1.1 The assay presented in this report
We here present the development and validation of the RTgill-W1 cell line assay to predict fish acute toxicity of chemicals in vitro. In this test, confluent monolayers of the gill cells are exposed in a completely defined, protein-free exposure medium, L-15/ex, for 24 h in 24-well tissue culture plates. Chemical concentrations in the exposure medium are quantified at the onset and upon termination of exposure. Cell viability after exposure is analysed by three fluorescent indicator dyes, measured on the same set of cells, and compared to unexposed cells, which serve as control. Combining the cell viability measurements and the measured chemical exposure concentrations, effective concentrations impacting 50 % of the gill cells (EC50 value) are calculated based on concentration-response modelling. [...]

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

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 which 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 emb ryos. The algorithm provided an NtC that is more protective than NOEC (No - Observed - Effect - Concentration), NEC (modeled No - Effect Concentration), EC10 and Benchmark Dose (BMD). Despite focusing on small scale bioassays here, this study indicates that the NtC algorithm could be used in various systems. Its application on the survival of zebrafish embryos and on 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 code, and as a free, user friendly online application.

Permanent fish cell lines constitute a promising complement or substitute for fish in the environmental risk assessment of chemicals. We demonstrate the potential of a set of cell lines originating from rainbow trout (Oncorhynchus mykiss) to aid in the prediction of chemical bioaccumulation in fish, using benzo[a]pyrene (BaP) as a model chemical. We selected three cell lines from different tissues to more fully account for whole-body biotransformation in vivo: the RTL-W1 cell line, representing the liver as major site of biotransformation, and the RTgill-W1 (gill) and RTgutGC (intestine) cell lines, as important environment-organism interfaces, which likely influence chemical uptake. All three cell lines were found to effectively biotransform BaP. However, rates of in vitro clearance differed, with the RTL-W1 cell line being most efficient, followed by RTgutGC. Co-exposures with α-naphthoflavone as potent inhibitor of biotransformation, assessment of CYP1A catalytic activity, and the progression of cellular toxicity upon prolonged BaP exposure revealed that BaP is handled differently in the RTgill-W1 compared to the other two cell lines. Application of the cell-line-derived in vitro clearance rates into a physiology-based toxicokinetic model predicted a BaP bioconcentration factor (BCF) of 909-1057 compared to 920 reported for rainbow trout in vivo.

