CEN/TC 444/WG 4 - Biological characterization

This document specifies a method for sampling, extracting and preserving macro-invertebrates from soils, including the litter zone.
The sampling and extraction methods in this document are applicable to almost all types of soil, with the exception of soils in extreme climatic conditions (hard, frozen or flooded soils) and matrices other than soil, e.g. tree trunks, plants or lichens.

This document specifies the crucial steps of a quantitative real-time polymerase chain reaction (qPCR) method to quantify the abundance of specific mRNA molecules extracted from Daphnia magna.
The method allows the identification of molecular responses to exposures for potentially toxic substances through the analysis of the abundance of specific mRNA molecules. In this document, the central genes involved in reproductive and toxic responses are included.
NOTE   The selection of genes can be adapted to specific exposure conditions, for example, exposure to known toxic substances, by adding genes known to respond to a specific insult.
The present method allows for rapid, robust and sensitive detection of molecular responses and can be used to analyse the toxic effects of water leachates from soil and waste. The method gives information of the concentration of a substance or test-liquid at which toxic effects begin to occur prior to observations of reproductive or toxic effects at higher levels of organization, which reduces the need for the use of safety factors in toxicity assessment.
The method is useful in several types of risk assessment. In this document, the genes studied are appropriate for the assessment of the risks when recycling materials and for the classification of waste, but the method can be adapted to other types of risk assessment by including other genes.

This document specifies a rapid method for assessing solid samples in an aerobic suspension, by determining the inhibition of dehydrogenase activity of Arthrobacter globiformis using the redox dye resazurin.
It is applicable for assessing the effect of water-soluble and solid matter bounded non-volatile contaminants in natural samples, such as soils and waste materials. Although not the main purpose, the contact test can additionally be used for testing the effect of chemicals, as described in the Annex C. The test yields a result within 6 h and can therefore be used for screening potentially contaminated test material.

This document specifies a method for sampling, extracting and preserving collembolans and mites from field soils as a prerequisite for using these animals as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms).
The sampling and extraction methods of this document are applicable to almost all types of soils. Exceptions can be soils from extreme climatic conditions (hard, frozen or flooded soils) and other matrices than soil, e.g. tree trunks, plants or lichens.

Ecotoxicity tests can be applied to wastes to identify their potential hazardous properties with respect to the environment or to assess the risk related to a site-specific exposure scenario. This document provides guidance for the selection and use of ecotoxicity tests for both applications.
This document focuses on the following selected field of applications:
a)   Basic ecotoxicological characterization;
b)   Site-specific exposure scenario;
c)   Landfill management:
1)   monitoring of leachates;
2)   mineral waste going to non-controlled landfill sites.
d)   Re-use of waste:
1)   use of sludge in agriculture;
2)   use of mineral waste in road construction.
The user should be aware that other fields of application can also be covered by ecotoxicological testing not being in the scope of the document. The ecotoxicological assessment of waste within other scenarios might need the development of other test strategies.
Depending on the waste type and the assessment goal, relevant criteria are described for the selection of a test strategy and the suitable ecotoxicity test(s).
This document also provides guidance for individual ecotoxicity test protocols to meet the specific demands of waste testing (e.g. limitations, test design, confounding factors).The tests recommended represent a minimum test battery that may be accomplished by additional tests or even be replaced by others according to the waste, the intended use or protection goal envisaged.

This document specifies one of the methods for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Folsomia candida Willem by dermal and alimentary uptake. This document also provides information on how to use this method for testing substances under temperate conditions.
The chronic test described is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, industrial, agricultural or other sites of concern and waste materials.
The method is not applicable to volatile substances, i.e. substances for which H (Henry's constant) or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 300 Pa at 25 °C.

