Predicting Fish Acute Toxicity Using a Fish Gill Cell Line-Based Toxicity Assay

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.

Photodegradation of Atrazine on TiO<sub>2</sub>―Products Toxicity Assessment

“…An in vitro cytotoxicity test with a RTgill-W1 cell line was performed according to the method described by Tanneberger et al [16]. RTgill-W1 cell line acquired from the American Type Culture Collection (ATCC) was cultured under standard conditions [17] in white L-15 medium (Sigma Aldrich, Czech Republic) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Czech Republic).…”

A validated algorithm for selecting non-toxic chemical concentrations_suppl1

“…Due to the simplicity of this approach, it is possible that the fitted 95%CI are so narrow that already very creasing chemical concentrations and all other columns are the biological replicates of the measured respective effects (in %) on testing objects. The regression-based dose-response curve (in our case Equation 1, chosen based on previous studies with fish cells and embryos (Knöbel et al, 2012;Tanneberger et al, 2013;Yue et al, 2015)) is fitted to the data by applying the Matlab "fit" function using the non-linear least-squares method.…”

“…All experiments with fish cell lines presented in this study were essentially performed as described previously (Tanneberger et al, 2013;Schirmer et al, 1997), but different well plates, cell number and medium volumes were used in some cases (see detailed experimental set-ups and procedure in Tab. S1 4 ).…”

“…The effects on survival of the determined NtCs were measured for the following twelve chemicals with the RTgill-W1 cell line according to the test procedures established by Tanneberger et al (2013): DFZ, TPZ, DEP, PAR, TCP, AAL, MED, DNP, 2AE, SDS, DBP and ANI (Tab. 1).…”

“…1). Moreover, the following five chemicals were tested in triplicates for impact on zebrafish embryo survival according to Knöbel et al (2012) (Knöbel et al, 2012) Data from Knöbel et al (2012), Tanneberger et al (2013) and Yue et al (2015) are based on the measured concentrations at the beginning and the end of experiments while for all other chemicals nominal concentrations (measured data not available) were used.…”