ISO 16623:2024

International
Standard
ISO 16623
First edition
Plastics — Marine biodegradation
2024-12
testing — Preparation of optimized
intertidal seawater and sediment
Plastiques — Essais de biodégradation en milieu marin —
Préparation d'eau de mer et de sédiments intertidaux optimisés
Reference number
© ISO 2024
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Apparatus . 4
6 Procedure . 4
6.1 General .4
6.2 Collection of sediment with less than 2 mm particle size and preparation of rinsed
seawater .5
6.3 Preparation of refined sediment .5
6.4 Purification of seawater for biodegradation test from suspended seawater .5
7 Validity of preparation . 6
7.1 Sediment for disintegration testing.6
7.2 Refined sediment for biodegradation testing .6
8 Preparation report of seawater and sediments . 6
Annex A (informative) Diagram of seawater and sediment preparation — Seawater and
sediment preparation by wet filtration and flotation . 8
Annex B (informative) Preparation of seawater and sediments from seafloor sediments: Wet
filtration and flotation of sediments . 9
Annex C (informative) Evolved carbon dioxide of seawater and sediment prepared from
seafloor sediments .11
Annex D (informative) Test validity of ISO 19679 — Validity of biodegradation test based on
carbon dioxide evolved .13
Annex E (informative) Example of biodegradation of reference material by ISO 19679 .15
Annex F (informative) Example of biodegradation of plastic films by ISO 19679 —
Biodegradation of plastic films using rinsed seawater and sand gravel sediments .16
Annex G (informative) Example of live and total cell count in sediment and seawater: Live and
total cell count in sediment and seawater by fluorescence method . 17
Annex H (informative) Example of biodegradation of plastic films by ISO 19679 . 19
Annex I (informative) Interlaboratory test ISO 19679 and ISO 18830 final report .20
Bibliography .21

iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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The procedures used to develop this document and those intended for its further maintenance are described
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This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 14, Environmental
aspects.
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
Introduction
The assessment of the degree of biodegradation of plastics in marine habitats is one effective measure
to understand and evaluate the impact of reuse, recycling and environmental pollution of plastics. The
biodegradation of plastics is the process, in which plastics are decomposed by heterotrophic microorganisms,
such as bacteria and fungi, through enzymatic hydrolysis and subsequent metabolization. Marine
biodegradation proceeds mainly in microbial consortia that form at the interface between seawater and
plastics. This is because marine microorganisms live aerobically within biofilms at the interface between
the liquid phase of seawater and solid phases such as gravel and shells.
The diversity of microbial consortia in the marine environment is high, depending on their natural
environmental conditions. The species and number of microorganisms vary depending on the climate, ocean
currents, tides, and topography. Considering the diverse habitats of these microorganisms, three types of
biodegradation assessment methods have been developed:
— one-phase systems consisting of seawater or sediment and
— two-phase systems consisting of seawater and seafloor sediments.
However, due to the diversity of microorganisms even a biodegradable material such as cellulose, which is
used as a reference material, gave biodegradation results that ranged from 0 to 100 percent in ring tests
of these test methods. From the perspective of biodegradable plastic specification, it is thus necessary to
optimize the preparation of natural inoculum for the biodegradation tests to avoid those fluctuations in
experimental outcomes.
In order to reduce the impact of seasonal and regional variation in the marine inoculum composition, this
document describes a method for preparing seawater and seafloor sediments. The prepared seawater
and sediment can be used for the test methods defined in ISO 19679, ISO 18830, ISO 22404, ISO 23977-1,
ISO 23977-2 and ISO 23832.
Prepared seawater for biodegradation tests is obtained by rinsing seafloor sediments with seawater.
Sand and gravel mixtures with particle sizes from 250 µm to 2 mm are used as sediments to provide pore
water flow, oxygen supply, seawater filtration and biofilm growth. Through the preparation of defined
compositions of seawater and sediments in marine tests, the number of microorganisms and aerobic
conditions are stabilized, and reproducibility and comparability of biodegradation experiments (including
curves, lag time, etc.) are improved.
This document specifies methods for preparing seawater and sediments in the intertidal zone for estimating
the aerobic biodegradation of plastics in pelagic to coastal marine environments.

