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.

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The present document specifies a method for direct extraction of DNA from soil samples to analyse
the abundance and composition of microbial communities by various techniques of molecular biology
including real-time quantitative PCR (qPCR). This method is mainly dedicated to agricultural and forest
soils. This method can possibly not be suitable for soils rich in organic matter (e.g. peat soils) or soils
heavily polluted with organic pollutants or heavy metals.
The direct extraction of DNA from soil samples provides unique insight into the α- and β-diversity
of microbial communities. Next-generation sequencing of amplicons obtained by PCR (polymerase
chain reaction) amplification of soil DNA constitutes a promising domain which will in the near future
contribute to the development of routine tools to monitor microbial communities in soil environments.

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This document specifies a method for the measurement of several hydrolase activities (arylamidase, arylsulfatase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, acid, alkaline and global phosphatases, urease) simultaneously (or not) in soil samples, using colorimetric substrates. Enzyme activities of soil vary seasonally and depend on soil chemical, physical and biological characteristics. This method can be applied either to detect harmful effects on soil enzyme activities derived from toxic substances or other anthropogenic agents in contaminated soils against a control soil, or to test chemicals.

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The present document specifies a method for direct extraction of DNA from soil samples to analyse the abundance and composition of microbial communities by various techniques of molecular biology including real-time quantitative PCR (qPCR). This method is mainly dedicated to agricultural and forest soils. This method can possibly not be suitable for soils rich in organic matter (e.g. peat soils) or soils heavily polluted with organic pollutants or heavy metals. The direct extraction of DNA from soil samples provides unique insight into the α- and β-diversity of microbial communities. Next-generation sequencing of amplicons obtained by PCR (polymerase chain reaction) amplification of soil DNA constitutes a promising domain which will in the near future contribute to the development of routine tools to monitor microbial communities in soil environments.

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This document specifies a chronic test method for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Hypoaspis aculeifer by ? mainly ? alimentary uptake. This method 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 under concern and waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed material, or manure, especially those for possible land disposal). The reproduction (= number of juveniles) is the measured parameter of the test. The test reflects the bioavailability of a mixture of contaminants in natural soils (contaminated site soils) to a species which represents a trophic level which is not covered by other ISO standards. This test is not intended to replace the earthworm (see ISO 11268-2) or Collembola (see ISO 11267) reproduction tests since this species belongs not only to a different trophic group but also a different taxonomic group (= mites; i.e. arachnids) than those used usually.
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. Depending on 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).
This document provides information on how to use this method for testing samples (soils or substances) under temperate conditions.
This document is not applicable 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.
NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the test method for monitoring the persistence of the substance under test.

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This International Standard gives guidance on the selection and method of appropriate tests for the determination of biodegradation of organic chemicals in soil samples under anaerobic conditions.

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This International Standard provides guidance on the selection and conduct of appropriate test methods for
the determination of biodegradation of organic chemicals in aerobic soils. lt does not describe any specific test method.

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ISO 17512-1:2008 specifies a rapid screening method for evaluating the habitat function of soils and the influence of contaminants and chemicals on earthworm behaviour.
The sublethal test is a rapid method that reflects the bioavailability of contaminant mixtures in natural soils and substances spiked into soils to Eisenia fetida and Eisenia andrei. The avoidance behaviour of the worms is the measurement endpoint of the test. This test is not intended to replace the earthworm reproduction test.
Two different designs (a two section unit and a six section unit) have been developed and successfully applied. Both designs are applicable to either single-concentration (e. g. for assessing the quality of a field soil) or multi-concentration (e. g. for assessing the toxicity of a spiked chemical) tests. In both cases, the earthworms are allowed to make the initial choice on which compartment, control and a treatment [in the two section test vessel between right and left side; in the six section test vessel between the (3 + 3) alternating compartments], to enter.

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ISO 14239:2017 specifies six suitable incubation systems for measuring the rates and extent of mineralization of organic compounds in soil by measurement of carbon dioxide (CO2) evolution. All incubation systems are applicable to soluble or insoluble compounds but choice of system depends on the overall purposes of the study.
ISO 14239:2017 does not apply to the use of such systems for material balance studies, which are often test-substance specific.

