ISO 21716-4:2025

International
Standard
ISO 21716-4
First edition
Ships and marine technology —
2025-08
Bioassay methods for screening
anti-fouling paints —
Part 4:
Algae
Reference number
© ISO 2025
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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 Test procedure . 2
5 Materials and apparatus . 2
6 Preparation of test organism and test seawater . 3
6.1 General .3
6.2 Preparation of test organism . .3
6.3 Preparation of test seawater . .4
7 Preparation of test/control panel . . 4
7.1 General .4
7.2 Preparation of test panels .4
7.3 Affixing algae on the substrates .5
8 Algae viability test . 5
8.1 General .5
8.2 Requirements .5
8.3 Operation of the algae viability test .6
9 Bioassay . 7
9.1 General .7
9.2 Operation .7
10 Colour space . 8
10.1 General .8
10.2 Calculation of the average value of the parameters of colour space .9
10.3 Data treatment and interpretation of the results .10
11 Test report . 10
Annex A (informative) Statistical analysis .12
Annex B (informative) Preparation of test organism.15
Annex C (informative) General information on macroalga, Ectocarpus sp. .16
Bibliography . 19

iii
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,
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
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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 8, Ships and marine technology, Subcommittee
SC 2, Marine environment protection.
A list of all parts in the ISO 21716 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
Introduction
Anti-fouling paints that contain biocides are widely used to prevent the fouling of ship hulls by marine
organisms. Effective anti-fouling technologies are critical for maintaining the fuel consumption efficiency of
ships and for minimizing possible translocation of aquatic species through maritime trade. The evaluation
of anti-fouling paints is generally undertaken by adopting a tiered approach whereby paint manufacturers
use a battery of laboratory, raft, patch tests and full vessel trials. Raft, patch tests and full vessel trials are
generally conducted over extended periods of time and are predominantly relied upon for the prediction of
coating performance when used commercially on in-service ships.
The results of raft, patch test and full vessel trials (field testing) can be used as part of the regulatory
process for pesticidal or biocidal products in certain countries in order to demonstrate the efficacy of an
anti-fouling paint. Laboratory testing alone is recognized as being unable to predict in-service performance
[1]
or efficacy. For example, guidance published by the European Chemical Agency (ECHA) on the assessment
and evaluation of the efficacy of anti-fouling products states clearly that laboratory testing of individual
anti-fouling paints is not performed as it is not considered to be a realistic evaluation of the product. Field
testing, which permits anti-fouling products to be tested under similar operating conditions and stresses as
[1]
those encountered when the anti-fouling products are in service, is routinely undertaken instead.
Although laboratory tests are unable to reliably predict in-service coating performance, they have merit in
the screening of experimental coatings for further evaluation during the research and development process.
Reproducible objective data obtained by following standardized screening methods, independent of the
test location or the season, can be a useful tool to support the selection of anti-fouling paints for higher
tier testing, e.g. raft or ship tests. The ISO 21716 series provides a compilation and description of in vitro
bioassay methods intended to aid the process of screening anti-fouling paints prior to higher tier raft or
ship tests. Toxicological screening methods included in each part of the ISO 21716 series can be used for
purposes such as early decision-making in research and product development, rapid feedback on potential
toxicological concerns, or for the preliminary assessment of anti-fouling paints. For instance, the ISO 21716
series provides information on methods that can be used to screen anti-fouling paints in order to determine
whether to continue development of either an experimental paint or a product, or both, that contains a
particular ingredient, or to determine whether to take on the cost of performing the remaining tiers within
a complete tiered-testing strategy.
The ISO 21716 series provides screening bioassays related to certain common genera of fouling organisms,
namely barnacles, mussels and algae. These screening tests are relatively simple and rapid laboratory
tests that can be performed to provide an indication of the toxicity of a painted surface towards selected
test organisms. The screening tests described in each part of the ISO 21716 series can be used as part of
a tiered approach to predict the ability of an anti-fouling paint to prevent fouling on ships. Alternatively,
these screening tests can be used to prevent the translocation of invasive marine species by progressively
involving subsequent semi-field (e.g. raft panels) and field testing (e.g. ship trials). On their own, the
screening tests described in each part of the ISO 21716 series do not reliably predict the ability of an anti-
fouling paint to prevent fouling on ships or the translocation of invasive marine species.
The ISO 21716 series is not intended to provide a list of validated tests for testing the efficacy of anti-fouling
paints; this can be covered by applicable legal regulations in each country or region. It is also not intended
to be used for predicting the ability of a fouling control paint to prevent fouling on ships or to prevent the
translocation of invasive marine species.
Algae are typical marine sessile organisms regarded as harmful fouling organisms because of their impact
on fuel consumption and the potential for translocation of non-indigenous species if they become attached
to ship hulls.
The test method specified in this document utilizes algae to assess their colour change in the presence of
treated panels. The brown macroalga is used because they are considered a major ship-fouling organism
and its colour change is easily measured compared to other algae. More information is provided in Annex C.

