ISO/FDIS 23611-6

Soil quality — Sampling of soil invertebrates — Part 6: Design of sampling programmes with soil invertebrates

This part of ISO 23611 provides guidance for the design of field studies with soil invertebrates (e.g. for the monitoring of the quality of a soil as a habitat for organisms). Detailed information on the sampling of the most important soil organisms is provided in the other parts of this International Standard (ISO 23611-1 to ISO 23611-5). This part of ISO 23611 is used for all terrestrial biotopes in which soil invertebrates occur. Basic information on the design of field studies in general is already laid down in ISO 10381-1. This information can vary according to the national requirements or the climatic/regional conditions of the site to be sampled. NOTE While this part of ISO 23611 aims to be applicable globally for all terrestrial sites that are inhabited by soil invertebrates, the existing information refers mostly to temperate regions. However, the (few) studies from other (tropical and boreal) regions, as well as theoretical considerations, allow the conclusion that the principles laid down in this part of ISO 23611 are generally valid, References [4], [6], [40], [21]. This part of ISO 23611 gives information on site-specific risk assessment of contaminated land, study of potential side effects of anthropogenic impacts (e.g. the application of chemicals or the building of roads), the biological classification and assessment of soils in order to determine the biological quality of soils, and longterm biogeographical monitoring in the context of nature protection or restoration, including global change (e.g. as in long-term ecological research projects).

Qualité du sol — Prélèvement des invertébrés du sol — Partie 6: Conception de programmes d'échantillonnage des invertébrés du sol

La présente partie de l'ISO 23611 spécifie les lignes directrices pour la conception de programmes d'échantillonnage des invertébrés du sol sur le terrain (par exemple la surveillance de la qualité d'un sol comme habitat pour les organismes). Des informations détaillées sur l'échantillonnage des organismes les plus importants vivant dans le sol sont fournies dans les autres parties de la présente Norme internationale (ISO 23611-1 à ISO 23611-5). La présente partie de l'ISO 23611 est utilisée pour tous les biotopes terrestres dans lesquels se trouvent les invertébrés du sol. Des informations fondamentales sur la conception des études sur le terrain en général sont déjà fournies dans l'ISO 10381-1. Ces informations peuvent varier selon les exigences nationales ou les conditions régionales/climatiques du site à prélever. NOTE Bien que la présente partie de l'ISO 23611 s'applique globalement à tous les sites terrestres habités par les invertébrés du sol, les informations existantes se réfèrent principalement aux régions tempérées. Cependant, les (quelques) études émanant d'autres régions (tropicales et boréales) ainsi que des considérations théoriques permettent de conclure que les principes établis dans la présente partie de l'ISO 23611 sont généralement valables (Références [4], [6], [40], [21]). La présente partie de l'ISO 23611 fournit des informations sur l'évaluation des risques spécifiques à un site contaminé, l'étude des effets secondaires potentiels des impacts anthropogéniques (par exemple l'utilisation de produits chimiques ou la construction de routes), la classification et l'évaluation biologiques des sols en vue de déterminer leur qualité biologique, la surveillance biogéographique à long terme dans le cadre de la protection ou restauration de la nature, y compris le changement climatique à l'échelle mondiale (par exemple comme dans les projets de recherche écologique à long terme).

General Information

• Status: Not Published
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: ISO/TC 190/SC 4 - Biological characterization
• Drafting Committee: ISO/TC 190/SC 4/WG 2 - Effects on soil fauna
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 30-Mar-2026
• Completion Date: 30-Mar-2026

Relations

• Consolidates: FprEN ISO 23611-6 - Soil quality - Sampling of soil invertebrates - Part 6: Design of sampling programmes with soil invertebrates (ISO/FDIS 23611-6:2026)
• Effective Date: 12-Feb-2026
• Consolidates: ISO 22854:2021 - Liquid petroleum products — Determination of hydrocarbon types and oxygenates in automotive-motor gasoline and in ethanol (E85) automotive fuel — Multidimensional gas chromatography method
• Effective Date: 04-Nov-2023
• Revises: ISO 23611-6:2012 - Soil quality — Sampling of soil invertebrates — Part 6: Guidance for the design of sampling programmes with soil invertebrates
• Effective Date: 28-Oct-2023

Overview

ISO/FDIS 23611-6: Soil quality - Sampling of soil invertebrates - Part 6: Design of sampling programmes with soil invertebrates is an international standard published by ISO. It provides comprehensive guidance for designing field studies involving soil invertebrates to monitor soil quality and assess soil as a habitat for organisms. This standard applies to all terrestrial biotopes where soil invertebrates are present and is relevant to environmental monitoring, risk assessment of contaminated sites, and ecological research.

While mainly based on data from temperate regions, the principles laid out are applicable globally, making this standard a critical resource for global soil quality studies and biodiversity assessments.

Key Topics

• Design of Sampling Programmes: Guidelines on planning and conducting soil invertebrate sampling, including objectives setting, sampling strategies, and statistical considerations.
• Objectives of Sampling: Covered purposes include site-specific risk assessments of contaminated land, evaluation of anthropogenic impacts, biological classification of soils, and long-term ecological monitoring.
• Types of Soil Invertebrates: Introduces practical classification by size (microfauna, mesofauna, macrofauna) to facilitate targeted monitoring.
• Sampling Patterns and Strategies: Recommends approaches such as random, systematic (e.g., grids or patterns like X or zig-zag), stratified sampling, and site-specific adaptations based on landscape or contamination gradients.
• Site Selection and Preparation: Guidance on selecting representative and uncontaminated reference soils, and accounting for soil heterogeneity and site-specific variables.
• Quality Assurance and Reporting: Outlines the importance of robust quality control measures and consistent, transparent reporting to enable comparable and defensible results.

Applications

ISO/FDIS 23611-6 is a valuable tool for various practical applications in the field of environmental science and soil management:

• Site-Specific Risk Assessment: Supports the assessment of contaminated land by indicating how to structure studies that reveal the impact of pollutants on soil-dwelling organisms.
• Monitoring Anthropogenic Effects: Used to detect and understand the biological implications of chemical application, construction, or other human interventions in terrestrial ecosystems.
• Soil Quality Classification: Facilitates biological classification and assessment of soils as part of broader land planning, regulatory compliance, or restoration efforts.
• Long-Term Ecological Monitoring: Vital for nature protection projects, restoration assessment, and tracking global change influences on soil health and biodiversity through longitudinal design.
• Legal and Regulatory Compliance: Supports the development of monitoring strategies to fulfill legal obligations concerning soil protection, environmental impact assessments, and ecological risk evaluations.

Related Standards

ISO/FDIS 23611-6 is part of the broader ISO 23611 series on soil invertebrate sampling and complements guidance found in other international standards:

• ISO 23611-1 to 23611-5: Detailed methods for the sampling of specific soil organism groups.
• ISO 10381-1: General guidelines for the design of soil sampling programmes.
• ISO 18400-101, ISO 18400-104, and ISO 18400-107: General principles and procedures for soil sampling.
• ISO 15799 and ISO 17616: Procedures for ecotoxicological testing and assessment of contaminated soils.
• ISO 18400-202 and ISO 18400-203: Quality assurance and control in soil sampling and data management.
• Other soil property standards such as ISO 10390 (pH), ISO 11277 (particle size), relevant for contextualizing biological sampling.

By aligning with these standards, users can ensure that their sampling programmes are scientifically robust, standardized, and internationally accepted, supporting environmental protection and sustainable soil management worldwide.

Keywords: ISO 23611-6, soil quality, soil invertebrates, sampling design, contaminated land assessment, soil monitoring, risk assessment, biological soil classification, ecological monitoring, soil biodiversity, environmental standards.

