Water quality - Guidelines for quantitative sampling and sample processing of marine soft-bottom macrofauna (ISO 16665:2014)

EN ISO 16665 provides guidelines on the quantitative collection and processing of subtidal soft-bottom macrofaunal samples in marine waters. This International Standard encompasses: a) development of the sampling programme; b) requirements for sampling equipment; c) sampling and sample treatment in the field; d) sorting and species identification; e) storage of collected and processed material.

Wasserbeschaffenheit - Anleitung für die quantitative Probenahme und Probenbearbeitung mariner Weichboden-Makrofauna (ISO 16665:2014)

Diese Internationale Norm enthält Anleitungen zur quantitativen Sammlung und Bearbeitung von Proben der sublitoralen Weichboden-Makrofauna in marinen Gewässern.
Diese Internationale Norm umfasst:
a) Entwicklung des Probenahmeprogrammes;
b) Anforderungen an die Probenahmegeräte;
c) Probenahme und Probenbehandlung vor Ort;
d) Sortierverfahren und Artenbestimmung;
e) Aufbewahrung von gesammeltem und bearbeitetem Material.
Diese Internationale Norm geht auf folgende Aspekte nicht speziell ein, wenn auch einige Elemente im Rahmen von Untersuchungen auftreten könnten:
- Entnahme von Teilproben für Biotests;
- Probenahme in Tiefenwasser (> 750 m) oder küstenfernen Bereichen;
- faunistische In-situ-Untersuchungen, z. B. Wiederbesiedlungsuntersuchungen;
- nicht benthische Organismen, die mit dem Probenahmegerät gefangen werden;
- Probenahme in Ästuaren;
- Probenahme in Tidebereichen;
- Probenahme und Analyse der Meiofauna [9];
- Probenahme mit Dredgen und Schlitten;
- Probenahme mit Hilfe von Tauchgeräten (SCUBA-Tauchen);
- Konzept für eine statistische Auswertung.
Die Genauigkeit bei der Festlegung der Position ist durch das geographische Gebiet, die verwendete Ausrüstung und das Ziel der Untersuchung vorgegeben.

Qualité de l'eau - Lignes directrices pour l'échantillonnage quantitatif et le traitement d'échantillons de la macrofaune marine des fonds meubles (ISO 16665:2014)

L'ISO 16665:2014 fournit des lignes directrices sur le prélèvement quantitatif et le traitement d'échantillons de la macrofaune des fonds meubles subtidaux en eaux marines.
L'ISO 16665:2014 englobe: a) le développement du programme d'échantillonnage; b) les exigences relatives à l'équipement nécessaire pour l'échantillonnage; c) l'échantillonnage et le traitement des échantillons sur le terrain; d) le tri et l'identification des espèces; e) le stockage des matériels collectés et traités.
L'ISO 16665:2014 ne traite pas spécifiquement de ce qui suit, bien qu'elle puisse s'appliquer à certains de ces éléments: le sous-échantillonnage pour les essais biologiques; l'échantillonnage en eaux profondes (>750 m) ou au large; les études faunistiques in situ, par exemple les essais de recolonisation; les organismes non benthiques capturés par le dispositif d'échantillonnage; l'échantillonnage en milieu estuarien; l'échantillonnage en zone intertidale; l'échantillonnage et l'analyse de la méiofaune; l'échantillonnage au moyen de dragues et de traîneaux; l'échantillonnage au moyen de scaphandres autonomes; la conception statistique.
L'exactitude de la détermination de la position dépend de la zone géographique, de l'équipement utilisé et de l'objectif de l'étude.

