ISO 23734:2021

INTERNATIONAL ISO
STANDARD 23734
First edition
2021-07
Marine technology — Marine
environment impact assessment
(MEIA) — On-board bioassay to
monitor seawater quality using
delayed fluorescence of microalga
Reference number
ISO 23734:2021(E)
©
ISO 2021

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ISO 23734:2021(E)

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© ISO 2021
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Published in Switzerland
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ISO 23734:2021(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Materials . 3
5.1 Test alga. 3
5.2 Reagents. 3
5.2.1 Water . 3
5.2.2 Growth medium . 3
5.2.3 Nutrient, metal and tris stock solutions . 3
6 Apparatus . 4
6.1 General . 4
6.2 High sensitivity luminometer . 4
6.3 Incubator and tube shaker . 4
6.4 Apparatus for determining algal cell density . 4
6.5 Culture tubes. 4
6.6 Clean bench . 4
7 Preparations at a land-based laboratory . 4
7.1 Preparation of the growth medium . 4
7.2 Preparation of the algal stock culture . 5
8 Test procedure on-board . 5
8.1 Preparation of the algal inoculum culture . 5
8.2 Choice of the test concentrations . 5
8.3 Preparation of the test medium . 6
8.4 Inoculation and incubation . 6
9 DF measurement . 6
10 Interpretation of data . 6
10.1 Plotting the DF decay curve . 6
10.2 Calculation of per cent inhibition . 7
11 Interpretation of the results . 7
12 Test report . 7
Annex A (informative) Schematic overview and procedures of the on-board bioassay .9
Annex B (informative) Preparation of the test medium in seawater .12
Annex C (informative) Practical procedures for the on-board bioassay and
schematic diagram of the high sensitivity luminometer .13
Annex D (informative) Cryopreservation procedure .14
Annex E (informative) Reference data for checking the appropriateness of the test procedures .15
Bibliography .16
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ISO 23734:2021(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).
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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 23734:2021(E)

Introduction
Mining of offshore mineral resources has attracted much interest. These resources can be utilized as
potential mineral resources. However, deep-sea mining of the seafloor can pose potential hazards to
deep-sea environments and ecosystems (see ISO 10253 and References [3], [19], [21]). One concern is
the toxicity of heavy metals released from excavated minerals. Such heavy metals can be released into
[7],[18]
the seawater of the deep marine ecosystem . Further, there is a risk of unexpected leakage of the
recovered minerals and mining wastewater from the mining plant, which can result in heavy metal
[8]
contamination of the surface seawater .
Considering the above, an appropriate scheme for the monitoring and evaluation of the quality of deep
and surface seawater can ideally be introduced at each deep-sea mining site. The International Seabed
Authority (ISA) states that environmental impact assessments should address not only areas directly
affected by mining, but also the wider region impacted by discharged plume and materials released
[8]
during mineral transport to the surface .
An on-board or onsite method for heavy metal evaluation is essential, as it would allow prompt action
in case of an unexpected pollution incident. Rapid evaluation of the nature and extent of pollution
provides an opportunity to prevent wider spread of the toxic contaminants and, consequently, minimize
idle periods of a mining plant.
Although many chemical analytical methods are available at land-based laboratories, few methods have
been developed for on-board application. Deep-sea mineral deposits are inhomogeneous and can be
the source of release of various types of metal elements. Therefore, evaluation of mining contaminants
requires simultaneous analysis of multiple elements. Special instruments are needed to perform such
analyses, such as inductively coupled plasma mass spectrometry. Further, these instruments have to
be operated by expert staff, require considerable laboratory space and are expensive to install. Such
instrument types are difficult to install at every mining site as standard equipment for environmental
monitoring.
Bioassays constitute an alternative approach to specialist equipment, and are commonly used to assess
ecological risks of chemical contaminations (see ISO 10253). Bioassays do not provide quantitative
information about the contaminating substances, but can be used to detect a wide spectrum of toxicants,
including unknown toxicants. This feature is advantageous for the monitoring of water quality during
deep-sea mining activities.
General bioassay test protocols that use a variety of aquatic organisms have been published by
organisations, such as ISO (see ISO 10253), the Organization for Economic Co-operation and
Development (OECD) and the United States Environmental Protection Agency (US-EPA). These
authorized protocols are accepted in various water quality management fields. However, similarly to
chemical analyses, they require a considerable amount of time and space, and are thus not suitable
for on-board monitoring. It should also be noted that most protocols have been developed for inland
freshwater quality assessments.
This document was developed to address the shortcomings of the currently available bioassays for
monitoring seawater quality on-board. It describes a bioassay specifically for on-board determinations.
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INTERNATIONAL STANDARD ISO 23734:2021(E)
Marine technology — Marine environment impact
assessment (MEIA) — On-board bioassay to monitor
seawater quality using delayed fluorescence of microalga
1 Scope
This document specifies a bioassay for the determination of the presence of unknown toxic contaminants
in test seawater (see Figure A.1). It is based on the inhibition of photosynthetic activity of the marine
cyanobacterium Cyanobium sp. (NIES-981) by such toxic contaminants. The inhibition is determined
based on delayed fluorescence (DF) intensity.
The method is rapid and requires less laboratory space than standard bioassays. Hence, it can be used
on-board to generate basic data for seawater quality management at deep-sea mining sites where time
and space are extremely limited.
2 Normative references
There are no normative references in this document.
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
delayed fluorescence
DF
delayed light emission
weak fluorescence signal from photosynthetically active cells that originates upon repopulation of the
excited energy states of chlorophyll by stored energy after charge separation
3.2
DF decay curve
time-course change of the DF (3.1) intensity of test algae (3.7) that had been left in darkness after
exposure to light
Note 1 to entry: See Annex E, Table E.1 and Figure E.1.
3.3
effective concentration
ECx
concentration of test substance that results in an x % reduction in specific growth rate relative to the
controls
3.4
no observed effect concentration
NOEC
tested concentration below the LOEC (3.5) that has no statistically significant effect
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ISO 23734:2021(E)

