Predicting Fish Acute Toxicity Using a Fish Gill Cell Line-Based Toxicity Assay Katrin Tanneberger, † Melanie Knö bel, † Frans J. M. Busser, ‡ Theo L. Sinnige, ‡ Joop L. M. Hermens, ‡ and Kristin Schirmer †,§,∥, * † Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dü bendorf, Switzerland ‡ Institute for Risk Assessment Sciences, University of Utrecht, 3508 TD Utrecht, The Netherlands § ETH Zü rich, Department of Environmental Systems Science, 8092 Zü rich, Switzerland ∥ EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015 Lausanne, Switzerland * S Supporting Information ABSTRACT: The OECD test guideline 203 for determination of fish acute toxicity requires substantial numbers of fish and uses death as an apical end point. One potential alternative are fish cell lines; however, several studies indicated that these appear up to several orders of magnitude less sensitive than fish. We developed a fish gill cell line-based (RTgill-W1) assay, using several measures to improve sensitivity. The optimized assay was applied to determine the toxicity of 35 organic chemicals, having a wide range of toxicity to fish, mode of action and physicochemical properties. We found a very good agreement between in vivo and in vitro effective concentrations. For up to 73% of the tested compounds, the difference between the two approaches was less than 5-fold, covering baseline toxicants but as well compounds with presumed specific modes of action, including reactivity, inhibition of acetylcholine esterase or uncoupling of oxidative phosphorylation. Accounting for measured chemical concentrations eliminated two outliers, the hydrophobic 4-decylaniline and the volatile 2,3-dimethyl-1,3- butadiene, with an outlier being operationally defined as a substance showing a more than 10-fold difference between in vivo/in vitro effect concentrations. Few outliers remained. The most striking were allyl alcohol (2700-fold), which likely needs to be metabolically activated, and permethrin (190-fold) and lindane (63-fold), compounds acting, respectively, on sodium and chloride channels in the brain of fish. We discuss further developments of this assay and suggest its use beyond predicting acute toxicity to fish, for example, as part of adverse outcome pathways to replace, reduce, or refine chronic fish tests. ■ INTRODUCTION The test for acute fish toxicity 1 is the most commonly used animal test in environmental risk assessment. This test requires a substantial number of fish, 42−60, takes five working days, produces liters of toxic waste and uses death as integrative but crude end point. In Europe alone, 140 000 fish were used in 2008 for regulatory requirements only to comply with REACh (Registration, Evaluation, Authorization and Restriction of Chemicals); 2 three millions of fish are used annually in North America for whole effluent testing (SE. Belanger, Procter & Gamble, personal communication). Therefore, an alternative, nonanimal approach has long been sought. The use of fish cell lines as surrogates for testing the toxicity of individual aquatic contaminants to fish was first proposed by Rachlin and Perlmutter. 3 Fish cell lines are preferred over mammalian cells because they should better reflect the properties of fish than mammalian cells do. Chemicals or water samples can be applied to fish cells at temperatures more typical of the temperatures to which fish would be exposed. 4 Moreover, fish cells are generally easier to maintain and more tolerant to simple culture conditions. 5 A considerable number of studies compared in vitro cytotoxicity of chemicals to fish cell lines with the in vivo fish acute toxicity and confirmed the general usability of fish cell lines (reviewed by ref 6; e.g., refs 7 and 8). Yet, fish cell lines frequently appeared to underestimate the in vivo toxicity by up to 3 orders of magnitude. Schirmer proposed several routes for advancing fish cell line-based toxicity assays to overcome the aforementioned hurdle: selecting cell lines derived from tissues that reflect the specific mode of action of a particular chemical; increasing sensitivity of the cellular response by modification of the culture environ- ment to more closely resemble the in vivo exposure; and by accounting for the chemical fraction available to the cells. 6 We here set out to explore these routes to improve the performance of a fish cell line-based toxicity assay toward its use as alternative method for the fish acute toxicity test. We selected a gill cell line from rainbow trout (Oncorhynchus Received: August 29, 2012 Revised: December 9, 2012 Accepted: December 11, 2012 Published: December 11, 2012 Article pubs.acs.org/est © 2012 American Chemical Society 1110 dx.doi.org/10.1021/es303505z | Environ. Sci. Technol. 2013, 47, 1110−1119