This document specifies a method for the sampling and laboratory preparation of benthic diatoms for ecological status and water quality assessments. The sampling and preparation procedures described can be used for later investigations using either light microscopy or molecular methods. Data produced by this method are suitable for production of indices based on the relative abundance of taxa.
Analysis using molecular methods is not within the scope of the document.

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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.

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This document specifies a method for the sampling and laboratory preparation of benthic diatoms for ecological status and water quality assessments. The sampling and preparation procedures described can be used for later investigations using either light microscopy or molecular methods. Data produced by this method are suitable for production of indices based on the relative abundance of taxa.
Analysis using molecular methods is not within the scope of the document.

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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.

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This document specifies the general requirements for the in vitro amplification of nucleic acid sequences (DNA or RNA). This includes polymerase chain reaction (PCR)-based methods like quantitative PCR, qualitative PCR, reverse transcription-PCR and digital PCR. The minimum requirements laid down in this document are intended to ensure that comparable and reproducible results are obtained in different organizations. It covers quality assurance aspects to be considered when working with PCR-based methods in a laboratory as well as validation and verification. In addition to laboratory PCR-based methods, this document is also applicable to on-site PCR-based methods. This document is applicable to PCR-based methods used for the analysis of microorganisms and viruses in different water matrices, including but not limited to: - drinking water; - groundwater; - pool water; - process water; - surface water; - wastewater. This document is applicable to the detection and quantification of nucleic acids (DNA or RNA) of microorganisms by PCR-based methods in water such as bacteria, yeasts, fungi but also parasites such as Cryptosporidium, Giardia, amoebas and multicellular organisms. In addition, this document is applicable to the detection and quantification of nucleic acids from viruses in water by PCR-based methods. NOTE In the context of this document, viruses are considered to be microorganisms. Clauses in this document can also specifically apply to viruses and not to other types of microorganisms. In these clauses, viruses are mentioned separately.

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This document specifies a method for assessing the effects of chemical and aqueous samples on the embryo-larval development of marine bivalves. This method 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 PSU (practical salinity unit) and 40 PSU for mussels, and - between 25 PSU and 35 PSU for oysters. This method in this document 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 or dilution is limited so that the aforementioned salinity ranges are respected, - aqueous extracts (pore water, elutriates, eluates and leachates) from sediments and petroleum products, and - samples of contaminated sediment or dredged material (see Annex C).

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

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This document describes a spectrophotometric method for determining the chlorophyll-a content corrected for phaeopigments as a measure of the amount of phytoplankton for all types of surface water including marine water. The determination limit is usually 2 µg/l to 5 µg/l and is calculated by each laboratory individually. It can be as low as 0,5 µg/l using 2 l of sample (or even more) and a 50 mm cuvette.
NOTE   In some measurement programs like marine studies on time series data and ecological status/classification no correction for phaeopigments is used and acidification is omitted, e.g. as recommended by OSPAR.

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This document 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.
NOTE            With modifications, as described in ISO 14442 and ISO 5667-16, the inhibitory effects of poorly soluble organic and inorganic materials, volatile compounds, metal compounds, effluents, marine water samples and elutriates of sediments can be tested.

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This document 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.
NOTE            With modifications, as described in ISO 14442 and ISO 5667-16, the inhibitory effects of poorly soluble organic and inorganic materials, volatile compounds, metal compounds, effluents, marine water samples and elutriates of sediments can be tested.

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This document describes a spectrophotometric method for determining the chlorophyll-a content corrected for phaeopigments as a measure of the amount of phytoplankton for all types of surface water including marine water. The determination limit is usually 2 µg/l to 5 µg/l and is calculated by each laboratory individually. It can be as low as 0,5 µg/l using 2 l of sample (or even more) and a 50 mm cuvette.
NOTE   In some measurement programs like marine studies on time series data and ecological status/classification no correction for phaeopigments is used and acidification is omitted, e.g. as recommended by OSPAR.