Die Identifizierung und Überwachung von Chemikalieneffekten auf Organismen in der Umwelt beruht auf einem breiten Spektrum von Herangehensweisen und Messpunkten. Allerdings wurden bisher viele der existierenden Untersuchungsmethoden lediglich für wenige Modellorganismen entwickelt. Die Berücksichtigung der Artenvielfalt und der Vielfalt an Reaktionen auf Umweltstressoren ist daher nach wie vor eine grosse Herausforderung. Die Analyse von messengerRNA (mRNA), welche für ausgewählte Biomarkermoleküle kodieren, ist dagegen ein vielversprechender Ansatz, da er auf ein weites Spektrum an Organismen und biologische Antworten übertragbar ist.
In diesem Projekt sollten die Einflüsse von Mikroschadstoffeintrag von Abwässern auf Flussökosysteme untersucht werden. Es wurde ein Biomarker-Genset für die Bachforelle (Salmo trutta) und die Regenbogenforelle (Oncorhynchus mykiss) etabliert, um die Wirkung von Mikroschadstoffen, wie Pharmaka und Pestizide, im Fisch nachweisen zu können. Ein typisches Biomarker-Set besteht aus 20-25 spezifischen Genen, welche verschiedene zelluläre Stressantworten, wie allgemeinen Stress, Metall- und oxidativen Stress, Biotransformation, Immunregulation usw. reflektieren. Die Regulation dieser Gene wurde in Leber und Nierengewebe in Bachforellen analysiert, welche ober- und unterströmig von vier verschieden Abwasserreinigungsanlagen (ARA) gefangen wurden. Zwei Standorte wurden doppelt untersucht: Herisau (Appenzell) vor und nach der Aufschaltung der Pulveraktivkohle (PAK)- Abwasserreinigungs-einheit und Steinach (St. Gallen) vor und nach dem Stopp der Abwassereinleitung. An diesen Standorten sollte überprüft werden ob mit dieser Methode Veränderungen der Wasserqualität und der Effekte auf die Fische erfasst werden können. Die Daten zeigen, dass die mRNA Regulation standortspezifisch ist. Fische, welche Unterhalb der Kläranalagen gefangen wurden, zeigen eine erhöhte Stressantwort an. Zudem konnten wir aufgrund der Genexpression das Einwirken von spezifischen Gruppen von Chemikalien identifizieren, wie Schwermetalle, endokrine Disruptoren, Pharmaka oder Pestizide. Tatsächlich bestätigte die chemische Analytik das Vorhandensein solcher Chemikalien im Wasser an den Probenahmestandorten. Ebenfalls konnten positive Effekte auf die Wasserqualität durch die Umbauten der ARAs in Herisau und Steinach mittels Genexpressionsanalyse in den untersuchten Fischen bestätigt werden.
Im Projekt ReTREAT auf der ARA Neugut wurden verschiedene Nachbehandlungen, im Anschluss an die Ozonung von Abwasser, auf deren Leistungsfähigkeit untersucht, toxische Transformations- oder Oxidationsnebenprodukte zu reduzieren. Zur Erfassung der ökotoxikologischen Wirkung wurden zahlreiche Biotests durchgeführt, darunter auch ein Fish Early Life Stage-Test (FELST) mit Regenbogenforellen (Oncorhynchus mykiss). Als Ergänzung zu den Standardendpunkten dieses Tests wurden normalerweise nicht erfassbare subletale Effekte mittels einem spezifischen Biomarker-Genset untersucht. Die Ergebnisse bestätigen die Effizienz der Ozonung, organische Mikroverunreinigungen aus dem Abwasser zu eliminieren und hervorgerufene subletale Effekte auf die Fische zu verringern. In Übereinstimmung mit den meisten anderen Tests konnte die gute Eignung der Nachbehandlung des ozonierten Abwassers durch den frischen Aktivkohlefilter (GAK2) bestätigt werden.
Im Projekt ReTREAT auf der ARA Neugut wurden verschiedene Nachbehandlungen, im Anschluss an die Ozonung von Abwasser, auf deren Leistungsfähigkeit untersucht, toxische Transformations- oder Oxidationsnebenprodukte zu reduzieren. Zur Erfassung der ökotoxikologischen Wirkung wurden zahlreiche Biotests durchgeführt, darunter auch ein Fish Early Life Stage-Test (FELST) mit Regenbogenforellen (Oncorhynchus mykiss). Als Ergänzung zu den Standardendpunkten dieses Tests wurden normalerweise nicht erfassbare subletale Effekte mittels einem spezifischen Biomarker-Genset untersucht. Die Ergebnisse bestätigen die Effizi-enz der Ozonung, organische Mikroverunreinigungen aus dem Abwasser zu eliminieren und hervorgerufene subletale Effekte auf die Fische zu verringern. In Übereinstimmung mit den meisten anderen Tests konnte die gute Eignung der Nachbehandlung des ozonierten Abwassers durch den frischen Aktivkohlefilter (GAK2) bestätigt werden.
Damit ist dies eine vielversprechende Methode für die Überwachung der Wasserqualität und der Fischgesundheit. Die Biomarker-Gensets sind sehr flexibel; weitere Anpassung für spezifische Effektmessungen, Übertragung auf andere Fischarten sowie weitere Anwendungen sind möglich.
Zudem wird versucht das Umweltmonitoring auf etablierte Zelllinien vom Fisch zu erweitern, welche eine tierfreie Alternative für die Umweltüberwachung darstellen könnten. Dabei werden permanente Zelllinien der Regenbogenforelle eingesetzt. Diese werden direkt im Labor mit Wasserproben, die an den Feldstandorten gewonnen wurden, exponiert. Die danach in diesen Zellen gemessene Expression wurde mit der Genexpression von im Fluss gefangenen Fischen und der chemischen Analytik verglichen. Stimmen die biologischen Antworten überein, könnten die Fischzellen zukünftig die Umweltüberwachung "übernehmen". Bisher konnte eine gute Übereinstimmung mit einigen Markergenen gefunden werden.

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 Cu²⁺ 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.

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 96 h 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 48/96 h 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.

In order to protect human health and the environment, chemicals have to undergo risk assessment before they come onto the market. For this purpose, thousands of animal tests are carried out every year. Eawag researchers have now shown that the acute toxicity of chemicals to fish can also be reliably predicted with a rainbow trout gill cell line. An international round robin test marks the next step on the long road to certification of the new 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.

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).