This document specifies one of the methods for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Enchytraeus sp. by dermal and alimentary uptake in a chronic test. It is applicable to soils and soil materials of unknown quality, for example, from contaminated sites, amended soils, soils after remediation, agricultural or other sites under concern and waste materials.
This document provides information on how to use this method for testing substances under temperate conditions.
The method is not applicable to substances, for which the air/soil partition coefficient is greater than 1, or to substances for which the vapour pressure exceeds 300 Pa at 25 °C.
NOTE      No provision is made in the test method for monitoring the persistence of the substance under test.

This document specifies one of the methods for evaluating the habitat function of soils and determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites concerned, and waste materials.
This method is designed mainly for determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method also includes technical information on how to use it with other environmentally relevant earthworm species: e.g. Dendrodrilus rubidus and Aporrectodea caliginosa (see Annexes B and C).
This method does not apply to substances for which the air/soil partition coefficient is greater than one, or to substances with vapour pressure exceeding 300 Pa, at 25 °C. This method does not take into account the persistence of the substance during the test.

This document is one of a family of International Standards providing guidance on soils and soil materials in relation to certain functions and uses including conservation of biodiversity. It applies in conjunction with these other standards. It provides guidance on the selection of experimental methods for the assessment of the ecotoxic potential of soils and soil materials (e.g. excavated and remediated soils, refills, embankments) with respect to their intended use and possible adverse effects on aquatic and soil dwelling organisms.
NOTE       This is a reflection of the maintenance of the habitat and retention function of the soil. In fact, the methods listed in this document are suitable for usage in a TRIAD approach, i.e. for an ecological assessment of potentially contaminated soils (see ISO 19204).
This document does not cover tests for bioaccumulation.
The ecological assessment of uncontaminated soils with a view to natural, agricultural or horticultural use is not within the scope of this document. Such soils can be of interest if they can serve as a reference for the assessment of soils from contaminated sites.
The interpretation of results gained by applying the proposed methods is not in the scope of this document.

ISO 19204:2017 describes in a general way the application of the soil quality TRIAD approach for the site-specific ecological risk assessment of contaminated soils. In detail, it presents in a transparent way three lines of evidence (chemistry, ecotoxicology and ecology) which together allow an efficient, ecologically robust but also practical risk assessment of contaminated soils. This procedure can also be applicable to other stress factors, such as acidification, soil compaction, salinization, loss of soil organic substance, and erosion. However, so far, no experience has been gained with these other applications. Therefore, this document focuses on soils contaminated by chemicals.
NOTE 1       This document focuses on ecological risk assessment. Thus, it does not cover human health end points.
In view of the nature of this document, the investigation procedure is described on a general level. It does not contain details of technical procedures for the actual assessment. However, this document includes references relating to technical standards (e.g. ISO 15799, ISO 17616) which are useful for the actual performance of the three lines of evidence.
In ecological risk assessment, the effects of soil contamination on the ecosystem are related to the intended land use and the requirements that this use sets for properly functioning soil. This document describes the basic steps relating to a coherent tool for a site-specific risk assessment with opportunities to work out site-specific details.
ISO 19204:2017 can also be used for the evaluation of clean-up operations, remediation processes or management measures (i.e. for the evaluation of the environmental quality after having performed such actions).
NOTE 2       This document starts when it has already been decided that an ecological risk assessment at a given site needs to be performed. In other words, the practical performance of the soil quality TRIAD and the evaluation of the individual test results will be described. Thus, nothing will be said about decisions whether (and if yes, how) the results of the assessment are included in soil management measures or not.
NOTE 3       The TRIAD approach can be used for different parts of the environment, but this document focuses mostly on the soil compartment. Comparable documents for other environmental compartments are intended to be prepared in addition (e.g. the terrestrial aboveground compartment) in order to perform a complete site assessment, based on the same principles and processes.

This document is one of the family of standards (ISO 15799, ISO 19204) providing guidance on the characterization of soils and soil materials in relation to their retention and habitat functions and uses. It is appropriate to use it in conjunction with the two other standards in this family. It provides guidance on the choice and evaluation of tests applied for ecotoxicological characterization of soils and soil materials. Recommendations for test strategies with respect to the protection of ground and surface waters and the maintenance of the habitat function of soil are included. The tests recommended represent a minimum test battery that can be complemented by additional tests, or even be replaced by others, according to the intended uses or protection goals envisaged. The effect values indicated in this document do not refer to regulation but represent the lowest level at which an adverse effect is considered likely to occur.