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

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SIGNIFICANCE AND USE
5.1 The enterococci are indicators of the bacteriological quality for potable water, shellfish growing waters, ambient, and recreational waters. A direct relationship between swimming, associated gastroenteritis, and enterococci has been established through epidemiological studies and marine and fresh water bathing beaches. These studies have led to the development of criteria that can be used to establish bathing water standards based on established health-water quality relationships.  
5.2 Since small or large volumes of water or dilutions thereof, can be analyzed by the membrane filter technique, a wide range of levels of enterococci in water can be enumerated and detected.
SCOPE
1.1 This test method covers a membrane filter (MF) procedure for the detection and enumeration of the enterococci bacteria in water. The enterococci, which include Entero-coccus faecalis (E. faecalis), E. faecium, and their varieties are commonly found in the feces of humans and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution, enterococci in water are an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit is one colony forming unit (CFU)/volume filtered.  
1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user’s responsibility to ensure the validity of this test method for waters of untested types.  
1.3 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are included for information only and are not considered standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For specific hazard statements, see Section 9.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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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.

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SIGNIFICANCE AND USE
5.1 Results from the test method suggest, within the confines of a controlled laboratory setting, the degree of aerobic aquatic biodegradation of a lubricant or components of a lubricant by measuring the evolved carbon dioxide upon exposure of the test material to an inoculum. The plateau level of CO2 evolution in this test method will suggest the degree of biodegradability of the lubricant. Test substances that achieve a high degree of biodegradation in this test may be assumed to easily biodegrade in many aerobic aquatic environments.  
5.2 Because of the stringency of this test, a low yield of CO2 does not necessarily mean that the test substance is not biodegradable under environmental conditions, but indicates that further testing is necessary to establish biodegradability.  
5.3 Information on toxicity to the inoculum of the test substance may be useful in the interpretation of low biodegradation results.  
5.4 Activated sewage-sludge from a sewage-treatment plant that principally treats domestic waste is considered an acceptable active aerobic inoculum available over a wide geographical area in which to test a broad range of lubricants. An inoculum derived from soil or natural surface waters, or both, or any combination of the three sources, is also appropriate for this test method.
Note 1: Allowance for various and multiple inoculum sources provides access to a greater diversity of biochemical competency and potentially represents more accurately the capacity for biodegradation.  
5.5 A reference or control substance known to biodegrade is necessary in order to verify the activity of the inoculum. The test must be regarded as invalid and should be repeated using a fresh inoculum if the reference does not demonstrate a biodegradation of >60 % of the theoretical CO2 evolution within 28 days.  
5.6 A total CO2 evolution in the blank at the end of the test exceeding 75 mg CO2 per 3 L of medium shall be considered as invalidating the test.  
5.7 The water...
SCOPE
1.1 This test method covers the determination of the degree of aerobic aquatic biodegradation of fully formulated lubricants or their components on exposure to an inoculum under laboratory conditions.  
1.2 This test method is intended to specifically address the difficulties associated with testing water insoluble materials and complex mixtures such as are found in many lubricants.  
1.3 This test method is designed to be applicable to all lubricants that are not volatile and are not inhibitory at the test concentration to the organisms present in the inoculum.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazards are discussed in Section 10.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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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.

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This document specifies a method for the determination of the inhibition of root re-growth in duckweeds (Lemna minor) by substances and mixtures contained in water or waste water. This method applies to environmental water samples including treated municipal wastewater and industrial effluents.

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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.

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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.

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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.

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This document specifies procedures for sampling, capture and preservation of environmental DNA (eDNA) in aquatic environments, stemming from organisms that are or have recently been present in a waterbody, have visited it or whose DNA has been introduced to the waterbody through some mechanism. This document also covers procedures for avoiding sample contamination and ensuring DNA quality, key properties of the filtering procedure and equipment and reporting standards.
This document does not include the collection of eDNA from biofilms, sediments or similar sample types and does not cover sampling designs.

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This document specifies procedures for sampling, capture and preservation of environmental DNA (eDNA) in aquatic environments, stemming from organisms that are or have recently been present in a waterbody, have visited it or whose DNA has been introduced to the waterbody through some mechanism. This document also covers procedures for avoiding sample contamination and ensuring DNA quality, key properties of the filtering procedure and equipment and reporting standards.
This document does not include the collection of eDNA from biofilms, sediments or similar sample types and does not cover sampling designs.

