ISO/DIS 23611‑6:2025(en)

ISO/TC 190/SC 4/WG 2

Secretariat: AFNOR

Date: 2025-05-11

Soil quality — Sampling of soil invertebrates — Part 6: Guidance for the design of sampling programmes with soil invertebrates

© ISO 2025

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Contents

Foreword iv

Introduction v

1 Scope 1

2 Normative references 1

3 Terms and definitions 2

3.1 Soil biology 2

3.2 Soil protection 3

3.3 Methods 4

4 Principle 4

4.1 General 4

4.2 Question to be answered when planning a field study 5

5 Objectives of sampling 6

5.1 General 6

5.2 General remarks 6

5.3 Pre-conditions 7

5.4 The performance of the site-specific assessment of contaminated land 7

5.5 The study of potential side effects of anthropogenic impacts 7

5.6 The biological classification and assessment of soils in order to determine the biological quality of soils 7

5.7 Biogeographical monitoring in nature protection or restoration 8

6 Samples and sampling points 8

6.1 General 8

6.2 Sampling patterns 8

6.3 Selecting and identifying the sampling location 9

6.4 Preparation of the sampling site 10

6.5 Further general advice on sampling performance 10

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 11

7.4 Preliminary survey 11

7.4.1 General 11

7.4.2 Desk-top study 11

7.4.3 Visiting the site 11

7.5 Main study 12

8 Design options for sampling soil invertebrates 13

8.1 Introduction 13

8.2 Description of possible sampling strategies 14

8.3 Recommendations from the European programme ENVASSO (Environmental Assessment of Soil for Monitoring) 14

9 Sampling report 16

10 Quality assurance and quality control (QA/QC) 17

Bibliography 18

Foreword

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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).

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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 shall be applied in site-specific risk assessment of contaminated land in section 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.

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, References [1], [2]. All terrestrial animal phyla can be found in soils, Reference [3]. 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 square metre of soil, References [4], [5]. This diversity can only be reliably estimated by investigation of the soil community itself, since other parameters like climate are not or only weakly correlated with species richness, Reference [6].

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 and conditions, climate, competition or biogeographical influences, Reference [7]. For this reason, the evaluation of the biodiversity of soil invertebrate communities becomes more and more important for the classification and assessment of biological soil quality, Reference [8]. 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 guidelines have been prepared covering the sampling of the most important soil organism groups.

In the individual parts of ISO 23611, 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 collecting data. References [9], [10]. Before identifying the optimal design, two issues have 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 has to be practical, e.g. the volume of soil to be sampled, depending on the size and distribution of the organisms, has 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 for this guidance on the design of field sampling methods for soil invertebrates takes into consideration the guidance provided in ISO 10381‑1 describing soil sampling in general.

The design of microbiological studies is already covered by ISO 10381‑6, ISO 14240‑1 and ISO 14240‑2.

Soil quality — Sampling of soil invertebrates — Part 6: Guidance for the 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 [1], [11], [12], [13].

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 long-term biogeographical monitoring in the context of nature protection or restoration, including global change (e.g. as in long-term ecological research projects).

The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10390, Soil, treated biowaste and sludge – Determination of pH

ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)

ISO 11074, Soil quality — Vocabulary

ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using barium chloride solution

ISO 11272, Soil quality — Determination of dry bulk density

ISO 11274, Soil quality — Determination of the water-retention characteristic — Laboratory methods

ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by sieving and sedimentation

ISO 11461, Soil quality — Determination of soil water content as a volume fraction using coring sleeves — Gravimetric method

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric method

ISO 11466, Soil quality — Extraction of trace elements soluble in aqua regia

ISO 13878, Soil quality — Determination of total nitrogen content by dry combustion ("elemental analysis")

ISO 14869‑1, Soil quality — Dissolution for the determination of total element content — Part 1: Dissolution with hydrofluoric and perchloric acids

ISO 15709, Soil quality — Soil water and the unsaturated zone — Definitions, symbols and theory

ISO 15799, Soil quality — Guidance on the ecotoxicological characterization of soils and soil materials

