Determination of the ultimate biodegradation of plastics materials in an aqueous system under anoxic (denitrifying) conditions - Method by measurement of pressure increase

This document specifies a method for the determination of the ultimate anoxic biodegradability of plastics made of organic compounds, where the amount of the produced nitrogen and carbon dioxide at the end of the test is measured.
The test substance is exposed to an inoculum stemming from the denitrification tank of a wastewater treatment plant. Testing is performed under defined laboratory conditions.

Bestimmung der vollständigen Bioabbaubarkeit von Kunststoff-Materialien in wässriger Phase unter anoxischen (denitrifizierenden) Bedingungen - Verfahren mittels Messung der Druckzunahme

Dieses Dokument legt ein Verfahren fest, mit dem durch Messung der Menge des produzierten Stickstoffs und des gebildeten Kohlenstoffdioxids am Ende der Prüfung die vollständige anoxische Bioabbaubarkeit von Kunststoffen aus organischen Verbindungen bestimmt wird.
Die Prüfsubstanz wird einem Inokulum ausgesetzt, das aus einem Denitrifikationsbecken einer Kläranlage stammt. Die Prüfung erfolgt unter definierten Bedingungen im Labor.
Qualitätsansprüche sind auf die in der Prüfung erhaltenen numerischen Ergebnisse begrenzt und werden nicht für unsachgemäße Aussagen wie „in Kläranlagen entsorgbar“ und dergleichen verwendet.

Détermination de la biodégradation ultime des matériaux plastiques dans un système aqueux dans des conditions anoxiques (dénitrifiantes) - Méthode par mesure de l'augmentation de pression

LLe présent document spécifie une méthode pour la détermination de la biodégradation anoxique ultime des plastiques constitués de composés organiques, où la quantité d’azote et de dioxyde de carbone produite à la fin de l’essai est mesurée.
La substance d’essai est exposée à un inoculum provenant du bassin de dénitrification d’une station d’épuration des eaux usées. Les essais sont effectués dans des conditions de laboratoire définies.
Les revendications de performance sont limitées au résultat numérique obtenu lors de l'essai et ne sont pas utilisées pour faire des affirmations non confirmées telles que «jetable dans les stations d'épuration» et similaires.

Ugotavljanje dokončne biorazgradnje plastičnih materialov v vodnem sistemu pri anoksičnih (denitrifikacijskih) pogojih - Metoda z meritvijo zviševanja tlaka

General Information

Status
Published
Public Enquiry End Date
31-Oct-2019
Publication Date
12-Nov-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
12-Nov-2020
Due Date
17-Jan-2021
Completion Date
13-Nov-2020

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SLOVENSKI STANDARD
SIST EN 17417:2021
01-januar-2021
Ugotavljanje dokončne biorazgradnje plastičnih materialov v vodnem sistemu pri
anoksičnih (denitrifikacijskih) pogojih - Metoda z meritvijo zviševanja tlaka

Determination of the ultimate biodegradation of plastics materials in an aqueous system

under anoxic (denitrifying) conditions - Method by measurement of pressure increase

Bestimmung der vollständigen Bioabbaubarkeit von Kunststoff-Materialien in wässriger

Phase unter anoxischen (denitrifizierenden) Bedingungen - Verfahren mittels Messung

der Druckzunahme

Détermination de la biodégradation ultime des matériaux plastiques dans un système

aqueux dans des conditions anoxiques (dénitrifiantes) - Méthode par mesure de
l'augmentation de pression
Ta slovenski standard je istoveten z: EN 17417:2020
ICS:
13.030.99 Drugi standardi v zvezi z Other standards related to
odpadki wastes
83.080.01 Polimerni materiali na Plastics in general
splošno
SIST EN 17417:2021 en,fr,de

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

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SIST EN 17417:2021
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SIST EN 17417:2021
EN 17417
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2020
EUROPÄISCHE NORM
ICS 13.030.99; 83.080.01
English Version
Determination of the ultimate biodegradation of plastics
materials in an aqueous system under anoxic
(denitrifying) conditions - Method by measurement of
pressure increase

Détermination de la biodégradation ultime des Bestimmung der vollständigen Bioabbaubarkeit von

matériaux plastiques dans un système aqueux dans des Kunststoff-Materialien in wässriger Phase unter

conditions anoxiques (dénitrifiantes) - Méthode par anoxischen (denitrifizierenden) Bedingungen -

mesure de l'augmentation de pression Verfahren mittels Messung der Druckzunahme
This European Standard was approved by CEN on 21 September 2020.

