysis of inorganic carbon in sealed vessels (CO2
ep

headspace test)
-R

[26] ISO 16221, Water quality — Guidance for determination of biodegradability in the marine
environment
RO

[27] ASTM D5864, Standard Test Method for Determining Aerobic Aquatic Biodegradation of
AS

Lubricants or Their Components

ru

[28] ASTM D6139, Standard Test Method for Determining the Aerobic Aquatic Biodegradation of
Lubricants or Their Components Using the Gledhill Shake Flask
uc
uz intern

el

[29] ASTM D6731, Standard Test Method for Determining the Aerobic, Aquatic Biodegradability of
Lubricants or Lubricant Components in a Closed Respirometer
td

[30] ASTM D6691, Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials
n

in the Marine Environment by a Defined Microbial Consortium or Natural Sea Water Inoculum
me

[31] OECD, Test No. 301: Ready Biodegradability, OECD Publishing, Paris
cu
Do

[32] OECD, Test No. 306: Biodegradability in Seawater, OECD Publishing, Paris

[33] OECD, Test No. 310: Ready Biodegradability - CO2 in sealed vessels (Headspace Test), OECD
Publishing, Paris

27