CEN/TS 17883:2024
(Main)Environmental characterization of eluates from leaching of waste and soil using reproductive and toxicological gene expression in Daphnia magna
Environmental characterization of eluates from leaching of waste and soil using reproductive and toxicological gene expression in Daphnia magna
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.
Umwelttechnische Charakterisierung von Sickerwässern aus Abfällen und Boden anhand der reproduktiven und toxikologischen Genexpression in Daphnia magna
Dieses Dokument legt die entscheidenden Schritte einer quantitativen Echtzeit-Polymerase-Kettenreaktion (qPCR) zur Quantifizierung der Anzahl spezifischer mRNA Moleküle fest, die aus Daphnia magna extrahiert wurden.
Dieses Verfahren ermöglicht durch die Analyse der Anzahl spezifischer mRNA Moleküle die Identifikation molekularer Reaktionen auf die Exposition gegenüber potenziell toxischen Stoffen. Dieses Dokument deckt auch die zentralen Gene ab, die an reproduktionsbezogenen und toxischen Reaktionen beteiligt sind.
ANMERKUNG Die Auswahl der Gene kann an spezifische Expositionsbedingungen angepasst werden, z. B. an die Exposition gegenüber bekannten toxischen Stoffen, indem Gene hinzugefügt werden, die eine bekannte Reaktion auf einen spezifischen Einfluss haben.
Das vorliegende Verfahren ermöglicht einen schnellen, robusten und empfindlichen Nachweis molekularer Reaktionen und kann zur Analyse der toxischen Wirkungen von Eluaten aus Böden und Abfällen verwendet werden. Das Verfahren liefert Informationen über die Verdünnung einer Substanz oder Prüfflüssigkeit, bei der erste toxische Wirkungen auftreten, bevor reproduktionsbezogene oder toxische Wirkungen auf höheren Organisationsebenen beobachtet werden, wodurch die Notwendigkeit der Verwendung von Sicherheitsfaktoren in der Toxizitätsbeurteilung verringert wird.
Das Verfahren ist für verschiedene Arten der Gefährdungseinschätzung nützlich. Die in diesem Dokument untersuchten Gene sind für die Beurteilung der Risiken beim Recycling von Stoffen und für die Klassifizierung von Abfall geeignet, das Verfahren kann durch Aufnahme anderer Gene jedoch auch an andere Arten der Gefährdungseinschätzung angepasst werden.
Caractérisation environnementale des éluats de lixiviats de déchets et de sols à l’aide de l’expression génétique reproductive et toxicologique chez Daphnia magna
Le présent document spécifie les étapes cruciales d’une méthode de réaction de polymérisation en chaîne (qPCR) quantitative en temps réel pour quantifier l’abondance de molécules d’ARNm spécifiques extraites de Daphnia magna.
La méthode permet d’identifier les réponses moléculaires aux expositions à des substances potentiellement toxiques par l’analyse de l’abondance de molécules d’ARNm spécifiques. Dans le présent document, les gènes principaux impliqués dans les réponses reproductives et toxiques sont inclus.
NOTE La sélection de gènes peut être adaptée à des conditions d’exposition spécifiques, par exemple l’exposition à des substances toxiques connues, en ajoutant des gènes connus pour répondre à une agression spécifique.
La présente méthode permet une détection rapide, fiable et sensible des réponses moléculaires et peut être utilisée pour analyser les effets toxiques des éluats aqueux des sols et des déchets. La méthode donne des informations sur la concentration d’une substance ou la dilution d’un liquide d’essai à laquelle les effets toxiques commencent à se manifester avant l’observation d’effets reproductifs ou toxiques à des niveaux d’organisation plus élevés, ce qui réduit la nécessité d’utiliser des facteurs de sécurité dans l’évaluation de la toxicité.
La présente méthode est utile dans plusieurs types d’évaluation des dangers. Dans le présent document, les gènes étudiés sont appropriés pour l’évaluation du risque lors du recyclage des matériaux et pour la classification des déchets, mais la méthode peut être adaptée à d’autres types d’évaluation en incluant d’autres gènes.
Karakterizacija izlužkov odpadkov in tal z reproduktivno in toksikološko ekspresijo genov pri Daphnia magna
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-maj-2024
Okoljska karakterizacija izlužkov iz odpadkov in tal z reproduktivno in
toksikološko ekspresijo genov pri Daphnia magna
Environmental characterization of eluates from leachates from waste and soil using
reproductive and toxicological gene expression in Daphnia magna
Umwelttechnische Charakterisierung von Sickerwässern aus Abfall und Boden mittels
reproduktiver und toxikologischer Genexpression in Daphnia magna
Caractérisation environnementale des éluats de lixiviats de déchets et de sols à l’aide de
l’expression génétique reproductive et toxicologique chez Daphnia magna
Ta slovenski standard je istoveten z: FprCEN/TS 17883
ICS:
13.030.01 Odpadki na splošno Wastes in general
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
13.080.99 Drugi standardi v zvezi s Other standards related to
kakovostjo tal soil quality
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17883
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
February 2024
ICS 13.030.01; 13.060.70; 13.080.99 Will supersede CEN/TS 17883:2022
English Version
Environmental characterization of eluates from leachates
from waste and soil using reproductive and toxicological
gene expression in Daphnia magna
Umwelttechnische Charakterisierung von
Sickerwässern aus Abfall und Boden mittels
reproduktiver und toxikologischer Genexpression in
Daphnia magna
This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 444.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17883:2024 E
worldwide for CEN national Members.