v
International Standard ISO 21716-4:2025(en)
Ships and marine technology — Bioassay methods for
screening anti-fouling paints —
Part 4:
Algae
1 Scope
This document specifies the laboratory test method for screening anti-fouling paints in a flow-through
system using algae as the test organism. It is intended to be used in conjunction with ISO 21716-1, which
specifies the general requirements of the test methods described in the other parts of the ISO 21716 series.
The purpose of the test specified in this document is to determine the difference in colour of algae on painted
test panels compared with algae on inert non-toxic control panels under the test conditions.
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 21716-1:2020, Ships and marine technology — Bioassay methods for screening anti-fouling paints — Part 1:
General requirements
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 21716-1 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
colour space
colour coordinate which is quantified as L*, a* and b* measured by a spectrocolorimeter
Note 1 to entry: See ISO/CIE 11664-4 for further details.
3.2
total colour difference
total colour difference between two colours each given in terms of colour space (3.1)
Note 1 to entry: See ISO/CIE 11664-4 for further details.
3.3
substrate
membrane made of nitrocellulose used for affixation (3.4) of algae
3.4
affixation
filtration of algal suspension through substrate (3.3) using a vacuum filtration system

3.5
purified water
water with an electric conductivity of 2 μS/cm or less, prepared by either by distillation or treatment with
ion exchange resin(s)
4 Test procedure
The test procedure consists of the following five sequential steps, summarized in Figure 1:
— preparation of the test organism and the test seawater;
— preparation of the test and control specimen for the test;
— operation of the test (algae viability test and bioassay);
— validation of the test; and
— data treatment and interpretation of the results.
Figure 1 — Composition and schematic procedure of the test
Each bioassay consists of three runs as a minimum. Each run shall consist of a test group of three or more
test panels and a control group of three or more control panels. Provided that the validation criteria are met,
each parameter of the colour space from the substrates for the test and control groups shall be compared.
5 Materials and apparatus
The items listed in Table 1 and Table 2 shall be used for the test.