v
International Standard ISO 16623:2024(en)
Plastics — Marine biodegradation testing — Preparation of
optimized intertidal seawater and sediment
1 Scope
This document specifies procedures for preparing seawater and sediments used in test methods to assess
the biodegradation of plastic materials in the marine environment. The screened sediment and sediment-
rinsed seawater are prepared to sustain aerobic testing at laboratory scale. The described method is
designed to separate sediment-rinsed seawater and sand-gravel sediments from intertidal sediments by wet
filtration and seawater flotation. This document does not include steps to enhance the biodegradation of
plastic materials by concentrating the natural seawater, adding nutrients to the seawater, and pre-culturing
the inoculum.
The methods described in this document are intended to be used in addition to issued ISO standard test
methods for evaluating the biodegradation and disintegration of plastic materials. The applicable evaluation
test methods are ISO 18830, ISO 19679, ISO 22404, ISO 23977-1, ISO 23977-2 and ISO 23832.
NOTE The conditions described in this document do not always correspond to the optimum conditions for
maximum biodegradation. This is a method of preparing test sediments from coastal seafloor sediments, not a method
of preparing sediments from deep-sea seafloors.
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 18830, Plastics — Determination of aerobic biodegradation of non-floating plastic materials in a seawater/
sandy sediment interface — Method by measuring the oxygen demand in closed respirometer
ISO 19679, Plastics — Determination of aerobic biodegradation of non-floating plastic materials in a seawater/
sediment interface — Method by analysis of evolved carbon dioxide
ISO 22404, Plastics — Determination of the aerobic biodegradation of non-floating materials exposed to marine
sediment — Method by analysis of evolved carbon dioxide
ISO 23977-1, Plastics — Determination of the aerobic biodegradation of plastic materials exposed to seawater
— Part 1: Method by analysis of evolved carbon dioxide
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 23832, Plastics — Test methods for determination of degradation rate and disintegration degree of plastic
materials exposed to marine environmental matrices under laboratory conditions
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 https:// www .electropedia .org/

3.1
intertidal zone
borderline between sea and land that extends from the high tide line, which is rarely inundated with water,
to the low tide line, which is typically always covered with water
Note 1 to entry: The tidal zone is frequently a sandy area that is kept constantly damp by the lapping of the waves.
Note 2 to entry: Stony and rocky shorelines also exist.
Note 3 to entry: They are also known as eulittoral zone, midlittoral zone, mediolittoral zone, intertidal zone, foreshore.
[SOURCE: ISO 22404:2019, 3.1]
3.2
biofilm
microbial cells and their metabolites, such as polysaccharides, proteins, lipids and nucleic acids, firmly
attached to the material surface of the product in water, and stained with crystal violet
[SOURCE: ISO 4768:2023, 3.1]
3.3
biodegradation
degradation (3.4) caused by biological activity, especially by enzymatic action, leading to a significant change
in the chemical structure of a material
[SOURCE: ISO 472:2013, 2.1680]
3.4
degradation
irreversible process leading to a significant change in the structure of a material, typically characterized
by a change of properties (e.g. integrity, molecular mass or structure, mechanical strength) and/or by
fragmentation, affected by environmental conditions, proceeding over a period of time and comprising one
or more steps
[SOURCE: ISO 472:2013, 2.262]
3.5
disintegration
physical breakdown of a material into very small fragments
[SOURCE: ISO 472:2013, 2.1757]
3.6
theoretical amount of evolved carbon dioxide
ThCO
maximum carbon dioxide evolved after completely oxidising a chemical compound, calculated from the
molecular formula or from determination of total organic carbon (TOC) (3.7)
[SOURCE: ISO 19679:2020, 3.1, modified — “theoretical amount of” removed after “maximum”.]
3.7
total organic carbon
TOC
amount of carbon bound in an organic compound
Note 1 to entry: Total organic carbon is expressed as milligrams of carbon per 100 mg of the compound.
[SOURCE: ISO 17556:2019, 3.14]