This document specifies a method for sampling and handling free-living nematodes from terrestrial field soils as a prerequisite for using them as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms).
This document applies to all terrestrial biotopes in which nematodes occur. The sampling design of field studies in general is specified in ISO 18400-101.
This document is not applicable to aquatic nematodes because of differences in the sample matrix (e.g. water column). Methods for some other soil organism groups such as earthworms, collembolans enchytraeids or macro-invertebrates are covered in ISO 23611-1, ISO 23611-2, ISO 23611-3 and ISO 23611-5.
This document does not cover the pedological characterization of the site which is highly recommendable when sampling soil invertebrates. ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 and ISO 11465 include suitable procedures for measuring pH, particle size distribution, C/N ratio, organic carbon content and water-holding capacity.

This document describes a method to assess the bioaccumulation of chemicals in snails, i.e. concentrations of metal(loid)s (ME) or organic compounds [e.g. polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)] accumulated in their tissues.
This document presents how to prepare snails for caging in situ for 28 days, the in situ test design and then how to collect and prepare the snails until conservation and further analysis. If a kinetic study of accumulation is necessary, sampling of snails at different time-points during exposure is possible as well [13],[19],[22].
This document excludes analytical methods. Preparation (extraction and mineralization) of the samples and quantification of chemicals are not in the scope of the present document.
The method is applicable for soils under different uses (agricultural, industrial, residential, forests, before and after remediation, on potentially contaminated sites, etc.) and waste materials [8],[10], preferably with vegetation and/or humus cover.
The method is applicable subject to certain limits of temperature (frost-free period, i.e. mainly from April to October in temperate region).
Optionally (see Annex I), the method can be used in the laboratory to evaluate the accumulation of contaminants [and optionally, the sum of excess of transfer (SET) index for ME, PAH, PCB] of snails exposed only to soil.

This document describes the necessary steps to be performed before carrying out ecotoxicity tests on wastes. The purpose of this document is to provide guidance on the taking of the sample, transport, storage of wastes and to define preparation, for the determination of ecotoxicological properties of wastes under the conditions specified in this document by biological testing either as raw wastes or water extracts from wastes. Sample preparation for other applications (e.g. assessment of waste effects on aquatic and terrestrial organisms in a disposal scenario) is not considered.
Specifying a test battery to characterize ecotoxicological properties of wastes is not in the scope of this document.
This document is applicable to solid and liquid wastes.

This document specifies one of the methods for evaluating the habitat function of soils and determining effects of soil contaminants and individual chemical substances on the reproduction of the oribatid mite Oppia nitens by dermal and alimentary uptake. This chronic (28-day) test is applicable to soils and soil materials of unknown quality (e.g., contaminated sites, amended soils, soils after remediation, agricultural or other sites under concern and waste materials). This method is not intended to replace the earthworm or Collembola tests since it represents another taxonomic group (= mites; i.e., arachnids), nor the predatory mite test since this species represents a different trophic level and ecological niche.
Effects of substances are assessed using standard soil, preferably a defined artificial soil substrate. For contaminated soils, the effects are determined in the test soil and in a control soil. According to the objective of the study, the control and dilution substrate (dilution series of contaminated soil) should be either an uncontaminated soil with similar properties to the soil sample to be tested (reference soil) or a standard soil (e.g., artificial soil).
Information is provided on how to use this method for testing substances under temperate conditions.
This document is not applicable to substances for which the air/soil partition coefficient is greater than 1, or to substances with vapour pressure exceeding 300 Pa at 25 °C.
NOTE    The stability of the test substance cannot be assured over the test period. No provision is made in the test method for monitoring the persistence of the substance under test.

