ISO 21716-3:2020

INTERNATIONAL ISO
STANDARD 21716-3
First edition
Ships and marine technology —
Bioassay methods for screening anti-
fouling paints —
Part 3:
Mussels
PROOF/ÉPREUVE
Reference number
ISO 21716-3:2020(E)
©
ISO 2020

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ISO 21716-3:2020(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
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Published in Switzerland
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ISO 21716-3:2020(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Material and apparatus . 3
6 Preparation of the test organism and the test seawater . 4
6.1 General . 4
6.2 Preparation of the test organism . 4
6.3 Preparation of test seawater . 4
7 Preparation of the test panels and control panels . 4
7.1 General . 4
7.2 Preparation of the test panels . 4
7.3 Affixing of mussels to the test panels and control panels . 4
8 Operation of the test . 5
9 Validation of the test . 6
10 Byssus threads formation . 6
10.1 General . 6
10.2 Calculation of the average number of byssus threads formed . 7
10.3 Data treatment and interpretation of the results. 7
11 Test report . 8
Annex A (informative) Statistical analysis — Examples .10
Annex B (informative) General information of mussel, Mytilus galloprovincialis .14
Annex C (informative) Identification of Mytilus galloprovincialis .17
Bibliography .19
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ISO 21716-3:2020(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 8, Ships and marine technology,
Subcommittee SC 2, Marine environmental protection, in collaboration with Technical Committee
ISO/TC 35, Paints and varnishes, Subcommittee SC 9, General test methods for paints and varnishes.
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.
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ISO 21716-3:2020(E)

Introduction
Anti-fouling paints that contain biocides are widely used to prevent fouling of ship hulls by marine
organisms. Effective anti-fouling technologies are critical for maintaining fuel consumption efficiency
of ships and also 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 or efficacy. For example, guidance published by the European Chemical Agency (ECHA) on
the assessment and evaluation of efficacy for anti-fouling products states clearly that laboratory testing
of individual anti-fouling paints is not undertaken 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 those encountered when the antifouling product are in service is routinely
undertaken instead (see Reference [35]).
Whilst 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. ISO 21716 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 ISO 21716 can be used for such
purposes 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, ISO 21716
provides information on methods that can be used to screen anti-fouling paints in order to determine
whether to continue development of an experimental paint and/or a product 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.
ISO 21716 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 ISO 21716 can be used as part of a tiered
approach to predict the ability of an anti-fouling coating to prevent fouling on ships. Alternatively, 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 ISO 21716 do not reliably predict the ability of an anti-fouling coating to prevent fouling on ships
or the translocation of invasive marine species.
ISO 21716 is not intended to provide a list of validated tests for testing the efficacy of anti-fouling; this
can be covered in regulations. It is not intended to provide a list of validated tests for this purpose,
nor for predicting the ability of a fouling control paint to prevent fouling on ships or to prevent the
translocation of invasive marine species.
Mussels are typical marine sessile organisms regarded as harmful fouling organisms because of
the impact on fuel consumption and the potential for translocation of non-indigenous species if they
become attached to ship hulls.
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ISO 21716-3:2020(E)

This test method utilizes young mussels to assess settling behaviour in the presence of treated panels.
Young mussels are used because they have higher byssus threads production activity as compared to
the adults. More information is provided in Annex B.
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INTERNATIONAL STANDARD ISO 21716-3:2020(E)
Ships and marine technology — Bioassay methods for
screening anti-fouling paints —
Part 3:
Mussels
1 Scope
This document specifies a laboratory test method for screening anti-fouling paints in a flow-through
system using mussels as the test organism. It is intended to be used in conjunction with ISO 21716-1,
which specifies the general requirements. The purpose of the test is to determine if there is a difference
in mussel settlement on painted test panels compared with mussel settlement on inert non-toxic control
panels under the conditions of the test. Examples of statistical analysis to determine if the difference in
mussel settlement is statistically significant are given in Annex A.
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 terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp;
— I EC E le c t r op e d i a : av a i l able at ht t p:// w w w . ele c t r op e d i a . or g / .