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ABSTRACT
This guide covers procedures for obtaining laboratory data concerning the adverse effects of aqueous ambient samples and effluents on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, during a short-term exposure, depending on the species, using the static, renewal, and flow-through techniques. These procedures will probably be useful for conducting acute toxicity tests on aqueous effluents with many other aquatic species, although modifications might be necessary. Static tests might not be applicable to effluents that have a high oxygen demand, or contain materials that (1) are highly volatile, (2) are rapidly biologically or chemically transformed in aqueous solutions, or (3) are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Results of acute toxicity tests should usually be reported in terms of a median lethal concentration (LC50) or median effective concentration (EC50). An acute toxicity test does not provide information about whether delayed effects will occur. Specified requirements involving the following are detailed: (1) hazards; (2) apparatus: facilities, special requirements, construction materials, metering system, test chambers, cleaning, and acceptability; (3) dilution water requirements, source, treatment, and characterization; (4) effluent sampling point, collection, preservation, treatment, and test concentrations; (5) test organism species, age, source, care and handling, feeding, disease treatment, holding, acclimation, and quality; (6) procedure: experimental design, dissolved oxygen, temperature, loading, beginning the test, feeding, duration of test, biological data, and other measurements; (7) analytical methodology; (8) acceptability of test; (9) calculation of results; and (1) report of results.
SIGNIFICANCE AND USE
5.1 An acute effluent toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term exposure to an effluent under specific experimental conditions. One can directly examine acute effects of complex mixtures of chemicals as occurs in effluents and some ambient waters. Acute effluent toxicity tests can be used to evaluate the potential for designated-use or aquatic life impairment in the receiving stream, lake, or estuary. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding if necessary, might provide such information.  
5.2 Results of acute effluent tests might be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms might avoid exposure when possible, (2) toxicity to benthic species might be dependent on sorption or settling of components of the effluent onto the substrate, and (3) the effluent might physically or chemically interact with the receiving water.  
5.3 Results of acute effluent tests might be used to compare the acute sensitivities of different species and the acute toxicities of different effluents, and to study the effects of various environmental factors on results of such tests.  
5.4 Acute tests are usually the first step in evaluating the effects of an effluent on aquatic organisms.  
5.5 Results of acute effluent tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors.
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of an aqueous effluent on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, usually during 2 day to 4 day exposures, depending on the species, using the static, renewal, and flow-through techniques. These procedures will probably be useful for conducting acute toxicity tests on ...

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This document aims to assist in designing and organizing trials for validation of biotests. The validation activities during the different steps of the standardization process are described. This document comprises the overall data evaluation and subsequent validation study conclusion. This document is intended for the validation of biotests which can differ in their experimental design and endpoints. It is possible that some of the requirements of this document are not applicable to all test methods.

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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.

  • Standard
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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.

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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.

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This document specifies a method for the enumeration of Pseudomonas aeruginosa in water. The method is based on the growth of target organisms in a liquid medium and calculation of the most probable number (MPN) of organisms by reference to MPN tables.
This document is applicable to a range of types of water. For example, hospital waters, drinking water and non‑carbonated bottled waters intended for human consumption, groundwater, swimming pool and spa pool waters including those containing high background counts of heterotrophic bacteria.
This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower waters or marine waters, for which the method has not been validated. These waters are, therefore, outside the scope of this document. Laboratories can employ the method presented in this document for these matrices by undertaking appropriate validation of performance of this method prior to use.
The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa through the hydrolysis of a 7‑amino‑4‑methylcoumarin aminopeptidase substrate present in a special reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme that cleaves the 7‑amido‑coumarin aminopeptidase substrate releasing a product which fluoresces under ultraviolet (UV) light. The test described in this document provides a confirmed result within 24 h with no requirement for further confirmation of positive wells.

