Viability assay with fish gill cell line to assess acute toxicity

Commonly used acronym: RTgill-W1 cell line assay

Scope of the method

The Method relates to
  • Environment
The Method is situated in
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Rainbow trout, Oncorhynchus mykiss
Type of cells/tissues/organs
Gill tissue


Method keywords
  • cell viability test
  • fish gill cell line
  • cell metabolic activity
  • lysosomal membrane integrity
  • cell membrane integrity
Scientific area keywords
  • fish acute toxicity
  • chemical exposure
Method description

The rainbow trout gill cell line assay quantifies cell viability using fluorescent measurements for metabolic activity (Alamar Blue, AB), cell membrane integrity (5-CarboxyFluorescein DiAcetate AcetoxyMethyl ester, CFDA-AM) and lysosomal membrane integrity (Neutral Red, NR). Chemicals are added to confluent RTgill-W1 cell monolayers in 24-well plates with L-15/ex medium (a simplified version of L-15 cell culture medium without serum). Cells are incubated for 24 hours in the incubator (19°C, normal atmosphere, in the dark). At the end of the exposure, cell viability measurements are performed with 3 fluorescent indicator dyes on the same set of exposed cells.

Lab equipment
  • Laminar flow ;
  • Incubator (room temperature, no CO2) ; 
  • Microplate reader for fluorescence detection.
Method status
  • History of use
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential

  • Cell line model with limited requirements ; 
  • Robust assay: repeatability and reproducibility is shown through inter- and intralaboratory studies ;
  • Alternative model to predict fish acute toxicity.

Exposure of chemicals (bioavailability).

References, associated documents and other information


Fischer, M ; Belanger, SE ; Berckmans, P ; Bernhard, MJ ; Blaha, L ; Schmid, DEC ; Dyer, SD ; Haupt, T ; Hermens, JLM ; Hultman, MT ; Laue, H ; Lillicrap, A ; Minarikova, M ; Natsch, A ; Novak, J ; Sinnige, TL ; Tollefsen, KE ; von Niederhausern, V ; Witters, H ; Zupanic, A ; & K. Schirmer (2019). Repeatability and reproducibility of the RTgill-W1 cell line assay for predicting fish acute toxicity. Toxicological Sciences, 169 (2), 353-3640.

Tanneberger, K., Knoebel, M., Busser, F. J. M., Sinnige, T. L.,Hermens, J. L. M., and Schirmer, K. (2013). Predicting fish acute toxicity using a fish gill cell line-based toxicity assay. Environ. Sci. Technol. 47, 1110–1119.

ISO 21115:2019. Water quality — Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1).


Vlaamse Instelling voor Technologisch Onderzoek (VITO)
Flemish Region


Swiss Federal Institute of Aquatic Science and Technology (EAWAG)