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

Poland, Portugal, Republic of North Macedonia, 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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17417:2020 E

worldwide for CEN national Members.
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SIST EN 17417:2021
EN 17417:2020 (E)
Contents Page

European foreword ...................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 9

5 Equipment and materials............................................................................................................................. 9

5.1 Pressure measurement system ................................................................................................................. 9

5.2 Stirring platform or single magnetic stirrers .....................................................................................10

5.3 Room or incubator with a constant temperature of (20 ± 2) °C ...................................................10

5.4 Argon for the elimination of oxygen from the medium and the gas space...............................10

5.5 Thermometer .................................................................................................................................................11

5.6 Membrane filter.............................................................................................................................................11

5.7 Activated sludge from the denitrification tank of a waste water treatment plant ...............11

5.8 KOH or used for sorption ...........................................................................................................................11

5.9 HCl used for titration ...................................................................................................................................11

5.10 pH meter with electrodes .........................................................................................................................11

5.11 Pipettes, pipette tips ....................................................................................................................................11

5.12 Photometric cuvette tests ..........................................................................................................................11

5.13 Photometer for the procedures or cuvette tests ...............................................................................12

5.14 Analytical balance .........................................................................................................................................12

6 Preparation .....................................................................................................................................................12

6.1 Determination of the volume of each reaction vessel .....................................................................12

6.2 Sample preparation .....................................................................................................................................12

6.3 Preparation of the medium .......................................................................................................................12

6.3.1 Reagents ...........................................................................................................................................................12

6.3.2 Distilled or deionized water .....................................................................................................................12

6.3.3 Preparation of the concentrates ..............................................................................................................12

6.3.4 Preparation of the medium .......................................................................................................................14

6.4 Preparation of the inoculum .....................................................................................................................14

7 Test procedure ...............................................................................................................................................14

7.1 Start of test ......................................................................................................................................................14

7.2 Determination of the initial concentrations (analytical sample) ................................................16

7.2.1 Determination of pH ....................................................................................................................................16

7.2.2 Determination of the suspended solids of the inoculum ................................................................16

7.2.3 Determination of ammonia-nitrogen, nitrite-nitrogen and nitrate-nitrogen (for a

nitrogen balance) ..........................................................................................................................................16

7.2.4 Protein determination (for a nitrogen and carbon balance) ........................................................16

7.2.5 Determination of DOC (for a carbon balance) ....................................................................................17

7.3 Incubation period .........................................................................................................................................17

7.4 End of test ........................................................................................................................................................17

7.5 Determination of CO absorbed in absorption vessels (for a carbon balance) ......................17

8 Calculation and evaluation ........................................................................................................................17

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SIST EN 17417:2021
EN 17417:2020 (E)

8.1 Calculation of the theoretical N production ...................................................................................... 17

8.2 Calculation of the present N production ............................................................................................ 18

8.3 Calculation of the level of degradation related to the nitrogen production ........................... 19

8.4 Generation of a nitrogen balance ............................................................................................................ 19

8.5 Evaluation and expression of results .................................................................................................... 20

9 Validity of results .......................................................................................................................................... 20

10 Test report ...................................................................................................................................................... 21

Annex A (informative) Test scheme — Calculation of the maximum permitted initial mass

of the test substance and the minimum nitrate concentration .................................................... 22

A.1 General ............................................................................................................................................................. 22

A.2 Upper pressure measurement limit ...................................................................................................... 22

A.3 Sorption capacity of the sorption solution (if applicable) ............................................................. 23

A.4 pH buffer capacity of the medium .......................................................................................................... 23

A.5 Minimum nitrate concentration .............................................................................................................. 24

Annex B (informative) Examples of degradation curves ............................................................................. 25

Annex C (informative) Calculation of the produced inorganic carbon and preparation of a

carbon balance .............................................................................................................................................. 28

C.1 Calculation of the produced inorganic carbon ................................................................................... 28

C.2 Generation of a carbon balance ............................................................................................................... 29

C.3 Calculation of the level of biodegradation related to carbon ....................................................... 30

Bibliography ................................................................................................................................................................. 31

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SIST EN 17417:2021
EN 17417:2020 (E)
European foreword

This document (EN 17417:2020) has been prepared by Technical Committee CEN/TC 249 “Plastics”,

the secretariat of which is held by NBN.

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 May 2021, and conflicting national standards shall be

withdrawn at the latest by May 2021.

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.

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, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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SIST EN 17417:2021
EN 17417:2020 (E)
Introduction

Biodegradation of a chemical substance strongly depends on environmental conditions. The presence

or the absence of oxygen is significant for the metabolic pathway on which the degradation by bacteria

can take place. At present, several test methods for the investigation of biodegradability of polymers

under aerobic conditions, but only a few test methods for the investigation of biodegradability under

anaerobic conditions exist. However, degradation under anoxic (denitrifying) conditions has barely

been considered yet. The concept “anoxic” has been created by engineers and designates conditions

under which denitrification can take place. This means that either a little amount of oxygen or no

oxygen at all (< 0,1 mg/l) but nitrate (> 0,1 mg/l NO -N) is present. During heterotrophic

denitrification, e.g. inside the denitrification tank of a wastewater treatment plant, nitrate is reduced to

nitrogen and at the same time organic substrate is oxidized to CO . In nature, anoxic conditions can be

present within the hypolimnion of eutrophic lakes or within the sediment at the transition zone

between the aerobic and the anaerobic zone.

A way to use biodegradable polymers after intended service life would be their addition as additional

carbon source to the denitrification unit of a wastewater treatment plant. In order to check if this way of

disposing a polymer is possible, the biodegradability under anoxic (denitrifying) conditions shall be

determined. Even if a substance shows good aerobic degradability, this does not necessarily apply

under anoxic conditions.

Furthermore, a distinction shall be made between biodegradable polymers that are soluble in water and

those not soluble in water.