Table 1 — List of materials used
Materials Remarks
Silicone rubber band Used for electronic devices for antistatic purposes
Control panels 50 mm × 50 mm of polyvinyl chloride (PVC) is recommended.
Cover film Transparent film (e.g. polyvinylidene film)
White plate A white plate (e.g. polyvinyl chloride plate) for measuring values of colour
space. Its size shall be larger than the control and test panels.
Cultured stock of algae Filamentous brown macroalga Ectocarpus sp. (See Reference [16])
Cultured stock of live algae Algal culture suspension of 100 µg/ml as dry weight
Natural seawater Specified in ISO 21716-1:2020, 3.8
Substrates It is recommended to use circular membrane made of nitrocellulose, with the
following characteristics:
Pore size: 8 µm or less
Thickness: 1,35 µm or less
Diameter: 47 mm or the size inscribed with the circumference of test plates
Porosity: more than 80 %
Test panels Specified in ISO 21716-1:2020, 4.2. 50 mm × 50 mm is recommended.
Glass microfibre filter (GF) with 1,2 μm Used to prepare test seawater
pore size grade C (GF/C)
Test seawater Natural seawater filtered with 1,2 µm GF/C unit boiled at 100 °C for 30 min
whose salinity is adjusted to 35,0 PSU ± 1,0 PSU and pH is adjusted to 7,8 - 8,2
using purified water, respectively
Culture medium Provasoli’s enriched seawater with iodine (ESI) (See Reference [16])
Table 2 — List of apparatus used
Apparatus Remarks
Light White fluorescence or LED
Light intensity meter Accuracy: ± 5,0 µmol photons/m /s
pH meter Accuracy: ± 0,1
Salinometer Accuracy: ± 0,1 ‰ in practical salinity units (PSU)
Thermometer Accuracy: ± 0,1 °C
Water flow-through system As specified in ISO 21716-1:2020, 5.2, with a means of maintaining the test seawater
tank at 20,0 °C ± 1,0 °C and alternately illuminating the test seawater tank with
a light irradiation of 20,0 µmol photons/m /s [see 8.3 a), light conditions] and
with a light irradiation of < 1,0 µmol photons/m /s [see 8.3 d) dark conditions]
Spectrocolorimeter Device measuring values of colour space (8 mm in diameter, illuminant D65)
Filtration system Device to affix the algae on membrane under vacuum condition
Heating device Device to heat liquid up to 100 °C
6 Preparation of test organism and test seawater
6.1 General
The cultured stock of live algae is used to conduct the bioassay test in test seawater.
6.2 Preparation of test organism
Live algae are generally provided by algal culture collections from universities or institutes and cultured in
the laboratory prior to testing. Guidance on the process of culturing and storing algae can be referred to in
Annex B.
Information on the life cycle and identification of macroalga Ectocarpus sp. can be found in Annex C.
6.3 Preparation of test seawater
Pass natural seawater through a 1,2 μm GF/C unit followed by boiling at 100 °C for 30 min. Adjust both the
salinity to 35,0 PSU ± 1,0 PSU and the pH to 8,1 ± 0,1 using purified water.
7 Preparation of test/control panel
7.1 General
The same combination of test and control groups shall be used throughout the whole test.
7.2 Preparation of test panels
Test panels shall be prepared following the specifications described in ISO 21716-1:2020, Clause 4. In order
to avoid any possible influence from the coating applied on extra edge of the test panel, the test paints should
be applied according to the size of substrate, as shown in Figure 2.
Key
1 test/control plate
2 coating (in the case of test plate)
3 substrate
4 silicone rubber band
5 algae affixed on the surface of the substrate
Figure 2 — Substrate with algae on the test or control panel

7.3 Affixing algae on the substrates
Prepare a certain concentration of algal suspension (i.e. 100 μg/ml ± 10 μg/ml) and take 10 ml of suspension
by a pipette. A substrate shall be set on a decompression filtration system followed by pouring the suspension
on the substrate. Start the vacuum operation and squeeze out the water for around 30 s. Place the substrate
on the paper to remove the test seawater for 5 min. The procedure for the affixation of the algae on the
substrates is illustrated in Figure 3.
NOTE If a substrate is floated on the surface of the purified water for 10 min before filtration, algae can be better
affixed on the substrate.
Figure 3 — Procedure for the affixation of algae on the substrates
8 Algae viability test
8.1 General
The bioassay is validated using arithmetic tests by confirming that the viability of the algae used in the
bioassay meets the criteria specified in 8.2. The results of the bioassay shall only be considered valid if the
criteria are met.
8.2 Requirements
The algae viability test is conducted to verify the health of the algae affixed for the bioassay and should be
performed in parallel with the bioassay specified in Clause 9. The duration of fixation algae should be at
least 5 days. L*, a* and b
...