Kakovost vode - Smernice za kvantitativno vzorčenje in obdelavo vzorcev morske makrofavne sedimentnega dna (ISO 16665:2014)

General Information

Status
Published
Public Enquiry End Date
19-Mar-2012
Publication Date
23-Feb-2014
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
31-Jan-2014
Due Date
07-Apr-2014
Completion Date
24-Feb-2014

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SLOVENSKI STANDARD
SIST EN ISO 16665:2014
01-marec-2014
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SIST EN ISO 16665:2005
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Water quality - Guidelines for quantitative sampling and sample processing of marine

soft-bottom macrofauna (ISO 16665:2014)
Wasserbeschaffenheit - Anleitung für die quantitative Probenahme und
Probenbearbeitung mariner Weichboden-Makrofauna (ISO 16665:2014)

Qualité de l'eau - Lignes directrices pour l'échantillonnage quantitatif et le traitement

d'échantillons de la macrofaune marine des fonds meubles (ISO 16665:2014)
Ta slovenski standard je istoveten z: EN ISO 16665:2013
ICS:
13.060.10 Voda iz naravnih virov Water of natural resources
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 16665:2014 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16665:2014
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SIST EN ISO 16665:2014
EUROPEAN STANDARD
EN ISO 16665
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2013
ICS 13.060.70; 13.060.10 Supersedes EN ISO 16665:2005
English Version
Water quality - Guidelines for quantitative sampling and sample
processing of marine soft-bottom macrofauna (ISO 16665:2014)

Qualité de l'eau - Lignes directrices pour l'échantillonnage Wasserbeschaffenheit - Anleitung für die quantitative

quantitatif et le traitement d'échantillons de la macrofaune Probenahme und Probenbearbeitung mariner Weichboden-

marine des fonds meubles (ISO 16665:2014) Makrofauna (ISO 16665:2014)
This European Standard was approved by CEN on 6 December 2013.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European

Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national

standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation

under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same

status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16665:2013 E

worldwide for CEN national Members.
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SIST EN ISO 16665:2014
EN ISO 16665:2013 (E)
Contents Page

Foreword ..............................................................................................................................................................3

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SIST EN ISO 16665:2014
EN ISO 16665:2013 (E)
Foreword

This document (EN ISO 16665:2013) has been prepared by Technical Committee ISO/TC 147 “Water quality”

in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by

DIN.

This European Standard shall be given the status of a national standard, either by publication of an identical

text or by endorsement, at the latest by June 2014, and conflicting national standards shall be withdrawn at

the latest by June 2014.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN ISO 16665:2005.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following

countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech

Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,

Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,

Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.

Endorsement notice

The text of ISO 16665:2014 has been approved by CEN as EN ISO 16665:2013 without any modification.

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SIST EN ISO 16665:2014
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SIST EN ISO 16665:2014
INTERNATIONAL ISO
STANDARD 16665
Second edition
2014-01-15
Water quality — Guidelines for
quantitative sampling and sample
processing of marine soft-bottom
macrofauna
Qualité de l’eau — Lignes directrices pour l’échantillonnage
quantitatif et le traitement d’échantillons de la macrofaune marine
des fonds meubles
Reference number
ISO 16665:2014(E)
ISO 2014
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SIST EN ISO 16665:2014
ISO 16665:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2014

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved
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SIST EN ISO 16665:2014
ISO 16665:2014(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Terms and definitions ..................................................................................................................................................................................... 1

2.1 Ecological or biological concepts ............................................................................................................................................ 2

2.2 Surveys and samples .......................................................................................................................................................................... 2

3 Strategies and objectives for soft-bottom faunal surveys .......................................................................................... 3

3.1 Sampling programme and plan ................................................................................................................................................ 3

3.2 Positioning of sampling stations.............................................................................................................................................. 3

3.3 Reference stations ................................................................................................................................................................................ 5

3.4 Types of survey ....................................................................................................................................................................................... 5

3.5 Change in sampling programme and intercalibration .......................................................................................... 8

4 Sampling ........................................................................................................................................................................................................................ 8

4.1 Documentation and field log ....................................................................................................................................................... 8

4.2 Sampling and sample processing in the field ............................................................................................................10

4.3 Sample fixation ....................................................................................................................................................................................13

4.4 Background environmental descriptors ........................................................................................................................14

5 Sample processing in the laboratory ............................................................................................................................................17

5.1 Sorting .........................................................................................................................................................................................................17

5.2 Sample residue .....................................................................................................................................................................................18

