ISO 23040:2021

INTERNATIONAL ISO
STANDARD 23040
First edition
2021-12
Marine environment impact
assessment (MEIA) — Specification for
marine sediments in seabed areas —
Survey of interstitial biota
Évaluation de l'impact environnemental marin — Spécifications
relatives aux sédiments marins dans les zones de fonds marins —
Étude du biote interstitiel
Reference number
© ISO 2021
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Contents Page
Foreword .viii
Introduction .ix
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General . 2
4.1 Technical design . 2
4.2 Basic recommendations for the surveys . 2
4.2.1 Survey object . 2
4.2.2 Auxiliary parameters . 3
4.2.3 Recommendations for the sampling equipment . 3
4.2.4 Auxiliary equipment on board ship . 3
4.2.5 Sampling method and scope of application . 3
4.3 Sampling . 3
4.3.1 Sediment sampling . 3
4.3.2 Trawl sampling. 3
4.3.3 Water sampling . 4
4.3.4 Records . 4
4.4 Sample analysis . . 4
4.4.1 Sample treatment . 4
4.4.2 Sample measurement . 4
4.4.3 Sample treatment and storage . 4
4.4.4 Sample identification and enumeration . 4
4.4.5 Sample data analysis . 4
4.5 Basic recommendations of data organization . 5
4.5.1 Organization of data . . 5
4.5.2 Data archiving, acceptance and achievements appraisal . 5
4.6 Survey results . 5
4.6.1 Original records . 5
4.6.2 Maps or drawings . 5
4.6.3 Investigation report . 5
4.7 Data archiving . 6
4.8 Program and quality control . 6
5 Survey of the sediment . 6
5.1 Principle . 6
5.2 General provisions . 7
5.3 Collection and preservation of the samples . 7
5.4 Measurement of environmental factors . 7
5.5 Measurement of the age of the sediments . 7
5.6 Measurement of the contents of heavy metals, organic pollutants and oils in the
sediments . 7
5.7 Measurement of the grain size of the sediments . 7
5.8 Analysis of the mineral compositions in the sediments . 8
5.9 Classification of the substrate type in offshore sediments . 8
5.10 Classification of the deep-sea sediments . 8
5.11 Survey of the biological components in the sediments . 8
5.11.1 Summary of the methods. 8
5.11.2 Technical recommendations . 8
5.11.3 Collection and preservation of the samples . 9
5.12 Organization of data . 9
6 Survey of foraminifera .9
6.1 Principle . 9
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6.2 General provisions . 9
6.3 Collection and preservation of the samples . 9
6.3.1 Sampling and sample processing . 9
6.3.2 Collection and preservation of the sedimentary tests . 10
6.3.3 Collection and preservation of living foraminifera . 10
6.4 Tools and reagents . 10
6.5 Processing and analysis of the samples . 10
6.5.1 Numbering and weighing the crystallizing dish or beaker . 10
6.5.2 Drying and weighing the sediment sample . 11
6.5.3 Sample soaking . 11
6.5.4 Washing and drying . 11
6.5.5 Suspension and concentration . 11
6.5.6 Bottling and sealing . 11
6.5.7 Specimen preparation and analysis . 11
7 Survey of ostracoda .12
7.1 Principle . 12
7.2 General provisions .12
7.3 Collection and preservation of the samples .12
7.3.1 Sampling and sample processing .12
7.3.2 Collection and preservation of dead ostracod tests .13
7.3.3 Collection and preservation of living ostracods .13
7.4 Tools and reagents .13
7.5 Processing and analysis of the samples . 13
7.5.1 Numbering and weighing the crystallizing dish or beaker .13
7.5.2 Drying and weighing. 13
7.5.3 Sample soaking . 14
7.5.4 Washing and drying . 14
7.5.5 Suspension and concentration . 14
7.5.6 Bottling and sealing . 14
7.5.7 Microscopic examination and enumeration . 14
8 Survey of radiolaria .15
8.1 Principle . 15
8.2 General provisions . 15
8.3 Collection and preservation of the samples . 15
