ISO/TC 147/SC 5 - Biological methods

This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in vitro bioassays which are based on measuring effects on a biological process such as enzyme induction or cellular growth. The concept described here can be used for any biological assay after the proof of its applicability. To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity of the sample – is expressed in terms of a concentration of a reference compound which results in an equivalent effect on the process. The term "sample" used in this document addresses environmental samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ concentrations can be derived for environmental water samples, extracts of environmental water samples including tap water or solutions of pure chemicals or mixtures of chemicals.

This document specifies a method for determining the toxicity of environmental samples on growth, fertility and reproduction of Caenorhabditis elegans. The method applies to contaminated whole freshwater sediment (maximum salinity 5 g/l), soil and waste, as well as to pore water, elutriates and aqueous extracts that were obtained from contaminated sediment, soil and waste.

This document specifies the determination of the biochemical oxygen demand of waters by dilution and seeding with suppression of nitrification after 5 d or 7 d incubation time. It is applicable to all waters having biochemical oxygen demands usually between 1 mg/l and 6 000 mg/l. It applies particularly to waste waters but also suits for the analysis of natural waters. For biochemical oxygen demands greater than 6 000 mg/l of oxygen, the method is still applicable, but special care is needed taking into consideration the representativeness of subsampling for preparation of the dilution steps. The results obtained are the product of a combination of biochemical and chemical reactions in presence of living matter which behaves only with occasional reproducibility. The results do not have the rigorous and unambiguous character of those resulting from, for example, a single, well‑defined, chemical process. Nevertheless, the results provide an indication from which the quality of waters can be estimated.

This document specifies a method for the determination of fish acute toxicity using the permanent cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. Cells in confluent monolayers in 24-well tissue culture plates are exposed to water samples, such as surface waters or different kinds of effluents, or to chemicals for 24 h and, thereafter, cell viability is assessed based on fluorescent cell viability indicator dyes (see 4.1). Data are then expressed as a percentage of unexposed control and toxicity quantified based on the percentage of cell viability versus the percentage of effluent or the chemical concentration in response curves (see Clause 9).

This document specifies techniques for preparing poorly water-soluble organic compounds (i.e. liquid and solid compounds) with a solubility in water of less than approximately 100 mg/l and introducing them into test vessels for a subsequent biodegradability test in an aqueous medium using standard methods. The subsequent tests on biodegradability are primarily methods using the analysis of the released carbon dioxide described in ISO 9439 and the determination of the oxygen described in ISO 9408 and following the usual precautions for ISO 10707. Thus, one can notice that the methods measuring the removal of dissolved organic carbon (DOC) are not appropriate. This document does not specify the biodegradation test methods. It is restricted to describing techniques for introducing the test compounds into the test medium and to keeping them in a dispersed state[4]. These techniques are implemented while observing the experimental conditions described in the standardized methods for evaluating biodegradability. ISO 9439, based on CO2 evolution, is not suitable for testing volatile compounds. Some of the preparation methods described in this document might not be accepted by regulators for making conclusions on the ready biodegradability of tested compounds. Examples of biodegradability curves are given in Annex A.

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay utilizing stably transfected human cells. This reporter gene assay is based on the activation of the human estrogen receptor alpha. This method is applicable to: — fresh water; — waste water; — aqueous extracts and leachates; — eluates of sediments (fresh water); — pore water; — aqueous solutions of single substances or of chemical mixtures; — drinking water; — the limit of quantification (LOQ) of this method for the direct analysis of water samples is between 0,3 ng/l and 1 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper working range was evaluated [based on the results of the international interlaboratory trial (see Table F.3)] up to a level of 75 ng EEQ/l. Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre concentration of water samples can prove necessary if their estrogenic potential is below the given LOQ.

