ISO 18957:2026

• Abstract:

  This document describes a laboratory test method to determine the aerobic biodegradation of plastic materials exposed to seawater using accelerated conditions. It describes the general requirements of the apparatus and the procedures for using this test method. This test method is designed to give an early indication of the potential biodegradability of plastic materials in marine environment. For the purpose of promoting the development of biodegradable plastics, this document also provides a method for measuring the biomass components produced by metabolizing the test material before it is completely mineralized to CO2, that enables to evaluate the potential biodegradability considered by carbon flow by the sum of the degree of biodegradation based on O2 or CO2 analysis and the degree of conversion to biomass in much shorter period of time than full mineralization. This method is not suitable to assess the degree of disintegration of plastic materials caused by abiotic factors like heat or UV radiation.

• Status: Published
• Publication Date: 04-Oct-2026
• ICS: 83.080.01 - Plastics in general
• Technical Committee: ISO/TC 61/SC 14 - Environmental aspects
• Drafting Committee: ISO/TC 61/SC 14 - Environmental aspects
• Current Stage: 6060 - International Standard published
• Start Date: 05-Oct-2026
• Due Date: 04-Aug-2026
• Completion Date: 05-Oct-2026

ISO 18957:2026 is an International Standard published by ISO for laboratory determination of the aerobic biodegradation of plastic materials exposed to seawater under accelerated conditions. It specifies a seawater-based test method, including apparatus, procedures and calculations, for checking the potential biodegradability of plastics in a marine environment. It is aimed at laboratories and organisations that need an early indication of biodegradation performance, not a test for disintegration caused by heat or UV.

What does ISO 18957:2026 specify?

ISO 18957:2026 specifies a laboratory method for measuring aerobic biodegradation of plastic materials in seawater under accelerated conditions. It also includes an optional way to estimate biodegradation by carbon flow, by looking at biomass formed from the test material before complete mineralization to CO2.

The document is organized into clauses on scope, normative references, terms and definitions, principle, test environment, reagents, apparatus, procedure, calculation and expression of results, validity of results, and test report. It then adds informative annexes with apparatus examples, worked examples and supporting background.

Annex	What it covers
A	Biodegradation test apparatus and stirring examples
B	Example BOD tests at different temperatures
C	Effect of ultrasonic treatment on microorganisms in seawater
D	Nitrification during biodegradation
E	Estimation of microorganism amount in tested seawater
F	Biodegradation results with activated seawater
G	Evaluation of potential biodegradation considered by carbon flow
H	H NMR analysis

What are the key requirements of ISO 18957:2026?

The test measures aerobic biodegradation in seawater

ISO 18957:2026 determines biodegradation either by biochemical oxygen demand (BOD) in a closed respirometer or by measuring evolved CO2. The result is expressed as percentage biodegradation relative to the theoretical oxygen demand (ThOD) or theoretical amount of evolved carbon dioxide (ThCO2).

In practice, this means the laboratory must choose one measurement route, follow it consistently, and interpret the result from the biodegradation curve rather than from a single end-point reading.

The incubation conditions are controlled and recorded

ISO 18957:2026 requires incubation in the dark or diffused light at a constant mesophilic temperature between 15 °C and 31 °C, preferably 27 °C ± 1 °C. The temperature must be monitored with a data logger and stated in the report.

This matters because temperature changes the biodegradation rate. A result obtained at one temperature should not be treated as directly transferable to real marine conditions.

Seawater quality and preparation are part of the method

ISO 18957:2026 requires seawater to be checked for TOC, pH, nitrogen content and salinity. Natural seawater can be used fresh or after limited storage under defined conditions, and artificial seawater may be prepared according to ASTM D1141-98. Coarse particles are removed before use.

This means the test medium is not just a background fluid - it is part of the test system and must be characterized so that differences in microbial activity can be explained and compared.

The test setup uses blanks, reference material and, when needed, a negative control

ISO 18957:2026 requires at least three test flasks for the test material, three blank flasks, and three flasks for a reference material. A negative control is recommended if biodegradation is expected to take longer than 3 months.