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This International Standard specifies a rapid method for the determination of the potential rate of ammonium
oxidation and inhibition of nitrification in soils. This method is suitable for all soils containing a population of
nitrifying microorganisms. It can be used as a rapid screening test for monitoring soil quality and quality of
wastes, and is suitable for testing the effects of cultivation methods, chemical substances [except volatiles,
i.e. H > 1 (Henry’s constant)], extracts of biosolids and pollution in soils.

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ISO 18763:2016 describes a technique for determining the effects of soil and soil-related materials on the seed germination and early growth of higher plants. These endpoints are useful indicators for the assessment of the quality of a soil as a habitat for organisms. It is applicable to all soils in which soil organisms are active and may be used to evaluate:
- the effects on plants due to toxicity of solid or liquid chemicals contaminating soil or materials (compost, sludge, waste) and chemicals added to soil;
- the changes in the soil effect on plants after restoration measures.

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This document describes a method to compare the quality of soils by determining the fatty acid composition of the leaves of plant species grown in these soils.
This method does not make it possible to determine an optimal value of the Omega-3 index and, therefore, cannot be used to determine the intrinsic quality of a soil from a specific area (regarded as homogeneous). The method can only be used to compare the quality of soils between various areas.
This method is applicable to:
— soils from contaminated sites;
— amended soils;
— soils after remediation;
? soil with waste products (e.g. slurry, manure, sludge or composts).
Alternatively, the quality of soils can be assessed by determining the Omega-3 index of Lactuca sativa seedlings grown in these soils under controlled conditions (i.e. phytotronic chamber) and by comparing these values to those obtained from control soils (see Annex B).

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This International Standard specifies a test method for determining the activity of active aerobic, eterotrophic
microbial biomass in soils. This method is applicable to the monitoring of soil quality and to the evaluation of
the ecotoxic potential of soils and soil materials. It is also applicable for soils sampled along contamination
gradients in the field and to soils that are contaminated experimentally in the field or in the laboratory.

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ISO 17512-2:2011 specifies a rapid screening method for evaluating the habitat function of soils based on the avoidance behaviour of springtails.
The test is a rapid method that reflects the bioavailability of contaminants in natural soils and substances spiked into soils to Folsomia candida. In both cases, it is possible to establish a dose-response-relationship. The avoidance behaviour of the springtails is the measurement endpoint of the test. This test is not intended to replace the Collembola reproduction test.

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The purpose of ISO 29200:2013 is to describe a method for assessing genotoxic effects (chromosome breakage or dysfunction of the mitotic spindle) of soils or soil materials on the secondary roots of a higher plant: Vicia faba (broad bean). This method allows the assessment of genotoxicity (toxicity for genetic material) of soils and soil materials like compost, sludge, waste, fertilizing matters, etc. Two ways of exposure can be considered: a direct exposure of plants to the soil (or soil material) which is relevant for the real genotoxic potential and an exposure of plants to the water extract of the soil (or soil material). This last way of exposure to a leachate or an eluate allows the detection of the mutagens which are not adsorbed to soils and which may be transferred to aquatic compartments. Moreover, this test may be used to evaluate genotoxic effects of chemical substances and to waters, effluents, etc.

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This document specifies a protocol to identify ecotoxicological test specimens (mainly invertebrates and plants) to the species level, based on the DNA barcoding technique. This protocol can be used by laboratories performing DNA barcoding in order to standardize both the wet-lab and data analysis workflows as much as possible, and make them compliant with community standards and guidelines.
This document does not intend to specify one particular strain for each test method, but to accurately document the species/strain which was used.
NOTE 1 This does not imply that DNA barcoding is performed in parallel to each test run, but rather regularly (e.g. once a year, such as reference substance testing) and each time a new culture is started or new individuals are added to an ongoing culture.
This document does not aim at duplicating or replacing morphological-based species identifications. On the contrary, DNA barcoding is proposed as a complementary identification tool where morphology is inconclusive, or to diagnose cryptic species, in order to ensure that the results obtained from different ecotoxicological laboratories are referring to the same species or strain.
This document is applicable to identifications of immature forms which lack morphological diagnostic characters (eggs, larvae, juveniles), as well as the streamline identification of specimens collected in field monitoring studies, where large numbers of organisms from diverse taxa are classified.
NOTE 2 In principle, all species regularly used in ecotoxicological testing can be analysed by DNA barcoding. Besides the earthwoms Eisenia fetida and E. andrei, further examples for terrestrial species are Lumbricus terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea, and A. caliginosa, Dendrodrilus rubidus, Enchytraeus albidus, and E. crypticus (Haplotaxida); Folsomia candida, F. fimetaria, Proisotoma minuta, and Sinella curviseta (Collembola); Hypoaspis aculeifer and Oppia nitens (Acari); Aleochara bilineata and Poecilus cupreus (Coleoptera); Scathophaga stercoraria, Musca autumnalis (Diptera) or Pardosa sp. (Arachnida). Nematodes or snails and even plants can also be added to this list.