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay with genetically modified yeast strains Saccharomyces cerevisiae. This reporter gene assay is based on the activation of the human estrogen receptor alpha. This method is applicable to: — fresh water; — waste water; — aqueous extracts and leachates; — eluates of sediments (fresh water); — pore water; — aqueous solutions of single substances or of chemical mixtures; — drinking water. The limit of quantification (LOQ) of this method for the direct analysis of water samples is between 8 ng/l and 15 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between 120 ng/l and 160 ng/l 17β-estradiol equivalents (EEQ). Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre-concentration of water samples can prove necessary, if their estrogenic potential is below the given LOQ.

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay with a genetically modified yeast strain Arxula adeninivorans. This reporter gene assay is based on the activation of the human estrogen receptor alpha. Arxula adeninivorans is a highly robust and salt- and temperature-tolerant test organism and is especially suitable for the analysis of samples with high salinity (conductivity up to 70 mS/cm). The test organism can be cultivated in medium with sodium chloride content up to 20 %. This method is applicable to: — fresh water; — waste water; — sea water; — brackish water; — aqueous extracts and leachates; — eluates of sediments (fresh water); — pore water; — aqueous solutions of single substances or of chemical mixtures; — drinking water. The limit of quantification (LOQ) of this method for the direct analysis of water samples is between 1,5 ng/l and 3 ng/l 17β-estradiol equivalents (EEQ). The upper threshold of the dynamic range for this test is between 25 ng/l and 40 ng/l 17β-estradiol equivalents (EEQ). Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre-concentration of water samples can prove necessary, if their estrogenic potential is below the given LOQ. An international interlaboratory trial for the validation of this document has been carried out. The results are summarized in Annex F. NOTE Extraction and pre-concentration of water samples can prove necessary.

ISO 20227:2017 specifies a method for the determination of the inhibition of the growth of the first fronds of Spirodela polyrhiza germinated from turions, by substances and mixtures contained in water or waste water, including treated municipal waste water and industrial effluents. The test is also applicable to pure chemicals and in particular, plant protection products and pesticides.

ISO 10253:2016 specifies a method for the determination of the inhibition of growth of the unicellular marine algae Skeletonema sp. and Phaeodactylum tricornutum by substances and mixtures contained in sea water or by environmental water samples (effluents, elutriates, etc.). The method can be used for testing substances that are readily soluble in water and are not significantly degraded or eliminated in any other way from the test medium.

ISO/TS 18220:2016 specifies an early-life stage procedure for determination of the toxic effects of chemicals and water samples on a cold-water brackish water copepod species under semi-static conditions. The biological test variables include survival and development of the early-life stages. The exposure starts with newly hatched ( The benthic living Nitocra complements the planktonic Acartia species in ISO 16778. These organisms represent different life-history strategies as Nitocra is egg-carrying, whereas Acartia is a broadcasting calanoid copepod and thus, different sensitivities of specific life stages. Nitocra is a fresh to brackish water species, which allows testing low salinity waters and is complementary to A. tonsa, which is of marine origin and poorly tolerates low salinities.

ISO 19820:2016 specifies a method for the determination of the lethal effects of toxicants to Brachionus plicatilis after 24 h or 48 h exposure. The method is applicable to the following: a) chemical substances which are soluble or which can be maintained as a stable suspension of dispersions under the conditions of the test; b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration, or centrifugation; c) marine or estuarine waters; d) sediment elutriates/eluates.

ISO 19827:2016(E) specifies a method for the determination of the lethal effects of toxicants to Brachionus calyciflorus after 24 h exposure. The method is applicable to: a) chemical substances which are soluble, or which can be maintained as a stable suspension of dispersions under the conditions of the test; b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or centrifugation; c) freshwaters; d) aqueous extracts.

ISO 17244:2015 specifies a method for determining the effects of chemical and aqueous samples on the embryo-larval development of marine bivalves. It allows the determination of the concentration levels that result in an abnormality in embryo-larval development. This test is suitable for salinity ranges between 20 and 40 for mussels and between 25 and 35 for oysters. This method applies to - chemical substances and preparations, - marine and brackish waters, - streams and aqueous effluents (urban, agricultural, industrial effluents, etc.) as long as the salinity is adjusted and/or dilution is limited so that the aforementioned salinity ranges are respected, and - aqueous extracts (pore water, elutriates, eluates, and leachates) from sediments and petroleum products.

