prEN ISO 23977-2

Plastics - Determination of the aerobic biodegradation of plastic materials exposed to seawater - Part 2: Method by measuring the oxygen demand in closed respirometer (ISO 23977-2:2020)

This document specifies a laboratory test method for determining the degree and rate of the aerobic biodegradation level of plastic materials. Biodegradation of plastic materials is determined by measuring the oxygen demand in a closed respirometer when exposed to seawater sampled from coastal areas under laboratory conditions.
The conditions described in this document might not always correspond to the optimum conditions for the maximum degree of biodegradation, however this test method is designed to give an indication of the potential biodegradability of plastic materials.
NOTE This document addresses plastic materials but can also be used for other materials.

Kunststoffe - Bestimmung des aeroben Bioabbaus von Meerwasser ausgesetzten Kunststoff-Materialien - Teil 2: Verfahren mittels Messung des Sauerstoffbedarfs in einem geschlossenen Respirometer (ISO 23977 2:2020)

Plastiques - Détermination de la biodégradation aérobie des matières plastiques exposées à l'eau de mer - Partie 2: Méthode par mesure de la demande en oxygène dans un respiromètre fermé (ISO 23977-2:2020)

Polimerni materiali - Določanje aerobne biorazgradljivosti polimernih materialov, izpostavljenih morski vodi - 2. del: Metoda z merjenjem porabe kisika v zaprtem respirometru (ISO 23977-2:2020)

General Information

• Status: Not Published
• ICS: 83.080.01 - Plastics in general
• Technical Committee: CEN/TC 249 - Plastics
• Drafting Committee: CEN/TC 249 - Plastics
• Current Stage: 4060 - Closure of enquiry - Enquiry
• Due Date: 07-Oct-2021
• Completion Date: 07-Oct-2021
• Ref Project: oSIST prEN ISO 23977-2:2021 - Plastics - Determination of the aerobic biodegradation of plastic materials exposed to seawater - Part 2: Method by measuring the oxygen demand in closed respirometer (ISO 23977-2:2020)

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 23977-2:2021
01-september-2021

Polimerni materiali - Določanje aerobne biorazgradljivosti polimernih materialov,

izpostavljenih morski vodi - 2. del: Metoda z merjenjem porabe kisika v zaprtem
respirometru (ISO 23977-2:2020)

Plastics - Determination of the aerobic biodegradation of plastic materials exposed to

seawater - Part 2: Method by measuring the oxygen demand in closed respirometer (ISO

23977-2:2020)
Kunststoffe - Bestimmung des aeroben Bioabbaus von Meerwasser ausgesetzten

Kunststoff-Materialien - Teil 2: Verfahren mittels Messung des Sauerstoffbedarfs in einem

geschlossenen Respirometer (ISO 23977 2:2020)
Plastiques - Détermination de la biodégradation aérobie des matières plastiques
exposées à l'eau de mer - Partie 2: Méthode par mesure de la demande en oxygène
dans un respiromètre fermé (ISO 23977-2:2020)
Ta slovenski standard je istoveten z: prEN ISO 23977-2
ICS:
13.020.40 Onesnaževanje, nadzor nad Pollution, pollution control
onesnaževanjem in and conservation
ohranjanje
83.080.01 Polimerni materiali na Plastics in general
splošno
oSIST prEN ISO 23977-2:2021 en,fr,de

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

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oSIST prEN ISO 23977-2:2021
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oSIST prEN ISO 23977-2:2021
INTERNATIONAL ISO
STANDARD 23977-2
First edition
2020-11
Plastics — Determination of the
aerobic biodegradation of plastic
materials exposed to seawater —
Part 2:
Method by measuring the oxygen
demand in closed respirometer
Plastiques — Détermination de la biodégradation aérobie des
matières plastiques exposées à l'eau de mer —
Partie 2: Méthode par mesure de la demande en oxygène dans un
respiromètre fermé
Reference number
ISO 23977-2:2020(E)
ISO 2020
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)
Contents Page

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

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

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

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 3

5 Test environment ................................................................................................................................................................................................. 3