Standards Content (Sample)

FINAL DRAFT
International
Standard
ISO/TC 190/SC 4
Soil quality — Sampling of soil
Secretariat: AFNOR
invertebrates —
Voting begins on:
2026-03-30
Part 6:
Design of sampling programmes
Voting terminates on:
2026-05-25
with soil invertebrates
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WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
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TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
International
Standard
ISO/TC 190/SC 4
Soil quality — Sampling of soil
Secretariat: AFNOR
invertebrates —
Voting begins on:
Part 6:
Design of sampling programmes
Voting terminates on:
with soil invertebrates
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2026
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
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TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
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Published in Switzerland Reference number
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 General terms .1
3.2 Terms related to soil protection .2
3.3 Terms related to methods .3
4 Principle . 3
4.1 General .3
4.2 Questions involved in planning a field study.3
5 Objectives of sampling . 5
5.1 General .5
5.2 General remarks .5
5.3 Pre-conditions . .6
5.4 Performance of the site-specific assessment of contaminated land.6
5.5 Study of potential side effects of anthropogenic impacts .6
5.6 Biological classification and assessment of soils in order to determine their biological
quality .6
5.7 Biogeographical monitoring in nature protection or restoration .6
6 Samples and sampling points . 7
6.1 General .7
6.2 Sampling patterns .7
6.3 Selecting and identifying the sampling location .8
6.4 Preparation of the sampling site .8
6.5 Further general advice on sampling performance .9
7 Practical considerations for the biological sampling of soils . 9
7.1 General .9
7.2 Formal preparations.9
7.3 Requirements on sampling personnel and safety precautions .9
7.4 Preliminary survey . .10
7.4.1 General .10
7.4.2 Desk-top study .10
7.4.3 Visiting the site .10
7.5 Main study .11
8 Design options for sampling soil invertebrates .11
8.1 General .11
8.2 Description of possible sampling strategies . 13
8.3 Recommendations and requirements from the European programme ENVASSO
(environmental assessment of soil for monitoring) . 13
9 Sampling report .15
10 Quality assurance and quality control (QA/QC) .16
Bibliography . 17

iii
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 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 190, Soil quality, Subcommittee SC 4, Biological
characterization, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 23611-6:2012), which has been technically
revised.
The main changes are:
— addition of detailed recommendations about the statistical methods that should be applied in site-
specific risk assessment of contaminated land in 7.5;
— removal of the informative Annex A with examples of case studies.
A list of all parts in the ISO 23611 series can be found on the ISO website.
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
Introduction
The biodiversity of soil fauna is tremendous. Soil harbours species-rich communities, which regulate
ecosystem processes such as organic matter decomposition, nutrient flows or soil fertility in general.
[1],[2] [3]
All terrestrial animal phyla can be found in soils. In addition to thousands of bacterial and fungal
“species”, more than 1 000 species of invertebrates in abundances of up to 1,5 million individuals can be
[4],[5]
found within a square metre of soil. This diversity can only be reliably estimated by investigation of the
soil community itself, since other parameters such as climate are not or only weakly correlated with species
[6]
richness.
The composition of this community, as well as the abundance and biomass of the individual species and
groups is a valuable source of information, since they integrate various abiotic and biotic effects such as
[7]
soil properties and conditions, climate, competition or biogeographical influences. For this reason, the
evaluation of the biodiversity of soil invertebrate communities becomes more and more important for the
[8]
classification and assessment of biological soil quality. However, this work is only possible if data collection
(i.e. sampling of the soil fauna) is carried out according to standardized methods. For this reason, a number
of ISO standards have been prepared covering the sampling of the most important soil organism groups.
[74]
In the individual parts of the ISO 23611 series, the practical work concerning the respective animal group
is described in detail. However, (nearly) nothing is said about how to plan the use of such methods or how
to evaluate the results. Despite the fact that sampling for any field study can be different depending on the
individual purpose, guidance is needed for monitoring studies in a legal context. Such studies can include
the following:
— site-specific risk assessment of contaminated land;
— study of potential side effects of anthropogenic impacts (e.g. the application of chemicals or the building
of roads);
— the biological classification and assessment of soils in order to determine the biological quality of soils;
— long-term biogeographical monitoring in the context of nature protection or restoration, including global
change [e.g. as in long-term ecological research projects (LTERs)].
Spatial studies focusing on environmental and ecological questions require a carefully designed strategy
[9],[10]
for collecting data. Before identifying the optimal design, two issues need to be clarified: what is the
objective of the study and what is already known about the survey area? Afterwards, one may select one
of the well-known design patterns (e.g. grid sampling, random sampling, clustered sampling or random
transects) or prepare a study-specific design. In any case, the field sampling design needs to be practical,
e.g. the volume of soil to be sampled, depending on the size and distribution of the organisms, needs to be
manageable (i.e. the smaller the individual animal, the smaller the size), and cost effective.
In studies focusing on soil invertebrates, it is not possible to observe the entire population. Therefore,
sampling is done only at a limited number of locations. The main reason for using statistical sound sampling
schemes is that such sampling guarantees scientific objectivity and avoids forms of bias such as those caused
by judgement sampling. This is especially valuable if the objective is to obtain data that are representative
for the whole area. At the same time, statistics-based sampling schemes ensure standardized sampling
methods over time, i.e. if the same area is to be re-sampled in the future, the results will be comparable.
The rationale provided in this document on the design of field sampling methods for soil invertebrates takes
[64] [67] [69]
into consideration the descriptions provided in ISO 18400-101, ISO 18400-104 and ISO 18400-107
describing soil sampling in general.
[65] [67]
The design of microbiological studies is already covered by ISO 18400-102, ISO 18400-104,
[68] [73] [58] [59]
ISO 18400-105, ISO 18400-206, ISO 14240-1 and ISO 14240-2 .

v
FINAL DRAFT International Standard ISO/FDIS 23611-6:2026(en)
Soil quality — Sampling of soil invertebrates —
Part 6:
Design of sampling programmes with soil invertebrates
1 Scope
This document provides requirements and recommendations for the design of field studies with soil
invertebrates (e.g. for the monitoring of the quality of a soil as a habitat for organisms). It applies to all
terrestrial biotopes inhabited by soil invertebrates, although this information can vary according to the
national requirements or the climatic and regional conditions of the site to be sampled.
NOTE While this document aims to be applicable globally, the existing information refers mostly to temperate
regions. However, the (few) studies from other (tropical and boreal) regions, as well as theoretical considerations,
[1],[11],[12],[13]
allow the conclusion that the principles laid down in this document are generally valid.
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 terminology 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 General terms
3.1.1
community
association of organisms, belonging to different species, families, etc. living at the same time at the same
place, i.e. the living portion of an ecosystem
Note 1 to entry: Adapted from Reference [14].
3.1.2
invertebrate
metazoan (Kingdom Animalia or Metazoa) without backbone (spine)
Note 1 to entry: This is not a taxonomic classification, but based on convenience and tradition.
Note 2 to entry: Adapted from Reference [15].
3.1.3
microfauna
soil fauna (invertebrate animals) including individuals with body sizes (length, diameter) between 20 µm
and 200 µm
EXAMPLE Water bears (Tardigrade), wheel animalcules (Rotifera) and roundworms (Nematoda).

