ISO 21115:2019
Water quality — Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1)
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).
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
4.1 Cell viability assay
4.2 Key differences in water sample and chemical testing procedure
5 Interferences
5.1 Matrix effects by effluent samples
5.2 Interferences of water constituents or chemicals with fluorescent dye assays
6 Reagents
6.1 General
6.2 Ready-for-use purchased reagents
6.3 Freshly prepared solutions
7 Apparatus and material
7.1 General equipment
7.2 Whole-water sample/ex preparation
7.3 Cell seeding and plate dosing
7.4 Preparation/dosing of stock solutions
7.5 Sampling for chemical analysis
7.6 Detection of cytotoxicity
8 Procedure
8.1 Cell line used
8.2 Seeding cells into 24-well plates
8.2.1 General
8.2.2 Preparation of working materials and solutions for cell seeding
8.2.3 Seeding cells: step-by-step performance
8.3 Cell exposure
8.3.1 General
8.3.2 Exposure of cells to water samples
8.3.3 Exposure of cells to chemicals
8.4 Determination of cytotoxicity
8.4.1 General
8.4.2 Visual control of cell damage
8.4.3 Preparation of working materials and solutions for cytotoxicity measurement
8.4.4 Measuring cytotoxicity — Step-by-step performance
8.4.5 Parameters for the fluorescence measurement
8.4.6 Chemical analyses of the test samples
9 Preparation and expression of results
10 Validity criteria of the test
10.1 No cells-control wells
10.2 Positive control in L-15/ex
10.3 Positive control in w-ws/ex
10.4 Solvent control
Annex A (informative) Routine cell culture of RTgill-W1
Annex B (informative) Performance of the RTgill-W1 cell line assay
Bibliography
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Principle
4.1 Cell viability assay
4.2 Key differences in water sample and chemical testing procedure
5 Interferences
5.1 Matrix effects by effluent samples
5.2 Interferences of water constituents or chemicals with fluorescent dye assays
6 Reagents
6.1 General
6.2 Ready-for-use purchased reagents
6.3 Freshly prepared solutions
7 Apparatus and material
7.1 General equipment
7.2 Whole-water sample/ex preparation
7.3 Cell seeding and plate dosing
7.4 Preparation/dosing of stock solutions
7.5 Sampling for chemical analysis
7.6 Detection of cytotoxicity
8 Procedure
8.1 Cell line used
8.2 Seeding cells into 24-well plates
8.2.1 General
8.2.2 Preparation of working materials and solutions for cell seeding
8.2.3 Seeding cells: step-by-step performance
8.3 Cell exposure
8.3.1 General
8.3.2 Exposure of cells to water samples
8.3.3 Exposure of cells to chemicals
8.4 Determination of cytotoxicity
8.4.1 General
8.4.2 Visual control of cell damage
8.4.3 Preparation of working materials and solutions for cytotoxicity measurement
8.4.4 Measuring cytotoxicity — Step-by-step performance
8.4.5 Parameters for the fluorescence measurement
8.4.6 Chemical analyses of the test samples
9 Preparation and expression of results
10 Validity criteria of the test
10.1 No cells-control wells
10.2 Positive control in L-15/ex
10.3 Positive control in w-ws/ex
10.4 Solvent control
Annex A (informative) Routine cell culture of RTgill-W1
Annex B (informative) Performance of the RTgill-W1 cell line assay
Bibliography