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 4

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Test material ............................................................................................................................................................................................. 5

8.2 Reference materials ............................................................................................................................................................................ 6

8.3 Test set up ................................................................................................................................................................................................... 6

8.4 Pre-conditioning phase .................................................................................................................................................................... 6

8.5 Start of the test ........................................................................................................................................................................................ 7

8.6 End of the test .......................................................................................................................................................................................... 7

9 Calculation and expression of results ............................................................................................................................................. 7

9.1 Calculation .................................................................................................................................................................................................. 7

9.2 Visual inspection ................................................................................................................................................................................... 8

9.3 Expression and interpretation of results ......................................................................................................................... 8

10 Validity of results ................................................................................................................................................................................................. 9

11 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Example of a respirometric system .......................................................................................................10

Bibliography .............................................................................................................................................................................................................................12

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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

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

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see

www .iso .org/ iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 14,

Environmental aspects.
A list of all parts in the ISO 23997 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)
Introduction

According to the United Nations Environment Program (UNEP), one of the most notable properties of

synthetic polymers and plastics is their durability which, combined with their accidental loss, deliberate

release and poor waste management has resulted in the ubiquitous presence of plastic in oceans (UNEP,

[15]
2015 ).

It is well known and documented that marine litter can pose risks and a negative impact on living

marine organisms and on human beings. Degradability of plastic materials exposed to the marine

environment is one of the factors affecting impact and strength of effects. The uncontrolled dispersion

of biodegradables plastics in natural environments is not desirable. The biodegradability of products

cannot be considered as an excuse to spread wastes that should be recovered and recycled. However,

test methods to measure rate and level of biodegradation in natural environments are of interest in

order to better characterize the behaviour of plastics in these very particular environments. Thus, the

degree and rate of biodegradation is of major interest in order to obtain an indication of the potential

biodegradability of plastic materials when exposed to different marine habitats.

ISO/TC 61/SC 14 has established several test methods for biodegradation testing of plastic materials

under laboratory conditions covering different environmental compartments and test conditions, as

shown in Table 1.
Table 1 — Test methods for biodegradation testing of plastics
Conditions
Test methods
Environmental compartment Presence/absence of oxygen
ISO 14855-1
Controlled composting conditions Aerobic conditions
ISO 14855-2
High-solids anaerobic-digestion
Anaerobic conditions ISO 15985
conditions
Controlled anaerobic slurry system Anaerobic conditions ISO 13975
Soil Aerobic conditions ISO 17556
ISO 14851
Aerobic conditions
Aqueous medium ISO 14852
Anaerobic conditions ISO 14853
ISO 18830
Seawater/sandy sediment interface Aerobic conditions
ISO 19679
Marine sediment Aerobic conditions ISO 22404
ISO 23977-1
Seawater Aerobic conditions
ISO 23977-2

Test method for measuring biodegradation of plastic materials when exposed to marine microbes.

All marine biodegradation test methods are based on exposure of plastic materials to marine samples

(seawater and/or sediment) taken from shoreline areas. By a quantitative viewpoint, these methods

are not equivalent, because, for example, the microbial density in seawater is generally lower compared

to the density determined in sediment. In addition, the microbial composition and diversity can be

different. Moreover, as a rule, the nutrient concentration found in sediment is normally higher compared

to the concentration in seawater.

This document provides a test method for determining the biodegradation level of plastic materials

exposed to the microbial population present in seawater from a pelagic zone under laboratory

conditions. The biodegradation is followed by measuring the oxygen demand in a closed respirometer.

The test is performed with either seawater only (“pelagic seawater test”) or with seawater to which

little sediment was added (“suspended sediment seawater test”).
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)

The pelagic seawater test simulates the conditions found in offshore areas with low water currents and

low tidal movements, whereas the suspended sediment seawater test simulates conditions which might

be found in coastal areas with stronger water currents and tidal movements.
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oSIST prEN ISO 23977-2:2021
INTERNATIONAL STANDARD ISO 23977-2:2020(E)
Plastics — Determination of the aerobic biodegradation of
plastic materials exposed to seawater —
Part 2:
Method by measuring the oxygen demand in closed
respirometer
1 Scope

This document specifies a laboratory test method for determining the degree and rate of the aerobic

biodegradation level of plastic materials. Biodegradation of plastic materials is determined by

measuring the oxygen demand in a closed respirometer when exposed to seawater sampled from

coastal areas under laboratory conditions.