Those biodegradable polymers that are soluble in water could be added systematically and

continuously to the denitrification unit as a solid substrate, which is quickly converted and which can

therefore replace the addition of an external liquid carbon source such as ethanol or acetic acid. Testing

their aerobic degradability, their water solubility and, if necessary, their water dispersibility can be

carried out in accordance with EN 14987 [1]. In addition to this, special testing regarding their use as a

carbon source for denitrification is done according to this document. As long as these biodegradable

polymers are present as a solid substance, it shall be ensured that they remain in the denitrification

tank in order to prevent operational failure during other phases of the wastewater treatment plant.

Those biodegradable polymers that are not soluble in water are discontinuously introduced as a solid

substance into a specially designed denitrification reactor, where they substantially remain because of

an appropriate process control. Induced by bacterial activity, they continuously release carbon for the

purpose of denitrification during a process of anoxic degradation, the duration of which depends on

their dimensions (surface/volume ratio). Special testing regarding their use as a water insoluble carbon

source for denitrification is described in this document.
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SIST EN 17417:2021
EN 17417:2020 (E)
1 Scope

This document specifies a method for the determination of the ultimate anoxic biodegradation of

plastics made of organic compounds, where the amount of the produced nitrogen and carbon dioxide at

the end of the test is measured.

The test substance is exposed to an inoculum stemming from the denitrification tank of a wastewater

treatment plant. Testing is performed under defined laboratory conditions.

Claims of performance are limited to the numerical result obtained in the test and not used for making

unqualified claims such as “disposable in waste water treatment plants” and similar.

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.

EN 872:2005, Water quality — Determination of suspended solids — Method by filtration through glass

fibre filters
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 https://www.iso.org/obp
3.1
ultimate anoxic biodegradation

degradation of an organic compound into carbon dioxide, water and mineral salts of any of the present

elements (mineralization) as well as new biomass by means of microorganisms in the presence of

oxidized nitrogen compounds (nitrate, nitrite) and in the absence of oxygen
3.2
suspended solids
solids obtained by filtration under specified conditions
[SOURCE: EN 872:2005, 3.1]
3.3
dissolved inorganic carbon
DIC

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

Note 1 to entry: Phase separation can be achieved for example by centrifugation at 40 000 m s for 15 min or by

membrane filtration using membranes with pores of 0,2 µm to 0,45 µm in diameter
[SOURCE: EN ISO 14852:2018, 3.4]
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SIST EN 17417:2021
EN 17417:2020 (E)
3.4
lag phase

time, measured in days, from the start of a test until adaptation and/or selection of the degrading

microorganisms is achieved and the level of biodegradation of a chemical compound or organic matter

has increased to about 10 % of the maximum level of biodegradation
[SOURCE: EN ISO 14855-1:2012, 3.7]
3.5
level of biodegradation related to the nitrogen production

measured level of biodegradation of a chemical compound or organic substance in a test, calculated

from the amount of actually produced nitrogen divided by the theoretical maximum amount of nitrogen

Note 1 to entry: It is expressed as a percentage.
3.6
level of biodegradation related to carbon

measured level of biodegradation of a chemical compound or organic substance in a test, calculated

from the final products of mineralization of the carbon fraction (amount of carbon from carbon dioxide

and biomass) divided by the carbon fraction of the amount of the test substance used

Note 1 to entry: It is expressed as a percentage.
3.7
maximum level of biodegradation

measured level of biodegradation of a chemical compound or organic substance in a test, above which

no further biodegradation takes places during the test
Note 1 to entry: It is expressed as a percentage.

[SOURCE: EN ISO 14855-1:2012, 3.8, modified — the unit “percentage” has been included in the Note]

3.8
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: It is expressed in days.

[SOURCE: EN ISO 14855-1:2012, 3.9, modified — the unit “days” has been included in the Note]

3.9
plateau phase
time from the end of the biodegradation phase until the end of a test
Note 1 to entry: It is expressed in days.

[SOURCE: EN ISO 14855-1:2012, 3.10, modified — the unit “days” has been included in the Note]

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SIST EN 17417:2021
EN 17417:2020 (E)
3.10
nitrogen recovery rate

sum of the mass concentrations of the nitrogen fractions of nitrate, nitrite, ammonium, protein and of

elementary nitrogen at the end of the test divided by the sum of the corresponding mass concentrations

at the beginning of the test
Note 1 to entry: It is expressed in percent.
3.11
carbon recovery rate

sum of the mass concentrations of the dissolved organic carbon (DOC), the carbon fractions of the test

substance, of carbon dioxide and of biomass at the end of the test divided by the sum of the

corresponding mass concentrations at the beginning of the test
Note 1 to entry: It is expressed in percent.
3.12
theoretical oxygen demand
ThOD