ISO 17616, Soil quality — Guidance on the choice and evaluation of bioassays for ecotoxicological characterization of soils and soil materials

ISO 18400‑102, Soil quality — Sampling — Part 102: Selection and application of sampling techniques

ISO 18400‑103, Soil quality — Sampling — Part 103: Safety

ISO 18400‑104, Soil quality — Sampling — Part 104: Strategies

ISO 18400‑202, Soil quality — Sampling — Part 202: Preliminary investigations

ISO 18400‑203, Soil quality — Sampling — Part 203: Investigation of potentially contaminated sites

ISO 18400‑206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory

ISO 23611‑1, Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and formalin extraction of earthworms

ISO 23611‑2, Soil quality — Sampling of soil invertebrates — Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina)

ISO 23611‑3, Soil quality — Sampling of soil invertebrates — Part 3: Sampling and extraction of enchytraeids

ISO 23611‑4, Soil quality — Sampling of soil invertebrates — Part 4: Sampling, extraction and identification of soil-inhabiting nematodes

ISO 23611‑5, Soil quality — Sampling of soil invertebrates — Part 5: Sampling and extraction of soil macro-invertebrates

For the purposes of this document, the terms and definitions given in ISO 11074 and the following 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/

3.1.1

biodiversity

variability among living organisms on the earth, including the variability within and between species, and within and between ecosystems

Note 1 to entry: Also often used as the number and variety of organisms found within a specified geographic region.

3.1.2

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

[SOURCE: See Reference [14].]

3.1.3

invertebrate

metazoans (Kingdom Animalia or Metazoa) without backbone (spine). This is not a taxonomic classification, but based on convenience and tradition”.

[SOURCE: See reference [15]]

3.1.4

microfauna, mesofauna and macrofauna

way of classifying the soil fauna (invertebrate animals) according to the size (length, diameter) of the individual animals

EXAMPLE Important examples of the microfauna are water bears (Tardigrade), wheel animalcules (Rotifera) and roundworms (Nematoda), for the mesofauna springtails (Collembola), mites (Acari) and potworms (Enchytraeid), and for the macrofauna earthworms (Crassiclitelata), ants (Formicidae), beetles (Coleoptera), termites (Isoptera), woodlice (Isopoda), millipedes (Chilopoda), centipedes (Diplopoda), spiders (Araneae) and snails (Gastropoda). Terms like micro-arthropods and macro-arthropods are also used to refer to part of mesofauna and macrofauna respectively.

[SOURCE: See Reference [16].]

3.1.5

taxocoenosis

total number of species belonging to the same higher taxonomic unit (e.g. family, order) within a community

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, as nutrient cycles, decomposition, retention and filtration, Reference [12].

[SOURCE: See References [3], [17].]

3.2.2

habitat

sum of the environment of a particular species or community (e.g. in terms of soil properties, land use, climate)

3.2.3

habitat function

ability of soils/soil materials to serve as a habitat for microorganisms, plants, and soil-living animals, and support their interactions (community or biocenosis)

3.2.4

contamination

substance(s) or agent(s) present in the soil as a result of human activity

Note 1 to entry: There is no assumption in this definition that harm results from the presence of the contaminant.

3.2.5

pollutant

substances which, due to their properties, amount or concentration, cause impacts on soil functions or soil use

3.2.6

reference soil

uncontaminated soil with comparable pedological properties to the soil being studied except that it is free of contamination

3.3.1

Geographical Information Systems

GIS

in the strictest sense, a 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 and/or physical methods

3.3.3

environmental risk assessment

process of identifying and quantifying risk (probability that an effect occurs) to non-human organisms and determining the acceptability of these risks

3.3.4

soil function

role performed by soil that support ecosystems, the biosphere, the water environment and human activities

3.3.5

soil organism function

activity provided by individual species or, more often, by interaction of several species or the whole soil community, e.g. nitrogen fixation, organic-matter breakdown or formation of soil structure

The design of field studies for the investigation of soil invertebrates differs significantly depending on the respective aim. However, in all cases, it is necessary to take samples since the site and biological populations to be studied are usually too large to be studied in total. In addition, most soil invertebrates live hidden within the soil and/or are too small to be studied directly. 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. It can often be necessary to carry out an exploratory sampling programme before the final study design can be defined in detail. The main points on which decisions shall be made are listed in 4.2, reflecting the logical order of how to proceed a study.

