ISO 23734:2021

Marine technology - Marine environment impact assessment (MEIA) - On-board bioassay to monitor seawater quality using delayed fluorescence of microalga

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.

Titre manque

General Information

• Status: Published
• Publication Date: 26-Jul-2021
• ICS: 13.020.30 - Environmental impact assessment
• ICS: 47.020.01 - General standards related to shipbuilding and marine structures
• Technical Committee: ISO/TC 8/SC 13 - Marine technology
• Drafting Committee: ISO/TC 8/SC 13/WG 4 - Marine environment impact assessment
• Current Stage: 6060 - International Standard published
• Start Date: 27-Jul-2021
• Due Date: 21-Jan-2022
• Completion Date: 27-Jul-2021

Overview

ISO 23734:2021 - Marine technology - Marine environment impact assessment (MEIA) - specifies an on‑board bioassay to monitor seawater quality by measuring the delayed fluorescence (DF) of the marine cyanobacterium Cyanobium sp. (NIES‑981). The method detects the presence of unknown toxic contaminants through inhibition of photosynthetic activity. It is designed for rapid, space‑efficient use on vessels or platforms (e.g., deep‑sea mining sites) where conventional laboratory resources are limited.

Key topics and technical requirements

• Test principle: Toxicants inhibit photosynthetic electron transport; inhibition is quantified via DF intensity and DF decay curves after transferring cells from light to darkness (DF signals occur over milliseconds to minutes).
• Test organism: Cyanobium sp. (NIES‑981) supplied as an algal stock culture (living or cryopreserved).
• Test medium: Growth medium (ASW‑SN) mixed with test seawater; a typical dilution used is test seawater : growth medium = 80 : 20 (v/v).
• Procedure highlights:
  • Prepare algal inoculum from land‑prepared stock cultures.
  • Set up control and test tubes (duplicate cultures in triplicate recommended).
  • Incubate for 24 h on‑board, then measure DF.
  • Compare test vs control using statistical tests; optionally determine ECx, NOEC, LOEC via serial dilutions.
• Apparatus & reagents: high‑sensitivity luminometer for DF, incubator/tube shaker, culture tubes, clean bench, reagents for growth medium (nutrients, trace metals, tris), and instruments for cell density measurement.
• Data interpretation: Plot DF decay curves, calculate percent inhibition, and assess significance to indicate contamination.
• Annexes: include schematic procedures, medium preparation, cryopreservation guidance, and reference check data.

Practical applications

• Rapid on‑site screening for seawater contamination at deep‑sea mining operations, discharge points, and other marine works.
• Early warning tool to support environmental safety actions, minimize spread of pollutants, and reduce operational downtime.
• Useful where onboard space, time, and specialist analytical instrumentation (e.g., ICP‑MS) are not available.
• Detects a broad spectrum of toxicants (including unknown contaminants), though it does not identify specific chemical species.

Who should use this standard

• Marine environmental scientists and technicians conducting shipboard monitoring
• Deep‑sea mining operators and environmental managers
• Regulatory bodies, environmental consultants, and research institutions performing MEIA
• Organizations needing rapid, on‑site toxicity screening for seawater quality management

Related standards and guidance

• ISO 10253 (general algal bioassay standards) - for extended laboratory protocols and comparisons
• International Seabed Authority (ISA) guidance on environmental impact assessment for seabed activities

Keywords: ISO 23734:2021, on‑board bioassay, delayed fluorescence, Cyanobium sp., seawater quality, marine environment impact assessment, deep‑sea mining, DF luminometer.

Standards Content (Sample)

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 2021
© 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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ii © ISO 2021 – All rights reserved

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
Foreword
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electrotechnical standardization.
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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.
iv © ISO 2021 – All rights reserved

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.
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
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
2 © ISO 2021 – All rights reserved

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
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
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 r
...