3.8
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, expressed as milligrams of oxygen uptake per
milligram or gram of test compound
[SOURCE: ISO 472:2013, 2.1723]
3.9
total dry solids
amount of solids obtained by taking a known volume of test material or inoculum and drying at about 105 °C
to constant mass
[SOURCE: ISO 13975:2019, 3.5]
3.10
volatile solids
amount of solids obtained by subtracting the residues of a known volume of test material or inoculum after
incineration at about 550 °C from the total dry solids (3.9) content of the same sample
Note 1 to entry: The volatile solids content is an indication of the amount of organic matter present.
[SOURCE: ISO 17088:2021, 3.9]
4 Principle
Biodegradable plastics in seawater are primarily degraded into water and inorganic carbon dioxide, and
partially assimilated into biomass by heterotrophic microorganisms in the marine food chain. In order to
evaluate aerobic biodegradation on a laboratory scale, the culture conditions such as nutrients, pH and
microbial species should be specified in the actual seawater and seafloor sediments used. Furthermore,
marine microorganisms survive aerobically as microbial communities in biofilms that form at the interface
between the liquid phase of seawater and solid phases, such as gravel, shells, and plastic films. Therefore,
marine biodegradation is dependent on the marine ecological environment, and the preparation methods of
seawater and sediment for marine biodegradation testing also need to be identified.
The pH of seawater is approximately 8,1, while the nutrient levels, biomass, microorganism abundance and
diversity are influenced by habitat and seasonal variation.
Interlaboratory tests according to ISO 19679 and ISO 18830 were conducted in nine laboratories in seven
countries, as shown in Annex I. At the end of the test, the average carbon dioxide production per gram of wet
sediment was 2,1 mg, with values ranging from 0,63 mg to 4,88 mg. The biodegradation value of reference
filter paper ranged from 5 % to 160 %, with an average value of 87 % and a coefficient of variation of
36 %. Similarly, an interlaboratory test was also conducted to improve the OECD 306 screening test, as the
[8]
biodegradation outcome varies depending on the abundance and composition of the microbial community .
In particular, the number of viable microorganisms in coastal areas is thousands of times higher than in
pelagic areas. On the other hand, sediments maintain aerobic conditions due to characteristics, such as
oxygen-saturated water flow, fine particle filtration, and pore water circulation. Sediments also serve as a
source of organic carbon, which is necessary for microbial growth, and source of calcium carbonate, which
helps buffer the pH of seawater.
This method of preparation of rinsed seawater and refined sediments significantly improves these values
for plastic test materials listed in Annex D and H.
In this preparation method, sediments in the sand-gravel area, including shells and corals, are selected from
the subseafloor sediments in the intertidal zone of the coastal area by sorting based on the particle size of
the object. Seawater for biodegradability testing is collected by washing the seafloor sediments and sand-
gravel surface overgrown with biofilms using seawater.

Seafloor sediments are wet filtered using a 2 mm sieve in a container filled with seawater. This sieve is used
for soil identification and classification according to ISO 14688-1, removing gravel and collecting clay, silt,
and sand based on particle size. Wet filtration separates the seafloor sediments into two layers: a lower
layer consisting of a sludge-like sediment including clay, silt, sand, gravel, benthic organisms, and eggs, and
an upper layer comprising a seawater suspension containing floating pieces of biofilm and microorganisms.
Larger aggregated floating particles are removed from the seawater using a filter paper having a pore size of
about 20 μm. This is to obtain filtered seawater containing microorganisms. The sludge-like sediments are
refined into sand-gravel sediments by flotation with seawater. By repeating flotation, as shown in Annex A
and B, more than 5 times, sediments with a particle size of 250 µm or more are prepared and can be used for
biodegradation testing. This sediment preparation method produces larger sediment particles than plastic
powder samples prepared according to ISO 10210.
Compared to unprepared pelagic seawater, this preparation method provides seawater with potentially
higher microbial diversity and cell count, which can lead to increased biodegradation rates. The prepared
seawater and sediment are effective in emulating the biodegradation of plastic materials in marine
environments based on BOD and carbon dioxide evolved in laboratory-scale testing.
5 Apparatus
5.1 Sieves, with 2 mm~3 mm opening and 250 µm or 300 µm mesh for filtering sand-gravel by wet
filtration method.
5.2 Bowls, two or more 15 l~20 l bowls (e.g. stainless steel) for the kitchen to prepare the sediments by
flotation and wet filtration of seawater and sediments.
5.3 Shovel, for collecting top sediment (the layer from surface till about 20 cm depth).
NOTE The type is a pointed digging shovel or gardening shovel about 1 m long.
5.4 Weight scale, capable of weighing 20 kg of seawater or sediment.
5.5 pH Meter, used for measurement of the pH of the marine test mixture. It shall be accurate to 0,1 pH-
units or better.
6 Procedure
6.1 General
In the intertidal zone, microorganisms form biofilms at the interface between sediments and seawater.
These biofilms exist in aerobic conditions. To collect biofilm-covered sediment and seawater rich in
microorganisms, the collected seafloor sediment is passed through a sieve with a pore size of 2 mm in the
seawater to remove gravel and seaweed (wet filtration). The filtrate is separated into a suspension and a
sludge-like sediment. The suspension separated by decantation is suction-filtered using a filter paper with
a pore size of 20 μm to prepare seawater for testing. The sludge-like sediment is washed away by seawater
flotation and becomes sand-gravel sediments covered with biofilm. These processes include the removal of
benthic organic matter.
Purified seawater and sediment shall be pre-incubated or stored according to the biodegradation or
disintegration test methods specified in ISO 18830, ISO 19679, ISO 22404, ISO 23977-1, ISO 23977-2, and
ISO 23832.
The purification and sieving steps can be performed outdoors at the sampling point or indoors in the
laboratory after transporting the samples taken at sea. In this case, artificial seawater can be used. Artificial
seawater formulation shall be in accordance with ISO 18830 or ISO 19679.
Collect top sediment layers from the surface to a depth of about 20 cm, suitable for lab-scale biodegradation tests.