NOTE This clause was written in close consideration with ISO 10381‑1.

The objective of a study can be established by 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 right 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?

— Are the requirements of ISO 10381-3, covering guidance on safety in sampling programmes, as well as those safety issues listed in other parts of this International Standard (ISO 23611‑1 to ISO 23611‑5) 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 part of ISO 23611?

— Is additional information required?

— How is it ensured that any later deviation from this part of ISO 23611 or the study plan is documented and distributed?

Answers to these questions are given in Clause 5 to Clause 8.

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 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 has currently been prepared by 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.

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 like being representative for certain regions or land-use forms or lack of contamination, Reference [18].

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 part of ISO 23611. In addition, capture-recapture methods – while often used in ecology for terrestrial above-ground invertebrates (e.g. spiders, Reference [19]) are rarely used in general monitoring schemes and thus will not be covered in this part of ISO 23611.

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 may or may not be separately sampled (samples shall be labelled accordingly).

To adequately support legal or regulatory action, particular attention should be paid to all aspects of quality assurance. The guidance given in ISO 10381-5 is particularly relevant. After clarifying the most important pre-conditions, the four groups of main objectives as given above are briefly presented in the following subclauses. However, it should be kept in mind that, in reality, one specific study can fit into more than one of these groups.

Before designing a field study with soil invertebrates, it is highly recommended to characterize the respective area pedologically, Reference [20]. 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 ISO 10390, ISO 10694, ISO 11260, ISO 11272, ISO 11274, ISO 11277, ISO 13878, ISO 11461, ISO 11465, ISO 15709, ISO 17616.

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 determine the nature and extent of contamination, to identify hazards associated with the contamination, to identify potential targets and routes of exposure, and to evaluate 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].

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 5.4).

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 activity). The information gained here can be used for the preparation of biological soil maps, Reference [21].

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 5.3) or of long-term changes such as global warming (5.7).

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).

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, Reference [20]. Geostatistics are used in the development of sampling strategies and are also used to analyse the data generated from the soil sampled, Reference [22]. In this clause which closely follows the terminology used in ISO 10381‑1:2002, Annex C, several (standard) options and issues to be considered are given.

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 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 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 an area of about 1 000 m² is divided into four squares of 250 m² 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 macro 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.

The selection of sampling locations depends upon the study objectives, preliminary information, and on-site conditions, Reference [20]. Examples of on-site conditions that need to 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) or the direction of contamination, Reference [13].

Identification of sampling points is not always necessary. However, where samples are taken at pre-defined points, their accurate location and identification is important for three 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 GPS (Global Positioning System) for geographic coordinates is highly recommended to identify the sampling points. The location of sampling points should be marked before sampling begins, using poles or markers of colour sprays.

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: establishment of safety measures or 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 may 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.

Details of the sampling performance are given in ISO 23611‑1 to ISO 23611‑5. However, some general advice can be given in the following:

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] 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 of animals) are given in ISO 23611‑1 to ISO 23611‑5 and, in particular for contaminated soils, in Reference [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 programmes (see Clause 10), meaning, for example, that in order to facilitate data documentation, specific forms (e.g. chain-of-custody forms used during transport of samples from the field to the laboratory) are used.

Specific attention is drawn to the requirements for sampling personnel and to the safety precautions necessary in various situations (see ISO 10381‑3).

All important information on the sampling programme should be laid down in a sampling plan which provides specific guidance for the methods and strategies for data and sample collection. The sampling plan should – at least – contain a description of the study objective, a characterization of the site, a description of the experimental design, the sampling procedure, and the end points to be measured. Already at this stage, besides personnel experienced in soil ecology, experts from other areas, like site managers, statisticians or soil scientists, should be consulted.