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SIGNIFICANCE AND USE
5.1 The ecological importance of bivalves, their wide geographic distribution, ease of handling in the laboratory and the field, and their ability to filter and ingest large volumes of water and sediment particles make them appropriate species for conducting field bioassays to assess bioaccumulation potential and associated biological effects. The test procedures in this guide are intended to provide guidance for conducting controlled experiments with caged bivalves under “natural,” site-specific conditions. It is important to acknowledge that a number of “natural” factors can affect bivalve growth and the accumulation of chemicals in their tissues (Section 6, Interferences). This field bioassay can also be conducted in conjunction with laboratory bioassays to help answer questions raised in the field exposures. The field exposures can also be used to validate the results of laboratory bioassays.  
5.2 The ultimate resources of concern are communities. However, it is often difficult or impossible to adequately assess the ecological fitness or condition of the community or identify and test the most sensitive species. Bivalves are recommended as a surrogate test species for other species and communities for the following reasons: (1) They readily accumulate many chemicals and show sublethal effects associated with exposure to those chemicals (2); (2) they accumulate many chemicals through multiple pathways of exposure, including water, sediment, and food  (24, 25, 26, 27, 28, 29), and (3) caged bivalves have been shown to represent effects on the benthos more accurately than traditional laboratory tests (30, 31). Although bivalve species might be considered insensitive because of their wide use as indicators of chemical bioavailability, it has been suggested that sensitivity is related to the type of test, end points being measured, and duration of exposure  (2). In relatively short-term toxicity assessments in which survival is typically determined as the measureme...
SCOPE
1.1 This guide describes procedures for conducting controlled experiments with caged bivalves under field conditions. The purpose of this approach is to facilitate the simultaneous collection of field data to help characterize chemical exposure and associated biological effects in the same organism under environmentally realistic conditions. This approach of characterizing exposure and effects is consistent with the US EPA ecological risk assessment paradigm. Bivalves are useful test organisms for in-situ field bioassays because they (1) concentrate and integrate chemicals in their tissues and have a more limited ability to metabolize most chemicals than other species, (2) exhibit measurable sublethal effects associated with exposure to those chemicals, (3) provide paired tissue chemistry and response data which can be extrapolated to other species and trophic levels, (4) provide tissue chemistry data which can be used to estimate chemical exposure from water or sediment, and (5) facilitate controlled experimentation in the field with large sample sizes because they are easy to collect, cage, and measure (1, 2)2. The experimental control afforded by this approach can be used to place a large number of animals of a known size distribution in specific areas of concern to quantify exposure and effects over space and time within a clearly defined exposure period. Chemical exposure can be estimated by measuring the concentration of chemicals in water, sediment, or bivalve tissues, and effects can be estimated with survival, growth, and other sublethal end points (3). Although a number of assessments have been conducted using bivalves to characterize exposure by measuring tissue chemistry or associated biological effects, relatively few assessments have been conducted to characterize both exposure and biological effects simultaneously (2, 4, 5). This guide is specifically designed to help minimize the variability in tissue che...

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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.

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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.