The conditions described in this document might not always correspond to the optimum conditions for

the maximum degree of biodegradation, however this test method is designed to give an indication of

the potential biodegradability of plastic materials.

NOTE This document addresses plastic materials but can also be used for other materials.

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 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples

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

organic carbon (DOC)

ISO 10210, Plastics — Methods for the preparation of samples for biodegradation testing of plastic

materials
ISO 10523, Water quality — Determination of pH

ISO 11261, Soil quality — Determination of total nitrogen — Modified Kjeldahl method

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:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
pelagic zone
water body above the seafloor
Note 1 to entry: It is also referred to as the open water or the water column.
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)

Note 2 to entry: The surface of the pelagic zone is moved by wind-driven waves, is in contact with the atmosphere

and exposed to sunlight. With increasing depth pressure increases, temperature decreases, and light and surface

wave energy are attenuated.
[SOURCE: ISO 22766:2020, 3.4]
3.2
biochemical oxygen demand
BOD

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

biological oxidation of a chemical compound or organic matter in water

Note 1 to entry: It is expressed as milligrams of oxygen uptake per milligram or gram of test compound.

[SOURCE: ISO 18830:2016, 3.1]
3.3
theoretical oxygen demand
ThOD

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

from the molecular formula

Note 1 to entry: It is expressed as milligrams of oxygen uptake per milligram or gram of test compound.

[SOURCE: ISO 18830:2016, 3.2]
3.4
total organic carbon
TOC
amount of carbon bound in an organic compound

Note 1 to entry: It is expressed as milligrams of carbon per 100 mg of the compound.

[SOURCE: ISO 17556:2019, 3.14]
3.5
dissolved organic carbon
DOC

part of the organic 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 diameter.
[SOURCE: ISO 14852:—, 3.7]
3.6
lag phase

time from the start of a test until adaptation and/or selection of the degrading microorganisms is

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

about 10 % of the maximum level of biodegradation (3.8)
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.8]
3.7
biodegradation phase

time from the end of the lag phase (3.6) of a test until the plateau phase has been reached

Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.10]
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ISO 23977-2:2020(E)
3.8
maximum level of biodegradation

degree of biodegradation of a chemical compound or organic matter in a test, above which no further

biodegradation takes place during the test
Note 1 to entry: It is measured in per cent.
[SOURCE: ISO 14852:—, 3.9]
3.9
plateau phase
time from the end of the biodegradation phase (3.7) until the end of a test
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.11]
3.10
pre-conditioning

pre-incubation of an inoculum under the conditions of the subsequent test in the absence of the chemical

compound or organic matter under test, with the aim of improving the test by acclimatization of the

microorganisms to the test conditions
[SOURCE: ISO 14852:—, 3.13]
4 Principle

This document describes two variations of a test method for determining the biodegradability of plastic

materials by the indigenous population of microorganisms in natural seawater using a static aqueous

test system. The test is performed under mesophilic test conditions for up to two years by incubating

plastic materials with either seawater only (“pelagic seawater test”) or with seawater to which low

amount of sediment has been added (“suspended sediment seawater test”), coming from the same site

as that from which the seawater was taken.

The system is contained in a closed flask, in a respirometer. The carbon dioxide evolved is absorbed

in a suitable absorber in the headspace of the flasks. The consumption of oxygen (BOD) is determined,

for example, by measuring the amount of oxygen required to maintain a constant volume of gas in the

respirometer flasks, or by measuring the change in volume or pressure (or a combination of the two)

either automatically or manually.