6 Taxon determination and quantification .................................................................................................................................18

6.1 Level of identification and taxon lists ......... ......................................................................................................................18

6.2 Quantification .......................................................................................................................................................................................19

6.3 Reference collection ........................................................................................................................................................................19

6.4 Biomass ......................................................................................................................................................................................................20

6.5 Data reporting ......................................................................................................................................................................................20

6.6 Storage and archiving .....................................................................................................................................................................20

7 Quality assurance and quality control ........................................................................................................................................21

7.1 General ........................................................................................................................................................................................................21

7.2 Auditing ......................................................................................................................................................................................................21

7.3 Equipment calibration and operating safety..............................................................................................................21

7.4 Training ......................................................................................................................................................................................................22

7.5 Checklists, sample log and anomaly reporting .........................................................................................................22

7.6 Quality of sample sorting ............................................................................................................................................................22

7.7 Quality of taxon identification ................................................................................................................................................22

7.8 Identification literature ................................................................................................................................................................23

7.9 Reference and collection..............................................................................................................................................................23

Annex A (informative) Sampling devices ......................................................................................................................................................24

Annex B (informative) Processing of samples with live specimens ..................................................................................29

Annex C (informative) Biomass measurements .....................................................................................................................................30

Annex D (informative) Processing particularly large samples ..............................................................................................32

Bibliography .............................................................................................................................................................................................................................33

© ISO 2014 – All rights reserved iii
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SIST EN ISO 16665:2014
ISO 16665:2014(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

organizations, 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, 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, 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 on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers

to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological

methods.

This second edition cancels and replaces the first edition (ISO 16665:2005), which has been technically

revised.
iv © ISO 2014 – All rights reserved
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SIST EN ISO 16665:2014
ISO 16665:2014(E)
Introduction

Analysis of macrofaunal communities in soft-bottom sediments is an integral part of marine

environmental assessment. The faunal composition, in terms of both the species present and their

relative abundance, reflects integrated environmental conditions in the survey area over a period of

time. The composition and structure of soft-bottom macrofaunal communities therefore can be used to

characterize environmental conditions and estimate the extent of environmental impact.

Characterization of environmental conditions is usually based on quantitative methods, in this case by

relating the numbers of species and individuals captured to a known area of sea floor. For accurate data

interpretation, it is essential to add information on the geophysicochemical characteristics or properties

of the water masses and bottom sediments, including nutrients, oxygenation, and redox state where

appropriate.

For effective data utilization and quality assurance (QA) of the work carried out, it is beneficial and may

be essential (depending on the individual survey aims) that surveys be intercomparable temporally,

spatially, and between operators. This International Standard contributes to ongoing work on QA of

data from soft-bottom macrofaunal surveys. These guidelines primarily aim to assist in standardizing

monitoring surveys carried out for commercial purposes or in connection with the EU Water

Framework Directive. For this reason, detailed specifications are given in areas of consequence for data

intercompatibility.

Where appropriate, cost–benefit issues have been taken into consideration, and accepted minimal

requirements for general environmental impact assessment have been given. The cited minimum

requirements for accuracy are not intended to satisfy research needs or to provide a full ecological

understanding of the sampling area. Designers of programmes for research or other studies requiring a

detailed knowledge of soft-bottom macrofauna should consult the guidelines given in Reference [13] for

decisions on survey design and sampling frequency.

This International Standard applies to all areas of the sea floor where it is possible to collect faunal

samples by a grab or coring device. For practical reasons, this applies to animals retained on a mesh

screen of 0,5 mm or 1 mm aperture size.

The sensitivity of the method, here defined as detection of faunal disturbance, change in taxon

composition or faunal mapping, is dependent on the survey design, the type of environmental influences

present in the area and on the level of competence or standardization of the personnel.