ISO 16778:2015 specifies an early-life stage test procedure for determination of the toxic effects of a chemical substance, effluent, or water sample on a cold-water marine and brackish water copepod species under semi-static conditions. The biological endpoints include survival and development of the early-life stages. The exposure starts with eggs and is continued until emergence of juvenile stages. Copepods occur widely in marine, brackish, and fresh water ecosystems. They represent important prey items for the larvae of many fish and larger invertebrates and are increasingly used as a live food source in aquaculture. They feed on phytoplankton and, thus, are an ecologically important energy-transfer link between primary producers and higher trophic levels.

ISO 16665:2014 provides guidelines on the quantitative collection and processing of subtidal soft-bottom macrofaunal samples in marine waters. ISO 16665:2014 encompasses: a) development of the sampling programme; b) requirements for sampling equipment; c) sampling and sample treatment in the field; d) sorting and species identification; e) storage of collected and processed material. ISO 16665:2014 does not specifically address the following, although some elements may be applicable: bioassay sub-sampling; deep water (>750 m) or offshore sampling; in situ faunal studies, e.g. recolonization assays; non-benthic organisms caught in the sampling device; estuarine sampling; intertidal sampling; meiofaunal sampling and analysis; sampling by dredge and sledge; self-contained underwater breathing apparatus (SCUBA) sampling; statistical design. Accuracy of position fixing is determined by the geographical area, equipment used and survey objective.

ISO 16303:2013 specifies a method for the determination of toxicity to young Hyalella azteca in whole sediment based on survival and growth inhibition after 14 d and/or 28 d. The method is applicable to a) samples of contaminated whole freshwater sediment, b) chemical, industrial, or municipal sludge, or other solid wastes that may combine with freshwater sediments, and c) chemicals or preparations spiked into clean sediment. ISO 16303:2013 is applicable to the testing of sediment samples from the freshwater environment. Hyalella azteca can be used in the testing of brackish waters up to a maximum of 15 ?, with careful acclimation. ISO 16303:2013 is not applicable to the testing of sediment samples from the marine and estuarine environment with a salinity of > 15 ?. This method is a 14 d and/or 28 d survival-and-growth test applicable to the sediment sample types described above.

ISO 16191:2013 specifies a method for determining the toxicity of environmental samples on the growth of Myriophyllum aquaticum. The method described is applicable to natural fresh water sediment and artificial sediment.

ISO 23893-3:2013 specifies a method for measuring vitellogenin (Vtg) concentrations in a fish plasma sample employing an enzyme-linked immunosorbent assay (ELISA) method. It applies to fish that are sampled in the environment (fresh, estuarine or salt water) and to fish exposed to substances or effluents in a laboratory. The method is quantitative when using Vtg antibodies and a Vtg standard well characterized with the species of choice.

ISO 6341.2012 specifies a method for the determination of the acute toxicity to Daphnia magna Straus (Cladocera, Crustacea). This method is applicable to: chemical substances which are soluble under the conditions of the test, or can be maintained as a stable suspension or dispersion under the conditions of the test; industrial or sewage effluents; treated or untreated waste water; aqueous extracts and leachates; fresh water (surface and ground water); eluates of fresh water sediment; pore water of fresh water sediment.

This International Standard specifies criteria for the selection of sampling methods and devices (operation and performance characteristics) used to evaluate benthic macroinvertebrate populations in fresh waters (rivers, canals, lakes, and reservoirs). The methods and devices considered in this International Standard are suitable for sampling all major components of the benthic assemblage. They are not suitable for sampling meiofauna.

This International Standard specifies a method for the determination of the genotoxic potential of water and waste water using the bacterial strains Salmonella enterica subsp. enterica serotype Typhimurium TA 98 and TA 100 in a fluctuation assay. This combination of strains is able to measure the genotoxicity of chemicals that induce point mutations (base pair substitutions and frameshift mutations) in genes coding for enzymes that are involved in the biosynthesis of the amino acid, histidine. NOTE 1 ISO 13829[8] applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents. This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water.
NOTE 2 When testing drinking water, extraction and pre-concentration of water samples can prove necessary.

