OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Predicting fish acute toxicity using a fish gill cell line-based toxicity assay.

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.

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

A validated algorithm for selecting non-toxic chemical concentrations.

The maximal chemical concentration that causes an acceptably small or no effect in an organism or isolated cells is an often-sought-after value in toxicology. Existing approaches to derive this value have raised several concerns; thus, it is often chosen case-by-case based on personal experience. To overcome this ambiguity, we propose an approach for choosing the non-toxic concentration (NtC) of a chemical in a rational, tractable way. We developed an algorithm that identifies the highest chemical concentration that causes no more than 10% effect (= EC10) including the modeled 95% confidence intervals and considering each of the measured biological replicates; and whose toxicity is not significantly different from no effect. The developed algorithm was validated in two steps: by comparing its results with measured and modeled data for 91 dose-response experiments with fish cell lines and/or zebrafish embryos; and by measuring actual effects caused by NtCs in a separate set of experiments using a fish cell line and zebrafish embryos. The algorithm provided an NtC that is more protective than NOEC (no-observed-effect-concentration), NEC (modeled no-effect concentration), EC10 and BMD (benchmark dose). Despite focusing on small-scale bioassays here, this study indicates that the NtC algorithm could be used in various systems. Its application to the survival of zebrafish embryos and to metabolic activity in cell lines showed that NtCs can be applied to different effect measurements, time points, and levels of biological organization. The algorithm is available as Matlab and R source code, and as a free, user-friendly online application.

Natural water as the test medium for Ag and CuO nanoparticle hazard evaluation: An interlaboratory case study.

Engineered nanoparticles (NPs) have realistic potential of reaching natural waterbodies and of exerting toxicity to freshwater organisms. The toxicity may be influenced by the composition of natural waters as crucial NP properties are influenced by water constituents. To tackle this issue, a case study was set up in the framework of EU FP7 NanoValid project, performing an interlaboratory hazard evaluation of NPs in natural freshwater. Ag and CuO NPs were selected as model NPs because of their potentially high toxicity in the freshwater. Daphnia magna (OECD202) and Danio rerio embryo (OECD236) assays were used to evaluate NP toxicity in natural water, sampled from Lake Greifen and Lake Lucerne (Switzerland). Dissolution of the NPs was evaluated by ultrafiltration, ultracentrifugation and metal specific sensor bacteria. Ag NP size was stable in natural water while CuO NPs agglomerated and settled rapidly. Ag NP suspensions contained a large fraction of Ag(+) ions and CuO NP suspensions had low concentration of Cu(2+) ions. Ag NPs were very toxic (48 h EC50 1-5.5 µg Ag/L) to D. magna as well as to D. rerio embryos (96 h EC50 8.8-61 µg Ag/L) in both standard media and natural waters with results in good agreement between laboratories. CuO NP toxicity to D. magna differed significantly between the laboratories with 48 h EC50 0.9-11 mg Cu/L in standard media, 5.7-75 mg Cu/L in Lake Greifen and 5.5-26 mg Cu/L in Lake Lucerne. No toxicity of CuO NP to zebrafish embryos was detected up to 100 mg/L independent of the medium used. The results show that Ag and CuO NP toxicity may be higher in natural water than in the standard media due to differences in composition. NP environmental hazard evaluation can and should be carried out in natural water to obtain more realistic estimates on the toxicity.