BSI PD CEN/TS 17883:2022:2023 Edition

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

Published By	Publication Date	Number of Pages
BSI	2023	26

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Categories: 13.030.01 - Wastes in general, 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
10	1 Scope 2 Normative references 3 Terms and definitions
11	4 Principle 4.1 General
12	4.2 Toxicogenomic qPCR method 4.3 Exposure of Daphnia magna
13	4.4 Choice of genes for study
14	5 Test materials 5.1 Daphnia magna reference water
15	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 6 Apparatus
16	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
17	7.3 Daphnia magna sampling for gene expression analysis 7.4 RNA extraction 7.4.1 General
18	7.4.2 Procedure 7.4.3 Determining RNA quality and quantity 7.5 DNA synthesis 7.5.1 General 7.5.2 Reagents
19	7.5.3 Procedure 7.6 qPCR procedure 7.6.1 General 7.6.2 Protocol
20	7.7 Analysis of results 7.8 Interpretation of results 7.8.1 General 7.8.2 Number of genes affected 7.8.3 Up- or down-regulated 7.8.4 Level of response
21	7.8.5 Statistical significance 7.8.6 Combined interpretation 7.8.7 Use of results in risk assessments
22	Annex A (informative)Presentation and interpretation of toxicogenomic data