3.1
byssus
attachment organ secreted by a mussel, consisting of stem, byssus threads and adhesive discs
Note 1 to entry: See Figure 1.
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Key
1 foot
2 root (in byssus gland)
3 stem
4 byssus thread
5 adhesive disc (or plaque)
Figure 1 — Attachment organ of a mussel
3.2
shell length
longest linear distance between two points on the outside edge of the shell of a mussel
3.3
purified water
water with an electric conductivity of 2 µS/cm or less, prepared by distillation and/or treatment with
ion exchange resin(s)
4 Principle
The test procedure consists of the following 5 sequential steps, summarized in Figure 2:
— preparation of the test organism and the test seawater;
— preparation of the test panel and control panel;
— operation of the test;
— validation of the test; and
— data treatment and interpretation of the results.
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Figure 2 — Scheme of the test procedure
Each bioassay shall consist 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, then the number of byssus threads for the test and control groups can be compared.
5 Material and apparatus
The items listed in Tables 1 and 2 shall be used for the test.
Table 1 — List of material used
Material Remarks
Adhesive of surgical grade E.g., cyano-acrylate adhesive.
Control panels 50 mm square of PVC is recommended.
Cultured stock of live young At least 30 live mussels, Mytilus galloprovincialis sp., with a shell length of
mussels 8 mm to 10 mm, that are attached to natural or artificial substrates.
Other mussel species may be used if Mytilus galloprovincialis sp. mussels are
not available.
Natural seawater Defined in ISO 21716-1:2020, 3.8.
Purified water Defined in 3.3.
Scissors
Small piece of paper Filter paper may be used as spacer.
Test panels Specified in ISO 21716-1:2020, 4.2. 50 mm square is recommended.
1 µm filters Used to prepare test seawater.
Table 2 — List of apparatus used
Apparatus Remarks
Light White fluorescence or LED.
Light intensity meter Accuracy: ±10 lx.
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Table 2 (continued)
Apparatus Remarks
pH meter Accuracy: ±0,1.
Salinometer Accuracy: ±0,1.
Stereo microscope Magnification: 5-30X with fiber light.
Thermometer Accuracy: ±0,1 °C.
Water flow-through system Specified in ISO 21716-1:2020, 5.2, with a means of maintaining the test sea-
water tank at 20 °C ± 1 °C and alternately illuminating the test seawater tank
with a light intensity of 3 000 lx [Clause 8, d), light conditions] and with a light
intensity of <5 lx [see Clause 8, d), dark conditions].
6 Preparation of the test organism and the test seawater
6.1 General
The cultured stock of live mussels is used to perform the bioassay test in sea water.
6.2 Preparation of the test organism
Live mussels are generally prepared by collecting wild mussels and acclimatizing them in the laboratory
prior to testing. Guidance on this process and on storing mussels can be found in Annex B.
Information on the life cycle of mussels can also be found in Annex B, and information on the
identification of M. galloprovincialis sp. mussels can be found in Annex C.
6.3 Preparation of test seawater
Pass natural seawater through a 1 µm filter unit and adjust to salinity 30,0 ± 0,5 using purified water.
7 Preparation of the test panels and control panels
7.1 General
The same test and control groups shall be used throughout the whole test.
7.2 Preparation of the test panels
Test panels shall be prepared following the specifications described in ISO 21716-1:2020, Clause 4.
7.3 Affixing of mussels to the test panels and control panels
Separate the live mussels from their substrate by cutting the byssus threads with scissors, taking care
to avoid damage to other tissue and organs. Affix five mussels to each test with the adhesive, using
filter paper as a spacer between the shell of the mussel and the surface of the panel, providing a 20 mm
diameter circular separation zone around each mussel [see Figure 3 a)].
NOTE A spacer of adhesive-infiltrated filter paper can be used to prevent excessive spreading of the adhesive
and improve adhesion of the mussel to the test and control panels [see Figure 3 b)].
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a) Top view
b) Side view
Key
1 mussel
2 test/ control panel
3 spacer of adhesive-infiltrated filter paper
Figure 3 — The mussels on the test or control panel
8 Operation of the test
The bioassay shall be simultaneously performed on the test group and the control group as follows (see
Figure 4).