© ISO 2014 – All rights reserved v
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SIST EN ISO 16665:2014
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SIST EN ISO 16665:2014
INTERNATIONAL STANDARD ISO 16665:2014(E)
Water quality — Guidelines for quantitative sampling and
sample processing of marine soft-bottom macrofauna

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the employer or user to establish appropriate safety and health

practices and to ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
1 Scope

This International Standard provides guidelines on the quantitative collection and processing of subtidal

soft-bottom macrofaunal samples in marine waters.
This International Standard encompasses:
a) development of the sampling programme;
b) requirements for sampling equipment;
c) sampling and sample treatment in the field;
d) sorting and species identification;
e) storage of collected and processed material.

This International Standard does not specifically address the following, although some elements may

be applicable:
— bioassay sub-sampling;
— deep water (>750 m) or offshore sampling;
— in situ faunal studies, e.g. recolonization assays;
— non-benthic organisms caught in the sampling device;
— estuarine sampling;
— intertidal sampling;
— meiofaunal sampling and analysis (see Reference [9]);
— sampling by dredge and sledge;
— self-contained underwater breathing apparatus (SCUBA) sampling;
— statistical design.

Accuracy of position fixing is determined by the geographical area, equipment used and survey objective.

2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2014 – All rights reserved 1
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SIST EN ISO 16665:2014
ISO 16665:2014(E)
2.1 Ecological or biological concepts
2.1.1
benthic
dwelling at the bottom of an aquatic environment
2.1.2
benthic macrofauna

bottom-dwelling animals retained on a mesh screen of 0,5 mm or 1 mm aperture size

2.1.3
receiving water body
recipient
recipient water body

water body which receives an input of material of either natural or anthropogenic origin

Note 1 to entry: The term often appears in the context of organic enrichment by, for example, effluent from

municipal waste water outlets or industrial processed water. The macrofaunal part of receiving water body

surveys describe the state of organic enrichment in a given area.

[SOURCE: ISO 5667-19:2004, definition 3.4, modified ― in the Note to entry, “contamination” has been

replaced by “organic enrichment”, and it is further specified that the terms apply to the macrofaunal

part of receiving water body surveys]
2.1.4
soft bottom

areas of sea floor consisting of loose deposited particles including clay, mud, sand and gravel, shells and

maerl, also including mixed substrata with gravels, small stones and pebbles scattered on a bed of finer

material, but excluding cobbles
2.1.5
soft-bottom fauna
animals living on, or completely or partially buried in, soft-bottom sediments
2.1.6
sublittoral

portion of the shore which is either totally immersed or only uncovered by the receding tide infrequently

and then for very short period (i.e. below the littoral zone)
2.2 Surveys and samples
2.2.1
baseline survey
environmental impact assessment

survey with emphasis on characterization and description of biotic and abiotic conditions in the survey

area, which provides the basis for future monitoring and/or follow-up surveys

[SOURCE: ISO 5667-19:2004, definition 3.2, modified — “classification” changed to “characterization”;

“biotic and abiotic” added]
2.2.2
reference station

one or more sampling stations chosen to represent environmental conditions in a given area, i.e. free

from direct anthropogenic influences
2 © ISO 2014 – All rights reserved
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SIST EN ISO 16665:2014
ISO 16665:2014(E)
2.2.3
reference collection
collection of identified specimens, used for reference purposes

Note 1 to entry: Institute reference collections are usually verified by an appropriate and approved taxonomic

specialist. In addition, individual identifiers may keep a personal collection and/or some surveys require contract-

or area-specific reference collections.
2.2.4
replicate samples

series of samples taken in the same time frame, at the same sampling station, in the same manner for

statistical validity and comparison

[SOURCE: ISO 5667-19:2004, definition 3.6, modified ― “simultaneously” has been changed to “in the

same time frame”; “for statistical validity and comparison” added]

Note 1 to entry: Replicate samples can include sets of sub-samples taken from a larger sample.