3.5
lowest observed effect concentration
LOEC
lowest tested concentration that is significantly different from control
3.6
test seawater
seawater that is tested
3.7
test algae
alga (Cyanobium sp., NIES-981) that is used for bioassay
3.8
growth medium
artificial seawater (ASW-SN) containing nutrients and trace metals, commonly used for the culture of
test algae (3.4)
3.9
test medium
mixture of the growth medium (3.8) and test seawater (3.6)
3.10
algal stock culture
living or cryopreserved culture of test algae (Cyanobium sp., NIES-981) that has been prepared at a
land-based laboratory and is carried on board
3.11
algal inoculum culture
culture used in the bioassay, prepared from the algal stock culture (3.10) immediately before testing
3.12
cryopreservation
preservation of cells, tissues or organs at a very low temperature for future use
Note 1 to entry: See Annex D.
4 Principle
The described on-board bioassay provides basic data for seawater quality management at deep-sea
mining sites using DF (3.1) of the marine cyanobacterium Cyanobium sp. (NIES-981). The method is
quicker, and requires less laboratory space and equipment, than a standard growth inhibition assay
[15]
using other algae . First, the test seawater is collected at the target site, e.g. the surface seawater
in the vicinity of the mining plant or mining wastewater generated by the mining plant. Then,
duplicate cyanobacterium cultures in triplicate are set up in control tubes (growth medium with no
test seawater) and tubes containing diluted test seawater [test seawater mixed with growth medium
at a volume fraction of 80:20] (see Annex A and B, Figure A.1, A.2 and B.1). After incubation of 24 h,
DF is determined using an appropriate detector system for luminescence (see 6.2). Finally, total DF
intensities of the test seawater are compared with those of the control (growth medium with no test
seawater) using an appropriate statistical test. Significant differences between the test seawater and
control indicate that the collected test seawater has been polluted by mining or other activities. The
results of the on-board bioassay would support the appropriate environmental safety actions. As an
option, effective concentration (ECx), no observed effect concentration (NOEC) and lowest observed
effect concentration (LOEC) values may be determined by assaying additional dilutions of the test
seawater in a geometric series (see ISO 10253).
[4],[20]
DF is measured as a delay (ms to min) after the cells are transferred from light to darkness .
The delay in emission is associated with the repopulation of excited states of chlorophyll by stored
energy after charge separation. More specifically, it is the back-reaction of accumulated charges across
[11]
the thylakoid membrane in the electron transport chain . Because DF is an indicator of the electron
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ISO 23734:2021(E)