The reference materials listed include poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate), poly(ε-caprolactone), microcrystalline cellulose, or ashless cellulose filters. A non-biodegradable polymer such as polyethylene may be used as negative control.

In practice, the blanks correct for background oxygen or CO2 changes in the seawater, while the reference material checks that the seawater and apparatus can show biodegradation as expected.

The test can be accelerated by activating the seawater

ISO 18957:2026 allows several ways to increase microbial activity in seawater: adding an organic nutrient source containing nitrogen, pretreating seawater with sediment, mixing seawater from multiple locations, or concentrating microorganisms by filtration. These methods can be used alone or in combination.

This is important because the standard is designed to shorten the time needed to obtain an indication of biodegradability. The laboratory must record which activation method was used, because it affects the microbial population and the result.

Sample form, loading and mixing are controlled

ISO 18957:2026 specifies a test material concentration of 75 mg/l to 300 mg/l of seawater, with enough headspace for oxygen transfer. The material may be tested as powder or film, and powder preparation should follow ISO 10210. Continuous stirring is used, and the sample may be protected from mechanical disintegration with non-biodegradable mesh.

In practice, the physical form of the plastic matters. The same polymer in powder and film form can behave differently, so the sample geometry must be chosen and reported carefully.

The result is taken from the plateau phase and must meet validity criteria

ISO 18957:2026 defines the maximum level of biodegradation as the mean value from the plateau phase, or the highest value in the plateau if the curve changes slightly. The test is valid only if the reference material reaches more than 60% biodegradation and replicate reference flasks differ by less than 20%.

This tells the user whether the test run is technically acceptable before any business or development decision is made from the result.

The document also includes an optional carbon-flow evaluation

ISO 18957:2026 provides an optional method for estimating biodegradation considered by carbon flow. This uses the biodegradation result together with analysis of residual material, degradation products and biomass, including H NMR analysis.

This option is useful when a simple CO2 or oxygen-demand result does not fully show where the carbon from the plastic has gone. It helps assess whether carbon has become biomass, soluble products or residual polymer.

What terms does ISO 18957:2026 define?

• Carbon flow - the series of carbon-containing compounds formed from the plastic during biodegradation, from lower-molecular-weight products to biomass and CO2.
• H NMR - proton nuclear magnetic resonance used to identify and quantify residual polymer and degradation products.
• Pelagic seawater - seawater above the seafloor, also called open water or the water column.
• Lag phase - the initial period before the biodegradation curve reaches about 10% of its maximum level.
• Biochemical oxygen demand (BOD) - the dissolved oxygen consumed under specified conditions by aerobic oxidation in water.
• Theoretical oxygen demand (ThOD) - the maximum oxygen required to completely oxidize a compound from its molecular formula.
• Dissolved organic carbon (DOC) - the organic carbon in water that remains after the specified phase separation.
• Theoretical amount of evolved carbon dioxide (ThCO2) - the maximum CO2 expected from complete oxidation of the compound.

Who uses ISO 18957:2026?

ISO 18957:2026 is used by plastics laboratories, material developers, environmental testing teams, and quality or technical managers who need to compare biodegradable plastic materials in seawater. It is also relevant to buyers and assessors who must review biodegradation claims or test data for marine-oriented materials.

Typical tasks include preparing the seawater, running BOD or CO2-based biodegradation tests, comparing test material with reference material and negative controls, and documenting whether the material shows potential biodegradability under the specified laboratory conditions.

Which standards are used with ISO 18957:2026?

Standard	Contribution to ISO 18957:2026
ISO 472	General plastics vocabulary
ISO 8245	TOC and DOC measurement in water
ISO 10210	Preparation of samples for biodegradation testing
ISO 10523	pH determination of seawater and test media
ISO 11261	Total nitrogen measurement
ISO 23977-1	CO2-based seawater biodegradation method used for related apparatus and calculations
ASTM D1141-98	Preparation of substitute ocean water
ISO 10304-1	Nitrate and nitrite analysis mentioned for nitrogen-containing samples
JIS K 0138:2018	Quantitative H NMR analysis used in the carbon-flow option

What does the ISO 18957:2026 document contain?

