Lubricants - Determination of aerobic biological degradation of fully formulated lubricants in an aqueous solution - Test method based on CO2-production

This standard 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. The above mentioned method applies to fully formulated lubricants which a) are water-soluble, non-water soluble or emulsifiable b) are not toxic and not inhibitory to the test microorganisms at the test concentration. The presence of inhibitory effects can be determined

Flüssige Mineralölerzeugnisse - Bestimmnung des aerobe biologischen Abbaus von vollständig formulierten Schmierstoffen in wässriger Lösung - Prüfverfahren mittels CO2-Produktion

Dieses Dokument legt ein Prüfverfahren für die Bestimmung des Grades des aeroben Bioabbaus fertig formulierter Schmierstoffe fest. Das organische Material in einem fertig formulierten Schmierstoff wird in einem künstlichen wässrigen Medium einem Inokulum aus Belebtschlamm ausgesetzt.
Der Bioabbau, der zur Mineralisierung des organischen Materials führt, kann durch Messung des frei-gesetzten CO2 in einem TOC-Analysator (en: total organic carbon) bestimmt werden. Im Gegensatz zu vorhandenen Prüfverfahren, die freigesetztes CO2 messen, nutzt dieses Verfahren eine präzise Probenvorbereitung für nicht-wasserlösliches organisches Material.
Das zuvor genannte Verfahren gilt für fertig formulierte Schmierstoffe, die:
a) wasserlöslich, nicht-wasserlöslich oder emulgierbar sind und
b) nicht toxisch und nicht hemmend gegenüber den Prüfmikroorganismen bei der Prüfkonzentration sind.
Das Vorhandensein von hemmenden Wirkungen wird nach diesem Dokument bestimmt.
Dieses Prüfverfahren ist auf Frischwasser als Prüfmedium fokussiert. Prüfungen in Meerwasser sind derzeit in diesem Verfahren nicht erfasst, können jedoch zu einem späteren Zeitpunkt eingeführt werden.

Bio-lubrifiants - Détermination de la biodégradabilité aérobie, en solution aqueuse, de lubrifiants complètement formulés - Méthode basée sur le dégagement de CO2

Le présent document définit une procédure pour la détermination du degré de dégradation aérobie de lubrifiants complètement formulés. La matière organique contenue dans un lubrifiant complètement formulé est placée en milieu aqueux synthétique dans des conditions de laboratoire et exposée à un inoculum de boues activées.
La dégradation biologique entraînant une minéralisation de la matière organique peut être déterminée en mesurant le dégagement de CO2 dans un analyseur de carbone organique total (COT). Contrairement aux méthodes d’essai existantes mesurant le dégagement de CO2, cette méthode s’appuie sur une procédure préparatoire précise pour une matière organique non soluble dans l’eau.
La méthode susmentionnée s’applique aux lubrifiants complètement formulés qui :
a)   sont solubles dans l’eau, non solubles dans l’eau ou émulsifiables ; et
b)   ne sont pas toxiques et n’ont aucun effet d’inhibition sur les microorganismes d’essai à la concentration d’essai.
La présence d’effets inhibiteurs est déterminée dans les conditions spécifiées par le présent document.
Cette méthode d’essai utilise de l’eau douce comme milieu d’essai. Les essais en eau de mer ne sont pas inclus actuellement dans cette méthode, mais peuvent être introduits à une date ultérieure.

Maziva - Določevanje aerobne biološke razgradnje polno formuliranih maziv v vodni raztopini - Preskusna metoda, ki temelji na proizvodnji CO2

Ta standard določa postopek za določanje stopnje aerobne razgradnje polno formuliranih maziv. Organski material v polno formuliranem mazivu je v sintetičnem vodnem mediju pod laboratorijskimi pogoji izpostavljen inokulumu iz aktivnega blata. Biološko razgradnjo, ki povzroča mineralizacijo organskega materiala, je mogoče določiti z merjenjem sproščenega CO2 v analizatorju skupnega organskega ogljika (TOC). Zgoraj navedena metoda velja za polno formulirana maziva, ki a) so topna v vodi, niso topna v vodi ali emulzivna; b) niso strupena in niso zaviralna za preskusne mikroorganizme pri preskusni koncentraciji. Določiti je mogoče prisotnost zaviralnih učinkov.