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SIGNIFICANCE AND USE
5.1 While federal criteria and state standards exist that define acute and chronic “safe” levels in the water column, effects levels in the sediment are poorly defined and may be dependent upon numerous modifying factors. Even where USEPA recommended Water Quality Criteria (WQC, (49)) are not exceeded by water-borne concentrations, organisms that live in or near the sediment may still be adversely affected (50). Therefore, simply measuring the concentration of a chemical in the sediment or in the water is often insufficient to evaluate its actual environmental toxicity. Concentrations of contaminants in sediment may be much higher than concentrations in overlying water; this is especially true of hydrophobic organic compounds as well as inorganic ions that have a strong affinity for organic ligands and negatively-charged surfaces. Higher chemical concentrations in sediment do not, however, always translate to greater toxicity or bioaccumulation (51), although research also suggests that amending sediment with organic matter actually increases the bioaccumulation of contaminant particles (52, 53). Other factors that can potentially influence sediment bioaccumulation and toxicity include pH mineralogical composition, acid-volatile sulfide (AVS) grain size, and temperature (54-56). Laboratory toxicity tests provide a direct and effective way to evaluate the impacts of sediment contamination on environmental receptors while providing empirical consideration of all of the physical, chemical and biological parameters that may influence toxicity.  
5.2 Amphibians are often a major ecosystem component of wetlands around the world, however limited data are available regarding the effects of sediment-bound contaminants to amphibians (39, 41, 43, 55, 57, 58). Laboratory studies such as the procedure described in this standard are one means of directly assessing sediment toxicity to amphibians in order to evaluate potential ecological risks in wetlands.  
5.3 Results from sed...
SCOPE
1.1 This standard covers procedures for obtaining laboratory data concerning the toxicity of test material (for example, sediment or hydric soil (that is, a soil that is saturated, flooded, or ponded long enough during the growing season to develop anaerobic (oxygen-lacking) conditions that favor the growth and regeneration of hydrophytic vegetation)) to amphibians. This test procedure uses larvae of the northern leopard frog (Lithobates pipiens). Other anuran species (for example, the green frog (Lithobates clamitans), the wood frog (Lithobates sylvatica), the American toad (Bufo americanus)) may be used if sufficient data on handling, feeding, and sensitivity are available. Test material may be sediments or hydric soil collected from the field or spiked with compounds in the laboratory.  
1.2 The test procedure describes a 10-d whole sediment toxicity test with an assessment of mortality and selected sublethal endpoints (that is, body width, body length). The toxicity tests are conducted in 300 to 500-mL chambers containing 100 mL of sediment and 175 mL of overlying water. Overlying water is renewed daily and larval amphibians are fed during the toxicity test once they reach Gosner stage 25 (operculum closure over gills). The test procedure is designed to assess freshwater sediments, however, R. pipiens can tolerate mildly saline water (not exceeding about 2500 mg Cl-/L, equivalent to a salinity of about 4.1 when Na+ is the cation) in 10-d tests, although such tests should always include a concurrent freshwater control. Alternative test durations and sublethal endpoints may be considered based on site-specific needs. Statistical evaluations are conducted to determine whether test materials are significantly more toxic than the laboratory control sediment or a field-collected reference sample(s).  
1.3 Where appropriate, this standard has been designed to be consistent with previously developed methods for assessing s...

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SIGNIFICANCE AND USE
5.1 Microbiological water testing procedures using membrane filtration are based on the premise that all bacteria within a specific size range will be retained by the membrane filter used. If the membrane filter does not retain these bacteria, false negative results or lowered density estimates may occur that could have serious repercussions due to the presence of unrecognized potential health hazards in the water being tested, especially in drinking water.  
5.2 This procedure as devised will enable the user to test each membrane filter lot number for its ability to retain all bacteria equal to, or larger than, the stated membrane pore size.
SCOPE
1.1 This test method covers a procedure to test membrane filters for their ability to retain bacteria whose diameter is equal to or slightly larger than membrane filters with pore size rated at 0.40 to 0.45 μm.  
1.2 The procedures described are for the use of user laboratories as differentiated from manufacturers’ laboratories.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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This document specifies a method for the enumeration of Pseudomonas aeruginosa in water. The method is based on the growth of target organisms in a liquid medium and calculation of the most probable number (MPN) of organisms by reference to MPN tables.
This document is applicable to a range of types of water. For example, hospital waters, drinking water and non‑carbonated bottled waters intended for human consumption, groundwater, swimming pool and spa pool waters including those containing high background counts of heterotrophic bacteria.
This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower waters or marine waters, for which the method has not been validated. These waters are, therefore, outside the scope of this document. Laboratories can employ the method presented in this document for these matrices by undertaking appropriate validation of performance of this method prior to use.
The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa through the hydrolysis of a 7‑amino‑4‑methylcoumarin aminopeptidase substrate present in a special reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme that cleaves the 7‑amido‑coumarin aminopeptidase substrate releasing a product which fluoresces under ultraviolet (UV) light. The test described in this document provides a confirmed result within 24 h with no requirement for further confirmation of positive wells.