FprCEN/TS 17883:2024 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 9
2 Normative references . 9
3 Terms and definitions . 10
4 Principle . 11
4.1 General. 11
4.2 Toxicogenomic qPCR method . 11
4.3 Exposure of Daphnia magna . 12
4.4 Choice of genes for study . 12
5 Test materials . 15
5.1 Daphnia magna reference water . 15
5.2 Daphnia magna culture . 15
5.2.1 Obtaining Daphnia magna from culture . 15
5.2.2 Obtaining Daphnia magna from dormant eggs (ephippia) . 15
5.3 Reagents . 15
6 Apparatus . 16
7 Procedure . 16
7.1 Leaching . 16
7.2 Protocol for exposure of the water flea Daphnia magna . 16
7.2.1 Introduction . 16
7.2.2 Acute immobilization test . 16
7.3 Daphnia magna sampling for gene expression analysis . 17
7.4 RNA extraction . 18
7.4.1 General. 18
7.4.2 Procedure . 18
7.4.3 Determining RNA quality and quantity. 18
7.5 DNA synthesis . 19
7.5.1 General. 19
7.5.2 Reagents . 19
7.5.3 Procedure . 19
7.6 qPCR procedure . 19
7.6.1 General. 19
7.6.2 Protocol . 20
7.7 Analysis of results . 20
7.8 Interpretation of results . 20
7.8.1 General. 20
7.8.2 Number of genes affected . 21
7.8.3 Up- or down-regulated . 21
7.8.4 Level of response . 21
7.8.5 Statistical significance . 21
7.8.6 Combined interpretation . 21
FprCEN/TS 17883:2024 (E)
7.8.7 Result assessment . 21
Annex A (informative) Presentation and interpretation of toxicogenomic data . 22
Bibliography . 25
FprCEN/TS 17883:2024 (E)
European foreword
This document (FprCEN/TS 17883:2024) has been prepared by Technical Committee CEN/TC 444
“Environmental characterization of solid matrices”, the secretariat of which is held by NEN.
This document is currently submitted to the Formal Vote.
This document will supersede CEN/TS 17883:2022.
FprCEN/TS 17883:2024 (E)
Introduction
The aim of this document is to describe the procedure used to set up and perform quantitative PCR to
quantify effects of eluates from waste and soil on reproductive and toxicological end points in Daphnia
magna. The presented method allows for rapid, robust and sensitive detection of molecular responses
and can be used to analyse the toxic effects of water eluates from soil and waste as well as the recipient
waters.
The study of messenger RNA (mRNA) from different living organisms, using different molecular
approaches, can be used to identify the responses of organisms to exposure to toxic substances.
Messenger RNA (mRNA) transfers information from the DNA, which stores all the necessary information
needed for life, to the cellular machinery that synthesizes proteins. Proteins are the working units in the
cell and their abundance is highly dependent on the RNA levels in the cell as all proteins are translation
products of mRNA. As mRNA is the first step in the response to toxic substances it is also a quick, precise
and sensitive biomarker of exposure that gives information on the mechanisms responsible for the
responses. The relationship between physiological impact and mechanistic resolution is shown in
Figure 1.
Figure 1 — Differences in physiological impact and mechanistic resolution at different
organizational levels
There is an important difference between determining an effect of a toxic substance and understanding
what it is that cause the effect. Effects that cause harm to entire ecosystems are usually identified by
changes in populations. Effects at the organism/individual level are easiest observed by changes in the
physiology, morphology or behaviour of an organism. However, as the environment is highly complex
and highly variable, the measurement of changes in ecosystems, population or individuals has very low
resolution when it comes to identifying the cause of any observed effect. To avoid “guilt by association”
it is important to identify the connection between an exposure and the observed effect. The most sensitive
methods for this are at the level of RNA and protein regulation at the cellular level. RNA forms the link
between proteins and chromosomal DNA (genes). The chromosomally located genes can be regulated in
several different ways, but independent of the regulatory mechanism, exposure to toxic substances will
result in a change in active RNA and thereby in the corresponding protein.
A toxic response is initiated by a molecular initiating event (MIE). This can be the exposure to an inorganic
or organic compound as well as to radioactivity. The MIE is followed by the molecular interaction
between the compound initiating the MIE and a molecule (receptor activation, protein binding, DNA
binding) that results in alterations in a set of key events (KE) starting with changes in gene expression
leading to changes in protein production. This in turn leads to an altered signal cascade that can, if it
overrides homoeostatic control, result in altered functions of cells, tissues and organs. These alterations
in function can then lead to adverse outcomes (AO) such as a malformation, organ dysfunction and
eventually lethality. This results in an AO of regulatory relevance for risk assessment that represents
overt adversity at either organism or population level (Figure 2).
FprCEN/TS 17883:2024 (E)
Figure 2 — The Adverse Outcome Pathway
An AOP (Adverse Outcome Pathway), starts with a molecular interaction between a chemical and a
molecule. This step is called the Molecular Initiating Event (MIE). This is followed by a set of Key Events
(KE) propagating the signal to higher levels of organization. This leads to an outcome on the physiology
of an organism and if this outcome is deleterious it is called and Adverse Outcome (AO). The complete
chain of events is therefore called AO Pathway (AOP) and connects the initial exposure, through a
molecular interaction to a physiological change in the organism. At sufficient concentrations of the
chemical and durations of exposure, a KE can increase to a level where it will trigger another KE to shut
down, overcoming cell defence mechanisms a
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