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 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 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 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 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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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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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.

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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.

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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.

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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.

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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.

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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).

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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.

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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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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 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.

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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.

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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.

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ISO 23611-3:2007 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). It applies to all terrestrial biotopes in which enchytraeids occur. ISO 23611-3:2007 is not applicable for semi-terrestrial soils and might be difficult to use under extreme climatic or geographical conditions.

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ISO 15952:2005 specifies a semi-static method for the determination of 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 or preparations; and soils (contaminated or of unknown quality) or waste materials. A static method is also described. ISO 15952:2005 does not apply to volatile substances, i.e. substances for which the Henry constant, H, or the air/water partition coefficient is over 1, or for which the vapour pressure is over 0,013 3 Pa at 25 °C. This test takes into account possible changes in the test substance, preparation, soil or waste material because the test mixture is prepared and renewed every 7 days during the 28-day test period.

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ISO 23611-1:2006 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). ISO 23611-1:2006 applies to all terrestrial biotopes in which earthworms occur. It 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).

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ISO 16387:2004 describes a method for determining the effects of substances or contaminated soils on reproduction and on survival of the worm Enchytraeus albidus (Enchytraeidae). The animals are exposed to the substances by dermal and alimentary uptake using a defined artificial soil substrate to which specified amounts of that substance are added, or by using a soil substrate of unknown quality. ISO 16387:2004 is applicable to test substances that are either insoluble or soluble in water, although the method of application differs. The method is not applicable to volatile test 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. For optimum applicability, the water solubility and the vapour pressure of the test substance should be known. Additionally, information on the persistence of the test substance in soil is desirable. Basic information on the ecology and ecotoxicology of Enchytraeidae in the terrestrial environment can be found in the bibliographic references. 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 test substance. Recommendations for adapting the method to comparing or monitoring soil quality are given in Annex B.

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The method described is based on placing adult earthworms in a defined substrate containing the test substance in different concentrations and determining the percent mortality after 7 d and 14 d. It 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 0,0133 Pa at 25 °C. It does not take into account the possible degradation of the test substance.

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