ISO 23732:2021

INTERNATIONAL ISO
STANDARD 23732
First edition
2021-07
Marine technology — Marine
environment impact assessment
(MEIA) — General protocol for
observation of meiofaunal community
Reference number
ISO 23732:2021(E)
©
ISO 2021

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ISO 23732:2021(E)

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© ISO 2021
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 23732:2021(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Sampling . 2
5.1 Method and equipment . 2
5.2 Sub-sampling . 3
5.3 Sample preservation . 3
5.3.1 Preservation method before analysis . 3
5.3.2 Preservation method after analysis . 3
5.4 Recording sample information. 3
6 Procedures for imaging flow cytometry (morphological analysis) .3
6.1 Pre-processing of samples . 3
6.2 Observation using imaging flow cytometer . 3
6.3 Image processing . 3
7 Procedures for metagenomic analysis (molecular analysis) . 4
7.1 General . 4
7.2 Nucleic acids (DNA/RNA) extraction . 4
7.3 PCR primers . 4
7.4 PCR protocol. 4
7.4.1 DNA . . 4
7.4.2 RNA (cDNA library production) . 4
7.5 Check and purification of PCR amplicon . 4
7.6 Index PCR . 5
7.7 Purification of amplicon by magnetic beads . 5
7.8 Concentration measurement . 5
7.9 Quality check for amplicon by an automated electrophoresis system using
microfluidic chip . 5
7.10 Sequencing by a next generation sequencer . 5
7.11 Data processing . 5
8 Data and sample management. 5
9 Combination of morphological analysis and molecular analysis (integrated analysis) .5
Annex A (informative) Example procedure for the analysis of the meiofaunal community
by imaging flow cytometry . 7
Annex B (informative) Example procedure for analysis of meiofaunal community by NGS .10
Annex C (informative) Example procedure for quantitative analysis
of meiofaunal community by NGS .13
Annex D (informative) Useful information for users .16
Bibliography .17
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ISO 23732:2021(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
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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
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on the ISO list of patent declarations received (see www .iso .org/ patents).
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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 13, Marine technology.
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 23732:2021(E)

Introduction
For environmental impact assessments (EIA) of plans for seabed mineral resource exploitation, objective,
comprehensive and easy-to-apply analysis techniques are required (see ISO 23730). Traditionally,
relatively large organisms have been used as indicators for environmental impact assessments, and
labour-intensive surveys using morphological characteristics were mainstream. Compared to larger
macrofauna and megafauna, meiofauna in the deep sea have high abundance and biomass and are an
[2]
important component of deep-sea ecosystems . In addition, meiofauna have a considerable influence
on the nutrient cycling in the sediments and sediment stability. Therefore, meiofauna are important
[3]
as biological indicators used to monitor natural or anthropogenic disturbances . The International
Seabed Authority (ISA) guidelines for contractors on the assessment of possible environmental
impacts due to exploration activities (see Reference ISBA/25/LTC/6) mandate the reporting of the
abundance and diversity of seafloor biotic communities, including meiofauna. Therefore, meiofauna,
being ubiquitous as well as sensitive to environmental perturbations, have been chosen as indicator
organisms for the analyses in this document. However, traditional methods for meiofaunal community
analysis are extremely time-consuming, which is economically problematic due to the costs of
conducting EIA as part of resource development. In addition, advanced expertise is required for the
identification of meiofauna to the species, genus, or even family level, and the number of experts
qualified to do this is limited. Also, if a technician does not have the training or knowledge to identify
meiofauna, a dissemination of inaccurate data could result. For these reasons, accurate, efficient and
objective analytical tools for the identification of meiofauna are needed.
Thus, the purpose of this document is to establish a convenient protocol for MEIA using meiofauna as
biological indicators. The role of EIA is the determination of fluctuation or change in the community
structure after environmental impacts. Data of species level composition and population size are
essential information to assess the effect of impacts.
Therefore, a meiofaunal analysis, following two methods is proposed, including:
1) imaging flow cytometry;
2) environmental metagenomic analysis.
By this protocol, the population density (number of individuals per unit area) is obtained by analysis
using an imaging flow cytometer, and the species composition is acquired by metagenomic analysis.
These methods obtain data faster than traditional analysis methods that have been done so far. Further,
it is possible to compensate for the disadvantages of both methods with each other. By using both
methods complementarily, it becomes possible to grasp communities of meiofauna in the environment
objectively, comprehensively, quickly and easily (it is a method aligned to the ISA recommendation, see
ISBA/25/LTC/6, mandating to obtain data on population density, biomass and species composition for
meiofauna).
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INTERNATIONAL STANDARD ISO 23732:2021(E)
Marine technology — Marine environment impact
assessment (MEIA) — General protocol for observation of
meiofaunal community
1 Scope
This document specifies a general protocol for the observation of the meiofaunal community in the
deep seabed.
The standardized method can be used in any phase [baseline data acquisition, monitoring during and
after mining (testing)] accompanying resource development, making it easier to compare data beyond
differences in workers and waters.
This document is intended for marine environment impact assessments and other occasions where
long-term image-based data are required.
2 Normative references
The following document is 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.
1)
ISA ISBA/25/LTC/6, Recommendations for the guidance of contractors for the assessment of the possible
environmental impacts arising from exploration for marine minerals in the Area, 2013. Available at https://
www .isa .org .jm
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISBA/25/LTC/6 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
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
meiofauna
animals of the benthic community that are intermediate in size between macrofauna and microfauna,
operationally defined as > 32 μm and < 250 μm
[SOURCE: ISBA/25/LTC/6:2013, Annex II.]
3.2
PCR
polymerase chain reaction
DNA sequence synthesis reaction repeated to amplify DNA fragments of target regions of hundreds of
thousands of times of genes using template DNA, and two types of short DNA fragments (primers), and
DNA polymerases
1) ISA: International seabed authority.
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ISO 23732:2021(E)