Note 1 to entry: Adapted from Reference [16].
3.1.4
mesofauna
soil fauna (invertebrate animals) including individuals with body sizes (length, diameter) between 0,2 µm
and 2 mm
EXAMPLE Springtails (Collembola), mites (Acari) and potworms (Enchytraeid). The term “microarthropods” is
also used to refer to part of mesofauna.
Note 1 to entry: Adapted from Reference [16].
3.1.5
macrofauna
soil fauna (invertebrate animals) including individuals with body sizes (length, diameter) greater than 2 mm
EXAMPLE Earthworms (Crassiclitelata), ants (Formicidae), beetles (Coleoptera), termites (Isoptera), woodlice
(Isopoda), millipedes (Chilopoda), centipedes (Diplopoda), spiders (Araneae) and snails (Gastropoda). The term
“macroarthropods” is also used to refer to part of macrofauna.
Note 1 to entry: Adapted from Reference [16].
3.1.6
taxocoenosis
total number of species belonging to the same higher taxonomic unit (e.g. family, order) within a community
3.2 Terms related to soil protection
3.2.1
soil quality
capacity of a specific kind of soil to function, within natural or managed ecosystem boundaries, to sustain
plant and animal productivity, maintain or enhance water and air quality, and support human health and
habitation
Note 1 to entry: In more recent definitions, the natural functions of soil are specifically listed: soil as a habitat for
organisms, as part of natural systems with particular functions, such as nutrient cycles, decomposition, retention and
[12]
filtration.
Note 2 to entry: See References [3] and [17].
3.2.2
habitat
sum of the environment of a particular species or community
EXAMPLE In terms of soil properties, land use, climate.
3.2.3
pollutant
substance which, due to their properties, amount or concentration, cause impacts on soil functions or soil
use
3.2.4
reference soil
uncontaminated soil with comparable pedological properties to the soil being studied except that it is free of
contamination
3.3 Terms related to methods
3.3.1
geographical information systems
GIS
computer system capable of assembling, storing, manipulating and displaying geographically referenced
information, i.e. data identified according to their locations
3.3.2
site-specific assessment
evaluation of the quality of a specific-site by using chemical, biological or physical methods, or a combination
of them
4 Principle
4.1 General
The design of field studies for the investigation of soil invertebrates differs significantly depending on the
respective aim. However, samples shall be taken in all cases, since the site and biological populations to be
studied are usually too large to be studied in total. In addition, most soil invertebrates either live hidden
within the soil or they are too small to be studied directly, or both. The samples collected should be as
representative as possible of the site to be characterized but destruction should be kept at a minimum. In
addition, the occurrence of material not naturally belonging to the study site (e.g. waste or chemicals) can
cause problems when taking samples in multiphase systems such as soils, which contains water, gases,
mineral solids and biological material.
The study design (e.g. the position and density of sampling points, time of sampling, and the sampling
method) depends mainly on the objectives of the study and on the amount and quality of information
already available from the study site (e.g. historical data, personal experience). The design also depends on
whether information is needed as an average value (sampling for the spatial mean, e.g. the average number
of nematodes) or as a spatial distribution (e.g. sampling for a map showing nematode abundances in relation
to soil properties). In addition, the sheer size and the heterogeneity of soil properties, as well as those of the
organisms to be sampled shall be taken into consideration. In any case, a list of measurement end points
should be compiled for the respective organism group(s) and the main limitations of the sampling method(s)
shall also be known. The latter refers mainly to the high natural variability of invertebrate data. The normal
statistical tests used by those who take composite samples (microflora, soil properties) or many samples
(soil properties) which can be processed more or less automatically, cannot be applied here.
Some consideration should also be given to the degree of detail and precision that is required and also the
manner in which the results are to be expressed (e.g. maximum and minimum values in a table, graphical
presentations or maps). Appropriate statistical methods for the evaluation of area-related data (including
the use of GIS methods) shall be identified as well. An exploratory sampling programme should often be
carry out before the final study design can be defined in detail. The main points on which decisions should
be made are listed in 4.2, reflecting the logical order of how to proceed a study.
[64] [67]
NOTE This clause was written in close consideration with ISO 18400-101, ISO 18400-104 and
[69]
ISO 18400-107 .
4.2 Questions involved in planning a field study
The objective of a study shall be established. This can be done by asking the following questions:
— Why is such a study going to be performed?
— What information is necessary to answer the questions asked and how can this information be clearly
presented?
— Which approach is used for the interpretation of the results?
— How can the study outcome be tailored to the needs of the study sponsor (or stakeholder)?

The preliminary information can be defined by the following questions:
— What is already known about present and historical (especially land-use, management) site and soil
characteristics?
— What information is missing? Can it be made available?
— Who is to be contacted for certain (e.g. historical) sources?
— Are there any legal problems such as entering the sites?
— Shall other than biological parameters be measured at the same site and time, i.e. are (negative)
interactions of the various sampling programmes to be expected?
— Has the site been visited already?
The strategy of a study can be developed by the following questions:
— How are the delineations in time and space of the area(s) to be investigated determined?
— Which organism groups and measurement end points are appropriate to reach the study objective?
— Which sampling patterns, sampling points, sampling times, depths of sampling should be used?
— Can methods specified in International Standards be employed for all activities?
The decision on sampling and analysis can be made by answering the following questions:
— Can the sampling be done according to the respective International Standard or is there any deviation?
— How is the communication with the personnel responsible for sample presentation and analysis
coordinated?
— Which statistical evaluation methods are being employed?
— Does sampling correspond to later data analyses?
— Is it possible to address the correct taxonomic level when studying the biological material?
— How is the documentation organized?
The following questions on safety should be answered:
— Are all necessary safety precautions at that site considered?
— Is information concerning landowners, local authorities, etc. secured?
[66]
— Are the requirements of ISO 18400-103, covering guidance on safety in sampling programmes, and
those on safety issues listed in other parts of the ISO 23611 series fulfilled?
The following questions on the sampling report should be answered:
— Is there any deviation from the basic content of a study report as specified in this document?
— Is additional information required?
— How is it ensured that any later deviation from this document or the study plan is documented and
distributed?
Answers to these questions are given in Clause 5 to Clause 8.

5 Objectives of sampling
5.1 General
Biological soil investigations address a number of different questions related to the status of invertebrates
living in or on the soil (including many different species belonging to different trophic, taxonomic,
physiological or functional groups and size classes), often after or under some kind of anthropogenic
impact. In the case of ecotoxicological questions, usually laboratory tests are used to study the effects of
the impact (e.g. chemicals added to the soil) on invertebrates and thus on the soil quality in general. Such
[62] [63]
methods are presented in ISO 15799, while the assessment of the test results is given in ISO 17616.
Further guidance on sampling, collection, handling and preparation of contaminated soil for biological (i.e.
ecotoxicological) testing can be found in Reference [13]. This is particularly important for the identification
and characterization of field reference soils which are necessary for the determination of biological reference
values.
5.2 General remarks
As stated in the Introduction, the principal objectives of sampling soil invertebrates can be distinguished as
follows:
— the performance of the site-specific characterization and assessment of contaminated land;
— the study of potential side effects of anthropogenic impacts (e.g. the application of chemicals or the
building of roads);
— the biological classification and assessment of soils in order to determine the biological condition of soils;
— long-term biogeographical monitoring in the context of nature protection or restoration, including global
change [e.g. as in long-term ecological research projects (LTERs)].
To a different degree, all four objectives include the determination of biological reference (or base-line)
values, meaning that it shall be clarified which community of soil organisms occurs in a specific soil
assuming that there is no anthropogenic impact. Since this precondition is, in many if not all soils, not
fulfilled any more, such a “normal” state shall be defined, e.g. by sampling of reference soils. These soils have
been selected based on criteria such as being representative for certain regions or land-use forms, or lack of
[18]
contamination.
The use of the soil and site are of varying importance depending on the primary objective of an investigation.
The results obtained from sampling can indicate a need for further investigation, e.g. detected contamination
can indicate a need for identification and assessment of potential hazards and risks. However, assessment
of such hazards or risks is not covered by this document. In addition, capture-recapture methods – while
[19]
often used in ecology for terrestrial above-ground invertebrates (e.g. spiders) ‒ are rarely used in general
monitoring schemes and thus are not covered in this document.
Often soil invertebrates are a part of an entire monitoring effort that includes other biological (mainly
microbial), as well as pedological, climatic and possibly also agricultural parameters. If such monitoring
programmes are performed at regular intervals, permanent sampling sites shall be set up. In such a case,
additional efforts are mandatory in order to secure an effective exchange of information. Sampling is
usually carried out within the main rooting zone (rarely at greater depths since most soil invertebrates live
within the uppermost 30 cm of the soil). Soil horizons or layers can be sampled separately or in combination
(samples shall be labelled accordingly).
To adequately support legal or regulatory action, particular attention should be paid to all aspects of quality
[67] [70] [71]
assurance. The guidance given in ISO 18400-104, ISO 18400-202 and ISO 18400-203 should be
followed. After clarifying the most important pre-conditions in 5.3, the four groups of main objectives as
given above are briefly presented in 5.4 to 5.7. However, it should be kept in mind that, in reality, one specific
study can fit into more than one of these groups.

