ISO/PRF 23734

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

Titre manque

General Information

• Status: Published
• 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

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 23734
First edition
Marine technology — Marine
environment impact assessment
(MEIA) — On-board bioassay to
monitor seawater quality using
delayed fluorescence of microalga
PROOF/ÉPREUVE
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

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

organisations, 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 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

[1],[3],[19],[21]

to deep-sea environments and ecosystems . One concern is the toxicity of heavy metals

released from excavated minerals. Such heavy metals can be released into the seawater of the deep

[7],[18]

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 contamination of the surface

[8]
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

[2]

ecological risks of chemical contaminations . 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
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 (3.4) 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

[2]
seawater in a geometric series .
[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

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 (6.1) 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.

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 6.1). Add a sufficient amount of cells from the algal stock culture to 100 ml of

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

phase shall be determined and adjusted to 10 algal cells per millilitre. If cell density at that stage is

much lower than 10 algal cells per millilitre, the culture is not at the exponential growth phase. In that

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

culture to the growth medium (7.1) for a cell density of 10 algal cells per millilitre. A three-day pre-

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

after pre-culturing is much lower than 10 algal cells per millilitre, the cells are not in the exponential

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 measur
...