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This document specifies an extraction method to determine the bioavailable (potential and environmental available) fraction and the non-bioavailable fraction of a contaminant in soil using a "receiver phase" for an organic contaminant with strong sorbing or complexing properties, for example, Tenax®[1] or cyclodextrin, respectively. NOTE 1 The bioavailable fraction is defined in ISO 17402 as environmental bioavailability. The method is applicable for non-polar organic contaminants with an aqueous solubility of NOTE 2 The method is theoretically applicable to non-polar organic contaminants with an aqueous solubility of 1 000 mg/l. The method has been often applied for compounds with a much lower solubility (Kow > 3) and less for compounds with a higher solubility. The applicability is therefore defined for compounds with an aqueous solubility of [1] Tenax® is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.

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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.

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This document provides guidance on the use of chemical methods establishing the bioavailability of trace elements in soil and soil-like materials and to stimulate the use of bioavailability in assessments. The methods themselves are not subject of this document.

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This Standard 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.

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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.

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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.

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This document specifies a method for the measurement of several enzyme activities (arylsulfatase, α −glucosidase, β -glucosidase, Cellubisidase, β -Xylosidase, phosphodiesterase (PDE), chitinase, phosphomonoesterase (PME), leucine-aminopeptidase, Alanine-aminopeptidase) simultaneously (or not) using fluorigenic substrates in soil samples. Enzyme activities of soil vary seasonally and depend on the chemical, physical and biological characteristics of soil. Its application for the detection of harmful effects of toxic chemicals or other anthropogenic impacts depends on the simultaneous comparison of enzyme activities in a control soil similar to the test soil, or on exposure tests with chemicals or treatments.

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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.

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

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This document describes a method to compare the quality of soils by determining the fatty acid composition of the leaves of plant species grown in these soils. This method does not make it possible to determine an optimal value of the Omega-3 index and, therefore, cannot be used to determine the intrinsic quality of a soil from a specific area (regarded as homogeneous). The method can only be used to compare the quality of soils between various areas. This method is applicable to: — soils from contaminated sites; — amended soils; — soils after remediation; ? soil with waste products (e.g. slurry, manure, sludge or composts). Alternatively, the quality of soils can be assessed by determining the Omega-3 index of Lactuca sativa seedlings grown in these soils under controlled conditions (i.e. phytotronic chamber) and by comparing these values to those obtained from control soils (see Annex B).

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This document specifies a chronic test method for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Hypoaspis aculeifer by ? mainly ? alimentary uptake. This method 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 under concern and waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed material, or manure, especially those for possible land disposal). The reproduction (= number of juveniles) is the measured parameter of the test. The test reflects the bioavailability of a mixture of contaminants in natural soils (contaminated site soils) to a species which represents a trophic level which is not covered by other ISO standards. This test is not intended to replace the earthworm (see ISO 11268-2) or Collembola (see ISO 11267) reproduction tests since this species belongs not only to a different trophic group but also a different taxonomic group (= mites; i.e. arachnids) than those used usually. 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. Depending on 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). This document provides information on how to use this method for testing samples (soils or substances) under temperate conditions. This document is not applicable 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. NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the test method for monitoring the persistence of the substance under test.

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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.