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

Note 1 to entry: It is calculated from the molecular formula of this compound and expressed in milligram of

oxygen uptake per milligram of the test compound.
3.13
theoretical nitrogen production
ThNP

theoretical maximum amount of nitrogen produced during biodegradation under denitrifying

conditions

Note 1 to entry: It is calculated from the molecular formula of the test compound and the stoichiometry of the

anoxic biodegradation of this compound (simplified, without biomass) and expressed in milligram of nitrogen

produced per milligram of the test compound.
3.14
theoretical nitrate demand
ThND

theoretical maximum amount of nitrate-nitrogen that is reduced during biodegradation under

denitrifying conditions

Note 1 to entry: It is calculated from the molecular formula of the test compound and the stoichiometry of the

anoxic biodegradation of this compound (simplified, without biomass) and is expressed in milligram of nitrate-

nitrogen produced per milligram of the test compound.
3.15
preadaptation

pre-incubation of an inoculum in the presence of the chemical compound or organic substance under

test, with the aim of enhancing the ability of the inoculum to biodegrade the test substance by

adaptation and/or selection of the microorganisms
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SIST EN 17417:2021
EN 17417:2020 (E)
4 Principle

In order to investigate anoxic degradation, the nitrogen production (N ) is monitored by means of

pressure measurement in a closed system.

A mineral salt medium, free of oxygen and containing nitrate, with the test substance being the only

carbon source, is placed in a pressure-tight bottle and inoculated with the inoculum from the

denitrification tank of a waste water treatment plant; The gas space of the bottle shall be gassed with

argon or nitrogen in order to prevent oxygen from entering. Subsequently, the vessel is closed by means

of a pressure measuring head. The use of KOH is favourable if the absorbed CO is to be measured by

means of titration in order to prepare a carbon balance.

NOTE KOH is used as its sorption capacity is higher than NaOH, especially if the solution cannot be stirred [4].

Therefore, the pressure increase inside the bottle is proportional to the nitrogen that is produced

during denitrification. Pressure measurement values of a sample are captured and recorded by

pressure sensors and can be transmitted to a personal computer any time. The analytical determination

− − +
NO NO NH

of the , , and protein content at the beginning and the end of the test allows a nitrogen

3 2 4

balance which provides information about the plausibility of results and, in particular, about the leak-

tightness of the system. Based on experience, the resulting concentrations of the metabolites NO and

N O will be very low, which is why they can be neglected.

The level of biodegradation under anoxic conditions is expressed as a percentage and determined by

comparison of the actual and the theoretical nitrogen production. The test result is the maximum level

of biodegradation determined from the plateau phase of the biodegradation curve. Optionally, a carbon

balance can be calculated in order to obtain additional information regarding biodegradation and to

improve the evaluation of biodegradation (see C.2).
5 Equipment and materials
5.1 Pressure measurement system

The following example represents the principle of a system for measuring the produced nitrogen by

means of pressure measurement.
The pressure measurement system (see Figure 1) consists of:

— pressure measuring heads, a combination of a screw cap with integrated pressure sensor;

— bottles capable of being sealed pressure-tight for being used as reaction vessels, nominal volume:

500 ml (or 1 000 ml) with side necks made of glass also capable of being sealed pressure-tight;

— sorption vessel for the purpose of CO sorption in the reaction vessels.
® ®

The OxiTop Control system of WTW, Weilheim, Germany, consisting of “OxiTop -C” pressure measuring

heads, sorption vessels, adequate bottles and accessories, is an example of a suitable product available

commercially. For this system, pressure measurement values of a sample are continuously recorded by the

OxiTop heads. At the start of the test, the value is automatically set to ambient pressure (relative zero). The

heads are controlled (e.g. mode, start, measurement duration, GLP monitoring, etc.) by means of a controller

(OxiTop OC110) using an infrared interface. The relevant pressure measurement values can be retrieved,

displayed and further processed by a software programme any time. This information is given for the convenience

of users of this standard and does not constitute an endorsement by CEN of the product named.

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SIST EN 17417:2021
EN 17417:2020 (E)
Key
1 pressure measuring head with IR interface
2 sorption vessel for CO sorption
3 volume of the gas space (V )
gas

4 reaction vessel containing the test mixture with medium, test substance and inoculum

5 magnetic stirrer bar
6 controller with IR interface
7 pH electrode (optional version with pH measurement)
Figure 1 — Pressure measurement system for measuring biodegradation
under anoxic conditions (example)
5.2 Stirring platform or single magnetic stirrers

Stirring platforms or magnetic stirrers with a heat emission as low as possible should be used. The

temperature inside the test vessels should largely remain constant because temperature variations

strongly influence the measurement results.
5.3 Room or incubator with a constant temperature of (20 ± 2) °C

The incubator should maintain the temperature setpoint as closely as possible because temperature

variations strongly influence the measurement results.
5.4 Argon for the elimination of oxygen from the medium and the gas space
Argon 5.0 with a purity of > 99,999 % shall be used.

The stirring platform IS 6-Var of WTW, Weilheim, Germany, for 6 test vessels is an example of a suitable

product available commercially. This information is given for the convenience of users of this standard and does

not constitute an endorsement by CEN of the product named.

The Thermostat Cabinets of WTW, Weilheim, Germany, are an example of a suitable product available

commercially. This information is given for the convenience of users of this standard and does not constitute an

endorsement by CEN of the product named.
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SIST EN 17417:2021
EN 17417:2020 (E)
5.5 Thermometer

A thermometer serves for measuring the temperature of the inoculum and for checking the set

temperature inside the incubator.
5.6 Membrane filter
The following membrane filters are used for suspended solids determination:
— cellulose acetate membrane filter (with a pore size of 0,45 µm);
— cellulose nitrate membrane filter (with a pore size of 0,45 µm).