ISO 18957:2026 contains the full laboratory procedure, from seawater preparation and apparatus selection through to calculation of biodegradation and reporting. It gives formulas for BOD-based and CO2-based biodegradation, and an optional carbon-flow route that uses residue separation and H NMR analysis.

The annexes add practical support: apparatus sketches, stirring examples, sonication effects, nitrification data, microbial counting approaches, activated seawater examples, carbon-flow evaluation and H NMR examples. The report section also requires recording method details, final observations and any deviations from the specified conditions.

The standard states that the results are informal only and that biodegradation times from this test cannot be used directly, or with a conversion factor, to predict behaviour in the real marine environment.

International
Standard
ISO 18957
First edition
Plastics — Determination of the
2026-10
aerobic biodegradation of plastic
materials exposed to seawater using
accelerated conditions in laboratory
Plastiques — Détermination de la biodégradation aérobie des
matières plastiques exposées à l'eau de mer en utilisant des
conditions accélérées en laboratoire
Reference number
© ISO 2026
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ii
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Test environment . 4
6 Reagents . 4
7 Apparatus . 5
7.1 Detection system for calculation of biodegradability .5
7.2 Analytical equipment for measuring nitrate and nitrite concentrations (optional) .6
7.3 Analytical equipment for measuring remaining test material and degradation products
(optional) .6
8 Procedure . 6
8.1 Test material .6
8.2 Reference materials .7
8.3 Test set-up .7
8.4 Preparation of seawater with high biodegradation capability .8
8.4.1 General .8
8.4.2 Addition of an organic nutrient source containing nitrogen components.8
8.4.3 Pre-treatment of seawater with sediment .8
8.4.4 Mixing of seawater samples from multiple locations .8
8.4.5 Concentration of microorganisms in seawater by filtration .8
8.5 Start of the test .9
8.5.1 General .9
8.5.2 BOD measurement system .9
8.5.3 CO production measurement system .9
8.6 End of the test .9
8.7 Chemicals for the test .10
9 Calculation and expression of results . 10
9.1 General .10
9.2 Calculation based on BOD .10
9.3 Calculation based on evolved CO .11
9.4 Calculation of potential of biodegradation considered by carbon flow (optional) . 12
9.4.1 General . 12
9.4.2 Judgment of continuation/termination of the biodegradation test of test material . 12
9.4.3 Calculation of carbon converted to biomass and potential biodegradation
considered by carbon flow . 12
9.4.4 H NMR analysis .14
9.5 Visual inspection . 15
9.6 Expression and interpretation of results. 15
10 Validity of results .15
11 Test report .15
Annex A (informative) Biodegradation test apparatus . 17
Annex B (informative) Example of biodegradation BOD test with different temperature . 19
Annex C (informative) Effect of sonic treatment using ultrasonic cleaner on microorganisms in
seawater .21
Annex D (informative) Nitrification of nitrogen in seawater during biodegradation process .22

iii
Annex E (informative) Estimation of amount of microorganisms in tested seawater .23
Annex F (informative) Biodegradation test results with activated seawater .25
Annex G (informative) Evaluation of potential biodegradation considered by carbon flow .28
Annex H (informative) H NMR analysis .30
Bibliography .33

iv
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
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This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 14, Environmental
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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.

