Department Environmental Toxicology

Predicting acute fish toxicity using in vitro models


Fish are the most frequently used vertebrates in regulatory ecotoxicology. Numerous OECD guidelines depend on the use of fish, including OECD 203 (acute fish test), OECD 210 (early life stage toxicity test) and OECD 305 (bioconcentration test). As vertebrates, fish are legally protected animals. Therefore, alternatives to reduce or replace fish tests for risk assessment of chemicals and industrial effluents are of high societal importance. 

In a transatlantic endeavor, the CEFIC-LRI/UK DEFRA funded CEllSens-Eco8 project, a strategy to predict fish acute lethality by industrial organic chemicals based on two model systems, fish cell lines and embryos of zebrafish, was developed. Upon establishment of a chemical list, which was derived based on pre-defined criteria to cover chemicals with a wide range of physico-chemical properties, different modes of action and toxicity, 34 chemicals were investigated in a rainbow trout gill (RTgill-W1) cell line assay and the zebrafish embryo toxicity test. 

Concentrations causing 50% toxic effects (EC50 cell line or LC50 embryo) were determined based on analytically confirmed exposure concentrations. Overall, a very good quantitative agreement was achieved for EC50 or LC50 values compared to LC50 from the acute fish lethality test, taken from the US EPA fathead minnow data base. The majority of test chemicals was within a 10-fold range from the line of unity of the alternatives vs. acute fish lethality tests if true exposure concentrations were accounted for (see Figure 1). 

While the zebrafish embryo test has meanwhile been accepted as the OECD test guideline 236 (see video below), the excellent performance of the RTgill-W1 cell line assay provided impetus to start bringing this potential animal replacement method to the same level of international acceptance. In addition to chemical regulation and potential application in effluent testing and product design, the cell line could also be implemented in the development of tiered approaches, e.g. along the lines of adverse outcome pathways to replace, reduce or refine chronic fish tests. 

Thus, with the support of CEFIC-LRI and UK NC3Rs, an Eawag-led round-robin test has been initiated involving six industrial and academic research laboratories from Europe and US. The overall goal is to test the robustness of the established methodology as a pre-requisite for acceptance by international organization. 


Strecker, R.; Busquet, F.; Rawlings, J. M.; Belanger, S. E.; Braunbeck, T.; Carr, G. J.; Cenijn, P.; Fochtman, P.; Gourmelon, A.; Hübler, N.; Kleensang, A.; Knöbel, M.; Kussatz, C.; Legler, J.; Lillicrap, A.; Martínez-Jerónimo, F.; Polleichtner, C.; Rzodeczko, H.; Salinas, E.; Schneider, K. E.; Scholz, S.; van den Brandhof, E.-J.; van der Ven, L. T. M.; Walter-Rohde, S.; Weigt, S.; Witters, H.; Halder, M. (2014) OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing, Regulatory Toxicology and Pharmacology, 69(3), 496-511, doi:10.1016/j.yrtph.2014.05.018, Institutional Repository
Schirmer, K.; Knöbel, M.; Tanneberger, K. (2013) Kiemenzellen statt Fische für Toxizitätstests, Eawag Newsletter [dtsch. Ausg.], 1-7, Institutional Repository
Tanneberger, K.; Knöbel, M.; Busser, F. J. M.; Sinnige, T. L.; Hermens, J. L. M.; Schirmer, K. (2013) Predicting fish acute toxicity using a fish gill cell line-based toxicity assay, Environmental Science and Technology, 47(2), 1110-1119, doi:10.1021/es303505z, Institutional Repository
Knöbel, M.; Busser, F. J. M.; Rico-Rico, Á.; Kramer, N. I.; Hermens, J. L. M.; Hafner, C.; Tanneberger, K.; Schirmer, K.; Scholz, S. (2012) Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis, Environmental Science and Technology, 46(17), 9690-9700, doi:10.1021/es301729q, Institutional Repository