This International Standard specifies a method for the determination of lethal as well as sublethal effects of contaminated sediments on the ostracod crustacean Heterocypris incongruens after 6 d exposure. The method is applicable not only to fresh water sediments, but also by extension to solid wastes and soils after addition of (uncontaminated) water. The method can also be applied to chemicals or preparations which are spiked into a reference sediment. This International Standard is not applicable to the testing of sediments from the estuarine or marine environment.

ISO 8692:2012 specifies a method for the determination of the growth inhibition of unicellular green algae by substances and mixtures contained in water or by waste water. This method is applicable for substances that are easily soluble in water. With modifications to this method, as specified in ISO 14442 and ISO 5667-16, the inhibitory effects of poorly soluble organic and inorganic materials, volatile compounds, heavy metals and waste water can be tested. A rapid algal growth inhibition screening test for waste water is described in an annex. An alternative test procedure with algae from algal beads, with direct measurement of algal growth in spectrophotometric cells, is described in an annex.

ISO 14380:2011 specifies a method for the determination of the lethal effects of toxicants to Thamnocephalus platyurus test organisms after 24 h exposure. A second method (rapid test) is described in an annex for the determination of sublethal effects after a very short exposure time (1 h). The methods are applicable to: a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions under the conditions of the test; b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or centrifugation; c) fresh waters; d) aqueous extracts; e) toxins of blue‑green algae. ISO 14380:2011 is not applicable to the testing of unstable chemicals (hydrolysing, absorbing, etc.) in water unless exposure concentration is measured, nor to the testing of aquatic samples from the estuarine or marine environment.

ISO 7827:2010 specifies a method for the evaluation of the “ready” and “ultimate” biodegradability of organic compounds at a given range of concentrations by aerobic microorganisms. In this context, ISO 7827:2010 also gives specific definitions for the terms “ready” and “ultimate”. The method applies to organic compounds which are: a) soluble at the concentration used under the conditions of the test [dissolved organic carbon (DOC) concentrations of 10 mg/l to 40 mg/l]; b) non‑volatile or having a negligible vapour pressure under the conditions of the test; c) not significantly adsorbable on glass and activated sludge; d) not inhibitory to the test microorganisms at the concentration chosen for the test. The method is not suitable for waste waters, as they usually contain significant amounts of water‑insoluble organic carbon, which is not included in DOC measurements.

ISO 21338:2010 specifies the kinetic direct‑contact method for determining the inhibitory effect of suspensions of sediment and other solid samples, and also for problematic turbid or coloured aqueous samples on the light emission of the marine bacterium Vibrio fischeri (NRRL B‑11177). This method is applicable to: a) sediment samples and water suspensions of sediments (fresh water, brackish, and seawater sediments); b) effluents (especially turbid and coloured); c) aqueous extracts (e.g. leachates, eluates, elutriates) of soil, solid waste, and other solid material (especially turbid and coloured).

ISO 10710:2010 specifies a method for the determination of the inhibition of growth of the macroalga Ceramium tenuicorne by substances and mixtures contained in seawater or by waste water with salinities between 4S and 32S. This method is applicable to substances that are easily soluble in water.

ISO 20665:2008 specifies a method for the determination of the chronic toxicity to Ceriodaphnia dubia (Cladocera, Crustacea), based on reproduction inhibition after (7 ± 1) d. The method is applicable to: a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions under the conditions of the test; b) industrial or sewage effluents, if appropriate after decantation, filtration or centrifugation; c) fresh waters; d) aqueous extracts (leachates, eluates, and elutriates). ISO 20665:2008 is not applicable to the testing of aquatic samples from the estuarine or marine environment.