This document specifies a simple method for the extraction of only phospholipid fatty acids (PLFA) from soils.

This document specifies a method for sampling, handling and extracting enchytraeids from terrestrial field soils as a prerequisite for using these animals as bioindicators (e.g. to assess the quality of a soil as a habitat for organisms).
Basic information on the ecology of enchytraeids and their use as bioindicators in the terrestrial environment is included in the Bibliography.
This document applies to all terrestrial biotopes in which enchytraeids occur. The sampling design of field studies in general is given in ISO 18400-101. These details can vary according to the climatic/regional conditions of the site to be sampled and an overview on the determination of effects of pollutants on enchytraeids in field situations is given in Reference [6].
Methods for some other soil organism groups such as earthworms or arthropods are given in ISO 23611-1, ISO 23611-2, ISO 23611-4 and ISO 23611-5.
This document is not applicable for very wet or flooded soils and might be difficult to use under extreme climatic or geographical conditions (e.g. in high mountains).
When sampling soil invertebrates, it is highly recommendable to characterize the site (e.g. concerning soil properties, climate and land use). However, such a characterization is not covered by this document. ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 and ISO 11465 are more suitable for measuring pH, particle size distribution, C/N ratio, organic carbon content and water-holding capacity.

This document specifies a method for determining the activity of dehydrogenases enzymes in soil using 2,3,5-triphenyltetrazolium chloride (TTC).

This document specifies a method for determining activity of dehydrogenases in soil, using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT)[1]-[5]. As the INT reduction is less sensitive to O2, the method is more robust than the TTC-method described in ISO 23753-1.

This document specifies a semi-static method for determining the effects of contaminants on growth and survival of young snails, usually Helix aspersa aspersa Müller. The animals are exposed via the cutaneous and digestive route using a test substrate (artificial or natural soil according to the objective of the study) to which defined amounts of the following are added:
-      substances, mixtures or preparations;
-      soils (contaminated or of unknown quality) or waste materials.
This test takes into account the possible changes in the test substance, preparation, soil or waste material because the test mixtures are prepared and renewed every week during the 28-day test period.
A static method may be implemented in addition to the semi-static method (optional). This method is described in Annex A.
This method does not apply to substances for which the air/soil partition coefficient is greater than one, or to substances with vapour pressure exceeding 300 Pa, at 25 °C.

This document specifies a method for sampling and handling earthworms from field soils as a prerequisite for using these animals as bioindicators (e.g. to assess the quality of a soil as a habitat for organisms).
This document applies to all terrestrial biotopes in which earthworms occur. The sampling design of field studies in general is given in ISO 18400‑101 and guidance on the determination of effects of pollutants on earthworms in field situations is given in ISO 11268‑3. These aspects can vary according to the national requirements or the climatic/regional conditions of the site to be sampled (see also Annex C).
This document is not applicable for semi-terrestrial soils and it can be difficult to use under extreme climatic or geographical conditions (e.g. in high mountains). Methods for some other soil organism groups, such as collembolans, are covered in other parts of ISO 23611.

ISO 17601:2016 specifies the crucial steps of a quantitative real-time polymerase chain reaction (qPCR) method to measure the abundance of selected microbial gene sequences from soil DNA extract which provides an estimation of selected microbial groups.
It is noteworthy that the number of genes is not necessarily directly linked to the number of organisms that are measured. For example, the number of ribosomal operon is ranging from one copy to 20 copies in different bacterial phyla. Therefore, the number of 16S rRNA sequences quantified from soil DNA extracts does not give an exact estimate of the number of soil bacteria. Furthermore, the number of sequences is not necessarily linked to living microorganisms and can comprise sequences amplified from dead microorganisms.