The level of biodegradation is determined by comparing the BOD with the theoretical amount (ThOD)

and expressed in percentage. The influence of possible nitrification processes on the BOD shall be

considered. The test result is the maximum level of biodegradation determined from the plateau phase

of the biodegradation curve.
5 Test environment

Incubation shall take place in the dark or in diffused light, in an enclosure which is free from vapours

inhibitory to marine microorganisms and which is maintained at a constant mesophilic temperature.

It should preferably be between 15 °C to 25 °C, but not exceeding 28 °C, to an accuracy of ±1 °C. Any

change in temperature shall be justified and clearly indicated in the test report.

NOTE Test results are obtained for temperatures that can be different from real conditions in marine

environment.
6 Reagents
Use only reagents of recognized analytical grade.
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)
6.1 Water

Distilled or deionized water, free of toxic substances (copper in particular) and containing less than

2 mg/l of TOC.
6.2 Natural seawater/sediment

Sampling, preservation, handling, transport and storage of natural seawater, and, if applicable,

sediment collected from the same site as that from which the seawater is taken, shall be in accordance

with ISO 5667-3.

Prior to use, remove coarse particles from the seawater and, if applicable, from the sediment by

appropriate means. The procedure used shall be reported.

Seawater can be filtered using a paper filter in order to remove coarse particles. It is recommended to

reduce the amount of coarse particles in sediment by means of at least two washing steps using filtered

seawater without coarse particles.

Measure TOC, pH and nitrogen content of seawater and, if applicable, of sediment samples according to

ISO 8245, ISO 10523 and ISO 11261, respectively.

If the TOC content of the seawater sample is found to be high, the seawater should be pre-conditioned

for about a week prior to use. If, for instance, the background concentration of TOC exceeds about 20 %

of the total TOC after addition of the test item, then pre-condition the seawater and, if applicable, the

sediment by stirring under aerobic conditions at the test temperature and in the dark or in diffuse light

in order to reduce the content of easily degradable organic material.

Provide the following information on the seawater, and, if applicable, on the sediment sample itself:

— date of collection;
— depth of collection (m);
— appearance of sample - turbid, clear, etc.;
— temperature at the time of collection (°C);
— salinity (PSU);
— total organic carbon (TOC; mg/l);
— nitrogen (total-N; mg/l);
— pH;
— description of the pre-conditioning process, if applicable.
7 Apparatus

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

Required is usual laboratory equipment, plus the following.
7.1 Closed respirometer.

Flasks of the volume of about 300 ml are appropriate. The vessels shall be located in a constant

temperature room or in an apparatus fitted with a thermostat (e.g. water-bath).

Reactors with higher or lower volumes can be used, if environmental conditions are not affected.

A suitable apparatus is shown in Annex A, Figure A.1.
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)

Any respirometer able to determine with sufficient accuracy the biochemical oxygen demand is

suitable, preferably an apparatus which measures and replaces automatically and continuously the

oxygen consumed so that no oxygen deficiency and no inhibition of the microbial activity occurs during

the degradation process. Analytical equipment to measure total organic carbon (TOC) and dissolved

organic carbon (DOC) is given in ISO 8245.
7.2 Analytical equipment for measuring nitrate and nitrite concentrations.

A qualitative test (for example test strips or photometric tests for nitrate and nitrite) is recommended

first to decide if any nitrification has occurred. If there is evidence of nitrate/nitrite in the medium, a

quantitative determination using a suitable method (for example ion chromatography) is required.

7.3 Analytical balance, which shall have a sensitivity of at least 0,1 mg.
7.4 Magnetic stirrer.
7.5 pH meter.
8 Procedure
8.1 Test material

The sample shall be of known mass and contain sufficient carbon to yield a BOD that can be adequately

measured by the chosen system [closed respirometer (7.1)].

Use a test material concentration of at least 100 mg/l of seawater plus sediment. The mass of the

samples should correspond to a ThOD of about 170 mg/l or a TOC of about 60 mg/l. The maximum mass

of sample per flask is limited by the oxygen supply to the respirometer.