In any case, it is recommended to prepare a sampling check list, as well as a field observation notes check list, Reference [13]. The former contains the sampling plan, as well as detailed information about the site location, the sampling locations, the sampling devices and procedures, documentation material and devices, packaging and storage material and general field equipment including health and safety equipment. The latter includes prepared forms etc. on soil sampling, sample handling, field measurements, on-site observations and storage and transportation forms.

The design of the sampling programme needs to take into account the sampling experience of the personnel and their ability to contribute to the design of the sampling programme relative to the investigation needs (see ISO 10381‑1). Sampling should preferably be carried out by an experienced scientist or another appropriately qualified person. The sampler should have a knowledge of the applied techniques and tools (see ISO 103812). Sampling depends on team work. Responsibilities should be made clear at all stages of the sampling campaign, both in the field and at the office. Staff working on the site should have detailed knowledge about necessary safety precautions, particularly when sampling contaminated sites (see ISO 10381‑3).

A preliminary survey should be carried out prior to any sampling programme, although the effort devoted to it depends on the objective of the investigation. It should always comprise a desk-top study (see 7.4.2) and a site visit. In addition, a limited amount of sampling may be carried out (see 7.4.3). The principal objectives of the preliminary study are to gain knowledge about the present condition of the site, and of past activities on the site and adjacent land which can have affected it in order to enable the sampling programme to be designed to be both technically effective and cost effective. In addition, measures shall be identified that protect the health and safety of the investigating personnel and of the environment.

Other information relevant to conduct the sampling programme may also be gathered (e.g. means of access, availability of power). It shall also be ensured that all necessary permits for carrying out the preliminary survey (e.g. for site access) have been obtained. Such information is of particular relevance when investigations for risk assessment shall be carried out.

This step includes collection of relevant information of the site, e.g. references to the location, infrastructure, utilization or historical information. Possible sources of this information are publications, maps, aerial photographs and satellite imagery from, for example, land surveyor’s offices, geological surveys, industrial inspection boards, mining companies, regional archives, or agriculture and forestry authorities. Particularly important is information on the physical and chemical properties and the possible spatial distribution of the soil parameter(s) relevant for the investigation. In addition, ecological information (such as geological, hydrogeological, botanical, and pedological classification of the site) shall be collected. In some cases it can even be possible to classify the site to a certain ecoregion or ecozone.

A visit of the site should preferably be done in conjunction with the desk-top study. Depending on the local situation and the objectives of the study, an experienced person should be chosen for this task. Such a visit gives a first impression about the correlation between existing maps and site reality and provides much additional information in a comparatively short time. Samples are not often taken during preliminary surveys. If they are, it is usually for obtaining an overview of the type of soil in order to choose the right equipment for later activities (see also ISO 10381‑4 to ISO 10381‑6). For example, screening soil samples by means of a Pürckhauer corer^[1] can be taken to become acquainted with the soil profile and the heterogeneity of soil properties. An inventory of plant indicator species as part of a vegetation survey can also be helpful. The output of the preliminary survey can include a first or additional map of the site, as well as a compilation of all available information in the form of a report.

Maps can be prepared by following either of two methods:

— The sampling grid is plotted onto an existing map. The sampling units are coloured according to the result of the respective sampling.

— Cartography programmes can be used that interpolate between adjacent sampling points. It is important to ascertain that the topography of the survey region and the density of sampling units allow the use of such algorithms.

As already mentioned, the aims of sampling soil invertebrates can be very different, leading to a high number and diversity of design options. Since it is by far not possible to cover these options here, in Clause 8 representative examples together with references are given for the most often used purposes.

As stated already in Clause 4, soil organism communities are characterized by high (but differing according to the individual group) variability of populations in time and space. While options for handling the spatial variability have already been discussed in Clause 6, addressing variability in time is difficult on a general level. Mainly it depends on the objective of the study (meaning that in many cases just one sampling is possible or necessary) but as a general rule it can be stated that – if possible – at least two samplings in different years should be performed.