v
Introduction
Plastics have been used in various fields because of their highly durable characteristics. Plastics are also
used intentionally in the environment or in applications where unintentional loss to the environment is
unavoidable. The degree of biodegradation of plastics in natural environment is of interest in order to better
characterize the behaviour of plastics in these very particular environments. Thus, the test methods to
measure the degree and rate of biodegradation are of major interest to obtain an indication of the potential
biodegradability of plastic materials when exposed to different marine habitats.
The biodegradability of plastics is defined in laboratory tests, for example ISO 18830, ISO 19679, ISO 23977-1
and ISO 23977-2, but these test methods in a laboratory basically use only seawater and sediment.
Since existing test methods are not intended to shorten the test period, the test period often extends to one
year and it is sometimes difficult to assess the degree of biodegradability of the sample in a short period of
time.
This document provides an accelerated test method for determining the biodegradation level of plastics
exposed to seawater in laboratory in shorter time by increasing the number of microorganisms in seawater
and enriching the flora, by adding nutrients, pre-treatment by sediment, and condensation of seawater.
The degree of biodegradation is calculated based on measurement of the oxygen (O ) demand in a closed
respirometer or carbon dioxide (CO ) evolution.
Test material is taken up by microorganisms, metabolized, and the contained carbon finally mineralized to
CO . Part of the carbon originating from the test material is temporarily converted to biomass. The biomass
cannot be detected unless the bacteria die and undergo self-digestion. It takes a longer period of time than
the conversion to biomass and it may require a long test period to complete the mineralization of the test
material. On the other hand, since biomass components are produced by the microbial activity, the total
biodegradation considered by carbon flow can be estimated by the sum of the degree of biodegradation
based on O and/or CO analysis and the degree of conversion to biomass. Therefore, for the purpose of
2 2
promoting the development of biodegradable plastics, this document also provides a method for measuring
the biomass components produced by metabolizing the test material before it is completely mineralized
to CO , that enables to evaluate the potential biodegradability considered by carbon flow in much shorter
period of time than full mineralization.

vi
International Standard ISO 18957:2026(en)
Plastics — Determination of the aerobic biodegradation
of plastic materials exposed to seawater using accelerated
conditions in laboratory
1 Scope
This document describes a laboratory test method to determine the aerobic biodegradation of plastic
materials exposed to seawater using accelerated conditions. It describes the general requirements of the
apparatus and the procedures for using this test method.
This test method is designed to give an early indication of the potential biodegradability of plastic materials
in marine environment.
For the purpose of promoting the development of biodegradable plastics, this document also provides
a method for measuring the biomass components produced by metabolizing the test material before it is
completely mineralized to CO , that enables to evaluate the potential biodegradability considered by carbon
flow by the sum of the degree of biodegradation based on O or CO analysis and the degree of conversion to
2 2
biomass in much shorter period of time than full mineralization.
This method is not suitable to assess the degree of disintegration of plastic materials caused by abiotic
factors like heat or UV radiation.
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 472, Plastics — Vocabulary
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
ISO 23977-1, Plastics — Determination of the aerobic biodegradation of plastic materials exposed to seawater
— Part 1: Method by analysis of evolved carbon dioxide
ASTM D1141-98, Standard Practice for Preparation of Substitute Ocean Water
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 472 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/