The experimental system specified in ISO 21716-1 shall be used for the test. The system is equipped
with the devices that maintain the specified water temperature and light irradiation of the test.
a) Fill the test seawater tank with test seawater and provide a continuous flow of test seawater from
the seawater storage tank. Maintain the temperature of the test seawater at 20 °C ± 1 °C for the
duration of the test. The flow rate should be set to achieve at least about 0,8 turnover per hour of
the water of test seawater tank.
NOTE If the flow rate is too low, the result can be affected by the concentration of biocide in seawater of
the test seawater tank.
b) Place the test and control panels in the test seawater tanks, ensuring all the panels are fully
immersed in the test seawater.
c) Measure and record the temperature, pH and salinity. Measure and record those parameters again
after 24 h from the beginning of the test.
d) Illuminate the test seawater tank for 12 h with a light intensity of 3 000 lx, and then leave the test
seawater tank in darkness for 12 h.
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e) Carefully remove the test and control panels from the test seawater tanks after 24 h from the
beginning of the test.
f) Using a stereo-microscope, count the number of byssus threads for each live and attached mussel
on each test and control panel, and record the results using e.g. Table 3.
Count the number of dead or detached mussels on each test and control panel, and record the results
using e.g. Table 3.
Figure 4 — Flow chart of the procedure for the test
9 Validation of the test
The results of the bioassay are validated by assessing if the results of the test meet the following four
criteria.
The results of the bioassay shall only be considered valid if all four criteria are met.
— The average number of byssus threads formed by living individual in the control panels in each run
is ten or more.
— The mortality of the mussels in the test panels in each run is 10 % or less.
— The mortality of the mussels in the control panels in each run is 10 % or less.
— The percentage of individuals detached from the test surface in each run is 5 or less.
10 Byssus threads formation
10.1 General
The evaluation procedure for screening anti-fouling paint is given in 10.2 and 10.3. The anti-fouling
paint is evaluated by comparing the number of byssus threads between the test and control groups.
The test data should be recorded using e.g. Table 4.
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10.2 Calculation of the average number of byssus threads formed
Calculate the average number of byssus threads formed by the mussels on the control group using
Formula (1) and record the results using e.g. Table 3 to one decimal place.
j
jj
j
()NN++N
∑ cn
cc12
j=1
A = (1)
c
j
j
n
∑
j=1
where
A is the average number of byssus threads in the control groups;
c
j is the run number;
j
is the number of byssus threads formed by living individual mussel attaching on the control
N
cn
panels on the j-th run;
j
is the number of living mussels attaching on the control panels on the j-th run.
n
Calculate the average number of byssus threads formed by the mussels on the test group using
Formula (2) and record the results using e.g. Table 3 to one decimal place.
j
jj j
()NN++N
∑ tt12 tn
j=1
A = (2)
t
j
j
n
∑
j=1
where
A is the average number of byssus threads in the test groups;
t
j is the run number;
j
is the number of byssus threads formed by living individual attaching on the test panels on
N
tn
the j-th run;
j
is the number of living mussels attaching on the test panels on the j-th run.
n
10.3 Data treatment and interpretation of the results
If A is less than A , this can indicate that the byssus settlement on the test group is less than that of the
t c
control group. However, further analysis of the results is required in order to determine if the difference
of the result between test and control groups is statistically significant. There are many possible ways
to perform the statistical analysis. Typical examples are shown in Annex A.