ISO 20666:2008 specifies a method for the determination of the chronic toxicity to rotifer Brachionus calyciflorus, based on population growth inhibition in 48 h. The method is applicable to: a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions under the conditions of the test; b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or centrifugation; c) fresh waters; d) aqueous extracts. ISO 20666:2008 is not applicable to the testing of unstable chemicals (hydrolysing, absorbing, etc.) in water unless exposure concentration is measured, nor to the testing of aquatic samples from the estuarine or marine environment.

ISO/TR 11044:2008 discusses scientific and technical aspects that have been considered in connection with the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and ISO 10253, for marine waters. Previously unpublished results of experiments performed at the Norwegian Institute for Water Research (NIVA) have been included to demonstrate various aspects.

ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B‑11177). ISO 11348-1:2007 specifies a method using freshly prepared bacteria. This method is applicable to: waste water; aqueous extracts and leachates; fresh water (surface and ground water); sea and brackish water; eluates of sediment (fresh water, brackish and sea water); pore water; single substances, diluted in water.

ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B‑11177). ISO 11348-2:2007 specifies a method using liquid‑dried bacteria. This method is applicable to: waste water; aqueous extracts and leachates; fresh water (surface water and ground water); sea water and brackish water; eluates of sediment (fresh water, brackish water and sea water); pore water; single substances, diluted in water.

ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B‑11177). ISO 11348-3:2007 specifies a method using freeze‑dried bacteria. This method is applicable to: waste water; aqueous extracts and leachates; fresh water (surface and ground water); sea and brackish water; eluates of sediment (freshwater, brackish and sea water); pore water; single substances, diluted in water.

ISO/TS 23893-2:2007 specifies a method for measuring the ethoxyresorufin-O-deethylase (EROD) enzyme activity on a post-mitochondrial fraction of fish liver homogenate (subcellular fraction in which the EROD activity is located) employing a cell or microplate fluorimetric method. It applies to fish that are sampled in their natural environment (fresh water or salt water) or exposed to substances or effluents in a laboratory. This method is applicable for EROD values greater than or equal to 1 pmol/(min mg) of proteins. A higher sensitivity may be achieved by using a cell (test tube) procedure.

ISO 23893-1:2007 provides guidance on how to sample fish for determination of biochemical and physiological characteristics, such as the composition and enzyme activities of blood, liver, muscle and other tissues in order to asses the health of fish in the field as well as in the laboratory. The biochemical and physiological variables used for this purpose are often called biomarkers. ISO 23893-1:2007 includes recommendations and methods for: obtaining a site‑specific sample of a representative number of fish; sampling fish tissues in the field and in the laboratory; and handling and preservation of samples prior to analysis of biochemical and physiological variables.

ISO 15088:2007 specifies a method for the determination of degrees of dilution or concentrations as a measure of the acute toxic effect of waste water to fish eggs within 48 h. It is also applicable to treated municipal waste water and industrial effluents.

ISO 19493:2007 provides guidance for marine biological surveys of supralittoral, eulittoral and sublittoral hard substrate for environmental impact assessment and monitoring in coastal areas. It comprises development of the sampling programme, survey methods, species identification and storage of data and collected material. ISO 19493:2007 specifies the minimum requirements for environmental monitoring. The methods are limited to surveys and semi-quantitative and quantitative recording techniques that cause little destruction of the fauna and flora. In practice, this refers to direct recording in the field and photography. Sampling by scraping off organisms, use of a suction sampler, etc. are not covered in ISO 19493:2007.

ISO 8192:2007 specifies a method for assessing the inhibitory effect of a test material on the oxygen consumption of activated sludge microorganisms. This method is intended to represent the conditions in biological waste-water treatment plants. It gives information on inhibitory or stimulatory effects after a short exposure (usually 30 min up to 180 min or even more ) of the test material on activated sludge microorganisms. This method is applicable for testing waters, waste waters, pure chemicals and mixtures of chemicals. Concerning the chemicals, the method refers to those which are soluble under the test conditions. Special care is necessary with materials of low water solubility or high volatility, and with materials abiotically consuming or producing oxygen.