3.1
carbon flow
series of compounds produced from carbon derived from plastics during biodegradation
Note 1 to entry: The series of compounds includes polymers with reduced molecular weight, water-soluble oligomers,
compounds in the process of biochemical metabolism, organic matter that makes up bacterial cells, and carbon dioxide
that is produced when plastics are completely biodegraded.
3.2
H NMR
proton NMR
nuclear magnetic resonance using proton as the observed nucleus
3.3
pelagic seawater
pelagic zone
water body above the seafloor
Note 1 to entry: It is also referred to as the open water or the water column.
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]
3.4
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 (3.10)
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:2021, 3.8]
3.5
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]
3.6
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]
3.7
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]
3.8
dissolved organic carbon
DOC
part of the organic carbon in water which cannot be removed by specified phase separation
−2
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 14852:2021, 3.7]
3.9
theoretical amount of evolved carbon dioxide
ThCO
maximum theoretical amount of carbon dioxide evolved after completely oxidizing a chemical compound,
calculated from the molecular formula
[SOURCE: ISO 23977-1:2020, 3.3, modified – Note 1 to entry was removed.]
3.10
biodegradation phase
time from the end of the lag phase (3.4) of a test until the plateau phase has been reached
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:2021, 3.10]
3.11
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 14852:2021, 3.9]
3.12
plateau phase
time from the end of the biodegradation phase (3.10) until the end of a test
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:2021, 3.11]
4 Principle
This document describes a test method for determination of the aerobic biodegradation of plastic materials
exposed in seawater using accelerated conditions in laboratory. The biodegradation is determined
by measuring the oxygen demand in a closed respirometer or by measuring the CO evolved during
mineralization of the plastic materials by microorganisms in the seawater.
The biochemical oxygen demand (BOD) is determined by consumption of oxygen, 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 calculated by dividing the BOD by the theoretical oxygen demand (ThOD)
and is 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.
To accelerate the biodegradation test using seawater, different methods are applied, such as adding nutrients
to the seawater, changing the incubation temperature, washing the seawater with marine sediment, and
concentrating the microorganisms via a filtration-based protocol.
In general, the smaller the test material/seawater ratio, the higher the biodegradation rate. To prevent a
limitation of the aerobic biodegradation rate of the test material due to low metabolic rates of microorganisms,
it is important to guarantee sufficient oxygen transfer into the seawater phase. The larger the surface area
of the sample, the faster the biodegradation. In addition, at higher test temperatures chemical reactions and
biological processes are faster, often resulting in higher biodegradation rates. However, the test temperature
should not exceed 31 °C since the bacterial strain composition in seawater might be negatively affected with
regard to biodegradation capability. See Annex B for examples of biodegradation BOD test with different
temperature.
Increase the capability of seawater microorganisms for plastic biodegradation is mainly achieved by
increasing the number of microorganisms in seawater and increasing diversity. However, even if the
microorganisms are enriched, their capability to degrade plastic materials might be limited, e.g. by nutrient
limitations in the seawater, which should be addressed by maintaining a minimum amount of inorganic
nutrients.
Since oxygen consumption and CO generation basically correspond to each other, the amount of CO evolved
2 2
may be measured instead of the BOD test.
Test material is taken up by microorganisms, metabolized, and the contained carbon finally mineralized to
CO . Part of the carbon originating from the test material is temporarily converted to biomass. The biomass
cannot be detected unless the bacteria die and undergo self-digestion, and it takes longer period of time
than the conversion to biomass and it may require long test period to complete the mineralization of the
test material. On the other hand, since biomass components are produced by the microbial activity, the total
biodegradation considered by carbon flow can be estimated by the sum of the degree of biodegradation
based on O and/or CO analysis and the degree of conversion to biomass.
2 2
For material development it is highly important to understand if a material is fully biodegradable.
Therefore, it is important to understand where the carbon goes if it cannot be detected by sole CO evolution
measurement. This document also provides a method for measuring the biomass components produced
by metabolizing the test material before it is completely mineralized to CO , that enables to evaluate the
potential biodegradability considered by carbon flow in much shorter period of time than full mineralization.
5 Test environment
Incubation shall take place in the dark or in diffused light, and which is maintained at a constant mesophilic
temperature. The temperature shall be between 15 °C to 31 °C, preferably at 27 °C to an accuracy of ±1 °C
and shall be monitored through a data logger 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.
6.1 Water, distilled or deionized, free of toxic substances and containing less than 2 mg/l of TOC.
6.2 Natural seawater/sediment
The seawater sample shall be used for testing preferably on the day the water is collected, or it can be stored
in dark between 4 °C to 13 °C and be used for testing within 3 days. If the seawater is kept at 4 °C, it can be
used for testing for 2 weeks. Sediment can also be stored if it is kept at 4 °C and regularly mixed and aerated
to avoid anaerobic conditions.
Prior to use, remove coarse particles from the seawater and, if applicable, from the sediment by appropriate
means, e.g., by sieving with a mesh of 3 mm opening. The procedure used shall be reported.