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

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This document specifies a protocol to identify ecotoxicological test specimens (mainly invertebrates and plants) to the species level, based on the DNA barcoding technique. This protocol can be used by laboratories performing DNA barcoding in order to standardize both the wet-lab and data analysis workflows as much as possible, and make them compliant with community standards and guidelines. This document does not intend to specify one particular strain for each test method, but to accurately document the species/strain which was used. NOTE 1 This does not imply that DNA barcoding is performed in parallel to each test run, but rather regularly (e.g. once a year, such as reference substance testing) and each time a new culture is started or new individuals are added to an ongoing culture. This document does not aim at duplicating or replacing morphological-based species identifications. On the contrary, DNA barcoding is proposed as a complementary identification tool where morphology is inconclusive, or to diagnose cryptic species, in order to ensure that the results obtained from different ecotoxicological laboratories are referring to the same species or strain. This document is applicable to identifications of immature forms which lack morphological diagnostic characters (eggs, larvae, juveniles), as well as the streamline identification of specimens collected in field monitoring studies, where large numbers of organisms from diverse taxa are classified. NOTE 2 In principle, all species regularly used in ecotoxicological testing can be analysed by DNA barcoding. Besides the earthwoms Eisenia fetida and E. andrei, further examples for terrestrial species are Lumbricus terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea, and A. caliginosa, Dendrodrilus rubidus, Enchytraeus albidus, and E. crypticus (Haplotaxida); Folsomia candida, F. fimetaria, Proisotoma minuta, and Sinella curviseta (Collembola); Hypoaspis aculeifer and Oppia nitens (Acari); Aleochara bilineata and Poecilus cupreus (Coleoptera); Scathophaga stercoraria, Musca autumnalis (Diptera) or Pardosa sp. (Arachnida). Nematodes or snails and even plants can also be added to this list.

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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.

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

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  • Standard
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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.

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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.

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This document deals with the assessment of human exposure from ingestion of soil and soil materials. It specifies a physiologically based test procedure for the estimation of the human bioaccessibility of metals from contaminated soil in connection with the evaluation of the exposure related to human oral uptake. The method is a sequential extraction using synthetic gastrointestinal fluids and can be used to estimate oral bioaccessibility. Soils or other geological materials, in sieved form, are extracted in an environment that simulates the basic physicochemical conditions of the human gastrointestinal tract. This document describes a method to simulate the release of metals from soil and soil materials after passage through three compartments of the human gastrointestinal tract (mouth, stomach and small intestine). It produces extracts that are representative of the concentration of potentially harmful elements in the human gastrointestinal tract for subsequent chemical characterization. NOTE 1 Bioaccessibility can be used to approximate oral bioavailability. NOTE 2 The test has been validated for arsenic, cadmium and lead in an interlaboratory trial. The method has been in vivo validated to assess the oral bioavailability of arsenic, cadmium and lead.

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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.

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This document specifies a method for the measurement of several hydrolase activities (arylamidase, arylsulfatase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, acid, alkaline and global phosphatases, urease) simultaneously (or not) in soil samples, using colorimetric substrates. Enzyme activities of soil vary seasonally and depend on soil chemical, physical and biological characteristics. This method can be applied either to detect harmful effects on soil enzyme activities derived from toxic substances or other anthropogenic agents in contaminated soils against a control soil, or to test chemicals.

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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.

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This document specifies a laboratory test for characterizing the ability (or inability) of soils to reduce the greenhouse gas N2O into N2 as it was previously shown that soils with a low ability to reduce N2O into N2 constitute situations with a risk of large emission of N2O[6], higher than those basically estimated by the use at the plot scale of the equations proposed in the IPCC guidelines for National Greenhouse Gas Inventories[10]. This test is performed in laboratory on a composite of sieved samples collected at the plot scale. It can be performed on all types of soils sampled all over the year except in very exceptional and extreme conditions of dryness. Results obtained are stable over time for situations that do not receive neither organic nor lime amendments.

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This document specifies a laboratory test for characterizing the denitrifying enzyme activities in soils[5]. It globally characterizes the transformation of the nitrate form to the nitrous oxide and dinitrogen forms. This method was first proposed by Reference [5] with the acronym DEA for Denitrifying Enzyme Activities. It is a standardized technique used in numerous scientific studies. DEA estimates the process of denitrification of fresh soil samples incubated under optimal conditions of substrates (nitrate and carbon sources) and environment (anaerobiosis, controlled temperature) for the denitrification process. The de novo enzyme synthesis is blocked by the use of chloramphenicol. DEA is believed to represent the size of the denitrifying enzyme pool present in the soil sample at the time of sample collection. This test is performed in laboratory on a composite of sieved samples collected at the plot scale. It can be performed on all types of soils sampled all over the year.

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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).

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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.

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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.

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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.

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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.

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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.

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