General Information

Status
Published
Public Enquiry End Date
30-Nov-2017
Publication Date
11-Mar-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
05-Mar-2019
Due Date
10-May-2019
Completion Date
12-Mar-2019

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SLOVENSKI STANDARD
SIST EN 17181:2019
01-april-2019
0D]LYD'RORþHYDQMHDHUREQHELRORãNHUD]JUDGQMHSROQRIRUPXOLUDQLKPD]LYY
YRGQLUD]WRSLQL3UHVNXVQDPHWRGDNLWHPHOMLQDSURL]YRGQML&2

Lubricants - Determination of aerobic biological degradation of fully formulated lubricants

in an aqueous solution - Test method based on CO2-production
Flüssige Mineralölerzeugnisse - Bestimmnung des aerobe biologischen Abbaus von

vollständig formulierten Schmierstoffen in wässriger Lösung - Prüfverfahren mittels CO2-

Produktion

Bio-lubrifiants - Détermination de la biodégradabilité aérobie, en solution aqueuse, de

lubrifiants complètement formulés - Méthode basée sur le dégagement de CO2
Ta slovenski standard je istoveten z: EN 17181:2019
ICS:
75.100 Maziva Lubricants, industrial oils and
related products
SIST EN 17181:2019 en,fr,de

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

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SIST EN 17181:2019
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SIST EN 17181:2019
EN 17181
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2019
EUROPÄISCHE NORM
ICS 75.100
English Version
Lubricants - Determination of aerobic biological
degradation of fully formulated lubricants in an aqueous
solution - Test method based on CO2-production

Bio-lubrifiants - Détermination de la biodégradabilité Schmierstoffe - Bestimmung der aeroben biologischen

aérobie, en solution aqueuse, de lubrifiants Abbaubarkeit ausformulierter Schmiermittel in

complètement formulés - Méthode basée sur le wässrigem Medium - Testmethode basierend auf CO2-

dégagement de CO2 Bestimmung
This European Standard was approved by CEN on 14 December 2018.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

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,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.
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.
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SIST EN 17181:2019
EN 17181:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 8

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

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

7 Apparatus ........................................................................................................................................................... 9

8 Procedure........................................................................................................................................................ 10

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

8.2 Preparation of the test volumes ............................................................................................................. 10

8.2.1 Test sample .................................................................................................................................................... 10

8.2.2 Reference compound .................................................................................................................................. 11

8.2.3 Inhibition control ......................................................................................................................................... 11

8.2.4 Elimination control ..................................................................................................................................... 11

8.3 Preparation of the inoculum .................................................................................................................... 11

8.3.1 General ............................................................................................................................................................. 11

8.3.2 Inoculum from an activated sludge plant ............................................................................................ 11

8.3.3 Pre-conditioning ........................................................................................................................................... 11

8.3.4 Pre-exposure ................................................................................................................................................. 11

8.4 Test .................................................................................................................................................................... 12

Table 1 — Overview on the desired final test matrix .................................................................................... 12

9 Calculation and reporting of results ..................................................................................................... 13

9.1 Theoretical amount of CO released from test sample .................................................................. 13

9.2 Percentage biodegradation ...................................................................................................................... 13

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

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

10.1 Validity ............................................................................................................................................................. 14

10.2 Precision .......................................................................................................................................................... 15

10.2.1 General ............................................................................................................................................................. 15

10.2.2 Repeatability .................................................................................................................................................. 15

10.2.3 Reproducibility ............................................................................................................................................. 15

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

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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SIST EN 17181:2019
EN 17181:2019 (E)
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.

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.

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

European Free Trade Association.

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

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
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SIST EN 17181:2019
EN 17181:2019 (E)
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.

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.

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

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

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.

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

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

confidence of the customer in this product group.
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EN 17181:2019 (E)
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 CO in a total organic carbon (TOC) analyser. In contrast to existing test methods measuring

released CO this method uses a precise preparation procedure for non-water soluble organic material.

The above mentioned method applies to fully formulated lubricants which
a) are water-soluble, non-water soluble or emulsifiable, and

b) are not toxic and not inhibitory to the test microorganisms at the test concentration.

The presence of inhibitory effects is determined as specified in this document.

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.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

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.

ISO 9439, Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in

aqueous medium — Carbon dioxide evolution test
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
organic material
total amount of all organic compounds a fully formulated lubricant consists of
3.2
aerobic biodegradation

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

resulting in cleavage of chemical bonds and CO 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
3.6
concentration of suspended solids of an activated sludge

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

drying at about 105 °C to constant mass
3.7
inoculum

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

described in this method
3.8
reference compound

organic compound of known biodegradability with a degradation degree of more than 60 %

3.9
dissolved organic carbon
DOC

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

membranes with pores of 0,2 μm to 0,45 μm diameter.
3.10
total inorganic carbon
TIC

total amount of inorganic carbon in a water sample arising from carbon dioxide and carbonates