8.3.1 Sample processing . 15
8.3.2 Collection and conservation of deposited empty shells . 16
8.4 Tools and reagents . 16
8.5 Processing and analysis of the samples . 16
8.5.1 Sample pretreatment. 16
8.5.2 Preparation of microscope slide specimens . 16
9 Survey of sedimentary diatom .17
9.1 Principle . 17
9.2 General provisions . 17
9.3 Collection and preservation of the samples . 17
9.3.1 Sampling and treatment . 17
9.3.2 Collection and preservation of sedimentary remains . 18
9.4 Tools and reagents . 18
9.5 Processing and analysis of the samples . 18
9.5.1 Sample pretreatment. 18
9.5.2 Preparation of microscope slide specimens . 19
9.6 Organization of data . 19
10 Survey of coccoliths .19
10.1 Principle . 19
10.2 General provisions . 19
10.3 Collection and preservation of the samples . 20
10.4 Tools and reagents .20
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10.5 Processing and analysis of the samples . 20
10.5.1 Pre-preparation of the equipment and the samples . 20
10.5.2 Method of preparing simple smear slides . 21
10.5.3 Pre-concentration of coccolith samples . 21
10.6 Organization of data . 21
11 Survey of sporopollen .22
11.1 Principle . 22
11.2 General provisions . 22
11.3 Collection and preservation of the samples . 23
11.4 Tools and reagents .23
11.5 Processing and analysis of the samples . 23
11.5.1 Sample disaggregation . 23
11.5.2 Alkali-soluble digestion . . 24
11.5.3 Sieving . . 24
11.5.4 Carbonate digestion . 24
11.5.5 Silicate digestion . 24
11.5.6 Ultrasonic cleaning and sieving . 24
11.5.7 Acetolysis . 25
11.5.8 Storage of processed sample. 25
11.5.9 Mounting sporopollen specimens . 25
11.5.10 .
Glycerine jelly preparation . 25
11.5.11 .
Precautions . 25
11.6 Organization of data . 26
12 Survey of benthic viruses .26
12.1 Principle . 26
12.2 General provisions . 26
12.3 Collection and preservation of the samples . 26
12.3.1 Samples for epifluorescence microscopy . 26
12.3.2 Samples for flow cytometry . 27
12.3.3 Samples for environmental measurements and molecular diversity of
viruses . 27
12.4 Tools and reagents . 27
12.4.1 Equipment and reagents for epifluorescence microscopy . 27
12.4.2 Equipment and reagents for flow cytometry .28
12.4.3 Equipment and tools for molecular diversity of viruses .28
12.5 Processing and analysis of samples .28
12.5.1 Enumeration of viruses by epifluorescence microscopy .28
12.5.2 Procedure for flow cytometry .30
12.5.3 Estimation of the molecular diversity of viruses .30
13 Survey of benthic microbes .32
13.1 Principle . 32
13.2 General provisions . 32
13.3 Collection and preservation of the samples . 33
13.3.1 Sampling stations and sample collection . 33
13.3.2 On-site sample process . 33
13.3.3 Sample storage . 33
13.4 Tools and reagents .34
13.4.1 Equipment .34
13.4.2 Reagents .34
13.5 Processing and analysis of the samples . 36
13.5.1 Assessment of molecular diversities of benthic microbes .36
13.5.2 Benthic microbial abundance . 41
14 Survey of benthic microalgae . 44
14.1 Principle .44
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14.2 General provisions .44
14.3 Collection and preservation of the samples . 45
14.3.1 Sampling design . 45
14.3.2 Sampling methods. 45
14.3.3 Sample preservation and fixation .46
14.4 Tools and reagents .46
14.5 Processing and analysis of the samples .46
14.5.1 Sample processing .46
14.5.2 Sample analysis .48
14.5.3 Abundance and biomass calculation .48
14.5.4 Cyst culture and identification .50
14.5.5 Fluorescence determination of demagnesium chlorophyll and chlorophyll a .50
14.5.6 Algal toxins determination . 51
15 Survey of benthic protozoa .51
15.1 Principle . 51
15.2 General provisions . 51
15.3 Collection and preservation of the samples . 52