transfer state within the photosynthetic apparatus, it can be used as a sensitive intrinsic index of
[12],[16],[17]
photosynthetic activity .
Bioassays that rely on alga and plant DF are comparable with conventional growth inhibition
[5],[6],[9],[13],[14],[22]
tests .The method described in this document is a new DF-based bioassay system
developed specifically for water quality monitoring in offshore environments. It relies on a marine
[22]
autotrophic cyanobacterium and a modified method . The DF-based bioassay is rapid and requires
less extensive sample handling than the standard growth inhibition test. Consequently, it can be used
on-board, where time and space are substantially limited.
5 Materials
5.1 Test alga
Axenic culture of the marine cyanobacterium Cyanobium sp. (NIES-981). Strain NIES-981 is closely
related to the genus Synechococcus that is one of the major primary producers in the marine
environment. It exhibits stable and high growth under the appropriate conditions. The complete
genome of strain NIES-981 has been sequenced. It encodes 3 268 proteins, and harbours 46 tRNA genes
[23]
and three sets of rRNA genes . These genetic features provide a basis for the development of the
ecotoxicological bioassay. Strain NIES-981 can be obtained from the Microbial Culture Collection at the
National Institute for Environmental Studies (NIES) (MCC-NIES, https:// mcc .nies .go .j).
5.2 Reagents
5.2.1 Water
Deionised, for the preparation of the medium and stocks (nutrient, metal and tris) for the medium.
5.2.2 Growth medium
ASW-SN, optimized to allow sufficient growth of Cyanobium (NIES-981) to meet the test quality
(Table 1), are used for pre-culture and testing (see 7.1).
5.2.3 Nutrient, metal and tris stock solutions
Stock solutions of nutrients, metals and tris for ASW-SN (see Table 1), are prepared at a land-based
laboratory. The stock solutions are also added to the test seawater so that their concentration is the
same as in ASW-SN (see Annex B).
Table 1 — Reagents for ASW-SN (left) and stock solutions
a) Stock solution b) Stock solution c) Stock solu-
ASW-SN g g mg g
of nutrients of trace metals tion of tris
NaCl 25,0 NaNO 75 Na EDTA·2(H O) 580 Tris 100
3 2 2
MgCl ·6(H O) 2,0 K HPO ·3H O 3,0 FeCl ·6(H O) 422 Deionised water 1 000 ml
2 2 2 4 2 3 2
KCl 0,5 Deionised water 1 000 ml ZnSO ·7(H O) 2,93
4 2
CaCl ·2(H O) 0,5 CoCl ·6(H O) 1,33
2 2 2 2
MgSO ·7(H O) 3,5 MnCl ·4(H O) 24,0
4 2 2 2
Nutrients a) 10 ml Na SeO 2,30
2 3
Trace metal b) 100 μl Na MoO ·2(H O) 0,839
2 4 2
Tris c) 10 ml NiCl ·6(H O) 0,37
2 2
Deionised
1 000 ml Deionised water 100 ml
water
pH 8,2  pH 8,2
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ISO 23734:2021(E)

6 Apparatus
6.1 General
All equipment that comes into contact with the test medium and all solutions used for its preparation
are made of glass or a chemically inert material. Glassware that is free of chemical contaminants and
sterile is used for culturing and testing. Use general laboratory apparatuses and the following (see 6.2
to 6.6).
6.2 High sensitivity luminometer
6
Sufficiently sensitive to detect DF of at least 10 NIES-981 cells per millilitre, able to count photons at
−2 −1
0,1-s intervals for at least 60 s, and equipped with a red light source (50 μE·m ·s ) for excitation. In
addition, the shutter controls the excitation time accurately (see Annex C and Figure C.1).
6.3 Incubator and tube shaker
An incubator that can maintain 23 °C ± 2 °C is recommended, although the assay may also be performed
using light equipment in a room controlled at 23 °C ± 2 °C. For culturing and testing, white fluorescent
−2 −1 −2 −1
light (60 μE·m ·s to 80 μE·m ·s ) is used. Although a white LED lamp can also be used instead of
the white fluorescent light, check that it contains two wavelength ranges, red (with a peak between
660 nm and 670 nm) and blue light (with a peak between 450 nm and 460 nm). These are the
appropriate wavelengths for algal photosynthesis. An orbital and wheel shaker with a speed controller
is recommended for the pre-culturing and testing.
6.4 Apparatus for determining algal cell density
The same device as that used for DF measurements (6.2) can be used for cell density determinations.
Algal cell density determination is needed for preparing a living stock culture at a land-based laboratory
and for determining the initial cell density at the beginning of testing on-board.
6.5 Culture tubes
Autoclaved or disposable sterile tubes with a capacity between 5 ml and 10 ml (glass tubes are
recommended) with air-permeable stoppers are used for pre-culture and testing.
6.6 Clean bench
Sub-culturing of the living stock cultures is ideally performed on a clean bench, but a portable bench
with apron covers and a fan unit is sufficient.
7 Preparations at a land-based laboratory
7.1 Preparation of the growth medium
ASW-SN is used for testing and pre-culture of strain NIES-981. ASW-SN is also used for the preparation
of the test seawater for the incubation of test algae. The growth medium is ideally prepared in advance
at a land-based laboratory, and sent to the offshore site in sterile containers.
— Dissolve ASW-SN reagents and stock solutions in 900 ml of deionised water, as indicated in the left
column of Table 1. Prepare the stock solutions of nutrients, metals and tris in advance, according to
Table 1.
— Adjust pH to 8,2 using 1,0 N HCl (this requires the addition of approximately 5,5 ml of 1,0 N HCl).
— Add deionised water until the volume equals 1 000 ml.
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ISO 23734:2021(E)