3.11
dissolved inorganic carbon
DIC

part of the inorganic 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

membranes with pores of 0,2 μm to 0,45 μm diameter.
3.12
theoretically released amount of carbon dioxide
ThCO

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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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

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

oxidation of a chemical compound or organic matter in water

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

3.15
chemical oxygen demand
COD

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

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

conditions

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

3.16
theoretical oxygen demand
ThOD

theoretical maximum amount of oxygen required to oxidize a chemical compound completely,

calculated from elemental analysis

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

3.17
lag phase

time from the start of a test until adaptation and selection of the degrading microorganisms are

achieved and the biodegradation degree of a chemical compound or organic matter has increased to

about 10 % of the theoretical maximum biodegradation
Note 1 to entry This is expressed in days.
3.18
maximum level of biodegradation

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

biodegradation takes place during the test
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
3.22
pre-exposure

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

and/or selection of the microorganisms
4 Principle

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

The evolution of carbon dioxide (CO ) is determined and compared with the theoretical carbon dioxide

evolution (ThCO ) using the CO evolution test as in ISO 9439.
2 2

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,

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).

This mixture is stirred in test flasks and aerated using CO -free air for a maximum time of 28 days. The

amount of carbon dioxide evolved from biological degradation will be collected in external flasks,

determined using a suitable analytical process, and expressed as a percentage of ThCO .

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.
5 Test environment

Incubation shall take place in the dark or diffused light, at a temperature within a range 20 °C to 25 °C

which shall not vary by more than ±1 °C during the test.
6 Reagents
Reagents of analytical grade should be used only.

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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EN 17181:2019 (E)
Stock solution (a)
Potassium dihydrogen orthophosphate, KH PO
8,50 g
2 4
Dipotassium hydrogen orthophosphate, K HPO
21,75 g
2 4
Disodium hydrogen orthophosphate dihydrate, Na HPO ⋅2H O
33,40 g
2 4 2
Ammonium chloride, NH Cl
0,50 g
Dissolve in water and make up to 1 l. The pH of the solution should be 7,4.
Stock solution (b)
Calcium chloride, anhydrous, CaCl
2 27,50 g
Calcium chloride dihydrate, CaCl ⋅2H2O 36,40 g
Dissolve in water and make up to 1 l.
Stock solution (c)
Magnesium sulphate heptahydrate, MgSO ⋅7H O
22,50 g
4 2
Dissolve in water and make up to 1 l.
Stock solution (d)
Iron (III) chloride hexahydrate, FeCl ⋅6H O
0,25 g
3 2
Dissolve in water and make up to 1 l.

NOTE Stock solution a) can be stabilized by the addition of one drop of concentrated HCl per litre. If any

precipitate is formed during storage the stock solution is replaced by a freshly prepared one.

7 Apparatus

Ensure that all glassware is thoroughly cleaned and free from organic or toxic matter.

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.

7.2 Water-bath or constant temperature room (to comply with Clause 5);

7.3 Equipment for production of CO -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. CO - or DIC-analysers or titrimetric determination of CO

2 2
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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EN 17181:2019 (E)

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.
8 Procedure
8.1 Preparation of mineral medium

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.
8.2 Preparation of the test volumes
8.2.1 Test sample

Add a suitable amount of the lubricant to the test flasks to obtain an organic carbon concentration of

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

completely, e.g. acetone, toluene, preferably hexane with regard to the complete removal of the

solvent.

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

remove any organic traces.

After complete evaporation of the solvent, (check weight before and after application of the stock

solution) the filter is incubated for 3 h at 40 °C to remove any traces of the solvent. Blank and reference

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

0,5 ml of stock solution is applied per litre of final medium.

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.

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

report.

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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EN 17181:2019 (E)
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

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

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

medium (8.1).
8.2.4 Elimination control

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

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

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

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).

8.3.2 Inoculum from an activated sludge plant

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

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

ISO 11923).

If it is suspected that the sludge contains inhibiting matter, centrifuge, wash with mineral medium (8.1),

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

respect to dry weight in the final mixture.
NOTE Based on experience, suitable volume means:

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

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

— 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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EN 17181:2019 (E)
8.4 Test

Set up the equipment for CO -free air (7.3) and assemble a sufficient number of test flasks in order to

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

only;
and optionally, if needed,
— 1 test flask (F ) for inhibition control (8.2.3);
— 1 test flask (F ) for abiotic elimination control (see 8.2.4).
Table 1 gives an overview on the desired final test matrix.

Add appropriate amounts of mineral medium (8.1) and inoculum (8.3) to the test flasks to obtain

...

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