5.3 Pre-conditions
Before designing a field study with soil invertebrates, the respective area should be characterized
[20]
pedologically. Depending on the principal objectives, it is usually necessary to determine for the body of
soil or part thereof:
— the nature, concentrations and distribution of naturally occurring substances;
— the nature, concentrations and distribution of contaminants;
— the physical and chemical properties and variations;
— the anthropogenic impact at that site, in particular the land use history (including vegetation cover).
It is often necessary to take into account changes in the above-mentioned variables with time and space
(vertically, horizontally), caused by either natural (e.g. climatic) or anthropogenic activities.
In addition, pH, particle size distribution, C/N ratio, organic matter and organic carbon content, total
nitrogen, cation exchange capacity and water holding capacity of the soil should be measured in accordance
[48] [49] [50] [51] [52] [53] [57]
with ISO 10390, ISO 10694, ISO 11260, ISO 11272, ISO 11274, ISO 11277, ISO 13878,
[54] [55] [61] [63]
ISO 11461, ISO 11465, ISO 15709, ISO 17616 .
5.4 Performance of the site-specific assessment of contaminated land
When land is contaminated with chemicals and other substances that are potentially acting as pollutants
to the environment, it can be necessary to carry out an investigation as a part of a hazard and/or risk
assessment. This includes determining the nature and extent of contamination, identifying hazards
associated with the contamination, identifying potential targets and routes of exposure, and evaluating the
environmental risks related to the current and future use of the site and neighbouring land. A sampling
programme for risk assessment can also comply with legal or regulatory requirements and careful attention
to sample integrity is recommended. An extensive overview of the benefits and limitations of biological
parameters as a component of contaminated land assessment is given in Reference [13].
5.5 Study of potential side effects of anthropogenic impacts
Sampling can be required following an anthropogenic effect such as the input of undesirable material
(mainly chemicals) which can be from a point source or from a diffuse source. Another example can be the
building of roads. The study design needs again to be developed on a site-specific basis. Sampling can also be
required to establish base-line conditions prior to an activity, which might affect the composition or quality
of soil.
NOTE Such base-line sampling can also be performed as part of a biological soil classification and assessment
(see 5.6).
5.6 Biological classification and assessment of soils in order to determine their biological
quality
This is typically carried out at (irregular) time intervals to determine the biological quality of a soil for a
particular purpose (e.g. as part of a large-scale screening programme or in the context of a local planning
[21]
activity). The information gained here can be used for the preparation of biological soil maps.
NOTE The study of the biological soil quality can also be used for the determination of “base-line conditions”
in the context of the assessment of anthropogenic impacts (see 5.5) or of long-term changes such as global warming
(5.7).
5.7 Biogeographical monitoring in nature protection or restoration
Finally, the information gained in sampling programmes extends the knowledge on the biogeography of soil
organisms, which is necessary in the context of nature protection and conservation, in particular concerning
long-term changes like global warming. So far, only few soil invertebrates (mainly beetles or other insects

which in their larval stage live in the soil) have been put on the Red List of endangered species (https://
www .iucnredlist .org/ statistics). Also, there is little proof that such species have been eradicated in modern
times. However, in both cases, this fact is mainly caused by the poor level of knowledge on these species;
many species can have died out without notice. Sampling programmes can also determine whether soil-
biological assemblages (site-specifically) expected in a region become established during nature restoration
or after remediation measures (control of success).
6 Samples and sampling points
6.1 General
The selection, location and preparation of the sampling points depend on the objectives of the investigation,
the preliminary information available and the on-site conditions. Soil properties, the occurrence of
organisms and contamination vary continuously in space; the values at locations close together are more
similar than those farther apart and this spatial dependence can be described by the use of geostatistics.
[20]
Geostatistics are used in the development of sampling strategies and are also used to analyse the
[22]
data generated from the soil sampled. In this clause, which closely follows the terminology used in
[64] [67] 1) [69]
ISO 18400-101, ISO 18400-104 and ISO 18400-107:2002 , Annex C, several (standard) options and
issues to be considered are given.
6.2 Sampling patterns
Sampling patterns are based on the estimation of the distribution of the soil invertebrates in the area to be
sampled. Several distribution patterns can be distinguished (of course with intermediate types, such as the
[67]
sampling strategies detailed in ISO 18400-104) :
— no specific distribution (i.e. random);
— homogenous distribution (probably very rare);
— clumped distribution;
— distribution varying according to an underlying gradient (linear or concentric).
Sampling design should be adjusted to the (theoretically expected) distribution pattern or observable
local conditions which make some patterns more probable. If the area to be sampled shows differences in
important properties, such as land use, soil conditions, geomorphology, vegetation patterns, the site should
be subdivided according to these differences and separate samples should be taken from “homogenous” sub-
areas (stratified sampling).
In agricultural or forestry sampling, a small number of convenient sampling patterns are established
in order to obtain information from larger areas. Examples of such patterns are briefly described in the
[67]
following (for details see ISO 18400-104) :
— Systematic patterns (irregular sampling):
— Assuming a relatively homogenous distribution, such sampling can be performed using patterns
resembling an “N”, “S”, “W” or “X”. In particular the diagonal sampling in form of an “X” is popular,
but one shall be aware that a serious bias towards the central area is obvious in this case. Traversing
the area in a “zig-zag pattern” is another way of applying a non-systematic pattern.
— For the purpose of permanently monitored areas, the diagonal X pattern was modified in a way that
2 2
an area of about 1 000 m is divided into four squares of 250 m each. In each of these four squares,
18 samples are taken following an X pattern. By rotating the X, the area can be sampled eight
1) Withdrawn.
times. This sampling pattern allows wide spatial coverage of the sampled location and collection of
composite samples (e.g. used for nematodes), however it is not usual for macrofauna and mesofauna.
— Circular grids: This rarely used pattern is performed when studying the influence of a regional emitting
source (e.g. precipitation from industrial plants). Sampling is carried out at the section of concentric
circles and the lines of the eight main points of the compass.
— Systematic sampling (regular grids): Samples are taken in the centre of a number of squares covering the
entire area of interest (sampling is also possible at the intersection of grid lines). Grid dimensions depend
on how much detail is required.
— Random sampling: Selection of sampling points by using a suitable randomization programme is easy, but
has the disadvantage of irregular coverage and makes interpolation between sampling points difficult. In
order to minimize this problem, sometimes a stratified randomized sampling is performed. Hereby, the
entire area is divided into a number of grid cells and a given number of randomly distributed sampling
points are chosen in each square. Finally, an unaligned random sampling on a regular grid, meaning that
only one of the two coordinates of each sampling point in the regular grid is chosen at random.
— Systematic sampling on a non-rectangular grid: In the case of an equilateral triangular grid, each grid
point is neighboured by three grid points at a unique distance.
6.3 Selecting and identifying the sampling location
The selection of sampling locations depends upon the study objectives, preliminary information and on-
[20]
site conditions. Examples of on-site conditions that should be considered when designing a sampling
strategy include local topography, climatic conditions, vegetation cover (especially trees), soil type and/or
soil physicochemical characteristics, and, if appropriate, the location of a contaminant source (point or non-
[13]
point) or the direction of contamination.
Identification of sampling points is not always necessary. However, where samples are taken at predefined
points, their accurate location and identification is important for four reasons:
— to enable actual sampling locations to be revisited if necessary (note that invertebrate sampling is usually
destructive, i.e. exact repetition is not possible);
— to avoid sample disturbance when taking further samples;
— to enable accurate plotting of data in relation to site features (e.g. soil properties or the concentration of
contaminants);
— to prepare maps or for modelling studies.
Both sketch maps and photographs (including a scale and a direction marker) should be prepared in the
field. Sampling locations should be determined with an appropriate degree of accuracy. The use of global
positioning system (GPS) for geographic coordinates is recommended to identify the sampling points. The
location of sampling points should be marked before sampling begins, using poles or markers of colour
sprays.
6.4 Preparation of the sampling site
Depending on the objective of the investigation, a sampling pattern is chosen at the design stage and is
then applied in the field. Afterwards, preparation of the site includes, for example, the establishment of
safety measures or the installation of markers for the exact sampling points. This work becomes very time
consuming if it is not possible to take a sample at the planned location due to a variety of reasons (e.g. trees,
rocks, or access difficulties). Contingency plans for dealing with such situations should be made in advance
(ad hoc decisions in the field can lead to a bias). The action taken depends on the circumstances: the point
can be ignored, or a nearby substitute location (e.g. within 10 % of grid spacing away from the original
location) can be chosen. In every case where a sampling point shall be re-located, this and the reason for
relocation shall be clearly indicated in the report.