Seawater can be filtered using a laboratory filter paper having particle retention between 1 µm to 5 µm to
remove particles.
Measure TOC, pH and nitrogen content of seawater and, if applicable, of sediment samples in accordance
with ISO 8245, ISO 10523 and ISO 11261, respectively, and measure salinity (PSU) by means of, for example,
refractometer-type and electrical conductivity-type salinity meter.
If the TOC content of the seawater sample is found to exceed about 20 % of the theoretical TOC after the
addition of the test item, the seawater should not be used, or be diluted with artificial seawater.
If artificial seawater is used, it shall be prepared in accordance with ASTM D1141-98.
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, including location and storage, if applicable.1
7 Apparatus
Ensure that all glassware shall be thoroughly cleaned and dry, in particular, free from organic or toxic
matter. Required is usual laboratory equipment, plus the following.
7.1 Detection system for calculation of biodegradability
7.1.1 General
In the case of using BOD measurement for calculation of biodegradability, a closed respirometer (7.1.2) is
used. In the case of using CO production measurement for calculation of biodegradability, a CO production
2 2
measurement system (7.1.3) is used.
7.1.2 Closed respirometer for static BOD measurement
Flasks of the volume of about 300 ml to 500 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).
Flasks with higher or lower volumes can be used, if it satisfies the volume condition specified in (see 8.1) and
the experimental conditions in the flask are not affected.
A suitable apparatus is shown in Annex A, Figure A.1.
Any closed 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.

7.1.3 Analytical equipment for determining producing carbon dioxide
Any test flask able to determine with sufficient accuracy the biochemical evolution of carbon dioxide is
suitable in accordance with ISO 14852, or ISO 23977-1, or ISO 19679. The evolved CO can be quantitatively
measured also using other suitable methods such as those based on infrared CO -analysers or those based
on TOC analysers equipped with an infrared photometer or on gravimetric analysis.
7.1.4 Analytical balance, which shall have a sensitivity of at least 0,1 mg.
7.1.5 Magnetic stirrer, which shape, and size are described in Annex A as example.
7.1.6 pH meter.
7.2 Analytical equipment for measuring nitrate and nitrite concentrations (optional)
In case the test material contains nitrogen, a quantitative test to determine nitrate/nitrite in the medium
before and after the test is recommended by means of ion chromatography in accordance with ISO 10304-1.
7.3 Analytical equipment for measuring remaining test material and degradation products
(optional)
To calculate potential of biodegradation considered by carbon flow (9.4), a quantitative test to determine
remaining test material and degradation products in the medium after the test is recommended by
1 1
means of H NMR which carrier frequency for proton ( H) should be 300 MHz or more in accordance with
JIS K 0138:2018.
8 Procedure
8.1 Test material
The test material 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 75 mg/l up to 300 mg/l of seawater. The mass of test material
should correspond to a ThOD of about 126 mg/l or a TOC of about 45 mg/l. The headspace of the flask should
be 100 ml and more per 10 mg sample to ensure that air is adequately supplied to the sample.
The ThOD (see ISO 14851:2019, Annex A) and the TOC (using ISO 8245, ISO 10694 or from the chemical
formula or determine by elemental analysis) shall be calculated.
Add KH PO (11,0 mg/l) and NH Cl (9,55 mg/l) to seawater at the beginning of a test.
2 4 4
If necessary, KH PO can be added up to 100 mg/l and NH Cl can be added up to 50 mg/l. However, when
2 4 4
ammonium chloride is added at a high concentration, the oxygen consumption due to nitrification may differ
between the blank and the test system, so after the test, NO and NO ions shall be analysed then corrected
2 3
as mentioned in 9.2 for BOD measurement.
An experimental example of the production of NOx ions after biodegradation in the presence of ammonium
chloride is shown in Annex D.
Following ratio of the test material is recommended:

— Seawater 200 ml
— Head space 300 ml or more
— Test material 15 mg ~ 60 mg (corresponding 75 mg/l ~ 300 mg/l of seawater)
(in a typical case, 30 mg of test sample and 200 ml of seawater)
— KH PO 2,2 mg (corresponding 11,0 mg/l of seawater)
2 4
— NH Cl 1,91 mg (corresponding 9,55 mg/l of seawater)
— Test flask 500 ml or more
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 as
small square pieces (0,5 cm × 0,5 cm) with 10 µm to 100 μm of thickness.
The form and shape of the test material can influence its biodegradability. Similar particle sizes of powder
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.
After the addition of the test material, mixing with a magnetic stirring bar for more than 10 min is
recommended to homogenously blend the test material. Adjust the stirring speed so that the powder sample
does not scatter above the liquid level. If the biodegradation rate of the sample is expected to be affected by
mechanical disintegration, it is recommended to adjust the stirring speed or to cover the sample with non-
biodegradable plastic mesh not to disintegrate the sample.
When powder or film pieces stick on the inner wall of the test flask above the seawater, a slight manual
shaking of the bottle is recommended to regain the powder or film pieces back to the seawater.
8.2 Reference materials
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate), poly(ε-caprolactone),
1)
microcrystalline cellulose or ashless cellulose filters should be used as a reference material . If possible,
weight, form, and size should be comparable to that of the test material. It is recommended to use a reference
polymer with a number average molecular weight (Mn) greater than 30 000 to eliminate molecular weight
effects.
As a negative control, a non-biodegradable polymer (e.g. polyethylene) in the same form as the test material
can be used.
Details of reference materials such as supplier, grade shall be reported. When possible, composition, and
molecular weight, are reported. If a commercially available product was used, describe to the extent of
available information.
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 );
T
b) three flasks for the blank (symbol F );
B
1) Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHBH (CAS RN® 147398-31-0) produced by Kaneka,
poly(3-hydroxybutyrate), P3HB (CAS RN® 26063-0-3) produced by Sigma-Aldrich, poly(ε-caprolactone) (Mn = 80
000), PCL (CAS RN® 24980-41-4) produced by Sigma-Aldrich, microcrystalline cellulose (CAS RN® 9004-34-6) "Avicel"
produced by Merck or Laboratory filter paper Whatman n° 42 have been found satisfactory for this purpose and are
examples of suitable products available commercially. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of these products.

c) three flasks for reference material (symbol F ).
C
In addition, if biodegradation is expected to take longer than 3 months, it is recommended that a negative
control is included:
d) three flasks for negative control (symbol F ).
N
8.4 Preparation of seawater with high biodegradation capability
8.4.1 General
For the purpose of increasing the biodegradation capability of natural seawater sample or for the purpose
of adding microorganisms to artificial seawater, the following methods shall be performed alone or in
combination (8.4.2, 8.4.3, 8.4.4 and/or 8.4.5).
8.4.2 Addition of an organic nutrient source containing nitrogen components
For the purpose of increasing the number of microorganisms in the seawater, 8 mg/kg peptone, yeast extract
2)
or nutrient broth are added to the blank flasks (only seawater), control flasks and test material flasks at
the beginning of the test. Although the addition of an organic nutrient source leads to an increased oxygen
consumption by microorganisms, the calculation of the biodegradation rate based on consumed oxygen is
not affected after correction using the oxygen consumption measured in the blank flask.
8.4.3 Pre-treatment of seawater with sediment
The following method can be used to add microorganisms adhering to the sediment to seawater in order
to enrich the microbial diversity and to increase the number of microorganisms. Approximately a mass
fraction of 10 % to 20 % of sediment is added to seawater, applied sonication by using ultrasonic cleaner at
28 ~ 100 kHz for 10 s to 30 s, and then allowed to stand for 30 min to achieve settling of sediment particles
and to obtain an enriched supernatant seawater. An enriched seawater can also be obtained by filtering
using a 10 µm. The sonication may be performed up to three times with an interval of 3 min or more on
the same seawater if necessary. Notably, the increase in the DOC value of seawater by sonication should
not exceed 20 mg/kg. The diversity index of the bacterial flora is recommended to measure and record
before and after the pre-treatment with sediment to evaluate whether microbial diversity has increased.
The diversity index of the bacterial flora can be calculated based on microbial community analysis targeting
the 16S ribosomal RNA (rRNA) gene as phylogenetic biomarker. The obtained sequence data are processed
in the QIIME2 platform to calculate the diversity indexes, including observed features, Chao1, Shannon’s H’,
and Simpson’s D. Record the mass of the sediment added to the seawater.
The effect of sonic treatment using ultrasonic cleaner on microorganisms in seawater is shown in Annex C.
8.4.4 Mixing of seawater samples from multiple locations
In order to diversify the microorganisms in the test seawater, seawater that is expected to have different
bacterial flora structures, such as estuary of different rivers and different points of ocean currents, can be
collected and mixed in each equal quantity.
8.4.5 Concentration of microorganisms in seawater by filtration
The number of microorganisms can be increased by collecting microorganisms by filtration and adding
them to the original seawater. A certain amount of seawater from which contaminants have been
removed through a 10 μm filter is passed through a filter of pore size between 0,2 μm to 0,45 μm, and the
microorganisms on the filter are added to the original seawater. Adding the microorganisms obtained from
filtering of 1 l of seawater to another 1 l of (unfiltered) seawater will result in doubling of the concentration
of microorganisms compared to the initial seawater sample. The concentration rate should be recorded.
TM TM
2) Difco Marine Broth2216 peptone, Bacto yeast extract and tryptic soy broth nutrient broth have been found
satisfactory for this purpose and are examples of suitable products available commercially. This information is given for
the convenience of users of this document and does not constitute an endorsement by ISO of these products.