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SIGNIFICANCE AND USE
5.1 The test procedure covered in this guide is not intended to simulate exactly the exposure of benthic polychaetes to chemicals under natural conditions, but rather to provide a conveniently rapid, standard toxicity test procedure yielding a reasonably sensitive indication of the toxicity of materials in marine and estuarine sediments.  
5.2 The protection of a community of organisms requires averting detrimental contaminant-related effects on the number and health of individuals and species within that population. Sediment toxicity tests provide information on the toxicity of test materials in sediments. Theoretically, projection of the most sensitive species within a community will protect the community as a whole.  
5.3 Polychaetes are an important component of the benthic community. They are preyed upon by many species of fish, birds, and larger invertebrate species, and they are predators of smaller invertebrates, larval stages of invertebrates, and, in some cases, algae, as well as organic material associated with sediment. Polychaetes are sensitive to both organic and inorganic chemicals (1, 2).5 The ecological importance of polychaetes, their wide geographical distribution and ability to be cultured in the laboratory, and sensitivity to chemicals, make them appropriate toxicity test organisms.  
5.4 An acute or 10-day toxicity test is conducted to obtain information concerning the immediate effects to a test material on a test organism under specified experimental conditions for a short period of time. An acute toxicity test does not necessarily provide information concerning whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, could provide such information.  
5.5 The results of acute sediment toxicity tests can be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of potentially contaminated sediment, or of a test material added experimentally to contaminated or uncontaminated sediment, on marine or estuarine infaunal polychaetes during 10-day or 20 to 28-day exposures. These procedures are useful for testing the effects of various geochemical characteristics of sediments on marine and estuarine polychaetes and could be used to assess sediment toxicity to other infaunal taxa, although modifications of the procedures appropriate to the test species might be necessary. Procedures for the 10-day static test are described for Neanthes arenaceodentata and Alitta virens 2 (formerly Nereis virens and Neanthes virens) and for the 20 to 28-day static-renewal sediment toxicity for  N. arenaceodentata.  
1.2 Modifications of these procedures might be appropriate for other sediment toxicity test procedures, such as flow-through or partial life-cycle tests. The methods outlined in this guide should also be useful for conducting sediment toxicity tests with other aquatic taxa, although modifications might be necessary. Other test organisms might include other species of polychaetes, crustaceans, and bivalves.  
1.3 Other modifications of these procedures might be appropriate for special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using unusual procedures are not likely to be comparable to those of many other tests. Comparisons of the results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting sediment tests with infaunal organisms.  
1.4 These procedures are applicable to sediments contaminated with most chemicals, either individually or in formulations, commercial products, and known or unknown...

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SIGNIFICANCE AND USE
5.1 The significance of measuring algal growth potential in water samples is that differentiation can be made between the nutrients of a sample determined by chemical analysis and the nutrients that are actually available for algal growth. The addition of nutrients (usually nitrogen and phosphorus singly or in combination) to the sample can give an indication of which nutrient(s) is (are) limiting for algal growth  (1,10,11,12,13,14).
SCOPE
1.1 This practice measures, by Pseudokirchnereilla subcapitata growth response, the biological availability of nutrients, as contrasted with chemical analysis of the components of the sample. This practice is useful for assessing the impact of nutrients, and changes in their loading, upon freshwater algal productivity. Other laboratory or indigenous algae can be used with this practice. However, Pseudokirchnereilla subcapitata must be cultured as a reference alga along with the alternative algal species.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For a specific precautionary statement, see Section 16.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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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.

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This document is focused on the structural features of rivers, on geomorphological and hydrological processes, and on river continuity. This document is focused on the structural features of rivers, on geomorphological and hydrological processes, and on river continuity. It provides guidance on the features and processes to be taken into account when characterizing and assessing the hydromorphology of rivers. The word ‘river’ is used as a generic term to describe flowing watercourses of all sizes, with the exception of artificial water bodies such as canals. The document is based on methods developed, tested, and compared in Europe, including the pan-European REFORM project (https://reformrivers.eu/). Its main aim is to improve the comparability of hydromorphological assessment methods, data processing and interpretation. It provides broad recommendations for the types of parameters that should be assessed, and the methods for doing this, within a framework that offers the flexibility to plan programmes of work that are affordable. Although this document does not constitute CIS guidance for the WFD, relevant references provided by the CIS expert group on hydromorphology have been included in the Bibliography.
Although it has particular importance for the WFD by providing guidance on assessing hydromorphological quality, this document has considerably wider scope for other applications. It does not attempt either to describe methods for defining high status for hydromorphology under the WFD, or to link broadscale hydromorphological classification to assessments of ecological status. In addition, while recognizing the important influence of hydromorphology on plant and animal ecology, no attempt is made to provide guidance in this area, but where the biota have an important influence on hydromorphology, these influences are included.
NOTE   A case study illustrating the application of this document is given in Gurnell and Grabowski[1].