6.5 Further general advice on sampling performance
[74]
Details of the sampling performance are given in the other parts of the ISO 23611-series . However, some
general advice is given here.
Mountain regions or hilly areas with pronounced slopes require special consideration before starting
sampling. No general recommendation can be given on the depths
...

ISO/DISFDIS 23611--6:2026(en)
ISO/TC 190/SC 4/WG 2
Secretariat: AFNOR
Date: 2026-02-1903-16
Soil quality — Sampling of soil invertebrates — —
Part 6:
Design of sampling programmes with soil invertebrates
FDIS stage
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All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
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ii
ISO/DISFDIS 23611-6:2026(en)
Contents
Foreword . iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 General terms . 1
3.2 Terms related to soil protection . 2
3.3 Terms related to methods . 3
4 Principle . 3
4.1 General . 3
4.2 Questions involved in planning a field study . 4
5 Objectives of sampling . 5
5.1 General . 5
5.2 General remarks . 5
5.3 Pre-conditions . 6
5.4 Performance of the site-specific assessment of contaminated land . 7
5.5 Study of potential side effects of anthropogenic impacts . 7
5.6 Biological classification and assessment of soils in order to determine their biological
quality . 7
5.7 Biogeographical monitoring in nature protection or restoration . 7
6 Samples and sampling points . 7
6.1 General . 7
6.2 Sampling patterns . 8
6.3 Selecting and identifying the sampling location . 9
6.4 Preparation of the sampling site . 9
6.5 Further general advice on sampling performance . 9
7 Practical considerations for the biological sampling of soils . 10
7.1 General . 10
7.2 Formal preparations . 10
7.3 Requirements on sampling personnel and safety precautions . 10
7.4 Preliminary survey . 10
7.5 Main study . 11
8 Design options for sampling soil invertebrates . 12
8.1 General . 12
8.2 Description of possible sampling strategies . 14
8.3 Recommendations and requirements from the European programme ENVASSO
(environmental assessment of soil for monitoring) . 14
9 Sampling report . 17
10 Quality assurance and quality control (QA/QC) . 17
Bibliography . 18

iii
Foreword
iv
ISO/DISFDIS 23611-6:2026(en)
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 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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'sISO’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 190, Soil quality, Subcommittee SC 4, Biological
characterization, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 23611--6:2012), which has been technically
revised.
The main changes are:
— — addition of detailed recommendations about the statistical methods that should be applied in site-
specific risk assessment of contaminated land in 7.57.5;
— — removal of the informative annexAnnex A with examples of case studies.
A list of all parts in the ISO 23611 series can be found on the ISO website.
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.
v
Introduction
The biodiversity of soil fauna is tremendous. Soil harbours species-rich communities, which regulate
[1][1], [2] ],[[2]
ecosystem processes such as organic matter decomposition, nutrient flows or soil fertility in general. .
[3] [3]
All terrestrial animal phyla can be found in soils . In addition to thousands of bacterial and fungal “species”,
more than 1 000 species of invertebrates in abundances of up to 1,5 million individuals can be found within a
[4][4], [5] ],[[5]
square metre of soil. . This diversity can only be reliably estimated by investigation of the soil
community itself, since other parameters likesuch as climate are not or only weakly correlated with species
[6] [6]
richness .
The composition of this community, as well as the abundance and biomass of the individual species and groups
is a valuable source of information, since they integrate various abiotic and biotic effects such as soil properties
[7] [7]
and conditions, climate, competition or biogeographical influences . For this reason, the evaluation of the
biodiversity of soil invertebrate communities becomes more and more important for the classification and
[8] [8]
assessment of biological soil quality . However, this work is only possible if data collection (i.e. sampling
of the soil fauna) is carried out according to standardized methods. For this reason, a number of ISO standards
have been prepared covering the sampling of the most important soil organism groups.
[74]
In the individual parts of the ISO 23611 series,, the practical work concerning the respective animal group
is described in detail. However, (nearly) nothing is said about how to plan the use of such methods or how to
evaluate the results. Despite the fact that sampling for any field study can be different depending on the
individual purpose, guidance is needed for monitoring studies in a legal context. Such studies can include the
following:
— — site-specific risk assessment of contaminated land;
— — study of potential side effects of anthropogenic impacts (e.g. the application of chemicals or the
building of roads);
— — the biological classification and assessment of soils in order to determine the biological quality of soils;
— — long-term biogeographical monitoring in the context of nature protection or restoration, including
global change [e.g. as in long-term ecological research projects (LTERs)].
Spatial studies focusing on environmental and ecological questions require a carefully designed strategy for
[9][9], [10] ],[[10]
collecting data. . Before identifying the optimal design, two issues need to be clarified: what is the
objective of the study and what is already known about the survey area? Afterwards, one may select one of
the well-known design patterns (e.g. grid sampling, random sampling, clustered sampling or random
transects) or prepare a study-specific design. In any case, the field sampling design needs to be practical, e.g.
the volume of soil to be sampled, depending on the size and distribution of the organisms, hasneeds to be
manageable (i.e. the smaller the individual animal, the smaller the size), and cost effective.
In studies focusing on soil invertebrates, it is not possible to observe the entire population. Therefore,
sampling is done only at a limited number of locations. The main reason for using statistical sound sampling
schemes is that such sampling guarantees scientific objectivity and avoids forms of bias such as those caused
by judgement sampling. This is especially valuable if the objective is to obtain data that are representative for
the whole area. At the same time, statistics-based sampling schemes ensure standardized sampling methods
over time, i.e. if the same area is to be re-sampled in the future, the results will be comparable.
The rationale provided in this document on the design of field sampling methods for soil invertebrates takes
[48] [64] [67][49]
into consideration the descriptions provided in ISO 18400-101 , , ISO 18400-104 and ISO 18400-
[69][50]
107 describing soil sampling in general.
vi
ISO/DISFDIS 23611-6:2026(en)
[51] [65] [49]
The design of microbiological studies is already covered by ISO 18400-102 , , ISO 18400-104 ,
[67] [52] [68] [53] [73] [58][54] [59][55]
, ISO 18400-105 , , ISO 18400-206 , , ISO 14240--1 and ISO 14240--2 .
vii
International Standard ISO/DIS 23611-6:2025(en)