15.3.1 Sampling equipment . 52
15.3.2 Sediment core collection . 52
15.3.3 Sediment layers . 52
15.3.4 Sample fixation and preservation . 52
15.3.5 Collection and treatment of living protozoa . 53
15.4 Tools and reagents .54
15.4.1 Equipment and reagents for silica sol centrifugation .54
15.4.2 Equipment and reagents of quantitative protargol staining .54
15.4.3 Equipment and reagents for molecular analysis .54
15.5 Processing and analysis of the samples . 55
15.5.1 Silica sol density centrifugation . 55
15.5.2 Quantitative protargol stain (QPS) .56
15.5.3 Estimation of molecular diversity . 59
16 Survey of metazoan meiobenthos .64
16.1 Principle .64
16.2 General provisions .65
16.3 Collection and preservation of the samples .65
16.3.1 Sampling in intertidal zones and shallow waters .65
16.3.2 Deep-sea sampling .65
16.3.3 Sample slicing .66
16.3.4 Sample fixation and preservation . 67
16.3.5 Labelling . 67
16.3.6 Record . 67
16.4 Tools and reagents . 67
16.5 Processing and analysis of the samples . 67
16.5.1 Quantification and taxa analysis . 67
16.5.2 Molecular analysis of biodiversity.68
16.6 Organization of data . 69
16.6.1 Precision . 69
16.6.2 Abundance .69
16.6.3 Biomass . 70
16.6.4 Complete the report . 71
16.6.5 Plotting data . 71
Annex A (informative) Photographs of several groups of communities of interstitial biota .72
Annex B (informative) Several stratified sampling devices for the survey of interstitial biota .74
Annex C (informative) Tables for sample labelling and sampling record — Examples .76
Annex D (informative) Tables of microscopy records — Examples .78
Annex E (informative) Calculation of community parameters.83
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Annex F (informative) Record sheet for sample treatment — Examples .86
Annex G (informative) Characteristics for the classification of coccoliths .87
Annex H (informative) Flow diagram for the enumeration of viruses by
epifluorescence microscopy .88
Annex I (informative) Filter unit used in microbenthos survey .89
Annex J (informative) Technical flowcharts for the investigation of microbenthos.90
Annex K (informative) Solutions and regents .93
Bibliography .99
vii
Foreword
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Introduction
Interstitial biota in marine sediments refers to the benthic life forms inhabited or deposited in the
interstitial spaces between sediment particles, including marine microorganisms, benthic virus,
microbenthos and meiobenthos. They cover the six “kingdoms” of life in the three-domain taxonomic
system: Archaea, Bacteria, Fungi, Protista, Plantae and Animalia. Interstitial biota in marine sediments
are so small that cannot be obtained and analysed by conventional methods for marine biological survey;
they are numerous and complex; they have diverse functions, remarkable ecological significances and
rich gene resources; they are ubiquitous and make up the basic components of the life system in marine
sediments. Sediment interstitial biotas are the most abundant and complex life groups in the estuaries,
intertidal zones, shelf shallow seas and deep sea. They play key roles in the regulation of material and
energy flows in benthic ecosystems.
In seabed areas, a number of large international research programs have been carried out, such as
the ocean drilling program (ODP) and the international ocean discovery program (IODP). Interstitial
biota in marine sediments surveys have been key to solve scientific problems in relevant fields, such
as marine biodiversity, oil and gas resource exploration, marine carbon cycle, global change, monsoon
rainfall, ice melting, ocean acidification and deep-sea bi
...