6.2 Collection of sediment with less than 2 mm particle size and preparation of rinsed
seawater
Prepare kitchen-use stainless steel mixing bowls (⌀ 45 cm) and sieves (2 mm, ⌀ 35 cm). Place the sieve in the
middle of the bowl containing about 10 l of seawater. Add the shovelled seafloor sediment to the sieve. Shake
the sieve to loosen and rinse the biofilm on the gravel surface. The biomass containing the biofilm disperses
into the seawater in the bowl to form a brown seawater suspension. Clays, silts, sands, gravel and benthic
organisms in the seafloor sediments are filtered and precipitated as a sludge-like sediment in the lower
layers of the seawater suspension. Remove the residual sediment in the sieve, add fresh sediment and repeat
this wet filtration 5 more times, moving the collection point.
If necessary, the collected mixture of suspended seawater and sludge-like sediment is further agitated
by hand, to rinse the biofilm from the sand-gravel surface. However, the collected seawater will be in an
anaerobic state, so be careful not to collect too much biomass.
6.3 Preparation of refined sediment
The sludge-like sediment prepared in 6.2 should undergo further purification through flotation with
seawater to eliminate clay, silt, organic particles, and eggs. Add five times as much seawater as the sediment
to the bowl and stir the sediments by hand about ten times. After one minute, remove suspended particles
along with the supernatant. Repeating this flotation process around five times will remove most of the
particles smaller than 250 μm. The resulting purified sediment is suitable for biodegradation testing. If
necessary, use a sieve with a smaller pore size (250 µm or 300 µm), to remove particles of desired dimensions.
Mix the prepared sediment with an equal volume of seawater and store at 4 °C for four months or aerobically
at room temperature for approximately 1 month. It is preferable to measure the pH of the mixed sediment-
seawater system under aerobic conditions where the sediment layer is 3 cm or less.
NOTE As shown in Annex D, 60 g of wet sediment in the test vessel contains 0,72 g of volatile solids (VS). Assuming
[14]
an algae with an organic carbon content of 50 %, the VS contains 0,31 g of TOC, which corresponds to 1,32 g of
ThCO . However, at the end of the 180-day test, the amount of carbon dioxide evolved from the sediment was 14 mg,
and the biodegradation rate of organic matter in the sediment was 1 %. On the other hand, the filter paper containing
73 mg of ThCO is 74 % biodegraded, and the carbon dioxide evolved is 54 mg. Furthermore, from the viable cell count
5 4
results in Annex G, the viable cell counts per ml of rinsed seawater decreased from 10 to 10 before and after the
test. However, the viable cell counts at the end of the test was the same as the viable cell counts (10 ) in the seawater
collected in May. On the other hand, the number of viable cell per gram of sand-gravel sediment was 10 , and there
was no change between before and after the test. Test results using two-phase system consisting of rinsed seawater
and sand-gravel sediments (carbon dioxide evolved, number of viable cell before and after the test, biodegradation
measurement of plastic materials, etc.) are shown in Annexes C to H. The optimized two-phase system maintains high
biodegradability and high reproducibility even in long-term studies.
Annex C presents the cumulative amount of carbon dioxide evolved in one-phase systems of rinsed seawater
and two-phase systems of rinsed seawater and sand-gravel sediments prepared in the intertidal zone.
Cumulative evolved carbon dioxide results showed that the usable lifespan of each test system depended
on the number of viable bacteria and nutrient biomass content. The active period of the seawater one-phase
system is less than one month. For biodegradation measurements over 180 days, a one-phase test system
with nutrients or a two-phase test system with sediment is recommended.
Annexes D to H demonstrate the optimality of this preparation method by demonstrating the biodegradation
of the test materials in a two-phase system consisting of rinsed seawater and sand-gravel sediments. The
validity of this evaluation was verified from the cumulative amount of carbon dioxide evolved from the
reference material and the blank, the dispersion, and the degree of biodegradation of the reference material.
After 180 days of testing, the number of viable bacteria decreased in seawater but not in sediments. The
absolute biodegradation of the test materials was over 70 % and the relative biodegradation was over 90 %.
The coefficient of variation is about 5 %, which is less than 2
...