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Table 3 — Example of compilation of the data
Number of byssus threads Number
Number of
of dead de-
Sample name
Mussel Mussel Mussel Mussel Mussel Subto- Aver-
mussel tached
Total
1 2 3 4 5 tal age
(%) mussel
(%)
Control panel 1 25 22 36 20 18 121
0
Control panel 2 21 31 11 34 19 116 341 22,73
(0,00)
1
Control panel 3 15 14 25 20 30 104
Run
1
Test panel 1 10 4 5 10 * 29
(3,3)
0
Test panel 2 12 5 8 10 5 40 112 8,00
(0,00)
Test panel 3 7 9 3 9 15 43
Control panel 1 25 17 * 20 31 93
0
Control panel 2 34 22 26 14 13 109 270 19,28
(0,00)
1
Control panel 3 15 15 8 15 15 68
Run
2
Test panel 1 8 * 4 8 3 23
(3,3)
1
Test panel 2 8 4 9 0 3 24 72 5,14
(6,7)
Test panel 3 2 2 8 11 2 25
Control panel 1 16 12 30 22 23 103
1
Control panel 2 * 8 14 10 22 54 241 18,54
(6,7)
1
Control panel 3 16 15 41 12 * 84
Run
3
Test panel 1 3 1 4 6 0 14
(3,3)
1
Test panel 2 6 * 2 4 2 14 42 3,00
(6,7)
Test panel 3 1 3 2 4 4 14
* Dead or detached mussel (excluded from the table).
Results:
A : 20,2
c
A : 5,4
t
11 Test report
Table 4 specifies the minimum required information for the test report. The test results shall be
reported using Table 4.
Table 4 — Minimum required information for the test report
Information Requirement
Materials and size of substrate to be painted ×
General specifications Biocides contained ×
and process for paint
Name of paint ×
Undercoat (×)
Surface treatment (×)
Dry film thickness ×
a
Methods and time (number of days) for aging test panels ×
x: required; (x): optional.
a
Information required for each run.
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Table 4 (continued)
Information Requirement
General information on Identification (species name and identifier) ×
a
the test organisms
Date of sampling/collection (×)
Place of sampling/collection (×)
Water quality parameters at the time of sampling (temperature, ×
salinity, pH)
Acclimatization condition of the mussel (temperature, salinity, pH, ×
light condition, acclimatization period, survival rate)
a
Initial test condition Starting date ×
Number of test and control panels ×
Size of the test seawater tank ×
Water quality parameters (temperature, pH, salinity), light ×
condition, rate of water exchange
Other information on the test procedure and test device (×)
Test condition Water quality (temperature, pH, salinity), light condition ×
a
after 24 h
The mortality of mussels in the test surface, the percentage of ×
individuals detached from the test surface
Accidental/unexpected items observed during the test (×)
Average number of byssus threads ×
Statistical analysis method used (×)
Is there a statistically significant difference between the bioassay results for the test and (×)
control groups, Yes/No?
x: required; (x): optional.
a
Information required for each run.
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Annex A
(informative)

Statistical analysis — Examples
A.1 Introduction
It is stated in the Scope that the purpose of the test is to determine if there is a difference in mussel
settlement on painted test panels compared with mussel settlement on inert non-toxic control panels
under the conditions of the test. It can be useful to perform statistical analysis to determine if the
difference in mussel settlement is statistically significant. A typical example of statistical analysis was
given in this Annex, based on Reference [1]. Selection of an optimal statistical analysis method from
different approaches should be made depending on the purpose and the data distributions. This annex
describes some statistical analysis methods as examples.
A.2 Statistical analysis to compare a test group and a control group
A.2.1 Introduction
This clause indicates some statistical analysis methods to calculate the difference between a control
group and a test group as examples. Figure A.1 shows an example for this process. Statistical analysis
of significance difference between the control and test groups is conducted using a one-way ANOVA
(analysis of variance) followed by multiple comparison test. In this case, Dunnett’s multiple comparison
test is recommended for comparing several treatments with the control groups (Reference [2]).
Probability values (p-value) less than 0,05 are considered significant.
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Figure A.1 — Example of a statistical analysis process
[3]–[7]
A.2.2 One-way analysis of variance
One-way analysis of variance (ANOVA) is a collection of statistical models and their associated
estimation procedures (such as the "variation" among and between groups) used to analyse the
differences among group means in a sample. The ANOVA is based on the law of total variance, where
the observed variance in a particular variable is partitioned into components attributable to different
sources of variation. In its simplest form, ANOVA provides a statistical test of whether two or more
population means are equal, and therefore generalizes the t-test beyond two means.
The one-way
...