ISO 21427-2:2006 specifies a method for the determination of genotoxicity of water and waste water using a mammalian in vitro test which detects damage, induced by water-soluble substances, to the chromosomes or the mitotic apparatus of V79 cells from the Chinese hamster. The micronucleus test allows the identification of substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments and/or whole chromosomes. The assay is based on the increase in the frequency of micronucleated cells after incubation with and without metabolic activation.

ISO 21427-1:2006 specifies a method for assessing genotoxicity using Amphibia larvae (Xenopus laevis and Pleurodeles waltl). The method is applicable to: aqueous effluents; aqueous leachates; eluates of soils; eluates of industrial waste; eluates of sludges from sewage treatment; surface and ground water; water-soluble substances.

ISO 9509:2006 specifies a method for assessing the short-term inhibitory effect of waters, waste waters or test substances on nitrifying bacteria in activated sludge. The inhibitory effect is estimated over an exposure period of usually 3 h or up to 24 h with weakly nitrifying sludge. The method is applicable to nitrifying activated sludge derived from domestic and synthetic sewage and also to sludges from industrial and mixed domestic and industrial waste waters. The nitrifying activity of the sludge is verified by testing in the presence and absence of a specific inhibitor. If the nitrification rate is within a suitable range for the test, i.e. 2 mg of nitrogen per gram of suspended solid and hour to 6,5 mg of nitrogen per gram of suspended solids and hour, the sludge may be used directly. If not, adjustments are necessary. The method is applicable to water-soluble, non-volatile chemicals, and to waste waters

ISO/TR 15462:2006 gives an overview of biodegradation tests for the aquatic environment standardized by ISO and provides recommendations on their use. The biodegradation tests listed are designed to determine the biodegradability of chemical substances or wastewaters under standardized conditions. Inhibitory tests with bacteria and mixed bacterial inocula are included because a possible toxicity on the inoculum is important information for the choice and performance of biodegradation tests.

ISO 14442:2006 provides procedures, not covered by the methods described in ISO 8692 and ISO 10253, for testing difficult substances for inhibition of algal growth. The main subjects covered by ISO 14442:2006 are the methods for preparing the test substance for testing and the procedures needed to carry out an appropriate test. The following test substances are covered by this guideline: poorly soluble pure organic compounds; poorly soluble mixtures of organic substances; poorly soluble inorganic materials; volatile substances; waste waters and environmental samples containing water and sediments; coloured and/or turbid samples; compounds of heavy metals. The following methods of addition are covered: direct; dispersion; water-soluble and water-accommodated fractions. Some guidelines related to the analytical procedures and to the interpretation of the results have been included.

ISO/TS 20281:2006 offers guidance on statistical methods used for the analysis of data of standardized ecotoxicity tests. It focuses on statistical methods for obtaining statistical estimates of parameters. The methods described are intended to cover laboratory ecotoxicity tests (aquatic, sediment and/or terrestrial tests), and may also be relevant for other toxicity tests. Hypothesis testing, concentration-response modelling and biology-based modelling are discussed for the different data types (quantal, continuous and discrete data, corresponding to mortality, growth or reproduction). In addition, some guidance on experimental design is given. Although the main focus is on giving assistance to the experimentalist, a secondary aim is to help those who are responsible for evaluating toxicity tests. Finally, ISO/TS 20281:2006 may be helpful in developing new toxicity test guidelines by giving information on experimental design and statistical analysis issues.

ISO 20079:2005 specifies a method for the determination of the growth-inhibiting response of duckweed (Lemna minor) to substances and mixtures contained in water, treated municipal wastewater and industrial effluents.

ISO 16240:2005 specifies a method for the determination of the genotoxic potential of water and wastewater using the bacterial strains Salmonella typhimurium TA 100 and TA 98. This method includes sterile filtration of water and wastewater prior to the test. ISO 16240:2005 is applicable only to the detection of genotoxic substances which are in the filtered aqueous phase. It is not applicable to the detection of genotoxic substances adsorbed by the retained particles.