— Filter the medium through a membrane filter with a pore size of less than 0,22 μm into a sterile
container. Alternatively, the medium may be autoclaved (121 °C for 20 min).
7.2 Preparation of the algal stock culture
Either a living stock culture or a cryopreserved stock culture of strain NIES-981 shall be prepared at
a land-based laboratory. The stock shall then be used to prepare an inoculum culture on-board before
testing. As soon as possible after the living stock culture arrives on-board, it should be placed under
culture condition. This step is important, to shorten the preparation time of the alga inoculum on-
board.
8
The density of the living stock culture should be 10 algal cells per millilitre in the exponential growth
phase. This corresponds to between 1 000 counts and 1 500 counts of photons at 0,1 s after the start of
the measurements. The living stock culture should be pre-cultured to obtain the inoculum culture for
the bioassay.
First, determine the cell density of original stock culture using a luminometer (the same device as that
for DF measurements, see 6.2). Add a sufficient amount of cells from the algal stock culture to 100 ml of
6
the growth medium (7.1) for the cell density to be 2 × 10 algal cells per millilitre, and pre-culture for 3
days. After pre-culturing, the cell density in 10 ml of the living stock culture in the exponential growth
8
phase shall be determined and adjusted to 10 algal cells per millilitre. If cell density at that stage is
8
much lower than 10 algal cells per millilitre, the culture is not at the exponential growth phase. In that
8
case, repeat the 3-day pre-culturing step until cell density exceeds 10 cells per millilitre. The living
stock culture shall be incubated under the same conditions as those for the test (8.4). Determine the
cell density of the living stock culture immediately before use, to calculate the required culture volume.
If the stock culture is in the stationary phase, at least two or three pre-culturing rounds are necessary
to obtain culture in the exponential phase of growth. When older stock cultures are used (more than 3
weeks old), more time may be required for recovery.
For the preparation of the cryopreserved stock culture, see Annex D.
All steps should be performed on a clean bench (6.6).
8 Test procedure on-board
8.1 Preparation of the algal inoculum culture
For reproducible results, the algal inoculum culture shall be in the exponential growth phase, with
the biomass increasing ca. 16-fold over 72 h. First, immediately before use, determine cell density of
the living stock culture using a luminometer, and add a sufficient amount of cells from the living stock
6
culture to the growth medium (7.1) for a cell density of 10 algal cells per millilitre. A three-day pre-
8
culture is necessary to obtain an algal inoculum culture with a cell density of over 10 algal cells per
millilitre (i.e. sufficient density for starting the test) in the exponential phase of growth. If cell density
8
after pre-culturing is much lower than 10 algal cells per millilitre, the cells are not in the exponential
8
growth phase. In that case, repeat the 3-day pre-culturing step until cell concentration exceeds 10
cells per millilitre.
8.2 Choice of the test concentrations
Two cyanobacterium cultures (Cyanobium sp. NIES-981) in triplicate shall be established in the control
tubes (growth medium with no test seawater) and tubes containing diluted test seawater [test seawater
mixed with the growth medium at a volume fraction of 80:20]. As an option, additional dilutions of the
test seawater in a geometric series may be tested to determine ECx based on the regression analysis.
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ISO 23734:2021(E)

8.3 Preparation of the test medium
First, test seawater shall be collected at an appropriate site (e.g. surface water, mining wastewater,
etc.). A filtrate shall be obtained using an appropriate filtering device and a filter with a pore size
of < 0,22 μm. The stock solutions of nutrients, metals and tris shall be added to the filtrate so that their
concentration is the same as that in ASW-SN. The mixture shall then be diluted to 80 % volume fraction
with the growth medium. As the test medium, aliquots of the diluted filtrate shall be dispensed into
three test tubes of appropriate dimensions and optical properties for DF measurements (see Annex B).
Additional aliquots of the growth medium shall be dispensed into three test tube
...