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This document is focused on the structural features of rivers, on geomorphological and hydrological processes, and on river continuity. This document is focused on the structural features of rivers, on geomorphological and hydrological processes, and on river continuity. It provides guidance on the features and processes to be taken into account when characterizing and assessing the hydromorphology of rivers. The word ‘river’ is used as a generic term to describe flowing watercourses of all sizes, with the exception of artificial water bodies such as canals. The document is based on methods developed, tested, and compared in Europe, including the pan-European REFORM project (https://reformrivers.eu/). Its main aim is to improve the comparability of hydromorphological assessment methods, data processing and interpretation. It provides broad recommendations for the types of parameters that should be assessed, and the methods for doing this, within a framework that offers the flexibility to plan programmes of work that are affordable. Although this document does not constitute CIS guidance for the WFD, relevant references provided by the CIS expert group on hydromorphology have been included in the Bibliography.
Although it has particular importance for the WFD by providing guidance on assessing hydromorphological quality, this document has considerably wider scope for other applications. It does not attempt either to describe methods for defining high status for hydromorphology under the WFD, or to link broadscale hydromorphological classification to assessments of ecological status. In addition, while recognizing the important influence of hydromorphology on plant and animal ecology, no attempt is made to provide guidance in this area, but where the biota have an important influence on hydromorphology, these influences are included.
NOTE   A case study illustrating the application of this document is given in Gurnell and Grabowski[1].

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SIGNIFICANCE AND USE
5.1 Protection of a species requires the prevention of detrimental effects of chemicals on the survival, growth, reproduction, health, and uses of individuals of that species. Behavioral toxicity tests provide information concerning the sublethal effects of chemicals and signal the presence of toxic test substances.  
5.1.1 The locomotory, feeding, and social responses of fish are adaptive and essential to survival. Major changes in these responses may result in a diminished ability to survive, grow, avoid predation, or reproduce and cause significant changes in the natural population (8). Fish behavioral responses are known to be highly sensitive to environmental variables as well as toxic substances.  
5.2 Results from behavioral toxicity tests may be useful for measuring injury resulting from the release of hazardous materials (9).  
5.3 Behavioral responses can also be qualitatively assessed in a systematic manner during toxicity tests to discern trends in sublethal contaminant effects (5).  
5.4 The assessment of locomotory, feeding, and social behaviors is useful for monitoring effluents and sediments from contaminated field sites as well as for defining no-effect concentrations in the laboratory or under controlled field conditions. Such behavioral modifications provide an index of sublethal toxicity and also indicate the potential for subsequent mortality.  
5.5 Behavioral toxicity data can be used to predict the effects of exposure likely to occur in the natural environment (10).  
5.6 Results from behavioral toxicity tests might be an important consideration when assessing the hazard of materials to aquatic organisms. Such results might also be used when deriving water quality criteria for fish and aquatic invertebrate organisms.  
5.7 Results from behavioral toxicity tests can be used to compare the sensitivities of different species, the relative toxicity of different chemical substances on the same organism, or the effect of various environmental...
SCOPE
1.1 This guide covers some general information on methods for qualitative and quantitative assessment of the behavioral responses of fish during standard laboratory toxicity tests to measure the sublethal effects of exposure to chemical substances. This guide is meant to be an adjunct to toxicity tests and should not interfere with those test procedures.  
1.2 Behavioral toxicosis occurs when chemical or other stressful conditions, such as changes in water quality or temperature, induce a behavioral change that exceeds the normal range of variability (1). Behavior includes all of the observable, recordable, or measurable activities of a living organism and reflects genetic, neurobiological, physiological, and environmental determinants (2).  
1.3 Behavioral methods can be used in biomonitoring, in the determination of no-observed-effect and lowest-observed-effect concentrations, and in the prediction of hazardous chemical impacts on natural populations (3).  
1.4 The behavioral methods described in this guide include locomotory activity, feeding, and social responses, which are critical to the survival of fish (4).  
1.5 This guide is arranged as follows:    
Section Number  
Scope  
1  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Interferences  
6  
Safety Precautions  
7  
Responses Measured  
8  
Test Organisms  
9  
Facility  
10  
Qualitative Behavioral Assessment Method  
11  
Quantitative Behavioral Measurements  
12  
Experimental Design  
13  
Calculation of Test Results  
14  
Report  
15  
1.6 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard. For an explanation of units and symbols, refer to IEEE/ASTM SI 10.  
1.7 This standard does ...