ISO 18311:2016 specifies a technique for determining the effects of anthropogenic impacts (e.g. substances) in the context of the prevailing environmental conditions on the feeding activity of soil organisms in the field. In addition, the use of this method for monitoring the biological quality of soil is described (see Annex A). The breakdown of organic matter by soil invertebrates and microorganisms is a crucial process that determines important soil functions such as nutrient availability for plants and the maintenance of soil fertility. In addition, decomposing plant litter provides habitats and food for a wide range of organisms, thus supporting biodiversity and ecosystem services [33][34].
ISO 18311:2016 is applicable to all soils in which soil organisms are active. The use of the bait-lamina test is independent from whether there is a litter layer or not. The sampling design of field studies in general is specified in ISO 23611‑6 (see also Reference [20]). The design can vary according to the aim of the study as well as conditions (e.g. soil properties, contamination, etc.) of the site to be investigated.
ISO 18311:2016 is not applicable for semi-terrestrial or very shallow soils. It can be difficult to use it under extreme climatic or geographical conditions (e.g. in high mountains).

ISO 11268-3: 2014 specifies techniques for determining the effects of substances on earthworms in the field and provides a basis for determining the effects of chemicals applied to or incorporated into soil, including soil injections or drilled pelleted formulations.

1 Scope
This part of ISO 11268 specifies one of the methods for evaluating the habitat function of soils and determining the acute toxicity of soil contaminants and chemicals to Eisenia fetida/Eisenia andrei by dermal and alimentary uptake. It is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites concerned, and waste materials.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For contaminated soils, the effects on survival are determined in the test soil and in a control soil. According to the objective of the study, the control and dilution substrate (dilution series of contaminated soil) should be either an uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard soil (e.g. artificial soil).
Information is provided on how to use this method for testing chemicals under temperate as well as under tropical conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry's constant) or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 Pa at 25 °C.
This method does not take into account the possible degradation of the substances or contaminants during the test.

ISO 16198:2015 specifies the plant-based test, called biotest, which enables estimation of the environmental bioavailability of trace elements to plants either basically as the concentration in shoots and roots or in a more integrative way as the net uptake flux in plants.

ISO/TS 29843‑1:2010 specifies an extended method for the extraction and determination of both phospholipid fatty acids (PLFA) and phospholipid ether lipids (PLEL) from soils.
ISO/TS 29843‑2 specifies a simple method for the extraction of only PLFA from soils.

This International Standard specifies laboratory procedures for measuring the mineralization and nitrification of nitrogen by the soil microbiota.
For investigations on the evaluation of soil quality or on effects of contamination, a procedure is given to measure the rates and extent of N-mineralization in soil or soils of known or unknown quality.
For investigation of the potential toxicity of chemicals to N-mineralization in soils, a simple procedure is given which allows the impact of single chemicals to be assessed and provides a basis for comparison of the toxicities of different chemicals.

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).

This Technical Specification specifies a test procedure for the determination of the content of unwanted viable weed seeds and plant propagules in growing media and soil improvers (see also Annex B for validation results).
The method in general is also applicable to soils and sludges.

This part of ISO 11269 describes a method to assess the quality of an unknown soil and the soil habitat function
by determining the emergence and early growth response of at least two terrestrial plant species compared
to reference or standard control soils. It is applicable to soils of unknown quality, e.g. from contaminated sites,
amended soils or soils after remediation.

ISO 11269-1:2012 describes a method for the determination of the effects of contaminated soils or contaminated samples on the root elongation of terrestrial plants.
This method is applicable to soils, soil materials, compost, sludge, waste or chemical testing. It is applicable to the comparison of soils of known and unknown quality and to the measurement of effects of materials (compost, sludge, waste) or chemicals deliberately added to the soil.
The method is not intended to be used as a measure of the ability of the soil to support sustained plant growth.

This Technical Report specifies three methods for the detection and enumeration of Escherichia coli in sludge, treated biowaste and soil:
-   Method A - Membrane filtration method for quantification (see Clause 6);
-   Method B - Miniaturised method (Most Probable Number, MPN) by inoculation in liquid medium (see Clause 7);
-   Method C - Macromethod (Most Probable Number) in liquid medium (see Clause 8).