The ThOD (see ISO 14851:2019, Annex A) and the TOC (using ISO 8245 or from the chemical formula or

determine by elemental analysis) shall be calculated.

The test material is added to a test flask, either as powder or in the form of a film. If the test material

is used in the form of powder, particles of known, narrow size distribution should be used. A particle-

size distribution with a maximum diameter of 250 µm is recommended. The preparation of powder

shall be performed in accordance with ISO 10210. If the test material is used in the form of a film, it

can be added either as pieces in the range of 0,2 cm × 0,2 cm to 0,5 cm × 0,5 cm or as a single plastic

strip (width: approximately 1,0 cm, length: depending on weight of the polymer and thickness of the

film). It is recommended that the plastic strip is fixed in, for example, a polytetrafluoroethylene (PTFE)

coated fibre net (size: approximately 4 cm × 9 cm, mesh size: 5 mm × 5 mm). The fibre net is folded

into 2 layers (approximately 2 cm × 9 cm) with the plastic strip test material fixed in between. Then, the

two ends of the fibre net are attached together. The test material fixed between the fibre net is placed

upright on the ground of a bottle base in the form of a cylinder (see Annex A, Figure A.2).

The form and shape of the test material can influence its biodegradability. Similar particle sizes of power

should preferably be used in the test. Similar shapes and thicknesses of the films should preferably be

used if different kinds of plastic materials are to be compared.

When powder or pieces of films are used in the test, particles or film pieces can stick on the inner

wall of the testing bottle above the seawater. In such cases, a slight manual shaking of the bottle is

recommended to regain the powder or film pieces back to the seawater sample. If the material is added

as a cylindrical plastic strip fixed between, for example, a polytetrafluoroethylene (PTFE) coated fibre

net (see Annex A, Figure A.2), it is immersed in the seawater most of the time.

1) PTFE Glass Fabric (product no 9002) produced by Fiberflon (https:// www .fiberflon .de/ Products/ PTFE -Coated

-Open -Mesh -Fabrics/ Page -307 -17 .aspx) has been found satisfactory for this purpose and is an example of a suitable

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

not constitute an endorsement by ISO of this product.
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oSIST prEN ISO 23977-2:2021
ISO 23977-2:2020(E)
8.2 Reference materials

Use microcrystalline cellulose or ashless cellulose filters as a reference material . If possible, the TOC,

form, and size should be comparable to that of the test material.

As a negative control, a non-biodegradable polymer (e.g. polyethylene) in the same form as the test

material can be used.
8.3 Test set up
Provide several flasks, so that the test includes at least the following:
a) three flasks for the test material (symbol F );
b) three flasks for the blank (symbol F );
c) three flasks for reference material (symbol F ).

In addition, if biodegradation is expected to take longer than 6 months, it is recommended that a

negative control is included:
d) three flasks for negative control (symbol F ).

Two flasks for test material, blank, reference material, and negative control may be used instead of

three for screening purposes.
8.4 Pre-conditioning phase
As a rule, use a test flask with a volume of 300 ml.

The test is performed in batch by incubating the test materials with either 90 ml of natural seawater

only (“pelagic seawater test”) or with 90 ml of natural seawater to which sediment of 0,1 g/l to 1,0 g/l

(wet weight) has added (“suspended sediment seawater test”).

Add carbon dioxide absorber to the absorber compartments of the test flask (see ISO 14851:2019,

Annex C). Place the sealed flasks on a magnetic stirrer (7.4) in a constant-temperature environment

and allow all vessels to reach the desired temperature. Agitation shall be continuous (e.g. 100 r/min

agitation) in order to maintain microorganisms and, if applicable, sediment in suspension.

The abrasion of sediment in coastal areas is a natural phenomenon caused by water currents and tidal

movements. Nevertheless, if a magnetic stirring bar is used to mix the seawater to which sediment has

added (“suspended sediment seawater test”), it is recommended that either a PTFE-coated dumbbell

shaped magnetic stirring bar be used or a PTFE-coated magnetic bar equipped with a pivot ring in

order to reduce excessive abrasion of sediment durin
...