From an assessment point of view, again different designs and statistical methods shall be applied depending on the respective objective. However, some very general recommendations can be given:

Site-specific risk assessment of contaminated land:

— e.g. after single events like an oil-spill: BACI-Design (before – after - control – impact), followed by GLM (generalized linear models) to compare single variables (e.g. species richness) or differences in community composition after using multivariate analysis (e.g. comparing control vs impacted similarities before and after impact). Data distributions like normal, Poisson for counts or binomial are acceptable for analysis using GLM. When existing correlation between sampling units in time or in space or when sampling units are hierarchically arranged (e.g. nested design), generalized-mixed effect models shall be applied to enable evaluation of autocorrelated data with the distributions covered with GLM. Alternatively, when more variables are evaluated together, PCA (Principal Component Analysis) or CA (correspondence analysis) can be used for exploratory analysis. In the case of a point source of impact and when explanatory variables are available (e.g. soil properties), gradient design followed by a regression type analysis (e.g. linear regression or generalised linear models) or canonical analyses (e.g. RDA – Redundancy analysis or CCA – canonical correspondence analysis) can be used, depending on character of data. In case of studies based on a randomized block design with difference degrees of impact/contamination (i.e. many treatments against one control), the closure principle computational approach test can be used to assess effects on soil communities (either using species richness or community composition^[23]).

Study of potential side effects of anthropogenic impacts (e.g. the application of chemicals or the building of roads):

— e.g. usually block or factorial design, followed by GLM (general linear models).

The biological classification and assessment of soils in order to determine the biological quality of soils or long-term biogeographical monitoring in the context of nature protection or restoration:

— e.g. representative sampling with determination of means and error estimation of species abundance/density or derivation of several diversity descriptors (e.g. diversity indices). Data should be compared with NOR values (Normative operating range) if existing, or contribute to its definition (coupling field data and modelling approaches).

Soil invertebrates cover a wide spectrum of life-form and life-history types, which inhabit a multitude of soil habitats and niches that themselves can be spatio-temporally variable, Reference [1]. For this reason, no one sampling method can assess the entire soil fauna and different standardized methods are necessary for evaluating the various taxonomic, functional or life-history soil-faunal groups, Reference [24].

The assessment of soil fauna often draws upon their significance as reaction- or impact-indicators, whereby changes in life-history patterns or the abundances of single species or entire communities indicate changes in soil biology, chemistry or physics, Reference [25]. This can take place through direct indication, which often utilizes single test or monitoring species to assess the impact of known contaminants, or through indirect indication, whereby single species or entire communities are used to indicate habitat conditions or their changes, References [26], [27], [28], [29]. Especially, indirect indication usually finds an application in environmental soil protection, as well as in the biological classification and assessment of soils. In this regard, entire communities of a higher-level taxon (multi-species assemblage or taxocoenosis) or guilds (functionally defined multi-taxon groups) are commonly assessed due to the integrative ability of different species to respond at different degrees to a similar impact or differentially to different habitat factors, References [17], [30], [31], [32]. This increases the amount of information available and allows an integrative evaluation, but also increases the complexity of assessment.

For a community-level biological assessment or characterization of soils, the investigated faunal indicator should ideally fulfil important prerequisites, References [25]:

— measurable (related to the availability of the necessary laboratory equipment and technical skills);

— efficient and cost-effective (considering capital and consumable costs as well as the labour intensiveness in the field and the laboratory) registration (collection) of the individuals of the taxon through standardized methods;

— fit for use (meaningful, spatiotemporally relevant, understandable and open to standardisation). medium to high densities and species richness;

— unambiguous ascertainability of active stages;

— unambiguous reference to a closely defined habitat due to low radius of activity;

— rapid response ability to environmental changes due to an advantageous voltinism (one to many generations per year);

— good characterization of life forms, nutritional demands, autecology, etc.;

— sensitivity to habitat changes such as land use and disturbance;

— policy relevant (to provide data on biodiversity and ecosystem functions for informed decision making).

Furthermore, for community-level use of soil fauna as reaction indicators in environmental soil-protection or biological classification and assessment of soils, field sampling shall allow the collected data to incorporate certain requirements:

— To allow a thorough biological soil assessment, the species assemblages (taxocoenosis or functional group) shall be sampled as representatively as possible, avoiding omission of important species.