In order to add the filtered microorganisms to the original seawater, the original seawater is divided into
three sterile beakers, and all the filter papers used are placed in the first beaker and applied sonication for
30 s. The filter papers are then placed in a second beaker and sonicated again for 30 s. This operation is also
carried out in a third beaker, a total of three times sonication. The seawater of the three beakers to which
the filtered microorganisms have been added is combined into one and subjected to testing.
Alternatively, tangential flow filtration can be used for concentration of microorganisms. The procedure
and the concentration rate shall be recorded.
3 4
For these methods, a concentration of microbes of minimally 1×10 cfu/ml, ideally more than 5×10 cfu/
ml, is recommended. Methods, e.g. based on the determination of colony-forming units (cfu), fluorescence
staining, or PCR are recommended for measuring the concentration of microorganisms.
Record details of the preparation procedure of seawater with high biodegradation activity.
Example of evaluation of amount of microorganisms in tested seawater is shown in Annex E.
8.5 Start of the test
8.5.1 General
Biodegradability is calculated either by BOD measurement (8.5.2) or CO production measurement (8.5.2).
8.5.2 BOD measurement system
Use the apparatus described in (7.1.2). The test is performed in batch using a test flask with a volume
preferably 500 ml with either 200 ml of natural seawater or the activated seawater described in (8.4) and
the test material according to the concentration specified in (8.1) at the temperature for the test.
Add 2 times the equivalent or more compared to the test sample of carbon dioxide absorber such as Ca(OH)
and KOH to the absorber compartments of the test flask (see ISO 14851:2019, Annex C). Place the sealed
flasks on a magnetic stirrer in a constant-temperature environment and allow all vessels to reach the desired
temperature. Agitation shall be continuous (e.g. 100 r/min agitation) to support oxygen transfer from the air
into the seawater and to guarantee sufficient mixing of the microorganisms and test material. The sample
can be covered with non-biodegradable plastic mesh to avoid mechanical disintegration, see Figure A.3.
Take the necessary readings on the manometers (if manual) and verify that the recorder of oxygen
consumption is functioning properly (automatic respirometer) (see ISO 18830).
8.5.3 CO production measurement system
Use the apparatus described in (7.1.3). The test is performed in accordance with ISO 14852, ISO 23977-1 or
ISO 19679 using a test flask with a volume, for example, 300 ml with either 90 ml of natural seawater or the
activated seawater described in (8.4) and the test material according to the concentration specified in (8.1)
at the temperature for the test.
The evolved CO can be quantitatively measured by using CO absorber such as Ba(OH) and KOH in an
2 2 2
amount of 2 times the equivalent or more compared to the test sample and periodically quantified by
titration or other methods. Alternatively, the evolved CO can be quantified by directly analysing the CO
2 2
concentration in the aeration gas using non-dispersive infrared detector and multiplying it by the total gas
volume.
8.6 End of the test
When the rate of biodegradation reached to a plateau and no further biodegradation is expected, the
test is considered complete. The test period should typically not exceed 6
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