5.7 Activated sludge from the denitrification tank of a waste water treatment plant

5.8 KOH or used for sorption
A freshly prepared aqueous standard solution wi
...

SLOVENSKI STANDARD
oSIST prEN 17417:2019
01-november-2019
Ugotavljanje dokončne biorazgradnje plastičnih materialov v vodnem sistemu pri
anoksičnih (denitrifikacijskih) pogojih - Metoda z meritvijo zviševanja tlaka

Determination of the ultimate biodegradation of plastics materials in an aqueous system

under anoxic (denitrifying) conditions - Method by measurement of pressure increase

Bestimmung der vollständigen Bioabbaubarkeit von Kunststoff-Materialien in wässriger

Phase unter anoxischen (denitrifizierenden) Bedingungen - Verfahren mittels Messung

der Druckzunahme

Détermination de la biodégradation ultime des matériaux plastiques dans un système

aqueux dans des conditions anoxiques (dénitrifiantes) - Méthode par mesure de
l'augmentation de pression
Ta slovenski standard je istoveten z: prEN 17417
ICS:
13.030.99 Drugi standardi v zvezi z Other standards related to
odpadki wastes
83.080.01 Polimerni materiali na Plastics in general
splošno
oSIST prEN 17417:2019 en,fr,de

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

---------------------- Page: 1 ----------------------
oSIST prEN 17417:2019
---------------------- Page: 2 ----------------------
oSIST prEN 17417:2019
DRAFT
EUROPEAN STANDARD
prEN 17417
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2019
ICS 13.030.99; 83.080.01
English Version
Determination of the ultimate biodegradation of plastics
materials in an aqueous system under anoxic
(denitrifying) conditions - Method by measurement of
pressure increase

Détermination de la biodégradation ultime des Bestimmung des endgültigen biologischen Abbaus von

matériaux plastiques dans un système aqueux dans des Kunststoffmaterialien in einem wässrigen System

conditions anoxiques (dénitrifiantes) - Méthode par unter anoxischen (denitrifizierenden) Bedingungen -

mesure de l'augmentation de pression Verfahren durch Messung des Druckanstiegs

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 249.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
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. prEN 17417:2019 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN 17417:2019
prEN 17417:2019 (E)
Contents Page

EUROPEAN FOREWORD ................................................................................................................................................ 5

INTRODUCTION............................................................................................................................................................. 6

1 SCOPE ................................................................................................................................................................... 7

2 NORMATIVE REFERENCES ..................................................................................................................................... 7

3 TERMS AND DEFINITIONS ..................................................................................................................................... 7

4 PRINCIPLE ............................................................................................................................................................. 9

5 EQUIPMENT AND MATERIALS ............................................................................................................................. 10

5.1 PRESSURE MEASUREMENT SYSTEM ............................................................................................................................. 10

FIGURE 1 — PRESSURE MEASUREMENT SYSTEM FOR MEASURING BIODEGRADATION UNDER ANOXIC CONDITIONS

(EXAMPLE) .................................................................................................................................................................. 11

5.2 STIRRING PLATFORM OR SINGLE MAGNETIC STIRRERS...................................................................................................... 11

5.3 ROOM OR INCUBATOR WITH A CONSTANT TEMPERATURE OF (20 ± 2) °C .......................................................................... 11

5.4 ARGON FOR THE ELIMINATION OF OXYGEN FROM THE MEDIUM AND THE GAS SPACE ............................................................. 11

5.5 THERMOMETER ...................................................................................................................................................... 12

5.6 MEMBRANE FILTER ................................................................................................................................................. 12

5.7 ACTIVATED SLUDGE FROM THE DENITRIFICATION TANK OF A WASTE WATER TREATMENT PLANT .............................................. 12

5.8 KOH OR NAOH USED FOR SORPTION ......................................................................................................................... 12

5.9 HCL USED FOR TITRATION ......................................................................................................................................... 12

5.10 PH METER WITH ELECTRODES ................................................................................................................................... 12

5.11 CHEMICALS, PIPETTES, PIPETTE TIPS ............................................................................................................................ 12

5.12 PHOTOMETRIC CUVETTE TESTS ................................................................................................................................... 12

5.13 PHOTOMETER FOR THE PROCEDURES OR CUVETTE TESTS ................................................................................................. 13

5.14 ANALYTICAL BALANCE .............................................................................................................................................. 13

6 PREPARATION .................................................................................................................................................... 13

6.1 DETERMINATION OF THE VOLUME OF EACH REACTION VESSEL .......................................................................................... 13

6.2 SAMPLE PREPARATION ............................................................................................................................................. 13

6.3 PREPARATION OF THE MEDIUM .................................................................................................................................. 13

6.3.1 Reagents ........................................................................................................................................................ 13

6.3.2 Distilled or deionized water ........................................................................................................................... 13

6.3.3 Preparation of the concentrates .................................................................................................................... 13

6.3.4 Preparation of the medium ........................................................................................................................... 15