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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.

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This document provides guidance for survey design, equipment specification, survey methods, sampling and data handling of macroalgae and marine angiosperms such as Zostera in the intertidal soft bottom environment. It does not include polyeuryhaline terrestrial angiosperms that are found in saltmarshes. Ruppia is a genus of angiosperms that can be found in brackish water. This document can also be applied to the study of Ruppia in these environments.
The document comprises:
-   development of a mapping and sampling programme;
-   requirements for mapping and sampling equipment;
-   procedures for remote sensing data collection;
-   procedures for direct mapping and sampling in the field;
-   recommendations for taxon identification and biomass determination;
-   data handling.

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This document specifies a method for the detection, semi-quantitative and quantitative (MPN) enumeration of thermotolerant Campylobacter species.  
The method can be applied to all kinds of waters including: drinking water, ground water and well water, fresh, brackish and saline surface water, swimming pools, spa and hydrotherapy pools, recreational waters, agricultural waters and runoff, untreated and treated wastewater and also sand and other sediments.  
This method can be used for the detection of Campylobacter species in a specified sample volume. Clean water samples with low turbidity can be membrane filtered for either a qualitative method, semi-quantitative or quantitative (MPN) method. Water samples with higher turbidity, such as primary and secondary wastewater effluents and sediments, are analysed using the same qualitative, semi-quantitative or quantitative MPN method by direct inoculation of material into bottles or tubes. Sediments can be suspended in a suitable diluent or inoculated directly into enrichment broths.
Users wishing to employ this method are expected to verify its performance for the particular matrix under their own laboratory conditions.

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This document specifies procedures for sampling of mesozooplankton using nets and continuous ribbon-sampling devices in marine and brackish waters for the purpose of water quality assessment and determination of ecological status of ecosystems.
Guidance on sampling procedures and the subsequent steps of preservation and storage are given. The sampling procedures allow estimates of species occurrence and their abundance (relative or absolute), including spatial distribution and seasonal and long-term temporal trends, for a given body of water.
The described methods are restricted to the sampling of mesozooplankton that inhabit marine and brackish waters and exclude the shallow littoral zones which require a different type of sampling (e.g. zooplankton in salt marshes).

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This document specifies a procedure for analysing mesozooplankton in marine and brackish waters. The procedure comprises how to identify and enumerate mesozooplankton to estimate quantitative information on diversity, abundance and biomass with regard to spatial distribution and long-term temporal trends for a given body of water.

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This document provides guidance for survey design, equipment specification, survey methods, sampling and data handling of macroalgae and marine angiosperms such as Zostera in the intertidal soft bottom environment. It does not include polyeuryhaline terrestrial angiosperms that are found in saltmarshes. Ruppia is a genus of angiosperms that can be found in brackish water. This document can also be applied to the study of Ruppia in these environments.
The document comprises:
-   development of a mapping and sampling programme;
-   requirements for mapping and sampling equipment;
-   procedures for remote sensing data collection;
-   procedures for direct mapping and sampling in the field;
-   recommendations for taxon identification and biomass determination;
-   data handling.

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This document specifies a method for the detection and quantification of Legionella spp. and L. pneumophila using a quantitative polymerase chain reaction (qPCR). It specifies general methodological requirements, performance evaluation requirements, and quality control requirements.
Technical details specified in this document are given for information only. Any other technical solutions complying with the performance requirements are suitable.
NOTE 1 For performance requirements, see Clause 9.
This document is intended to be applied in the bacteriological investigation of all types of water (hot or cold water, cooling tower water, etc.), unless the nature and/or content of suspended matter and/or accompanying flora interfere with the determination. This interference can result in an adverse effect on both the detection limit and the quantification limit.
NOTE 2 For validation requirements, see 9.7.
The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per litre of sample.
The method described in this document is applicable to all types of water. However, some additives, such as chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method.
The qPCR methods do not give any information about the physiological state of the Legionella.

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