Soil quality — Sampling of soil invertebrates — —
Part 6:
Design of sampling programmes with soil invertebrates
1 Scope
This document provides requirements and recommendations for the design of field studies with soil
invertebrates (e.g. for the monitoring of the quality of a soil as a habitat for organisms). It applies to all
terrestrial biotopes inhabited by soil invertebrates, although this information can vary according to the
national requirements or the climatic/ and regional conditions of the site to be sampled.
NOTE While this document aims to be applicable globally, the existing information refers mostly to temperate
regions. However, the (few) studies from other (tropical and boreal) regions, as well as theoretical considerations, allow
[1]],[[11]],[[12][1], [11], [12], [13] ],[[13]
the conclusion that the principles laid down in this document are generally valid. .
This document gives information on site-specific risk assessment of contaminated land, study of potential side
effects of anthropogenic impacts (e.g. the application of chemicals or the building of roads), the biological
classification and assessment of soils in order to determine the biological quality of soils, and long-term
biogeographical monitoring in the context of nature protection or restoration, including global change (e.g. as
in long-term ecological research projects).
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 terminology 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 3.1.1General terms
3.1.1
community
association of organisms, belonging to different species, families, etc. living at the same time at the same place,
i.e. the living portion of an ecosystem
Note 1 to entry: Adapted from Reference [14] [14]. .
3.1.2 3.1.2
invertebrate
metazoansmetazoan (Kingdom Animalia or Metazoa) without backbone (spine). )
Note 1 to entry: This is not a taxonomic classification, but based on convenience and tradition.
Note 2 to entry: Adapted from Reference [15]reference [15].
3.1.3
microfauna
3.1.3
Microfauna
way of classifying the soil fauna (invertebrate animals) based onincluding individuals with body sizes (length,
diameter) between 20 µm and 200 µm.
EXAMPLE Important examples of the Watermicrofauna are water bears (Tardigrade), wheel animalcules (Rotifera)
and roundworms (Nematoda).
Note 1 to entry: Adapted from Reference [16] [16].
3.1.4
mesofauna
3.1.4
Mesofauna
way of classifying the soil fauna (invertebrate animals) based onincluding individuals with body sizes (length,
diameter) between 0,2 µm and 2 mm.
EXAMPLE Important examples of the Springtailsmesofauna are springtails (Collembola), mites (Acari) and
potworms (Enchytraeid). The term micro-arthropods“microarthropods” is also used to refer to part of mesofauna.
Note 1 to entry: Adapted from Reference [16] [16].
3.1.5
macrofauna
3.1.5
Macrofauna
way of classifying the soil fauna (invertebrate animals) based onincluding individuals with body sizes (length,
diameter) greater than 2 mm.
EXAMPLE Important examples of the Earthwormsmacrofauna are earthworms (Crassiclitelata), ants (Formicidae),
beetles (Coleoptera), termites (Isoptera), woodlice (Isopoda), millipedes (Chilopoda), centipedes (Diplopoda), spiders
(Araneae) and snails (Gastropoda). The term macro-arthropods“macroarthropods” is also used to refer to part of
macrofauna.
Note 1 to entry: Adapted from Reference [16] [16].
3.1.6 3.1.6
taxocoenosis
total number of species belonging to the same higher taxonomic unit (e.g. family, order) within a community
3.2 SoilTerms related to soil protection
3.2.1 3.2.1
soil quality
capacity of a specific kind of soil to function, within natural or managed ecosystem boundaries, to sustain plant
and animal productivity, maintain or enhance water and air quality, and support human health and habitation
Note 1 to entry: In more recent definitions, the natural functions of soil are specifically listed: soil as a habitat for
organisms, as part of natural systems with particular functions, such as nutrient cycles, decomposition, retention and
[12] [12]
filtration .
Note 2 to entry: See References [3]and [17] [3], [17]. .
ISO/DISFDIS 23611-6:2026(en)
3.2.2 3.2.2
habitat
sum of the environment of a particular species or community
EXAMPLE In terms of soil properties, land use, climate).
3.2.3 3.2.3
pollutant
substancessubstance which, due to their properties, amount or concentration, cause impacts on soil functions
or soil use
3.2.4 3.2.4
reference soil
uncontaminated soil with comparable pedological properties to the soil being studied except that it is free of
contamination
3.3 Methods
3.3.1
3.3 Terms related to methods
3.3.1
geographical information systems
GIS
computer system capable of assembling, storing, manipulating, and displaying geographically referenced
information, i.e. data identified according to their locations
3.3.13.3.2 3.3.2
site-specific assessment
evaluation of the quality of a specific-site by using chemical, biological or physical methods, or a combination
of them
4 Principle
4.1 General
The design of field studies for the investigation of soil invertebrates differs significantly depending on the
respective aim. However, in all cases, samples shall be taken in all cases, since the site and biological
populations to be studied are usually too large to be studied in total. In addition, most soil invertebrates either
live hidden within the soil or they are too small to be studied directly, or both. The samples collected should
be as representative as possible of the site to be characterized but destruction should be kept at a minimum.
In addition, the occurrence of material not naturally belonging to the study site (e.g. waste or chemicals) can
cause problems when taking samples in multiphase systems such as soils, which contains water, gases,
mineral solids and biological material.
The study design (e.g. the position and density of sampling points, time of sampling, and the sampling method)
depends mainly on the objectives of the study and on the amount and quality of information already available
from the study site (e.g. historical data, personal experience). The design also depends on whether information
is needed as an average value (sampling for the spatial mean, e.g. the average number of nematodes) or as a
spatial distribution (e.g. sampling for a map showing nematode abundances in relation to soil properties). In
addition, the sheer size and the heterogeneity of soil properties, as well as those of the organisms to be
sampled shall be taken into consideration. In any case, a list of measurement end points should be compiled
for the respective organism group(s) and the main limitations of the sampling method(s) shall also be known.
The latter refers mainly to the high natural variability of invertebrate data. The normal statistical tests used
by those who take composite samples (microflora, soil properties) or many samples (soil properties) which
can be processed more or less automatically, cannot be applied here.
Some consideration should also be given to the degree of detail and precision that is required and also the
manner in which the results are to be expressed (e.g. maximum and minimum values in a table, graphical
presentations or maps). Appropriate statistical methods for the evaluation of area-related data (including the
use of GIS methods) shall be identified as well. An exploratory sampling programme should often be carry out
before the final study design can be defined in detail. The main points on which decisions should be made are
listed in 4.24.2,, reflecting the logical order of how to proceed a study.
[48] [64] [67][49]
NOTE This clause was written in close consideration with ISO 18400-101 , , ISO 18400-104 and
[69][50]
ISO 18400-107 .
4.2 Questions involved in planning a field study
The objective of a study shall be established. This can be done by asking the following questions:
— — Why is such a study going to be performed?
— — What information is necessary to answer the questions asked and how can this information be clearly
presented?
— — Which approach is used for the interpretation of the results?
— — How can the study outcome be tailored to the needs of the study sponsor (or stakeholder)?
The preliminary information can be defined by the following questions:
— — What is already known about present and historical (especially land-use, management) site and soil
characteristics?
— — What information is missing? Can it be made available?
— — Who is to be contacted for certain (e.g. historical) sources?
— — Are there any legal problems such as entering the sites?
— — Shall other than biological parameters be measured at the same site and time, i.e. are (negative)
interactions of the various sampling programmes to be expected?
— — Has the site been visited already?
The strategy of a study can be developed by the following questions:
— — How are the delineations in time and space of the area(s) to be investigated determined?
— — Which organism groups and measurement end points are appropriate to reach the study objective?
— — Which sampling patterns, sampling points, sampling times, depths of sampling should be used?
— — Can methods specified in International Standards be employed for all activities?
The decision on sampling and analysis can be made by answering the following questions:
— — Can the sampling be done according to the respective International Standard or is there any deviation?
ISO/DISFDIS 23611-6:2026(en)
— — How is the communication with the personnel responsible for sample presentation and analysis
coordinated?
— — Which statistical evaluation methods are being employed?
— — Does sampling correspond to later data analyses?
— — Is it possible to address the rightcorrect taxonomic level when studying the biological material?
— — How is the documentation organized?
The following questions on safety should be answered:
— — Are all necessary safety precautions at that site considered?
— — Is information concerning landowners, local authorities, etc. secured?
[56] [66]
— — Are the requirements of ISO 18400-103 ,, covering guidance on safety in sampling programmes,
[57]
as well asand those on safety issues listed in other parts of this International Standard (the ISO 23611-1
[58]
to ISO 23611-5 ) series fulfilled?
The following questions on the sampling report should be answered:
— — Is there any deviation from the basic content of a study report as specified in this document?
— — Is additional information required?
— — How is it ensured that any later deviation from this document or the study plan is documented and
distributed?
Answers to these questions are given in Clause 5Clause 5 to Clause 8Clause 8.
5 Objectives of sampling
5.1 General
Biological soil investigations address a number of different questions related to the status of invertebrates
living in or on the soil (including many different species belonging to different trophic, taxonomic,
physiological or functional groups and size classes), often after or under some kind of anthropogenic impact.
In the case of ecotoxicological questions, usually laboratory tests are used to study the effects of the impact
(e.g. chemicals added to the soil) on invertebrates and thus on the soil quality in general. Such methods are
[59] [62] [60] [63]
presented in ISO 15799 ,, while the assessment of the test results is given in ISO 17616 . Further
guidance on sampling, collection, handling and preparation of contaminated soil for biological (i.e.
ecotoxicological) testing can be found in Reference [13] [13]. This is particularly important for the