6.4 PREPARATION OF THE INOCULUM ............................................................................................................................... 15

7 TEST PROCEDURE ............................................................................................................................................... 15

7.1 START OF TEST ........................................................................................................................................................ 15

TABLE 1 — EXAMPLE OF ADDITION INTO THE REACTION VESSELS .............................................................................. 16

7.2 DETERMINATION OF THE INITIAL CONCENTRATIONS (ANALYTICAL SAMPLE) ......................................................................... 17

7.2.1 Determination of pH ...................................................................................................................................... 17

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7.2.2 Determination of the suspended solids of the inoculum ............................................................................... 17

7.2.3 Determination of ammonia-nitrogen, nitrite-nitrogen and nitrate-nitrogen (for a nitrogen balance) ......... 17

7.2.4 Protein determination (for a nitrogen and carbon balance) ......................................................................... 17

7.2.5 Determination of DOC (for a carbon balance) ............................................................................................... 17

7.3 INCUBATION PERIOD ................................................................................................................................................ 17

7.4 END OF TEST .......................................................................................................................................................... 18

7.5 DETERMINATION OF CO ABSORBED IN ABSORPTION VESSELS (FOR A CARBON BALANCE) ..................................................... 18

8 CALCULATION AND EVALUATION ........................................................................................................................ 18

8.1 CALCULATION OF THE THEORETICAL N PRODUCTION .................................................................................................... 18

8.2 CALCULATION OF THE PRESENT N PRODUCTION .......................................................................................................... 19

8.3 CALCULATION OF THE LEVEL OF DEGRADATION RELATED TO THE NITROGEN PRODUCTION ...................................................... 20

8.4 GENERATION OF A NITROGEN BALANCE ....................................................................................................................... 20

TABLE 2 — EXAMPLE OF A NITROGEN BALANCE FOR TESTING THE ANOXIC DEGRADATION WITH PHB AS THE TEST

SUBSTANCE (INITIAL MASS: M = 100 MG) ................................................................................................................... 20

8.5 EVALUATION AND EXPRESSION OF RESULTS .................................................................................................................. 21

9 VALIDITY OF RESULTS ......................................................................................................................................... 21

10 TEST REPORT....................................................................................................................................................... 21

ANNEX A (INFORMATIVE) TEST SCHEME — CALCULATION OF THE MAXIMUM PERMITTED INITIAL MASS OF THE TEST

SUBSTANCE AND THE MINIMUM NITRATE CONCENTRATION ..................................................................................... 23

A.1 GENERAL......................................................................................................................................................... 23

A.2 UPPER PRESSURE MEASUREMENT LIMIT ........................................................................................................ 23

A.3 SORPTION CAPACITY OF THE SORPTION SOLUTION (IF APPLICABLE) .............................................................. 24

A.4 PH BUFFER CAPACITY OF THE MEDIUM .......................................................................................................... 24

A.5 MINIMUM NITRATE CONCENTRATION ........................................................................................................... 25

ANNEX B (INFORMATIVE) EXAMPLES OF DEGRADATION CURVES .............................................................................. 26

FIGURE B.1 — PRESSURE INCREASE DURING A BIODEGRADATION TEST UNDER ANOXIC CONDITIONS WITH PHB

(POLY-Β-HYDROXYBUTYRIC ACID) AND BLANK VALUE ................................................................................................ 27

FIGURE B.2 — PERCENTAGE BIODEGRADATION RELATED TO THE NITROGEN PRODUCTION OF A TEST SUBSTANCE

(PHB IN THIS CASE) IN A DEGRADATION TEST UNDER ANOXIC CONDITIONS — SAME TEST AS IN FIGURE B.1 ............ 28

ANNEX C (INFORMATIVE) CALCULATION OF THE PRODUCED INORGANIC CARBON AND PREPARATION OF A CARBON

BALANCE ..................................................................................................................................................................... 29

C.1 CALCULATION OF THE PRODUCED INORGANIC CARBON ................................................................................ 29

FIGURE C.1 — EXAMPLES OF TITRATION CURVES OF THE ORIGINAL KOH (NEW, ORIGINAL, 25 MMOL) AND OF THE

SORPTION SOLUTIONS OF A BLANK TEST AND A TEST WITH PHB (100 MG) AFTER COMPLETION OF THE TEST

(STANDARD TEST CONDITIONS) .................................................................................................................................. 29

C.2 GENERATION OF A CARBON BALANCE ............................................................................................................ 30

TABLE 3 — EXAMPLE OF A CARBON BALANCE FOR PHB (INITIAL MASS: M = 100 MG) ................................................ 31

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C.3 CALCULATION OF THE LEVEL OF BIODEGRADATION RELATED TO CARBON..................................................... 31

BIBLIOGRAPHY ............................................................................................................................................................ 32

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European foreword

This document (prEN 17417:2019) has been prepared by Technical Committee CEN/TC 249 “Plastics”,

the secretariat of which is held by NBN.
This document is currently submitted to the CEN Enquiry.
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Introduction

Biodegradation of chemical substance strongly depends on environmental conditions. The presence or

the absence of oxygen is significant for the metabolic pathway on which the degradation of bacteria can

take place. At present, several test methods for the investigation of biodegradability of polymers under

aerobic conditions, but only a few test methods for the investigation of biodegradability under anaerobic

conditions exist. However, degradation under anoxic (denitrifying) conditions has barely been

considered yet. The concept “anoxic” has been created by engineers and designates conditions under

which denitrification can take place. This means that either a little amount of oxygen or no oxygen at all

(< 0,1 mg/l) but nitrate (> 0,1 mg/l NO -N) is present. During heterotrophic denitrification, e.g. inside

the denitrification tank of a wastewater treatment plant, nitrate is reduced to nitrogen and at the same

time organic substrate is oxidized to CO . In nature, anoxic conditions can be present within the

hypolimnion of eutrophic lakes or within the sediment at the transition zone between the aerobic and

the anaerobic zone.