3.3
NGS
next generation sequencing/sequencer
device capable of reading nucleotide
sequences of huge numbers of genes at high speed
Note 1 to entry: High-throughput or pyro-sequence.
4 Principle
A collected sediment sample is divided into two sub-samples and an analysis is performed by the two
methods as illustrated in Figure 1. The results obtained by each method are integrated and analysed.
Information on the meiofaunal community at the sampling point can be obtained.
Key
a
Sediment samples.
b
Morphological data [imaging flow cytometry].
c
Molecular sequence data [next-generation sequencing].
d
Integrated analysis.
e
Result of meiofaunal community.
Figure 1 — Schematic overview of the meiofaunal community analysis procedure
5 Sampling
5.1 Method and equipment
Marine sediments should be quantitatively collected for the analysis of the meiofaunal density and
community composition. Examples of equipment (corers) for quantitative sampling include box corers,
[4]
grab corers, and multiple corers .
The corers are deployed from a research ship to the seafloor; thus, it is not suitable for surveys that
require a selection of sampling points while observing seafloor conditions. In areas where pin-point
sampling is required, e.g. hydrothermal fields where chimneys and mounds exist, sampling using push
corers operated by manned or unmanned submersibles is recommended.
In addition, if samples from different deployments, different samplers or corers with different diameters
[5, 6, 7]
are used, the community composition and vertical distribution of the meiofauna can be changed .
Thus, for the same EIA program, identical samplers should be employed, and when comparing results
with other programs, differences in sampling gear shall also be considered.
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5.2 Sub-sampling
Sediment samples are sub-sampled by cutting into several layers from the surface layer. ISA guidelines
suggest the following cutting layers (depths) for meiofaunal investigation: 0 cm to 0,5 cm, 0,5 cm to
1,0 cm, 1 cm to 2 cm, 2 cm to 3 cm, 3 cm to 4 cm, 4 cm to 5 cm.
5.3 Sample preservation
5.3.1 Preservation method before analysis
The collected samples should be preserved by suitable ways to avoid affecting the analytical results.
— Morphological observation; fix by formaldehyde or suitable fixative reagents.
— DNA/RNA analysis; deep-freeze (−80 °C), or suitable buffers for the preservation of DNA/RNA.
5.3.2 Preservation method after analysis
The sample after analysis should be stored in a way that can maintain the same state.
— Morphological observation, samples are preserved by 5 % neutral formalin.
— Extracted DNA/RNA are preserved in the deep-freezer (−80 °C).
5.4 Recording sample information
Sample information shall be recorded, such as core colour, texture, a photograph of the core, sampling
place (latitude, longitude, depth), date, sampling method, core number, layer, usage method, storage
method, etc.
6 Procedures for imaging flow cytometry (morphological analysis)
6.1 Pre-processing of samples
Before any observation using an imaging flow cytometer, the sediment sample shall be sieved to
extract a meiofaunal size fraction. Superfluous sediment particles should be removed as far as possible,
because sediment particles obstruct to obtain clear images of meiofauna in most other cases. These
qualifications can be realized with the method described by References [8], [9] and [10].
6.2 Observation using imaging flow cytometer
Observation shall be done with instruments suitable for imaging flow cytometry, such as given in
Annex A. Examples of imaging flow cytometer are shown in Clause D1. Perform the following:
— operate an imaging flow cytometer in accordance with the manufacture’s manual;
— use a colloidal silica solution as a sample flowing medium;
— put the sample and retrieve the image.
6.3 Image processing
The taxon should be identified based on the obtained images with reference to Reference [11] and other
relevant data sources. Alternatively, the obtained images can be referred to the database created as
necessary or open to the public.
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ISO 23732:2021(E)