identification and characterization of field reference soils which are necessary for the determination of
biological reference values.
5.2 General remarks
As stated in the Introduction, the principal objectives of sampling soil invertebrates can be distinguished as
follows:
— — the performance of the site-specific characterization and assessment of contaminated land;
— — the study of potential side effects of anthropogenic impacts (e.g. the application of chemicals or the
building of roads);
— — the biological classification and assessment of soils in order to determine the biological condition of
soils;
— — long-term biogeographical monitoring in the context of nature protection or restoration, including
global change [e.g. as in long-term ecological research projects (LTERs)].
To a different degree, all four objectives include the determination of biological reference (or base-line) values,
meaning that it shall be clarified which community of soil organisms occurs in a specific soil assuming that
there is no anthropogenic impact. Since this precondition is, in many if not all soils, not fulfilled any more, such
a “normal” state shall be defined, e.g. by sampling of reference soils. These soils have been selected based on
[18] [18]
criteria likesuch as being representative for certain regions or land-use forms, or lack of contamination .
The use of the soil and site are of varying importance depending on the primary objective of an investigation.
The results obtained from sampling can indicate a need for further investigation, e.g. detected contamination
can indicate a need for identification and assessment of potential hazards and risks. However, assessment of
such hazards or risks is not covered by this document. In addition, capture-recapture methods – while often
[19] [19]
used in ecology for terrestrial above-ground invertebrates (e.g. spiders) ) ‒ are rarely used in general
monitoring schemes and thus are not covered in this document.
Often soil invertebrates are a part of an entire monitoring effort that includes other biological (mainly
microbial), as well as pedological, climatic and possibly also agricultural parameters. If such monitoring
programmes are performed at regular intervals, permanent sampling sites shall be set up. In such a case,
additional efforts are mandatory in order to secure an effective exchange of information. Sampling is usually
carried out within the main rooting zone (rarely at greater depths since most soil invertebrates live within the
uppermost 30 cm of the soil). Soil horizons or layers can be sampled separately or in combination (samples
shall be labelled accordingly).
To adequately support legal or regulatory action, particular attention should be paid to all aspects of quality
[49] [67] [70][61] [71][62]
. , ISO 18400-202 and ISO 18400-203 should
assurance. The guidance given in ISO 18400-104
be followed. After clarifying the most important pre-conditions in 5.35.3,, the four groups of main objectives
as given above are briefly presented in 5.45.4 to 5.75.7. However, it should be kept in mind that, in reality, one
specific study can fit into more than one of these groups.
5.3 Pre-conditions
Before designing a field study with soil invertebrates, the respective area should be characterized
[20] [20]
pedologically . Depending on the principal objectives, it is usually necessary to determine for the body of
soil or part thereof:
— — the nature, concentrations and distribution of naturally occurring substances;
— — the nature, concentrations and distribution of contaminants;
— — the physical and chemical properties and variations;
— — the anthropogenic impact at that site, in particular the land use history (including vegetation cover).
It is often necessary to take into account changes in the above-mentioned variables with time and space
(vertically, horizontally), caused by either natural (e.g. climatic) or anthropogenic activities.
In addition, pH, particle size distribution, C/N ratio, organic matter and organic carbon content, total nitrogen,
cation exchange capacity and water holding capacity of the soil should be measured in accordance with
[63] [48] [64] [49] [65] [50] [66] [51] [67] [52] [68]
ISO 10390 , , ISO 10694 , , ISO 11260 , , ISO 11272 , , ISO 11274 , , ISO 11277 ,
[53] [69] [57] [70] [54] [71] [55] [72] [61] [63][60]
, ISO 13878 , , ISO 11461 , , ISO 11465 , , ISO 15709 , , ISO 17616 .
ISO/DISFDIS 23611-6:2026(en)
5.4 Performance of the site-specific assessment of contaminated land
When land is contaminated with chemicals and other substances that are potentially acting as pollutants to
the environment, it can be necessary to carry out an investigation as a part of a hazard and/or risk assessment.
This includes to determinedetermining the nature and extent of contamination, to identifyidentifying hazards
associated with the contamination, to identifyidentifying potential targets and routes of exposure, and to
evaluateevaluating the environmental risks related to the current and future use of the site and neighbouring
land. A sampling programme for risk assessment can also comply with legal or regulatory requirements and
careful attention to sample integrity is recommended. An extensive overview of the benefits and limitations
of biological parameters as a component of contaminated land assessment is given in Reference [13] [13].
5.5 Study of potential side effects of anthropogenic impacts
Sampling can be required following an anthropogenic effect such as the input of undesirable material (mainly
chemicals) which can be from a point source or from a diffuse source. Another example can be the building of
roads. The study design needs again to be developed on a site-specific basis. Sampling can also be required to
establish base-line conditions prior to an activity, which might affect the composition or quality of soil.
NOTE Such base-line sampling can also be performed as part of a biological soil classification and assessment
(see 5.6 5.6).).
5.6 Biological classification and assessment of soils in order to determine their biological
quality
This is typically carried out at (irregular) time intervals to determine the biological quality of a soil for a
particular purpose (e.g. as part of a large-scale screening programme or in the context of a local planning
[21] [21]
activity). The information gained here can be used for the preparation of biological soil maps .
NOTE The study of the biological soil quality can also be used for the determination of “base-line conditions” in the
context of the assessment of anthropogenic impacts (see 5.55.5)) or of long-term changes such as global warming
(5.7(5.7).).
5.7 Biogeographical monitoring in nature protection or restoration
Finally, the information gained in sampling programmes extends the knowledge on the biogeography of soil
organisms, which is necessary in the context of nature protection and conservation, in particular concerning
long-term changes like global warming. So far, only few soil invertebrates (mainly beetles or other insects
which in their larval stage live in the soil) have been put on the Red List of endangered species
(https://www.iucnredlist.org/statistics). Also, there is little proof that such species have been eradicated in
modern times. However, in both cases, this fact is mainly caused by the poor level of knowledge on these
species; many species can have died out without notice. Sampling programmes can also determine whether
soil-biological assemblages (site-specifically) expected in a region become established during nature
restoration or after remediation measures (control of success).
6 Samples and sampling points
6.1 General
The selection, location and preparation of the sampling points depend on the objectives of the investigation,
the preliminary information available and the on-site conditions. Soil properties, the occurrence of organisms
and contamination vary continuously in space; the values at locations close together are more similar than
[20] [20]
those farther apart and this spatial dependence can be described by the use of geostatistics . Geostatistics
are used in the development of sampling strategies and are also used to analyse the data generated from the
[22] [22] [48]
soil sampled . In this clause, which closely follows the terminology used in ISO 18400-101 ,
[64] [67][49] [50] 1) [69]
, ISO 18400-104 and ISO 18400-107 ,:2002 , Annex C, several (standard) options and issues to
be considered are given.
6.2 Sampling patterns
Sampling patterns are based on the estimation of the distribution of the soil invertebrates in the area to be
sampled. Several distribution patterns can be distinguished (of course with intermediate types, such as the
[67][49]
sampling strategies detailed in ISO 18400--104) )::
— — no specific distribution (i.e. random);
— — homogenous distribution (probably very rare);
— — clumped distribution;
— — distribution varying according to an underlying gradient (linear or concentric).
Sampling design should be adjusted to the (theoretically expected) distribution pattern or observable local
conditions which make some patterns more probable. If the area to be sampled shows differences in important
properties, such as land use, soil conditions, geomorphology, vegetation patterns, the site should be
subdivided according to these differences and separate samples should be taken from “homogenous” sub-
areas (stratified sampling).
In agricultural or forestry sampling, a small number of convenient sampling patterns are established in order
to obtain information from larger areas. Examples of such patterns are briefly described in the following (for
[67][49]
details see ISO 18400--104) )::
— — Systematic patterns (irregular sampling):
— — Assuming a relatively homogenous distribution, such sampling can be performed using
patterns resembling an “N”, “S”, “W” or “X”. In particular the diagonal sampling in form of an “X” is
popular, but one shall be aware that a serious bias towards the central area is obvious in this case.
Traversing the area in a “zig-zag pattern” is another way of applying a non-systematic pattern.
— — For the purpose of permanently monitored areas, the diagonal X pattern was modified in a way
2 2
that an area of about 1 000 m is divided into four squares of 250 m each. In each of these four
squares, 18 samples are taken following an X pattern. By rotating the X, the area can be sampled eight
times. This sampling pattern allows wide spatial coverage of the sampled location and collection of
composite samples (e.g. used for nematodes), however it is not usual for macrofauna and mesofauna.
— — Circular grids: This rarely used pattern is performed when studying the influence of a regional emitting
source (e.g. precipitation from industrial plants). Sampling is carried out at the section of concentric circles
and the lines of the eight main points of the compass.
— — Systematic sampling (regular grids): Samples are taken in the centre of a number of squares covering
the entire area of interest (sampling is also possible at the intersection of grid lines). Grid dimensions
depend on how much detail is required.
— — Random sampling: Selection of sampling points by using a suitable randomization programme is easy,
but has the disadvantage of irregular coverage and makes interpolation between sampling points difficult.
In order to minimize this problem, sometimes a stratified randomized sampling is performed. Hereby, the
entire area is divided into a number of grid cells and a given number of randomly distributed sampling