A way to use biodegradable polymers after intended service life would be their addition as additional

carbon source to the denitrification unit of a wastewater treatment plant. In order to check if this way of

disposing a polymer is possible, the biodegradability under anoxic (denitrifying) conditions shall be

determined. Even if a substance shows good aerobic degradability, this does not necessarily apply under

anoxic conditions.

Furthermore, a distinction shall be made between biodegradable polymers that are soluble in water and

those not soluble in water.

Those biodegradable polymers that are soluble in water could be added systematically and continuously

to the denitrification unit as a solid substrate, which is quickly converted and which can therefore replace

the addition of an external liquid carbon source such as ethanol or acetic acid. Testing their aerobic

degradability, their water solubility and, if necessary, their water dispersibility can be carried out in

accordance with EN 14987. In addition to this, special testing regarding their use as a carbon source for

denitrification is done according to this standard. As long as these biodegradable polymers are present

as a solid substance, it shall be ensured that they remain in the denitrification tank in order to prevent

operational failure during other phases of the wastewater treatment plant.

Those biodegradable polymers that are not soluble in water are discontinuously introduced as a solid

substance into a specially designed denitrification reactor, where they substantially remain because of

an appropriate process control. Induced by bacterial activity, they continuously release carbon for the

purpose of denitrification during a process of anoxic degradation, the duration of which depends on their

dimensions (surface/volume ratio). Special testing regarding their use as a water insoluble carbon source

for denitrification is described in this standard.
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1 Scope

This document specifies a method for the determination of the ultimate anoxic biodegradability of

plastics made of organic compounds, where the amount of the produced nitrogen and carbon dioxide at

the end of the test is measured.

The test substance is exposed to an inoculum stemming from the denitrification tank of a wastewater

treatment plant. Testing is performed under defined laboratory conditions.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

EN 872:2005, Water quality — Determination of suspended solids - Method by filtration through glass fibre

filters
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
ultimate anoxic biodegradation

degradation of an organic compound into carbon dioxide, water and mineral salts of any of the present

elements (mineralization) as well as new biomass by means of microorganisms in the presence of

oxidized nitrogen compounds (nitrate, nitrite) and in the absence of oxygen
3.2
suspended solids
solids removed by filtration under specified conditions
[SOURCE: EN 872:2005, 3.1]
3.3
dissolved inorganic carbon
DIC

that part of the inorganic carbon in water which cannot be removed by specified phase separation, for

example by centrifugation at 40 000 m s for 15 min or by membrane filtration using membranes with

pores of 0,2 µm to 0,45 µm in diameter
[SOURCE: EN ISO 14852:2004, 3.4] [1]
3.4
lag phase

time, measured in days, from the start of a test until adaptation and/or selection of the degrading

microorganisms is achieved and the level of biodegradation of a chemical compound or organic matter

has increased to about 10 % of the maximum level of biodegradation
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[SOURCE: EN ISO 14855-1:2013, 3.7] [2]
3.5
level of biodegradation related to the nitrogen production

measured level of biodegradation of a chemical compound or organic substance in a test, calculated from

the amount of actually produced nitrogen divided by the theoretical maximum amount of nitrogen

Note 1 to entry: It is expressed as a percentage.
3.6
level of biodegradation related to carbon

measured level of biodegradation of a chemical compound or organic substance in a test, calculated from

the final products of mineralization of the carbon fraction (amount of carbon from carbon dioxide and

biomass) divided by the carbon fraction of the amount of the test substance used
Note 1 to entry: It is expressed as a percentage.
3.7
maximum level of biodegradation

measured level of biodegradation of a chemical compound or organic substance in a test, above which no

further biodegradation takes places during the test
Note 1 to entry: It is expressed as a percentage.

[SOURCE: EN ISO 14855-1:2013, 3.8, modified — the unit “percentage” has been included in the Note]

[2]
3.8
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: It is expressed in days.

[SOURCE: EN ISO 14855-1:2013, 3.9, modified — the unit “days” has been included in the Note] [2]

3.9
plateau phase
time from the end of the biodegradation phase until the end of a test
Note 1 to entry: It is expressed in days.