7 Procedures for metagenomic analysis (molecular analysis)
7.1 General
For metagenomic analysis, DNA or RNA shall be targeted. When RNA is used, it can be easily matched
with the results of imaging flow cytometry. RNA is subjected to reverse transcription reaction and
treated as cDNA (complementary DNA) (see 7.4.2). Examples of metagenomic analysis are shown in
Annex B.
7.2 Nucleic acids (DNA/RNA) extraction
A DNA/RNA extraction kit by using beads beating method for soil sample is recommended.
The same DNA/RNA extraction method for the sample collected at the same sampling point during time
course observation should be used.
For RNA extraction, purification with DNase should be performed to digest all DNA fragments.
7.3 PCR primers
For metagenomic analysis by NGS, the target gene that is used for taxonomic identification is
recommended [e.g. 18S rRNA, ITS (internal transcribed spacer), 28S rRNA (large subunit rRNA), and
mitochondrial COI (cytochrome oxidase subunit I)]. The target gene should be chosen depending on
the purpose. The primers for this step, the sequence should consist of specific sequence for target gene
sequence and overhang adaptor sequence for NGS.
7.4 PCR protocol
7.4.1 DNA
In PCR (amplicon PCR), DNA fragments are amplified by repeated cycles (thermal cycling), which
consists of 3 steps, (1) denaturation (denaturing a double stranded DNA into two single stranded
DNA molecules by heat), (2) annealing (annealing of a primer with a single stranded complementary
DNA), and (3) elongation (elongation of a single stranded DNA from the site where a primer annealed).
PCR conditions (number of cycles and reaction temperature) shall be designated for appropriate
amplification.
The amplifications should be done in 3 replicate PCR reactions for each sample, after electrophoresis to
check the amplicon, combined triplicate PCR reactions of the same sample into a single volume.
7.4.2 RNA (cDNA library production)
A reverse transcription shall be performed for preparing a cDNA library using the extracted RNA.
Usually, reverse primer is used to extend the target region. Only one reaction should be done for a single
primer extension. The generated cDNA library is used as templates for amplicon PCR (see 7.4.1).
At the same time, to confirm extracted RNA quality (to check the DNA contamination), the same PCR
should be performed by RNA before reverse transcription. If the amplified fragments are seen on the
agarose gel as a band by RNA template PCR, the RNA may contain co-extracted DNA fragments. In that
case, it should purify the RNA.
7.5 Check and purification of PCR amplicon
The amplicons should be checked by agarose gel electrophoresis. If the DNA fragment is amplified with
appropriate PCR conditions, the expected length amplicon is seen as a band on the agarose gel.
After checking the amplicons, 3 replicate PCR reactions of the same sample should be combined into the
single volume, and proceed to the purification step.
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ISO 23732:2021(E)