— Since the most abundant soil mesofauna species are often eurytopic and euryoecous species, Reference [33], thus not allowing sufficient indicative site differentiation, not only these species, but especially also secondary or corollary species, shall be included in the sampled communities (for the discussion of specific problems when using mesofauna, see Reference [34]).

— The spatial distribution of the individual samples within a plot shall take into account the patchy distribution of the communities and species (see below).

— Despite the above requirements, sampling shall remain cost effective.

Sampling is complicated by the strongly aggregated occurrence of the soil mesofauna, causing a large spatial and temporal heterogeneity and thus non-normal distribution including many gaps and patches, References [35], [36], [37], [38], [39]. For example, on average 25 to 35 soil cores (each 76 cm²) are necessary to obtain representative data on the taxonomic diversity of large soil invertebrates of Russian forest sites, Reference [40]. However, there are indications that in some regions no such detailed numbers can be given yet: for example, when sampling micro-arthropod communities in Amazonian savannahs, it seems that as more plots were sampled, more species were recorded, Reference [41]. This heterogeneous horizontal distribution of soil organisms creates high demands on the sampling design to achieve the above requirements, mostly in terms of sampling intensity (number of samples). Most studies involve a compromise between sampling intensity (data precision) and cost effectiveness. Differences in sampling design, however, can cause methods-based discrepancies within the data, thereby limiting the value of temporal (within site) or spatial (between site) comparisons. Thus, the use of standardized methods for data collection is as important as that for data analysis or evaluation methods.

Two main categories of sampling strategies can be distinguished: deterministic and probabilistic ones. In deterministic (often called judgmental) sampling, sample locations are selected based on expert knowledge of the site or on professional judgment. With probabilistic sampling strategies, sampling locations are selected by applying statistical theory and the application of random chance to location selection. Judgmental sampling strategies can be less expensive and more efficient than probabilistic strategies, however they depend heavily on expert knowledge, there is no way to measure the precision of the data, and the data cannot be interpreted statistically. In contrast, probabilistic sampling strategies are more difficult to implement (often requiring the assistance of a statistician), but, when used, the uncertainty in the data can be measured and quantitative conclusions can be made.

When choosing the adequate strategy (either deterministic or probabilistic), the selected sampling points shall be identified and marked in situ to facilitate field work using GPS (Global Positioning System) or similar methods. In fact, statistical methods shall be used on any scale, i.e. for the selection of sampling points on a sample plot but also for the identification of sampling sites. Once the results of sampling are obtained, they should be evaluated regarding their representativity for the plot, site or landscape under investigation using geostatistical methods (for further advice see Reference [13] where this issue is discussed in detail).

The EU project ENVASSO aimed to design a single, integrated and operational set of EU-wide criteria and indicators to provide the basis for a harmonised comprehensive soil and land information system for monitoring in Europe, References [42], [43]. For the purpose of long-term monitoring of soil biodiversity the following recommendations were proposed by a large working group consisting of experts experienced in soil biology sampling and monitoring, References [44], [45], [46], [47]. While this proposal was originally developed in the context of EU-wide monitoring, as proposed in the Draft Soil Framework Directive of the European Union, References [48], [49], the indicators proposed can also be used for other purposes, such as the study of potential side effects of anthropogenic impacts or the biological classification and assessment of soils. It can be divided into three steps. The biological indicators were selected for three different levels (Triad approach) and should always be used in combination with a detailed site and soil characterization.

a) Step 1: Site description and soil characterization according to

— ISO 23611‑1;

— Land management, land use and vegetation type should follow FAO 2006 classification (https://www.fao.org/4/a0541e/a0541e.pdf).

— Soil type should follow WRB 2022 (https://www.isric.org/sites/default/files/WRB_fourth_edition_2022-12-18.pdf), or a referred international soil classification as FAO 2006.

b) Step 2: Installation of the sampling area (surface definition, localization, replicates)

— Sampling area shall be about 100 m².

— If there is an existing monitoring network which assesses the site and soil characteristics (“conventional” monitoring area), in order to use the collected data (e.g. climatic, land use, physicochemical analysis) the biological sampling area should be located inside the ‘conventional’ monitoring area or, nearby the ‘conventional’ monitoring area (5 m from the conventional area at the most).