2.2.5
sampling station
precise location where samples are collected

Note 1 to entry: A sampling station is defined by its geographical position (latitude, longitude), its depth (relative

to chart datum and normalized to mean low water as given in tide tables) and any other invariant or physical

conditions. The station is delineated using the given level of precision. In cases of doubt, when revisiting sampling

stations, emphasis should be placed on landmarks and water depth.
2.2.6
sub-sample

ideally representative portion removed from a sample, taken for separate analysis

[SOURCE: ISO 5667-19:2004, definition 3.7, modified ― “ideally” and “taken for separate analysis”

added]
3 Strategies and objectives for soft-bottom faunal surveys
3.1 Sampling programme and plan

The design of the sampling programme depends on the detailed aims of the survey and the required

power of the data. The programme should be developed with regard to local topographical and

hydrographic conditions in the survey area, information on local contamination sources, and knowledge

from previous surveys, if any. The number of sampling stations, their positions and numbers of replicate

samples to be taken at each sampling station should be established prior to the initiation of the survey.

The design of the programme has a strong influence on the options for data treatment and statistical

analyses. Prior considerations about data treatment and reporting should therefore be made. Quality

assurance (QA) procedures should be incorporated at this stage.

For guidance and considerations of sampling and statistical design, see Reference [13].

3.2 Positioning of sampling stations
3.2.1 General

Sampling stations should be located to satisfy predefined requirements, bearing in mind the objectives

of the study and the likely scale of natural variability in the biota.

Sampling stations should, for monitoring purposes (except for biodiversity studies — see the following),

preferably be positioned in areas of homogenous sandy or muddy bottom sediments. Certain bottom

types where it is difficult to obtain good quality samples, e.g. in sediments containing large amounts of

stones, hard gravel, twigs and similar objects, should be avoided. However, it may be possible for a diver

© ISO 2014 – All rights reserved 3
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SIST EN ISO 16665:2014
ISO 16665:2014(E)

to sample pockets of sediment in such areas. Alternatively, supplementary semiquantitative techniques

may be used, e.g. underwater photography, video, remotely operated vehicle (ROV) or benthic dredging.

[6]
Consult EN 16260 for guidance on visual seabed surveys.

In special cases, where habitats within the sampling area vary strongly, different sampling techniques

may be combined, but generally the same gear should be used for all sampling in one survey.

For biodiversity studies, various bottom types should be included, as appropriate to the aims of the

programme.

Sampling stations can be positioned according to one, or combinations of, the following strategies:

— station network, see 3.2.2;
— randomly, see 3.2.3;
— stratified, see 3.2.4;
— transect, see 3.2.5;
— single-spot sampling, see 3.2.6.
3.2.2 Station network

Sampling stations are arranged in a regular grid-like pattern. This arrangement is appropriate for

overview surveys and for mapping of distribution of factors of interest, e.g. zone of influence around point

source discharges. The survey area should be one of topographic homogeneity, but some adjustments

can be made according to local conditions, e.g. in fjords and coastal waters with smaller variations in

depth.
3.2.3 Random or scattered sampling

In special circumstances, sampling stations may be positioned randomly or scattered. An example of

this is when no previous knowledge of the area is available as a guide to appropriate stratification, or

when an unbiased value for a whole area is desired.
3.2.4 Stratified sampling

Sampling stations are arranged within locally homogeneous subdivisions of the survey area. The

subdivisions (strata) may be delineated according to depth, sediment types or other factors that vary

across the survey area. Stratification is appropriate in cases where habitat variability can confound

patterns of interest. Within-strata stations may be placed in a network, e.g. for zone-of-influence

mapping, or randomized for description of “average“ characteristics of the strata. Echo-sounders or

appropriate ground discrimination tools should be used.
3.2.5 Transect sampling

Sampling stations are arranged along linear transects. One approach is to place stations along a known

or anticipated gradient of a factor of interest in a sub-area of minimum habitat variability. Such sampling

is, for example, applicable to trace effects of point-source discharges by establishing the transect in the

main current direction from the source. Another rather different approach is to place stations across

possible habitat gradients when it is not feasible or appropriate to work in strata.

3.2.6 Single-spot (station) sampling

This applies when a small number of stations are placed according to individual assessment. In fjordic or

sill-influenced systems, where eutrophication or chemical contamination is suspected or investigated,

sampling stations may be positioned in the deepest parts of the survey area (depressions, basins), where

the earliest signs of disturbance are oft
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