Amplicons shall be purified using magnetic beads to remove residual PCR primers and reagents,
according to the supplier’s manual. Magnetic beads can purify the large DNA fragments (such as
amplicons) without excess primers, non-specific short fragments, salts, and enzymes by paramagnetic
bead technology.
7.6 Index PCR
Performance of NGS depends on the type of a sequencer; however, it is known to read 10 million
sequences (called reads) in one analysis. So, it is possible to analyse multiple samples simultaneously
and be often used to sequence all of mixed multiple amplicons. Therefore, to distinguish the amplicon
which derived from different source, tag sequence is added to each amplicon by index PCR. Primers for
index PCR shall be comprised overhang adaptor sequence which is the same sequence of amplicon PCR,
tag sequence (index sequence), and sequence adaptor for NGS.
7.7 Purification of amplicon by magnetic beads
A purification shall be performed for index PCR products after checking by agarose gel electrophoresis
as described in 7.5.
7.8 Concentration measurement
In order to equalize the DNA concentration of each amplicon used in NGS, the concentration shall be
measured. For this purpose, quantitative PCR or fluorometric quantitation can be used.
7.9 Quality check for amplicon by an automated electrophoresis system using
microfluidic chip
If necessary, a quality check for created amplicons should be performed using an automated
electrophoresis system.
7.10 Sequencing by a next generation sequencer
Follow the protocol of each manufacturer as to how to use the equipment.
The samples mixed to equal concentrations shall be denatured beforehand and applied for sequencing.
7.11 Data processing
The same program shall be used to compare the samples. Some analysing methods (programs) for
NGS data are available as freeware softs. Examples of metagenomic analysis programs are shown in
Clause D2. The parameters affect analysed data, especially taxon assignment. Analysing programs
used, database, and other related information shall be recorded.
8 Data and sample management
DNA sequence and its metadata should be submitted to the DNA database (GenBank/DDBJ/ENA).
Appearance data of organisms should be described and registered in compliance with Darwin Core
[12]
(DwC ) (also adopted in GIF and CoML) including image files.
9 Combination of morphological analysis and molecular analysis (integrated
analysis)
The morphological data derived from imaging cytometry provide information on the taxonomical group
and its population size (density) of the meiofaunal community. The nucleotide sequence data derived
from metagenomic analysis provide also the community composition and diversity. By conducting these
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ISO 23732:2021(E)

two analyses for the same sample and by integrating the two data, the analytical method complement
each other (see Annexes B and C).
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ISO 23732:2021(E)

Annex A
(informative)

Example procedure for the analysis of the meiofaunal community
by imaging flow cytometry
A.1 General
Although few instruments are known for imaging flow cytometry, this Annex provides an example of
2)
the procedure by FlowCam , see References [10] and [13].
A.2 Sampling and sub-sampling
Core sampling is the same method as described in Clause 5. The following procedure provides details
for sub-sampling.
1. Make filtered seawater using a 0,22 µm filter and keep it in a wash bottle.
2. Place the collected sediment core onto an extruder.
3. Using a siphon tube, remove the seawater above the sediment to approximately 1 cm above the
sediment surface.
4. Using a syringe, remove the remaining seawater 1 cm above the sediment and place in a plastic bag.
5. Using a spatula, slice the sediment cores into layers: 0 cm to 0,5 cm, 0,5 cm to 1 cm, 1 cm to 2 cm,
2 cm to 3 cm, 3 cm to 4 cm, and 4 cm to 5 cm from the surface. Place each layer into separate plastic
bags.
6. Use filtered seawater to rinse off any sediments remaining on the spatula into the same plastic bag
holding the layer.
7. Use pure water or clean tap water to completely wash the spatula and dry it thoroughly with paper
wipes each time the spatula is used.
A.3 Fixation and storage of samples
Each sediment layer (sub-sampling of core sample) should be fixed and preserved separately. Add the
neutral buffered formalin to the samples for a final concentration of about 5 %. Seal tightly and store in
room temperature. Fixation with formalin is suitable for preservation of the morphology of meiofauna.
2) FlowCam is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product.
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A.4 Analysis using flow cytometer
A.4.1 Pre-processing
Based on differences in density of meiofaunal specimens and sediment particles, a centrifugal
[8, 9]
separation method using colloidal silica solution has been devised . This method was modified to
[10]
apply the imaging flow cytometry observation for meiofauna . The procedures are as follows.
1. Add rose bengal staining solution into the sediment samples (final concentration, 0,05 g/l) and
leave to stand for at least one night.
2. Sequentially pass the sediment samples through 1 mm, 250 µm and 63 µm mesh size sieves. Rinse
away the seawater using pure water or clean tap water as much as possible.
3. Using pure water or clean tap water, transfer the fractions retained on the 1 mm and 250 µm mesh
size sieves to 50 ml conical tubes. Add formalin for storage.
4. Use a spoon to transfer the fraction retained on the 63 µm mesh size sieve to a 50 ml conical tube.
Keep the tube as dry as possible.
5. Rinse any sediments remaining on the sieve into a conical tube using a wash bottle containing
3)
Ludox HS-40 .
6. Centrifuge the conical tube containing sedimen
...