1)
Withdrawn.
ISO/DISFDIS 23611-6:2026(en)
points are chosen in each square. Finally, an unaligned random sampling on a regular grid, meaning that
only one of the two coordinates of each sampling point in the regular grid is chosen at random.
— — Systematic sampling on a non-rectangular grid: In the case of an equilateral triangular grid, each grid
point is neighboured by three grid points at a unique distance.
6.3 Selecting and identifying the sampling location
The selection of sampling locations depends upon the study objectives, preliminary information, and on-site
[20] [20]
conditions . Examples of on-site conditions that should be considered when designing a sampling
strategy include local topography, climatic conditions, vegetation cover (especially trees), soil type and/or soil
physicochemical characteristics, and, if appropriate, the location of a contaminant source (point or non-point)
[13] [13]
or the direction of contamination .
Identification of sampling points is not always necessary. However, where samples are taken at pre-
definedpredefined points, their accurate location and identification is important for threefour reasons:
— — to enable actual sampling locations to be revisited if necessary (note that invertebrate sampling is
usually destructive;, i.e. exact repetition is not possible);
— — to avoid sample disturbance when taking further samples;
— — to enable accurate plotting of data in relation to site features (e.g. soil properties or the concentration
of contaminants);
— — to prepare maps or for modelling studies.
Both sketch maps and photographs (including a scale and a direction marker) should be prepared in the field.
Sampling locations should be determined with an appropriate degree of accuracy. The use of Global
Positioning Systemglobal positioning system (GPS) for geographic coordinates is recommended to identify the
sampling points. The location of sampling points should be marked before sampling begins, using poles or
markers of colour sprays.
6.4 Preparation of the sampling site
Depending on the objective of the investigation, a sampling pattern is chosen at the design stage and is then
applied in the field. Afterwards, preparation of the site includes, for example, the establishment of safety
measures or the installation of markers for the exact sampling points. This work becomes very time consuming
if it is not possible to take a sample at the planned location due to a variety of reasons (e.g. trees, rocks, or
access difficulties). Contingency plans for dealing with such situations should be made in advance (ad hoc
decisions in the field can lead to a bias). The action taken depends on the circumstances: the point can be
ignored, or a nearby substitute location (e.g. within 10 % of grid spacing away from the original location) can
be chosen. In every case where a sampling point shall be re-located, this and the reason for relocation shall be
clearly indicated in the report.
6.5 Further general advice on sampling performance
[74] [57]
Details of the sampling performance are given in the other parts of the ISO 23611-series -1 to
[58]
ISO 23611-5 . However, some general advice is given here.
Mountain regions or hilly areas with pronounced slopes require special consideration before starting
sampling. No general recommendation can be given on the depths at which samples should be taken. This
depends on the objectives of the study and the respective organism groups to be sampled. The same is true
for the timing and frequency of sampling. In addition, the sample quantity varies considerably according to
the method used (approximately 100 g to 5 kg, see Reference [13] [13] for a general overview). In most
sampling guidelines for agricultural (including microbial) investigations, composite samples are
recommended, while for the study of soil invertebrates, single samples are usually taken. Other information
relevant to conduct the sampling (e.g. sample containers, transport and storage of samples and preservation
[57] [58] [74]
of animals) are given in the ISO 23611-1 to ISO 23611-5 -series and, in particular for contaminated
soils, in Reference [13] [13]. In any case, each sample shall be clearly and unmistakably marked and their
location in the field noted. Preferably, labelling should be done both within and outside of the containers.
Finally, if the sampling programme is performed for legal purposes, all raw data gained should be collected in
accordance with local quality assurance/quality control programme
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