BSI PD CEN/TS 17883:2024

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Environmental characterization of eluates from leaching of waste and soil using reproductive and toxicological gene expression in Daphnia magna

Published By	Publication Date	Number of Pages
BSI	2024	28

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Categories: 13.060.70 - Examination of biological properties of water, BSI

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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.

PDF Catalog

PDF Pages	PDF Title
2	undefined
11	1 Scope 2 Normative references
12	3 Terms and definitions
13	4 Principle 4.1 General 4.2 Toxicogenomic qPCR method
14	4.3 Exposure of Daphnia magna 4.4 Choice of genes for study
17	5 Test materials 5.1 Daphnia magna reference water 5.2 Daphnia magna culture 5.2.1 Obtaining Daphnia magna from culture 5.2.2 Obtaining Daphnia magna from dormant eggs (ephippia) 5.3 Reagents
18	6 Apparatus 7 Procedure 7.1 Leaching 7.2 Protocol for exposure of the water flea Daphnia magna 7.2.1 Introduction 7.2.2 Acute immobilization test 7.2.2.1 General 7.2.2.2 Procedure for acute immobilization test
19	7.3 Daphnia magna sampling for gene expression analysis
20	7.4 RNA extraction 7.4.1 General 7.4.2 Procedure 7.4.3 Determining RNA quality and quantity
21	7.5 DNA synthesis 7.5.1 General 7.5.2 Reagents 7.5.3 Procedure 7.6 qPCR procedure 7.6.1 General
22	7.6.2 Protocol 7.7 Analysis of results 7.8 Interpretation of results 7.8.1 General
23	7.8.2 Number of genes affected 7.8.3 Up- or down-regulated 7.8.4 Level of response 7.8.5 Statistical significance 7.8.6 Combined interpretation 7.8.7 Result assessment
24	Annex A (informative)Presentation and interpretation of toxicogenomic data