ISO 22030:2005 describes a method for determining the inhibition of the growth and reproductive capability of higher plants by soils under controlled conditions. Two species are recommended: a rapid-cycling variant of turnip rape (Brassica rapa CrGC syn. Rbr) and oat (Avena sativa). The duration of test should be sufficient to include chronic endpoints that demonstrate the reproductive capability of the test plants.
By using natural test soils, e.g. from contaminated sites or remediated soils, and by comparing the development of the test plants in these soils with reference or standard control soils, the test is applicable to assess soil quality, especially the function of the soil as a habitat for plants.
This method can be modified to allow use of the chronic plant assay for the testing of chemicals incorporated into soil. By preparing a dilution series of a test substance in standard control soils, the same endpoints can be measured to assess the chronic toxicity of chemicals.
This method is not applicable to volatile substances, i.e. substances for which Henry's constant or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 Pa at 25 °C.

ISO 20963:2005 describes a method for the determination of the effects of contaminated soils and substances on the survival of the larvae of Oxythyrea funesta. The larvae are exposed to the pollutants by cuticular and alimentary uptake.
For contaminated soils, the effects on the survival are determined in the test soil and in a control soil. Depending on the objectives of the study, the control and dilution substrates (dilution series of contaminated soil) are either uncontaminated soil comparable to the soil sample to be tested or artificial soil substrate. Effects of substances are assessed using a defined artificial soil substrate.
ISO 20963:2005 is not applicable to volatile substances, i.e. substances for which Henry's constant or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,001 33 Pa at 25 °C.
This method does not take into account the possible degradation of the substances or pollutants during the test.

ISO 16702:2002 describes methods for the determination of soil microbial respiration of aerobic, unsaturated soils. The methods are suitable for the determination of O2 uptake or CO2 release, either after addition of a substrate (substrate-induced respiration), or without substrate addition (basal respiration).
ISO 16702:2002 is applicable to the measurement of soil respiration in order to:
determine the microbial activity in soil (see [2]);establish the effect of additives (nutrients, pollutants, soil improvers, etc.) on the metabolic performance of microorganisms;determine the microbial biomass (see [3]);determine the metabolic quotient qCO2.

Gives a method for the determination of microbial biomass of soils by measurement of total extractable organic biomass material mainly from freshly killed microorganisms. It is also applicable to the estimation of microbial nitrogen and ninhydrin-reactive nitrogen in soil.

Contains a method for the determination of the active aerobic, heterotrophic microbial biomass in aerated agricultural and mineral soils.

ISO 23611-5:2011 specifies a method for sampling, extracting and preserving macro-invertebrates from soils, including the litter zone. The proposed method is a prerequisite for using these animals as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms). The main premise of this method is rapid assessment (completing the sampling of a plot in one or two days with only basic equipment and a small number of field assistants), in order to be able to address all the taxonomic groups of soil macro-invertebrates at the same time and in the same place. The Tropical Soil Biology and Fertility (TSBF) method has evolved and some modifications have been introduced in order to use it in temperate regions.
The sampling and extraction methods in ISO 23611-5:2011 are applicable to almost all types of soils, with the exception of soils in extreme climatic conditions (hard, frozen or flooded soils) and matrices other than soil, e.g. tree trunks, plants or lichens.
Sampling design is specified in ISO 23611-6.

This document specifies the crucial steps of a quantitative real-time polymerase chain reaction (qPCR) method to quantify the abundance of specific mRNA molecules extracted from Daphnia magna.
The method allows the identification of molecular responses to exposures for potentially toxic substances through the analysis of the abundance of specific mRNA molecules. In this document, the central genes involved in reproductive and toxic responses are included.
NOTE   The selection of genes can be adapted to specific exposure conditions, for example, exposure to known toxic substances, by adding genes known to respond to a specific insult.
The present method allows for rapid, robust and sensitive detection of molecular responses and can be used to analyse the toxic effects of water leachates from soil and waste. The method gives information of the concentration of a substance or test-liquid at which toxic effects begin to occur prior to observations of reproductive or toxic effects at higher levels of organization, which reduces the need for the use of safety factors in toxicity assessment.
The method is useful in several types of risk assessment. In this document, the genes studied are appropriate for the assessment of the risks when recycling materials and for the classification of waste, but the method can be adapted to other types of risk assessment by including other genes.