[SOURCE: EN ISO 14855-1:2013, 3.10, modified — the unit “days” has been included in the Note] [2]

3.10
nitrogen recovery rate

sum of the mass concentrations of the nitrogen fractions of nitrate, nitrite, ammonium, protein and of

elementary nitrogen at the end of the test divided by the sum of the corresponding mass concentrations

at the beginning of the test
Note 1 to entry: It is expressed in percent.
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3.11
carbon recovery rate

sum of the mass concentrations of the dissolved organic carbon (DOC), the carbon fractions of the test

substance, of carbon dioxide and of biomass at the end of the test divided by the sum of the corresponding

mass concentrations at the beginning of the test
Note 1 to entry: It is expressed in percent.
3.12
theoretical oxygen demand
ThOD

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

Note 1 to entry: It is calculated from the molecular formula of this compound and expressed in milligram of oxygen

uptake per milligram of the test compound.
3.13
theoretical nitrogen production
ThNP

theoretical maximum amount of nitrogen produced during biodegradation under denitrifying conditions

Note 1 to entry: It is calculated from the molecular formula of the test compound and the stoichiometry of the

anoxic biodegradation of this compound (simplified, without biomass) and expressed in milligram of nitrogen

produced per milligram of the test compound.
3.14
theoretical nitrate demand
ThND

theoretical maximum amount of nitrate-nitrogen that is reduced during biodegradation under

denitrifying conditions

Note 1 to entry: It is calculated from the molecular formula of the test compound and the stoichiometry of the

anoxic biodegradation of this compound (simplified, without biomass) and is expressed in milligram of nitrate-

nitrogen produced per milligram of the test compound.
3.15
preadaptation

pre-incubation of an inoculum in the presence of the chemical compound or organic substance under test,

with the aim of enhancing the ability of the inoculum to biodegrade the test substance by adaptation

and/or selection of the microorganisms
4 Principle

In order to investigate anoxic degradation, the nitrogen production (N ) is monitored by means of

pressure measurement in a closed system.

A mineral salt medium, free of oxygen and containing nitrate, with the test substance being the only

carbon source, is placed in a pressure-tight bottle and inoculated with the inoculum from the

denitrification tank of a waste water treatment plant; The gas space of the bottle shall be gassed with

argon or nitrogen in order to prevent oxygen from entering. Subsequently, the vessel is closed by means

of a pressure measuring head. The use of KOH or NaOH is favourable if the absorbed CO is to be

measured by means of titration in order to prepare a carbon balance.

Therefore, the pressure increase inside the bottle is proportional to the nitrogen that is produced during

denitrification. Pressure measurement values of a sample are captured and recorded by pressure sensors

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− −

and can be transmitted to a personal computer any time. The analytical determination of the , ,

NO NO
3 2

NH and protein content at the beginning and the end of the test allows a nitrogen balance which

provides information about the plausibility of results and, in particular, about the leak-tightness of the

system. Based on experience, the resulting concentrations of the metabolites NO and N O will be very

low, which is why they can be neglected.

The level of biodegradation under anoxic conditions is expressed as a percentage and determined by

comparison of the actual and the theoretical nitrogen production. The test result is the maximum level of

biodegradation determined from the plateau phase of the biodegradation curve. Optionally, a carbon

balance can be calculated in order to obtain additional information regarding biodegradation and to

improve the evaluation of biodegradation (see C.2).
5 Equipment and materials
5.1 Pressure measurement system

The following example represents the principle of a system for measuring the produced nitrogen by

means of pressure measurement.
The pressure measurement system (see Figure 1) consists of:

— pressure measuring heads, a combination of a screw cap with integrated pressure sensor;

— bottles capable of being sealed pressure-tight for being used as reaction vessels, nominal volume:

500 ml (or 1 000 ml) with side neck made of glass also capable of being sealed pressure-tight;

— sorption vessel for the purpose of CO sorption in the reaction vessels.

The OxiTop Control system of WTW, Weilheim, Germany, consisting of “OxiTop -C” pressure measuring

heads, sorption vessels, adequate bottles and accessories, is an example of a suitable product available

commercially. For this system, pressure measurement values of a sample are continuously recorded by the OxiTop

heads. At the start of the test, the value is automatically set to ambient pressure (relative zero). The heads are

controlled (e.g. mode, start, measurement duration, GLP monitoring, etc.) by means of a controller

(OxiTop OC110) using an infrared interface. The relevant pressure measurement values can be retrieved,

displayed and further processed by a software programme any time. This information is given for the convenience

of users of this standard and does not constitute an endorsement by CEN of the product named.

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Key
1 pressure measuring head with IR interface
2 sorption vessel for CO sorption
3 volume of the gas space (V )
gas

4 reaction vessel containing the test mixture (medium, test substance and inoculum)

5 magnetic stirrer
6 controller with IR interface
7 pH electrode (optional version with pH measurement)
Figure 1 — Pressure measurement system for measuring biodegradation
under anoxic conditions (example)
5.2 Stirring platform or single magnetic stirrers

Stirring platforms or magnetic stirrers with a heat emission as low as possible should be used. The

temperature inside the test vessels should largely remain constant because temperature variations

strongly influence the measurement results.
5.3 Room or incubator with a constant temperature of (20 ± 2) °C

The incubator should maintain the temperature setpoint as closely as possible because temperature

variations strongly influence the measurement results.
5.4 Argon for the elimination of oxygen from the medium and the gas space
Argon 5.0 with a purity of > 99,999 % shall be used.
...

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