ISO/TC 190/SC 4/WG 4 - Effects on soil micro-organisms

This document specifies a test procedure for the evaluation of the habitat function of soils by determining effects of soil contaminants and substances on organic matter decomposition. This test is applicable to natural soils and soil materials of unknown quality (e.g. contaminated sites, amended soils, soils after remediation, agricultural or other sites under concern). This document also specifies 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. It is not applicable 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.

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

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.

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.

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 provides standard procedures for the collection, handling and storage of soil for subsequent biological testing under aerobic conditions in the laboratory. It applies to the collection, handling and storage for assessing the effects of soil on microorganisms, invertebrates (e.g. survival, reproduction, growth, behaviour) and plants (e.g. development, growth). This document is not applicable to the handling of soil where anaerobic conditions need to be maintained throughout. This document describes how to minimize the effects of differences in temperature, water content, and availability of oxygen on aerobic processes as well as the fractionation of soil particles to facilitate reproducible laboratory determinations[1][2]. This document is mainly applicable to temperate soils. Soils collected from extreme climates (e.g. permafrost, tropical soils) can require special handling. NOTE This document does not provide standard procedures on the collection, handling and storage of soil organisms when assessing the structure and function of soil organism communities in the field. Such standard procedures are provided in ISO 23611‑1 to ISO 23611‑6.

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.

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.

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.

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.

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 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.

1 Scope This International Standard specifies a test method for determining the activity of active aerobic, heterotrophic 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.

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.

ISO 15685:2012 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.

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.

ISO/TS 10832:2009 specifies a method to evaluate the effects of pollutants on spore germination of a mycorrhizal fungus, Glomus mosseae. This direct acute toxicity bioassay allows the evaluation of potential effects of pollutants and contaminated soils on beneficial soil microorganisms important for plant growth within the concept of sustainable agriculture. ISO/TS 10832:2009 is applicable to chemical substances, and contaminated soils, waste and soil-waste mixture and sludge.

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.

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. NOTE For methods intended for tests in the framework of the registration of chemicals, an OECD Guideline on soil degradation gives useful guidance on additional test requirements.

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.

Provides guidance on the selection and conduct of appropriate test methods for the determination of biodegradation of organic chemicals in aerobic soils. Does not describe any specific test method.

This International Standard specifies a method for direct extraction of DNA from soil samples to analyse the global structure and the abundance of soil bacterial communities using PCR-based technologies. 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 richness and structure of microbial communities which are key parameters to estimate the biodiversity of soil microbiota. Molecular approaches based on PCR (polymerase chain reaction) amplification of soil DNA constitute a promising domain and can contribute in the near future to the development of routine tools to monitor the microbiota of soil environments. Users of the method ought to be aware that although soil submitted to the DNA extraction procedure is sieved thoroughly (2 mm mesh, procedure described in 5.1), plant residues can still remain in soil samples and, as a result, traces of plant DNA can contaminate the soil DNA extract.

ISO/TS 29843-2:2011 specifies a simple method for the extraction of only phospholipid fatty acids (PLFA) from soils.

ISO/TS 22939:2010 specifies a method for the measurement of several enzyme activities simultaneously 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.

ISO 10381-6:2009 provides guidance on the collection, handling and storage of soil for subsequent testing under aerobic conditions in the laboratory. The recommendations in this document are not applicable to the handling of soil where anaerobic conditions are to be maintained throughout. ISO 10381-6:2009 is mainly applicable to temperate soils. Soils collected from extreme climates (e.g. permafrost, tropical soils) may require special handling.

ISO 23753-2:2005 specifies a method for determining soil dehydrogenase activity using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT). As the INT reduction is less sensitive to O2, the method is more reproducible than the TTC-method described in ISO 23753-1. It is not applicable for determining the dehydrogenase activity in upper layers (L, F, H horizons) of forest humus forms with low microbial activity (e.g. mor), or in soils showing reducing properties (e.g. waterlogged soils).

ISO 23753-1:2005 specifies a method for determining the dehydrogenase activity in soil using 2,3,5-triphenyltetrazolium chloride (TTC). It is not applicable for determining the dehydrogenase activity in the upper layers (L, F, H horizons) of forest humus forms with low microbial activity (e.g. mor), or in soils showing reducing properties (e.g. waterlogged soils).

ISO 15685:2004 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 and pollution in soils.

ISO 17155:2002 specifies a test method for determining the activity of the active aerobic, heterotrophic 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 to soils that are contaminated experimentally in the field or in the laboratory (chemical testing) and for soils sampled along contamination gradients in the field.

Specifies laboratory procedures for measuring the mineralization and nitrification of nitrogen by the soil microflora. Gives a simple procedure for investigation of the potential toxicity of chemicals to N-mineralization in soils.