ISO 18187:2016 specifies a rapid method for assessing solid samples in an aerobic suspension, by determining the inhibition of dehydrogenase activity of Arthrobacter globiformis using the redox dye resazurin.
It is applicable for assessing the effect of water-soluble and solid matter bounded non-volatile contaminants of natural samples, such as soils and waste materials. The test yields a result within 6 h and can therefore be used for screening potentially contaminated material.

ISO 23611-2:2006 specifies a method for sampling, extracting and preserving collembolans and mites from field soils as a prerequisite for using these animals as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms).
The sampling and extraction methods of ISO 23611-2:2006 are applicable to almost all types of soils. Exceptions may be soils from extreme climatic conditions (hard, frozen or flooded soils) and other matrices than soil, e.g. tree trunks, plants or lichens.

Ecotoxicity tests can be applied to wastes to identify their potential hazardous properties with respect to the environment or to assess the risk related to a site-specific exposure scenario. This document provides guidance for the selection and use of ecotoxicity tests for both applications.
This document focuses on the following selected field of applications:
a)   Basic ecotoxicological characterization;
b)   Site-specific exposure scenario;
c)   Landfill management:
1)   monitoring of leachates;
2)   mineral waste going to non-controlled landfill sites.
d)   Re-use of waste:
1)   use of sludge in agriculture;
2)   use of mineral waste in road construction.
The user should be aware that other fields of application can also be covered by ecotoxicological testing not being in the scope of the document. The ecotoxicological assessment of waste within other scenarios might need the development of other test strategies.
Depending on the waste type and the assessment goal, relevant criteria are described for the selection of a test strategy and the suitable ecotoxicity test(s).
This document also provides guidance for individual ecotoxicity test protocols to meet the specific demands of waste testing (e.g. limitations, test design, confounding factors).The tests recommended represent a minimum test battery that may be accomplished by additional tests or even be replaced by others according to the waste, the intended use or protection goal envisaged.

ISO 11267:2014 specifies one of the methods for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Folsomia candida Willem by dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, industrial, agricultural or other sites of concern and waste materials.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For contaminated soils, the effects are determined in the soil to be tested and in a control soil. According to the objective of the study, the control and dilution substrate (dilution series of contaminated soil) are either an uncontaminated soil comparable to the soil to be tested (reference soil) or a standard soil (e.g. artificial soil).
ISO 11267:2014 provides information on how to use this method for testing substances under temperate conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry's constant) or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 Pa at 25 °C.

ISO 16387:2014 specifies one of the methods for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Enchytraeus sp. by dermal and alimentary uptake in a chronic test. It is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites under concern and waste materials.
ISO 16387:2014 provides information on how to use this method for testing substances under temperate conditions.

ISO 11268-2:2012 specifies one of the methods for evaluating the habitat function of soils and determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites concerned, and waste materials.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For contaminated soils, the effects are determined in the test soil and in a control soil. According to the objective of the study, the control and dilution substrate (dilution series of contaminated soil) should be either an uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard soil (e.g. artificial soil).
Information is provided on how to use this method for testing chemicals under temperate as well as under tropical conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry's constant) or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 Pa at 25 °C.
This method does not take into account the persistence of the substance during the test.

ISO 23611-4:2007 specifies a method for sampling and handling free-living nematodes from terrestrial field soils as a prerequisite for using them as bio-indicators (e.g. to assess the quality of a soil as a habitat for organisms).
ISO 23611-4:2007 applies to all terrestrial biotopes in which nematodes occur. It is not applicable to aquatic nematodes because these nematodes do not pass through the filter.