— If there is no monitoring network, complementary analyses shall be performed on a composite sample from the investigated area to explain/interpret biodiversity data (required parameters).

— Localization of the sampling area in a homogeneous area (based on pedological characteristics and soil cover).

— Record the location of the sampling area position with a differential GPS device.

— Sampling strategy: minimum of three replicates, with equal distance between subplot/replicates.

c) Step 3: Soil sampling area preparation

— Cut the vegetation or take off the soil cover as mulch without damaging the soil surface.

— In the case of forest: take the litter and put it in a plastic sample bag in order to assess the fauna in the laboratory.

To interpret the biological data, several soil analyses are generally required as follows:

— pH in accordance with ISO 10390;

— soil moisture content in accordance with ISO 11465;

— organic carbon, total carbon in accordance with ISO 10694;

— heavy metal analysis in accordance with ISO 14869-1, and ISO 11466;

— texture in accordance with ISO 11277.

NOTE 1 These recommendations made for monitoring purposes can be relevant for other purposes, too.

A proposal was also made for a set of suitable indicators for monitoring soil biodiversity, Reference [45] (see Table 1). Indicators were selected both from a literature review and an inventory of national monitoring programmes. Soil biodiversity was defined as the forms of life (genes, species and, rarely, higher level) living in soils (both in terms of quantity and variety) and of related functions. To select level I indicators, three stringent criteria were applied: an indicator should:

1) have a standardized sampling and/or measuring methodology,

2) be complementary to other indicators, and

3) be easy to interpret at both scientific and policy levels.

The level I indicators were chosen as representative of three different taxonomical groups and functional levels:

— abundance, biomass and species diversity of earthworms – macrofauna (see Note 2);

— abundance and species diversity of Collembola – mesofauna;

— microbial respiration.

Biodiversity (species level), as well as ecological functions of soil organisms, are covered by these groups and levels. In principle, when considering soil biodiversity, all soil organisms and the biological functions which they provide are important and should be assessed. However, for priority level I (Table 1) three indicators were selected to act as surrogate measures for overall biodiversity. Depending on the availability of resources and specific requirements, this minimum set of indicators could be extended to include priority levels II and III (Table 1), e.g. all macrofauna, nematode diversity, bacteria and fungi diversity and activity, faunal activity as biogenic structures or feeding activity. The three priority levels I selected indicators are as given in Table 1.

NOTE 2 When earthworms are not supposed to be found (e.g. in acidic soils) the diversity and abundance of enchytraeids should preferably be measured.

Table 1 — Priority level of indicators for decline in soil biodiversity (ENVASSO)

The sampling report shall refer to this part of ISO 23611 and shall contain a summary of the methods and parameters used during the study and the results obtained. It shall provide the following information:

a) detailed description of the study objective used;

b) characterization of the study site (especially soil properties), including the coordinates of the sample location(s);

c) a full description of the experimental design and procedures;

d) sampling procedure;

e) all modifications or changes compared to the methods described in ISO guidelines (in particular ISO 23611‑1 to ISO 23611‑5);

f) description of the sampling conditions, including date and duration of sampling in the field and climatic parameters like air temperature;

g) unambiguous sample identification numbers;

h) all information, including all measured raw data and all problems that might have occurred, developed during all phases of the study;

i) discussion of the results.

NOTE In addition, chain-of-custody forms can be important when samples are required for legal purposes.

The goal of QA/QC programmes is to identify, measure, and control the errors associated with every component of a sampling study, including planning, sampling, testing and reporting, Reference [13]. Because of the various reasons for and objectives of sampling, there can be no single set of quality control and quality assurance procedures to be followed by all organizations offering sampling services under all circumstances. However, it is strongly recommended that, as far as practicable, the guidelines set out in ISO 9000 should be followed. In particular, the preparation of a sampling plan, the inclusion of qualified personnel in planning and performing the work, as well as the detailed documentation of all steps of the study (and here especially of the field work), is of utmost importance.

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1. Pürckhauer corer is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. ↑